Anti-TMPRSS2 antibodies and antigen binding fragments

Abstract

The present invention includes antibodies, or antigen-binding fragments thereof, that specifically bind to TMPRSS2 and methods of using such antibodies and fragments to treat or prevent viral infections (e.g., influenza virus infections).

Description

[0001] This application is a divisional application of Chinese application No. 201980021929.3, with an application date of January 24, 2019 and an invention title of "Anti-TMPRSS2 Antibodies and Antigen-Binding Fragments".

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 622,292, filed on January 26, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to antibodies and antigen-binding fragments that specifically bind to TMPRSS2 and methods of treating or preventing viral infections with such antibodies and fragments. Background of the Invention

[0005] Influenza viruses have acquired resistance to currently used drugs targeting the viral neuraminidase (NA) or the ion channel protein matrix protein 2 (M2). The emergence of resistance highlights the need to develop new antiviral strategies. Host cell targeting can reduce or avoid the emergence of escape mutants, but may create "traps" due to widespread expression and raise concerns about toxicity. It has been shown that the fusion proteins of multiple respiratory viruses require cleavage by host proteases for activation (Shirato et al., Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology, 91, e01387–16 (2017); Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017); Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015); Zmora et al., TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015)), including influenza (Zmor et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017); et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011); Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology, 84, 10016–10025 (2010); Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice, Journal of Virology, (2014), May; 88(9):4744-51).

[0006] For activation, the influenza A hemagglutinin precursor (HA0) needs to be cleaved into HA1 and HA2 by host serine proteases. For example, transmembrane serine protease 2; TMPRSS2, TMPRSS4, and TMPRSS11D, as well as human airway trypsin-like protease (HAT), have been implicated in HA cleavage (Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology, 84, 10016–10025 (2010); Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium, Journal of Virology, 2006 Oct; 80(19):9896-8; International Patent Application Publication No. WO2017 / 151453). Additionally, TMPRSS2 is a target for anti-cancer therapies. See, for example, WO2008127347 and WO2002004953. The fusion between TMPRSS2 and ERG (TMPRSS2:ERG) is a gene fusion known to primarily drive prostate cancer development triggered by ERα and repressed by ERβ. Bonkhoff, Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression, Prostate 78(1):2-10 (2018). Brief Description of the Invention

[0008] Although there are small molecule inhibitors of TMPRSS2 and research antibodies, such as those useful for immunohistochemistry, there is a need in the art for neutralizing therapeutic anti-TMPRSS2 antibodies and their use for treating or preventing viral infections. See, e.g., Shen et al., Biochimie 142:1-10 (2017); WO2008127347; WO2002004953; US9498529; antibody ab92323 available from Abcam (Cambridge, MA) or antibodies c-515727 and c-101847 available from Santa Cruz Biotech (Dallas, TX). The present invention partly meets this need by providing human anti-human TMPRSS2 antibodies, such as H1H7017N and combinations thereof (e.g., including, anti-influenza HA antibodies (e.g., group I HA or group II HA)) and methods of using the same to treat viral infections.

[0009] The present invention provides neutralizing human antigen-binding proteins that specifically bind to human TMPRSS2, e.g., antibodies or antigen-binding fragments thereof. For example, in one embodiment of the invention, the antigen-binding protein comprises: (a) CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulin heavy chain comprising the amino acid sequences set forth in SEQ ID NO:2, 17, or 19; and / or

[0010] (b) The CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 4 or 18. In one embodiment of the present invention, the antigen-binding protein comprises: (a) a light chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 or 18; and / or (b) a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19. In one embodiment of the present invention, the present invention provides an antigen-binding protein comprising: (a) the CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin, the light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 4 or 18 and having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 or 18; and / or (b) the CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin, the heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19 and having at least 90% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19. For example, in one embodiment of the present invention, the antigen-binding protein comprises a light chain immunoglobulin variable region comprising: (a) CDR-H1 comprising the amino acid sequence: G F T F S S Y G (SEQ ID NO: 6); (b) CDR-H2 comprising the amino acid sequence: I W N D G S Y V (SEQ ID NO: 8); (c) CDR-H3 comprising the amino acid sequence: A R E G E W V L Y Y F D Y (SEQ ID NO: 10); and a heavy chain immunoglobulin variable region comprising: (a) CDR-L1 comprising the amino acid sequence: Q S I S S W (SEQ ID NO: 12); (b) CDR-L2 comprising the amino acid sequence: K A S (SEQ ID NO: 14); and / or (c) CDR-L3 comprising the amino acid sequence: Q Q Y N S Y S Y T (SEQ ID NO: 16). The present invention also provides an antigen-binding protein comprising: (a) a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19; and / or (b) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0011] The present invention also provides any anti-TMPRSS2 antigen-binding protein that competes with any of the antigen-binding proteins described herein for binding to TMPRSS2 (e.g., as measured using a real-time, label-free biolayer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBioCorp.)); or that binds to the same or overlapping epitopes on TMPRSS2 (or a fragment thereof) as any of the antigen-binding proteins described herein.

[0012] The present invention also provides a multispecific antigen-binding protein that binds to TMPRSS2 and another antigen or binds to TMPRSS2 at a different epitope. For example, the multispecific molecule comprises (a) a first antigen-binding domain that specifically binds to TMPRSS2; and (b) a second antigen-binding domain that specifically binds to another antigen or to TMPRSS2 or to an epitope different from the epitope of the first antigen-binding domain.

[0013] The present invention also provides any anti-TMPRSS2 antigen-binding protein (e.g., an antibody or antigen-binding fragment, e.g., that comprises a sequence described herein) that comprises one or more of the following characteristics:

[0014] · inhibits the growth of influenza virus (e.g., A / Puerto Rico / 08 / 1934 (H1N1)) in cells expressing TMPRSS2 (e.g., Calu-3 cells);

[0015] · binds to the surface of cells expressing TMPRSS (e.g., MDCK / Tet-on) with an EC 50 value of 440 pM or 1.06 nM;

[0016] · does not significantly bind to MDCK / Tet-on cells that do not express TMPRSS2;

[0017] · binds to human TMPRSS2 with a K -9 of about 2.81×10 D M at about 25°C;

[0018] · binds to human TMPRSS2 with a K -9 of about 9.31×10 D M at about 37°C;

[0019] · binds to cynomolgus monkey TMPRSS2 with a K -8 of about 5.60×10 D M at about 25°C;

[0020] · binds to cynomolgus monkey TMPRSS2 with a K -7 of about 1.40×10 D M at about 37°C;

[0021] ·Restrict the spread of influenza virus infection of cells in vitro; and / or

[0022] ·Protect mice engineered to express human TMPRSS2 protein from death caused by influenza virus infection.

[0023] The present invention also provides a complex comprising any of the antigen-binding proteins described herein, which antigen-binding proteins bind to TMPRSS2 polypeptide, for example, in vitro or in a subject.

[0024] The present invention also provides a method for producing an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) or an immunoglobulin chain thereof described herein, the method comprising: (a) introducing one or more polynucleotides encoding an immunoglobulin light chain and / or heavy chain of the antigen-binding protein; (b) culturing a host cell (e.g., a CHO cell, a Pichia cell or a Pichia pastoris cell) under conditions favorable for expression of the polynucleotide; and (c) optionally, isolating the antigen-binding protein or immunoglobulin chain from the host cell and / or the medium in which the host cell is cultured. The antigen-binding protein or immunoglobulin chain as a product of this method is part of the present invention.

[0025] A polypeptide (e.g., an immunoglobulin) comprising the following also forms part of the present invention: (a) CDR1, CDR2 and CDR3 of the V H domain of an immunoglobulin chain comprising the amino acid sequence set forth in SEQ ID NO:2; or (b) CDR1, CDR2 and CDR3 of the V L domain of an immunoglobulin chain comprising the amino acid sequence set forth in SEQ ID NO:4 (e.g., wherein the polypeptide is in a host cell).

[0026] The present invention also provides polynucleotides (e.g., DNA or RNA) encoding the polypeptides of the present invention. In one embodiment of the present invention, the polynucleotide encodes two different immunoglobulin chains (e.g., a heavy chain and a light chain). In one embodiment of the present invention, one polynucleotide encodes an immunoglobulin light chain and another polynucleotide encodes an immunoglobulin heavy chain, e.g., wherein the chains are in a host cell or in a container. For example, the polynucleotide is in a vector (e.g., a plasmid) and / or integrated into the host cell chromosome.

[0027] The host cells of the present invention (e.g., CHO cells, Pichia cells or Pichia pastoris cells) can comprise an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N), a polypeptide thereof or a polynucleotide encoding such a polypeptide and / or a vector comprising such a polynucleotide.

[0028] The present invention also provides a composition or a kit, which comprises an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) described herein in combination with other therapeutic agents (e.g., antiviral drugs and / or vaccines). For example, the composition can be a pharmaceutical composition comprising the antigen-binding protein, a pharmaceutically acceptable carrier, and optionally other therapeutic agents. The other therapeutic agents can be ledipasvir, sofosbuvir, the combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-α2b, interferon-α2a, and / or an antibody or an antigen-binding fragment thereof that specifically binds to influenza HA. In one embodiment of the present invention, the other therapeutic agent is an antibody or an antigen-binding fragment thereof selected from the following: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B;H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B;H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B;H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B: H1H18237B; H1H18238B: H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B: H1H18286B: H1H18287B;H1H18288B: H1H18289B: H1H18290B: H1H18291B: H1H18292B: H1H18293B: H1H18294B: H1H18295B: H1H18297B: H1H18298B: H1H18299B: H1H18300B: H1H18301B: H1H18302B: H1H18303B: H1H18304B: H1H18305B; H1H18306B; H1H18307B: H1H18308B: H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.;

[0029] In one embodiment of the invention, other therapeutic agents provided in combination with the anti-TMPRSS2 antigen-binding protein are antibodies or antigen-binding fragments that bind to influenza group II HA proteins, such as H1H14611N2; or antibodies or fragments comprising V H and V L of H1H14611N2; or heavy chain immunoglobulins comprising CDR-H1, CDR-H2, and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs: 25-27) and light chain immunoglobulins comprising CDR-L1, CDR-L2, and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31).

[0030] In one embodiment of the invention, other therapeutic agents provided in combination with the anti-TMPRSS2 antigen-binding protein are antibodies or antigen-binding fragments that bind to influenza group II HA proteins, such as H1H14612N2; or antibodies or fragments comprising V H and V Lantibodies or fragments thereof; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs: 41-43) and a light chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47).

[0031] In one embodiment of the present invention, other therapeutic agents provided in combination with the anti-TMPRSS2 antigen-binding protein are antibodies or antigen-binding fragments that bind to influenza group I HA protein, such as H1H11729P; or comprise V H and V L antibodies or fragments thereof; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39).

[0032] The present invention also provides a container or injection device comprising an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) or a composition thereof (e.g., a pharmaceutical composition).

[0033] The present invention also provides a method for treating or preventing viral infections other than influenza virus infection in a subject in need thereof (e.g., a human), the method comprising administering a therapeutically effective amount of the anti-TMPRSS2 antigen-binding protein described herein (e.g., H1H7017N).

[0034] The present invention also provides a method for treating or preventing cancer (e.g., prostate cancer) or infection (e.g., viral infection, such as influenza virus, coronavirus, SARS-CoV virus, MERS-CoV virus, parainfluenza virus, human metapneumovirus, or hepatitis C virus (HCV) infection) in a subject in need thereof (e.g., a human), the method comprising administering a therapeutically effective amount of the anti-TMPRSS2 antigen-binding protein described herein (e.g., H1H7017N). For example, the antigen-binding protein is administered in combination with one or more other therapeutic agents (e.g., antiviral drugs and / or vaccines).

[0035] In one embodiment of the present invention, the other therapeutic agent is a member selected from the following: ledipasvir, sofosbuvir, the combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-α2b, interferon-α2a, or an antibody or antigen-binding fragment thereof that specifically binds to influenza HA. In one embodiment of the present invention, the other therapeutic agent is an antibody or antigen-binding fragment thereof selected from the following: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B;H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B; H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B;H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B; H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B; H1H18196B; H1H18197B; H1H18198B; H1H18199B; H1H18200B; H1H18201B; H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B;H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B; H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B;H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B and H1H18335B.;

[0036] The present invention also provides a method of administering an anti-TMRPSS2 antigen-binding protein (e.g., H1H7017N) described herein to a subject (e.g., a human) body, the method comprising parenterally (e.g., subcutaneously, intravenously or intramuscularly) injecting the antigen-binding protein into the body of the subject. Brief Description of the Drawings

[0038] Figure 1 (A - B). Progression of the spread of A / Puerto Rico / 08 / 1934 (H1N1)–GFP virus in different cell lines at an initial multiplicity of infection of 0.01 (A) or 0.001 (B) in the absence of exogenous trypsin. Calu3 cells (circles), A549 cells (squares), MDCK cells (triangles), and HepG2 cells (inverted triangles).

[0039] Figure 2 . Administration of H1H7017N reduces the number of focus forming units (FFU) of A / Puerto Rico / 08 / 1934 (H1N1) at 72 hours post-infection during the infection cycle compared to isotype control antibody, no antibody, anti-HA antibody, and uninfected control.

[0040] Figure 3 (A - B). Anti-TMPRSS2, H1H7017N, binds to human and cynomolgus monkey TMPRSS2 expressed on the cell surface. (A) H1H7017N binds to MDCK / Tet-on / hTMPRSS2 and MDCK / Tet-on / mfTMPRSS2 with EC 50 values of 460 pM and 1.06 nM respectively and does not show significant binding to MDCK / Tet-on cells. (B) Control mAb1, an irrelevant isotype control antibody, does not show binding to any of the tested cell lines.

[0041] Figure 4. Survival curves of mice engineered to express human TMPRSS2 protein treated with 5 mg / kg H1H7017N on day -1 post - injection (PI) (inverted triangles, dashed lines) or day 0 post - injection (circles, solid lines), showing protection against H1N1 in a prophylactic model. Mice treated with isotype control H1H1238N (triangles, solid lines) did not show protection.

[0042] Figure 5 . Survival curves of H1N1 - infected mice engineered to express human TMPRSS2 protein treated with 10 mg / kg H1H7017N, showing protection. Mice were treated on day 0 (diamonds, dotted lines), day 1 (circles, solid lines), day 2 (inverted triangles, solid lines), or day 3 (squares, dashed lines) post - injection. Isotype control H1H1238N (triangles, solid lines) had partial protection with a survival rate of 25%.

[0043] Figure 6 . Survival curves of hTPMRSS2 mice treated with 10 mg / kg H1H7017N on day 1 (triangles) or day 2 (circles) post - injection, showing protection against H3N2. Untreated mice (squares) showed no protection.

[0044] Figure 7 (A - B). Survival curves of wild - type mice (A) or mice engineered to express human TMPRSS2 protein (B) infected with A / Puerto Rico / 08 / 1934 (H1N1) at 150 PFU (triangles), 750 PFU (squares), or 1,500 PFU (circles). Mice were weighed daily until day 14 post - injection.

[0045] Figure 8 . Survival curves of mice engineered to express human TMPRSS2 protein, which were infected with A / Aichi / 2 / 68 (HA, NA) x A / PR / 8 / 34 (H3N2) on day 0 and treated with a combination of 2.5 mg / kg each of H1H7017N and H1H14611N2 (diamonds), 10 mg / kg H1H7017N (triangles), 10 mg / kg H1H14611N2 (squares), 5 mg / kg each of H1H7017N and H1H14611N2, or 10 mg / kg hIgG1 isotype control (circles). Mice were weighed daily until day 14 post - injection.

[0046] Figure 9Survival curves of mice engineered to express human TMPRSS2 protein, which were infected with A / Puerto Rico / 08 / 1934 (H1N1) on day 1 and treated with a combination of 1 mg / kg H1H7017N and 2 mg / kg H1H11729P (circles), 2.5 mg / kg each of H1H7017N and H1H11729P (inverted triangles), 5 mg / kg H1H11729P (diamonds), 5 mg / kg H1H7017N (squares), or 5 mg / kg hIgG1 isotype control (triangles). Mice were weighed daily until day 14 post-injection. DETAILED DESCRIPTION OF THE INVENTION

[0048] Before describing the methods of the invention, it is to be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described in this invention may be used in the practice or testing of the invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0050] The term "influenza hemagglutinin", also referred to as "influenza HA", is a trimeric glycoprotein present on the surface of influenza virions that mediates virus attachment (by binding of HA1 to α-2,3- and α-2,6-sialic acid) and entry (through a conformational change) into host cells. HA consists of two domains: a globular head domain containing the receptor-binding site (which is susceptible to high-frequency antigenic mutations) and a stalk region (which is more conserved among various strains of influenza virus). Influenza HA is synthesized as a precursor (HA0), which undergoes proteolytic processing to yield two subunits (HA1 and HA2) that associate with each other to form the stalk / globular head structure. Viral HA is the most variable antigen on the virus and the stalk (HA2) is highly conserved within each group.

[0051] The term "influenza neuraminidase", also referred to as "influenza NA", is a sialidase (EC 3.2.1.18) that cleaves the α-ketosidic bond between sialic acid (N-acetylneuraminic acid) and adjacent sugar residues.

[0052] The amino acid sequence of full-length influenza HA is exemplified by the amino acid sequence of influenza isolate H1N1 A / California / 04 / 2009 provided as accession number FJ966082.1 in GenBank. The term "influenza-HA" also includes protein variants of influenza HA isolated from different influenza isolates, e.g., GQ149237.1, NC_002017, KM972981.1, etc. The term "influenza-HA" also includes recombinant influenza HA or fragments thereof. This term also encompasses influenza HA or fragments thereof conjugated with, e.g., a histidine tag, murine or human Fc, or a signal sequence.

[0053] An anti-TMPRSS2 "antigen-binding protein" is a polypeptide or a complex of more than one polypeptide (e.g., a tetrameric IgG antibody) that specifically binds to a TMPRSS2 polypeptide, e.g., an anti-TMPRSS2 antibody or an antigen-binding fragment, whether monospecific or multispecific.

[0054] TMPRSS2

[0055] TMPRSS2 (transmembrane serine protease 2) is a protein located on human chromosome 21 and belongs to the serine protease family (type II transmembrane serine protease (TTSP)) that is important for influenza virus infectivity. It has been shown that TMPRSS2 mediates the cleavage of influenza virus HA0 into HA1 and HA2.

[0056] The human TMPRSS2 gene encodes a predicted protein of 492 amino acids that is anchored to the cytoplasmic membrane. This protein is converted into its mature form by autocatalytic cleavage between Arg255 and Ile256. After cleavage, the mature protease is mostly membrane-bound, however, a portion of them can be released into the extracellular environment.

[0057] In one embodiment of the invention, human TMPRSS2 (V160M) comprises the following amino acid sequence:

[0058]

[0059] (SEQ ID NO:22; methionine 160 in bold). In one embodiment of the invention, the TMPRSS2 polypeptide does not comprise the V160M mutation. See also NM_005656.3.

[0060] In one embodiment of the invention, Macaca mulatta TMPRSS2 (S129L, N251S, I415V, R431Q, D492G) comprises the following amino acid sequence:

[0061]

[0062] (SEQ ID NO:23) In one embodiment of the present invention, the TMPRSS2 polypeptide does not contain the S129L, N251S, I415V, R431Q, and / or D492G mutations.

[0063] In one embodiment of the present invention, Mus musculus TMPRSS2 mRNA contains the nucleotide sequence described in NM_015775.2.

[0064] Virus

[0065] The present invention includes methods for treating or preventing viral infections in a subject. The term "virus" includes any virus whose infection in a subject can be treated or prevented by administering an anti-TMPRSS2 antibody or an antigen-binding fragment thereof (e.g., wherein the infectivity of the virus is at least partially dependent on TMPRSS2). In one embodiment of the present invention, the "virus" is any virus that expresses HA0 or another substrate that expresses TMPRSS2, wherein the complete infectivity of the virus for cells in a host requires proteolytic cleavage of the other substrate. The term "virus" also includes TMPRSS2-dependent respiratory viruses, which are viruses that infect the respiratory system tissues of a subject (e.g., the upper and / or lower respiratory tract, trachea, bronchi, lungs) and can be treated or prevented by administering anti-TMPRSS2. For example, in one embodiment of the present invention, the virus includes influenza virus, coronavirus, SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome (MERS) CoV), parainfluenza virus, Sendai virus (SeV), human metapneumovirus, and / or hepatitis C virus (HCV). "Viral infection" refers to the invasion and proliferation of a virus in a subject. The present invention includes multiple embodiments where the "virus" does not include influenza virus, for example, where the viral infection does not include influenza virus infection.

[0066] There are now two genera of human parainfluenza viruses (HPIV): Respirovirus (HPIV-1 and HPIV-3) and Rubulavirus (HPIV-2 and HPIV-4). Both genera (paramyxoviruses) can be morphologically separated from influenza viruses.

[0067] Sendai virus, also known as murine parainfluenza virus, is the type species in the genus Respirovirus, which also contains the following species: human parainfluenza virus 3, bovine parainfluenza virus 3, and human parainfluenza virus 1. TMPRSS2 is an activating protease for respiratory parainfluenza viruses such as parainfluenza virus and Sendai virus (SeV). See Abe et al., J. Virol. 87(21):11930-11935 (2013).

[0068] Human metapneumovirus (HMPV) is classified as the first human member of the genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae. It is an enveloped, negative-sense, single-stranded RNA virus. The RNA genome consists of 8 genes encoding 9 different proteins. HMPV is genetically related to avian metapneumovirus (AMPV), which also belongs to the genus Metapneumovirus. TMPRSS2 is expressed in human lung epithelium, efficiently cleaves the HMPV F protein and supports HMPV replication, and may be involved in the development of lower respiratory tract disease in patients infected with HMPV. See Shirogane et al., J Virol. 82(17):8942–8946 (2008).

[0069] Hepatitis C virus (HCV) is a small, enveloped, positive-sense, single-stranded RNA virus of the family Flaviviridae in humans. HCV, which has at least 6 genotypes and numerous subtypes, is a member of the genus Hepacivirus. TMPRSS2 can activate HCV infection at the post-binding and entry stages. Esumi et al., Hepatology 61(2):437-446 (2015).

[0070] Influenza viruses are members of the family Orthomyxoviridae in humans. This family represents enveloped viruses with segmented, negative-sense, single-stranded RNA segments in their genomes. There are four genera in this family: A, B, C, and Thogotovirus. Influenza virus types A, B, and C are based on the core proteins and are further divided into subtypes determined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) (e.g., subtype A / H1N1). There are at least 18 influenza hemagglutinin (“HA”) protein subtypes (H1-H18 or HA1-HA18) and at least 11 influenza neuraminidase (NA) protein subtypes (N1-N11 or NA1-NA11) used to define influenza subtypes. Group 1 influenza has subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 and NA8, NA5, Na4, and NA1 subtypes. Group 2 has subtypes H3, H4, H7, H10, H14, and H15 and NA6, NA9, NA7, NA2, and NA3 subtypes. Influenza A viruses infect a range of mammalian and avian species, while influenza B and C infections are mainly limited to humans. The eight genomic segments of influenza A and B viruses are loosely encapsidated by nucleoproteins into capsids.

[0071] Coronavirus virions are spherical, approximately 125 nm in diameter. The most prominent feature of coronaviruses is the club-shaped spike projections that emanate from the virion surface. These spikes are defining features of the virion and give it a corona-like appearance, suggesting the name, coronavirus. Inside the envelope of the virion is the nucleocapsid. Coronaviruses have a helical symmetric nucleocapsid, which is uncommon among positive-sense RNA viruses but much more common for negative-sense RNA viruses. MERS-CoV (Middle East Respiratory Syndrome Coronavirus) and SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) both belong to the Coronaviridae family. The initial attachment of the virion to the host cell is triggered by the interaction between the S protein and its receptor. The site of the receptor-binding domain (RBD) within the S1 region of the coronavirus S protein varies according to the virus, with some viruses having the RBD at the C-terminus of S1. The S-protein / receptor interaction is the main determinant of coronavirus infection of host species and also determines the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptors. After receptor binding, the virus must then enter the host cell cytosol. This is typically achieved as follows: acid-dependent proteolytic cleavage of the S protein by cathepsin, TMPRRS2, or another protease, followed by fusion of the viral membrane and the cell membrane.

[0072] Anti-TMPRSS2 antibodies and antigen-binding fragments

[0073] The present invention provides antigen-binding proteins that specifically bind to the TMPRSS2 protein or an antigenic fragment thereof, such as antibodies and antigen-binding fragments thereof.

[0074] As used herein, the term "antibody" refers to an immunoglobulin molecule (i.e., a "complete antibody molecule") comprising four chains, two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM), such as H1H7017N. Each heavy chain comprises a heavy chain variable region ("HCVR" or "V H ") (e.g., SEQ ID NO 2) and a heavy chain constant region (composed of domains C H 1, C H 2, and C H 3). Each light chain is composed of a light chain variable region ("LCVR" or "V L ") (e.g., SEQ ID NO4) and a light chain constant region (C L ). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each V H and V LComprising three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen-binding fragment thereof) are identical to the human germline sequences or are modified naturally or artificially.

[0075] Generally, the variable domains of both immunoglobulin heavy and light chains each contain three hypervariable regions, also called complementarity-determining regions (CDRs), within relatively conserved framework regions (FRs). Typically, from the N-terminus to the C-terminus, the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In one embodiment of the invention, amino acids are assigned to each domain according to the definition of Sequences of Proteins of Immunological Interest (Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia et al., (1987) J Mol. Biol. 196:901-917 or Chothia et al., (1989) Nature 342:878-883).

[0076] The present invention includes monoclonal anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" refers to a population of antibodies that are substantially homogeneous, i.e., the antibody molecules comprising the population are substantially identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. The "plurality" of such monoclonal antibodies and fragments in the composition refers to a concentration of identical (i.e., as discussed above, identical in amino acid sequence, excluding possible naturally occurring mutations that may be present in trace amounts) antibodies and fragments that is higher than the concentration that would normally be present in nature (e.g., in the blood of a host organism such as a mouse or a human).

[0077] In one embodiment of the invention, an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment, comprises a heavy-chain constant domain of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), or IgM type. In one embodiment of the invention, the antigen-binding protein, e.g., an antibody or antigen-binding fragment, comprises a light-chain constant domain of the κ or λ type.

[0078] As used herein, the term "human" antigen-binding protein, such as an antibody, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences, whether the human germline immunoglobulin sequences are in human cells or transplanted into non-human cells, e.g., mouse cells. See, e.g., US8502018, US6596541, or US5789215. The human mAbs of the invention may include, e.g., amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs and particularly in CDR3 (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, as used herein, the term "human mAb" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in human subjects. See below.

[0079] The invention includes anti-TMPRSS2 chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of using the same. As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, wherein the first and second antibodies are from different species. (US4816567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0080] The term "recombinant" antigen-binding protein, such as an antibody or antigen-binding fragment thereof, refers to such molecules produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, such as DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammalian (including transgenic non-human mammals, e.g., transgenic mice) or cell (e.g., CHO cell) expression systems or isolated from recombinant human antibody combinatorial libraries.

[0081] The recombinant anti-TMPRSS2 antigen-binding proteins disclosed herein, e.g., antibodies and antigen-binding fragments, can also be produced in an E. coli / T7 expression system. In this embodiment, a nucleic acid encoding an immunoglobulin molecule of an anti-TMPRSS2 antibody of the invention (e.g., H1H7017N) can be inserted into a pET-based plasmid and expressed in the E. coli / T7 system. For example, the invention includes a method of expressing an antibody or its antigen-binding fragment or its immunoglobulin chain in a host cell (e.g., a bacterial host cell such as E. coli such as BL21 or BL21DE3), the method comprising expressing T7 RNA polymerase in a cell further comprising a polynucleotide encoding an immunoglobulin chain operably linked to a T7 promoter. For example, in one embodiment of the invention, a bacterial host cell, such as E. coli, comprises a polynucleotide encoding a T7 RNA polymerase gene operably linked to a lac promoter, and expression of the polymerase and the chain is induced by incubating the host cell with IPTG (isopropyl-β-D-thiogalactopyranoside). See US4952496 and US5693489 or Studier and Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5;189(1):113-30.

[0082] There are several methods known in the art by which recombinant antibodies can be generated. An example of a method for the recombinant production of antibodies is disclosed in US4816567.

[0083] Transformation can be by any known method of introducing polynucleotides into a host cell. Methods of introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, biolistic injection, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, e.g., U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461, and 4,959,455.

[0084] Accordingly, the present invention includes recombinant methods for generating an anti-TMPRSS2 antigen-binding protein (such as an antibody or antigen-binding fragment thereof of the present invention) or an immunoglobulin chain thereof, said recombinant methods comprising (i) introducing one or more polynucleotides encoding an immunoglobulin light chain and / or heavy chain of an antigen-binding protein (e.g., H1H7017N or H4H7017N), e.g., a nucleotide sequence included in any one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15, e.g., wherein the polynucleotide is in a vector; and / or integrating into the host cell chromosome and / or being operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable for expression of the polynucleotide; and (iii) optionally, isolating the antigen-binding protein (e.g., antibody or fragment) or chain from the host cell and / or the medium in which the host cell is cultured. When generating an antigen-binding protein that comprises more than one immunoglobulin chain (e.g., an antibody or antigen-binding fragment), e.g., an antibody that comprises two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell results in association of the chains, e.g., association in the cell or on the cell surface or outside the cell (if such chains are secreted), thereby forming the antigen-binding protein (e.g., antibody or antigen-binding fragment). These methods include those in which only an immunoglobulin heavy chain or only an immunoglobulin light chain is expressed (e.g., any of the chains discussed herein, including mature fragments and / or variable domains thereof). Such chains can, for example, be used as intermediates when expressing an antibody or antigen-binding fragment that comprises such a chain. For example, the present invention also includes such anti-TMPRSS2 antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that comprise a heavy chain immunoglobulin (or variable domain thereof or comprising its CDRs) encoded by a polynucleotide that comprises the nucleotide sequence set forth in SEQ ID NO: 1, and a light chain immunoglobulin (or variable domain thereof or comprising its CDRs) encoded by the nucleotide sequence set forth in SEQ ID NO: 3, said antigen-binding protein being a product of such production methods and optionally the purification methods described herein. For example, in one embodiment of the present invention, the product of the method is an anti-TMPRSS2 antigen-binding protein as an antibody or fragment, said antibody or fragment comprising a V H and a V comprising the amino acid sequence set forth in SEQ ID NO: 4 L , or comprising an HC comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19 and an LC comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0085] Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for expressing anti-TMPRSS2 antigen-binding proteins. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, in particular, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocarcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and numerous other cell lines. Mammalian host cells include human cells, mouse cells, rat cells, dog cells, monkey cells, pig cells, goat cells, bovine cells, horse cells, and hamster cells. Other cell lines that can be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells.Fungal cells include yeast and filamentous fungal cells, such as, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. The present invention includes isolated host cells (e.g., CHO cells) that contain an antigen-binding protein, such as H1H7017N; or a polynucleotide encoding such a polypeptide.

[0086] The term "specifically binds" refers to those that have a stated K for an antigen such as the TMPRSS2 protein (e.g., human TMPRSS2) D of at least about 10 -8 M (e.g., 2.81X 10-9 M; 9.31X10 -9 M; 10 -9 M; 10 -10 M, 10 -11 M or 10 -12 An antigen-binding protein (e.g., mAb) having a binding affinity for M), said binding affinity being determined by real-time, label-free biolayer interferometry analysis (e.g., at 25 °C or 37 °C, e.g., by HTX biosensor) or by surface plasmon resonance (e.g., BIACORE TM ), or by solution affinity ELISA. The present invention includes antigen-binding proteins that specifically bind to the TMPRSS2 protein.

[0087] As used herein, terms such as "antigen-binding portion" or "antigen-binding fragment" of an antibody or antigen-binding protein include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues that mimic the hypervariable regions of an antibody (e.g., individual complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, microantibodies, nanobodies (e.g., as defined in WO08 / 020079 or WO09 / 138519) (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment" as used herein. In one embodiment of the present invention, the antigen-binding fragment comprises three or more CDRs of H1H7017N (e.g., CDR-H1, CDR-H2, and CDR-H3; or CDR-L1, CDR-L2, and CDR-L3).

[0088] In one embodiment of the present invention, the antigen-binding fragment of an antibody will comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to or in the same reading frame as one or more framework sequences. In an antigen-binding fragment having a V H domain and an associated V L domain, V H and VL The domains can be positioned relative to each other in any suitable arrangement. For example, the variable regions can be dimers and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0089] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) VH-CL; (viii) VL-CH1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L。In any configuration including variable and constant domains of any of the exemplary configurations listed above, the variable and constant domains can be directly connected to each other or can be connected by a full or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, antigen-binding fragments of the antibodies of the present invention can comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently (e.g., via disulfide bonds) bound to each other and / or non-covalently (e.g., via disulfide bonds) bound to one or more monomeric V H or V L domains.

[0090] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are further discussed herein.

[0091] In a specific embodiment, an antibody or antibody fragment of the present invention can be conjugated to a moiety, such as a ligand or a therapeutic moiety (“immunoconjugate”), such as an antiviral drug, a second anti-influenza antibody, or any other therapeutic moiety useful for treating a viral infection (e.g., an influenza viral infection). See below.

[0092] The present invention also provides complexes comprising an anti-TMPRSS2 antigen-binding protein as discussed herein, e.g., an antibody or antigen-binding fragment, complexed with a TMPRSS2 polypeptide or an antigenic fragment thereof and / or with a second antibody or an antigen-binding fragment thereof that specifically binds to the anti-TMPRSS2 antibody or fragment (e.g., a detectably labeled second antibody). In one embodiment of the present invention, the antibody or fragment is in vitro (e.g., immobilized to a solid substrate) or in a subject. In one embodiment of the present invention, TMPRSS2 is in vitro (e.g., immobilized to a solid substrate) or on the cell surface or in a subject. Immobilized anti-TMRPSS2 antibodies and antigen-binding fragments thereof covalently linked to an insoluble matrix material (e.g., glass or a polysaccharide such as agarose or agarose gel, e.g., its beads or other particles) are also part of the present invention; optionally, wherein the immobilized antibody is complexed with TMPRSS2 or an antigenic fragment thereof or a second antibody or fragment thereof.

[0093] "Isolated" antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides and vectors are at least partially free of other biomolecules from the cells or cell cultures from which the former are produced. Such biomolecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, sugars or other materials such as cell debris and growth media. Isolated antibodies or antigen-binding fragments may also be at least partially free of expression system components such as biomolecules from the host cell or its growth media. Generally, the term "isolated" does not mean the complete absence of such biomolecules or does not mean the absence of water, buffers or salts or does not mean the components of a pharmaceutical formulation containing the antibody or fragment.

[0094] The term "epitope" refers to an antigenic determinant (e.g., on a TMPRSS2 polypeptide) that interacts with a specific antigen-binding site (e.g., the variable region of an antibody molecule), called a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. The term "epitope" also refers to a site on an antigen that elicits a B and / or T cell response. It also refers to the region of an antigen that is bound by an antibody. Epitopes can be defined as structural epitopes or functional epitopes. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity interaction. Epitopes can be linear or conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope can include a determinant, which is a chemically active molecular surface group such as an amino acid, sugar side chain, phosphoryl or sulfonyl group, and in certain embodiments, can have specific three-dimensional structural features, and / or specific charge features.

[0095] Methods for determining epitopes of antigen-binding proteins (e.g., antibodies or fragments or polypeptides) include alanine scanning mutagenesis analysis, peptide blotting analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide that interact with an antigen-binding protein (e.g., an antibody or fragment or polypeptide) (e.g., coversin) is the hydrogen / deuterium exchange method detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of an antigen-binding protein, e.g., an antibody or fragment or polypeptide, to the deuterium-labeled protein. Next, the TMPRSS2 protein / antigen-binding protein complex is transferred to water and the exchangeable protons within the amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a lower rate than the exchangeable protons within the amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antigen-binding protein interface may retain deuterium and thus show a relatively higher mass compared to the amino acids not incorporated into the interface. After dissociation of the antigen-binding protein (e.g., an antibody or fragment or polypeptide), the target protein is subjected to protease cleavage and mass spectrometry, thus revealing the deuterium-labeled residues corresponding to the specific amino acids that interact with the antigen-binding protein. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0096] As used herein, the term "compete" refers to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) that binds to an antigen (e.g., TMPRSS2) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) to the antigen. The term also includes competition between two antigen-binding proteins (e.g., antibodies) in two orientations, i.e., the first antibody binds and blocks the binding of the second antibody and vice versa. In certain embodiments, the first antigen-binding protein (e.g., an antibody) and the second antigen-binding protein (e.g., an antibody) can bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) can bind to different, but e.g., overlapping epitopes, where the binding of one inhibits or blocks the binding of the second antibody, e.g., by steric hindrance. The competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, e.g., by real-time, label-free biolayer interferometry assay. In one embodiment of the invention, the competition between the first and second anti-TMPRSS2 antigen-binding proteins (e.g., antibodies) is determined by measuring the ability of a immobilized first anti-TMPRSS2 antigen-binding protein (e.g., an antibody) (initially not complexed with the TMPRSS2 protein) to bind to a soluble TMPRSS2 protein complexed with a second anti-TMPRSS2 antigen-binding protein (e.g., an antibody). A decrease in the ability of the first anti-TMPRSS2 antigen-binding protein (e.g., an antibody) to bind to the complexed TMPRSS2 protein relative to the uncomplexed TMPRSS2 protein indicates competition between the first and second anti-TMPRSS2 antigen-binding proteins (e.g., antibodies). The degree of competition can be expressed as a percentage decrease in binding. This competition can be measured using real-time, label-free biolayer interferometry assay (e.g., on an OctetRED384 biosensor (Pall ForteBio Corp.)), ELISA (enzyme-linked immunosorbent assay), or SPR (surface plasmon resonance).

[0097] Binding competition between anti-TMPRSS2 antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using real-time, label-free biolayer interferometry assays on an Octet RED384 biosensor (Pall ForteBio Corp.). For example, to determine the competition between two anti-human TMPRSS2 monoclonal antibodies, the anti-TMPRSS2 mAb can first be captured onto the tip of an Octet biosensor (Pall ForteBio Corp., #18-5060) coated with an anti-hFc antibody by immersing the tip into a solution of the anti-human TMPRSS2 mAb (subsequently referred to as "mAb1"). As a positive control for blocking, the biosensor tip with the captured antibody can then be saturated with a blocking mAb by immersing it into a solution of a known blocking isotype control mAb (subsequently referred to as the "blocking mAb"). To determine whether mAb2 competes with mAb1, the biosensor tip can then be immersed into a ternary complex solution of human TMPRSS2 polypeptide and a second anti-human TMPRSS2 mAb (subsequently referred to as "mAb2"), where the ternary complex solution has been pre-incubated for a period of time, and the binding of mAb1 to the TMPRSS2 polypeptide can be determined. The biosensor tip can be washed in buffer between each step of the experiment. The real-time binding response can be monitored during the experiment and the binding response at the end of each step can be recorded.

[0098] For example, in one embodiment of the invention, the competition assay is conducted at 25 °C and a pH of about 7 (e.g., 7.4), for example, in the presence of a buffer, salts, surfactants, and non-specific proteins (e.g., bovine serum albumin).

[0099] Generally, an antibody or antigen-binding fragment of the invention that is modified in some way retains the ability to specifically bind to TMPRSS2, e.g., when activity is expressed on a molar basis, retains at least 10% of its TMPRSS2 binding activity (when compared to the parental antibody). Preferably, the antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the TMPRSS2 binding affinity of the parental antibody. It is also contemplated that the antibody or antigen-binding fragment of the invention may contain conservative or non-conservative amino acid substitutions that substantially do not alter its biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

[0100] Polypeptides such as immunoglobulin chains (e.g., H1H7017N V H 、V L, a "variant" of HC or LC)) refers to a polypeptide comprising an amino acid sequence that is at least about 70 - 99.9% identical or similar (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) to the reference amino acid sequence described herein (e.g., SEQ ID NO: 2, 4, 17, 18 or 19); at this time, comparison is carried out by the BLAST algorithm, and the parameters of this algorithm are selected to produce the maximum match within the entire length range of the corresponding reference sequence between the corresponding sequences (e.g., expected threshold: 10; word length: 3; maximum match within the query range: 0; BLOSUM 62 matrix; gap cost: existence 11, extension 1; conditional composition scoring matrix adjustment).

[0101] , a "variant" of a polynucleotide refers to a polynucleotide comprising a nucleotide sequence that is at least about 70 - 99.9% identical (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) to the reference nucleotide sequence described herein (e.g., SEQ ID NO: 1 or 3); at this time, comparison is carried out by the BLAST algorithm, and the parameters of this algorithm are selected to produce the maximum match within the entire length range of the corresponding reference sequence between the corresponding sequences (e.g., expected threshold: 10; word length: 28; maximum match within the query range: 0; match / mismatch score: 1, -2; gap cost: linear).

[0102] In one embodiment of the present invention, an anti-TMPRSS2 antigen-binding protein, e.g., an antibody of the present invention and its antigen-binding fragment, comprises a heavy-chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity to the amino acids described in SEQ ID NO: 2, 17 or 19 and / or a light-chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity to the amino acids described in SEQ ID NO: 4 or 18.

[0103] In addition, the variant anti-TMPRSS2 antigen-binding proteins can include polypeptides that contain the amino acid sequences described herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. For example, the present invention includes antigen-binding proteins that contain immunoglobulin light chain variants that contain the amino acid sequences described in SEQ ID NO: 4 or 18, but with one or more such mutations, and / or immunoglobulin heavy chain variants that contain the amino acid sequences described in SEQ ID NO: 2, 17, or 19, but with one or more such mutations. In one embodiment of the present invention, the variant anti-TMPRSS2 antigen-binding proteins include immunoglobulin light chain variants and / or immunoglobulin heavy chain variants, where one or more (e.g., 1 or 2 or 3) of the CDRs (e.g., CDR-L1, CDR-L2, and CDR-L3) of the immunoglobulin light chain variant have one or more such mutations (e.g., conservative substitutions), and the immunoglobulin heavy chain variant has one or more (e.g., 1 or 2 or 3) of the CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3) with one or more such mutations (e.g., conservative substitutions).

[0104] The present invention also provides variant anti-TMPRSS2 antigen-binding proteins, such as, for example, antibodies or antigen-binding fragments thereof, that contain one or more variant CDRs (e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3) that have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity or similarity to, for example, SEQ ID NO: 12, 14, 16, 6, 8, and / or 10 as described herein.

[0105] Embodiments of the present invention also include variant antigen-binding proteins, such as, for example, anti-TMPRSS2 antibodies and antigen-binding fragments thereof, where the variant antigen-binding proteins contain such immunoglobulin V H and V L ; or HC and LC that contain the corresponding V H 、V LAn amino acid sequence with 70% or more (e.g., 80%, 85%, 90%, 95%, 97% or 99%) overall amino acid sequence identity or similarity to the amino acid sequence of HC or LC, provided that the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of such immunoglobulins are not variants and contain the amino acid sequences set forth in SEQ ID NOs: 12, 14, 16, 6, 8 and 10, respectively. Thus, in such embodiments, the CDRs within the variant antigen-binding protein itself are not variants.

[0106] Conservative modified variant anti-TMPRSS2 antibodies and antigen-binding fragments thereof are also part of the present invention. "Conservative modified variant" or "conservative substitution" refers to variants in which one or more substitutions of amino acids in a polypeptide are made with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). Such changes can often be made without significantly disrupting the biological activity of the antibody or fragment. Those skilled in the art recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., page 224 (4th ed.)). In addition, substitutions of amino acids with similar structure or function are not likely to significantly disrupt biological activity.

[0107] Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any changes having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., (1992) Science 256:1443 - 45.

[0108] Functional conservative variants of anti-TMPRSS2 antibodies and antigen-binding fragments thereof are also part of the present invention. Any variant of an anti-TMPRSS2 antibody and antigen-binding fragment thereof (as discussed herein) can be a "functionally conservative variant". In some cases, such functionally conservative variants can also be characterized as conservatively modified variants. As used herein, "functionally conservative variant" refers to a variant of an anti-TMPRSS2 antibody or antigen-binding fragment thereof, in which one or more amino acid residues have been altered while not significantly altering one or more functional properties of the antibody or fragment. In one embodiment of the present invention, the functionally conservative variant of the anti-TMPRSS2 antibody or antigen-binding fragment thereof of the present invention comprises a variant amino acid sequence and exhibits one or more of the following functional properties:

[0109] · Inhibit the growth of influenza virus (e.g., A / Puerto Rico / 08 / 1934 (H1N1)) in cells expressing TMPRSS2 (e.g., Calu-3 cells);

[0110] · For example, bind to the surface of cells expressing TMPRSS (e.g., MDCK / Tet-on) with an EC 50 value of 440 pM or 1.06 nM, respectively;

[0111] · Do not significantly bind to MDCK / Tet-on cells that do not express TMPRSS2;

[0112] · Bind to human TMPRSS2 with a K -9 of approximately 2.81 X 10 D M at about 25°C;

[0113] · Bind to human TMPRSS2 with a K -9 of approximately 9.31 X 10 D M at about 37°C;

[0114] · Bind to cynomolgus monkey TMPRSS2 with a K -8 of approximately 5.60 X 10 D M at about 25°C;

[0115] · Bind to cynomolgus monkey TMPRSS2 with a K -7 of approximately 1.40 X 10 D M at about 37°C;

[0116] · Restrict the spread of influenza virus infection (e.g., H1_PR34; H1_CA09; H1_Bris; H9N2 or H3N2 influenza virus) in cells (e.g., Calu-3) in vitro; and / or

[0117] · Optionally in combination with an anti-HA antibody, protects mice engineered to express human TMPRSS2 protein from death caused by influenza virus infection (e.g., H1N1 or H3N2), e.g., where the mice are infected with a virus dose that would otherwise be lethal.

[0118] The invention includes mice engineered to express human TMPRSS2 protein, which contain in the mouse body anti-TMPRSS2 antigen-binding proteins (e.g., antibodies or antigen-binding fragments) such as H1H7017N and H4H7017N. See International Patent Application Publication No. WO2017 / 151453.

[0119] A "neutralizing" or "antagonist" anti-TMPRSS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment, refers to a molecule that inhibits the activity of TMPRSS2 to any detectable extent, e.g., inhibits the protease activity of TMPRSS2, e.g., against a substrate such as HA; Cbz-Gly-Gly-Arg-AMC (Sigma), where Cbz is benzyloxycarbonyl and AMC is 7-amino-4-methylcoumarin; influenza virus HA0; coronavirus S protein; or precursor TMPRSS2 that autocatalytically cleaves between Arg255 and Ile256, and / or inhibits influenza virus entry into cells and / or inhibits influenza virus replication in a subject.

[0120] "H1H7017N" and "H4H7017N" refer to antigen-binding proteins such as antibodies and their antigen-binding fragments, which contain a heavy chain or V H (or its variant) and a light chain or V L (or its variant); or contain a V that contains its CDRs (CDR-H1 (or its variant), CDR-H2 (or its variant), and CDR-H3 (or its variant)) H and a V that contains its CDRs (CDR-L1 (or its variant), CDR-L2 (or its variant), and CDR-L3 (or its variant)) L , e.g., where the immunoglobulin chains, variable regions, and / or CDRs contain the specific amino acid sequences described below.

[0121] In one embodiment of the invention, "H1H7017N" or "H4H7017N" refers to an antibody or its antigen-binding fragment that contains CDR-H1, CDR-H2, and CDR-H3 of an immunoglobulin heavy chain that contains the amino acid sequences described in SEQ ID NO: 2, 17, or 19; and CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulin light chain that contains the amino acid sequences described in SEQ ID NO: 4 or 18.

[0122] In one embodiment of the present invention, "H1H7017N" or "H4H7017N" refers to an antibody or an antigen-binding fragment thereof that comprises a V that comprises the amino acid sequence set forth in SEQ ID NO:2 H ; and a V that comprises the amino acid sequence set forth in SEQ ID NO:4 L .

[0123] In one embodiment of the present invention, "H1H7017N" refers to an antibody or antigen-binding fragment that comprises a heavy-chain immunoglobulin and a light-chain immunoglobulin, wherein the heavy-chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:17 and the light-chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:18.

[0124] In one embodiment of the present invention, "H4H7017N" refers to an antibody or antigen-binding fragment that comprises a heavy-chain immunoglobulin and a light-chain immunoglobulin, wherein the heavy-chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:19 and the light-chain immunoglobulin comprises the amino acid sequence set forth in SEQ ID NO:18. The term "H4H7017N" also includes embodiments in which V H is fused to wild-type IgG4 (e.g., wherein residue 108 is S).

[0125] Anti-TMRPS22 antibodies or antigen-binding fragments H1H7017N and H4H7017N

[0126] H1H7017N and H4H7017N heavy-chain variable regions (DNA)

[0127]

[0128] H1H7017N and H4H7017N heavy-chain variable regions (polypeptide)

[0129]

[0130] H1H7017N and H4H7017N light-chain variable regions (DNA)

[0131]

[0132] H1H7017N and H4H7017N light-chain variable regions (polypeptide)

[0133]

[0134] H1H7017N and H4H7017N CDR-H1 (DNA)

[0135]

[0136] H1H7017N and H4H7017N CDR-H1 (polypeptide)

[0137] G F T F S S Y G

[0138] (SEQ ID NO:6 (or a variant thereof having 1, 2, 3, or 4 point mutations and / or point deletions))

[0139] H1H7017N and H4H7017N CDR-H2 (DNA)

[0140]

[0141] H1H7017N and H4H7017N CDR-H2 (polypeptide)

[0142] I W N D G S Y V

[0143] (SEQ ID NO:8 (or a variant thereof having 1, 2, 3, or 4 point mutations and / or point deletions))

[0144] H1H7017N and H4H7017N CDR-H3 (DNA)

[0145]

[0146] H1H7017N and H4H7017N CDR-H3 (polypeptide)

[0147] A RE G E W V L Y Y F D Y

[0148] (SEQ ID NO:10 (or a variant thereof having 1, 2, 3, or 4 point mutations and / or point deletions))

[0149] H1H7017N and H4H7017N CDR-L1 (DNA)

[0150]

[0151] H1H7017N and H4H7017N CDR-L1 (polypeptide)

[0152] Q S I S S W

[0153] (SEQ ID NO:12 (or a variant thereof having 1, 2, 3, or 4 point mutations and / or point deletions))

[0154] H1H7017N and H4H7017N CDR-L2 (DNA)

[0155] AAG GCG TCT

[0156] (SEQ ID NO:13)

[0157] H1H7017N and H4H7017N CDR-L2 (polypeptide)

[0158] K A S

[0159] (SEQ ID NO:14 (or a variant thereof with point mutations and / or point deletions))

[0160] H1H7017N and H4H7017N CDR-L3 (DNA)

[0161]

[0162] H1H7017N and H4H7017N CDR-L3 (polypeptide)

[0163] Q Q Y N S Y S Y T

[0164] (SEQ ID NO:16 (or a variant thereof with 1, 2, 3, or 4 point mutations and / or point deletions))

[0165] H1H7017N

[0166] Full-length heavy chain - human IgG1

[0167]

[0168] Full-length light chain - human kappa

[0169]

[0170] H4H7017N

[0171] Full-length heavy chain - human IgG4 (S108P)

[0172]

[0173] Full-length light chain - human kappa

[0174]

[0175] The antibodies and antigen-binding fragments of the present invention comprise immunoglobulin chains that contain the amino acid sequences described herein, as well as cellular modifications and post-translational modifications of the antibody in vitro. For example, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to TMPRSS2 and contain the heavy chain and / or light chain amino acid sequences described herein (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3), and antibodies and fragments in which one or more amino acid residues are glycosylated, one or more Asn residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, the N-terminal Gln is pyroglutamic acid (pyroE), and / or the C-terminal lysine is lost.

[0176] The present invention provides containers (e.g., plastic or glass vials, e.g., with caps or chromatography columns, hollow needles, or syringe cylinders) containing the anti-TMPRSS2 antigen-binding proteins of the present invention (e.g., H1H7017N or H4H7017N).

[0177] The present invention also provides an injection device, comprising one or more antigen-binding proteins (e.g., antibodies or antigen-binding fragments) that specifically bind to TMPRSS2 (e.g., H4H7017N or H1H7017N) or a pharmaceutical composition thereof. The injection device may be packaged as a kit. The injection device is a device for introducing a substance into a subject's body via a parenteral (e.g., intramuscular, subcutaneous, or intravenous) route. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an autoinjector), which includes, for example, a barrel or cartridge that houses the fluid to be injected (e.g., containing an antibody or fragment or a pharmaceutical composition thereof), a needle that pierces the skin and / or blood vessel to inject the fluid; and a plunger that pushes the fluid out of the barrel and through the needle aperture. In one embodiment of the present invention, the injection device comprising an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof or a pharmaceutical composition thereof) from the combination of the present invention is an intravenous (IV) injection device. Such a device may include an antigen-binding protein or a pharmaceutical composition thereof in a cannula or a stylet / needle that can be joined to a tube, wherein the tube can be joined to a bag or reservoir that holds a fluid (e.g., saline), and the fluid is introduced into the subject's body through the cannula or the stylet / needle. In one embodiment of the present invention, once the stylet and the cannula are inserted into the subject's vein and the stylet is removed from the inserted cannula, an antibody or a fragment or a pharmaceutical composition thereof can be introduced into the device. The IV device can be inserted, for example, into a peripheral vein (e.g., in the palm or arm); the superior vena cava or the inferior vena cava, or inside the right atrium of the heart (e.g., central IV); or into the subclavian vein, the internal jugular vein, or the femoral vein, and advanced, for example, towards the heart until it reaches the superior vena cava or the right atrium (e.g., central venous catheter). In one embodiment of the present invention, the injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid that penetrates the epidermis to introduce an antibody or fragment or a pharmaceutical composition thereof into the subject's body. An external infusion pump is a medical device that delivers an antibody or fragment or a pharmaceutical composition thereof to the subject's body in a controlled amount. The external infusion pump can be electrically or mechanically driven. Different pumps operate in different ways. For example, an injection pump holds a fluid in a reservoir of a syringe, and a movable plunger controls the fluid delivery. An elastomeric pump holds a fluid in an expandable balloon reservoir, and the pressure from the elastic wall of the balloon drives the fluid delivery. In a peristaltic pump, a set of rollers gradually constricts along the length of a flexible tubing to push the fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.

[0178] The present invention also provides methods of administering the anti-TMPRSS2 antigen-binding proteins of the present invention (e.g., H4H7017N or H1H7017N), which methods include introducing the antigen-binding protein into the body of a subject (e.g., a human). For example, the method includes puncturing the body of the subject with the needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g., injecting into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0179] Producing human antibodies

[0180] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to generate human antibodies that specifically bind to TMPRSS2. An immunogen comprising any of the following can be used to generate antibodies against TMPRSS2. In certain embodiments of the present invention, the antibodies of the present invention are obtained from mice that are immunized with full-length, native TMPRSS2 or with live attenuated or inactivated virus or with DNA encoding a protein or a fragment thereof. Alternatively, standard biochemical techniques can be used to produce the TMPRSS2 protein or a fragment thereof and modify and use it as an immunogen. In one embodiment of the present invention, the immunogen is a recombinantly produced TMPRSS2 protein or a fragment thereof. In certain embodiments of the present invention, the immunogen can be a TMPRSS2 polypeptide vaccine. In certain embodiments, one or more booster injections can be administered. In certain embodiments, the immunogen can be a recombinant TMPRSS2 polypeptide expressed in Escherichia coli or any other eukaryotic or mammalian cell such as Chinese hamster ovary (CHO) cells.

[0181] Using the VELOCIMMUNE TM technology (e.g., see US 6,596,541, Regeneron Pharmaceuticals, ) or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against TMPRSS2 was initially isolated, which has human variable regions and murine constant regions. The technology includes generating transgenic mice that have a genome comprising human heavy and light chain variable regions operably linked to the endogenous murine constant region locus such that the mice produce antibodies comprising human variable regions and murine constant regions in response to antigen stimulation. The DNA encoding the variable regions of the antibody heavy and light chains is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing full-length human antibodies.

[0182] Typically, upon challenge with the antigen of interest Mice, lymphocytes (such as B cells) are recovered from the antibody-expressing mice. The lymphocytes can be fused with a myeloma cell line to prepare an immortalized hybridoma cell line, and these hybridoma cell lines are screened and selected to identify the hybridoma cell lines that produce antibodies specific to the antigen of interest. The DNA encoding the variable regions of the heavy and light chains is isolated and ligated to the desired heavy and light chain isotype constant regions. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, the DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be directly isolated from antigen-specific lymphocytes.

[0183] Initially, high-affinity chimeric antibodies with human variable regions and murine constant regions are isolated. As in the experimental section below, the antibodies are characterized and the desired features, including affinity, selectivity, epitope, etc., are selected. Then the murine constant regions are replaced with the desired human constant regions to produce the fully human antibodies of the present invention, such as wild-type or modified IgG1 or IgG4. Although the selected constant regions may vary depending on the specific use, the high-affinity antigen-binding and target-specific features reside in the variable regions.

[0184] Anti-TMPRSS2 antibody comprising an Fc variant

[0185] According to certain embodiments of the present invention, there is provided an anti-TMPRSS2 antigen-binding protein comprising an Fc domain, such as an antibody or an antigen-binding fragment, wherein the Fc domain comprises one or more mutations, for example, the mutations enhance or weaken the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention includes mutations in the C H 2 region or C HRegion 3 contains a mutant anti-TMPRSS2 antibody, wherein the mutation increases the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at the following positions: 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at the following positions: 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modifications at the following positions: 250 and / or 428; or modifications at the following positions: 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0186] For example, the present invention includes an anti-TMPRSS2 antigen-binding protein comprising an Fc domain, such as an antibody or antigen-binding fragment, wherein the Fc domain comprises one or more pairs or sets of mutations selected from the following: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F).

[0187] Within the scope of the present invention, anti-TMPRSS antigen-binding proteins are contemplated, such as antibodies and antigen-binding fragments thereof, that comprise any possible combination of the V that includes the aforementioned Fc domain mutations as described herein H and / or V L .

[0188] The present invention also includes such anti-TMPRSS2 antigen-binding proteins, antibodies or antigen-binding fragments that comprise the V H and a chimeric heavy chain constant (C H ) region, wherein the chimeric C H region comprises segments derived from C H regions of more than one immunoglobulin isotype. For example, an antibody of the present invention can comprise a chimeric C H region, the chimeric C H region comprising a partial or complete C H 2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, the partial or complete being combined with a partial or complete C H 3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, an antibody of the present invention comprises a chimeric C H region having a chimeric hinge region. For example, the chimeric hinge region can comprise an "upper hinge" amino acid sequence (amino acid residues from position 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region, which is combined with a "lower hinge" sequence (amino acid residues from position 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. In certain embodiments, an antibody comprising a chimeric C H region as described herein can exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., WO2014 / 022540).

[0189] Immunoconjugates

[0190] The present invention encompasses anti-TMPRSS2 antigen-binding proteins, e.g., antibodies or antigen-binding fragments, conjugated to another moiety, e.g., a therapeutic moiety (“immunoconjugate”), such as a toxoid or an antiviral agent, for treating influenza virus infection. In one embodiment of the invention, an anti-TMPRSS2 antibody or fragment is conjugated to any other therapeutic agent described herein. As used herein, the term “immunoconjugate” refers to an antigen-binding protein, e.g., an antibody or antigen-binding fragment, that is chemically or biologically linked to a radioactive substance, cytokine, interferon, target or reporter molecule moiety, enzyme, peptide or protein, or therapeutic agent. The antigen-binding protein can be linked to the radioactive substance, cytokine, interferon, target or reporter molecule moiety, enzyme, peptide or therapeutic agent at any position of the molecule as long as it is capable of binding its target (TMPRSS2). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the invention, the agent can be a different second antibody that specifically binds to TMPRSS2. The type of therapeutic moiety that can be conjugated to an anti-TMPRSS2 antigen-binding protein (e.g., an antibody or fragment) will take into consideration the condition to be treated and the desired therapeutic effect to be achieved.See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, Monoclonal Antibodies 1984: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[0191] Multispecific antibodies

[0192] The present invention includes anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of using and producing such antigen-binding proteins. The term "anti-TMPRSS2" antigen-binding protein, e.g., an antibody or antigen-binding fragment, includes multispecific (e.g., bispecific or bivalent) molecules that comprise at least a first antigen-binding domain that specifically binds to TMPRSS2 (e.g., an antigen-binding domain from H1H7017N or H4H7017N) and at least a second antigen-binding domain that binds to a different antigen or to an epitope in TMPRSS2, wherein the epitope is different from the epitope of the first antigen-binding domain (e.g., influenza HA, such as an antigen-binding domain from H1H14611N2, H1H14612N2, or H1H11729P). In one embodiment of the invention, the first and second epitopes overlap. In another embodiment of the invention, the first and second epitopes do not overlap. For example, in one embodiment of the invention, the multispecific antibody is a bispecific IgG antibody (e.g., IgG1 or IgG4) that comprises a first antigen-binding domain that specifically binds to TMPRSS2, the first antigen-binding domain comprising the immunoglobulin heavy and light chains of H1H7017N or H4H7017N, and a second antigen-binding domain that specifically binds to influenza HA (which comprises different immunoglobulin light and heavy chains such as from H1H14611N2, H1H14612N2, or H1H11729P).

[0193] "H1H7017N" includes such multispecific molecules, e.g., antibodies or antigen-binding fragments, that comprise the HCDR and LCDR of H1H7017N, V H and V L , or HC and LC (including variants thereof as described herein).

[0194] "H4H7017N" includes such multispecific molecules, e.g., antibodies or antigen-binding fragments, that comprise the HCDR and LCDR of H4H7017N, V H and V L , or HC and LC (including variants thereof as described herein).

[0195] In one embodiment of the invention, the antigen-binding domains that specifically bind to TMPRSS that can be incorporated into the multispecific molecule include: (1)

[0197] (i) A heavy chain variable domain sequence comprising CDR-H1 of the amino acid sequence set forth in SEQ ID NO:6, CDR-H2 of the amino acid sequence set forth in SEQ ID NO:8, and CDR-H3 of the amino acid sequence set forth in SEQ ID NO:10, and

[0198] (ii) A light chain variable domain sequence comprising CDR-L1 of the amino acid sequence set forth in SEQ ID NO:12, CDR-L2 of the amino acid sequence set forth in SEQ ID NO:14, and CDR-L3 of the amino acid sequence set forth in SEQ ID NO:16;

[0199] or (2)

[0201] (i) A heavy chain variable domain sequence of the amino acid sequence set forth in SEQ ID NO:2, and;

[0202] (ii) A light chain variable domain sequence of the amino acid sequence set forth in SEQ ID NO:4;

[0203] or (3)

[0205] (i) A heavy chain immunoglobulin sequence of the amino acid sequence set forth in SEQ ID NO:17 or 19, and

[0206] (ii) A light chain immunoglobulin sequence of the amino acid sequence set forth in SEQ ID NO:18.

[0207] In one embodiment of the invention, the multispecific antibody or fragment comprises more than two different binding specificities (e.g., a trispecific molecule), e.g., one or more additional antigen-binding domains that are the same as or different from the first and / or second antigen-binding domains.

[0208] In one embodiment of the present invention, in addition to the antigen-binding site that specifically binds to TMPRSS2, the multispecific molecule further comprises an antigen-binding site that specifically binds to influenza HA obtained from an antibody selected from the following: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B;H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B; H1H18032B; H1H18033B; H1H18034B; H1H18035B; H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B; H1H18091B; H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B; H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B; H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B; H1H18112B; H1H18113B; H1H18114B; H1H18115B; H1H18116B; H1H18117B;H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B: H1H18127B: H1H18128B: H1H18129B: H1H18130B: H1H18131B: H1H18132B: H1H18133B: H1H18134B: H1H18135B: H1H18136B: H1H18137B: H1H18138B: H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B: H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B: H1H18194B: H1H18195B: H1H18196B: H1H18197B: H1H18198B: H1H18199B: H1H18200B: H1H18201B: H1H18202B; H1H18203B; H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B;H1H18219B; H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B;H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B and H1H18335B; as described in International Patent Application Publication No. WO2016 / 100807 (e.g., its CDR-H, V; H or heavy chain; and its CDR-L, V L or light chain).

[0209] In one embodiment of the present invention, in addition to the antigen-binding site that specifically binds to TMPRSS2, the multispecific molecule further comprises an antigen-binding site that specifically binds to the influenza group II HA protein. For example, the antigen-binding site comprises the V of H1H14611N2 H and V L (e.g., SEQ ID NoS; 24 and 28); or a heavy-chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs: 25-27) and a light-chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31).

[0210] In one embodiment of the present invention, in addition to the antigen-binding site that specifically binds to TMPRSS2, the multispecific molecule further comprises an antigen-binding site that specifically binds to the influenza group II HA protein. For example, the antigen-binding site comprises the V of H1H14612N2 H and V L (e.g., SEQ ID NoS; 40 and 44); or a heavy-chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs: 41-43) and a light-chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47).

[0211] In one embodiment of the present invention, in addition to the antigen-binding site that specifically binds to TMPRSS2, the multispecific molecule further comprises an antigen-binding site that specifically binds to the influenza group I HA protein. For example, the antigen-binding site comprises the V of H1H11729P H and V L(e.g., SEQ ID Nos: 32 and 36); or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39).

[0212] In one embodiment of the invention, the bispecific antigen-binding fragment comprises a first scFv having binding specificity for a first epitope (e.g., TMPRSS2) (e.g., comprising V of H1H7017N or H4H7017N H and V L ), and a second scFv having binding specificity for a different second epitope (e.g., comprising V of an anti-influenza HA antibody H and V L ). For example, in one embodiment of the invention, the first and second scFvs are tethered by a linker (e.g., a peptide linker (e.g., a GS linker such as (GGGGS) n (SEQ ID NO: 48), where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)). Other bispecific antigen-binding fragments include F(ab)2 of a bispecific IgG antibody comprising the heavy chain CDRs and light chain CDRs of H1H7017N or H4H7017N and another antibody that binds to a different epitope.

[0213] Methods of Treatment

[0214] The invention provides methods for treating or preventing viral infections or cancers (e.g., prostate cancer) by administering to a subject (e.g., a human) in need of such treatment or prevention a therapeutically effective amount of an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N).

[0215] The influenza virus infection can be treated or prevented in a subject by administering to the subject an anti-TMPRSS2 antigen-binding protein of the present invention. Influenza viruses are classified into types A, B, and C based on their core proteins. Subtypes of influenza A virus are determined by the envelope glycoproteins having hemagglutinin (HA) or neuraminidase (NA) activity. There are several HA subtypes of influenza A virus (e.g., HA1, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA11, HA12, HA13, HA14, HA15, HA16, HA17, or HA18, which subtypes may be designated as H1, H2, H3, etc.), and NA subtypes of influenza A virus (e.g., NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, NA9, NA10, or NA11, which subtypes may be designated as N1, N2, N3, etc.), which are used to name the influenza A subtypes. For example, influenza A viruses H1N1 and H3N2 are well-known human pathogens. Humans are generally infected with viruses of the H1, H2, or H3 subtypes and the N1 or N2 subtypes. The present invention includes methods for treating or preventing infection with the influenza virus subtypes discussed herein. In one embodiment of the present invention, a multispecific antibody that binds to TMPRSS2 and its antigen-binding fragments also binds to HA / and / or NA (e.g., of the subtypes described herein).

[0216] An effective dose or a therapeutically effective dose of an anti-TMPRSS2 antigen-binding protein (e.g., an antibody or an antigen-binding fragment (e.g., H1H7017N or H4H7017N)) for treating or preventing a viral infection refers to an amount of such an antibody or fragment that is sufficient to alleviate one or more signs and / or symptoms of infection in a treated individual, whether by causing such signs and / or symptoms to regress or be eliminated or by inhibiting the progression of such signs and / or symptoms. The dosage can vary depending on the age and physical build of the subject to be administered, the target disease condition, the route of administration, etc. In one embodiment of the invention, (e.g., in adult subjects) an effective dose or a therapeutically effective dose of an antibody or an antigen-binding fragment thereof of the invention for treating or preventing a viral infection is from about 0.01 to about 200 mg / kg, e.g., up to about 150 mg / kg. In one embodiment of the invention, the dose is up to about 10.8 or 11 grams (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 grams). Depending on the severity of the infection, the frequency and duration of treatment can be adjusted. In certain embodiments, the antigen-binding protein of the invention can be administered at an initial dose, followed by one or more secondary doses. In certain embodiments, following the initial dose can be an antibody or an antigen-binding fragment thereof administered at an amount that can be substantially equivalent to or less than the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0217] As used herein, the term "subject" refers to, for example, a mammal (e.g., a rat, a mouse, a cat, a dog, a cow, a sheep, a horse, a goat, a rabbit), preferably a human, in need of preventing and / or treating a disease or disorder such as a viral infection or cancer. A subject can have a viral infection, e.g., an influenza infection, or be susceptible to forming an infection. Subjects susceptible to forming an infection or potentially at an increased risk of exposure to an infection (e.g., an influenza virus) include subjects whose immune systems are impaired due to an autoimmune disease, subjects receiving immunosuppressive therapy (e.g., after an organ transplant), subjects suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), subjects having anemic forms that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects suffering from an inflammatory disease. Additionally, very young (e.g., 5 years old or younger) or elderly (e.g., 65 years old or older) subjects are at increased risk. Additionally, a subject may be at risk of exposure to a viral infection due to proximity to an outbreak of a disease (e.g., the subject lives in a densely populated city or adjacent to a subject with a confirmed or suspected viral infection) or an employment choice (e.g., a hospital worker, a drug researcher, a traveler to an infected area, or a frequent flyer).

[0218] "Treatment" or "therapy" refers to administering an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N) of the invention, to a subject having one or more signs or symptoms of a disease or infection (e.g., a viral infection), wherein the antigen-binding protein is effective for the subject when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0219] The invention also encompasses prophylactic administration of an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N) of the invention, to a subject at risk of viral infection, thereby preventing such infection. Antibody-based passive immunoprophylaxis has proven to be an effective strategy for preventing a subject from suffering from viral infection. See, e.g., Berry et al., Passive broad-spectrum influenza immunoprophylaxis, Influenza Res Treat. 2014;2014:267594. Epub 2014 Sep 22; and Jianqiang et al., Passive immuneneutralization strategies for prevention and control of influenza A infections, Immunotherapy. 2012 February;4(2):175–186; Prabhu et al., Antivir Ther. 2009;14(7):911-21, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza. "Prevention" or "prophylaxis" refers to administering an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N) of the invention, to a subject to inhibit the manifestation of a disease or infection (e.g., a viral infection) in the subject, wherein the antigen-binding protein is effective for the subject when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0220] In one embodiment of the invention, a sign or symptom of viral infection in a subject is viral survival or proliferation in the subject, as determined, for example, by viral titer analysis (e.g., influenza virus proliferation in embryonated chicken eggs or influenza virus hemagglutination assay). Other signs and symptoms of viral infection are discussed herein.

[0221] The present invention provides a method for treating or preventing a viral infection (e.g., influenza virus or coronavirus infection) or causing regression, elimination or inhibition of progression of at least one of the following signs or symptoms of a viral infection in a subject in need (e.g., a human), by:

[0222] · Fever or feeling feverish / chilly;

[0223] · Cough;

[0224] · Sore throat;

[0225] · Runny nose or nasal congestion;

[0226] · Sneezing

[0227] · Muscle or body aches;

[0228] · Various types of headache;

[0229] · Fatigue (tiredness);

[0230] · Vomiting;

[0231] · Diarrhea;

[0232] · Respiratory tract infection;

[0233] · Chest discomfort;

[0234] · Shortness of breath;

[0235] · Bronchitis; and / or

[0236] · Pneumonia,

[0237] wherein the signs or symptoms are secondary to a viral infection: administering to the subject a therapeutically effective amount of an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N or H4H7017N), e.g., injecting the protein into the body of the subject.

[0238] The present invention also includes a method of treating or preventing cancer, such as metastatic cancer, such as prostate cancer (e.g., characterized by the expression of a TMPRSS2:ERG fusion), colon cancer, lung cancer, pancreatic cancer, urinary tract cancer, breast cancer, ovarian cancer, prostate adenocarcinoma, renal cell carcinoma, colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and / or pleural mesothelioma, in a subject by administering to the subject a therapeutically effective amount of a TMPRSS2 antigen-binding protein (e.g., H1H7017N or H4H7017N), for example, injecting the protein into the body of the subject. In one embodiment of the present invention, a TMPRSS2 antigen-binding protein is also administered to the subject in combination with other therapeutic agents (e.g., anti-cancer therapeutic agents). In one embodiment of the present invention, the cancer is a tumor whose cells express TMPRSS2 or a variant thereof.

[0239] Combinations and pharmaceutical compositions

[0240] To prepare a pharmaceutical composition comprising an anti-TMPRSS2 antigen-binding protein (e.g., an antibody and its antigen-binding fragments (e.g., H1H7017N or H4H7017N)), the antigen-binding protein is mixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and The United States Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y. In one embodiment of the invention, the pharmaceutical composition is sterile. Such compositions are part of the present invention.

[0241] The scope of the present invention includes dehydrated (e.g., lyophilized) compositions comprising an anti-TMPRSS2 antigen-binding protein (e.g., an antibody or its antigen-binding fragments (e.g., H1H7017N or H4H7017N)) or pharmaceutical compositions thereof that comprise a pharmaceutically acceptable carrier but are substantially free of water.

[0242] In yet another embodiment of the invention, other therapeutic agents administered in combination with the anti-TMPRSS2 antigen-binding proteins disclosed herein (e.g., an antibody or an antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N)) are administered to a subject according to the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th Edition (November 1, 2002)).

[0243] The mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intraarterial.

[0244] The invention provides a method for administering an anti-TMPRSS2 antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N)), the method comprising introducing the protein into the body of a subject. For example, the method comprises puncturing the body of the subject with the needle of a syringe and injecting the antigen-binding protein into the body of the subject, e.g., injecting into a vein, artery, tumor, muscle tissue or subcutaneous tissue of the subject.

[0245] The invention provides a container (e.g., a plastic or glass vial, e.g., with a cap or a chromatographic column, a hollow needle or a syringe cylinder), the container containing any of the anti-TMPRSS2 antigen-binding proteins described herein, e.g., an antibody or an antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N), a polypeptide (e.g., the HC, LC, V H or V L ) or a polynucleotide or a vector or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.

[0246] In one embodiment of the present invention, the anti-TMPRSS2 antigen-binding protein (e.g., an antibody of the present invention or an antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N)) is combined with one or more other therapeutic agents. For example, in one embodiment of the present invention, the other therapeutic agent is an antiviral drug and / or a vaccine. As used herein, the term "antiviral drug" refers to any anti-infective drug or therapy used to treat, prevent, or mitigate a viral infection in a subject. The term "antiviral drug" includes, but is not limited to, cationic steroid antimicrobials, leupeptin, antipain, amantadine, rimantadine, oseltamivir, zanamivir, ribavirin, or interferon-α2b. A method of treating or preventing a viral (e.g., influenza) infection by co-administering H1H7017N or H4H7017N with one or more other therapeutic agents in a subject in need of such treatment or prevention is part of the present invention.

[0247] For example, in one embodiment of the present invention, the other therapeutic agent is a vaccine, e.g., an influenza vaccine. In one embodiment of the present invention, the vaccine is an inactivated / killed virus vaccine, a live attenuated virus vaccine, or a virus subunit vaccine.

[0248] For example, in one embodiment of the present invention, the other therapeutic agent is:

[0249]

[0250] (Camostat mesylate);

[0251]

[0252] (Nafamostat mesylate);

[0253]

[0254] (Bromhexine hydrochloride (BHH));

[0255]

[0256] (4-(2-Aminomethyl)benzenesulfonyl fluoride hydrochloride (AEBSF));

[0257]

[0258]

[0259] (Polyamide). See Shen et al., Biochimie 142:1-10 (2017).

[0260] In one embodiment of the present invention, the antiviral agent is an antibody or antigen-binding fragment that specifically binds to influenza virus (e.g., influenza HA). For example, in one embodiment of the present invention, the anti-HA antibody is any one of the following: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B: H1H17962B: H1H17963B: H1H17964B: H1H17965B: H1H17966B: H1H17967B: H1H17968B: H1H17969B: H1H17970B: H1H17971B: H1H17972B: H1H17973B: H1H17974B: H1H17975B: H1H17976B: H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B: H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009B; H1H18010B; H1H18011B; H1H18012B; H1H18013B; H1H18014B;H1H18015B; H1H18016B; H1H18017B; H1H18018B; H1H18019B; H1H18020B; H1H18021B; H1H18022B; H1H18023B; H1H18024B; H1H18025B; H1H18026B; H1H18027B; H1H18028B; H1H18029B; H1H18030B; H1H18031B: H1H18032B: H1H18033B; H1H18034B: H1H18035B: H1H18037B; H1H18038B; H1H18039B; H1H18040B; H1H18041B; H1H18042B; H1H18043B; H1H18044B; H1H18045B; H1H18046B; H1H18047B; H1H18048B; H1H18049B; H1H18051B; H1H18052B; H1H18053B; H1H18054B; H1H18055B; H1H18056B; H1H18057B; H1H18058B; H1H18059B; H1H18060B; H1H18061B; H1H18062B; H1H18063B; H1H18064B; H1H18065B; H1H18066B; H1H18067B; H1H18068B; H1H18069B; H1H18070B; H1H18071B; H1H18072B; H1H18073B; H1H18074B; H1H18075B; H1H18076B; H1H18077B; H1H18078B; H1H18079B; H1H18080B; H1H18081B; H1H18082B; H1H18083B; H1H18084B; H1H18085B; H1H18086B; H1H18087B; H1H18088B; H1H18089B; H1H18090B: H1H18091B: H1H18092B; H1H18093B; H1H18094B; H1H18095B; H1H18096B; H1H18097B: H1H18098B; H1H18099B; H1H18100B; H1H18101B; H1H18102B: H1H18103B; H1H18104B; H1H18105B; H1H18107B; H1H18108B; H1H18109B; H1H18110B; H1H18111B: H1H18112B: H1H18113B: H1H18114B: H1H18115B; H1H18116B; H1H18117B;H1H18118B; H1H18119B; H1H18120B; H1H18121B; H1H18122B; H1H18123B; H1H18124B; H1H18125B; H1H18126B; H1H18127B; H1H18128B; H1H18129B; H1H18130B; H1H18131B; H1H18132B; H1H18133B; H1H18134B; H1H18135B; H1H18136B; H1H18137B; H1H18138B; H1H18139B; H1H18140B; H1H18141B; H1H18142B; H1H18143B; H1H18144B; H1H18145B; H1H18146B; H1H18147B; H1H18148B; H1H18149B; H1H18150B; H1H18151B; H1H18152B; H1H18153B; H1H18154B; H1H18155B; H1H18156B; H1H18157B; H1H18158B; H1H18159B; H1H18160B; H1H18161B; H1H18162B; H1H18163B; H1H18164B; H1H18165B; H1H18166B; H1H18167B; H1H18168B; H1H18169B; H1H18170B; H1H18171B; H1H18172B; H1H18173B; H1H18174B; H1H18175B; H1H18176B; H1H18177B; H1H18178B; H1H18179B; H1H18180B; H1H18181B; H1H18182B; H1H18183B; H1H18184B; H1H18185B; H1H18186B; H1H18187B; H1H18188B; H1H18189B; H1H18190B; H1H18191B; H1H18192B; H1H18193B; H1H18194B; H1H18195B: H1H18196B: H1H18197B: H1H18198B: H1H18199B: H1H18200B: H1H18201B: H1H18202B: H1H18203B: H1H18204B; H1H18205B; H1H18206B; H1H18207B; H1H18208B; H1H18209B; H1H18210B; H1H18211B; H1H18212B; H1H18213B; H1H18214B; H1H18216B; H1H18217B; H1H18218B;H1H18219B: H1H18220B; H1H18221B; H1H18222B; H1H18223B; H1H18224B; H1H18225B; H1H18226B; H1H18227B; H1H18228B; H1H18229B; H1H18230B; H1H18231B; H1H18232B; H1H18233B; H1H18234B; H1H18235B; H1H18236B; H1H18237B; H1H18238B; H1H18239B; H1H18240B; H1H18241B; H1H18242B; H1H18243B; H1H18244B; H1H18245B; H1H18246B; H1H18247B; H1H18248B; H1H18249B; H1H18250B; H1H18251B; H1H18252B; H1H18253B; H1H18254B; H1H18255B; H1H18256B; H1H18257B; H1H18258B; H1H18259B; H1H18261B; H1H18262B; H1H18263B; H1H18264B; H1H18265B; H1H18266B; H1H18267B; H1H18268B; H1H18269B; H1H18270B; H1H18271B; H1H18272B; H1H18274B; H1H18275B; H1H18276B; H1H18277B; H1H18278B; H1H18279B; H1H18280B; H1H18281B; H1H18282B; H1H18283B; H1H18284B; H1H18285B; H1H18286B; H1H18287B; H1H18288B; H1H18289B; H1H18290B; H1H18291B; H1H18292B; H1H18293B; H1H18294B; H1H18295B; H1H18297B; H1H18298B; H1H18299B; H1H18300B; H1H18301B; H1H18302B; H1H18303B; H1H18304B; H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H18312B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B;H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B or H1H18335B; as described in International Patent Application Publication No. WO2016 / 100807; or an antigen-binding fragment thereof, e.g., wherein the antibody or fragment comprises a light chain immunoglobulin that comprises CDR-L1, CDR-L2, and CDR-L3 of any of the foregoing anti-influenza HA antibodies (e.g., its V; L or light chain); and a heavy chain that comprises its CDR-H1, CDR-H2, and CDR-H3 (e.g., its V H or heavy chain).

[0261] In one embodiment of the invention, the other therapeutic agent is an antibody or antigen-binding fragment that binds to an influenza group II HA protein, such as H1H14611N2; or an antibody or fragment that comprises the V H and V L of H1H14611N2; or a heavy chain immunoglobulin that comprises CDR-H1, CDR-H2, and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs: 25-27) and a light chain immunoglobulin that comprises CDR-L1, CDR-L2, and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs: 29-31). "H1H14611N2" refers to any anti-group II HA antibody that comprises such sequences.

[0262] H1H14611N2

[0263] Heavy chain variable region

[0264]

[0265] CDR-H1: GFTFSGFS (SEQ ID NO: 25)

[0266] CDR-H2: ISTSGNYM (SEQ ID NO: 26)

[0267] CDR-H3: ARGGGYNWNLFDY (SEQ ID NO: 27)

[0268] Light chain variable region

[0269]

[0270] CDR-L1: QSLNSNY (SEQ ID NO: 29)

[0271] CDR-L2: GAS (SEQ ID NO: 30)

[0272] CDR-L3: QQYGNSPLI (SEQ ID NO: 31)

[0273] In one embodiment of the invention, the other therapeutic agent is an antibody or antigen-binding fragment that binds to an influenza Group II HA protein, such as H1H14612N2; or an antibody or fragment comprising VH H and VL L of H1H14612N2; or a heavy-chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs: 41-43) and a light-chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs: 45-47). "H1H14612N2" refers to any Group II HA antibody comprising such sequences.

[0274] H1H14612N2

[0275] Heavy-chain variable region

[0276]

[0277] CDR-H1: GFSFSGFS (SEQ ID NO: 41)

[0278] CDR-H2: ISTSGNYM (SEQ ID NO: 42)

[0279] CDR-H3: ARGGGYNWNLFDY (SEQ ID NO: 43)

[0280] Light-chain variable region

[0281]

[0282] CDR-L1: QSLNSNY (SEQ ID NO: 45)

[0283] CDR-L2: GAS (SEQ ID NO: 46)

[0284] CDR-L3: QQYGNSPLT (SEQ ID NO: 47)

[0285] In one embodiment of the present invention, the other therapeutic agent is an antibody or antigen-binding fragment that binds to influenza group 1 HA protein, such as H1H1 1729P; or an antibody or fragment comprising V H and V L ; or a heavy chain immunoglobulin comprising CDR-H1, CDR-H2, and CDR-H3 of H1H11729P (e.g., SEQ ID NOs: 33-35) and a light chain immunoglobulin comprising CDR-L1, CDR-L2, and CDR-L3 of H1H11729P (e.g., SEQ ID NOs: 37-39). "H1H11729P" refers to any group I HA antibody comprising such sequences.

[0286] H1H1 1729P

[0287] Heavy chain variable region

[0288]

[0289] CDR-H1: GGTFSSYA (SEQ ID NO: 33)

[0290] CDR-H2: IIPIFGTP (SEQ ID NO: 34)

[0291] CDR-H3: ARQQPVYQYNMDV (SEQ ID NO: 35)

[0292] Light chain variable region

[0293]

[0294] CDR-L1: QGIRNN (SEQ ID NO: 37)

[0295] CDR-L2: AAS (SEQ ID NO: 38)

[0296] CDR-L3; LQYNNYPWT (SEQ ID NO: 39)

[0297] In certain embodiments of the present invention, the other therapeutic agent is not amantadine, rimantadine, oseltamivir, zanamivir, antipain, leupeptin, cationic steroid antimicrobial, influenza vaccine (e.g., killed, live, attenuated whole virus or subunit vaccine) or an antibody against influenza virus (e.g., anti-hemagglutinin antibody).

[0298] The term "in combination with" means that a component (an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof of the invention) can be formulated with another therapeutic agent (such as oseltamivir) into a single composition, e.g., for co-delivery, or separately into two or more compositions (e.g., a kit). Each component can be administered at a time different from when the other component is administered; e.g., each administration can be given non-simultaneously (e.g., separately or sequentially) at intervals over a given period of time. Additionally, the separate components can be administered to a subject by the same or by different routes (e.g., where the anti-TMPRSS2 antibody or its antigen-binding fragment).

[0299] Kit

[0300] Further provided is a kit comprising one or more components in combination with one or more additional components, said components including but not limited to an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof as discussed herein (e.g., H1H7017N or H4H7017N), and said additional components including but not limited to other therapeutic agents as discussed herein. The antigen-binding protein and / or other therapeutic agents can be formulated as a single composition or separately in two or more compositions, e.g., formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.

[0301] In one embodiment of the invention, the kit comprises in one container (e.g., a sterile glass or plastic vial) an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof of the invention (e.g., H1H7017N or H4H7017N) or a pharmaceutical composition thereof and in another container (e.g., a sterile glass or plastic vial) comprises another therapeutic agent.

[0302] In another embodiment, the kit comprises in a single, common container a combination of the invention, said combination comprising an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof of the invention (e.g., H1H7017N or H4H7017N), or a pharmaceutical composition thereof formulated together with one or more other therapeutic agents, optionally formulated in a pharmaceutical composition.

[0303] If the kit comprises a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for such administration (e.g., an injection device). For example, the kit can include one or more subcutaneous injection needles or other injection devices as discussed above, said other injection devices containing an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof of the invention (e.g., H1H7017N or H4H7017N).

[0304] The kit may include a medicine instruction manual, which contains information related to the pharmaceutical composition and dosage form in the kit. Generally, such information assists patients and physicians in effectively and safely using the included pharmaceutical composition and dosage form. For example, the following information about the combination of the present invention may be provided in the instruction manual: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and utilization rates, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, how to supply, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0305] Diagnostic use of the antibody

[0306] An anti-TMPRSS2 antigen-binding protein, such as an antibody of the present invention or an antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N), can be used to detect and / or measure TMPRSS2 in a sample. Exemplary TMPRSS2 assays may include, for example, contacting the sample with an anti-TMPRSS2 antigen-binding protein of the present invention, wherein the anti-TMPRSS2 antigen-binding protein is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate TMPRSS2 from the sample. The presence of the anti-TMPRSS2 antigen-binding protein complexed with TMPRSS2 indicates the presence of TMRPSS2 in the sample. Alternatively, an unlabeled anti-TMPRSS2 antibody can be used in combination with a second antibody that is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I; a fluorescent moiety or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure TMPRSS2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Accordingly, the present invention includes a method for detecting the presence of a TMPRSS2 polypeptide in a sample, the method comprising contacting the sample with an anti-TMPRSS2 antigen-binding protein and detecting the presence of TMPRSS / anti-TMPRSS2 antigen-binding protein, wherein the presence of the complex indicates the presence of TMPRSS2.

[0307] The present invention includes a cell-based ELISA method that uses an anti-TMPRSS2 antigen-binding protein (e.g., an antibody of the present invention and its antigen-binding fragment (e.g., H1H7017N)) to detect the presence of TMPRSS2 on cells. In one embodiment of the present invention, the method comprises the steps of:

[0308] (i) Contact cells with the anti-TMPRSS2 antigen-binding protein of the present invention in which the presence of TMPRSS2 protein to be tested is immobilized on a solid surface (e.g., a microplate).

[0309] (ii) Optionally wash the mixture to remove unbound anti-TMPRSS2 antigen-binding protein.

[0310] (iii) Contact the anti-TMPRSS2 antigen-binding protein with a labeled secondary antibody or an antigen-binding fragment thereof that binds to the anti-TMPRSS2 antigen-binding protein.

[0311] (iv) Optionally wash the complex to remove unbound antigen-binding protein; and

[0312] (v) Detect the presence of a label on the secondary antibody or fragment, where detection of the label indicates that the cells contain TMPRSS2. For example, the present invention includes such a cell-based ELISA method for identifying TMPRSS2 in a sample. + A cell-based ELISA method for cells.

[0313] The anti-TMPRSS2 antigen-binding proteins of the present invention (e.g., H1H7017N or H4H7017N) can be used in a Western blot or immunoblotting assay for detecting the presence of TMPRSS2 or a fragment thereof in a sample. This method forms part of the present invention and, for example, includes the steps of:

[0314] (1) Provide a membrane or other solid substrate that contains a sample for which the presence of TMPRSS2 is to be examined. For example, optionally including the step of using a method known in the art (e.g., semi-dry blotting or slot blotting) to transfer proteins from a sample for which the presence of TMPRSS2 is to be examined (e.g., PAGE or SDS-PAGE electrophoresis separation of proteins from the sample) to the membrane or other solid substrate; and contact the membrane or other solid substrate for which the presence of TMPRSS2 or a fragment thereof is to be examined with the anti-TMPRSS2 antigen-binding protein of the present invention.

[0315] Such a membrane can take the form of, for example, a nitrocellulose membrane or a vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to which proteins for which the presence of TMPRSS2 is to be examined in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel (e.g., after electrophoretic separation in the gel) are transferred. Before contacting the membrane with the anti-TMPRSS2 antigen-binding protein, the membrane is optionally blocked (e.g., with skim milk, etc.) to bind non-specific protein-binding sites on the membrane.

[0316] (2) Wash the membrane one or more times to remove unbound anti-TMPRSS2 antigen-binding protein and other unbound substances; and

[0317] (3) Detect the bound anti-TMPRSS2 antigen-binding protein.

[0318] Detection of the bound antigen-binding protein indicates the presence of TMPRSS2 protein on the membrane or substrate and in the sample. The bound antigen-binding protein can be detected by: binding a second antibody (anti-immunoglobulin antibody) labeled in a detectable manner to the antigen-binding protein and then detecting the presence of the second antibody label.

[0319] The anti-TMPRSS2 antigen-binding proteins disclosed herein (e.g., antibodies and antigen-binding fragments (e.g., H1H7017N or H4H7017N)) can also be used in immunohistochemistry. This method forms part of the present invention and includes, for example,

[0320] (1) Contacting a tissue to be tested for the presence of TMPRSS2 protein with the anti-TMPRSS2 antigen-binding protein of the present invention; and

[0321] (2) Detecting the antigen-binding protein on or in the tissue.

[0322] If the antigen-binding protein itself is detectably labeled, it can be directly detected. Alternatively, the antigen-binding protein can be bound by a second antibody labeled in a detectable manner, where the label is then detected. Examples

[0323] The following examples are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope that the inventors regard as their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, room temperature is about 25 °C, and pressure is at or near atmospheric pressure.

[0324] Example 1 : In vitro multi-cycle replication.

[0325] The ability of influenza virus A / Puerto Rico / 08 / 1934 (H1N1)-GFP to replicate in Calu3, A549, MDCK, and HepG2 cells was evaluated.

[0326] Table 1 Reagents used.

[0327] Description Supplier Calu-3 cells American Type Culture Collection (ATCC) A549 cells American Type Culture Collection (ATCC) MDCK (London) cells IRR HepG2 cells American Type Culture Collection (ATCC) A / Puerto Rico / 08 / 1934 (H1N1)-GFP N / A DMEM Gibco F12 Gibco Penicillin / Streptomycin Gibco Low IgG BSA Sigma PBS Life Technologies Fetal bovine serum Life Technologies

[0328] Experimental methods

[0329] In 96-well plates, Calu-3 cells (ATCC HTB55), A549 cells (ATCC CCL-185), MDCK cells (IRR FR-58), and HepG2 cells (ATCC HB-8065) were diluted to 40,000 cells / well in DMEM:F12 medium containing 5% FBS. The next day, after three washes, A / Puerto Rico / 08 / 1934 (H1N1) (B. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus, Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11531-6) carrying a GFP reporter gene in the NS segment was formulated in DMEM:F12 containing low IgG BSA at MOIs (multiplicity of infection) of 0.1 and 0.01. The virus was incubated on the cells at 37 °C for 1 hour, after which the virus was removed and the wells were washed an additional three times. The number of infected cells was quantified on a S6 Universal Analyzer (Cellular Technology Limited, Cleveland, OH) at 24, 48, 72, and 142 hours post-infection.

[0330] Results summary and conclusions

[0331] Calu-3 is an immortalized human airway epithelial cell line that has been shown to permit multi-cycle replication of human influenza virus in the absence of exogenous trypsin (Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology, 81, 12439–12449 (2007)). In addition, Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at least at the mRNA level, but not TMPRSS11D (HAT) ( et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011)). To confirm that Calu-3 cells can support proteolytic activation of influenza viruses possessing a monobasic cleavage site in hemagglutinin, the growth of the H1N1 GFP reporter virus in Calu-3 cells was analyzed and compared to its replication over time in the absence of trypsin in A549 (human alveolar basal epithelial), MDCK (Madin Darby canine kidney), and HepG2 (human liver carcinoma) cells. Cells were infected at a low MOI, and at the indicated time points, virus titers were determined by counting fluorescent foci. Tables 2 and 1 show low levels of infection in A549, MDCK, and HepG2 cells, while Calu-3 cells showed a significant increase in titer at each time point. Although Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at least at the mRNA level, knockdown of TMPRSS2 reduced influenza virus titers 100- to 1,000-fold ( et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011)). The low virus titer levels in A549, MDCK, and HepG2 cells in the absence of trypsin may be attributed to the addition of cleaved virus (harvested from embryonated eggs or from MDCK cultures with trypsin), but the presence of another HA-activating protease may be an explanation.

[0332] Table 2. Number of infected cells represented by fluorescent focus units (FFU) at different days post-infection with A / Puerto Rico / 08 / 1934 (H1N1)-GFP at an MOI of 0.1 or 0.01 in different cell types.

[0333]

[0334] References

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[0341] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology, 84, 10016–10025 (2010). PMID: 20631123.

[0342] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice (TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice), Journal of Virology (Journal of Virology) (2014), May; 88(9):4744-51. doi:10.1128 / JVI.03799-13. PMID:24522916.

[0343] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium (Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium), Journal of Virology (Journal of Virology). 2006 Oct; 80(19):9896-8. PMID:16973594.

[0344] 10. B. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus (Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus), Proc Natl Acad Sci U S A. 2010 Jun 22; 107(25):11531-6. doi:10.1073 / pnas.0914994107. PMID:20534532.

[0345] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type I interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007). PMID: 17855549.

[0346] Example 2 : The anti-TMPRSS2 antibody H1H7017N blocks influenza spread in vitro.

[0347] The ability of multiple antibodies to reduce the titer of influenza virus A / Puerto Rico / 08 / 1934 (H1N1) in Calu-3 cells was evaluated.

[0348] Table 3. All reagents.

[0349]

[0350]

[0351] Experimental methods

[0352] In a 96-well plate, Calu-3 cells (ATCC HTB55) were diluted to 40,000 cells / well in DMEM:F12 medium containing 5% FBS. The next day, monoclonal antibodies were diluted to 166.7 nM in DMEM:F12 containing low IgG BSA and added to the cells and incubated for 3 hours at 37 °C and 5% CO2. The mAb solution was removed and the cells were infected with A / Puerto Rico / 08 / 1934 (H1N1) at an MOI of 0.001. The virus was incubated on the cells at 37 °C in 5% CO2 for 1 hour, after which the virus was removed and the medium was replaced with DMEM:F12 containing 166.7 nM mAb. After 24 hours and 48 hours, the medium was replaced with fresh medium containing mAb and the cells were washed twice with PBS at 72 hours. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS and the virus was detected using the anti-NP primary antibody at a dilution of 1:1000. The cells were incubated for 1 hour and then washed and the secondary antibody at a dilution of 1:2000 was added. The number of infected cells was in Quantification was performed on a S6 Universal Analyzer (Cellular Technology Limited, Cleveland, OH).

[0353] Results summary and conclusions

[0354] Calu-3 is an immortalized human airway epithelial cell line that has been shown to allow multi-cycle replication of human influenza virus in the absence of exogenous trypsin (Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology, 2007 Nov; 81(22):12439-49). In addition, Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at least at the mRNA level, but not TMPRSS11D (HAT)( et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011)). It has previously been shown that Calu-3 cells support proteolytic activation of influenza virus - but in this study, the inhibition of TMPRSS2 was examined using the TMPRSS2-specific monoclonal antibody H1H7017N. The growth of A / Puerto Rico / 08 / 1934 (H1N1) was analyzed within 72 hours after treating the cells with 166.7 nM H1H7017N. Virus titers were determined by counting fluorescent foci. Tables 4 and Figure 2 show a decrease in titer after treatment with the antibody H1H7017N. Although Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at least at the mRNA level, knockdown of TMPRSS2 reduces the influenza virus titer by 100 to 1,000-fold( et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2. Journal of Virology. 85, 1554–1562 (2011)). The low virus titer levels in the absence of mAb may be attributed to the addition of cleaved virus (harvested from embryonated eggs or from MDCK cultures with trypsin), but the presence of another HA-activating protease may also explain the presence of virus, even after treatment with anti-TMPRSS2 mAb.

[0355] Table 4. Number of focus-forming units (FFUs) of A / Puerto Rico / 08 / 1934 (H1N1) at 72 h post-infection following application of H1H7017N during the infection cycle.

[0356]

[0357] References

[0358] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology. 91, e01387–16 (2017). PMID: 27733646.

[0359] 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017). PMID: 27733646.

[0360] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0361] 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015). PMID: 26379044.

[0362] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017). PMID: 28493964.

[0363] 6. E. D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology. 85, 1554–1562 (2011).). PMID: 21123387.

[0364] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology, 84, 10016–10025 (2010). PMID: 20631123.

[0365] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice, Journal of Virology (2014), May; 88(9):4744-51. doi:10.1128 / JVI.03799-13. PMID: 24522916.

[0366] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium, Journal of Virology. 2006 Oct; 80(19):9896-8. PMID: 16973594.

[0367] 10. B. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus, Proc Natl Acad Sci U S A. 2010 Jun 22; 107(25):11531-6. doi:10.1073 / pnas.0914994107. PMID: 20534532.

[0368] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type I interferon response in polarized human bronchial epithelial cells (Highly pathogenic avian influenza H5N1 viruses stimulate an attenuated type I interferon response in polarized human bronchial epithelial cells), Journal of Virology (Journal of Virology). 81, 12439–12449 (2007). PMID: 17855549.

[0369] Example 3 : FACS analysis using MDCK / Tet-on, MDCK / Tet-on / hTMPRSS2, and MDCK / Tet-on / MfTMPRSS2 cells.

[0370] The ability of the anti-TMPRSS2 antibody H1H7017N to bind to MDCK cells expressing TMPRSS2 or not expressing TMPRSS2 was evaluated.

[0371] Table 5. All reagents.

[0372]

[0373]

[0374] Experimental methods

[0375] Cell lines expressing human and cynomolgus monkey TMPRSS2 (hTMPRSS2 and mfTMPRSS2) in MDCK (Madin Darby canine kidney) cells upon doxycycline induction were developed. MDCK cells were transduced to stably express a modified tetracycline-controlled transactivator protein (Clontech), and the resulting cell line was designated as the MDCK / Tet-on cell line. The MDCK / Tet-on cell line was transduced with constructs containing hTMPRSS2 (NP_005647.3 with V160M) or mfTMPRSS2 (Ref seq XP_015302311.1 with S129L, N251S, I415V, R431Q, D492G) under the control of an inducible promoter, and the cell lines were designated as MDCK / Tet-on / hTMPRSS2 and MDCK / Tet-on / mfTMPRSS2. The stable cell lines were maintained in growth medium containing DMEM supplemented with 10% FBS, sodium pyruvate, penicillin / streptomycin / glutamine, 500 μg / mL G418, with or without 2 μg / mL puromycin.

[0376] For cell binding assays by flow cytometry, cells were seeded in growth medium and incubated with 1 μg / mL doxycycline for 16 h to induce TMPRSS2 expression. Cells were detached using Accutase and resuspended in 1% FBS in PBS. Antibodies were serially diluted from 500 nM to 25 pM, and each concentration of antibody was incubated with 1X 10 6 cells for 30 min at 4 °C. A condition without adding antibody to the cells was included. After incubation with the primary antibody, cells were stained with an allophycocyanin-conjugated anti-human IgG secondary antibody at 1:1000 for 30 min at 4 °C. Cells were fixed using BD CytoFixTM and analyzed using a CytoFLEX flow cytometer. For all cell lines, unstained controls and secondary antibody alone controls were also included. The geometric mean fluorescence values of live cells were determined using FlowJo software, and the results were analyzed using non-linear regression (4-parameter log) with Prism7 software (GraphPad) to obtain the EC 50 values.

[0377] As Figure 3 shown, the anti-hTMPRSS2 antibody H1H7017N of the present invention had EC 50Values bind to MDCK / Tet-on / hTMPRSS2 and MDCK / Tet-on / mfTMPRSS2. H1H7017N did not show significant binding to MDCK / Tet-on cells. Control mAb1, an irrelevant isotype control antibody, did not show binding to any of the cell lines tested.

[0378] Example 4 : Biacore binding kinetics of anti-TMPRSS2 monoclonal antibodies binding to different TMPRSS2 reagents measured at 25 °C and 37 °C.

[0379] The equilibrium dissociation constant (K D ) for the binding of different TMPRSS2 reagents to purified anti-TMPRSS2 monoclonal antibodies was determined using a real-time surface plasmon resonance-based Biacore4000 biosensor. All binding studies were performed at 25 °C and 37 °C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET) running buffer. The surface of a Biacore CM5 sensor chip was first derivatized with a rabbit anti-mouse Fc-specific polyclonal antibody (GE Healthcare catalog number BR100838) by amine coupling to capture the anti-TMPRSS2 monoclonal antibody. Binding studies were performed on the extracellular domain of human TMPRSS2 (hTMPRSS2.mmh) expressed with a C-terminal myc-myc-hexahistidine tag and the extracellular domain of simian TMPRSS2 (mfTMPRSS2.mmh) expressed with a C-terminal myc-myc-hexahistidine tag. Different concentrations of HMM-hTMPRSS2 and HMM-mfTMPRSS2 (100 nM–6.25 nM; 4-fold serial dilutions) were first prepared in HBS-ET running buffer and injected at a flow rate of 30 μL / min for 2.5 min over the surface of the anti-mouse Fc-captured anti-TMPRSS2 monoclonal antibody, while the dissociation of the TMPRSS2 reagent bound to the monoclonal antibody was monitored for 7 min in HBS-ET running buffer. The association rate (ka) and dissociation rate (kd) were determined using Scrubber 2.0c curve-fitting software by fitting the real-time binding sensorgrams with mass transfer limitations to a 1:1 binding model. The binding dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

[0380] and

[0381] Table 6 to Table 9 show the binding kinetic parameters of HMM-hTMPRSS2 or HMM-mfTMPRSS2 with different anti-TMPRSS2 monoclonal antibodies of the present invention at 25°C and 37°C.

[0382] At 25°C, the anti-TMPRSS2 monoclonal antibody binds to HMM-hTMPRSS2 with a K D value of 2.81 nM, as shown in Table 6. At 37°C, the anti-HMM-hTMPRSS2 monoclonal antibody binds to HMM-hTMPRSS2 with a K D value of 9.31 nM, as shown in Table 7.

[0383] At 25°C, the anti-TMPRSS2 monoclonal antibody binds to HMM-mfTMPRSS2 with a K D value of 56.0 nM, as shown in Table 8. At 37°C, the anti-TMPRSS2 monoclonal antibody binds to HMM-mfTMPRSS2 with a K D value of 140 nM, as shown in Table 9.

[0384] TMPRSS2 protein

[0385] hTMPRSS2knob_mmh(W106-R255).mmh:

[0386] Amino acids 1-150: Amino acids 106 to 255 of human TMPRSS2 (Accession No. NP_005647.3, with V160M)

[0387] Amino acids: 151-178: myc-myc-hexahistidine tag

[0388]

[0389] (SEQ ID NO:20; myc tag underlined, His6 tag double underlined)

[0390] mfTMPRSS2knob_mmh(W106-R255).mmh:

[0391] Amino acids 1-150: Amino acids 106-255 of monkey TMPRSS2 (Accession No. XP_005548700.1, with S129L, N251S)

[0392] Amino acids 151-178: myc-myc-hexahistidine tag

[0393]

[0394] (SEQ ID NO:21; myc tag underlined, His6 tag double underlined)

[0395] Results

[0396] Table 6. Binding kinetic parameters of HMM-hTMPRSS2 binding to TMPRSS2 monoclonal antibodies at 25°C.

[0397]

[0398] *H2aM7017N is an antibody with the H1H7017N variable domain described herein and a murine IgG2a Fc.

[0399] Table 7. Binding kinetic parameters of HMM-hTMPRSS2 binding to TMPRSS2 monoclonal antibodies at 37°C.

[0400]

[0401] Table 8. Binding kinetic parameters of HMM-mfTMPRSS2 binding to TMPRSS2 monoclonal antibodies at 25°C.

[0402]

[0403] Table 9. Binding kinetic parameters of HMM-mfTMPRSS2 binding to MSR1 monoclonal antibodies at 37°C.

[0404]

[0405] Example 5 : In vitro influenza spread of influenza H1, H3, and FluB strains.

[0406] In this example, the ability of various types of influenza to spread across in vitro cultures of Calu-3 cells and the effect of anti-TMPRSS2 antibodies on this spread were determined.

[0407] Table 10. Reagents and lot numbers used.

[0408]

[0409]

[0410] Experimental methods

[0411] In a 96-well plate, Calu-3 cells were seeded at 40,000 cells / well in DMEM:F12 medium containing 5% FBS. The next day, the influenza virus strain was diluted to a pre-determined MOI (see Table 11) and the antibodies were diluted to 100 μg / mL. In these experiments, the anti-HA and anti-TMPRSS2 antibodies have different mechanisms of action, and therefore, these antibodies were assayed differently to properly detect them. The anti-HA antibody was pre-incubated with the respective influenza virus strain in a separate plate at 37 °C for one hour. After the pre-incubation phase, the antibody / virus mixture was added to the Calu-3 cells for one hour. The anti-TMPRSS2 antibody was pre-incubated with uninfected Calu-3 cells at 37 °C for three hours. After the pre-incubation phase, the virus was added to the Calu-3 cells pre-incubated with the anti-TMPRSS2 antibody for one hour. After one hour of infection, the cells were washed three times with PBS and fresh antibody was added to each well together with fresh medium. Additional antibody was added at 24 hours and 48 hours post-infection. At 72 hours post-infection, the cells were stained with anti-NP and quantified on a S6 Universal Analyzer (Cellular Technology Limited, Cleveland, OH).

[0412] Table 11A. Experiment 1.

[0413] Influenza virus strains Final MOI H1_PR34 0.001 H1_CA09 0.001 H1_Bris 0.001 H9N2 0.01 H3N2 0.001

[0414] Table 11B. Experiment 2.

[0415] Influenza virus strains Final MOI H1_PR34 0.01 Florida 0.01 Malaysia 0.001

[0416] Results summary and conclusions

[0417] Calu-3 is an immortalized human airway epithelial cell line that has been shown to permit multiple cycles of human influenza virus replication in the absence of exogenous trypsin (Zeng et al., Journal of Virolog, 81:12439–12449 (2007)). In addition, Calu-3 cells have been shown to express TMPRSS2( et al., Journal of Virology 85:1554–1562 (2011)), the former is necessary for these experiments because anti-TMPRSS2 antibodies are being tested. In these experiments, it was investigated whether H1H7017N (anti-TMPRSS2 antibody) could prevent the spread of different influenza strains. In addition, the corresponding anti-HA antibodies of different strains were run as positive controls. As expected, in the presence of anti-TMPRSS2 antibodies, there was an initial infection, but H1H7017N successfully prevented the spread of infection of H1_PR34, H1_CA09, H1_Bris, H9N2, and H3N2. This can be observed by examining the difference in the number of infected cells between anti-TMPRSS2 treated cells and infected controls (Table 12). It is concluded that anti-TMPRSS2 antibodies are unable to prevent the spread of any influenza B strain because the number of infected cells in the control wells and the treated wells is the same. In contrast, anti-HA antibodies are pre-incubated with the virus and prevent initial infection. This can also be seen by comparing the number of infected cells. Infected cells were counted on a CTL meter and reported in the table below.

[0418] Table 12A. Experiment 1.

[0419]

[0420] Table 12B. Experiment 2.

[0421]

[0422] References

[0423] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology. 91, e01387–16 (2017). PMID: 27733646.

[0424] 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017). PMID: 28636671.

[0425] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0426] 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015). PMID: 26379044.

[0427] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017). PMID: 28493964.

[0428] 6. E. D.A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011). PMID: 21123387.

[0429] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology. 84, 10016–10025 (2010). PMID: 20631123.

[0430] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice, Journal of Virology (2014), doi:10.1128 / JVI.03799-13. PMID: 24522916.

[0431] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium, Journal of Virology, 80, 9896–9898 (2006). PMID: 16973594.

[0432] 10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus, Proc Natl Acad Sci U S A. 2010 Jun 22; 107(25):11531-6. doi: 10.1073 / pnas.0914994107. Epub 2010 Jun 7. PMID: 20534532.

[0433] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007), PMID: 17855549.

[0434] Example 6 : Effect of treatment with H1H7017N alone in TMPRS22 humanized mice.

[0435] The ability of an anti-TMPRSS2 antibody to protect mice engineered to express the human TMPRSS2 protein from H1N1 influenza virus infection was evaluated.

[0436] Table 13. Reagents and lot numbers used.

[0437]

[0438]

[0439] Table 14. mAb Clone ID.

[0440] AbPID Description H1H7017N Anti-TMPRSS2 mAb H1H1238N IgG1 isotype control

[0441] Experimental methods

[0442] These experiments were conducted in 5 - 8 - week - old male and female mice engineered to express human TMPRSS2 protein. Mice were challenged with 150 plaque - forming units (PFU) of H1N1. The mice were sedated by intraperitoneal injection with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) and then infected intranasally with 20 μL of virus. Antibodies were delivered subcutaneously (SC) one day before infection or intravenously (IV) several days post - infection (PI). Antibody dosing regimens varied between experiments (Table 15). Body weights were collected daily until PI day 14, and when the mice lost 20% of their initial body weight, they were sacrificed. Results were reported as percent survival.

[0443] Table 15A. Antibody administration (Experiment 1).

[0444] Antibody PI days Dose Delivery H1H1238N -1 5 mg / kg SC H1H7017N -1,0 5 mg / kg SC, IV

[0445] Table 15B. Antibody administration (Experiment 2).

[0446] Antibody PI days Dose Delivery H1H1238N 0 10 mg / kg IV H1H7017N 0,1,2,3 10 mg / kg IV

[0447] Results summary and conclusions

[0448] Mice engineered to express human TMPRSS2 protein have been shown to be susceptible to lethal doses of influenza virus. The purpose of these experiments was to show that H1H7017N could protect mice engineered to express human TMPRSS2 protein against influenza A group 1. Antibodies were tested in prophylactic and therapeutic modes. In both trials, H1H7017N treatment resulted in higher survival rates compared to mice treated with an isotype control (H1H1238N) ( Figure 4 and Figure 5 ). In the prophylactic experiment, the survival rate of mice treated with H1H1238N was 0%, the survival rate of mice treated on PI day - 1 was 85.7%, and the survival rate of mice treated with H1H7017N on PI day 0 was 100%. For the therapeutic mode, the H1H1238N - treated group had a 25% survival rate, while the group treated with H1H7017N on PI days 0 - 3 had a 100% survival rate. The data are summarized in Table 16. H1H7017N showed efficacy in mice engineered to express human TMPRSS2 protein.

[0449] Table 16A. Tabular data summary (Experiment 1)

[0450]

[0451] Table 16B. Tabular data summary (Experiment 2)

[0452]

[0453] References

[0454] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology. 91, e01387–16 (2017), PMID: 27733646.

[0455] 2. LM Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017), PMID: 28636671.

[0456] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0457] 4.P.Zmora,A.-S.Moldenhauer,H.Hofmann-Winkler,S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015), PMID: 26379044.

[0458] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017). PMID: 28493964.

[0459] 6. E. D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011), PMID: 21123387.

[0460] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology, 84, 10016–10025 (2010). PMID: 20631123.

[0461] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice (TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice), Journal of Virology (Journal of Virology) (2014), doi:10.1128 / JVI.03799-13. PMID:24522916.

[0462] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium (Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium), Journal of Virology (Journal of Virology), 80, 9896–9898 (2006). PMID:16973594.

[0463] 10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus (Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus), Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11531-6. doi:10.1073 / pnas.0914994107. Epub 2010 Jun 7. PMID:20534532.

[0464] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type I interferon response in polarized human bronchial epithelial cells, Journal of Virology, 81, 12439–12449 (2007), PMID: 17855549.

[0465] Example 7 : Activity of anti-TMPRSS2 mAb (H1H7017N) in a TMPRSS2 humanized mouse model.

[0466] The ability of anti-TMPRSS2 antibodies to protect mice engineered to express human TMPRSS2 protein from H3N2 influenza virus infection was evaluated.

[0467] Table 17. mAb clone ID.

[0468] AbPID Description H1H7017N Anti-TMPRSS2 antibody

[0469] Table 18: Reagents and lot numbers used.

[0470]

[0471] Experimental methods

[0472] Eleven-week-old male and female mice engineered to express human TMPRSS2 protein were challenged with 20,000 plaque-forming units (PFU) of H3N2. The mice were sedated by intraperitoneal injection with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) and then infected intranasally with 20 μL of virus. On day 1 or 2 post-infection (PI), the mice were injected intravenously with antibody. The mice were weighed and observed daily until day 14 post-infection (PI). The mice were euthanized when they lost 25% of their initial body weight.

[0473] Results summary and conclusions

[0474] When considering influenza therapies, breadth is an important quality. The anti-TMPRSS2 antibody H1H7017N has been shown to be effective against Group 1 of influenza A. The purpose of this experiment was to show that H1H7017N could protect mice engineered to express the human TMPRSS2 protein against Group 2 of influenza A. Mice engineered to express the human TMPRSS2 protein were infected with a lethal dose of H3N2 and treated on Day 1 or Day 2 post-infection (PI). The two treatment groups had higher survival rates compared to the infected control. Mice treated on Day 1 PI had a 100% survival rate, which was higher than the group treated on Day 2 PI with a 50% survival rate, while the untreated mice had a 0% survival rate. All mice died between Days 5 - 6 PI. Figure 6 Survival curves are shown and the % survival is summarized in Table 19. These results show that H1H7017 improved the outcome in the H3N2 lethal model.

[0475] Table 19. Tabulated data summary.

[0476]

[0477] References

[0478] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology. 91, e01387–16 (2017). PMID: 27733646.

[0479] 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017). PMID: 28636671.

[0480] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015), PMID: 25666761.

[0481] 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015). PMID: 26379044.

[0482] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017). PMID: 28493964.

[0483] 6. E. D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011). PMID: 21123387.

[0484] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology, 84, 10016–10025 (2010). PMID: 20631123.

[0485] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice, Journal of Virology (2014), doi:10.1128 / JVI.03799-13. PMID: 24522916.

[0486] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium, Journal of Virology. 80, 9896–9898 (2006). PMID: 16973594.

[0487] 10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus, Proc Natl Acad Sci U S A. 2010 Jun 22; 107(25):11531-6. doi:10.1073 / pnas.0914994107. Epub 2010 Jun 7. PMID: 20534532.

[0488] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells (Highly pathogenic avian influenza H5N1 virus stimulates an attenuated type I interferon response in polarized human bronchial epithelial cells), Journal of Virology (Journal of Virology). 81, 12439–12449 (2007). PMID: 17855549.

[0489] Example 8 : Infection of mice engineered to express human TMPRSS2 protein (relative to WT).

[0490] The survival rate of mice engineered to express human TMPRSS2 protein infected with H1N1 influenza virus was evaluated and compared with wild-type (WT) mice.

[0491] Table 20: Reagents and batch numbers used.

[0492]

[0493] Experimental methods

[0494] Experiments were conducted in 7.5 - 8-week-old male and female mice engineered to express human TMPRSS2 protein or wild-type littermates. Mice were challenged with 150, 750, or 1,500 plaque-forming units (PFU) of A / Puerto Rico / 08 / 1934 (H1N1). Mice were sedated by intraperitoneal injection with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) and then infected intranasally with 20 μL of virus. Body weights were collected daily until day 14 post-infection (PI), and when mice lost 20% of their initial body weight, they were sacrificed. Results are reported as percent survival ( Figure 7 ).

[0495] Results summary and conclusions

[0496] Mice engineered to express human TMPRSS2 protein were generated to test the therapeutic efficacy of anti-TMPRSS2 antibodies in an in vivo influenza model. In this experiment, the survival rates of mice engineered to express human TMPRSS2 protein and wild-type mice infected with 150, 750, or 1,500 PFU of a historical H1N1 strain were compared. In all three infected groups, the survival rates of mice engineered to express human TMPRSS2 protein and wild-type mice were 0%. All mice died between days 5 and 8 post-infection (PI), and those receiving higher virus doses became moribund faster than those receiving lower virus doses. The survival pattern of mice engineered to express human TMPRSS2 protein was similar to that of wild-type mice. This indicates that mice engineered to express human TMPRSS2 protein can be used as an in vivo influenza model to evaluate the efficacy of TMPRSS2-specific antibodies. See Table 21.

[0497] Table 21. Tabular data summary.

[0498]

[0499] References

[0500] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology. 91, e01387–16 (2017). PMID: 27733646.

[0501] 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017). PMID: 28636671.

[0502] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0503] 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015), PMID: 26379044.

[0504] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017), PMID: 28493964.

[0505] 6. E. D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology. 85, 1554–1562 (2011). PMID: 21123387.

[0506] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. Journal of Virology, 84, 10016–10025 (2010), PMID: 20631123.

[0507] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice, Journal of Virology (2014), doi:10.1128 / JVI.03799-13. PMID: 24522916.

[0508] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium, Journal of Virology, 80, 9896–9898 (2006), PMID: 16973594.

[0509] 10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus, Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11531-6. doi:10.1073 / pnas.0914994107, Epub 2010 Jun 7, PMID: 20534532.

[0510] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells (Highly pathogenic avian influenza H5N1 virus elicits an attenuated type i interferon response in polarized human bronchial epithelial cells), Journal of Virology (Journal of Virology), 81, 12439–12449 (2007), PMID: 17855549.

[0511] Example 9 : Therapeutic effects of H1H14611N2 and H1H7017N combinations in mice after H3N2 infection.

[0512] The ability of the combination of anti-TMPRSS2 and anti-influenza antibodies to protect mice engineered to express the human TMPRSS2 protein from H3N2 influenza virus infection was evaluated.

[0513] Table 22. mAb clone ID.

[0514] AbPID Description H1H7017N Anti-TMPRSS2 antibody H1H14611N2 Anti-influenza group 2 antibody H1H1238N IgG1 isotype control

[0515] Table 23. Reagents and batch numbers used.

[0516]

[0517] Experimental methods

[0518] Eight-week-old male and female mice engineered to express the human TMPRSS2 protein were challenged with 20,000 plaque-forming units (PFU) of A / Aichi / 2 / 68 (HA, NA) x A / PR / 8 / 34, Re* reassortant X-31 (H3N2). The mice were sedated by intraperitoneal injection with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) and then infected intranasally with 20 μL of virus. On day 4 post-infection (PI), the mice were injected intravenously with the antibody. Body weights were collected daily until PI day 14, and the mice were sacrificed when they had lost 25% of their initial body weight. Results are reported as percent survival.

[0519] Results summary and conclusions

[0520] It has been shown that the TMPRSS2 antibody H1H7017N and the broad-spectrum group 2 influenza A antibody H1H14611N2 each have therapeutic efficacy against lethal challenge with historical H3N2 strains in mice. It has also been shown that, by means of the combination of H1H7017N and the broad-spectrum group 1 influenza A antibody H1H11729P, the survival rate of mice infected with lethal H1N1 challenge can be significantly increased after treatment with a lower total amount of antibody compared to either agent alone. The purpose of this experiment was to evaluate the synergistic effect of the combination of H1H7017N and H1H14611N2. As Figure 8 shown, 3 out of 4 mice treated with the hIgG1 isotype control antibody on day 4 post-infection (PI) died by day 7 PI. When 10 mg / kg of H1H14611N2 was administered, 3 out of 5 animals survived, and when 10 mg / kg of H1H7017N was administered, 4 out of 5 animals survived. When the two antibodies, H1H14611N2 and H1H7017N, were administered in combination at 5 mg / kg each, there was a 40% survival rate. Mice treated with a combination of 2.5 mg / kg of each of the antibodies H1H14611N2 and H1H7017N after challenge survived 100%. The survival rate of mice infected with lethal H3N2 challenge was increased by means of the combination of lower concentrations of H1H7017N and H1H14611N2 compared to the higher concentration of the combined antibodies or either antibody alone. The percentage of survival is summarized in Table 24.

[0521] Table 24. Tabular data summary.

[0522]

[0523]

[0524] References

[0525] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology, 91, e01387–16 (2017), PMID: 27733646.

[0526] 2. L.M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017). PMID: 28636671.

[0527] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0528] 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE. 10, e0138380 (2015). PMID: 26379044.

[0529] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017). PMID: 28493964.

[0530] 6. E. D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011), PMID: 21123387.

[0531] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology, 84, 10016–10025 (2010). PMID: 20631123.

[0532] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice, Journal of Virology (2014), doi: 10.1128 / JVI.03799-13. PMID: 24522916.

[0533] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium, Journal of Virology. 80, 9896–9898 (2006). PMID: 16973594.

[0534] 10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus, Proc Natl Acad Sci U S A. 2010 Jun 22; 107(25):11531-6. doi: 10.1073 / pnas.0914994107. Epub 2010 Jun 7. PMID: 20534532.

[0535] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007). PMID: 17855549.

[0536] Example 10 : Therapeutic effects of the H1H11729P and H1H7017N combinations in mice after H1N1 infection.

[0537] The ability of combinations of anti-TMPRSS2 and anti-influenza antibodies to protect mice engineered to express the human TMPRSS2 protein from H1N1 influenza virus infection was evaluated.

[0538] Table 25. mAb clone ID.

[0539] AbPID Description H1H7017N Anti-TMPRSS2 antibody H1H11729P Anti-influenza group 1 antibody H1H1238N IgG1 isotype control

[0540] Table 26. Reagents and batch numbers used.

[0541]

[0542] Experimental methods

[0543] Five-week-old male and female mice engineered to express human TMPRSS2 protein were challenged with 1,500 plaque-forming units (PFU) of H1N1. The virus was delivered by sedating the mice with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) and intranasally delivering 20 μL of virus. On day 3 post-infection (PI), the mice were injected intravenously with antibody. Body weights were collected daily until PI day 14, and the mice were sacrificed when they lost 25% of their initial body weight.

[0544] Results summary and conclusions

[0545] It has been shown that the TMPRSS2 antibody H1H7017N and the broad-spectrum group 1 influenza A antibody H1H11729P have therapeutic efficacy against lethal challenge of mice with historical H1N1 strains, respectively. However, the purpose of this experiment was to evaluate the synergistic effect of the antibodies when used in combination. By PI day 6, all mice treated with the hIgG1 isotype control antibody on PI day 3 had died. When animals received 5 mg / kg of H1H11729P or H1H7017N, 40% and 0% of the animals survived the infection, respectively. However, a combination of 2.5 mg / kg of each of the H1H11729P and H1H7017N antibodies resulted in a 60% survival rate. Eighty percent of the mice treated with a combination of 1 mg / kg of H1H7017N and 2 mg / kg of H1H11729P (total 3 mg / kg) survived after challenge. With the combination of H1H7017N and H1H11729P, the survival rate of mice infected with lethal H1N1 challenge was significantly increased after treatment with a lower total amount of antibody compared to either alone (see Figure 9 and Table 27).

[0546] Table 27. Tabular data summary.

[0547]

[0548] References

[0549] 1. K. Shirato, K. Kanou, M. Kawase, S. Matsuyama, Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry, Journal of Virology. 91, e01387–16 (2017). PMID: 27733646.

[0550] 2. LM Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2, PLoS ONE. 12, e0179177 (2017). PMID: 28636671.

[0551] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0552] 4.P.Zmora,A.-S.Moldenhauer,H.Hofmann-Winkler,S. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells, PLoS ONE.10, e0138380 (2015). PMID: 26379044.

[0553] 5. P. Zmora et al., Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin, PLoS ONE. 12, e0176597 (2017), PMID: 28493964.

[0554] 6. E. D. A. Stein, H.-D. Klenk, W. Garten, Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011). PMID: 21123387.

[0555] 7. S. Bertram et al., TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells, Journal of Virology. 84, 10016–10025 (2010). PMID: 20631123.

[0556] 8. C. Tarnow et al., TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice (TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice), Journal of Virology (Journal of Virology) (2014), doi:10.1128 / JVI.03799-13. PMID:24522916.

[0557] 9. E. Bottcher et al., Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium (Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium), Journal of Virology (Journal of Virology). 80, 9896–9898 (2006). PMID:16973594.

[0558] 10. Manicassamy et al., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus (Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus), Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11531-6. doi:10.1073 / pnas.0914994107. Epub 2010 Jun 7. PMID:20534532.

[0559] 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells (Highly pathogenic avian influenza H5N1 virus stimulates an attenuated type I interferon response in polarized human bronchial epithelial cells), Journal of Virology (Journal of Virology). 81, 12439–12449 (2007). PMID: 17855549.

[0560] *****************

[0561] All references cited herein are incorporated herein by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent were specifically and individually incorporated by reference. This statement of incorporation by reference by the applicant is intended to refer to each and every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, even if such reference is not immediately adjacent to the specific statement of incorporation by reference. The inclusion of a specific statement of incorporation by reference within the scope of this specification in no way weakens the general status of such incorporation by reference. The citation of references herein is not intended to admit that such reference is relevant prior art, nor does it constitute any admission as to the content or date of these publications or documents.

Claims

1. A group of polynucleotides comprising: (A) a first polynucleotide encoding an immunoglobulin heavy chain variable region (HCVR) of a human antigen-binding protein that specifically binds to human TMPRSS2; and (B) a second polynucleotide encoding an immunoglobulin light chain variable region (LCVR) of said human antigen-binding protein, Among them, wherein said human antigen-binding protein that specifically binds to human TMPRSS2 comprises: (a) an immunoglobulin HCVR that comprises: a CDR-H1 consisting of the amino acid sequence G F T F S S Y G (SEQ ID NO:6), a CDR-H2 consisting of the amino acid sequence I W N D G S Y V (SEQ ID NO:8), and a CDR-H3 consisting of the amino acid sequence A R E G E W V L Y Y F D Y (SEQ ID NO:10); and (b) an immunoglobulin LCVR that comprises a CDR-L1 consisting of the amino acid sequence Q S I S S W (SEQ ID NO:12), a CDR-L2 consisting of the amino acid sequence KA S (SEQ ID NO:14), and a CDR-L3 consisting of the amino acid sequence Q Q Y N S Y S Y T (SEQ ID NO:16).

2. An antibody or an antigen-binding fragment thereof that binds to influenza group I HA protein, which comprises: a heavy chain immunoglobulin that comprises: (a) a CDR-H1 consisting of the amino acid sequence shown in SEQ ID NO:33, (b) a CDR-H2 consisting of the amino acid sequence shown in SEQ ID NO:34, and (c) a CDR-H3 consisting of the amino acid sequence shown in SEQ ID NO:35; and a light chain immunoglobulin that comprises: (a) a CDR-L1 consisting of the amino acid sequence shown in SEQ ID NO:37, (b) a CDR-L2 consisting of the amino acid sequence shown in SEQ ID NO:38, and (c) a CDR-L3 consisting of the amino acid sequence shown in SEQ ID NO:

39.

3. The group of polynucleotides according to claim 1, wherein said antigen-binding protein is an antibody or an antigen-binding fragment thereof.

4. The group of polynucleotides according to claim 1, wherein said antigen-binding protein comprises: (a) an immunoglobulin HCVR that comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:2; and (b) an immunoglobulin LCVR that comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

5. The group of polynucleotides according to claim 1, wherein said antigen-binding protein comprises: (a) an immunoglobulin heavy chain that comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:17 or 19; and (b) An immunoglobulin light chain comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

18.

6. The polynucleotide set according to claim 1, wherein the antigen-binding protein comprises: (a) An immunoglobulin HCVR consisting of the amino acid sequence shown in SEQ ID NO: 2; and (b) An immunoglobulin LCVR consisting of the amino acid sequence shown in SEQ ID NO:

4.

7. The polynucleotide set according to claim 1, wherein the antigen-binding protein comprises: (a) An immunoglobulin heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 17 or 19; and (b) An immunoglobulin light chain consisting of the amino acid sequence shown in SEQ ID NO:

18.

8. A set of vectors comprising: (A) A first vector comprising a polynucleotide encoding an immunoglobulin HCVR of a human antigen-binding protein that specifically binds to human TMPRSS2; and (B) A second vector comprising a polynucleotide encoding an immunoglobulin LCVR of the human antigen-binding protein, Among them, The human antigen-binding protein that specifically binds to human TMPRSS2 comprises: (a) An immunoglobulin HCVR comprising CDR-H1 consisting of the amino acid sequence G F T F S S Y G (SEQ ID NO: 6), CDR-H2 consisting of the amino acid sequence I W N D G S Y V (SEQ ID NO: 8), and CDR-H3 consisting of the amino acid sequence A R E G E W V L Y Y F D Y (SEQ ID NO: 10); and (b) An immunoglobulin LCVR comprising: CDR-L1 consisting of the amino acid sequence Q S I S S W (SEQ ID NO: 12), CDR-L2 consisting of the amino acid sequence K A S (SEQ ID NO: 14), and CDR-L3 consisting of the amino acid sequence Q Q Y N S Y S Y T (SEQ ID NO: 16).

9. The set of vectors according to claim 8, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

10. The set of vectors according to claim 8, wherein the antigen-binding protein comprises: (a) An immunoglobulin HCVR comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2; and (b) An immunoglobulin LCVR comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

11. The set of vectors according to claim 8, wherein the antigen-binding protein comprises: (a) An immunoglobulin heavy chain comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or 19; and (b) An immunoglobulin light chain comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:

18.

12. The set of vectors according to claim 8, wherein the antigen-binding protein comprises: (a) An immunoglobulin HCVR consisting of the amino acid sequence shown in SEQ ID NO: 2; and (b) An immunoglobulin LCVR consisting of the amino acid sequence shown in SEQ ID NO:

4.

13. The set of vectors according to claim 8, wherein the antigen-binding protein comprises: (a) An immunoglobulin heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 17 or 19; and (b) An immunoglobulin light chain consisting of the amino acid sequence shown in SEQ ID NO:

18.

14. A host cell comprising the set of polynucleotides according to any one of claims 1 and 3 - 7.

15. The host cell according to claim 14, wherein the host cell is a Chinese hamster ovary cell.

16. A host cell comprising the set of vectors according to any one of claims 8 - 13.

17. The host cell according to claim 16, wherein the host cell is a Chinese hamster ovary cell.

18. A method for preparing an antigen-binding protein that specifically binds to human TMPRSS2, comprising: (a) Culturing the host cell according to claim 14 under conditions conducive to the expression of the antigen-binding protein; and (b) Optionally, isolating the antigen-binding protein from the host cell and / or the culture medium in which the host cell grows.

19. A method for preparing an antigen-binding protein that specifically binds to human TMPRSS2, comprising: (a) Culturing the host cell according to claim 16 under conditions conducive to the expression of the antigen-binding protein; and (b) Optionally, isolating the antigen-binding protein from the host cell and / or the culture medium in which the host cell grows.

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