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 (eg, influenza virus infections).

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 622,292, filed January 26, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to antibodies and antigen-binding fragments that specifically bind to TMPRSS2 and methods of treating or preventing viral infections using the antibodies and fragments. Background of the Invention

[0004] Influenza viruses have acquired resistance to currently used drugs that target viral neuraminidase (NA) or the ion channel protein matrix protein 2 (M2). The emergence of resistance highlights the need for new antiviral strategies. Host cell targeting can reduce or avoid the emergence of escape mutants, but may create "traps" due to widespread expression and bring concerns about toxicity. It has been shown that multiple respiratory virus fusion proteins require host protease cleavage 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).

[0005] In order to activate, 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 and human airway trypsin-like protease (HAT) have been involved 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). In addition, TMPRSS2 is a target for anticancer therapy. See, for example, WO2008127347 and WO2002004953. Fusion between TMPRSS2 and ERG (TMPRSS2:ERG) is a gene fusion known to primarily drive prostate cancer development that is 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).

[0006] Brief description of the invention

[0007] Despite the existence of small molecule inhibitors of TMPRSS2 and research antibodies that can be used, for example, for immunohistochemistry, there is a need in the art for neutralizing therapeutic anti-TMPRSS2 antibodies and their uses for treating or preventing viral infections. See, for example, 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 partially satisfies 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.

[0008] The present invention provides a neutralizing human antigen binding protein, such as an antibody or an antigen binding fragment thereof, that specifically binds to human TMPRSS2. For example, in one embodiment of the present invention, the antigen binding protein comprises: (a) CDR-H1, CDR-H2 and CDR-H3 of an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 2, 17 or 19; and / or

[0009] (b) 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) CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin, wherein the light chain immunoglobulin comprises an amino acid sequence as described in SEQ ID NO:4 or 18 and has at least 90% amino acid sequence identity with the amino acid sequence described in SEQ ID NO:4 or 18; and / or (b) CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin, wherein the heavy chain immunoglobulin comprises an amino acid sequence as described in SEQ ID NO:2, 17 or 19 and has at least 90% amino acid sequence identity with the amino acid sequence described in SEQ ID NO:2, 17 or 19. For example, in one embodiment of the invention, the antigen binding protein comprises a light chain immunoglobulin variable region, which comprises (a) a CDR-H1 comprising the amino acid sequence: GFTFSSYG (SEQ ID NO:6); (b) a CDR-H2 comprising the amino acid sequence: IWNDGSYV (SEQ ID NO:8); (c) a CDR-H3 comprising the amino acid sequence: AREGEWVLY YF DY (SEQ ID NO:10); and a heavy chain immunoglobulin variable region, which comprises (a) a CDR-L1 comprising the amino acid sequence: QSISSW (SEQ ID NO:12); (b) a CDR-L2 comprising the amino acid sequence: K AS (SEQ ID NO:14); and / or (c) a CDR-L3 comprising the amino acid sequence: QQYNSYSYT (SEQ ID NO:16). The present invention also provides an antigen-binding protein comprising: (a) a heavy chain immunoglobulin comprising the amino acid sequence described in SEQ ID NO: 17 or 19; and / or (b) a light chain immunoglobulin comprising the amino acid sequence described in SEQ ID NO: 18.

[0010] The invention also provides any anti-TMPRSS2 antigen binding protein that competes with any antigen binding protein described herein for binding to TMPRSS2 (e.g., as measured using real-time, label-free bio-bilayer interferometry analysis, e.g., on an Octet RED384 biosensor (Pall ForteBioCorp.)); or that binds to the same or overlapping epitope on TMPRSS2 (or a fragment thereof) as any antigen binding protein described herein.

[0011] The present invention also provides multispecific antigen-binding proteins that bind to TMPRSS2 and another antigen or bind to different epitopes of TMPRSS2. 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 that is different from the epitope of the first antigen-binding domain.

[0012] The present invention also provides any anti-TMPRSS2 antigen binding protein (e.g., an antibody or antigen binding fragment, e.g., comprising a sequence described herein) comprising one or more of the following properties:

[0013] Inhibits the growth of influenza virus (e.g., A / Puerto Rico / 08 / 1934 (H1N1)) in cells expressing TMPRSS2 (e.g., Calu-3 cells);

[0014] For example, with EC 50 Value 440pM or 1.06nM for surface binding to cells expressing TMPRSS (e.g., MDCK / Tet-on);

[0015] No significant binding to MDCK / Tet-on cells that do not express TMPRSS2;

[0016] At about 25°C, about 2.81X 10 -9 M of K D Binds to human TMPRSS2;

[0017] At about 37°C, about 9.31 X 10 -9 M of K D Binds to human TMPRSS2;

[0018] At about 25°C, about 5.60X 10 -8 M of K D Binds to cynomolgus monkey TMPRSS2;

[0019] At about 37°C with a temperature of about 1.40 X 10 -7 M of K D Binds to cynomolgus monkey TMPRSS2;

[0020] Limiting the spread of influenza virus infection of cells in vitro; and / or

[0021] • Protection of mice engineered to express human TMPRSS2 protein from mortality caused by influenza virus infection.

[0022] The invention also provides a complex comprising any of the antigen binding proteins described herein that binds to a TMPRSS2 polypeptide, eg, in vitro or in vivo in a subject.

[0023] The present invention also provides a method for producing an anti-TMPRSS2 antigen binding protein (e.g., H1H7017N) or an immunoglobulin chain thereof as described herein, the method comprising: (a) introducing one or more polynucleotides encoding the 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 conducive to the expression of the polynucleotide; and (c) optionally, isolating the antigen binding protein or immunoglobulin chain from the host cell and / or the culture 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.

[0024] A polypeptide (eg, an immunoglobulin) comprising: (a) a V region of an immunoglobulin chain comprising the amino acid sequence set forth in SEQ ID NO: 2; H or (b) a V domain of an immunoglobulin chain comprising the amino acid sequence set forth in SEQ ID NO: 4. L CDR1, CDR2 and CDR3 of a domain (e.g., where the polypeptide is in a host cell).

[0025] The present invention also provides polynucleotides (e.g., DNA or RNA) encoding polypeptides of the present invention. In one embodiment of the present invention, the polynucleotides encode two different immunoglobulin chains (e.g., heavy chain and light chain). In one embodiment of the present invention, one polynucleotide encodes an immunoglobulin light chain and another polynucleotide encodes an immunoglobulin heavy chain, for example, wherein the chains are in a host cell or in a container. For example, the polynucleotides are in a vector (e.g., a plasmid) and / or integrated into a host cell chromosome.

[0026] The host cells of the present invention (eg, CHO cells, Pichia cells or Pichia pastoris cells) may contain an anti-TMPRSS2 antigen binding protein (eg, H1H7017N), a polypeptide thereof or a polynucleotide encoding such a polypeptide and / or a vector comprising such a polynucleotide.

[0027] The present invention also provides a composition or kit comprising 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 an antigen binding protein and a pharmaceutically acceptable carrier and optionally other therapeutic agents. The other therapeutic agent can be ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin and interferon-α2b, interferon-α2a and / or an antibody or antigen-binding fragment thereof that specifically binds to influenza HA. In one embodiment of the invention, the additional therapeutic agent is an antibody or antigen-binding fragment thereof selected from the group consisting of: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H1171 4P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; ;H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17 958B; H1H17959B; H1H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; 17968B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; 1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B;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;<h2 style=";text-align:left;direction:ltr">H1H18201B;H1H18202B;H1H18203B;H1H18204B;H1H18205B;H1H18206B;H1 H18207B;H1H18208B;H1H18209B;H1H18210B;H1H18211B;H1H18212B;H1H18 213B;H1H18214B;H1H18216B;H1H18217B;H1H18218B;H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18225B;H1H18226B; H1H18227B;H1H18228B;H1H18229B;H1H18230B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18 239B;H1H18240B;H1H18241B;H1H18242B;H1H18243B;H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B;H1H18251B; H1H18252B;H1H18253B;H1H18254B;H1H18255B;H1H18256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18 265B;H1H18266B;H1H18267B;H1H18268B;H1H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B; H1H18279B;H1H18280B;H1H18281B;H1H18282B;H1H18283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B;H1H18290B;H1H18 291B;H1H18292B;H1H18293B;H1H18294B;H1H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18303B;H1H18304B;H1H18305B; H1H18306B; H1H18307B; H1H18308B; H1H18309B; H1H18310B; H1H18311B; H1H1831 2B; H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18 320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B. ;

[0028] In one embodiment of the present invention, other therapeutic agents provided in combination with anti-TMPRSS2 antigen binding proteins are antibodies or antigen binding fragments that bind to influenza group II HA proteins, such as H1H14611N2; or V comprising H1H14611N2. H and V L 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).

[0029] In one embodiment of the present invention, other therapeutic agents provided in combination with anti-TMPRSS2 antigen binding proteins are antibodies or antigen binding fragments that bind to influenza group II HA proteins, such as H1H14612N2; or V comprising H1H14612N2. H and V L 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).

[0030] In one embodiment of the present invention, other therapeutic agents provided in combination with anti-TMPRSS2 antigen binding proteins are antibodies or antigen binding fragments that bind to influenza group I HA proteins, such as H1H11729P; or V comprising H1H11729P. H and VL 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).

[0031] The present invention also provides a container or injection device comprising an anti-TMPRSS2 antigen binding protein (eg, H1H7017N) or a composition thereof (eg, a pharmaceutical composition).

[0032] The present invention also provides a method for treating or preventing a viral infection other than influenza virus infection in a subject (eg, a human) in need thereof, the method comprising administering a therapeutically effective amount of an anti-TMPRSS2 antigen binding protein described herein (eg, H1H7017N).

[0033] The present invention also provides a method for treating or preventing cancer (e.g., prostate cancer) or infection (e.g., viral infection, e.g., influenza virus, coronavirus, SARS-Co virus, MERS-Co virus, parainfluenza virus, human metapneumovirus or hepatitis C virus (HCV) infection) in a subject (e.g., human) in need thereof, the method comprising administering a therapeutically effective amount of an 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).

[0034] In one embodiment of the present invention, the additional therapeutic agent is a member selected from the group consisting of ledipasvir, sofosbuvir, a 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 invention, the additional therapeutic agent is an antibody or antigen-binding fragment thereof selected from the group consisting of: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; 1727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; 2B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H17975B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; 986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; 7B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B;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;<h2 style=";text-align:left;direction:ltr">H1H18212B;H1H18213B;H1H18214B;H1H18216B;H1H18217B;H1H18218B;H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18 225B;H1H18226B;H1H18227B;H1H18228B;H1H18229B;H1H18230B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B; H1H18238B;H1H18239B;H1H18240B;H1H18241B;H1H18242B;H1H18243B;H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18 250B;H1H18251B;H1H18252B;H1H18253B;H1H18254B;H1H18255B;H1H18256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B; H1H18264B;H1H18265B;H1H18266B;H1H18267B;H1H18268B;H1H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18 277B;H1H18278B;H1H18279B;H1H18280B;H1H18281B;H1H18282B;H1H18283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B; H1H18290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B;H1H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18 303B;H1H18304B;H1H18305B;H1H18306B;H1H18307B;H1H18308B;H1H1830 9B;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. ;

[0035] The present invention also provides a method of administering the anti-TMRPSS2 antigen binding protein described herein (e.g., H1H7017N) into the body of a subject (e.g., a human), the method comprising injecting the antigen binding protein into the body of the subject parenterally (e.g., subcutaneously, intravenously, or intramuscularly). BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 (AB). Progression of A / Puerto Rico / 08 / 1934(H1N1)–GFP virus spread in different cell lines at an initial MOI 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).

[0038] Figure 2 Application of H1H7017N reduced the number of fluorescent focus 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 controls.

[0039] Figure 3 (AB). Anti-TMPRSS2, H1H7017N, binds to human and cynomolgus monkey TMPRSS2 expressed on cells. (A) H1H7017N binds to human and cynomolgus monkey TMPRSS2 expressed on cells with EC values ​​of 460 pM and 1.06 nM, respectively. 50 (B) Control mAb1, an irrelevant isotype control antibody, showed no binding to any of the cell lines tested.

[0040] Figure 4Survival curves of mice engineered to express human TMPRSS2 protein treated with 5 mg / kg H1H7017N at day -1 post-injection (PI) (inverted triangles, dashed lines) or day PI 0 (circles, solid lines), showing protection against H1N1 in a prevention model. Mice treated with the isotype control H1H1238N (triangles, solid lines) showed no protection.

[0041] 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 line), day 1 (circles, solid line), day 2 (inverted triangles, solid line), or day 3 (squares, dashed line) post-injection. The isotype control H1H1238N (triangles, solid line) had partial protection with a survival rate of 25%.

[0042] 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) show no protection.

[0043] Figure 7 (AB). Survival curves of wild-type mice (A) or mice engineered to express human TMPRSS2 protein (B) infected with 150 PFU (triangles), 750 PFU (squares), or 1,500 PFU (circles) of A / Puerto Rico / 08 / 1934(H1N1). Mice were weighed daily until day 14 after injection.

[0044] Figure 8 Survival curves of mice engineered to express human TMPRSS2 protein, 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.

[0045] Fig. 9Survival curves of mice engineered to express human TMPRSS2 protein, infected with A / Puerto Rico / 08 / 1934 (H1N1) on day 1 and treated with 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

[0047] Before describing the method of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used in the text are only for the purpose of describing specific embodiments, and are not intended to be limiting, because the scope of the present invention will only be limited by the appended claims.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described in the present invention may be used in the practice or testing of the present invention, preferred methods and materials are described herein. All publications mentioned herein are fully incorporated herein by reference.

[0049] The term "influenza hemagglutinin", also known as "influenza HA", is a trimeric glycoprotein present on the surface of influenza virions that mediates viral attachment (by virtue of HA1 binding to α-2,3- and α-2,6-sialic acid) and entry into host cells (through conformational changes). HA consists of two domains: a globular head domain containing a receptor binding site (susceptible to high-frequency antigenic mutations) and a handle region (more conserved between various strains of influenza virus). Influenza HA is synthesized as a precursor (HA0) that undergoes proteolytic processing to produce two subunits (HA1 and HA2) that associate with each other to form a handle / globular head structure. Viral HA is the most variable antigen on the virus and the handle (HA2) is highly conserved within each group.

[0050] The term "influenza neuraminidase", also known as "influenza NA", is an exosialidase (EC 3.2.1.18) that cleaves the α-ketosidic bond between sialic acid (N-acetylneuraminic acid) and an adjacent sugar residue.

[0051] 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, for example, GQ149237.1, NC_002017, KM972981.1, etc. The term "influenza-HA" also includes recombinant influenza HA or its fragment. This term also encompasses influenza HA or its fragment coupled with, for example, histidine tags, mouse or human Fc or signal sequences.

[0052] An anti-TMPRSS2 "antigen binding protein" is a polypeptide or a complex of more than one polypeptide (eg, a tetrameric IgG antibody) that specifically binds to a TMPRSS2 polypeptide, eg, an anti-TMPRSS2 antibody or antigen-binding fragment, whether monospecific or multispecific.

[0053] TMPRSS2

[0054] TMPRSS2 (transmembrane serine protease 2) is a protein located on human chromosome 21 that belongs to a family of serine proteases important for influenza virus infectivity (type II transmembrane serine proteases (TTSPs)). TMPRSS2 has been shown to mediate the cleavage of influenza virus HA0 into HA1 and HA2.

[0055] The human TMPRSS2 gene encodes a predicted protein of 492 amino acids anchored to the cytoplasmic membrane. The protein is converted into its mature form by autocatalytic cleavage between Arg255 and Ile256. After cleavage, the mature protease is mostly membrane-bound, but a portion of it can be released into the extracellular environment.

[0056] In one embodiment of the present invention, human TMPRSS2 (V160M) comprises the following amino acid sequence:

[0057]

[0058] (SEQ ID NO:22; methionine 160 in bold). In one embodiment of the invention, the TMPRSS2 polypeptide does not comprise a V160M mutation. See also NM_005656.3.

[0059] In one embodiment of the present invention, the TMPRSS2 of macaque (Macaca mulatta) (S129L, N251S, I415V, R431Q, D492G) comprises the following amino acid sequence:

[0060]

[0061] (SEQ ID NO: 23) In one embodiment of the invention, the TMPRSS2 polypeptide does not comprise S129L, N251S, I415V, R431Q and / or D492G mutations.

[0062] In one embodiment of the present invention, the Mus musculus TMPRSS2 mRNA comprises the nucleotide sequence set forth in NM_015775.2.

[0063] Virus

[0064] The present invention includes methods for treating or preventing viral infections in subjects. The term "virus" includes any virus that can be treated or prevented from infecting a subject 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, a "virus" is any virus that expresses HAO or expresses another substrate of TMPRSS2, wherein the complete infectivity of the virus to cells in the host requires protease cleavage of the other substrate. The term "virus" also includes TMPRSS2-dependent respiratory viruses, which are viruses that infect the respiratory tissues of the subject (e.g., upper and / or lower respiratory tract, trachea, bronchi, lungs) and are treatable or preventable by administering anti-TMPRSS2. For example, in one embodiment of the present invention, viruses include influenza viruses, coronaviruses, SARS-Co viruses (severe acute respiratory syndrome coronavirus), MERS-Co viruses (Middle East respiratory syndrome (MERS) CoV), parainfluenza viruses, Sendai viruses (SeV), human metapneumoviruses, and / or hepatitis C viruses (HCV). "Viral infection" refers to the invasion and proliferation of viruses in a subject. The present invention includes embodiments wherein "virus" does not include influenza virus, e.g., wherein viral infection does not include influenza virus infection.

[0065] There are currently two genera of human parainfluenza viruses (HPIVs): respirovirus (HPIV-1 and HPIV-3) and rubulavirus (HPIV-2 and HPIV-4). Both genera (paramyxoviruses) can be separated morphologically from influenza viruses.

[0066] Sendai virus, also known as murine parainfluenza virus, is the type species in the genus Respirovirus, which also includes the following species: human parainfluenza virus 3, bovine parainfluenza virus 3, and human parainfluenza virus 1. TMPRSS2 is an activating protease of respiratory parainfluenza viruses such as parainfluenza virus and Sendai virus (SeV). See Abe et al., J. Virol. 87(21): 11930-11935 (2013).

[0067] Human metapneumovirus (HMPV) is classified as the first human member of the genus Metapneumovirus in the subfamily Pneumovirinae within the family Paramyxoviridae. It is an enveloped negative-sense single-stranded RNA virus. The RNA genome includes 8 genes encoding 9 different proteins. HMPV is genetically identical to avian pneumovirus (AMPV), which also belongs to the genus Metapneumovirus. TMPRSS2 is expressed in human lung epithelium, efficiently cleaves HMPV F protein and supports HMPV proliferation, and may be involved in the development of lower respiratory tract disease in HMPV-infected patients. See Shirogane et al., J Virol. 82(17):8942–8946 (2008).

[0068] Hepatitis C virus (HCV) is a small, enveloped, positive-sense, single-stranded RNA virus of the family Flaviviridae. HCV, with at least six genotypes and numerous subtypes, is a member of the genus hepacivirus. TMPRSS2 can activate HCV infection during post-binding and entry stages. Esumi et al., Hepatology 61(2):437-446 (2015).

[0069] Influenza virus is a member of the human Orthomyxoviridae family. This family represents enveloped viruses whose genomes have segmented negative-sense single-stranded RNA segments. There are four genera in this family: Type A, Type B, Type C, and Thogotovirus. The influenza virus categories A, B, and C are based on the core protein and are further divided into subtypes (e.g., subtype A / H1N1) determined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). 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 subtypes NA8, NA5, Na4 and NA1. Group 2 has subtypes H3, H4, H7, H10, H14 and H15 and subtypes NA6, NA9, NA7, NA2 and NA3. Influenza A viruses infect a range of mammalian and avian species, while infections of types B and C are primarily limited to humans. The eight genome segments of influenza A and B viruses are loosely encapsidated by nucleoproteins.

[0070] The coronavirus virion is spherical, with a diameter of about 125nm. The most prominent feature of the coronavirus is the stick-shaped thorny protrusions from the surface of the virion. These spikes are the defining characteristics of the virion and give it a corona appearance, suggesting the name, coronavirus. Inside the envelope of the virion is the nucleocapsid. The coronavirus has a helical symmetric nucleocapsid, which is uncommon between positive RNA viruses, but much more common for negative 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) inside the S1 region of the coronavirus S protein varies according to the virus, and some viruses have RBD at the C-terminus of S1. The S-protein / receptor interaction is the main determinant of the coronavirus infection host species and also determines the tissue tropism of the virus. Many coronaviruses use peptidases as their cell receptors. After receptor binding, the virus must then enter the host cell cytosol. This is typically achieved by acid-dependent proteolytic cleavage of the S protein by cathepsins, TMPRRS2, or another protease, followed by fusion of the viral and cellular membranes.

[0071] Anti-TMPRSS2 antibodies and antigen-binding fragments

[0072] The present invention provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to TMPRSS2 protein or antigenic fragments thereof.

[0073] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds (i.e., a "complete antibody molecule"), as well as 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 the heavy chain constant region (composed of domain C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region ("LCVR" or "V L ”) (e.g., SEQ ID NO 4) and a light chain constant region (C L ). The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V LIt comprises 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 present invention, the FRs of the antibody (or its antigen-binding fragment) are identical to the human germline sequence, or are naturally or artificially modified.

[0074] Generally, the variable domains of immunoglobulin heavy and light chains include three hypervariable regions located within relatively conservative framework regions (FRs), also known as complementarity determining regions (CDRs). Typically, from N-terminus to C-terminus, the light chain variable domain and the heavy chain variable domain include 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 Edition; 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).

[0075] 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 substantially homogeneous population of antibodies, i.e., the antibody molecules comprising this population are substantially identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. A "plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., identical in amino acid sequence, excluding possible naturally occurring mutations that may be present in minor amounts, as discussed above) antibodies and fragments that is higher than the concentration that would normally exist in nature (e.g., in the blood of a host organism such as a mouse or a human).

[0076] In one embodiment of the invention, the anti-TMPRSS2 antigen binding protein, e.g., an antibody or antigen binding fragment, comprises, for example, 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, for example, a light chain constant domain of the κ or λ type.

[0077] As used herein, the term "human" antigen-binding proteins, such as antibodies, include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences, whether or not the human germline immunoglobulin sequences are in human cells or transplanted into non-human cells, for example, mouse cells. See, for example, US8502018, US6596541 or US5789215. The human mAb of the present invention may include, for example, amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo) that are not encoded by human germline immunoglobulin sequences in CDR and especially CDR3. 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 transplanted to 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 separated from human subjects or generated therein. See below.

[0078] The present invention includes anti-TMPRSS2 chimeric antigen binding proteins, e.g., antibodies and antigen binding fragments thereof, and methods of use thereof. As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain 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).

[0079] 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 technology, including, for example, DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice) or cell (e.g., CHO cells) expression systems or isolated from recombinant human antibody combinatorial libraries.

[0080] 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 anti-TMPRSS2 antibody immunoglobulin molecule of the invention (e.g., H1H7017N) can be inserted into a pET-based plasmid and expressed in an E. coli / T7 system. For example, the present invention includes a method for expressing an antibody or an antigen binding fragment thereof or an immunoglobulin chain thereof in a host cell (e.g., a bacterial host cell such as E. coli such as BL21 or BL21DE3), the method comprising expressing a T7 RNA polymerase in a cell that also contains a polynucleotide encoding an immunoglobulin chain operably linked to a T7 promoter. For example, in one embodiment of the present invention, a bacterial host cell, such as E. coli, contains a polynucleotide encoding a T7 RNA polymerase gene operably linked to a lac promoter, and the expression of the polymerase and 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.

[0081] There are several methods known in the art by which recombinant antibodies are produced. An example of a method for recombinantly producing antibodies is disclosed in US4816567.

[0082] Transformation can be performed by any known method for introducing a polynucleotide into a host cell. Methods for introducing a heterologous polynucleotide 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 for 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.

[0083] Thus, the present invention includes a recombinant method for producing an anti-TMPRSS2 antigen binding protein (e.g., an antibody or antigen binding fragment thereof of the present invention) or an immunoglobulin chain thereof, the recombinant method 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), for example, wherein the polynucleotide is in a vector; and / or integrated into a host cell chromosome and / or operably linked to a promoter; (ii) culturing a 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 culture medium in which the host cell is cultured. When producing an antigen binding protein (e.g., an antibody or antigen binding fragment) comprising more than one immunoglobulin chain, e.g., an antibody comprising two heavy immunoglobulin chains and two light immunoglobulin chains, co-expression of the chains in a single host cell results in association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, thereby forming an antigen binding protein (e.g., an antibody or antigen binding fragment). These methods include those in which only heavy immunoglobulin chains are expressed or only light immunoglobulin chains (e.g., any of those chains discussed herein, including mature fragments and / or variable domains thereof) are expressed. Such chains can be used, for example, as intermediates in the expression of antibodies or antigen binding fragments comprising such chains. For example, the present invention also includes such anti-TMPRSS2 antigen binding proteins, such as antibodies and antigen binding fragments thereof, which comprise a heavy chain immunoglobulin (or a variable domain thereof or comprising a CDR thereof) encoded by a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:1, and a light chain immunoglobulin (or a variable domain thereof or comprising a CDR thereof) encoded by the nucleotide sequence set forth in SEQ ID NO:3, the antigen binding protein being the 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, the antibody or fragment comprising a V sequence comprising the amino acid sequence set forth in SEQ ID NO:2. H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L , or comprises the HC comprising the amino acid sequence described in SEQ ID NO: 17 or 19 and the LC comprising the amino acid sequence described in SEQ ID NO: 18.

[0084] Eukaryotic host cells 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 Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma 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, cattle 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, including, 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. 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 (eg, CHO cells) comprising an antigen binding protein, such as H1H7017N; or a polynucleotide encoding such a polypeptide.

[0085] The term "specific binding" refers to those proteins that have a specific binding affinity expressed as K for an antigen such as TMPRSS2 protein (e.g., human TMPRSS2). D 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 M), as measured by real-time, label-free bio-bilayer 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 measured by solution affinity ELISA. The present invention includes antigen binding proteins that specifically bind to TMPRSS2 protein.

[0086] As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody or antigen binding protein, etc., includes any naturally occurring, enzymatically obtained, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to 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) the minimum recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., independent 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, tetrabodies, minibodies, 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 scope of the expression "antigen-binding fragment" used herein. In one embodiment of the 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).

[0087] In one embodiment of the invention, the antigen-binding fragment of an antibody will contain at least one variable domain. The variable domain can be of any size or amino acid composition and will generally contain at least one CDR that is adjacent to or in the same reading frame as one or more framework sequences. H Domains and related V L In the antigen-binding fragment of the structural domain, V H and VL The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V L Structural domain.

[0088] In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of an antibody 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 of variable domains and constant domains including any of the exemplary configurations listed above, the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may be composed 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, the antigen-binding fragment of the antibody of the present invention may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently bound to each other (e.g., by disulfide bonds) and / or to one or more monomer V H or V L The domains are non-covalently associated (eg, via disulfide bonds).

[0089] Antigen binding proteins (eg, antibodies and antigen binding fragments) can be monospecific or multispecific (eg, bispecific). Multispecific antigen binding proteins are discussed further herein.

[0090] In specific embodiments, the antibodies or antibody fragments of the 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 viral infections (e.g., influenza viral infections). See below.

[0091] The present invention also provides a complex comprising an anti-TMPRSS2 antigen binding protein discussed herein, e.g., an antibody or antigen binding fragment, complexed with a TMPRSS2 polypeptide or antigenic fragment thereof and / or with a second antibody or 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 invention, the antibody or fragment is in vitro (e.g., fixed to a solid substrate) or in vivo in a subject. In one embodiment of the invention, TMPRSS2 is in vitro (e.g., fixed to a solid substrate) or on a cell surface or in vivo 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 sepharose, e.g., beads or other particles thereof) 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.

[0092] "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 they 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 medium. An isolated antibody or antigen-binding fragment may also be at least partially free of expression system components such as biomolecules from host cells or their growth medium. In general, the term "isolated" does not connote the complete absence of such biomolecules or the absence of water, buffers or salts or components of a pharmaceutical preparation comprising the antibody or fragment.

[0093] The term "epitope" refers to an antigenic determinant (e.g., on a TMPRSS2 polypeptide) that interacts with a specific antigen binding site (e.g., a variable region of an antibody molecule) (called a paratope) of an antigen binding protein. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen that responds to B and / or T cells. It also refers to a region of an antigen that is bound by an antibody. An epitope may be defined as a structural epitope or a functional epitope. Functional epitopes are typically subsets of structural epitopes and have those residues that directly contribute to affinity interactions. An epitope may be linear or conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant that is a chemically active molecular surface group such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0094] Methods for determining the epitope of an antigen binding protein (e.g., an antibody or fragment or polypeptide) include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide shear 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 hydrogen / deuterium exchange detected by mass spectrometry. In general, hydrogen / deuterium exchange methods involve deuterium labeling of a target protein, 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 interchangeable protons within the amino acids protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a lower rate than the interchangeable protons within the amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antigen binding protein interface may retain deuterium and therefore exhibit a relatively higher mass than amino acids that are not incorporated into the interface. After the antigen binding protein (e.g., antibody or fragment or polypeptide) is dissociated, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing deuterium-labeled residues corresponding to 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.

[0095] As used herein, the term "competition" 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) may bind to the same epitope. Alternatively, the first and second antigen binding proteins (e.g., antibodies) may bind to different, but for example overlapping epitopes, the binding of one of which inhibits or blocks the binding of the second antibody, for example, by steric hindrance. Competition between antigen binding proteins (e.g., antibodies) can be measured by methods known in the art, for example, by real-time, label-free biobilayer interferometry analysis. In one embodiment of the invention, competition between a first and a second anti-TMPRSS2 antigen binding protein (e.g., antibody) is determined by measuring the ability of an immobilized first anti-TMPRSS2 antigen binding protein (e.g., antibody) (not initially complexed with a TMPRSS2 protein) to bind to a soluble TMPRSS2 protein complexed with a second anti-TMPRSS2 antigen binding protein (e.g., antibody). A reduction in the ability of a first anti-TMPRSS2 antigen binding protein (e.g., antibody) to bind to a complexed TMPRSS2 protein relative to an 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 reduction in binding. Such competition can be measured using real-time, label-free biobilayer interferometry analysis (e.g., on an OctetRED384 biosensor (Pall ForteBio Corp.)), ELISA (enzyme-linked immunosorbent assay), or SPR (surface plasmon resonance).

[0096] Binding competition between anti-TMPRSS2 antigen binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined on an Octet RED384 biosensor (Pall ForteBio Corp.) using real-time, label-free biobilayer interferometry analysis. For example, to determine competition between two anti-human TMPRSS2 monoclonal antibodies, an anti-TMPRSS2 mAb can first be captured onto an anti-hFc antibody-coated Octet biosensor tip (Pall ForteBio Corp., #18-5060) by immersing the tip in a solution of anti-human TMPRSS2 mAb (hereinafter referred to as "mAb1"). As a positive control for blocking, the antibody-captured biosensor tip can then be saturated with a blocking mAb by immersing the tip in a solution of a known blocking isotype control mAb (hereinafter referred to as "blocking mAb"). To determine whether mAb2 competes with mAb1, the biosensor tip can then be immersed in a secondary complex solution of human TMPRSS2 polypeptide and a second anti-human TMPRSS2 mAb (hereinafter referred to as "mAb2"), wherein the secondary 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.

[0097] For example, in one embodiment of the invention, the competition assay is performed at 25°C and pH about 7 (eg, 7.4), for example, in the presence of buffers, salts, surfactants, and nonspecific proteins (eg, bovine serum albumin).

[0098] Typically, an antibody or antigen-binding fragment of the invention that has been modified in some manner retains the ability to specifically bind to TMPRSS2, for example, retaining at least 10% of its TMPRSS2 binding activity (when compared to the parent antibody) when the activity is expressed on a molar basis. Preferably, the antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more TMPRSS2 binding affinity as the parent antibody. It is also intended that the antibody or antigen-binding fragment of the invention may contain conservative or non-conservative amino acid substitutions that do not substantially change its biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies).

[0099] Polypeptide (such as immunoglobulin chain (for example, H1H7017N V H 、V LA "variant" of 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 a reference amino acid sequence described herein (e.g., SEQ ID NO: 2, 4, 17, 18 or 19); when compared using the BLAST algorithm, wherein the parameters of the algorithm are selected to produce maximum matches between corresponding sequences over the entire length of the corresponding reference sequence (e.g., Expected threshold: 10; Word length: 3; Maximum match in query: 0; BLOSUM 62 matrix; Gap penalty: presence 11, extension 1; conditional composition scoring matrix adjustment).

[0100] 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 a reference nucleotide sequence described herein (e.g., SEQ ID NO: 1 or 3); when compared using the BLAST algorithm, wherein the parameters of the algorithm are selected to produce maximum matches between corresponding sequences over the entire length of the corresponding reference sequence (e.g., Expected threshold: 10; Word length: 28; Maximum match within query range: 0; Match / mismatch score: 1, -2; Gap penalty: linear).

[0101] In one embodiment of the invention, the anti-TMPRSS2 antigen binding protein, e.g., an antibody of the invention and an antigen-binding fragment thereof, 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 set forth 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 set forth in SEQ ID NO: 4 or 18.

[0102] In addition, variant anti-TMPRSS2 antigen binding proteins can include polypeptides comprising an amino acid sequence 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 invention includes antigen binding proteins comprising an immunoglobulin light chain variant comprising an amino acid sequence set forth in SEQ ID NO: 4 or 18, but having one or more such mutations, and / or an immunoglobulin heavy chain variant comprising an amino acid sequence set forth in SEQ ID NO: 2, 17 or 19, but having one or more such mutations. In one embodiment of the present invention, the variant anti-TMPRSS2 antigen binding protein includes an immunoglobulin light chain variant and / or an immunoglobulin heavy chain variant, wherein the immunoglobulin light chain variant comprises CDR-L1, CDR-L2 and CDR-L3 in which one or more (e.g., 1 or 2 or 3) such CDRs have one or more such mutations (e.g., conservative substitutions), and the immunoglobulin heavy chain variant comprises CDR-H1, CDR-H2 and CDR-H3 in which one or more (e.g., 1 or 2 or 3) such CDRs have one or more such mutations (e.g., conservative substitutions).

[0103] The present invention also provides variant anti-TMPRSS2 antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, comprising one or more variant CDRs described herein (e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or CDR-H3) having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.9% sequence identity or similarity to, e.g., SEQ ID NO: 12, 14, 16, 6, 8 and / or 10.

[0104] Embodiments of the present invention also include variant antigen binding proteins, for example, anti-TMPRSS2 antibodies and antigen binding fragments thereof, wherein the variant antigen binding protein comprises such immunoglobulin V H and V L ; or HC and LC, which contain the corresponding V as specifically described herein H 、V LIn some embodiments, the amino acid sequences of the variant antigen binding proteins have 70% or more (e.g., 80%, 85%, 90%, 95%, 97% or 99%) overall amino acid sequence identity or similarity to the amino acid sequences of the immunoglobulins, HC or LC, but wherein the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 of such immunoglobulins are not variants and comprise 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 proteins themselves are not variants.

[0105] Conservatively modified variant anti-TMPRSS2 antibodies and antigen-binding fragments thereof are also part of the present invention. "Conservatively modified variants" or "conservative substitutions" refer to variants in which there are one or more substitutions of amino acids in a polypeptide with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.). Such changes can often be made without significantly destroying the biological activity of the antibody or fragment. Those skilled in the art recognize that, in general, 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., p. 224 (4th ed.)). In addition, substitutions of amino acids that are similar in structure or function are unlikely to significantly destroy biological activity.

[0106] Examples of amino acid groups with side chains of 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 conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., (1992) Science 256:144345.

[0107] 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) may be a "functional conservative variant". In some cases, such functional conservative variants may also be characterized as conservatively modified variants. As used herein, "functional 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 changed without significantly changing one or more functional properties of the antibody or fragment. In one embodiment of the present invention, a functional conservative variant of an 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:

[0108] Inhibits the growth of influenza virus (e.g., A / Puerto Rico / 08 / 1934 (H1N1)) in cells expressing TMPRSS2 (e.g., Calu-3 cells);

[0109] For example, with EC 50 Values ​​of 440 pM or 1.06 nM, respectively, bind to the surface of cells expressing TMPRSS (e.g., MDCK / Tet-on);

[0110] No significant binding to MDCK / Tet-on cells that do not express TMPRSS2;

[0111] At about 25°C, about 2.81X 10 -9 M of K D Binds to human TMPRSS2;

[0112] At about 37°C, about 9.31 X 10 -9 M of K D Binds to human TMPRSS2;

[0113] At about 25°C, about 5.60X 10 -8 M of K D Binds to cynomolgus monkey TMPRSS2;

[0114] At about 37°C with a temperature of about 1.40 X 10 -7 M of K D Binds to cynomolgus monkey TMPRSS2;

[0115] Limiting 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

[0116] • Protecting mice engineered to express human TMPRSS2 protein from mortality resulting from influenza virus infection (eg, H1N1 or H3N2), optionally in combination with an anti-HA antibody, for example, where the mice are infected with an otherwise lethal dose of the virus.

[0117] The present invention includes mice engineered to express human TMPRSS2 protein, which contain anti-TMPRSS2 antigen binding proteins (e.g., antibodies or antigen binding fragments) such as H1H7017N and H4H7017N in the mouse. See International Patent Application Publication No. WO2017 / 151453.

[0118] 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., on a substrate such as HA; Cbz-Gly-Gly-Arg-AMC (Sigma), wherein Cbz is benzyloxycarbonyl and AMC is 7-amino-4-methylcoumarin; influenza virus HA0; coronavirus S protein; or a precursor TMPRSS2 that is autocatalytically cleaved between Arg255 and Ile256, and / or inhibits influenza virus entry into a cell and / or inhibits influenza virus multiplication in a subject.

[0119] "H1H7017N" and "H4H7017N" refer to antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain or a V chain as described below. H (or its variant) and light chain or V L (or its variant); or comprising a V comprising its CDRs (CDR-H1 (or its variant), CDR-H2 (or its variant) and CDR-H3 (or its variant)) H and a V comprising its CDRs (CDR-L1 (or a variant thereof), CDR-L2 (or a variant thereof) and CDR-L3 (or a variant thereof)) L , for example, wherein the immunoglobulin chains, variable regions and / or CDRs comprise the specific amino acid sequences described below.

[0120] In one embodiment of the present invention, "H1H7017N" or "H4H7017N" refers to an antibody or an antigen-binding fragment thereof, which comprises CDR-H1, CDR-H2 and CDR-H3 of an immunoglobulin heavy chain comprising the amino acid sequence described in SEQ ID NO:2, 17 or 19; and CDR-L1, CDR-L2 and CDR-L3 of an immunoglobulin light chain comprising the amino acid sequence described in SEQ ID NO:4 or 18.

[0121] In one embodiment of the present invention, "H1H7017N" or "H4H7017N" refers to an antibody or antigen-binding fragment thereof comprising a V sequence comprising the amino acid sequence set forth in SEQ ID NO: 2. H and V comprising the amino acid sequence described in SEQ ID NO:4 L .

[0122] In one embodiment of the present invention, "H1H7017N" refers to an antibody or antigen-binding fragment comprising 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.

[0123] In one embodiment of the present invention, "H4H7017N" refers to an antibody or antigen-binding fragment comprising a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 18. The term "H4H7017N" also includes an antibody or antigen-binding fragment wherein V H Embodiments fused to wild-type IgG4 (eg, wherein residue 108 is S).

[0124] Anti-TMRPS22 antibodies or antigen-binding fragments H1H7017N and H4H7017N

[0125] H1H7017N and H4H7017N heavy chain variable region (DNA)

[0126]

[0127] H1H7017N and H4H7017N heavy chain variable region (peptide)

[0128]

[0129] H1H7017N and H4H7017N light chain variable region (DNA)

[0130]

[0131] H1H7017N and H4H7017N light chain variable region (peptide)

[0132]

[0133] H1H7017N and H4H7017N CDR-H1 (DNA)

[0134]

[0135] H1H7017N and H4H7017N CDR-H1 (peptide)

[0136] GFTFSSYG

[0137] (SEQ ID NO: 6 (or variants thereof having 1, 2, 3 or 4 point mutations and / or point deletions))

[0138] H1H7017N and H4H7017N CDR-H2 (DNA)

[0139]

[0140] H1H7017N and H4H7017N CDR-H2 (peptide)

[0141] IU

[0142] (SEQ ID NO: 8 (or variants thereof having 1, 2, 3 or 4 point mutations and / or point deletions))

[0143] H1H7017N and H4H7017N CDR-H3 (DNA)

[0144]

[0145] H1H7017N and H4H7017N CDR-H3 (peptide)

[0146] AREGEWVLYYFDY

[0147] (SEQ ID NO: 10 (or variants thereof having 1, 2, 3 or 4 point mutations and / or point deletions))

[0148] H1H7017N and H4H7017N CDR-L1 (DNA)

[0149]

[0150] H1H7017N and H4H7017N CDR-L1 (peptide)

[0151] QSISSW

[0152] (SEQ ID NO: 12 (or variants thereof having 1, 2, 3 or 4 point mutations and / or point deletions))

[0153] H1H7017N and H4H7017N CDR-L2 (DNA)

[0154] AAG GCG TCT

[0155] (SEQ ID NO:13)

[0156] H1H7017N and H4H7017N CDR-L2 (peptide)

[0157] KAS

[0158] (SEQ ID NO: 14 (or a variant thereof having a point mutation and / or a point deletion))

[0159] H1H7017N and H4H7017N CDR-L3 (DNA)

[0160]

[0161] H1H7017N and H4H7017N CDR-L3 (peptide)

[0162] QQYNSYSYT

[0163] (SEQ ID NO: 16 (or variants thereof having 1, 2, 3 or 4 point mutations and / or point deletions))

[0164] H1H7017N

[0165] Full length heavy chain - human IgG1

[0166]

[0167] Full length light chain - human K

[0168]

[0169] H4H7017N

[0170] Full length heavy chain - human IgG4 (S108P)

[0171]

[0172] Full length light chain - human K

[0173]

[0174] The antibodies and antigen-binding fragments of the present invention include such immunoglobulin chains, which include the amino acid sequences described herein and the cell modifications and in vitro post-translational modifications of the antibodies. For example, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to TMPRSS2, including 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 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. Antibodies and fragments.

[0175] The invention provides a container (eg, a plastic or glass vial, eg, with a cap or chromatography column, hollow needle or syringe cylinder) comprising an anti-TMPRSS2 antigen binding protein of the invention (eg, H1H7017N or H4H7017N).

[0176] The present invention also provides an injection device comprising one or more antigen-binding proteins (e.g., antibodies or antigen-binding fragments) (e.g., H4H7017N or H1H7017N) that specifically bind to TMPRSS2 or a pharmaceutical composition thereof. The injection device can be packaged as a kit. The injection device is a device that introduces a substance into a subject's body by means of a parenteral (e.g., intramuscular, subcutaneous or intravenous) route. For example, the injection device can be a syringe (e.g., prefilled with a pharmaceutical composition, such as an automatic syringe), the syringe comprising, for example, a barrel or barrel containing a fluid to be injected (e.g., containing an antibody or fragment or a pharmaceutical composition thereof), a needle that pierces the skin and / or a blood vessel to inject the fluid; and a plunger that pushes the fluid out of the barrel and through the needle hole. 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 contain an antigen binding protein or a pharmaceutical composition thereof in a cannula or trocar / needle that may be engaged with a tube, wherein the tube may be engaged with a bag or reservoir containing a fluid (e.g., saline) that is introduced into the subject's body through the cannula or trocar / needle. In one embodiment of the invention, once the trocar and cannula are inserted into the subject's vein and the trocar is removed from the inserted cannula, the antibody or fragment thereof or the pharmaceutical composition may be introduced into the device. The IV device may be inserted, for example, into a peripheral vein (e.g., in the palm of the hand or arm); into the superior vena cava or the inferior vena cava, or inside the right atrium of the heart (e.g., a central IV); or into the subclavian vein, the internal jugular vein, or the femoral vein, and, for example, advanced toward the heart until it reaches the superior vena cava or the right atrium (e.g., a central venous catheter). In one embodiment of the invention, the injection device is an automatic injector; 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 the antibody or fragment or its pharmaceutical composition into the subject's body. An external infusion pump is a medical device that delivers an antibody or fragment or its pharmaceutical composition to the subject's body in controlled amounts. External infusion pumps can be electrically or mechanically driven. Different pumps operate in different ways, for example, syringe pumps contain fluid in a reservoir in a syringe, and a moving plunger controls fluid delivery, elastomeric pumps contain fluid in a stretchable balloon reservoir, and pressure from the elastic wall of the balloon drives fluid delivery. In peristaltic pumps, a set of rollers gradually narrow along the length of a flexible tube, pushing the fluid forward. In multichannel pumps, fluid can be delivered from multiple reservoirs at multiple rates.

[0177] The present invention also provides a method of administering an anti-TMPRSS2 antigen binding protein of the present invention (e.g., H4H7017N or H1H7017N), the method comprising introducing the antigen binding protein into the body of a subject (e.g., a human). For example, the method comprises puncturing the body of the subject with a needle of a syringe and injecting the antigen binding protein into the subject's body, for example, into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0178] Preparation of human antibodies

[0179] Methods for producing 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 produce human antibodies that specifically bind to TMPRSS2. An immunogen comprising any of the following can be used to produce 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 viruses or with DNA encoding a protein or a fragment thereof. Alternatively, standard biochemical techniques can be used to produce TMPRSS2 proteins or fragments thereof and to modify and use them as immunogens. In one embodiment of the present invention, the immunogen is a recombinantly produced TMPRSS2 protein or 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 a Chinese hamster ovary (CHO) cell.

[0180] Using VELOCIMMUNE TM Technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, ) or any other known method for producing monoclonal antibodies, a high affinity chimeric antibody against TMPRSS2 was initially isolated that had a human variable region and a mouse constant region. The technology includes producing transgenic mice, which have a genome comprising human heavy and light chain variable regions operably connected to endogenous mouse constant region loci, so that the mice produce antibodies comprising human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding antibody heavy and light chain variable regions is separated and operably connected to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing complete human antibodies.

[0181] Usually, the target antigen is challenged Mouse, reclaim lymphocytes (such as B cells) from the mouse expressing the antibody. Lymphocytes can be fused with myeloma cell lines to prepare immortal hybridoma cell lines, screen and select these hybridoma cell lines to identify hybridoma cell lines that produce antibodies specific to the target antigen. Separate the DNA encoding the heavy chain and light chain variable regions and connect to the desired heavy chain and light chain isotype constant regions. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, the DNA encoding the variable domains of antigen-specific chimeric antibodies or light and heavy chains can be directly separated from antigen-specific lymphocytes.

[0182] Initially, a high-affinity chimeric antibody with a human variable region and a mouse constant region is isolated. As in the following experimental section, the antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is then replaced with the desired human constant region to produce a fully human antibody of the present invention, such as a wild-type or modified IgG1 or IgG4. Although the selected constant region may vary according to specific use, high-affinity antigen binding and target-specific characteristics are present in the variable region.

[0183] Anti-TMPRSS2 antibodies comprising Fc variants

[0184] According to certain embodiments of the present invention, there is provided an anti-TMPRSS2 antigen binding protein, e.g., an antibody or antigen binding fragment, comprising an Fc domain, wherein the Fc domain comprises one or more mutations, e.g., mutations that enhance or weaken the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention comprises a C in the Fc domain H Zone 2 or C HRegion 3 comprises an anti-TMPRSS2 antibody with a mutation, wherein the mutation increases the affinity of the Fc domain for FcRn under an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modifications at 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 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 positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification includes 428L (e.g., M428L) and 434S (e.g., N434S) modification; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modification; 433K (e.g., H433K) and 434 (e.g., 434Y) modification; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; 250Q and 428L modification (e.g., T250Q and M428L); and 307 and / or 308 modification (e.g., 308F or 308P). In another embodiment, the modification includes 265A (e.g., D265A) and / or 297A (e.g., N297A) modification.

[0185] For example, the present invention includes an anti-TMPRSS2 antigen binding protein, e.g., an antibody or antigen binding fragment, comprising an Fc domain comprising one or more pairs or one or more groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M42 8L 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).

[0186] Anti-TMPRSS antigen binding proteins, e.g., antibodies and antigen binding fragments thereof, comprising a V domain comprising any possible combination of the aforementioned Fc domain mutations as described herein are contemplated within the scope of the present invention. H and / or V L .

[0187] The present invention also includes such anti-TMPRSS2 antigen binding proteins, antibodies or antigen binding fragments, which contain V described herein. H and chimeric heavy chain constant (C H ) region, wherein the chimeric C H The region contains C from more than one immunoglobulin isotype H For example, the antibody of the present invention may comprise a chimeric C H region, the chimeric C H The region comprises a C region derived from a human IgG1, human IgG2 or human IgG4 molecule. H 2 domain, said part or all of the C domain derived from human IgG1, human IgG2 or human IgG4 molecule H According to certain embodiments, the antibodies of the present invention comprise a chimeric C having a chimeric hinge region. H For example, the chimeric hinge region may comprise an "upper hinge" amino acid sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues from positions 216 to 227 according to EU numbering), combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues from positions 228 to 236 according to EU numbering). According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2 as described herein. H Antibodies with modified Fc regions can exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibodies. (See, e.g., WO2014 / 022540).

[0188] Immunoconjugates

[0189] 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 drug, to treat influenza virus infection. In one embodiment of the invention, the 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, chemically or biologically linked to a radioactive substance, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein, or a therapeutic agent. The antigen binding protein can be linked to a radioactive substance, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or a therapeutic agent at any position of the molecule, as long as it is able to bind to its target (TMPRSS2). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the invention, the agent may 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 (eg, antibody or fragment) will take into account 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: A Review”, Monoclonal Antibodies 1984: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); 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).

[0190] Multispecific Antibodies

[0191] 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., antibody or antigen binding fragment, includes multispecific (e.g., bispecific or biparatopic) molecules comprising at least one first antigen binding domain that specifically binds to TMPRSS2 (e.g., an antigen binding domain from H1H7017N or H4H7017N) and at least one 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 epitope and the second epitope overlap. In another embodiment of the invention, the first epitope and the second epitope do not overlap. For example, in one embodiment of the invention, the multispecific antibody is a bispecific IgG antibody (e.g., IgG1 or IgG4), which comprises a first antigen binding domain that specifically binds to TMPRSS2, wherein the first antigen binding domain comprises an immunoglobulin heavy chain and a light chain of H1H7017N or H4H7017N, and a second antigen binding domain that specifically binds to influenza HA (which comprises a different immunoglobulin light chain and heavy chain, such as from H1H14611N2, H1H14612N2 or H1H11729P).

[0192] "H1H7017N" includes multispecific molecules, for example, antibodies or antigen-binding fragments, comprising the HCDRs and LCDRs, V H and V L , or HC and LC (including variants thereof as described herein).

[0193] "H4H7017N" includes multispecific molecules, e.g., antibodies or antigen-binding fragments, comprising the HCDRs and LCDRs, V H and V L , or HC and LC (including variants thereof as described herein).

[0194] In one embodiment of the present invention, antigen binding domains that specifically bind to TMPRSS that can be incorporated into multispecific molecules include: (1)

[0196] (i) a heavy chain variable domain sequence comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:6, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:10, and

[0197] (ii) a light chain variable domain sequence comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16;

[0198] or (2)

[0200] (i) a heavy chain variable domain sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, and;

[0201] (ii) a light chain variable domain sequence comprising the amino acid sequence set forth in SEQ ID NO:4;

[0202] or (3)

[0204] (i) a heavy chain immunoglobulin sequence comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19, and

[0205] (ii) a light chain immunoglobulin sequence comprising the amino acid sequence set forth in SEQ ID NO:18.

[0206] In one embodiment of the invention, a multispecific antibody or fragment comprises more than two different binding specificities (eg, a trispecific molecule), eg, one or more additional antigen binding domains that are the same or different from the first and / or second antigen binding domain.

[0207] In one embodiment of the invention, in addition to an antigen binding site that specifically binds to TMPRSS2, the multispecific molecule further comprises an antigen binding site that specifically binds to influenza HA taken from an antibody selected from the group consisting of:

[0208] <h2 style=";text-align:left;direction:ltr">H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N; H1H11829N2;H2aM11829N;H2M11830N;H1H11830N2;H1H11903N;H1H14571N ;H2a14571N;H1H11704P;H1H11711P;H1H11714P;H1H11717P;H1H11724P;H 1H11727P;H1H11730P2;H1H11731P2;H1H11734P2;H1H11736P2;H1H11742P 2;H1H11744P2;H1H11745P2;H1H11747P2;H1H11748P2;H1H17952B;H1H17953B;H1H17954B;H1H17955B;H1H17956B;H1H17957B;H1H17958B;H1H17959 B;H1H17960B;H1H17961B;H1H17962B;H1H17963B;H1H17964B;H1H17965B;H1H17966B;H1H17967B;H1H17968B;H1H17969B;H1H17970B;H1H17971B;H1H 17972B;H1H17973B;H1H17974B;H1H17975B;H1H17976B;H1H17977B;H1H17978B;H1H17979B;H1H17980B;H1H17981B;H1H17982B;H1H17983B;H1H1798 4B;H1H17985B;H1H17986B;H1H17987B;H1H17988B;H1H17989B;H1H17990B ;H1H17991B;H1H17992B;H1H17993B;H1H17994B;H1H17995B;H1H17996B;H 1H17997B;H1H17998B;H1H17999B;H1H18000B;H1H18001B;H1H18002B;H1H 18003B;H1H18004B;H1H18005B;H1H18006B;H1H18007B;H1H18008B;H1H18 009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H1801 5B;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;<h2 style=";text-align:left;direction:ltr">H1H18226B;H1H18227B;H1H18228B;H1H18229B;H1H18230B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18 238B;H1H18239B;H1H18240B;H1H18241B;H1H18242B;H1H18243B;H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B; H1H18251B;H1H18252B;H1H18253B;H1H18254B;H1H18255B;H1H18256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18 264B;H1H18265B;H1H18266B;H1H18267B;H1H18268B;H1H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B; H1H18278B;H1H18279B;H1H18280B;H1H18281B;H1H18282B;H1H18283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B;H1H18 290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B;H1H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18303B; H1H18304B;H1H18305B;H1H18306B;H1H18307B;H1H18308B;H1H18309B;H1 H18310B;H1H18311B;H1H18312B;H1H18313B;H1H18314B;H1H18315B;H1H18 316B;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., 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 influenza group II HA protein, for example, the antigen binding site comprises V1H14611N2. 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 influenza group II HA protein, for example, the antigen binding site comprises V1H14612N2. 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 influenza group I HA protein, for example, the antigen binding site comprises V1H11729P. 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 (e.g., a V comprising H1H7017N or H4H7017N) having binding specificity for a first epitope (e.g., TMPRSS2). H and V L ) and a second scFv having binding specificity for a different second epitope (e.g., comprising a V H and V L For example, in one embodiment of the invention, the first and second scFvs are linked by a linker (e.g., a peptide linker (e.g., a GS linker such as (GGGGS) n (SEQ ID NO: 48), wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10)) tethered. Other bispecific antigen-binding fragments include F(ab)2 of a bispecific IgG antibody comprising the heavy chain CDR and light chain CDR of H1H7017N or H4H7017N and another antibody that binds to a different epitope.

[0213] Treatment

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

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

[0216] An effective dose or therapeutically effective dose of an anti-TMPRSS2 antigen binding protein (e.g., an antibody or antigen binding fragment (e.g., H1H7017N or H4H7017N)) for treating or preventing viral infection refers to an amount of the antibody or fragment that is sufficient to alleviate one or more signs and / or symptoms of infection in the treated individual, whether by causing such signs and / or symptoms to subside or disappear or by inhibiting the progression of such signs and / or symptoms. The dosage may vary depending on the age and size of the subject to be administered, the target disease condition, the route of administration, etc. In one embodiment of the invention, an effective dose or therapeutically effective dose of an antibody or antigen binding fragment thereof of the invention for treating or preventing viral infection (e.g., in an adult subject) is about 0.01 to about 200 mg / kg, for example, 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 may be adjusted. In certain embodiments, the antigen-binding proteins of the invention may be administered in an initial dose, followed by one or more secondary doses. In certain embodiments, the initial dose may be followed by a second or more subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be roughly equivalent to or less than the initial dose, wherein the subsequent doses are separated by at least 1 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., rat, mouse, cat, dog, cattle, sheep, horse, goat, rabbit), preferably a human, that needs to prevent and / or treat a disease or condition such as a viral infection or cancer. The subject may have a viral infection, for example, an influenza infection, or be susceptible to infection. Subjects susceptible to infection or subjects who may be exposed to elevated risk of contact infection (e.g., influenza virus) include subjects whose immune system is impaired due to autoimmune disease, subjects receiving immunosuppressive therapy (e.g., after organ transplantation), subjects suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), subjects suffering from anemia forms of loss or destruction of white blood cells, subjects receiving radiation or chemotherapy, or subjects suffering from inflammatory diseases. In addition, very young (e.g., 5 years old or less) or old (e.g., 65 years old or more) subjects face increased risks. Additionally, subjects may be at risk for exposure to viral infections due to proximity to disease outbreaks (e.g., subjects living in densely populated cities or living in close proximity to subjects with confirmed or suspected viral infections) or employment choices (e.g., hospital workers, pharmaceutical researchers, travelers to infected areas, or frequent travelers).

[0218] "Treatment" or "treatment" means administering an anti-TMPRSS2 antigen binding protein, e.g., an antibody or antigen binding fragment of the invention (e.g., H1H7017N or H4H7017N), 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 against the subject when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0219] The present invention also encompasses the prophylactic administration of an anti-TMPRSS2 antigen binding protein, e.g., an antibody or antigen binding fragment thereof (e.g., H1H7017N or H4H7017N) to a subject at risk of viral infection, thereby preventing such infection. Antibody-based passive immunoprophylaxis has been shown 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 ResTreat. 2014; 2014:267594. Epub 2014 Sep 22; and Jianqiang et al., Passive immuneneutralization strategies for prevention and control of influenza Ainfections, Immunotherapy. 2012February; 4(2):175–186; Prabhu et al., AntivirTher. 2009; 14(7):911-21, Prophylactic and therapeutic efficacy of a chimericmonoclonal antibody specific for H5 hemagglutinin against lethal H5N1influenza. "Preventing" or "prevention" means administering an anti-TMPRSS2 antigen binding protein, e.g., an antibody or antigen binding fragment of the invention (e.g., H1H7017N or H4H7017N), 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 against 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, the signs or symptoms of viral infection in a subject are viral survival or proliferation in the subject, such as determined by a viral titer assay (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., an influenza virus or coronavirus infection) or causing at least one of the following signs or symptoms of a viral infection to subside or be eliminated or inhibit its progression in a subject (e.g., a human) in need thereof by:

[0222] Fever or feeling hot / chilled;

[0223] ·cough;

[0224] Sore throat

[0225] Rhinorrhea or nasal congestion;

[0226] ·sneeze

[0227] Muscle or body pain;

[0228] Various headaches;

[0229] Fatigue (tiredness);

[0230] ·Vomit;

[0231] ·diarrhea;

[0232] Respiratory tract infection;

[0233] Chest discomfort

[0234] Shortness of breath

[0235] Bronchitis; and / or

[0236] ·pneumonia,

[0237] The signs or symptoms are secondary to a viral infection: A therapeutically effective amount of an anti-TMPRSS2 antigen binding protein (eg, H1H7017N or H4H7017N) is administered to the subject, for example, by injecting the protein into the subject's body.

[0238] The present invention also includes methods of treating or preventing cancer, e.g., metastatic cancer, e.g., prostate cancer (e.g., characterized by 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 a therapeutically effective amount of a TMPRSS2 antigen binding protein (e.g., H1H7017N or H4H7017N) to the subject, e.g., injecting the protein into the subject's body. In one embodiment of the invention, a TMPRSS2 antigen binding protein in combination with other therapeutic agents (e.g., anticancer therapeutic agents) is also administered to the subject. In one embodiment of the 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 of an anti-TMPRSS2 antigen binding protein (eg, an antibody and an antigen binding fragment thereof (eg, 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, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; 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, NY. In one embodiment of the invention, the pharmaceutical composition is sterile. Such compositions are part of the present invention.

[0241] Included within the scope of the present invention are dehydrated (eg, lyophilized) compositions comprising an anti-TMPRSS2 antigen binding protein (eg, an antibody or antigen binding fragment thereof (eg, H1H7017N or H4H7017N)) or pharmaceutical compositions thereof comprising a pharmaceutically acceptable carrier but substantially lacking water.

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

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

[0244] The present invention provides methods for administering an anti-TMPRSS2 antigen binding protein (e.g., an antibody or 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 a needle of a syringe and injecting the antigen binding protein into the body of the subject, for example, into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

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

[0246] In one embodiment of the invention, an anti-TMPRSS2 antigen binding protein (e.g., an antibody or antigen binding fragment thereof of the invention (e.g., H1H7017N or H4H7017N)) is combined with one or more other therapeutic agents. For example, in one embodiment of the 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 alleviate a viral infection in a subject. The term "antiviral drug" includes, but is not limited to, cationic steroid antimicrobials, leupeptin, aprotinin, amantadine, rimantadine, oseltamivir, zanamivir, ribavirin or interferon-α2b. Methods of treating or preventing viral (e.g., influenza) infections by administering H1H7017N or H4H7017N in combination with other therapeutic agents in a subject in need of such treatment or prevention are part of the invention.

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

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

[0249]

[0250] (camostat mesylate);

[0251]

[0252] (nefamostat 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 invention, the antiviral drug is an antibody or antigen-binding fragment that specifically binds to an influenza virus (e.g., influenza HA). For example, in one embodiment of the invention, the anti-HA antibody is any 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; 17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; 964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; 75B; H1H17976B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H18009B; 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;<h2 style=";text-align:left;direction:ltr">H1H18218B;H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18225B;H1H18226B;H1H18227B;H1H18228B;H1H18229B;H1H18 230B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1H18241B;H1H18242B; H1H18243B;H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B;H1H18251B;H1H18252B;H1H18253B;H1H18254B;H1H18 255B;H1H18256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1H18267B;H1H18268B; H1H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B;H1H18279B;H1H18280B;H1H18281B;H1H18 282B;H1H18283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B;H1H18290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B; H1H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18303B;H1H18304B;H1H18305B;H1H18306B;H1H18307B;H1H18 308B;H1H18309B;H1H18310B;H1H18311B;H1H18312B;H1H18313B;H1H1831 4B;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, for example, wherein the antibody or fragment comprises CDR-L1, CDR-L2 and CDR-L3 (e.g., V; L or light chain) of a light chain immunoglobulin that; and a CDR-H1, CDR-H2 and CDR-H3 thereof (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 influenza group II HA protein, such as H1H14611N2; or a V comprising H1H14611N2. H and V L 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). "H1H14611N2" refers to any anti-group II HA antibody comprising 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: QQYGNSPLT (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 influenza group II HA protein, such as H1H14612N2; or a V comprising H1H14612N2. H and V L 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 anti-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 invention, the other therapeutic agent is an antibody or antigen-binding fragment that binds to influenza group I HA protein, such as H1H11729P; or a V comprising H1H11729P. 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 anti-group I HA antibody comprising such sequences.

[0286] H1H11729P

[0287] Heavy chain variable region

[0288]

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

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

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

[0292] Light chain variable region

[0293]

[0294] CDH-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 invention, the additional therapeutic agent is not amantadine, rimantadine, oseltamivir, zanamivir, aprotinin, leupeptin, a cationic steroid antimicrobial, an influenza vaccine (e.g., killed, live, attenuated whole virus or subunit vaccine), or an antibody against influenza virus (e.g., an anti-hemagglutinin antibody).

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

[0299] Kits

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

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

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

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

[0304] The kit may include a drug package insert containing information relating to the pharmaceutical composition and dosage form in the kit. Typically, 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 package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and utilization, 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 Uses of Antibodies

[0306] Anti-TMPRSS2 antigen binding proteins, e.g., antibodies or antigen binding fragments thereof of the invention (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 a sample with an anti-TMPRSS2 antigen binding protein of the 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 separate TMPRSS2 from the sample. The presence of an anti-TMPRSS2 antigen binding protein complexed with TMPRSS2 indicates the presence of TMRPSS2 in the sample. Alternatively, an unlabeled anti-TMPRSS2 antibody may be used in combination with a second antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S, or 125 I; a fluorescent 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). Therefore, 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 the 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 an antigen-binding fragment thereof (e.g., H1H7017N)) to detect the presence of TMPRSS2 on a cell. In one embodiment of the present invention, the method comprises the steps of:

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

[0309] (ii) optionally washing the mixture to remove unbound anti-TMPRSS2 antigen binding protein;

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

[0311] (iv) optionally washing the complex to remove unbound antigen binding protein; and

[0312] (v) detecting the presence of a marker on the second antibody or fragment, wherein detection of the marker indicates that the cell contains TMPRSS2. For example, the present invention includes such a method for identifying TMPRSS2 in a sample. + Cell-based ELISA method.

[0313] The anti-TMPRSS2 antigen binding proteins of the invention (e.g., H1H7017N or H4H7017N) can be used in Western blot or immuno-Western blot methods for detecting the presence of TMPRSS2 or fragments thereof in a sample. Such methods form part of the invention and, for example, comprise the steps of:

[0314] (1) providing a membrane or other solid substrate comprising a sample to be tested for the presence of TMPRSS2, for example, optionally comprising the steps of: transferring proteins from the sample to be tested for the presence of TMPRSS2 (e.g., separated by PAGE or SDS-PAGE electrophoresis of proteins in the sample) to a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or slot blotting); and contacting the membrane or other solid substrate to be tested for the presence of TMPRSS2 or a fragment thereof with an 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 the protein to be tested for the presence of TMPRSS2 has been transferred (e.g., after electrophoretic separation in a gel) in a native PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel. Prior to contacting the membrane with the anti-TMPRSS2 antigen binding protein, the membrane is optionally blocked (e.g., with skim milk, etc.) to bind nonspecific protein binding sites on the membrane.

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

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

[0318] Detection of bound antigen binding protein indicates the presence of TMPRSS2 protein on the membrane or substrate and in the sample. Bound antigen binding protein can be detected by binding the antigen binding protein with a detectably labeled second antibody (anti-immunoglobulin antibody) 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 for immunohistochemistry. Such methods form part of the present invention and include, for example,

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

[0321] (2) Detection of antigen-binding proteins on or in tissues.

[0322] If the antigen binding protein itself is detectably labeled, it can be detected directly. Alternatively, the antigen binding protein can be bound by a detectably labeled second antibody, whereupon the label is detected. Example

[0323] The following examples are provided to provide a person of ordinary skill in the art with 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 of what 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 considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric pressure.

[0324] Example 1 : Multi-cycle replication in vitro.

[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] describe 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] 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 in a 96-well plate. 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 US 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 MOI (multiplicity of infection) of 0.1 and 0.01. The virus was incubated on the cells for 1 hour at 37°C, after which the virus was removed and the wells were washed an additional three times. The number of infected cells was compared at 24, 48, 72 and 142 hours post infection. The data were quantified on an S6 universal analyzer (Cellular Technology Limited, Cleveland, OH).

[0330] Summary of results and conclusion

[0331] Calu-3 is an immortalized human airway epithelial cell line that has been shown to allow multicycle 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, but not TMPRSS11D (HAT), at least at the mRNA level ( et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholinooligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011)). In order to confirm that Calu-3 cells can support proteolytic activation of influenza viruses that have hemagglutinin containing a monovalent shearing site, the growth of H1N1 GFP reporter virus in the Calu-3 cells was analyzed and compared in the absence of trypsin, and the replication of A549 (human alveolar basal epithelium), MDCK (Madin Darby canine kidney) and HepG2 (human liver cancer) cells was adopted over time. Cells were infected by low MOI, and at the indicated time point, viral titer was determined by counting fluorescent focus spots. Table 2 and Fig. 1 are presented at low levels of infection in A549, MDCK and HepG2 cells, and Calu-3 cells are presented at each time point and significantly increase titer. 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 morpholinooligomer 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. The number of infected cells represented by fluorescent focus units (FFU) in different cell types after infection with A / Puerto Rico / 08 / 1934(H1N1)-GFP at different days after infection at MOI 0.1 or 0.01.

[0333]

[0334] References

[0335] 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.

[0336] 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: 27733646.

[0337] 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Research, 116, 76–84 (2015). PMID: 25666761.

[0338] 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.

[0339] 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.

[0340] 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.

[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. 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, 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, Proc Natl Acad Sci US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007). PMID: 17855549.

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

[0347] The ability of various 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] Calu-3 cells (ATCC HTB55) were diluted to 40,000 cells / well in DMEM:F12 medium containing 5% FBS in a 96-well plate. The next day, the monoclonal antibody was diluted to 166.7 nM in DMEM:F12 containing low IgG BSA and added to the cells and continued 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 MOI 0.001. The virus was incubated on the cells at 37°C at 5% CO2 for 1 hour, after which the virus was removed and the culture medium was replaced with DMEM:F12 containing 166.7 nM mAb. After 24 hours and 48 hours, the culture medium was replaced with fresh culture medium containing mAb and the cells were washed twice with PBS at 72 hours. The cells were subsequently fixed with 4% paraformaldehyde in PBS and the anti-NP first antibody at a dilution of 1:1000 was used to detect the virus. The cells were incubated for 1 hour and then washed and the secondary antibody was added at a dilution of 1:2000. The quantification was performed on an S6 universal analyzer (Cellular Technology Limited, Cleveland, OH).

[0353] Summary of results and conclusion

[0354] Calu-3 is an immortalized human airway epithelial cell line that has been shown to allow multiple cycles of human influenza virus replication 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, but not TMPRSS11D (HAT), at least at the mRNA level ( et al., Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholinooligomer targeting the hemagglutinin-activating protease TMPRSS2, Journal of Virology, 85, 1554–1562 (2011). Calu-3 cells have been shown previously to support proteolytic activation of influenza virus—but here inhibition of TMPRSS2 was examined using the TMPRSS2-specific monoclonal antibody H1H7017N. Growth of A / Puerto Rico / 08 / 1934 (H1N1) was analyzed over a 72-hour period after treatment of cells with 166.7 nM H1H7017N. Virus titers were determined by counting fluorescent foci. Table 4 and Figure 2 The titers were shown to be reduced after treatment with antibody H1H7017N. 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 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. Administration of H1H7017N during the infection cycle reduces the number of fluorescent focus units (FFU) of A / Puerto Rico / 08 / 1934 (H1N1) at 72 hours post infection.

[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. 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: 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 US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007). PMID: 17855549.

[0369] Example 3 : FACS analysis of 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 that do or do not express 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) from 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 lines were referred to as MDCK / Tet-on cell lines. The MDCK / Tet-on cell lines were 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 referred to as MDCK / Tet-on / hTMPRSS2 and MDCK / Tet-on / mfTMPRSS2. 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 analysis by flow cytometry, cells were plated in growth medium and incubated with 1 μg / mL of doxycycline for 16 hours 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 mixed with 1×10 6 The cells were incubated at 4°C for 30 minutes. Conditions were included in which no antibody was added to the cells. After incubation with the primary antibody, the cells were stained with an anti-human IgG secondary antibody conjugated to allophycocyanin at 1:1000 for 30 minutes at 4°C. The 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 nonlinear regression (4-parameter logarithm) and Prism7 software (GraphPad) to obtain the EC values ​​of antibody-bound cells. 50 value.

[0377] like Figure 3 As shown in Figure 2, the anti-hTMPRSS2 antibody H1H7017N of the present invention has an EC of 460 pM and 1.06 nM, respectively. 50Values ​​for binding 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 constants (K) of different TMPRSS2 reagents binding to purified anti-TMPRSS2 monoclonal antibodies were determined using a real-time surface plasmon resonance-based Biacore 4000 biosensor. D All binding studies were performed 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 at 25°C and 37°C. The surface of the Biacore CM5 sensor chip was first derivatized by amine coupling with a rabbit anti-mouse Fc-specific polyclonal antibody (GE Healthcare catalog number BR100838) to capture the anti-TMPRSS2 monoclonal antibody. Binding studies were performed on the human TMPRSS2 extracellular domain expressed with a C-terminal myc-myc-hexa-histidine tag (hTMPRSS2.mmh) and the monkey TMPRSS2 extracellular domain expressed with a C-terminal myc-myc-hexa-histidine tag (mfTMPRSS2.mmh). 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 over the anti-mouse Fc-captured anti-TMPRSS2 mAb surface at a flow rate of 30 μL / min for 2.5 min, while the dissociation of mAb-bound TMPRSS2 reagent was monitored for 7 min in HBS-ET running buffer. The association rates (ka) and dissociation rates (kd) were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transfer limitations using Scrubber 2.0c curve fitting software. The binding-dissociation equilibrium constant (Kd) was calculated from the kinetic rates. D ) and dissociation half-life (t1 / 2) are:

[0380] and

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

[0382] At 25°C, the anti-TMPRSS2 monoclonal antibody had a K of 2.81 nM. D The values ​​were shown in Table 6 for binding to HMM-hTMPRSS2. At 37°C, the anti-HMM-hTMPRSS2 monoclonal antibody had a K of 9.31 nM. D The values ​​were combined with HMM-hTMPRSS2 as shown in Table 7.

[0383] At 25°C, the anti-TMPRSS2 monoclonal antibody had a K of 56.0 nM. D The values ​​were shown in Table 8 for binding to HMM-mfTMPRSS2. At 37°C, the anti-TMPRSS2 monoclonal antibody had a K of 140 nM. D The values ​​were combined with HMM-mfTMPRSS2 as shown in Table 9.

[0384] TMPRSS2 protein

[0385] hTMPRSS2 knob_mmh(W106-R255).mmh:

[0386] Amino acids 1-150: amino acids 106 to 255 of human TMPRSS2 (accession number NP_005647.3, with V160M)

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

[0388]

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

[0390] mfTMPRSS2 knob_mmh(W106-R255).mmh:

[0391] Amino acids 1-150: amino acids 106-255 of monkey TMPRSS2 (accession number XP_005548700.1, with S129L, N251S)

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

[0393]

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

[0395] result

[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 domains described herein and mouse 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 antibody at 37°C.

[0404]

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

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

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

[0408]

[0409]

[0410] Experimental methods

[0411] Calu-3 cells were seeded to 40,000 cells / well in a 96-well plate in DMEM:F12 medium containing 5% FBS. The next day, the influenza strains were diluted to a pre-determined MOI (see Table 11) and the antibodies were diluted to 100 μg / mL. In these experiments, anti-HA and anti-TMPRSS2 antibodies have different mechanisms of action, and therefore, the experimental methods for these antibodies are different to properly detect them. The anti-HA antibodies were pre-incubated with individual influenza strains in separate plates at 37°C for one hour. After the pre-incubation period, the antibody / virus mixture was added to the Calu-3 cells for one hour. The anti-TMPRSS2 antibodies were pre-incubated with uninfected Calu-3 cells at 37°C for three hours. After the pre-incubation period, the viruses were added to the Calu-3 cells pre-incubated with the anti-TMPRSS2 antibodies for one hour. After one hour of infection, the cells were washed three times with PBS and fresh antibodies were added to each well along with new medium. Additional antibodies were added 24 hours and 48 hours after infection. 72 h after infection, cells were stained with anti-NP and The data were quantified on an S6 universal analyzer (Cellular Technology Limited, Cleveland, OH).

[0412] Table 11A. Experiment 1.

[0413] Flu 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] Flu strains Final MOI H1_PR34 0.01 Florida 0.01 Malaysia 0.001

[0416] Summary of results and conclusion

[0417] Calu-3 is an immortalized human airway epithelial cell line that has been shown to allow 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. 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.

[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 US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007), PMID: 17855549.

[0434] Example 6 : Effects of single treatment with H1H7017N in TMPRS22 humanized mice.

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

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

[0437]

[0438]

[0439] Table 14. mAb clone IDs.

[0440] AbPID describe 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. Mice were sedated with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) by intraperitoneal injection and then infected intranasally with 20 μL of virus. Antibodies were delivered subcutaneously (SC) one day before infection or intravenously (IV) a few days after infection (PI). Antibody dosing regimens varied between experiments (Table 15). Body weights were collected daily until day 14 PI, and mice were sacrificed when they lost 20% of their initial body weight. Results are reported as percent survival.

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

[0444] Antibody PI Days dose deliver H1H1238N -1 5mg / kg SC H1H7017N -1,0 5mg / kg SC, IV

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

[0446] Antibody PI Days dose deliver H1H1238N 0 10mg / kg IV H1H7017N 0,1,2,3 10mg / kg IV

[0447] Summary of results and conclusion

[0448] It has been shown that mice engineered to express human TMPRSS2 protein can be infected with a lethal dose of influenza virus. The purpose of these experiments was to show that H1H7017N can protect mice engineered to express human TMPRSS2 protein against influenza A group 1. The antibody was tested in both a preventive and a therapeutic mode. In both experiments, H1H7017N treatment resulted in a higher survival rate ( 1.5% vs 1.5% survival) than mice treated with the isotype control (H1H1238N). Figure 4 and Figure 5 ). In the prevention experiment, the survival rate of mice treated with H1H1238N was 0%, the survival rate of mice treated on day -1 PI was 85.7%, and the survival rate of mice treated with H1H7017N on day 0 PI was 100%. For the treatment mode, the H1H1238N treatment group produced a 25% survival rate, while the group treated with H1H7017N on days 0-3 PI produced a 100% survival rate. The data are summarized in Table 16. H1H7017N showed effectiveness 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. 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, 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, Proc Natl Acad Sci US 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 elicitan 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 infection with H3N2 influenza virus was evaluated.

[0467] Table 17. mAb clone IDs.

[0468] AbPID describe H1H7017N Anti-TMPRSS2 Antibodies

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

[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. Mice were sedated with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) by intraperitoneal injection and then infected intranasally with 20 μL of virus. On day 1 or 2 post infection (PI), mice were injected intravenously with antibodies. Mice were weighed and observed daily until day 14 post infection (PI). When mice lost 25% of their initial body weight, they were sacrificed.

[0473] Summary of results and conclusion

[0474] Breadth is an important quality when considering influenza therapy. The anti-TMPRSS2 antibody H1H7017N has been shown to be effective against influenza A group 1. The purpose of this experiment was to show that H1H7017N can protect mice engineered to express human TMPRSS2 protein against influenza A group 2. Mice engineered to express human TMPRSS2 protein were infected with a lethal dose of H3N2 and treated on day 1 or day 2 PI. Both treatment groups had higher survival rates than infected controls. Mice treated on day 1 PI had 100% survival, which was higher than the group treated on day 2 PI with 50% survival, while untreated mice had 0% survival. All mice died between days 5-6 PI. Figure 6 Survival curves are shown in and % survival is summarized in Table 19. These results show that H1H7017 improves outcomes in the H3N2 lethal model.

[0475] Table 19. Tabular 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. 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.

[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 US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, 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 of H1N1 influenza virus infected mice engineered to express human TMPRSS2 protein was assessed and compared to wild-type (WT) mice.

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

[0492]

[0493] Experimental methods

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

[0495] Summary of results and conclusion

[0496] Mice engineered to express human TMPRSS2 protein were generated to test the therapeutic effectiveness of anti-TMPRSS2 antibodies in an in vivo model of influenza. 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 H1N1 historical strains were compared. In all three infection 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 PI, and those mice that received higher viral doses died faster than mice that received lower viral doses. The survival pattern of mice engineered to express human TMPRSS2 protein was similar to that of wild-type mice. This shows that mice engineered to express human TMPRSS2 protein can be used as an in vivo model of influenza to evaluate the effectiveness 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. 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.

[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 US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology, 81, 12439–12449 (2007), PMID: 17855549.

[0511] Example 9 : Therapeutic effect of the combination of H1H14611N2 and H1H7017N in mice after H3N2 infection.

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

[0513] Table 22. mAb clone IDs.

[0514] AbPID describe H1H7017N Anti-TMPRSS2 Antibodies H1H14611N2 Anti-Influenza A Group 2 Antibodies H1H1238N IgG1 Isotype Control

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

[0516]

[0517] Experimental methods

[0518] Eight-week-old male and female mice engineered to express 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 with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) by intraperitoneal injection and then infected intranasally with 20 μL of virus. On day 4 post-infection (PI), mice were injected intravenously with antibodies. Body weights were collected daily until day 14 PI, and when the mice lost 25% of their initial body weight, they were sacrificed. Results are reported as percent survival.

[0519] Summary of results and conclusion

[0520] It has been shown that the TMPRSS2 antibody H1H7017N and the broad-spectrum influenza A group 2 antibody H1H14611N2 have therapeutic effectiveness against lethal challenge of mice with H3N2 historical strains. It has also been shown that the survival rate of mice infected with lethal H1N1 challenge can be significantly increased with the combination of H1H7017N and the broad-spectrum influenza A group 1 antibody H1H11729P compared to either alone, after less total antibody treatment. The purpose of this experiment is to evaluate the synergistic effect of H1H7017N and H1H14611N2 when combined. Figure 8 As shown in , 3 of 4 mice treated with the hIgG1 isotype control antibody on day 4 PI died by day 7 PI. 3 of 5 animals survived when 10 mg / kg H1H14611N2 was administered, and 4 of 5 animals survived when 10 mg / kg H1H7017N was administered. When H1H14611N2 and H1H7017N were administered in combination at 5 mg / kg each, there was a 40% survival rate. One hundred percent of mice treated with a combination of H1H14611N2 and H1H7017N at 2.5 mg / kg each after challenge survived. The survival rate of mice infected with lethal H3N2 challenge was increased with the combination of lower concentrations of H1H7017N and H1H14611N2 compared to higher concentrations of the combined antibodies or any single antibody. The survival percentages are 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. 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.

[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 US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, Journal of Virology. 81, 12439–12449 (2007). PMID: 17855549.

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

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

[0538] Table 25. mAb clone IDs.

[0539] AbPID describe H1H7017N Anti-TMPRSS2 Antibodies H1H11729P Anti-Influenza A Group 1 Antibodies H1H1238N IgG1 isotype control

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

[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. Virus was delivered by sedating the mice with 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml) and delivering 20 μL of virus intranasally. On day 3 post infection (PI), mice were injected intravenously with antibodies. Body weights were collected daily until day 14 PI, and mice were sacrificed when they lost 25% of their initial body weight.

[0544] Summary of results and conclusion

[0545] It has been shown that the TMPRSS2 antibody H1H7017N and the broad-spectrum influenza A group 1 antibody H1H11729P have therapeutic effectiveness against lethal challenge of mice with historical strains of H1N1, respectively. However, the purpose of this experiment was to evaluate the synergistic effect of the antibodies when used in combination. As of day 6 PI, all mice treated with the hIgG1 isotype control antibody on day 3 PI died. When the animals received 5 mg / kg H1H11729P or H1H7017N, 40% and 0% of the animals survived infection, respectively. However, the combination of 2.5 mg / kg of each antibody H1H11729P and H1H7017N resulted in a 60% survival rate. Eighty percent of the mice treated with a combination of 1 mg / kg H1H7017N and 2 mg / kg H1H11729P (3 mg / kg in total) survived after the challenge. With the combination of H1H7017N and H1H11729P, the survival rate of mice infected with lethal H1N1 challenge was significantly increased after less total antibody treatment than with either alone (see Fig. 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. 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, 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, Proc Natl Acad Sci US 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 elicitan attenuated type i interferon response in polarized human bronchial epithelial cells, 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 was specifically and solely 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 citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of a dedicated statement of incorporation by reference (if any) within the scope of this specification does not in any way impair this overall status of incorporation by reference. The citation of a reference herein is not intended to admit that the reference is relevant prior art, nor does it constitute any admission of the contents or dates of these publications or documents.

Claims

1. A human antigen binding protein that specifically binds to human TMPRSS2, comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region: The immunoglobulin heavy chain variable region comprises: (a) CDR-H1 consisting of the amino acid sequence GFTFSSYG (SEQ ID NO: 6), (b) CDR-H2 consisting of the amino acid sequence IWNDGSYV (SEQ ID NO: 8) and (c) CDR-H3 consisting of the amino acid sequence AREGEWVLYYFDY (SEQ ID NO: 10); and The immunoglobulin light chain variable region comprises: (a) CDR-L1 consisting of the amino acid sequence QSISSW (SEQ ID NO: 12), (b) CDR-L2 consisting of the amino acid sequence KAS (SEQ ID NO: 14) and (c) CDR-L3 consisting of the amino acid sequence QQYNSYSYT (SEQ ID NO: 16). The antigen-binding protein according to claim 1 , which is an antibody or an antigen-binding fragment thereof.

3. The antigen-binding protein according to claim 1, comprising: (a) an immunoglobulin heavy chain 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; and (b) an immunoglobulin light chain variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

4. The antigen binding protein according to claim 1, comprising: (a) an immunoglobulin heavy chain comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth 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 set forth in SEQ ID NO:

18.

5. The antigen binding protein according to claim 1, comprising: (a) an immunoglobulin heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2; and (b) an immunoglobulin light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:

4.

6. The antigen binding protein according to claim 1, comprising: (a) an immunoglobulin heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 17 or 19; and (b) an immunoglobulin light chain consisting of the amino acid sequence set forth in SEQ ID NO:

18. The antigen-binding protein according to claim 1 , comprising an immunoglobulin chain thereof.

8. The antigen binding protein according to any one of claims 1 to 7, which is multispecific.

9. The antigen binding protein according to any one of claims 1 to 7, comprising one or more of the following characteristics: Inhibits influenza virus growth in cells expressing TMPRSS2; Binds to the surface of cells expressing TMPRSS; No significant binding to MDCK / Tet-on cells that do not express TMPRSS2; ·2.81X 10 at 25℃ -9 M of K D Binds to human TMPRSS2; · At 37°C, 9.31X 10 -9 M of K D Binds to human TMPRSS2; ·5.60X 10 at 25℃ -8 M of K D Binds to cynomolgus monkey TMPRSS2; · At 37°C at 1.40X 10 -7 M of K D Binds to cynomolgus monkey TMPRSS2; Limiting the spread of influenza virus infection of cells in vitro; and / or • Protection of mice engineered to express human TMPRSS2 protein from mortality caused by influenza virus infection.

10. A complex comprising the antigen binding protein according to any one of claims 1 to 7 bound to a TMPRSS2 polypeptide.

11. A polynucleotide encoding the antigen binding protein or immunoglobulin chain thereof according to any one of claims 1 to 7.

12. A vector comprising the polynucleotide according to claim 11.

13. A host cell comprising the polynucleotide according to claim 11.

14. A host cell comprising the vector according to claim 12.

15. Host cells, including: (a) a first polynucleotide encoding an immunoglobulin heavy chain variable region of the antigen binding protein according to claim 1; and (b) a second polynucleotide encoding the immunoglobulin light chain variable region of the antigen binding protein.

16. Host cells, including: (a) a first polynucleotide encoding the immunoglobulin heavy chain variable region of the antigen binding protein according to claim 5; and (b) a second polynucleotide encoding the immunoglobulin light chain variable region of the antigen binding protein.

17. A host cell comprising: (a) a first polynucleotide encoding an immunoglobulin heavy chain of the antigen binding protein according to claim 6; and (b) a second polynucleotide encoding an immunoglobulin light chain of the antigen binding protein.

18. A method for producing an antigen binding protein or an immunoglobulin chain thereof that specifically binds to human TMPRSS2, comprising: (a) culturing the host cell according to any one of claims 13 to 17 under conditions conducive to the expression of the one or more polynucleotides to produce an antigen binding protein or an immunoglobulin chain thereof; and (b) optionally, isolating the antigen binding protein or immunoglobulin chain thereof from the host cell and / or the culture medium in which the host cell is cultivated.

19. The host cell according to any one of claims 13-17, wherein the host cell is a Chinese hamster ovary cell.

20. The method of claim 18, wherein the host cell is a Chinese hamster ovary cell.

21. An antigen binding protein or immunoglobulin chain thereof, which is the product of the method according to claim 18.

22. A composition or kit comprising an antigen binding protein according to any one of claims 1 to 7 in combination with other therapeutic agents.

23. A pharmaceutical composition comprising an antigen binding protein according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier and optionally other therapeutic agents.

24. The composition or kit of claim 22, wherein the other therapeutic agent is an antiviral drug or a vaccine.

25. The composition or kit of claim 22, wherein the other therapeutic agent is a member selected from the group consisting of ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-α2b, interferon-α2a, an anticancer drug, and an antibody or antigen-binding fragment thereof that specifically binds to influenza HA.

26. The composition or kit of claim 25, wherein the antibody or antigen-binding fragment thereof that specifically binds to influenza HA is selected from the following antibodies or antigen-binding fragments thereof: <h2 style=";text-align:left;direction:ltr">H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N; H1H11829N2;H2aM11829N;H2M11830N;H1H11830N2;H1H11903N;H1H14571N ;H2a14571N;H1H11704P;H1H11711P;H1H11714P;H1H11717P;H1H11724P;H 1H11727P;H1H11730P2;H1H11731P2;H1H11734P2;H1H11736P2;H1H11742P 2;H1H11744P2;H1H11745P2;H1H11747P2;H1H11748P2;H1H17952B;H1H17953B;H1H17954B;H1H17955B;H1H17956B;H1H17957B;H1H17958B;H1H17959 B;H1H17960B;H1H17961B;H1H17962B;H1H17963B;H1H17964B;H1H17965B;H1H17966B;H1H17967B;H1H17968B;H1H17969B;H1H17970B;H1H17971B;H1H 17972B;H1H17973B;H1H17974B;H1H17975B;H1H17976B;H1H17977B;H1H17978B;H1H17979B;H1H17980B;H1H17981B;H1H17982B;H1H17983B;H1H1798 4B;H1H17985B;H1H17986B;H1H17987B;H1H17988B;H1H17989B;H1H17990B ;H1H17991B;H1H17992B;H1H17993B;H1H17994B;H1H17995B;H1H17996B;H 1H17997B;H1H17998B;H1H17999B;H1H18000B;H1H18001B;H1H18002B;H1H 18003B;H1H18004B;H1H18005B;H1H18006B;H1H18007B;H1H18008B;H1H18 009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H1801 5B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1 H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18 041B;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; <h2 style=";text-align:left;direction:ltr">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.

27. The pharmaceutical composition of claim 23, wherein the other therapeutic agent is an antiviral drug or a vaccine.

28. The pharmaceutical composition of claim 23, wherein the other therapeutic agent is a member selected from the group consisting of ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-α2b, interferon-α2a, an anticancer drug, and an antibody or antigen-binding fragment thereof that specifically binds to influenza HA.

29. The pharmaceutical composition of claim 28, wherein the antibody or antigen-binding fragment thereof that specifically binds to influenza HA is selected from the following antibodies or antigen-binding fragments thereof: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11717P; H1H11724P; H1 H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2 ;H1H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H1795 3B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B ;H1H17960B;H1H17961B;H1H17962B;H1H17963B;H1H17964B;H1H17965B;H1 H17966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H1 7972B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H179 78B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984 B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H 1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H 17997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H18 003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H1800 9B; 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;<h2 style=";text-align:left;direction:ltr">H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">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; <h2 style=";text-align:left;direction:ltr"> 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.

30. A container or injection device comprising an antigen binding protein or immunoglobulin chain thereof according to any one of claims 1 to 7.

31. Use of an antigen binding protein according to any one of claims 1 to 7 in the preparation of a medicament for treating or preventing influenza virus infection in a subject in need thereof.

32. The use according to claim 31, wherein one or more other therapeutic agents are administered to the subject.

33. The use according to claim 32, wherein the one or more other therapeutic agents are antiviral drugs or vaccines.

34. The use according to claim 31, wherein one or more other therapeutic agents are administered to the subject, wherein the other therapeutic agent is a member selected from the following: ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-α2b, interferon-α2a, and an antibody or antigen-binding fragment thereof that specifically binds to influenza HA.

35. The use according to claim 34, wherein the antibody or antigen-binding fragment thereof that specifically binds to influenza HA is selected from the following antibodies or antigen-binding fragments thereof: H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H11711P; H1H11714P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; 727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1 H11744P2; H1H11745P2; H1H11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B; H1H17958B; H1H17959B; H1 H17960B; H1H17961B; H1H17962B; H1H17963B; H1H17964B; H1H17965B; H1H1 7966B; H1H17967B; H1H17968B; H1H17969B; H1H17970B; H1H17971B; H1H179 72B; H1H17973B; H1H17974B; H1H17975B; H1H17976B; H1H17977B; H1H17978 B; H1H17979B; H1H17980B; H1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H17987B; H1H17988B; H1H17989B; H1H17990B; H1 H17991B; H1H17992B; H1H17993B; H1H17994B; H1H17995B; H1H17996B; H1H1 7997B; H1H17998B; H1H17999B; H1H18000B; H1H18001B; H1H18002B; H1H180 03B; H1H18004B; H1H18005B; H1H18006B; H1H18007B; H1H18008B; H1H18009 B; 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;<h2 style=";text-align:left;direction:ltr">H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">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; <h2 style=";text-align:left;direction:ltr"> 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.

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