Anti-TMPRSS2 antibody and antigen-binding fragment

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 infection (eg, influenza virus infection). [Selected Figure] Figure 2

Description

[Technical Field]

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

[0002] The present invention relates to antibodies and antigen-binding fragments that specifically bind to TMPRSS2, and to methods for treating or preventing viral infection using the antibodies and fragments. [Background technology]

[0003] Influenza viruses have acquired resistance to currently used drugs that target viral neuraminidase (NA) or matrix protein 2 (M2), an ion channel protein. The emergence of drug resistance highlights the need for the development of new antiviral strategies. Targeting host cells may reduce or avoid the emergence of escape mutants, but it may create a “sync” for widespread expression and increase toxicity concerns. Numerous respiratory virus fusion proteins have been shown to require cleavage by host proteases (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3; Non-Patent Literature 4), including influenza (Non-Patent Literature 5; Non-Patent Literature 6; Non-Patent Literature 7; Non-Patent Literature 8).

[0004] Influenza A hemagglutinin precursor (HA0) requires cleavage into HA1 and HA2 by host serine proteases for activation. For example, the transmembrane proteases serine 2; TMPRSS2, TMPRSS4, and TMPRSS11D, as well as human airway trypsin-like proteases (HAT), have been suggested to be involved in HA cleavage (Non-Patent Document 7; Non-Patent Document 9; Patent Document 1). TMPRSS2 is also a target for anti-cancer therapy. See, for example, Patent Documents 2 and 3. The fusion of TMPRSS2 and ERG (TMPRSS2:ERG) is a gene fusion known to be a major driving factor of prostate carcinogenesis, induced by ERα and suppressed by ERβ. Non-Patent Document 10. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. WO2017 / 151453 [Patent Document 2] International Publication No. WO2008 / 127347 [Patent Document 3] International Publication No. WO2002 / 004953 [Non-patent literature]

[0006] [Non-Patent Document 1] Shirato et al., “Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry,” Journal of Virology, 91, e01387-16 (2017) [Non-Patent Document 2] 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) [Non-Patent Document 3] Zhou et al., "Protease inhibitors targeting coronavirus and filovirus entry," Antiviral Research, Vol. 116, pp. 76-84 (2015). [Non-Patent Document 4] Zmora et al., “TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells,” PLoS ONE, 10, e0138380 (2015) [Non-Patent Document 5] Zmora et al., “Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin,” PLoS ONE, 12, e0176597 (2017) [Non-Patent Document 6] Bottcher-Friebertshauser 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, vol. 85, pp. 1554-1562 (2011) [Non-Patent Document 7] Bertram et al., "TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells," Journal of Virology, Vol. 84, pp. 10016-10025 (2010). [Non-Patent Document 8] 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; Vol. 88 (No. 9): pp. 4744-4751. [Non-Patent Document 9] Bottcher et al., "Proteolytic Activation of Influenza Viruses by Serine Proteases TMPRSS2 and HAT from Human Airway Epithelium," Journal of Virology, October 2006; Vol. 80 (No. 19): pp. 9896-988. [Non-Patent Document 10] Bonkhoff, “Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression,” Prostate, Vol. 78 (Issue 1): pp. 2-10 (2018) [Overview of the project] [Means for solving the problem]

[0007] For example, there are small molecule inhibitors and research antibodies of TMPRSS2 that are useful in immunohistochemistry, but there is a need in the art for neutralizing therapeutic anti-TMPRSS2 antibodies and their use to treat or prevent viral infections. See, for example, Shen et al., Biochimie vol. 142: pp. 1-10 (2017), International Publication No. WO2008 / 127347; International Publication No. WO2002 / 004953; U.S. Patent No. 9498529; antibody ab92323 available from Abcam (Cambridge, MA), or antibodies sc-515727 and sc-101847 available from Santa Cruz Biotech (Dallas, TX). The present invention addresses this need in part by providing human anti-human TMPRSS2 antibodies such as H1H7017N, and combinations thereof including, for example, anti-influenza HA antibodies (e.g., group I HA or group II HA), and methods of using them 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 containing the amino acid sequence shown in SEQ ID NO: 2, 17, or 19; and / or (b) CDR-L1, CDR-L2, and CDR-L3 of an immunoglobulin light chain containing the amino acid sequence shown 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 containing an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 18; and / or (b) a heavy chain immunoglobulin variable region containing an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown 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) light chain immunoglobulins CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 4 or 18 and at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 4 or 18; and / or (b) heavy chain immunoglobulins CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 2, 17, or 19 and at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 17, or 19. For example, in one embodiment of the present invention, the antigen-binding protein comprises a light chain immunoglobulin variable region comprising (a) CDR-H1 comprising the amino acid sequence: GFTFSSYG (SEQ ID NO: 6); (b) CDR-H2 comprising the amino acid sequence: IWNDGSYV (SEQ ID NO: 8); (c) CDR-H3 comprising the amino acid sequence: AREGEWVLYYFDY (SEQ ID NO: 10); and a heavy chain immunoglobulin variable region comprising (a) CDR-L1 comprising the amino acid sequence: QSISSW (SEQ ID NO: 12); (b) CDR-L2 comprising the amino acid sequence: KAS (SEQ ID NO: 14); and / or (c) CDR-L3 comprising the amino acid sequence: QQYNSYSYT (SEQ ID NO: 16).The present invention also provides antigen-binding proteins comprising (a) a heavy chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO: 17 or 19; and / or (b) a light chain immunoglobulin comprising the amino acid sequence shown in SEQ ID NO: 18.

[0009] The present invention also provides any anti-TMPRSS2 antigen-binding protein that competes with any antigen-binding protein described herein for binding to TMPRSS2 (as determined, for example, by using a real-time label-free biolayer interference assay on an Octet RED384 biosensor (Pall ForteBio Corp.)); or binds to an epitope (or fragment thereof) on TMPRSS2 that is identical to or overlaps with any antigen-binding protein described herein.

[0010] The present invention also provides a multispecific antigen-binding protein that binds to TMPRSS2 and another antigen, or to a different epitope. For example, the multispecific molecule comprises (a) a first antigen-binding domain that specifically binds to TMPRSS2; and (b) a second antigen-binding domain that specifically binds to another antigen or to TMPRSS2 or an epitope different from the first antigen-binding domain.

[0011] The present invention also has the following characteristics: • Inhibits the proliferation of influenza virus (e.g., A / Puerto Rico / 08 / 1934(H1N1)) in TMPRSS2-expressing cells (e.g., Calu-3 cells); For example, EC of 440 pM or 1.06 nM 50 The value indicates that it binds to the surface of TMPRSS-expressing cells (e.g., MDCK / Tet-on); • Does not significantly bind to MDCK / Tet-on cells that do not express TMPRSS2; • Approximately 2.81 × 10 at approximately 25℃ -9 M's K D Binds to human TMPRSS2; • Approximately 9.31 × 10 at approximately 37℃ -9 M's K Dand binds to human TMPRSS2; · At about 25°C, about 5.60×10 -8 M of K D and binds to cynomolgus TMPRSS2; · At about 37°C, about 1.40×10 -7 M of K D and binds to cynomolgus TMPRSS2; · Limits the spread of influenza virus infection of cells in vitro; and / or · Protects mice engineered to express the human TMPRSS2 protein from death caused by influenza virus infection Provided is any anti-TMPRSS2 antigen-binding protein (e.g., including the sequences described herein, e.g., an antibody or antigen-binding fragment) that includes one or more of the following:

[0012] The present invention also provides a complex comprising any antigen-binding protein described herein that is bound to a TMPRSS2 polypeptide, e.g., in vitro or in the subject's body.

[0013] The present invention also provides a method for producing the anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) described herein or an immunoglobulin chain thereof, the method comprising: (a) introducing one or more polynucleotides encoding the light and / or heavy immunoglobulin chains of the antigen-binding protein; (b) culturing a host cell (e.g., a CHO cell, a Pichia cell or a Pichia pastoris cell) under conditions favorable for the expression of the polynucleotide; and (c) optionally, isolating the antigen-binding protein or immunoglobulin chain from the host cell and / or the medium in which the host cell is grown. The antigen-binding protein or immunoglobulin chain that is the product of such a method is part of the present invention.

[0014] (a) The V of an immunoglobulin chain comprising the amino acid sequence set forth in SEQ ID NO: 2 HCDR1, CDR2, and CDR3 of the domain; or (b) V of the immunoglobulin chain containing the amino acid sequence shown in SEQ ID NO: 4 L A polypeptide (e.g., immunoglobulin) containing the domains CDR1, CDR2, and CDR3 (e.g., the polypeptide is present in a host cell) also forms part of the present invention.

[0015] 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 polynucleotide encodes two different immunoglobulin chains (e.g., a heavy chain and a light chain). In one embodiment of the present invention, one polynucleotide encodes a light immunoglobulin chain and another polynucleotide encodes a heavy immunoglobulin chain, and for example, those chains are present in a host cell or container. For example, the polynucleotide is present in a vector (e.g., a plasmid) and / or incorporated into a host cell chromosome.

[0016] The host cells of the present invention (e.g., CHO cells, Pichia cells, or Pichia pastris cells) may include an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N), its polypeptide, or a polynucleotide encoding such polypeptide, and / or a vector comprising such polynucleotide.

[0017] The present invention also provides compositions or kits comprising the anti-TMPRSS2 antigen-binding protein described herein (e.g., H1H7017N) in conjunction with further therapeutic agents (e.g., antiviral agents and / or vaccines). For example, a composition may be a pharmaceutical composition comprising the antigen-binding protein and a pharmaceutically acceptable carrier, and optionally further therapeutic agents. The further therapeutic agents may be ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-alpha 2b, interferon-alpha 2a, and / or antibodies or antigen-binding fragments thereof that specifically bind to influenza HA. In one embodiment of the present invention, further therapeutic agents include H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N;H1H11829N2;H2aM11829N;H2M11830N;H1H11830N2;H1H11903N;H1H14571N;H2a14571N;H1H11704P;H1H1171 1P;H1H11714P;H1H11717P;H1H11724P;H1H11727P;H1H11730P2;H1H11731P2;H1H11734P2;H1H11736P2; H1H11742P2;H1H11744P2;H1H11745P2;H1H11747P2;H1H11748P2;H1H17952B;H1H17953B;H1H17954B;H1 H17955B;H1H17956B;H1H17957B;H1H17958B;H1H17959B;H1H17960B;H1H17961B;H1H17962B;H1H17963B ;H1H17964B;H1H17965B;H1H17966B;H1H17967B;H1H17968B;H1H17969B;H1H17970B;H1H17971B;H1H179 72B;H1H17973B;H1H17974B;H1H17975B;H1H17976B;H1H17977B;H1H17978B;H1H17979B;H1H17980B;H1H 17981B;H1H17982B;H1H17983B;H1H17984B;H1H17985B;H1H17986B;H1H17987B;H1H17988B;H1H17989B;H1H17990B;H1H17991B;H1H17992B;H1H17993B;H1H17994B;H1H17995B;H1H17996B;H1H17997B;H1H17998B;H1H17999B;H1H18000B;H1H18001B;H1H18002B;H1H18003B;H1H18004B;H1H18005B;H1H18006B;H1H18007B;H1H18008B;H1H18009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H18015B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18041B;H1H18042B;H1H18043B;H1H18044B;H1H18045B;H1H18046B;H1H18047B;H1H18048B;H1H18049B;H1H18051B;H1H18052B;H1H18053B;H1H18054B;H1H18055B;H1H18056B;H1H18057B;H1H18058B;H1H18059B;H1H18060B;H1H18061B;H1H18062B;H1H18063B;H1H18064B;H1H18065B;H1H18066B;H1H18067B;H1H18068B;H1H18069B;H1H18070B;H1H18071B;H1H18072B;H1H18073B;H1H18074B;H1H18075B;H1H18076B;H1H18077B;H1H18078B;H1H18079B;H1H18080B;H1H18081B;H1H18082B;H1H18083B;H1H18084B;H1H18085B;H1H18086B;H1H18087B;H1H18088B;H1H18089B;H1H18090B;H1H18091B;H1H18092B;H1H18093B;H1H18094B;H1H18095B;H1H18096B;H1H18097B;H1H18098B;H1H18099B;H1H18100B;H1H18101B;H1H18102B;H1H18103B;H1H18104B;H1H18105B;H1H18107B;H1H18108B;H1H18109B;H1H18110B;H1H18111B;H1H18112B;H1H18113B;H1H18114B;H1H18115B;H1H18116B;H1H18117B;H1H18118B;H1H18119B;H1H18120B;H1H18121B;H1H18122B;H1H18123B;H1H18124B;H1H18125B;H1H18126B;H1H18127B;H1H18128B;H1H18129B;H1H18130B;H1H18131B;H1H18132B;H1H18133B;H1H18134B;H1H18135B;H1H18136B;H1H18137B;H1H18138B;H1H18139B;H1H18140B;H1H18141B;H1H18142B;H1H18143B;H1H18144B;H1H18145B;H1H18146B;H1H18147B;H1H18148B;H1H18149B;H1H18150B;H1H18151B;H1H18152B;H1H18153B;H1H18154B;H1H18155B;H1H18156B;H1H18157B;H1H18158B;H1H18159B;H1H18160B;H1H18161B;H1H18162B;H1H18163B;H1H18164B;H1H18165B;H1H18166B;H1H18167B;H1H18168B;H1H18169B;H1H18170B;H1H18171B;H1H18172B;H1H18173B;H1H18174B;H1H18175B;H1H18176B;H1H18177B;H1H18178B;H1H18179B;H1H18180B;H1H18181B;H1H18182B;H1H18183B;H1H18184B;H1H18185B;H1H18186B;H1H18187B;H1H18188B;H1H18189B;H1H18190B;H1H18191B;H1H18192B;H1H18193B;H1H18194B;H1H18195B;H1H18196B;H1H18197B;H1H18198B;H1 H18199B;H1H18200B;H1H18201B;H1H18202B;H1H18203B;H1H18204B;H1H18 205B;H1H18206B;H1H18207B;H1H18208B;H1H18209B;H1H18210B;H1H1821 1B;H1H18212B;H1H18213B;H1H18214B;H1H18216B;H1H18217B;H1H18218B; H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18225B;H1H18226B;H1H18227B;H1H18228B;H1H18229B;H1H18230B;H1H18 231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1H18241B;H1H18242B;H1H18243B; H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B;H1H18251B;H1H18252B;H1H18253B;H1H18254B;H1H18255B;H1H18 256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1H18267B;H1H18268B;H1H18269B; H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B;H1H18279B;H1H18280B;H1H18281B;H1H18282B;H1H18 283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B;H1H18290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B;H1H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18303B;H1H18304B;H1H18305B;H1H18306B;H1H18 307B;H1H18308B;H1H18309B;H1H18310B;H1H18311B;H1H18312B;H1H18313B;H1H18314B;H1H18315B;H1H18316B;H1H18317B; An antibody or its antigen-binding fragment selected from the group consisting of H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B.

[0018] In one embodiment of the present invention, a further therapeutic agent provided in conjunction with the anti-TMPRSS2 antigen-binding protein is an antibody or antigen-binding fragment that binds to an influenza group II HA protein such as H1H14611N2; or a V of H1H14611N2. H and V L Antibodies or fragments containing; or heavy chain immunoglobulins containing CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs. 25-27) and light chain immunoglobulins containing CDR-L1, CDR-L2 and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs. 29-31).

[0019] In one embodiment of the present invention, a further therapeutic agent provided in conjunction with the anti-TMPRSS2 antigen-binding protein is an antibody or antigen-binding fragment that binds to an influenza group II HA protein such as H1H14612N2; or a V of H1H14612N2. H and V LAntibodies or fragments containing; or heavy chain immunoglobulins containing CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs. 41-43) and light chain immunoglobulins containing CDR-L1, CDR-L2 and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs. 45-47).

[0020] In one embodiment of the present invention, a further therapeutic agent provided in conjunction with the anti-TMPRSS2 antigen-binding protein is an antibody or antigen-binding fragment that binds to an influenza group I HA protein such as H1H11729P; or a V of H1H11729P. H and V L An antibody or fragment containing; or heavy chain immunoglobulins containing H1H11729P CDR-H1, CDR-H2 and CDR-H3 (e.g., SEQ ID NOs. 33-35) and light chain immunoglobulins containing H1H11729P CDR-L1, CDR-L2 and CDR-L3 (e.g., SEQ ID NOs. 37-39).

[0021] The present invention also provides a container or injection device comprising an anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) or a composition thereof (e.g., a pharmaceutical composition).

[0022] The present invention also provides a method for treating or preventing viral infections other than influenza virus infection in subjects in need (e.g., humans), which comprises administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) as described herein.

[0023] The present invention also provides a method for treating or preventing cancer (e.g., prostate cancer) or infection, such as infection by influenza virus, coronavirus, SARS-Co virus, MERS-Co virus, parainfluenza virus, human metapneumovirus, or hepatitis C virus (HCV), in a subject (e.g., human) in need thereof, comprising administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N) as described herein. For example, the antigen-binding protein is administered in conjunction with one or more further therapeutic agents (e.g., antiviral drugs and / or vaccines). In one embodiment of the present invention, the further therapeutic agent is a member selected from the group consisting of ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-alpha 2b, interferon-alpha 2a, and antibodies or antigen-binding fragments that specifically bind to influenza HA. In one embodiment of the present invention, further therapeutic agents include H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N;H1H11829N2;H2aM11829N;H2M11830N;H1H11830N2;H1H11903N; H1H14571N;H2a14571N;H1H11704P;H1H11711P;H1H11714P;H1H11717P;H1H11724 P;H1H11727P;H1H11730P2;H1H11731P2;H1H11734P2;H1H11736P2;H1H11742P2;H 1H11744P2;H1H11745P2;H1H11747P2;H1H11748P2;H1H17952B;H1H17953B;H1H17 954B;H1H17955B;H1H17956B;H1H17957B;H1H17958B;H1H17959B;H1H17960B;H1H 17961B;H1H17962B;H1H17963B;H1H17964B;H1H17965B;H1H17966B;H1H17967B;H 1H17968B;H1H17969B;H1H17970B;H1H17971B;H1H17972B;H1H17973B;H1H17974B;H1H17975B;H1H17976B;H1H17977B;H1H17978B;H1H17979B;H1H17980B;H1H17981B;H1H17982B;H1H17983B;H1H17984B;H1H17985B;H1H17986B;H1H17987B;H1H17988B;H1H17989B;H1H17990B;H1H17991B;H1H17992B;H1H17993B;H1H17994B;H1H17995B;H1H17996B;H1H17997B;H1H17998B;H1H17999B;H1H18000B;H1H18001B;H1H18002B;H1H18003B;H1H18004B;H1H18005B;H1H18006B;H1H18007B;H1H18008B;H1H18009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H18015B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18041B;H1H18042B;H1H18043B;H1H18044B;H1H18045B;H1H18046B;H1H18047B;H1H18048B;H1H18049B;H1H18051B;H1H18052B;H1H18053B;H1H18054B;H1H18055B;H1H18056B;H1H18057B;H1H18058B;H1H18059B;H1H18060B;H1H18061B;H1H18062B;H1H18063B;H1H18064B;H1H18065B;H1H18066B;H1H18067B;H1H18068B;H1H18069B;H1H18070B;H1H18071B;H1H18072B;H1H18073B;H1H18074B;H1H18075B;H1H18076B;H1H18077B;H1H18078B;H1H18079B;H1H18080B;H1H18081B;H1H18082B;H1H18083B;H1H18084B;H1H18085B;H1H18086B;H1H18087B;H1H18088B;H1H18089B;H1H18090B;H1H18091B;H1H18092B;H1H18093B;H1H18094B;H1H18095B;H1H18096B;H1H18097B;H1H18098B;H1H18099B;H1H18100B;H1H18101B;H1H18102B;H1H18103B;H1H18104B;H1H18105B;H1H18107B;H1H18108B;H1H18109B;H1H18110B;H1H18111B;H1H18112B;H1H18113B;H1H18114B;H1H18115B;H1H18116B;H1H18117B;H1H18118B;H1H18119B;H1H18120B;H1H18121B;H1H18122B;H1H18123B;H1H18124B;H1H18125B;H1H18126B;H1H18127B;H1H18128B;H1H18129B;H1H18130B;H1H18131B;H1H18132B;H1H18133B;H1H18134B;H1H18135B;H1H18136B;H1H18137B;H1H18138B;H1H18139B;H1H18140B;H1H18141B;H1H18142B;H1H18143B;H1H18144B;H1H18145B;H1H18146B;H1H18147B;H1H18148B;H1H18149B;H1H18150B;H1H18151B;H1H18152B;H1H18153B;H1H18154B;H1H18155B;H1H18156B;H1H18157B;H1H18158B;H1H18159B;H1H18160B;H1H18161B;H1H18162B;H1H18163B;H1H18164B;H1H18165B;H1H18166B;H1H18167B;H1H18168B;H1H18169B;H1H18170B;H1H18171B;H1H18172B;H1H18173B;H1H18174B;H1H18175B;H1H18176B;H1H18177B;H1H18178B;H1H18179B;H1H18180B;H1H18181B;H1H18182B;H1H18183B;H1 H18184B;H1H18185B;H1H18186B;H1H18187B;H1H18188B;H1H18189B;H1H18 190B;H1H18191B;H1H18192B;H1H18193B;H1H18194B;H1H18195B;H1H1819 6B;H1H18197B;H1H18198B;H1H18199B;H1H18200B;H1H18201B;H1H18202B; H1H18203B;H1H18204B;H1H18205B;H1H18206B;H1H18207B;H1H18208B;H1 H18209B;H1H18210B;H1H18211B;H1H18212B;H1H18213B;H1H18214B;H1H18 216B;H1H18217B;H1H18218B;H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18225B;H1H18226B;H1H18227B;H1H18228B; H1H18229B;H1H18230B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1H18 241B;H1H18242B;H1H18243B;H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B;H1H18251B;H1H18252B;H1H18253B; H1H18254B;H1H18255B;H1H18256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1H18 267B;H1H18268B;H1H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B;H1H18279B;H1H18280B;H1H18281B;H1H18282B;H1H18283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H 1H18288B;H1H18289B;H1H18290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B;H1 H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H 18303B;H1H18304B;H1H18305B;H1H18306B;H1H18307B;H1H18308B;H1H18309B;H1H18 310B;H1H18311B;H1H18312B;H1H18313B;H1H18314B;H1H18315B;H1H18316B;H1H183 17B;H1H18318B;H1H18319B;H1H18320B;H1H18321B;H1H18322B;H1H18323B;H1H18324 An antibody or its antigen-binding fragment selected from the group consisting of B;H1H18325B;H1H18326B;H1H18327B;H1H18328B;H1H18329B;H1H18330B;H1H18331B;H1H18332B;H1H18333B;H1H18334B; and H1H18335B.

[0024] The present invention also provides a method for administering the anti-TMRPSS2 antigen-binding protein described herein (e.g., H1H7017N) to a living body of a subject (e.g., a human), which comprises the step of parenterally injecting the antigen-binding protein into the living body of the subject (e.g., subcutaneously, intravenously, or intramuscularly). [Brief explanation of the drawing]

[0025] [Figure 1] This figure shows the progression of the A / Puerto Rico / 08 / 1934(H1N1)-GFP virus to different cell lines with an initial multiplicity of infection of 0.01(A) or 0.001(B) in the absence of exogenous trypsin. Calu3 (circular), A549 (square), MDCK (triangular), and HepG2 (inverted triangle) cells. [Figure 2]Application of H1H7017N during the infection cycle reduces the number of fluorescent focus units (FFUs) of A / Puerto Rico / 08 / 1934(H1N1) at 72 hours post-infection compared to isotype control antibody, no antibody, anti-HA antibody, and non-infection control. [Figure 3] Anti-TMPRSS2, H1H7017N, binds to human and cynomolgus monkey TMPRSS2 expressed on cells. (A) H1H7017N bound to MDCK / Tet-on / hTMPRSS2 and MDCK / Tet-on / mfTMPRSS2 at EC50 values ​​of 460 pM and 1.06 nM, respectively, and did not show significant binding to MDCK / Tet-on cells. (B) Control mAb1, an unrelated isotype control antibody, did not show binding to any of the cell lines tested. [Figure 4] The survival curves of mice engineered to express human TMPRSS2 protein treated with 5 mg / kg of H1H7017N on day -1 of PI (inverted triangle, dashed line) or day 0 of PI (circle, solid line) showed protection against H1N1 in the prevention model. Mice treated with the isotype control H1H1238N (triangle, solid line) did not show protection. [Figure 5] The survival curves of mice engineered to express the H1N1-infected human TMPRSS2 protein and treated with 10 mg / kg of H1H7017N showed protection. Mice were treated on day 0 (diamond, dotted line), day 1 (circle, solid line), day 2 (inverted triangle, solid line), or day 3 (square, dashed line) of PI. The isotype control H1H1238N (triangle, solid line) showed partial protection with a 25% survival rate. [Figure 6] The survival curves of hTPMRSS2 mice treated with 10 mg / kg of H1H7017N on day 1 of PI (triangle) or day 2 of PI (circle) showed protection against H3N2. Untreated mice (square) did not show protection. [Figure 7]Survival curves of wild-type mice (A) or mice engineered to express human TMPRSS2 protein infected with 150 PFU (triangle), 750 PFU (square), or 1,500 PFU (circular) of A / Puerto Rico / 08 / 1934 (H1N1) (B). Mice were weighed daily until day 14 of the PI. [Figure 8] Survival curves of mice manipulated to express human TMPRSS2 protein infected with A / Aichi / 2 / 68(HA,NA)×A / PR / 8 / 34(H3N2) on day 0, and treated with a combination of H1H7017N and H1H14611N2 at 2.5 mg / kg (diamond), H1H1H7017N at 10 mg / kg (triangle), H1H14611N2 at 10 mg / kg (square), H1H7017N and H1H14611N2 at 5 mg / kg each, or a 10 mg / kg hIgG1 isotype control (circle). Mice were weighed daily until day 14 of the PI. [Figure 9] Survival curves of mice engineered to express human TMPRSS2 protein infected with A / Puerto Rico / 08 / 1934(H1N1) on day 1 of PI, and treated with a combination of 1 mg / kg H1H7017N and 2 mg / kg H1H11729P (circular), 2.5 mg / kg H1H7017N and H1H11729P respectively (inverted triangle), 5 mg / kg H1H11729P (diamond), 5 mg / kg H1H7017N (square), or 5 mg / kg hIgG1 isotype control (triangle). Mice were weighed daily until day 14 of PI. [Modes for carrying out the invention]

[0026] Before describing this method, it should be understood that the present invention is not limited to specific methods and experimental conditions described, and for example, methods and conditions may vary. Furthermore, the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit them, and it should be understood that the scope of the present invention is limited only by the appended claims.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications mentioned herein are incorporated herein in their entirety by reference.

[0028] The term "influenza hemagglutinin," also known as "influenza HA," refers to a trimeric glycoprotein found on the surface of influenza virions, mediating viral adhesion (via the binding of HA1 to α-2,3- and α-2,6-sialic acids) and entry into host cells (via conformational changes). HA consists of two structural domains: a globular head domain containing the receptor binding site (which undergoes frequent antigenic mutations) and a stem region (which is often more conserved across various influenza virus strains). Influenza HA is synthesized as a precursor (HA0), which undergoes proteolytic processing to produce two subunits (HA1 and HA2), which associate with each other to form the stem / globular head structure. Viral HA is the most variable antigen on the virus, and the stem (HA2) is highly conserved within each group.

[0029] The term "influenza neuraminidase," also known as "influenza NA," refers to an exosialidase (EC3.2.1.18) that cleaves the α-ketoside linkage between sialic acid (N-acetylneuraminic acid) and an adjacent sugar residue.

[0030] 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 to GenBank under accession number FJ966082.1. The term “influenza-HA” also includes protein variants of influenza HA isolated from different influenza isolates, e.g., GQ149237.1, NC_002017, KM972981.1, etc. The term “influenza-HA” also includes recombinant influenza HA or fragments thereof. This term also includes influenza HA or fragments thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence.

[0031] An anti-TMPRSS2 "antigen-binding protein" is a complex of TMPRSS2 polypeptides, such as a polypeptide that specifically binds to an anti-TMPRSS2 antibody or antigen-binding fragment, whether monospecific or multispecific, or one or more polypeptides (e.g., a tetrameric IgG antibody).

[0032] TMPRSS2 TMPRSS2 (transmembrane protease serine 2) is a protein located on human chromosome 21 that belongs to the serine protease family (type II transmembrane serine proteases (TTSPs)) which are important for influenza virus infectivity. TMPRSS2 has been shown to mediate the cleavage of influenza virus HA0 into HA1 and HA2.

[0033] The human TMPRSS2 gene encodes a 492-amino acid putative protein that adheres to the cell membrane. This protein is converted to its mature form via autocatalytic cleavage between Arg255 and Ile256. After cleavage, the mature protease is mostly membrane-bound, but some of it is released into the extracellular environment.

[0034] In one embodiment of the present invention, human TMPRSS2(V160M) has the amino acid sequence: [ka] (Sequence ID 22; methionine 160 is in bold) is included. In one embodiment of the present invention, the TMPRSS2 polypeptide does not contain the V160M mutation. See also NM_005656.3.

[0035] In one embodiment of the present invention, the rhesus macaque (Macaca mulatta) TMPRSS2 (S129L, N251S, I415V, R431Q, D492G) has the following amino acid sequence: [ka] (SEQ ID NO: 23) is included. In one embodiment of the present invention, the TMPRSS2 polypeptide does not contain the S129L, N251S, I415V, R431Q and / or D492G mutations.

[0036] In one embodiment of the present invention, the mouse (Mus musculus) TMPRSS2 mRNA contains the nucleotide sequence shown in NM_015775.2.

[0037] virus The present invention includes methods for treating or preventing viral infection in a subject. The term “virus” includes any virus whose infection in the subject's organism is treatable or preventable by administration of an anti-TMPRSS2 antibody or its antigen-binding fragment (e.g., the infectivity of the virus is at least partially dependent on TMPRSS2). In one embodiment of the present invention, “virus” is any virus expressing HA0 or another substrate of TMPRSS2, the proteolytic cleavage of which is required for the virus to be fully infectable to cells in the host. 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 tracts, bronchi, lungs) and are treatable or preventable by administration of anti-TMPRSS2. For example, in one embodiment of the present invention, the virus includes influenza virus, coronavirus, SARS-Co virus (Severe Acute Respiratory Syndrome Coronavirus), MERS-Co virus (Middle East Respiratory Syndrome (MERS)CoV), parainfluenza virus, Sendai virus (SeV), human metapneumovirus and / or hepatitis C virus (HCV). "Viral infection" refers to the invasion and reproduction of a virus in the target organism. The present invention includes embodiments subject to the condition that "virus" excludes influenza viruses, for example, that viral infection excludes influenza virus infection.

[0038] Currently, human parainfluenza viruses (HPIV) comprise two genera: respiroviruses (HPIV-1 and HPIV-3) and rubraviruses (HPIV-2 and HPIV-4). Both genera (paramyxoviruses) can be morphologically isolated from influenza viruses.

[0039] Sendai virus, also known as the mouse parainfluenza virus, is the type species of the respirovirus genus, which also contains human parainfluenza virus 3, bovine parainfluenza virus 3, and human parainfluenza virus 1. TMPRSS2 is an activating protease for parainfluenza viruses and respiratory parainfluenza viruses such as Sendai virus (SeV). See Abe et al., J. Virol., Vol. 87 (No. 21): pp. 11930-11935 (2013).

[0040] Human metapneumovirus (HMPV) is classified as the first human member of the genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae. It is an enveloped, negative-sense single-stranded RNA virus. Its RNA genome contains eight genes encoding nine proteins. HMPV has the same gene order as tri pneumovirus (AMPV), which belongs to the genus Metapneumovirus. TMPRSS2 is expressed in human lung epithelium, efficiently cleaves the HMPV F protein, supports HMPV reproduction, and may be involved in the development of lower respiratory tract disease in HMPV-infected patients. See Shirogane et al., J Virol., vol. 82(no. 17): pp. 8942-8946 (2008).

[0041] Hepatitis C virus (HCV) is a small, enveloped, positive-sense single-stranded RNA virus belonging to the family Flaviviridae. HCV has at least six genotypes and numerous subtypes and is a member of the genus hepacivirus. TMPRSS2 can activate HCV infection post-binding and during the entry phase. Esumi et al., Hepatology, vol. 61 (no. 2): pp. 437-446 (2015).

[0042] Influenza viruses belong to the family Orthomyxoviridae. This family exhibits enveloped viruses whose genomes contain segmented negative-sense single-stranded RNA segments. This family comprises four genera: type A, B, C, and Togotovirus. Influenza virus classes A, B, and C are classified into subtypes (e.g., subtype A / H1N1) based on core proteins and further determined by viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). The definition of influenza subtypes uses 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). Group 1 influenza viruses include the H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 subtypes, as well as the NA8, NA5, Na4, and NA1 subtypes. Group 2 includes the H3, H4, H7, H10, H14, and H15 subtypes, as well as the NA6, NA9, NA7, NA2, and NA3 subtypes. Influenza A viruses infect a variety of mammalian and avian species, while infections with influenza B and C viruses are primarily limited to humans. The eight genomic segments of influenza A and B viruses are loosely capsidized by nucleoproteins.

[0043] Coronavirus virions are spherical, with a diameter of approximately 125 nm. The most prominent feature of coronaviruses is the club-like spike protrusions emanating from the surface of the virion. These spikes are a defining characteristic of virions, giving rise to the appearance of the solar corona and prompting the name coronavirus. The nucleocapsid is located within the envelope of the virion. Coronaviruses have a helically symmetrical nucleocapsid, which is less common in positive-sense RNA viruses but much more common in negative-sense RNA viruses. MERS-CoV (Middle East Respiratory Syndrome Coronavirus) and SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) both belong to the Coronaviridae family. The initial binding of the virion to the host cell is initiated by the interaction of the S protein with its receptor. The location of the receptor-binding domain (RBD) within the S1 region of the coronavirus S protein varies among viruses, with some having the RBD at the C-terminus of S1. The S protein / receptor interaction is a major determinant of coronavirus infection in host species and also governs the virus's tissue affinity. Many coronaviruses utilize peptidases as their cell receptors. After receptor binding, the virus must then approach the cytoplasm of the host cell. This is generally achieved by acid-dependent proteolytic cleavage of the S protein by cathepsin, TMPRS2, or another protease, followed by fusion of the viral membrane and the cell membrane.

[0044] Anti-TMPRSS2 antibody and antigen-binding fragment The present invention provides an antigen-binding protein, such as an antibody that specifically binds to the TMPRSS2 protein or its antigenic fragment, and the antigen-binding fragment thereof.

[0045] The term “antibody,” as used herein, refers to an immunoglobulin molecule (i.e., a “complete antibody molecule”) comprising two heavy chains (HC) and two light chains (LC), which are four polypeptide chains interconnected by disulfide bonds, as well as their polymers (e.g., IgM), e.g., H1H701N. Each heavy chain has a heavy chain variable region ("HCVR" or "V").H ) (e.g., Sequence ID 2) and heavy chain constant region (domain C H 1. C H 2 and C H It includes a light chain variable region ("LCVR" or "V"). L ) (e.g., Sequence ID 4) and light chain constant region (C L ) is composed of V H and V L The region is further subdivided into hypervariable regions called complementary determination regions (CDRs), with more conserved regions called framework regions (FRs) scattered throughout. H and V L It comprises three CDRs and four FRs, aligned from the amino terminus to the carboxyl terminus in the 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 those of the human germline sequence, or are naturally or artificially modified.

[0046] Typically, the variable domains of both heavy and light immunoglobulin chains contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within a relatively conserved framework region (FR). Generally, from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In one embodiment of the present invention, the assignment of amino acids to each domain follows the definitions in 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. Vol. 32: pp. 1-75; Kabat et al., (1977) J. Biol. Chem., Vol. 252: pp. 6609-6616; Chothia et al., (1987) J Mol. Biol., Vol. 196: pp. 901-917; or Chothia et al., (1989) Nature, Vol. 342: pp. 878-883.

[0047] The present invention includes monoclonal anti-TMPRSS2 antigen-binding proteins, such as antibodies and their antigen-binding fragments, and monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. The term “monoclonal antibody,” as used herein, refers to a substantially homogeneous population of antibodies; that is, antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. “Pluried” of such monoclonal antibodies and fragments in a composition refers to concentrations of identical antibodies and fragments (i.e., in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts, as discussed above), which are typically higher than those naturally present in the blood of a host organism, such as a mouse or human.

[0048] In one embodiment of the present invention, the anti-TMPRSS2 antigen-binding protein, for example, an antibody or antigen-binding fragment, includes a heavy chain constant domain, for example, type IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4), or IgM. In another embodiment of the present invention, the antigen-binding protein, for example, an antibody or antigen-binding fragment, includes a light chain constant domain, for example, type κ or λ.

[0049] The term “human” antigen-binding protein, e.g., antibody, as used herein, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences, whether in human cells or transplanted into non-human cells, e.g., mouse cells. See, for example, U.S. Patent No. 8502018, U.S. Patent No. 6596541, or U.S. Patent No. 5789215. The human mAbs of the present invention may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, e.g., by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo) in the CDR and particularly CDR3. However, the term “human antibody,” as used herein, is not intended to include mAbs in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is transplanted onto a human FR sequence. The term includes antibodies recombinantly produced in or in non-human mammals. The term is not intended to include antibodies isolated from or produced in human subjects. See below.

[0050] The present invention includes anti-TMPRSS2 chimeric antigen-binding proteins, such as antibodies and their antigen-binding fragments, and methods of using them. As used herein, “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 derived from different species (U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, Vol. 81: pp. 6851-6855).

[0051] The term “recombinant” antigen-binding protein, e.g., an antibody or its antigen-binding fragment, refers to such molecules produced, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA techniques, including DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice) or cells (e.g., CHO cells) expression systems, or isolated from recombinant combinatorial human antibody libraries.

[0052] The recombinant anti-TMPRSS2 antigen-binding proteins disclosed herein, e.g., antibodies and antigen-binding fragments, are also generated in an E. coli (E. coli) / T7 expression system. In this embodiment, the nucleic acid encoding the anti-TMPRSS2 antibody immunoglobulin molecule of the present 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 its antigen-binding fragment or its immunoglobulin chain in a host cell (e.g., a bacterial host cell such as E. coli, e.g., BL21 or BL21DE3), comprising expressing 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, e.g., 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 incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). See U.S. Patent No. 4,952,496 and U.S. Patent No. 5,693,489, or Studier & Moffatt, "Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes," J.Mol.Biol., 1986, May 5; Vol. 189 (No. 1): pp. 113-130.

[0053] There are several methods known in the art for producing recombinant antibodies. One example of a method for recombinant antibody production is disclosed in U.S. Patent No. 4,816,567.

[0054] Transformation can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of one or more polynucleotides in liposomes, biolistek injection, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461 and 4,959,455.

[0055] Accordingly, the present invention comprises a recombinant method for producing an anti-TMPRSS2 antigen-binding protein, for example, the antibody of the present invention or its antigen-binding fragment, or its immunoglobulin chain, the recombinant method comprising: (i) introducing one or more polynucleotides (e.g., including one or more nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15) encoding an antigen-binding protein, for example, a light and / or heavy immunoglobulin chain of H1H7017N or H4H7017N, for example, the polynucleotide being in a vector; and / or incorporated into a host cell chromosome and / or operably ligated to a promoter; (ii) culturing host cells (e.g., CHO or Pichia or Pichia pastris) under conditions suitable for polynucleotide expression; and (iii) optionally isolating the antigen-binding protein (e.g., antibody or fragment) or chain from the host cells and / or the culture medium in which the host cells grow. When producing antigen-binding proteins (e.g., antibodies or antigen-binding fragments) containing more than one immunoglobulin chain, such as an antibody containing two heavy immunoglobulin chains and two light immunoglobulin chains, co-expression of the chains in a single host cell results in chain binding, for example, intracellular, on the cell surface, or extracellularly, when such chains are secreted to form an antigen-binding protein (e.g., an antibody or antigen-binding fragment). This method includes the expression of heavy immunoglobulin chains only or light immunoglobulin chains only (e.g., any of those considered herein, including mature fragments and / or their variable domains). Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments containing such chains.For example, the present invention also includes an anti-TMPRSS2 antigen-binding protein, such as an antibody and its antigen-binding fragment, and the products of such production methods and optionally the purification methods described herein, a heavy chain immunoglobulin (including its variable domain or its CDR) encoded by a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1, and a light chain immunoglobulin (including its variable domain or its CDR) encoded by the nucleotide sequence shown in SEQ ID NO: 3. For example, in one embodiment of the present invention, the product of the method is V, which contains the amino acid sequence shown in SEQ ID NO: 2. H , and V containing the amino acid sequence shown in Sequence ID No. 4 L Alternatively, it is an anti-TMPRSS2 antigen-binding protein which is an antibody or fragment containing an HC containing the amino acid sequence shown in SEQ ID NO: 17 or 19, and an LC containing the amino acid sequence shown in SEQ ID NO: 18.

[0056] Eukaryotic and prokaryotic host cells, such as mammalian cells, are used as hosts for the expression of anti-TMPRSS2 antigen-binding proteins. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, in particular, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and numerous other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Other cell lines used include insect cell lines (e.g., fall armyworm (Spodoptera frugiperda) or nettle moth (Trichoplusia ni)), amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, and Pichia güercuum. Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.), Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum Examples include *Venetum*, *Physcomitrella patens*, and *Neurospora crassa*. The present invention comprises isolated host cells (e.g., CHO cells) containing an antigen-binding protein such as H1H7017N; or a polynucleotide encoding such a polypeptide.

[0057] The term "specifically binds" means, for example, that when measured in a real-time label-free biolayer interference assay at 25°C or 37°C, for example by an Octet® HTX biosensor, or by surface plasmon resonance, for example by BIACORE®, or by solution-affinity ELISA, it binds at least about 10 times. -8 M (for example, 2.81 × 10 -9 M; 9.31 × 10 -9 M;10 -9 M;10 -10 M, 10 -11 M, or 10 -12 M)'s K DThis refers to an antigen-binding protein (e.g., mAb) that has binding affinity to an antigen such as the TMPRSS2 protein (e.g., human TMPRSS2) and is expressed as such. The present invention includes an antigen-binding protein that specifically binds to the TMPRSS2 protein.

[0058] The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody or antigen-binding protein, as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen in order to form a complex. Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated CDRs such as the complementarity-determining region (CDR)3 peptide), or constrained FR3-CDR3-FR4 peptides. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (as defined, e.g., in International Publication No. WO08 / 020079 or International Publication No. WO09 / 138519) (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunotherapy drugs (SMIPs), and shark variable IgNAR domains are also included within the expression “antigen-binding fragment” as used herein. In one embodiment of the present invention, the antigen-binding fragment comprises three or more CDRs of H1H7017N (e.g., CDR-H1, CDR-H2, and CDR-H3; or CDR-L1, CDR-L2, and CDR-L3).

[0059] In one embodiment of the present invention, the antigen-binding fragment of the antibody includes at least one variable domain. The variable domain may be of any size or amino acid composition and generally includes at least one CDR adjacent to or within one or more framework sequences. L V associated with the domainH In antigen-binding fragments having a domain, V H Domain and V L Domains are arranged relative to one another in any appropriate configuration. For example, the variable region is a dimer, V H -V H , V H -V L or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may contain monomer V H or V L It may include a domain name.

[0060] In certain embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains found within the antigen-binding fragment of the antibody of the present invention include (i)V H -C H 1;(ii)V H -C H 2;(ii)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)V H -C L ;(vii)V L -C H 1;(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 H3; and (xiv)V L -C L This includes: In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains are linked to each other directly or by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage between the variable and / or constant domains adjacent to a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention is linked to each other and / or one or more monomer V H or V L The domain may contain, non-covalently (e.g., by disulfide bonds(s)) homodimers or heterodimers (or other polymers) of any of the variable and constant domain configurations listed above.

[0061] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are further discussed herein.

[0062] In specific embodiments, the antibody or antibody fragment of the present invention is conjugated to a portion such as a ligand, or to a therapeutic portion such as an antiviral drug, a secondary anti-influenza antibody, or any other therapeutic portion useful for treating a viral infection, such as influenza virus infection ("immunoconjugate"). See below.

[0063] The present invention also provides a complex comprising the antibody or antigen-binding fragments discussed herein, which is complexed with an anti-TMPRSS2 antigen-binding protein, e.g., a TMPRSS2 polypeptide or its antigenic fragment, and / or a secondary antibody or its antigen-binding fragment that specifically binds to the TMPRSS2 polypeptide or its fragment (e.g., a detectably labeled secondary antibody). In one embodiment of the present invention, the antibody or fragment is in vitro (e.g., immobilized on a solid substrate) or present in the living organism of the subject. In one embodiment of the present invention, TMPRSS2 is in vitro (e.g., immobilized on a solid substrate), or on the surface of a cell, or present in the living organism of the subject. Also, an immobilized anti-TMPRSS2 antibody and its antigen-binding fragment, covalently linked to an insoluble matrix material (e.g., glass or polysaccharide, e.g., agarose or Sepharose, e.g., beads or other particles thereof), is part of the present invention; optionally, the immobilized antibody is complexed with TMPRSS2 or its antigenic fragment, or a secondary antibody or its fragment.

[0064] "Isolated" antigen-binding proteins, antibodies or their antigen-binding fragments, polypeptides, polynucleotides, and vectors do not contain, at least partially, other biological molecules from the cells or cell cultures from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth media. Isolated antibodies or antigen-binding fragments may also not contain, at least partially, expression system components such as biological molecules from the host cells or their growth media. Generally, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of pharmaceutical formulations containing antibodies or fragments.

[0065] The term “epitope” refers to an antigenic determinant (e.g., on the TMPRSS2 polypeptide) that interacts with a specific antigen-binding site on an antigen-binding protein, such as a variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on an antigen and have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to the region of the antigen to which an antibody binds. Epitopes are defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes are linear or steric, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule, such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and in certain embodiments may have specific three-dimensional structural properties and / or specific charge properties.

[0066] Methods for determining the epitopes of antigen-binding proteins, such as antibodies, fragments, or polypeptides, include alanine scanning mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol. Biol., Vol. 248: pp. 443-463), peptide cleavage analysis, crystallographic studies, and NMR analysis. Furthermore, methods such as epitope removal, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci., Vol. 9: pp. 487-496). Another method that can be used to identify amino acids within polypeptides that interact with antigen-binding proteins (e.g., antibodies, fragments, or polypeptides) (e.g., Coversin) is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, hydrogen / deuterium exchange involves deuterizing the protein of interest and subsequently conjugating the antigen-binding protein, such as antibodies, fragments, or polypeptides, to the deuterium-labeled protein. Next, the TMPRSS2 protein / antigen-binding protein complex is transferred to water, and the exchangeable protons within amino acids protected by the antibody complex undergo deuterium-hydrogen reverse exchange at a slower rate than the exchangeable protons within amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antigen-binding protein interface retain deuterium and therefore exhibit a relatively higher mass compared to amino acids not included in the interface. After dissociation of the antigen-binding protein (e.g., antibody, fragment, or polypeptide), the target protein is subjected to protease cleavage and mass spectrometry to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antigen-binding protein interacts. See, for example, Ehring (1999) Analytical Biochemistry, Vol. 267: pp. 252-259; Engen and Smith (2001) Anal. Chem., Vol. 73: pp. 256A-265A.

[0067] As used herein, the term “competing” refers to an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) that binds to an antigen (e.g., TMPRSS2) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or its antigen-binding fragment) to that antigen. The term also includes competition in both directions between two antigen-binding proteins, e.g., antibodies, i.e., a first antibody binds and blocks the binding of a second antibody, and vice versa. In certain embodiments, a first antigen-binding protein (e.g., an antibody) and a 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 overlapping epitopes, e.g., the binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) is measured by methods known in the art, e.g., by real-time label-free biolayer interference assays. In one embodiment of the present invention, competition between first and second anti-TMPRSS2 antigen-binding proteins (e.g., antibodies) is determined by measuring the ability of an immobilized first anti-TMPRSS2 antigen-binding protein (e.g., antibody) (not initially complexed with the TMPRSS2 protein) to bind to a soluble TMPRSS2 protein complexed with the second anti-TMPRSS2 antigen-binding protein (e.g., antibody). A decrease in the ability of the first anti-TMPRSS2 antigen-binding protein (e.g., antibody) to bind to the complexed TMPRSS2 protein compared to the uncomplexed TMPRSS2 protein indicates competition between the first and second anti-TMPRSS2 antigen-binding proteins (e.g., antibodies). The degree of competition can be expressed as a percentage of the decrease in binding. Such competition can be measured using real-time label-free biolayer interference assays, e.g., Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assay), or SPR (surface plasmon resonance).

[0068] Binding competition between anti-TMPRSS2 antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using a real-time, label-free biolayer interference assay on an Octet RED384 biosensor (Pall ForteBio Corp.). For example, to determine competition between two anti-human TMPRSS2 monoclonal antibodies, the anti-TMPRSS2 mAb can be initially captured on an anti-hFc antibody-coated Octet biosensor chip (Pall ForteBio Corp., #18-5060) by immersing its tip in a solution of the anti-human TMPRSS2 mAb (hereafter referred to as "mAb1"). Next, as a positive control for blocking, the antibody-captured biosensor chip can be saturated with a known blocking isotype control mAb (hereafter referred to as "blocking mAb") by immersing it in a solution of the blocking mAb. Next, to determine whether mAb2 competes with mAb1, the biosensor chip can be immersed in a co-complexing solution of human TMPRSS2 polypeptide and a second anti-human TMPRSS2 mAb (hereafter referred to as "mAb2"), which is pre-incubated for a certain period to determine the binding of mAb1 to the TMPRSS2 polypeptide. The biosensor chip can be washed in buffer between each step of the experiment. Real-time binding response can be monitored throughout the experiment, and the binding response at the end of each step can be recorded.

[0069] For example, in one embodiment of the present invention, the competitive assay is performed at 25°C and pH approximately 7, e.g., 7.4, in the presence of a buffer, salt, surfactant, and a nonspecific protein (e.g., bovine serum albumin).

[0070] Typically, an antibody or antigen-binding fragment of the present invention, modified in any way, retains the ability to specifically bind to TMPRSS2, for example, retaining at least 10% of its TMPRSS2 binding activity (compared to the parent antibody) when its activity is expressed in molar terms. Preferably, the antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the TMPRSS2 binding affinity of the parent antibody. Furthermore, the antibody or antigen-binding fragment of the present invention is intended to contain conserved or non-conserved amino acid substitutions (referred to as “conserved variants” or “functionally conserved variants” of the antibody) that do not substantially alter its biological activity.

[0071] Immunoglobulin chains (e.g., H1H7017N, V) H , V L A “variant” of a polypeptide such as HC or LC refers to a polypeptide that contains at least about 70–99.9% (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%) identical or similar amino acid sequences to a reference amino acid sequence described herein (e.g., SEQ ID NOs: 2, 4, 17, 18, or 19); if the comparison is performed by the BLAST algorithm, the algorithm parameters are selected to give the maximum match between each sequence over the full length of each reference sequence (e.g., expected threshold: 10; word size: 3; maximum match within query range: 0; BLOSUM 62 matrix; gap cost: present 11, extended 1; conditional score matrix adjustment).

[0072] A “variant” of a polynucleotide refers to a polynucleotide containing a nucleotide sequence that is at least approximately 70–99.9% (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%) identical to a reference nucleotide sequence described herein (e.g., SEQ ID NO: 1 or 3); if the comparison is performed by the BLAST algorithm, the algorithm parameters are selected to give the maximum match between each sequence over the full length of each reference sequence (e.g., expected threshold: 10; word size: 28; maximum match within query range: 0; match / mismatch score: 1, -2; gap cost: linear).

[0073] An anti-TMPRSS2 antigen-binding protein, for example, the antibody and antigen-binding fragment of the present invention, in one embodiment of the present invention, includes a heavy-chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 95%, 99%) amino acid sequence identity with the amino acids shown 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 with the amino acids shown in SEQ ID NO: 4 or 18.

[0074] Furthermore, the mutant anti-TMPRSS2 antigen-binding protein may include a polypeptide comprising the amino acid sequences described herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. For example, the present invention includes an immunoglobulin light chain mutant comprising the amino acid sequence shown in SEQ ID NO: 4 or 18, provided that it has one or more such mutations, and / or an immunoglobulin heavy chain mutant comprising the amino acid sequence shown in SEQ ID NO: 2, 17, or 19, provided that it has one or more such mutations. In one embodiment of the present invention, the mutant anti-TMPRSS2 antigen-binding protein is an immunoglobulin light chain mutant comprising CDR-L1, CDR-L2, and CDR-L3 (one or more of such CDRs (e.g., 1, 2, or 3) have one or more such mutations (e.g., conservative substitutions)) and / or an immunoglobulin heavy chain mutant comprising CDR-H1, CDR-H2, and CDR-H3 (one or more of such CDRs (e.g., 1, 2, or 3) have one or more such mutations (e.g., conservative substitutions)).

[0075] The present invention further provides an antibody or an antigen-binding fragment thereof comprising a mutant anti-TMPRSS2 antigen-binding protein, such as one or more mutant CDRs described herein (e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3), and having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% sequence identity or similarity to, for example, SEQ ID NOs: 12, 14, 16, 6, 8, and / or 10.

[0076] Embodiments of the present invention also include mutant antigen-binding proteins, such as immunoglobulin V H and V L ; or comprising HC and LC, and the corresponding V specifically described hereinH , V L The present embodiment includes an anti-TMPRSS2 antibody and its antigen-binding fragment, which comprises an amino acid sequence having 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, or 99%) total amino acid sequence identity or similarity to the amino acid sequence of HC or LC, wherein CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of such immunoglobulins are not variants, but rather contain the amino acid sequences shown in SEQ ID NOs. 12, 14, 16, 6, 8, and 10, respectively. Therefore, in such embodiments, the CDR within the variant antigen-binding protein is not a variant in itself.

[0077] A conservatively modified mutant anti-TMPRSS2 antibody and its antigen-binding fragment are also part of the present invention. “Conservatively modified mutant” or “conservative substitution” refers to a mutant in which one or more amino acid substitutions exist in the polypeptide with other amino acids having similar properties (e.g., charge, side chain size, hydrophobic / hydrophilicity, skeletal structure, and rigidity). Such changes are frequently made without significantly disrupting the biological activity of the antibody or fragment. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does 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 edition)). Furthermore, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt biological activity.

[0078] Examples of amino acid groups with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl 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 conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science vol. 256: pp. 1443-45.

[0079] Functionally conserved variants of anti-TMPRSS2 antibodies and their antigen-binding fragments are also part of the present invention. Any of the variants of anti-TMPRSS2 antibodies and their antigen-binding fragments (discussed herein) may be “functionally conserved variants.” Such functionally conserved variants are also, in some cases, characterized as conservatively modified variants. As used herein, “functionally conserved variant” refers to a variant of an anti-TMPRSS2 antibody or its antigen-binding fragment in which one or more amino acid residues are altered without significantly changing one or more functional properties of the antibody or fragment. In one embodiment of the present invention, a functionally conserved variant anti-TMPRSS2 antibody or its antigen-binding fragment comprises a variant amino acid sequence and exhibits one or more of the following functional properties: • Inhibits the proliferation of influenza virus (e.g., A / Puerto Rico / 08 / 1934(H1N1)) in TMPRSS2-expressing cells (e.g., Calu-3 cells); For example, EC of 440 pM or 1.06 nM respectively 50 The value indicates that it binds to the surface of TMPRSS-expressing cells (e.g., MDCK / Tet-on); ·Does not significantly bind to MDCK / Tet-on cells that do not express TMPRSS2; ·Binds to human TMPRSS2 with a K -9 of approximately 2.81×10 D M at about 25°C; ·Binds to human TMPRSS2 with a K -9 of approximately 9.31×10 D M at about 37°C; ·Binds to cynomolgus TMPRSS2 with a K -8 of approximately 5.60×10 D M at about 25°C; ·Binds to cynomolgus TMPRSS2 with a K -7 of approximately 1.40×10 D M at about 37°C; ·Limits the spread of influenza virus infection (e.g., H1_PR34; H1_CA09; H1_Bris; H9N2 or H3N2 influenza virus) in cells, such as Calu-3, in vitro; and / or ·Protects mice engineered to express human TMPRSS2 protein, optionally in combination with anti-HA antibodies, from death caused by, for example, influenza virus infection, such as H1N1 or H3N2 (e.g., infecting the mice with a normally lethal dose of the virus).

[0080] The present invention includes mice engineered to express human TMPRSS2 protein, including anti-TMPRSS2 antigen-binding proteins (e.g., antibodies or antigen-binding fragments), such as H1H7017N and H4H7017N, in vivo in the mice. See International Patent Application Publication No. WO2017 / 151453.

[0081] 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 degree, inhibits the protease activity of substrates such as HA;Cbz-Gly-Gly-Arg-AMC(Sigma) (where Cbz is benzyloxycarbonyl and AMC is 7-amino-4-methylcoumarin); influenza virus HA0; coronavirus S protein; or precursor TMPRSS2 autocatalytically cleaved between Arg255 and Ile256, and / or inhibits the entry of influenza virus into cells and / or inhibits influenza virus replication in the organism of interest.

[0082] "H1H7017N" and "H4H7017N" refer to antigen-binding proteins, such as the heavy chain or V described below. H (or its variants) and light chain or V L (or its variants) or V containing its CDR (CDR-H1 (or its variants), CDR-H2 (or its variants), and CDR-H3 (or its variants)) H and its CDR (CDR-L1 (or its variant), CDR-L2 (or its variant), or CDR-L3 (or its variant) including V L This refers to an antibody and its antigen-binding fragment, including, for example, an immunoglobulin chain, variable region, and / or CDR, which include the specific amino acid sequence described below.

[0083] In one embodiment of the present invention, "H1H7017N" or "H4H7017N" refers to an antibody or antigen-binding fragment comprising CDR-H1, CDR-H2, and CDR-H3 of immunoglobulin heavy chains containing the amino acid sequence shown in SEQ ID NO: 2, 17, or 19, and CDR-L1, CDR-L2, and CDR-L3 of immunoglobulin light chains containing the amino acid sequence shown in SEQ ID NO: 4 or 18.

[0084] In one embodiment of the present invention, "H1H7017N" or "H4H7017N" refers to a V containing the amino acid sequence shown in SEQ ID NO: 2. H ; and V containing the amino acid sequence shown in SEQ ID NO: 4 L This refers to an antibody containing or an antigen-binding fragment of that antibody.

[0085] In one embodiment of the present invention, "H1H7017N" refers to an antibody or antigen-binding fragment containing a heavy chain immunoglobulin containing the amino acid sequence shown in SEQ ID NO: 17, and a light chain immunoglobulin containing the amino acid sequence shown in SEQ ID NO: 18.

[0086] In one embodiment of the present invention, "H4H7017N" refers to an antibody or antigen-binding fragment comprising a heavy chain immunoglobulin containing the amino acid sequence shown in SEQ ID NO: 19, and a light chain immunoglobulin containing the amino acid sequence shown in SEQ ID NO: 18. The term "H4H7017N" also refers to V H This includes embodiments in which the compound is fused to wild-type IgG4, for example, an embodiment in which residue 108 is S.

[0087] Anti-TMS22 antibody or antigen-binding fragments H1H7017N and H4H7017N H1H7017N and H4H7017N heavy chain variable regions (DNA) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTCCTATGGCATGCACTGGGTCCGCCAGTCTCCAGGCAAGGGGCTCGAGTGGGTGGCAGTTATATGGAATGATGGAAGTTATGTATACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACATTTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGGGAGTGGGTACTTTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (Sequence ID 1)

[0088] H1H7017N and H4H7017N heavy chain variable regions (polypeptides) QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQSPGKGLEWVAV IWNDGSYV YYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYC AREGEWVLYYFDY WGQGTLVTVSS (Sequence 2)

[0089] H1H7017N and H4H7017N light chain variable regions (DNA) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCGTCTACTT TAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (Sequence ID 3)

[0090] H1H7017N and H4H7017N light chain variable regions (polypeptides) DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS TLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYSYT FGQGTKLEIK (Sequence No. 4)

[0091] H1H7017N and H4H7017N CDR-H1(DNA) GGA TTC ACC TTC AGT TCC TAT GGC (Sequence ID 5)

[0092] H1H7017N and H4H7017N CDR-H1 (polypeptide) GFTFSSYG (Sequence ID 6 (or its variants having point mutations and / or point deletions of 1, 2, 3, or 4))

[0093] H1H7017N and H4H7017N CDR-H2(DNA) ATA TGG AAT GAT GGA AGT TAT GTA (Sequence ID 7)

[0094] H1H7017N and H4H7017N CDR-H2 (polypeptide) IWNDGSYV (Sequence ID 8 (or its variants having point mutations and / or point deletions of 1, 2, 3, or 4))

[0095] H1H7017N and H4H7017N CDR-H3(DNA) GCG AGA GAG GGG GAG TGG GTA CTT TAC TAC TTT GAC TAC (Sequence ID 9)

[0096] H1H7017N and H4H7017N CDR-H3 (polypeptide) AREGEWVLYYFDY (Sequence ID 10 (or its variants having point mutations and / or point deletions of 1, 2, 3, or 4))

[0097] H1H7017N and H4H7017N CDR-L1(DNA) CAG AGT ATT AGT AGC TGG (Sequence ID 11)

[0098] H1H7017N and H4H7017N CDR-L1 (polypeptide) QSISSW (Sequence ID 12 (or its variants having point mutations and / or point deletions of 1, 2, 3, or 4))

[0099] H1H7017N and H4H7017N CDR-L2(DNA) AAG GCG TCT (Sequence ID 13)

[0100] H1H7017N and H4H7017N CDR-L2 (polypeptide) KAS (Sequence ID 14 (or its variant with a point mutation and / or point deletion))

[0101] H1H7017N and H4H7017N CDR-L3(DNA) CAA CAG TAT AAT AGT TAT TCG TAC ACT (Sequence ID 15)

[0102] H1H7017N and H4H7017N CDR-L3 (polypeptide) QQYNSYSYT (Sequence ID 16 (or its variants having point mutations and / or point deletions of 1, 2, 3, or 4))

[0103] H1H7017N Full-length heavy chain - human IgG1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAVIWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREGEWVLYYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 17)

[0104] Total length light chain - Hitokappa DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence No. 18)

[0105] H4H7017N Full-length heavy chain - human IgG4 (S108P) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAVIWNDGSYVYYADSVKGRFTISRDISKNTLFLQMNSLRAEDTAVYYCAREGEWVLYYFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 19)

[0106] Total length light chain - Hitokappa DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence No. 18)

[0107] The antibody and antigen-binding fragments of the present invention include immunoglobulin chains comprising amino acid sequences described herein, as well as cellular and in vitro post-translational modifications of the antibody. For example, the present invention includes antibody and antigen-binding fragments that specifically bind to TMPRSS2 comprising 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), as well as antibodies and fragments in which one or more amino acid residues are glycosylated, one or more Asn residues are deamidated, one or more residues (e.g., Met, Trp and / or His) are oxidized, the N-terminal Gln is pyroglutamic acid (pyro-E), and / or a C-terminal lysine is deleted.

[0108] The present invention provides a container (for example, a plastic or glass vial, having a cap or chromatography column, a hollow-hole needle or syringe cylinder) containing the anti-TMPRSS2 antigen-binding protein of the present invention, for example, H1H7017N or H4H7017N.

[0109] The present invention also provides an infusion device comprising one or more antigen-binding proteins (e.g., antibodies or antigen-binding fragments) or a pharmaceutical composition thereof that specifically bind to TMPRSS2, e.g., H4H7017N or H1H7017N. The infusion device is packaged in a kit. The infusion device is a device for introducing a substance into a target organism via parenteral routes, e.g., intramuscular, subcutaneous, or intravenous. For example, the infusion device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an autoinjector) comprising, for example, a cylinder or barrel for holding the fluid to be injected (e.g., containing an antibody or fragment or a pharmaceutical composition thereof), a needle for puncturing the skin and / or blood vessels for injecting the fluid; and a plunger for pushing the fluid out of the cylinder and injecting it through the needle hole. In one embodiment of the present invention, an infusion 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) infusion device. Such a device may contain an antigen-binding protein or its pharmaceutical composition within a cannula or trocar / needle and may be attached to a tube that can be attached to a bag or reservoir for holding a fluid (e.g., saline) introduced into the target organism via the cannula or trocar / needle. In one embodiment of the present invention, the antibody or fragment or its pharmaceutical composition is introduced into the device when the trocar and cannula are inserted into the target vein and the trocar is removed from the inserted cannula. The IV device may be inserted, for example, into a peripheral vein (e.g., hand or arm); into the superior or inferior vena cava or into the right atrium of the heart (e.g., central IV); or into the subclavian vein, internal jugular vein or femoral vein, and is advanced toward the heart until it reaches, for example, the superior vena cava or right atrium (e.g., central venous line). In one embodiment of the present invention, the infusion device is an automatic injector; a jet injector or an external infusion pump. A jet injector uses a narrow, high-pressure jet of liquid penetrating the epidermis to introduce the antibody or fragment or its pharmaceutical composition into the target organism. An external injection pump is a medical device that delivers antibodies, fragments, or pharmaceutical compositions into a target organism in a controlled amount.External injection pumps are powered electrically or mechanically. Different pumps operate in different ways; for example, a syringe pump holds fluid in a syringe reservoir, with a movable piston controlling fluid delivery, while an elastic pump holds fluid in an expandable balloon reservoir, with pressure from the balloon's elastic wall driving fluid delivery. In a peristaltic pump, a pair of rollers pinch down the length of a flexible tube that pushes the fluid forward. In a multi-channel pump, fluid is delivered from multiple reservoirs at multiple speeds.

[0110] The present invention further provides a method for administering the anti-TMPRSS2 antigen-binding protein of the present invention, for example, H4H7017N or H1H7017N, which comprises the step of introducing the antigen-binding protein into the living body of a subject (e.g., a human). For example, this method comprises the step of puncturing the living body of the subject with a syringe needle and injecting the antigen-binding protein into the living body of the subject, for example, into the subject's vein, artery, tumor, muscle tissue or subcutaneous tissue.

[0111] Preparation of human antibodies Methods for producing human antibodies in transgenic mice are known in the art. Such known methods can be used in connection with the present invention to produce human antibodies that specifically bind to TMPRSS2. Antibodies against TMPRSS2 can be produced using an immunogen comprising any one of the following: In certain embodiments of the present invention, the antibody of the present invention is obtained from a mouse immunized with full-length, natural TMPRSS2, or live attenuated or inactivated virus, or DNA encoding its protein or fragment. Alternatively, the TMPRSS2 protein or fragment is produced and modified using standard biochemical techniques and used as an immunogen. In one embodiment of the present invention, the immunogen is a recombinantly produced TMPRSS2 protein or fragment. In certain embodiments of the present invention, the immunogen may be a TMPRSS2 polypeptide vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, the immunogen may be a recombinant TMPRSS2 polypeptide expressed in Escherichia coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0112] High-affinity chimeric antibodies against TMPRSS2 having human variable regions and mouse constant regions can be initially isolated using VELOCIMMUNE® technology (see, for example, U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies. VELOCIMMUNE® technology involves the creation of transgenic mice having a genome containing human heavy and light chain variable regions operably ligated to an endogenous mouse constant region locus, so that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigen stimulation. The DNA encoding the variable regions of the antibody's heavy and light chains is isolated and operably ligated to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing complete human antibodies.

[0113] Generally, VELOCIMMUNE® mice are loaded with the target antigen, and lymphocytes (such as B cells) are recovered from mice that express antibodies. Immortalized hybridoma cell lines can be produced by fusing lymphoid cells with myeloma cell lines. Such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins are produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains can be directly isolated from antigen-specific lymphocytes.

[0114] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As in the experimental section below, the antibody is characterized and selected for desired properties, including affinity, selectivity, and epitope. The mouse constant region is replaced with the desired human constant region to produce a fully human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. While the selected constant region may vary depending on the specific use, the high-affinity antigen-binding and target-specific properties reside in the variable region.

[0115] Anti-TMPRSS2 antibody containing Fc variant According to certain embodiments of the present invention, for example, an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment, is provided, comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present invention provides an Fc domain with C H 2 or C HThe anti-TMPRSS2 antibody contains mutations in three regions, and these mutations (one or more) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-restrictive examples of such Fc modifications include, for example, 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 modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0116] For example, the present invention includes 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 43 The anti-TMPRS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment, comprises an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 4H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F).

[0117] The V described herein includes any possible combination of the aforementioned Fc domain mutations. H and / or V L Anti-TMPRSS antigen-binding proteins, such as antibodies and their antigen-binding fragments, which include the above, are intended to be included within the scope of the present invention.

[0118] The present invention also relates to V described herein. H and chimeric heavy chain constant (C H The anti-TMPRSS2 antigen-binding protein, antibody, or antigen-binding fragment containing the ) region, and the chimeric C H The region is C, which contains more than 1 immunoglobulin isotype. H It includes segments derived from a region. For example, the antibody of the present invention is derived from human IgG1, human IgG2, or human IgG4 molecules. H C, which contains part or all of two domains and is derived from human IgG1, human IgG2, or human IgG4 molecules. H Chimera C, which is a combination of some or all of the three domains. H It may include a region. According to a particular embodiment, the antibody of the present invention is a chimeric C having a chimeric hinge region. HThe region includes. For example, the chimeric hinge may include an "upper hinge" amino acid sequence (amino acid residues from EU numbering positions 216 to 227) derived from the human IgG1, human IgG2, or human IgG4 hinge region, combined with an "lower hinge" sequence (amino acid residues from EU numbering positions 228 to 236) derived from the human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region includes 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. Chimeric C described herein H Antibodies containing the region can, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, for example, International Publication WO2014 / 022540).

[0119] Immunoconjugate The present invention encompasses an anti-TMPRSS2 antigen-binding protein, e.g., an antibody or antigen-binding fragment, conjugated to another part, e.g., a therapeutic part ("immunoconjugate") such as a toxoid or antiviral drug for treating influenza virus infection. In one embodiment of the present invention, an anti-TMPRSS2 antibody or fragment is conjugated to one of the further therapeutic agents described herein. As used herein, the term "immunoconjugate" means an antigen-binding protein, e.g., an antibody or antigen-binding fragment, chemically or biologically linked to a radioactive substance, cytokine, interferon, target or reporter part, enzyme, peptide or protein, or therapeutic agent. The antigen-binding protein is linked to a radioactive substance, cytokine, interferon, target or reporter part, enzyme, peptide or therapeutic agent at any position along its molecule, as long as it can bind to its target (TMPRSS2). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the present invention, the drug may be a second different antibody that specifically binds to TMPRSS2. The type of therapeutic portion conjugated to the anti-TMPRSS2 antigen-binding protein (e.g., antibody or fragment) should be determined considering the condition to be treated and the desired therapeutic effect to be achieved.For example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-256 (Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (2nd edition), Robinson et al. (eds.), pp. 623-253 (Marcel Dekker, Inc., 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies 1984: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And See Therapy, Baldwin et al. (eds.), pp. 303-3016 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., Vol. 62: pp. 119-158 (1982).

[0120] multispecific antibodies The present invention includes anti-TMPRSS2 antigen-binding proteins, e.g., antibodies and their antigen-binding fragments, as well as methods for using them and methods for producing such antigen-binding proteins. The term “anti-TMPRSS2” antigen-binding protein, e.g., antibody or antigen-binding fragment, includes a multispecific (e.g., bispecific or biparatopic) molecule comprising at least one first antigen-binding domain that specifically binds to TMPRSS2 (e.g., an antigen-binding domain derived from H1H7017N or H4H7017N), and at least one second antigen-binding domain that binds to an antigen or epitope in TMPRSS2 different from the first antigen-binding domain (e.g., influenza HA such as an antigen-binding domain derived from H1H14611N2, H1H14612N2 or H1H11729P). In one embodiment of the present invention, the first and second epitopes overlap. In another embodiment of the present invention, the first and second epitopes do not overlap. For example, in one embodiment of the present invention, the multispecific antibody is a bispecific IgG antibody (e.g., IgG1 or IgG4) comprising a first antigen-binding domain that specifically binds to TMPRSS2 containing heavy and light immunoglobulin chains of H1H7017N or H4H7017N, and a second antigen-binding domain that specifically binds to influenza HA (containing different light and heavy immunoglobulin chains such as H1H14611N2, H1H14612N2, or H1H11729P).

[0121] "H1H7017N" is for HCDR and LCDR, V H and V L The present invention includes a multispecific molecule, such as an antibody or antigen-binding fragment, comprising HC and LC of H1H7017N (including their variants as described herein).

[0122] "H4H7017N" is for HCDR and LCDR, V H and V L The present invention includes a multispecific molecule, such as an antibody or antigen-binding fragment, comprising HC and LC of H4H7017N (including their variants as described herein), or an antibody or antigen-binding fragment.

[0123] In one embodiment of the present invention, the antigen-binding domain that specifically binds to TMPRSS contained in the multispecific molecule is (1) (i) Heavy chain variable domain sequences including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 6, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 8, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 10, and (ii) A light chain variable domain sequence comprising CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 12, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 14, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16; or (2) (i) A heavy chain variable domain sequence containing the amino acid sequence shown in Sequence ID No. 2, and (ii) A light chain variable domain sequence containing the amino acid sequence shown in Sequence ID No. 4; or (3) (i) A heavy chain immunoglobulin sequence comprising the amino acid sequence shown in SEQ ID NO: 17 or 19, and (ii) Light chain immunoglobulin sequence containing the amino acid sequence shown in Sequence ID No. 18 Includes.

[0124] In one embodiment of the present invention, the multispecific antibody or fragment comprises two or more different binding specificities (e.g., trispecific molecules), for example, one or more additional antigen-binding domains that are identical or different from the first and / or second antigen-binding domains.

[0125] In one embodiment of the present invention, the multispecific molecule has an antigen-binding site that specifically binds to TMPRSS2, as well as the following: H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N;H1H11829N2;H2aM11829N;H2M11830N;H1H11830N2;H1H11903N;H1H14571N;H2a14571N;H1H11704P;H1H11711P;H1H11714P;H1H11717P;H1H11724P;H1H11727P;H1H 11730P2;H1H11731P2;H1H11734P2;H1H11736P2;H1H11742P2;H1H11744P2 ;H1H11745P2;H1H11747P2;H1H11748P2;H1H17952B;H1H17953B;H1H17954 B;H1H17955B;H1H17956B;H1H17957B;H1H17958B;H1H17959B;H1H17960B; H1H17961B;H1H17962B;H1H17963B;H1H17964B;H1H17965B;H1H17966B;H1H 17967B;H1H17968B;H1H17969B;H1H17970B;H1H17971B;H1H17972B;H1H17 973B;H1H17974B;H1H17975B;H1H17976B;H1H17977B;H1H17978B;H1H1797 9B;H1H17980B;H1H17981B;H1H17982B;H1H17983B;H1H17984B;H1H17985B ;H1H17986B;H1H17987B;H1H17988B;H1H17989B;H1H17990B;H1H17991B;H 1H17992B;H1H17993B;H1H17994B;H1H17995B;H1H17996B;H1H17997B;H1H 17998B;H1H17999B;H1H18000B;H1H18001B;H1H18002B;H1H18003B;H1H18 004B;H1H18005B;H1H18006B;H1H18007B;H1H18008B;H1H18009B;H1H1801 0B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H18015B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18041B;H1H18042B;H1H18043B;H1H18044B;H1H18045B;H1H18046B;H1H18047B;H1H18048B;H1H18049B;H1H18051B;H1H18052B;H1H18053B;H1H18054B;H1H18055B;H1H18056B;H1H18057B;H1H18058B;H1H18059B;H1H18060B;H1H18061B;H1H18062B;H1H18063B;H1H18064B;H1H18065B;H1H18066B;H1H18067B;H1H18068B;H1H18069B;H1H18070B;H1H18071B;H1H18072B;H1H18073B;H1H18074B;H1H18075B;H1H18076B;H1H18077B;H1H18078B;H1H18079B;H1H18080B;H1H18081B;H1H18082B;H1H18083B;H1H18084B;H1H18085B;H1H18086B;H1H18087B;H1H18088B;H1H18089B;H1H18090B;H1H18091B;H1H18092B;H1H18093B;H1H18094B;H1H18095B;H1H18096B;H1H18097B;H1H18098B;H1H18099B;H1H18100B;H1H18101B;H1H18102B;H1H18103B;H1H18104B;H1H18105B;H1H18107B;H1H18108B;H1H18109B;H1H18110B;H1H18111B;H1H18112B;H1H18113B;H1H18114B;H1H18115B;H1H18116B;H1H18117B;H1H18118B;H1H18119B;H1H18120B;H1H18121B;H1H18122B;H1H18123B;H1H18124B;H1H18125B;H1H18126B;H1H18127B;H1H18128B;H1H18129B;H1H18130B;H1H18131B;H1H18132B;H1H18133B;H1H18134B;H1H18135B;H1H18136B;H1H18137B;H1H18138B;H1H18139B;H1H18140B;H1H18141B;H1H18142B;H1H18143B;H1H18144B;H1H18145B;H1H18146B;H1H18147B;H1H18148B;H1H18149B;H1H18150B;H1H18151B;H1H18152B;H1H18153B;H1H18154B;H1H18155B;H1H18156B;H1H18157B;H1H18158B;H1H18159B;H1H18160B;H1H18161B;H1H18162B;H1H18163B;H1H18164B;H1H18165B;H1H18166B;H1H18167B;H1H18168B;H1H18169B;H1H18170B;H1H18171B;H1H18172B;H1H18173B;H1H18174B;H1H18175B;H1H18176B;H1H18177B;H1H18178B;H1H18179B;H1H18180B;H1H18181B;H1H18182B;H1H18183B;H1H18184B;H1H18185B;H1H18186B;H1H18187B;H1H18188B;H1H18189B;H1H18190B;H1H18191B;H1H18192B;H1H18193B;H1H18194B;H1H18195B;H1H18196B;H1H18197B;H1H18198B;H1H18199B;H1H18200B;H1H18201B;H1H18202B;H1H18203B;H1H18204B;H1H18205B;H1H18206B;H1H18207B;H1H18208B;H1H18209B;H1H18210B;H1H18211B;H1H18212B;H1H18213B;H1H18214B;H1H18216B;H1H18217B;H1H18218B;H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18225B;H1H18226B;H1H18227B;H1H18228B;H1H18229B;H1H18230B;H1H18231B;H1H18232B;H1H18 233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1H18241B;H1H18242B;H1H18243B;H1H18244B;H1H18245B; H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B;H1H18251B;H1H18252B;H1H18253B;H1H18254B;H1H18255B;H1H18256B;H1H18257B;H1H18 258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1H18267B;H1H18268B;H1H18269B;H1H18270B;H1H18271B; H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B;H1H18279B;H1H18280B;H1H18281B;H1H18282B;H1H18283B;H1H18284B;H1H18 285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B;H1H18290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B;H1H18295B;H1H18297B;H1H18298B; H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18303B;H1H18304B;H1 H18305B;H1H18306B;H1H18307B;H1H18308B;H1H18309B;H1H18310B;H1H18 311B;H1H18312B;H1H18313B;H1H18314B;H1H18315B;H1H18316B;H1H18317B;H1H18318B;H1H18319B;H1H18320B;H1H18321B;H1H18322B;H1H18323B;The antigen-binding sites include antigen-binding sites that specifically bind to influenza HA taken from antibodies selected from the group consisting of H1H18324B;H1H18325B;H1H18326B;H1H18327B;H1H18328B;H1H18329B;H1H18330B;H1H18331B;H1H18332B;H1H18333B;H1H18334B; and H1H18335B, which are described in International Patent Application Publication WO2016 / 100807 (e.g., CDR-H, V; H or its heavy chain; and CDR-L, V L (or its light chain)

[0126] In one embodiment of the present invention, the multispecific molecule has an antigen-binding site that specifically binds to TMPRSS2, as well as an antigen-binding site that specifically binds to influenza group II HA proteins, for example, the V of H1H14611N2. H and V L The formulation includes antigen-binding sites (e.g., SEQ ID NOs. 24 and 28); or heavy chain immunoglobulins containing CDR-H1, CDR-H2 and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs. 25-27); and light chain immunoglobulins containing CDR-L1, CDR-L2 and CDR-L3 of H1H14611N2 (e.g., SEQ ID NOs. 29-31).

[0127] In one embodiment of the present invention, the multispecific molecule has an antigen-binding site that specifically binds to TMPRSS2, as well as an antigen-binding site that specifically binds to influenza group II HA proteins, for example, the V of H1H14612N2. H and V L The formulation includes antigen-binding sites (e.g., SEQ ID NOs. 40 and 44); or heavy chain immunoglobulins containing CDR-H1, CDR-H2 and CDR-H3 of H1H14612N2 (e.g., SEQ ID NOs. 41-43); and light chain immunoglobulins containing CDR-L1, CDR-L2 and CDR-L3 of H1H14612N2 (e.g., SEQ ID NOs. 45-47).

[0128] In one embodiment of the present invention, the multispecific molecule has an antigen-binding site that specifically binds to TMPRSS2, as well as an antigen-binding site that specifically binds to influenza group I HA proteins, for example, the V of H1H11729P. H and V L The formulation includes antigen-binding sites (e.g., SEQ ID NOs. 32 and 36); or heavy chain immunoglobulins containing CDR-H1, CDR-H2 and CDR-H3 of H1H11729P (e.g., SEQ ID NOs. 33-35); and light chain immunoglobulins containing CDR-L1, CDR-L2 and CDR-L3 of H1H11729P (e.g., SEQ ID NOs. 37-39).

[0129] In one embodiment of the present invention, the two specific antigen-binding fragments are a first scFv (e.g., V of H1H7017N or H4H7017N) having binding specificity to a first epitope (e.g., TMPRSS2). H and V L (including), and a second scFv having binding specificity to a second different epitope (e.g., V of an anti-influenza HA antibody) H and V L (including) n (Sequence ID 48) (where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)) is linked. Other bispecific antigen-binding fragments include F(ab)2 of a bispecific IgG antibody containing the heavy and light chain CDRs of H1H7017N or H4H7017N, as well as another antibody that binds to a different epitope.

[0130] Treatment method The present invention provides a method for treating or preventing a viral infection or cancer (e.g., prostate cancer) by administering a therapeutically effective amount of anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N), to a subject (e.g., a human) in need of treatment or prevention.

[0131] 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. Subtypes of influenza A viruses are determined by envelope glycoproteins having either hemagglutinin (HA) or neuraminidase (NA) activity. There are several HA subtypes of influenza A viruses used to designate influenza A subtypes (e.g., HA1, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA11, HA12, HA13, HA14, HA15, HA16, HA17, or HA18 - these subtypes are designated as H1, H2, H3, etc.), and NA subtypes (e.g., NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, NA9, NA10, or NA11 - these subtypes are designated as N1, N2, N3, etc.). For example, influenza A viruses H1N1 and H3N2 are commonly known human pathogens. Humans are generally infected with viruses of subtypes H1, H2, or H3, and N1 or N2. The present invention includes methods for treating or preventing infection by influenza virus subtypes discussed herein. A multispecific antibody that binds to TMPRSS2 and its antigen-binding fragment also binds to HA and / or NA of, for example, the subtypes described herein, in one embodiment of the present invention.

[0132] An effective 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 a viral infection refers to an amount of antibody or fragment sufficient to alleviate one or more signs and / or symptoms of infection in the subject, whether by inducing regression or disappearance of such signs and / or symptoms, or by inhibiting the progression of such signs and / or symptoms. The dosage may vary depending on the age and size of the subject, the target disease, the condition, the route of administration, etc. In one embodiment of the present invention, for example, an effective or therapeutically effective dose of the antibody or antigen-binding fragment of the present invention for treating or preventing a viral infection in an adult human subject is about 0.01 to about 200 mg / kg, e.g., up to about 150 mg / kg. In one embodiment of the present invention, the dosage is up to about 10.8 or 11 grams (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 grams). Depending on the severity of the infection, the frequency and duration of treatment can be adjusted. In certain embodiments, the antigen-binding protein of the present 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 the administration of a second or more subsequent doses of antibody or antigen-binding fragments in an amount that may be approximately the same as or less than the initial dose, with the subsequent doses spaced at least 1 to 3 days; at least 1 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 apart.

[0133] As used herein, the term “subject” refers to mammals (e.g., rats, mice, cats, dogs, cattle, sheep, horses, goats, rabbits), preferably humans, that require prevention and / or treatment of a disease or disorder, such as a viral infection or cancer. Subjects may have a viral infection, such as influenza infection, or may be predisposed to developing an infection. Subjects predisposed to developing an infection, or at high risk of contracting an infection (e.g., influenza virus), include subjects with immunodeficiency due to autoimmune disease, subjects undergoing immunosuppressive therapy (e.g., post-organ transplantation), subjects with human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), subjects with a form of anemia that depletes or destroys white blood cells, subjects undergoing radiation or chemotherapy, or subjects with inflammatory disorders. Furthermore, subjects who are very young (e.g., under 5 years of age) or elderly (e.g., over 65 years of age) are at higher risk. Furthermore, individuals may be at risk of developing the virus due to proximity to disease outbreaks, for example, by living in a densely populated city, by coming into contact with individuals who have been confirmed or suspected of having the virus, or by their employment choices (e.g., hospital workers, pharmaceutical researchers, travelers to infected areas, or frequent flyers).

[0134] "To treat" or "to treat" means administering an anti-TMPRSS2 antigen-binding protein, such as the antibody or antigen-binding fragment of the present invention (e.g., H1H7017N or H4H7017N), to a subject having one or more signs or symptoms of a disease or infection, such as a viral infection, to which the antigen-binding protein is effective when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0135] The present invention also encompasses the prophylactic administration of an anti-TMPRSS2 antigen-binding protein, such as the antibody of the present invention or its antigen-binding fragment (e.g., H1H7017N or H4H7017N), to subjects at risk of viral infection to prevent such infection. Passive antibody-based immunoprevention has proven to be an effective strategy for preventing subjects from viral infection. For example, see Berry et al., Passive broad-spectrum influenza immunoprophylaxis. Influenza Res Treat., 2014;2014:267594.Epub 2014 Sep 22; Jianqiang et al., Passive immune neutralization strategies for prevention and control of influenza A infections, Immunotherapy., 2012 February;Vol. 4 (No. 2):pp. 175-186; Prabhu et al., Antivir Ther., 2009;Vol. 14 (No. 7):pp. 911-921, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza. "To prevent" or "to prevent" means administering an anti-TMPRSS2 antigen-binding protein, such as the antibody or antigen-binding fragment of the present invention (e.g., H1H7017N or H4H7017N), to a subject in order to inhibit the emergence of a disease or infection (e.g., viral infection) within the subject's body, and for this purpose, the antigen-binding protein is effective when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0136] In one embodiment of the present invention, signs or symptoms of viral infection in a subject are the survival or replication of the virus within the subject's body, which is determined, for example, by a viral titer assay (e.g., influenza virus replication in fetal chicken eggs or influenza virus hemagglutination assay). Other signs and symptoms of viral infection are discussed herein.

[0137] The present invention provides a therapeutically effective dose of anti-TMPRSS2 antigen-binding protein (e.g., H1H7017N or H4H7017N) to be administered to a subject, for example, by injecting the protein into the subject's body, thereby treating or preventing viral infections (e.g., influenza virus or coronavirus infection) or complications following viral infections in a subject requiring such treatment (e.g., a human), as follows: • Fever or feeling feverish / chills; ·cough; ·Sore throat; • Runny nose or nasal congestion; ·sneeze; • Muscle or body pain; ·headache; • Lethargy (fatigue); ·vomiting; ·diarrhea; • Respiratory tract infection; ·Chest discomfort; ·shortness of breath; • Bronchitis; and / or ·pneumonia, The present invention provides a method for inducing regression or disappearance, or inhibiting the progression, of at least one sign or symptom of a viral infection.

[0138] Furthermore, the present invention includes a method for treating or preventing cancer in a subject, such as metastatic cancer, such as prostate cancer (e.g., characterized by the expression of a TMPRSS2:ERG fusion), colon cancer, lung cancer, pancreatic cancer, urinary tract cancer, breast cancer, ovarian cancer, prostate adenocarcinoma, renal cell carcinoma, colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and / or pleural mesothelioma, by administering a therapeutically effective amount of TMPRSS2 antigen-binding protein (e.g., H1H7017N or H4H7017N) to the subject, for example by injecting the protein into the subject's body.

[0139] Combination agents and pharmaceutical compositions To produce a pharmaceutical composition of an anti-TMPRSS2 antigen-binding protein, such as an antibody and its antigen-binding fragment (e.g., H1H7017N or H4H7017N), the antigen-binding protein is mixed with a pharmaceutically acceptable carrier or excipient. For example, Remington's Pharmaceutical Sciences and USPharmacopeia: 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. and See Kotkoskie (2000), Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In one embodiment of the present invention, the pharmaceutical composition is sterile. Such a composition is part of the present invention.

[0140] The scope of the present invention includes a dry, for example, lyophilized composition, or a pharmaceutically acceptable carrier, comprising an anti-TMPRSS2 antigen-binding protein, for example, an antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N), but substantially lacking water.

[0141] In further embodiments of the present invention, the further therapeutic agents administered to a subject in conjunction with the anti-TMPRSS2 antigen-binding protein disclosed herein, for example, an antibody or its antigen-binding fragment (e.g., H1H7017N or H4H7017N), are administered to the subject in accordance with Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)).

[0142] The mode of administration may vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, infusion, topical, cutaneous, percutaneous, or intra-arterial.

[0143] The present invention provides a method for administering an anti-TMPRSS2 antigen-binding protein, such as an antibody or its antigen-binding fragment (e.g., H1H7017N or H4H7017N), comprising the step of introducing the protein into a target organism. For example, the method includes the step of puncturing the target organism with a syringe needle and injecting the antigen-binding protein into the target organism, for example, into the target's vein, artery, tumor, muscle tissue, or subcutaneous tissue.

[0144] The present invention relates to anti-TMPRSS2 antigen-binding proteins, such as antibodies or their antigen-binding fragments (e.g., H1H70N or H4H7017N), and polypeptides (e.g., HC, LC, V of H1H7017N or H4H7017N). H Or V L The present invention provides a container (for example, a plastic or glass vial, for example, having a cap or chromatography column, a hollow-hole needle or syringe cylinder) containing any of the following: a polynucleotide or vector described herein, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0145] In one embodiment of the present invention, an anti-TMPRSS2 antigen-binding protein, for example, the antibody of the present invention or its antigen-binding fragment (e.g., H1H7017N or H4H7017N), is accompanied by one or more further therapeutic agents. For example, in one embodiment of the present invention, the further therapeutic agents are antiviral agents and / or vaccines. As used herein, the term “antiviral agent” means any anti-infective agent or treatment used to treat, prevent or improve a viral infection in a subject. The term “antiviral agent” includes, but is not limited to, cationic steroid antibacterial agents, leupeptin, aprotinin, amantadine, rimantadine, oseltamivir, zanamivir, ribavirin, or interferon-alpha 2b. A method of treating or preventing a viral (e.g., influenza) infection in a subject requiring the above treatment or prevention by administering H1H7017N or H4H7017N in conjunction with further therapeutic agents is part of the present invention.

[0146] For example, in one embodiment of the present invention, the further therapeutic agent is a vaccine, such as an influenza vaccine. In one embodiment of the present invention, the vaccine is an inactivated / dead virus vaccine, a live attenuated virus vaccine, or a virus subunit vaccine.

[0147] For example, in one embodiment of the present invention, further therapeutic agents are as follows: [ka]

[0148] [ka]

[0149] [ka]

[0150] [ka]

[0151] [ka] See Shen et al., Biochimie, Vol. 142: pp. 1-10 (2017).

[0152] In one embodiment of the present invention, the antiviral drug is an antibody or antigen-binding fragment that specifically binds to the influenza virus, for example, influenza HA. ​​For example, in one embodiment of the present invention, the anti-HA antibody is H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N;H1H11829N2;H2aM11829N;H2M11830N;H1H11830N2;H1H11903N;H1H14571N;H2a14571N;H1H11704P;H1H11711P;H1H11714P;H1H11717P;H1H11724P;H1H11727P;H1H11730P2;H1 H11731P2;H1H11734P2;H1H11736P2;H1H11742P2;H1H11744P2;H1H11745P2;H1H11747P2;H1H11748P2;H1H17952B;H1H17953B;H1H17954B; H1H17955B;H1H17956B;H1H17957B;H1H17958B;H1H17959B;H1H17960B;H1H17961B;H1H17962B;H1H17963B;H1H17964B;H1H17965B;H1H179 66B;H1H17967B;H1H17968B;H1H17969B;H1H17970B;H1H17971B;H1H1 7972B;H1H17973B;H1H17974B;H1H17975B;H1H17976B;H1H17977B;H1 H17978B;H1H17979B;H1H17980B;H1H17981B;H1H17982B;H1H17983B;H1H17984B;H1H17985B;H1H17986B;H1H17987B;H1H17988B;H1H17989 B;H1H17990B;H1H17991B;H1H17992B;H1H17993B;H1H17994B;H1H17995B;H1H17996B;H1H17997B;H1H17998B;H1H17999B;H1H18000B;H1H1 8001B;H1H18002B;H1H18003B;H1H18004B;H1H18005B;H1H18006B;H1 H18007B;H1H18008B;H1H18009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H18015B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18041B;H1H18042B;H1H18043B;H1H18044B;H1H18045B;H1H18046B;H1H18047B;H1H18048B;H1H18049B;H1H18051B;H1H18052B;H1H18053B;H1H18054B;H1H18055B;H1H18056B;H1H18057B;H1H18058B;H1H18059B;H1H18060B;H1H18061B;H1H18062B;H1H18063B;H1H18064B;H1H18065B;H1H18066B;H1H18067B;H1H18068B;H1H18069B;H1H18070B;H1H18071B;H1H18072B;H1H18073B;H1H18074B;H1H18075B;H1H18076B;H1H18077B;H1H18078B;H1H18079B;H1H18080B;H1H18081B;H1H18082B;H1H18083B;H1H18084B;H1H18085B;H1H18086B;H1H18087B;H1H18088B;H1H18089B;H1H18090B;H1H18091B;H1H18092B;H1H18093B;H1H18094B;H1H18095B;H1H18096B;H1H18097B;H1H18098B;H1H18099B;H1H18100B;H1H18101B;H1H18102B;H1H18103B;H1H18104B;H1H18105B;H1H18107B;H1H18108B;H1H18109B;H1H18110B;H1H18111B;H1H18112B;H1H18113B;H1H18114B;H1H18115B;H1H18116B;H1H18117B;H1H18118B;H1H18119B;H1H18120B;H1H18121B;H1H18122B;H1H18123B;H1H18124B;H1H18125B;H1H18126B;H1H18127B;H1H18128B;H1H18129B;H1H18130B;H1H18131B;H1H18132B;H1H18133B;H1H18134B;H1H18135B;H1H18136B;H1H18137B;H1H18138B;H1H18139B;H1H18140B;H1H18141B;H1H18142B;H1H18143B;H1H18144B;H1H18145B;H1H18146B;H1H18147B;H1H18148B;H1H18149B;H1H18150B;H1H18151B;H1H18152B;H1H18153B;H1H18154B;H1H18155B;H1H18156B;H1H18157B;H1H18158B;H1H18159B;H1H18160B;H1H18161B;H1H18162B;H1H18163B;H1H18164B;H1H18165B;H1H18166B;H1H18167B;H1H18168B;H1H18169B;H1H18170B;H1H18171B;H1H18172B;H1H18173B;H1H18174B;H1H18175B;H1H18176B;H1H18177B;H1H18178B;H1H18179B;H1H18180B;H1H18181B;H1H18182B;H1H18183B;H1H18184B;H1H18185B;H1H18186B;H1H18187B;H1H18188B;H1H18189B;H1H18190B;H1H18191B;H1H18192B;H1H18193B;H1H18194B;H1H18195B;H1H18196B;H1H18197B;H1H18198B;H1H18199B;H1H18200B;H1H18201B;H1H18202B;H1H18203B;H1H18204B;H1H18205B;H1H18206B;H1H18207B;H1H18208B;H1H18209B;H1H18210B;H1H18211B;H1H18212B;H1H18213B;H1H18214B;H1H18216B;H1H18217B;H1H18218B;H1H18219B;H1H18220B;H1H18221B;H1H18222B;H1H18223B;H1H18224B;H1H18225B;H1H18226B;H1H18227B;H1H18228B;H1H18 229B;H1H18230B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1H18241B; H1H18242B;H1H18243B;H1H18244B;H1H18245B;H1H18246B;H1H18247B;H1H18248B;H1H18249B;H1H18250B;H1H18251B;H1H18252B;H1H18253B;H1H18 254B;H1H18255B;H1H18256B;H1H18257B;H1H18258B;H1H18259B;H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1H18267B; H1H18268B;H1H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B;H1H18279B;H1H18280B;H1H18 281B;H1H18282B;H1H18283B;H1H18284B;H1H18285B;H1H18286B;H1H18287B;H1H18288B;H1H18289B;H1H18290B;H1H18291B;H1H18292B;H1H18293B; H1H18294B;H1H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18301B;H1H18302B;H1H18303B;H1H18304B;H1H18305B;H1H18306B;H1H18 307B;H1H18308B;H1H18309B;H1H18310B;H1H18311B;H1H18312B;H1H1831 3B;H1H18314B;H1H18315B;H1H18316B;H1H18317B;H1H18318B;H1H18319B;H1H18320B;H1H18321B;H1H18322B;H1H18323B;H1H18324B;H1H18325B;H1H18326B;H1H18327B;H1H18328B;H1H18329B;H1H18330B;H1H18331B;H1H18332B;H1H18333B;H1H18334B;or any one of H1H18335B; described in International Patent Application Publication No. WO2016 / 100807; or its antigen-binding fragment, for example, the antibody or fragment is any of the above-mentioned anti-influenza HA antibodies CDR-L1, CDR-L2 and CDR-L3 (e.g., V; L Light chain immunoglobulins containing or their light chains; and CDR-H1, CDR-H2 and CDR-H3 (e.g., V H Includes a heavy chain (or its heavy chain).

[0153] In one embodiment of the present invention, further therapeutic agents include antibodies or antigen-binding fragments that bind to influenza group II HA proteins such as H1H14611N2; or V of H1H14611N2. H and V L An antibody or fragment containing; or heavy chain immunoglobulins containing CDR-H1, CDR-H2, and CDR-H3 of H1H14611N2 (e.g., SEQ ID NOs. 25-27) and light chain immunoglobulins containing 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 containing such a sequence.

[0154] H1H14611N2 Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAAS GFTFSGFS MNWVRQVPGKGLEWVSS ISTSGNYM YYADSVKGRFTISRDNAKKSFSLQMNSLRAEDSAIYYC ARGGGYNWNLFDY WGQGSLVTVSS (Sequence ID 24) CDR-H1:GFTFSGFS (Sequence ID 25) CDR-H2:ISTSGNYM (Sequence ID 26) CDR-H3:ARGGGYNWNLFDY (Sequence ID 27)

[0155] Light chain variable region EIVLTQSPGTLSLSPGERATLSCRAS QSLNSNY LAWYQQKPGQAPRLLIY GAS SRATGIP DRFSGSGSGTDFTLTITRLESEDFAVYYC QQYGNSPLT FGGGTKVEIK (Sequence ID 28) CDR-L1:QSLNSNY (Sequence ID 29) CDR-L2:GAS (Sequence ID 30) CDR-L3:QQYGNSPLT(Sequence ID 31)

[0156] In one embodiment of the present invention, a further therapeutic agent is an antibody or antigen-binding fragment that binds to an influenza group II HA protein such as H1H14612N2; or a V of H1H14612N2. H and V L An antibody or fragment containing H1H14612N2; or a heavy chain immunoglobulin containing H1H14612N2 CDR-H1, CDR-H2, and CDR-H3 (e.g., SEQ ID NOs. 41-43) and a light chain immunoglobulin containing H1H14612N2 CDR-L1, CDR-L2, and CDR-L3 (e.g., SEQ ID NOs. 45-47). "H1H14612N2" refers to any anti-group II HA antibody containing such a sequence.

[0157] H1H14612N2 Heavy chain variable region EVQLVESGGGLVKPGGSLRLSCAAS GFSFSGFS MNWVRQAPGKGLEWVSS ISTSGNYM YY ADSVKGRFTISRDNAKKSFSLQMNSLRAEDSAIYYC ARGGGYNWNLFDY WGQGSLVTVSS (Sequence ID 40) CDR-H1:GFSFSGFS (Sequence ID 41) CDR-H2:ISTSGNYM (Sequence ID 42) CDR-H3:ARGGGYNWNLFDY (Sequence ID 43)

[0158] Light chain variable region EIVLTQSPGTLSLSPGERATLSCRAS QSLNSNY LAWYQQKPGQAPRLLIY GAS SRATGIP DRFSGSGSGADFTLTISRLESEDFAVYYC QQYGNSPLT FGGGTKVEIK (Sequence ID 44) CDR-L1:QSLNSNY (Sequence ID 45) CDR-L2:GAS (Sequence ID 46) CDR-L3:QQYGNSPLT(Sequence ID 47)

[0159] In one embodiment of the present invention, further therapeutic agents include antibodies or antigen-binding fragments that bind to influenza group I HA proteins such as H1H11729P; or V of H1H11729P. H and V L An antibody or fragment containing H1H11729P; or a heavy chain immunoglobulin containing H1H11729P CDR-H1, CDR-H2, and CDR-H3 (e.g., SEQ ID NOs. 33-35) and a light chain immunoglobulin containing H1H11729P CDR-L1, CDR-L2, and CDR-L3 (e.g., SEQ ID NOs. 37-39). "H1H11729P" refers to any anti-group I HA antibody containing such a sequence.

[0160] H1H11729P Heavy chain variable region QVQLVQSGAEVKKSGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTP SY AQKFQDRVTITTDESTSTVYMELSSLRSEDTAVYYC ARQQPVYQYNMDV WGQGTTVTVSS (Sequence ID 32) CDR-H1:GGTFSSYA (Sequence ID 33) CDR-H2: IIPIFGTP (SEQ ID NO: 34) CDR-H3: ARQQPVYQYNMDV (SEQ ID NO: 35)

[0161] Light chain variable region DIQMTQSPSSLSASVGDRVTITCRAS QGIRNN LGWYQQKPLKAPKRLIY AAS SLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYC LQYNNYPWT FGQGTKVEIK (SEQ ID NO: 36) CDR-L1: QGIRNN (SEQ ID NO: 37) CDR-L2: AAS (SEQ ID NO: 38) CDR-L3: LQYNNYPWT (SEQ ID NO: 39)

[0162] In certain embodiments of the invention, the additional therapeutic agent is not amantadine, rimantadine, oseltamivir, zanamivir, aprotinin, leupeptin, cationic steroid antibacterial agents, influenza vaccine (e.g., inactivated, live, attenuated whole virus or subunit vaccine), and antibodies against influenza virus (e.g., anti-hemagglutinin antibodies).

[0163] The term "accompanied by" indicates that a component, an anti-TMPRSS2 antigen-binding protein, e.g., an antibody of the invention or an antigen-binding fragment thereof, and another agent, e.g., oseltamivir, can be formulated into a single composition, e.g., for co-delivery, or separately formulated into two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than when the other component is administered; e.g., each administration can be given non-concurrently (e.g., separately or sequentially) at intervals over a given period of time. Further, the separate components can be administered to the subject by the same or different routes (e.g., an anti-TMPRSS2 antibody or an antigen-binding fragment thereof).

[0164] Kit Furthermore, without limitation, kits are provided that include one or more additional components, including but not limited to additional therapeutic agents, considered herein, and one or more components including but not limited to an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N) considered herein. The antigen-binding protein and / or additional therapeutic agent can be formulated in a pharmaceutical composition, as a single composition, or separately in two or more compositions, e.g., with a pharmaceutically acceptable carrier.

[0165] In one embodiment of the invention, the kit includes an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N) of the invention, or a pharmaceutical composition thereof in one container (e.g., a sterile glass or plastic vial), and an additional therapeutic agent in a separate container (e.g., a sterile glass or plastic vial).

[0166] In another embodiment, the kit includes a combination of the invention, including an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment (e.g., H1H7017N or H4H7017N) of the invention, or a pharmaceutical composition thereof, in a single common container, optionally combined with one or more additional therapeutic agents formulated together in the pharmaceutical composition.

[0167] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device (e.g., an infusion device) for such administration. For example, the kit can include one or more subcutaneous injection needles or other infusion devices considered above, including an anti-TMPRSS2 antigen-binding protein, such as an antibody or antigen-binding fragment thereof (e.g., H1H7017N or H4H7017N) of the invention.

[0168] The kit may include a package insert containing information about the pharmaceutical composition and dosage form contained within the kit. Generally, such information helps patients and physicians in effectively and safely using the enclosed pharmaceutical composition and dosage form. For example, the following information relating to the combination of the present invention: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, symptoms and usage, contraindications, warnings, precautions, side effects, overdose, appropriate dose and administration, supply method, appropriate storage conditions, references, manufacturer / distributor information, and patent information may be provided in the package insert.

[0169] Diagnostic use of antibodies Anti-TMPRSS2 antigen-binding proteins, such as the antibody or antigen-binding fragment of the present invention (e.g., H1H7017N or H4H7017N), are used to detect and / or measure TMPRSS2 in a sample. An exemplary assay for TMPRSS2 may include, for example, contacting a sample with the anti-TMPRSS2 antigen-binding protein of the present invention, which is labeled with a detectable label or reporter molecule or used as a capture ligand for selective isolation of TMPRSS2 from the sample. The presence of an anti-TMPRSS2 antigen-binding protein complexed with TMPRSS2 indicates the presence of TMPRSS2 in the sample. Alternatively, an unlabeled anti-TMPRSS2 antibody can be used in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a radioisotope, e.g., 3 H, 14 C, 32 P, 35 S, or 125I; a fluorescent or chemiluminescent moiety, e.g., fluorescein isothiocyanate or rhodamine; or an enzyme, e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure TMPRSS2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Accordingly, the present invention includes a method for detecting the presence of a TMPRSS2 polypeptide in a sample, comprising contacting the sample with an anti-TMPRSS2 antigen-binding protein to detect the presence of the TMPRSS / anti-TMPRSS2 antigen-binding protein, the presence of which indicates the presence of TMPRSS2.

[0170] The present invention includes a cell-based ELISA method that uses an anti-TMPRSS2 antigen-binding protein, such as the antibody of the present invention and its antigen-binding fragment (e.g., H1H7017N), to detect the presence of TMPRSS2 on cells. In one embodiment of the present invention, the method is (i) A step of contacting cells immobilized on a solid surface (e.g., a microplate) and testing for the presence of TMPRSS2 using the anti-TMPRSS2 antigen-binding protein of the present invention; (ii) If applicable, a step of washing the mixture to remove unbound anti-TMPRSS2 antigen-binding proteins; (iii) A step of contacting an anti-TMPRSS2 antigen-binding protein with a labeled secondary antibody or its antigen-binding fragment that binds to the anti-TMPRSS2 antigen-binding protein; (iv) If applicable, a step of washing the complex to remove unbound antigen-binding proteins; and (v) A step of detecting the presence of a secondary antibody or label on a fragment, wherein the detection of the label indicates that the cells contain TMPRSS2. This includes, for example, TMPRSS2 in a sample + This includes cell-based ELISA methods for identifying cells.

[0171] The anti-TMPRSS2 antigen-binding protein of the present invention (e.g., H1H7017N or H4H7017N) is used in Western blotting or immunoprotein blotting to detect the presence of TMPRSS2 or fragments thereof in a sample. Such a procedure forms part of the present invention and includes, for example, the following steps: (1) Provide a membrane or other solid substrate containing a sample to be tested for the presence of TMPRSS2, and optionally transfer a protein from the sample to be tested for the presence of TMPRSS2 (e.g., from PAGE or SDS-PAGE electrophoresis separation of proteins in the sample) onto the membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or tank blotting); and contact the membrane or other solid substrate to be tested for the presence of TMPRSS2 or a fragment thereof with the anti-TMPRSS2 antigen-binding protein of the present invention.

[0172] Such membranes can take the form of nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membranes to which the protein to be tested for the presence of TMPRSS2 has been transferred (e.g., after electrophoretic separation in the gel) in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Before contacting the membrane with the anti-TMPRSS2 antigen-binding protein, the membrane may be blocked, for example, with skim milk powder, to bind to nonspecific protein-binding sites on the membrane.

[0173] (2) Wash the membrane once or more to remove unbound anti-TMPRSS2 antigen-binding protein and other unbound substances. (3) Detect the bound anti-TMPRSS2 antigen-binding protein.

[0174] Detection of bound antigen-binding proteins indicates the presence of the TMPRSS2 protein on the membrane or substrate and in the sample. Detection of bound antigen-binding proteins can be performed by conjugating the antigen-binding protein with a detectably labeled secondary antibody (anti-immunoglobulin antibody) and then detecting the presence of the secondary antibody label.

[0175] 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, for example, (1) A step of contacting a tissue to be tested for the presence of TMPRSS2 protein with the anti-TMPRSS2 antigen-binding protein of the present invention; and; (2) Steps for detecting antigen-binding proteins on or within tissues Includes.

[0176] If the antigen-binding protein itself is detectably labeled, it can be detected directly. Alternatively, a detectably labeled secondary antibody can be bound to the antigen-binding protein, and then the label can be detected. [Examples]

[0177] The following examples are provided to give a complete disclosure and explanation of the methods and compositions of the present invention for the manufacture and use of the present invention, and are not intended to limit the scope of what the inventors consider to be the invention. Although efforts have been made to ensure the accuracy of the numbers used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise specified, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or close to atmospheric pressure. [Examples]

[0178] Multiple replications in vitro The replication ability of influenza virus A / Puerto Rico / 08 / 1934 (H1N1)-GFP in Calu3, A549, MDCK and HepG2 cells was evaluated.

[0179] [Table 1]

[0180] Experimental procedure 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 96-well plates in DMEM:F12 medium containing 5% FBS. The next day, A / Puerto Rico / 08 / 1934 (H1N1) (B. Manicassamy et al., "Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus", Proc Natl Acad Sci U S A., 2010 Jun 22;107(25):11531-6), which has a GFP reporter gene in the NS segment, was prepared at MOIs (multiplicity of infection) of 0.1 and 0.01 in DMEM:F12 with low IgG BSA after three washes. The virus was incubated on the cells for 1 hour at 37 °C, then the virus was removed and the wells were washed three more times. The number of infected cells was quantified at 24, 48, 72 and 142 hours post-infection using a CTL-ImmunoSpot® S6 Universal Analyzer (Cellular Technology Limited, Cleveland, OH).

[0181] Summary of results and conclusions Calu-3 is an immortalized human airway epithelial cell line that has been shown to enable multiple replications 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, Vol. 81, pp. 12439-12449 (2007)). Furthermore, it has been shown that Calu-3 cells express both TMPRSS2 and TMPRSS4 at least at the mRNA level, but not TMPRSS11D(HAT) (Bottcher-Friebertshauser 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, Vol. 85, pp. 1554-1562 (2011)). To confirm that Calu-3 cells can support the proteolytic activation of influenza viruses with hemagglutinins containing monobasic cleavage sites, the proliferation of H1N1 GFP reporter virus in Calu-3 cells was analyzed, and replication over time was compared using A549 (human alveolar basal epithelium), MDCK (Madin Darby canine kidney), and HepG2 (human liver cancer) cells in the absence of trypsin. Cells were infected with a low MOI, and viral titers were determined by counting the fluorescence focus spot at indicated time points. Table 2 and Figure 1 show low levels of infection in A549, MDCK, and HepG2 cells, while Calu-3 cells showed significantly increased titers at all time points.Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at the mRNA level, and knockdown of TMPRSS2 reduced influenza virus titer by 100 to 1,000 times (Bottcher-Friebertshauser 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, Vol. 85, pp. 1554-1562 (2011)). The low levels of viral titer in A549, MDCK, and HepG2 cells in the absence of trypsin are likely due to the addition of cleaved virus (recovered from chicken embryo eggs or from MDCK cultures using trypsin), but the presence of another HA-activating protease may explain this.

[0182] [Table 2]

[0183] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 27733646. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID:26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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 . 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0184] The anti-TMPRSS2 antibody H1H7017N inhibits the spread of influenza in vitro. We evaluated the ability of various antibodies to reduce the titer of influenza virus A / Puerto Rico / 08 / 1934(H1N1) in Calu-3 cells.

[0185] [Table 3]

[0186] Experimental Procedure Calu-3 cells (ATCC HTB55) were diluted to 40,000 cells / well in 96-well plates of DMEM:F12 medium containing 5% FBS. The following day, a monoclonal antibody was diluted to 166.7 nM in DMEM:F12 containing low IgG BSA and added to the cells at 37°C and 5% CO2 for 3 hours. The mAb solution was removed, and the cells were infected with A / Puerto Rico / 08 / 1934(H1N1) at an MOI of 0.001. The virus was incubated on the cells at 37°C for 1 hour in 5% CO2, then the virus was removed, and the medium was replaced with DMEM:F12 containing 166.7 nM mAb. After 24 and 48 hours, the medium was replaced with fresh medium containing mAb, and after 72 hours, the cells were washed twice with PBS. Next, the cells were fixed with 4% paraformaldehyde in PBS, and the virus was detected using a 1:1000 dilution of anti-NP primary antibody. The cells were incubated for 1 hour, then washed, and a 1:2000 dilution of secondary antibody was added. The number of infected cells was quantified using a CTL-ImmunoSpot® S6 Universal Analyzer (Cellular Technology Limited, Cleveland, OH).

[0187] Summary of Results and Conclusions Calu-3 is an immortalized human airway epithelial cell line that has been shown to enable multiple replications of human influenza virus in the absence of exogenous trypsin (Zeng et al., "Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells," Journal of Virology, 2007 Nov; Vol. 81 (No. 22): pp. 12439-49). Furthermore, Calu-3 cells have been shown to express both TMPRSS2 and TMPRSS4 at least at the mRNA level, but not TMPRSS11D(HAT) (Bottcher-Friebertshauser 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, Vol. 85, pp. 1554-1562 (2011)). While Calu-3 cells support the proteolytic activation of influenza virus, inhibition of TMPRSS2 was tested herein using the TMPRSS2-specific monoclonal antibody, H1H7017N. After treating cells with 166.7 nM H1H7017N, the proliferation of A / Puerto Rico / 08 / 1934(H1N1) over 72 hours was analyzed. Viral titers were determined by counting fluorescent focus spots. Table 4 and Figure 2 show the decrease in titers after treatment with antibody H1H7017N.Calu-3 cells have been shown to express TMPRSS2 and TMPRSS4 at the mRNA level, and knockdown of TMPRSS2 reduced influenza virus titers by 100 to 1,000 times (Bottcher-Friebertshauser 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, Vol. 85, pp. 1554-1562 (2011)). The low levels of pre-existing viral titers in the absence of mAbs were likely due to the addition of cleaved virus (recovered from chicken embryo eggs or from MDCK cultures using trypsin), but the presence of another HA-activating protease may also explain the presence of the virus despite anti-TMPRSS2 mAb treatment.

[0188] [Table 4]

[0189] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 27733646. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID:25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID:26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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 . 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0190] FACS analysis using MDCK / Tet-on, MDCK / Tet-on / hTMPRSS2, and MDCK / Tet-on / MfTMPRSS2 cells. The ability of the anti-TMPRSS2 antibody, H1H7017N, to bind to MDCK cells expressing TMPRSS2 or MDCK cells that do not express TMPRSS2 was evaluated.

[0191] [Table 5]

[0192] Experimental Procedure To induce doxycycline-induced tetrocycline activity, we developed a cell line to express human and cynomolgus monkey TMPRSS2 (hTMPRSS2 and mfTMPRSS2) in MDCK (Madin Darby canine kidney) cells. MDCK cells were transduced to stably express a modified tetracycline-regulating transactivator protein (Clontech), and the resulting cell line was named the MDCK / Tet-on cell line. MDCK / Tet-on cell lines were transduced under the control of an inducible promoter using constructs containing hTMPRSS2 (NP_005647.3 with V160M) or mfTMPRSS2 (Ref seq XP_015302311.1 with S129L, N251S, I415V, R431Q, D492G), and the resulting cell lines were named 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, and 500 μg / mL G418, with or without 2 μg / mL puromycin.

[0193] For cell binding analysis by flow cytometry, cells were seeded in growth medium and incubated with 1 μg / mL doxycycline for 16 hours to induce TMPRSS2 expression. Cells were desorbed using Accutase and resuspended in 1% FBS in PBS. Antibodies were serially diluted from 500 nM to 25 pM, and 1 × 10⁶ of each antibody concentration were added. 6 Cells were incubated with the primary antibody at 4°C for 30 minutes. This included conditions where no antibody was added to the cells. After incubation with the primary antibody, the cells were stained with 1:1000 allophycocyanin-conjugated anti-human IgG secondary antibody at 4°C for 30 minutes. Cells were fixed using BD CytoFix® and analyzed using a CytoFLEX flow cytometer. Unstained and secondary antibody-only controls were also included for all cell lines. Geometric mean fluorescence was measured for live cells using FlowJo software, and the results were used to determine the EC of antibody-mediated cell binding. 50To obtain the values, we performed an analysis using nonlinear regression (4-parameter logistics) with Prism 7 software (GraphPad).

[0194] As shown in Figure 3, the anti-hTMPRSS2 antibody H1H7017N of the present invention has EC2 levels of 460 pM and 1.06 nM, respectively. 50 H1H7017N bound to MDCK / Tet-on / hTMPRSS2 and MDCK / Tet-on / mfTMPRSS2 at a value. H1H7017N did not show significant binding to MDCK / Tet-on cells. The unrelated isotype control antibody, control mAb1, did not show binding to any of the cell lines tested. [Examples]

[0195] Biacore binding kinetics of anti-TMPRSS2 monoclonal antibodies to different TMPRSS2 reagents, measured at 25°C and 37°C. Equilibrium dissociation constants (K) of various TMPRSS2 reagents that bind to purified anti-TMPRSS2 monoclonal antibodies DThe following parameters were measured using a real-time surface plasmon resonance-based Biacore 4000 biosensor. All coupling 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) electrophoresis buffer (25°C and 37°C). First, the Biacore CM5 sensor chip surface was derivatized by amine coupling with a rabbit anti-mouse Fc-specific polyclonal antibody (GE Healthcare catalog no. BR100838) to capture an anti-TMPRSS2 monoclonal antibody. Coupling studies were performed on the human TMPRSS2 extracellular domain (hTMPRSS2.mmh) expressed with a C-terminal myc-myc-hexahistidine tag, and the monkey TMPRSS2 extracellular domain (mfTMPRSS2.mmh) expressed with a C-terminal myc-myc-hexahistidine tag. First, HMM-hTMPRSS2 and HMM-mfTMPRSS2 at different concentrations (100 nM-6.25 nM; 4-fold serial dilution) were prepared in HBS-ET electrophoresis buffer and injected onto the surface of anti-mouse Fc-captured anti-TMPRSS2 monoclonal antibody at a flow rate of 30 μL / min for 2.5 minutes. Meanwhile, the dissociation of the TMPRSS2 reagent conjugated to the monoclonal antibody was monitored in HBS-ET electrophoresis buffer for 7 minutes. The binding rate (ka) and dissociation rate (kd) were determined by fitting a real-time binding sensorgram to a 1:1 binding model with mass transport limitations using Scrubber 2.0c curve fitting software. The binding-dissociation equilibrium constant (K) was determined. D The ) and dissociation half-life (t1 / 2) were calculated from the kinetic velocity as follows:

number

[0196] Tables 6-9 show the binding kinetics parameters of HMM-hTMPRSS2 or HMM-mfTMPRSS2 to different anti-TMPRSS2 monoclonal antibodies of the present invention at 25°C and 37°C.

[0197] At 25°C, the anti-TMPRSS2 monoclonal antibody showed a K2 level of 2.81 nM, as shown in Table 6. D The antibody bound to HMM-hTMPRSS2 at a value of 9.31 nM. At 37°C, the anti-HMM-hTMPRSS2 monoclonal antibody was found to have a K value of 9.31 nM, as shown in Table 7. D It was bound to HMM-hTMPRSS2 by value.

[0198] At 25°C, the anti-TMPRSS2 monoclonal antibody was 56.0 nM K, as shown in Table 8. D The antibody bound to HMM-mfTMPRSS2 at a value of 140 nM. At 37°C, the anti-TMPRSS2 monoclonal antibody was found to be 140 nM, as shown in Table 9. D The HMM-mfTMPRSS2 was bound to the specified value.

[0199] TMPRSS2 protein hTMPRSS2 knob_mmh(W106-R255).mmh: Amino acids 1-150: Amino acids 106-255 of human TMPRSS2 (acceptance number NP_005647.3 with V160M) Amino Acids: 151-178: myc-myc-hexahistidine tag [ka] (Array No. 20; the myc tag is underlined, and the His6 tag is double-underlined.) mfTMPRSS2 knob_mmh(W106-R255).mmh: Amino acids 1-150: Amino acids 106-255 of monkey TMPRSS2 (Accession number XP_005548700.1, S129L, N251S) Amino acids 151-178: myc-myc-hexahistidine tag [ka] (Sequence number 21; the myc tag is underlined, and the His6 tag is double-underlined.)

[0200] result

[0201] [Table 6]

[0202] [Table 7]

[0203] [Table 8]

[0204] [Table 9] [Examples]

[0205] In vitro influenza spread of influenza H1, H3, and FluB strains In this example, we determined the ability of various types of influenza to spread across Calu-3 cell in vitro cultures and the effect of anti-TMPRSS2 antibodies on this spread.

[0206] [Table 10]

[0207] Experimental Procedure Calu-3 cells were seeded at 40,000 cells / well in 96-well plates in DMEM:F12 medium containing 5% FBS. The following day, influenza virus strains were diluted to a predetermined MOI (see Table 11), and antibodies were diluted to 100 μg / mL. In these experiments, anti-HA and anti-TMPRSS2 antibodies had different mechanisms of action, and therefore, the experimental procedures for appropriately testing these antibodies were different. Anti-HA antibodies were pre-incubated with individual influenza virus strains in separate plates at 37°C for 1 hour. After the pre-incubation period, the antibody / virus mixture was added to Calu-3 cells for 1 hour. Anti-TMPRSS2 antibodies were pre-incubated with uninfected Calu-3 cells at 37°C for 3 hours. After the pre-incubation period, the virus was added to Calu-3 cells pre-incubated with anti-TMPRSS2 antibody for 1 hour. After 1 hour of infection, cells were washed three times with PBS, and fresh antibody was added to each well along with fresh medium. Additional antibody was added at 24 and 48 hours post-infection. At 72 hours post-infection, cells were stained with anti-NP and quantified using a CTL-ImmunoSpot® S6 Universal Analyzer (Cellular Technology Limited, Cleveland, OH).

[0208] [Table 11]

[0209] [Table 12]

[0210] Summary of Results and Conclusions Calu-3 is an immortalized human airway epithelial cell line that has been shown to enable multiple replications of human influenza virus in the absence of exogenous trypsin (Zeng et al., Journal of Virology, Vol. 81: pp. 12439-12449 (2007)). Furthermore, Calu-3 cells have been shown to express TMPRSS2, which is essential for these experiments as an anti-TMPRSS2 antibody has been tested (Bottcher-Friebertshauser et al., Journal of Virology, Vol. 85: pp. 1554-1562 (2011)). These experiments investigated whether the anti-TMPRSS2 antibody H1H7017N could prevent the spread of different influenza strains. In addition, corresponding anti-HA antibodies against different strains were used as positive controls. As expected, initial infection occurred in the presence of anti-TMPRSS2 antibody, but H1H7017N successfully prevented the spread of infection with 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). Since the number of infected cells in the control wells and treated wells was the same, it was concluded that anti-TMPRSS2 antibody could not prevent the spread of any influenza B strain. In contrast, pre-incubation with anti-HA antibody with the virus prevented initial infection. This can also be observed by comparing the number of infected cells. The count of infected cells was performed using a CTL device and reported in the table below.

[0211] [Table 13]

[0212] [Table 14]

[0213] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017).PMID: 28636671. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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. 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0214] Efficacy of H1H7017N monotherapy in TMPRS22 humanized mice We evaluated the ability of anti-TMPRSS2 antibodies to protect mice engineered to express the human TMPRSS2 protein from H1N1 influenza virus infection.

[0215] [Table 15]

[0216] [Table 16]

[0217] Experimental Procedure These experiments were performed on 5-8 week old male and female mice engineered to express the human TMPRSS2 protein. Mice were loaded with 150 plaque-forming units (PFUs) of H1N1. Mice were sedated by intraperitoneal infusion of 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml), followed by intranasal infection with 20 μL of the virus. Antibodies were delivered subcutaneously (SC) one day before infection or intravenously (IV) on various days after infection (PI). Antibody administration schedules differed between experiments (Table 15). Body weight was measured daily until day 14 of PI, and mice were sacrificed when their starting body weight decreased by 20%. Results are reported as survival rates.

[0218] [Table 17]

[0219] [Table 18]

[0220] Summary of Results and Conclusions Mice engineered to express the human TMPRSS2 protein have been shown to be susceptible to lethal doses of influenza. The aim of these experiments was to demonstrate that H1H7017N can protect mice engineered to express the human TMPRSS2 protein against influenza A group 1. This antibody was tested in prophylactic and therapeutic models. Treatment with H1H7017N resulted in higher survival rates than mice treated with the isotype control (H1H1238N) in both experiments (Figures 4 and 5). In the prophylactic experiment, the survival rate of mice treated with H1H1238N was 0%, the survival rate of mice treated on day -1 of the PI was 85.7%, and the survival rate of mice treated with H1H7017N on day 0 of the PI was 100%. In the therapeutic model, the H1H1238N-treated group had a survival rate of 25%, while the group treated with H1H7017N on days 0–3 of the PI had a survival rate of 100%. The data is summarized in Table 16. H1H7017N shows efficacy in mice engineered to express the human TMPRSS2 protein.

[0221] [Table 19]

[0222] [Table 20]

[0223] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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. 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0224] Activity of anti-TMPRSS2 mAb, H1H7017N in a TMPRSS2 humanized mouse model We evaluated the ability of anti-TMPRSS2 antibodies to protect mice engineered to express the human TMPRSS2 protein from H3N2 influenza virus infection.

[0225] [Table 21]

[0226] [Table 22]

[0227] Experimental Procedure Eleven-week-old male and female mice engineered to express the human TMPRSS2 protein were loaded with 20,000 plaque-forming units (PFUs) of H3N2. Mice were sedated by intraperitoneal infusion of 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml), followed by intranasal infection with 20 μL of the virus. Antibodies were intravenously administered to the mice on day 1 or 2 post-infection (PI). Mice were weighed and observed daily until day 14 post-infection (PI). Mice were sacrificed when their weight decreased by 25% from their starting weight.

[0228] Summary of Results and Conclusions The range of effectiveness is an important quality to consider when evaluating influenza treatment. The anti-TMPRSS2 antibody H1H7017N has already been demonstrated to be effective against influenza A group 1. The objective of this experiment was to demonstrate that H1H7017N could protect mice engineered to express the human TMPRSS2 protein against influenza A group 2. Mice engineered to express the human TMPRSS2 protein were infected with a lethal dose of H3N2 and treated on day 1 or day 2 of the PI (Patient Infection). Both treatment groups had higher survival rates than the infected controls. The survival rate of mice treated on day 1 of the PI was 100%, higher than the group treated on day 2 of the PI, which had a 50% survival rate, while the survival rate of untreated mice was 0%. All mice died between days 5 and 6 of the PI. A survival rate graph is shown in Figure 6, and the survival rate percentages are summarized in Table 19. These results demonstrate that H1H7017 improves outcomes in the H3N2 lethal model.

[0229] [Table 23]

[0230] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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. 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0231] Infection of mice (vs. wild) that were generated to express the human TMPRSS2 protein. We evaluated the survival of mice infected with the H1N1 influenza virus and engineered to express the human TMPRSS2 protein, and compared it to the survival of wild-type (WT) mice.

[0232] [Table 24]

[0233] Experimental Procedure The experiments were performed on 7.5–8 week old male and female mice engineered to express either human TMPRSS2 protein or wild-type littermates. Mice were loaded with 150, 750, or 1,500 plaque-forming units (PFUs) of A / Puerto Rico / 08 / 1934(H1N1). Mice were sedated by intraperitoneal infusion of 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml), followed by intranasal infection with 20 μL of the virus. Body weight was measured daily until day 14 of the PI, and mice were sacrificed when their starting body weight decreased by 20%. Results are reported as survival rates (Figure 7).

[0234] Summary of Results and Conclusions To test the therapeutic efficacy of anti-TMPRSS2 antibodies in an in vivo model of influenza, mice were engineered to express the human TMPRSS2 protein. In this experiment, the survival rates of mice engineered to express the human TMPRSS2 protein were compared to those of wild-type mice infected with 150, 750, or 1,500 PFUs of the historical H1N1 strain. The survival rates of both the human TMPRSS2-engineered mice and wild-type mice were 0% in all three infection groups. All mice died between day 5 and day 8 of the PI (infection phase), with mice receiving high viral doses dying earlier than those receiving low viral doses. The survival pattern of the human TMPRSS2-engineered mice was similar to that of wild-type mice. This indicates that mice engineered to express the human TMPRSS2 protein can be used as an in vivo model of influenza to evaluate the efficacy of TMPRSS2-specific antibodies. See Table 21.

[0235] [Table 25]

[0236] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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. 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0237] Therapeutic effects of the combination of H1H14611N2 and H1H7017N in mice infected with H3N2. We evaluated the ability of a combination of anti-TMPRSS2 antibodies and anti-influenza antibodies to protect mice engineered to express the human TMPRSS2 protein from H3N2 influenza virus infection.

[0238] [Table 26]

[0239] [Table 27]

[0240] Experimental Procedure Eight-week-old male and female mice engineered to express the human TMPRSS2 protein were loaded with 20,000 plaque-forming units (PFUs) of A / Aichi / 2 / 68(HA,NA)×A / PR / 8 / 34, resorted X-31(H3N2). Mice were sedated by intraperitoneal infusion of 200 μL of ketamine:xylazine (12 mg / ml:0.5 mg / ml), and then intranasal infection with 20 μL of the virus. Four days post-infection (PI), mice were intravenously injected with antibodies. Body weight was measured daily until 14 days post-PI, and mice were sacrificed when their starting body weight decreased by 25%. Results are reported as survival rates.

[0241] Summary of Results and Conclusions Individually, the TMPRSS2 antibody, H1H7017N, and the broad influenza A group 2 antibody, H1H14611N2, have been shown to have therapeutic efficacy against lethal mice loaded with historical strains of H3N2. Furthermore, combining H1H7017N with the broad influenza A group 1 antibody, H1H11729P, has been shown to significantly increase the survival of mice infected with lethal H1N1 loading after treatment with less total antibody than either antibody alone. The objective of this experiment was to evaluate the synergistic effect of using H1H7017N and H1H14611N2 together. As shown in Figure 8, three of the four mice treated with the hIgG1 isotype control antibody on day 4 of the PI died by day 7 of the PI. When administered 10 mg / kg of H1H14611N2, 3 out of 5 animals survived, and when administered 10 mg / kg of H1H7017N, 4 out of 5 animals survived. When administered with a combination of 5 mg / kg of each antibody (H1H14611N2 and H1H7017N), the survival rate was 40%. 100% of mice treated with a combination of 2.5 mg / kg of each antibody (H1H14611N2 and H1H7017N) survived the load. Survival of mice infected with a lethal H3N2 load was increased by the low-concentration combination of H1H7017N and H1H14611N2 compared to high concentrations of the combined antibody or either antibody alone. Survival rates are summarized in Table 24.

[0242]

Table 28

[0243] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044. 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 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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. 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. [Examples]

[0244] Efficacy of treatment with a combination of H1H11729P and H1H7017N in mice infected with H1N1 We evaluated the ability of a combination of anti-TMPRSS2 antibodies and anti-influenza antibodies to protect mice engineered to express the human TMPRSS2 protein from infection by the H1N1 influenza virus.

[0245] [Table 29]

[0246] [Table 30]

[0247] Experimental Procedure Five-week-old male and female mice engineered to express the human TMPRSS2 protein were loaded with 1,500 plaque-forming units (PFUs) of H1N1. The 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. Antibodies were intravenously injected into the mice on day 3 post-infection (PI). Body weight was measured daily until day 14 of PI, and mice were sacrificed when they had lost 25% of their starting body weight.

[0248] Summary of Results and Conclusions Individually, the TMPRSS2 antibody, H1H7017N, and the broad influenza A group 1 antibody, H1H11729P, have been shown to have therapeutic efficacy against lethal mice loaded with historical strains of H1N1. However, the objective of this experiment was to evaluate the synergistic effect of combined antibodies. All mice treated with the hIgG1 isotype control antibody on day 3 of PI died by day 6 of PI. When animals received 5 mg / kg of H1H11729P or H1H7017N, 40% and 0% of the animals survived the infection, respectively. However, the combination of H1H11729P and H1H7017N, at 2.5 mg / kg of each antibody, resulted in a 60% survival rate. 80% of mice treated with the combination of 1 mg / kg of H1H7017N and 2 mg / kg of H1H11729P (total 3 mg / kg) survived the loading. The survival of mice infected with a lethal H1N1 load was significantly increased after treatment with a lower total antibody dose via the combination of H1H7017N and H1H11729P compared to treatment with either antibody alone (see Figure 9 and Table 27).

[0249] [Table 31]

[0250] References 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. 2. L. M. Reinke et al., Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 12, e0179177 (2017). PMID: 28636671. 3. Y. Zhou et al., Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Research. 116, 76-84 (2015). PMID: 25666761. 4. P. Zmora, A.-S. Moldenhauer, H. Hofmann-Winkler, S. Pohlmann, TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells. PLoS ONE. 10, e0138380 (2015). PMID: 26379044. 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. 6. E. Bottcher-Friebertshauser, 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. 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. 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. 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. 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. 11. H. Zeng et al., Highly pathogenic avian influenza H5N1 viruses elicit an attenuated type i interferon response in polarized human bronchial epithelial cells. Journal of Virology. 81, 12439-12449 (2007). PMID: 17855549. *****************

[0251] All references cited herein are incorporated by reference to the same extent as each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent would be specifically and individually indicated as being incorporated by reference. The applicant intends that such statements of incorporation by reference relate to each and all of the individual publications, database entries (e.g., Genbank sequence or GeneID entries), patent applications, or patents identified, even if such citations are not immediately adjacent to a dedicated statement of incorporation by reference. Including a dedicated statement of incorporation by reference, if any, in the specification does not weaken this general statement of incorporation by reference in any way. The citations of references herein are not intended to acknowledge that the references are relevant prior art, nor do they constitute an acknowledgment of the content or date of these publications or documents.

Claims

1. (a) Amino acid sequence: CDR-H1 containing GTFFSSYG (SEQ ID NO: 6) (b) Amino acid sequence: CDR-H2 containing IWNDGSYV (SEQ ID NO: 8) (c) Amino acid sequence: Heavy chain immunoglobulin variable region containing CDR-H3 containing AREGEWVLYYFDY (SEQ ID NO: 10); and (a) Amino acid sequence: CDR-L1 containing QSISSW (SEQ ID NO: 12), (b) Amino acid sequence: CDR-L2 containing KAS (SEQ ID NO: 14) (c) Amino acid sequence: CDR-L3 containing QQYNSYSYT (SEQ ID NO: 16) Light chain immunoglobulin variable region including A human antigen-binding protein that specifically binds to human TMPRSS2, including [specific component].

2. The antigen-binding protein according to claim 1, wherein the antibody or an antigen-binding fragment thereof.

3. (a) Heavy chain immunoglobulin containing an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or 19, or a variable region of heavy chain immunoglobulin containing an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2; and (b) The antigen-binding protein according to claim 1 or 2, comprising a light chain immunoglobulin having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 18, or a light chain immunoglobulin variable region having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

4. (a) an immunoglobulin heavy chain containing an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 17 or 19, or an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2; and (b) The antigen-binding protein according to any one of claims 1 to 3, comprising an immunoglobulin light chain having at least 95% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 18, or an immunoglobulin light chain variable region having at least 95% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

5. (a) The immunoglobulin heavy chain variable region containing the amino acid sequence shown in Sequence ID No. 2; and (b) The immunoglobulin light chain variable region containing the amino acid sequence shown in SEQ ID NO: 4 An antigen-binding protein according to any one of claims 1 to 4, comprising:

6. (a) Heavy chain immunoglobulins containing the amino acid sequence shown in Sequence ID No. 17; and (b) Light chain immunoglobulin containing the amino acid sequence shown in Sequence ID No. 18 An antigen-binding protein according to any one of claims 1 to 4, comprising:

7. (a) Heavy chain immunoglobulins containing the amino acid sequence shown in SEQ ID NO: 19; and (b) Light chain immunoglobulin containing the amino acid sequence shown in Sequence ID No. 18 An antigen-binding protein according to any one of claims 1 to 4, comprising:

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

9. The following characteristics: - Inhibit the proliferation of influenza virus in TMPRSS2-expressing cells; - Binds to the surface of TMPRSS-expressing cells; - Does not significantly bind to MDCK / Tet-on cells that do not express TMPRSS2; - Limit the spread of influenza virus infection in cells in vitro; and / or - Protect mice engineered to express the human TMPRSS2 protein from death caused by influenza virus infection. An antigen-binding protein according to any one of claims 1 to 8, comprising one or more of the above.

10. A complex comprising an antigen-binding protein according to any one of claims 1 to 9, bound to the TMPRSS2 polypeptide.

11. A method for producing an antigen-binding protein or its immunoglobulin chain according to any one of claims 1 to 9, (a) the step of introducing one or more polynucleotides encoding the immunoglobulin chain of the antigen-binding protein into a host cell; and (b) A step of culturing host cells under conditions suitable for the expression of the polynucleotide; The method comprising the above.

12. (c) The step of isolating the antigen-binding protein or immunoglobulin chain from the host cells and / or the culture medium in which the host cells are grown. The method according to claim 11, further comprising:

13. The method according to claim 11, wherein the host cells are Chinese hamster ovary cells.

14. (a) Amino acid sequence: CDR-H1 containing GTFFSSYG (SEQ ID NO: 6) (b) Amino acid sequence: CDR-H2 containing IWNDGSYV (SEQ ID NO: 8) (c) Amino acid sequence: Heavy chain variable region including CDR-H3 containing AREGEWVLYYFDY (SEQ ID NO: 10); and (a) Amino acid sequence: CDR-L1 containing QSISSW (SEQ ID NO: 12), (b) Amino acid sequence: CDR-L2 containing KAS (SEQ ID NO: 14) (c) Amino acid sequence: CDR-L3 containing QQYNSYSYT (SEQ ID NO: 16) Light chain variable region including A polynucleotide that codes for [something].

15. (a) an immunoglobulin heavy chain containing the amino acid sequence shown in SEQ ID NO: 17 or 19; or (b) Immunoglobulin light chain containing the amino acid sequence shown in Sequence ID No. 18 The polynucleotide according to claim 14, which codes for a polynucleotide.

16. (a) V of an immunoglobulin chain containing the amino acid sequence shown in Sequence ID No. 2 H domain; also teeth (b) V of an immunoglobulin chain containing the amino acid sequence shown in SEQ ID NO: 4 L domain The polynucleotide according to claim 14, which codes for a polynucleotide.

17. A vector comprising a polynucleotide according to any one of claims 14 to 16.

18. A host cell comprising an antigen-binding protein or immunoglobulin chain or polynucleotide or vector according to any one of claims 1 to 9 and 14 to 17.

19. A composition or kit comprising, together with a further therapeutic agent, an antigen-binding protein according to any one of claims 1 to 9 or a polynucleotide according to any one of claims 14 to 16.

20. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 9 or a polynucleotide according to any one of claims 14 to 16, and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, comprising a further therapeutic agent.

22. A composition or kit according to any one of claims 19 to 21, accompanied by a further therapeutic agent which is an antiviral drug or vaccine.

23. Further therapeutic agents include members selected from the group consisting of redipacivir, sofosbuvir, a combination of redipacivir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-alpha 2b, interferon-alpha 2a, anticancer agents, and antibodies or antigen-binding fragments that specifically bind to influenza HA; and / or H1H14611N2;H1H14612N2;H1H11723P;H1H11729P;H1H11820N;H1H11829N;H1H11829N2;H2aM11829N;H2M11 830N; H1H11830N2; H1H11903N; H1H14571N; H2a14571N; H1H11704P; H1H117 11P; H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H1173 1P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H 11747P2; H1H11748P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H 17956B; H1H17957B; H1H17958B; H1H17959B; H1H17960B; H1H17961B; H1H17 962B; H1H17963B; H1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H1796 8B; H1H17969B; H1H17970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B ;H1H17975B;H1H17976B;H1H17977B;H1H17978B;H1H17979B;H1H17980B;H 1H17981B; H1H17982B; H1H17983B; H1H17984B; H1H17985B; H1H17986B; H1H 17987B; H1H17988B; H1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17 993B; H1H17994B; H1H17995B; H1H17996B; H1H17997B; H1H17998B; H1H1799 9B; H1H18000B; H1H18001B; H1H18002B; H1H18003B; H1H18004B; H1H18005B;H1H18006B;H1H18007B;H1H18008B;H1H18009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H18015B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18041B;H1H18042B;H1H18043B;H1H18044B;H1H18045B;H1H18046B;H1H18047B;H1H18048B;H1H18049B;H1H18051B;H1H18052B;H1H18053B;H1H18054B;H1H18055B;H1H18056B;H1H18057B;H1H18058B;H1H18059B;H1H18060B;H1H18061B;H1H18062B;H1H18063B;H1H18064B;H1H18065B;H1H18066B;H1H18067B;H1H18068B;H1H18069B;H1H18070B;H1H18071B;H1H18072B;H1H18073B;H1H18074B;H1H18075B;H1H18076B;H1H18077B;H1H18078B;H1H18079B;H1H18080B;H1H18081B;H1H18082B;H1H18083B;H1H18084B;H1H18085B;H1H18086B;H1H18087B;H1H18088B;H1H18089B;H1H18090B;H1H18091B;H1H18092B;H1H18093B;H1H18094B;H1H18095B;H1H18096B;H1H18097B;H1H18098B;H1H18099B;H1H18100B;H1H18101B;H1H18102B;H1H18103B;H1H18104B;H1H18105B;H1H18107B;H1H18108B;H1H18109B;H1H18110B;H1H18111B;H1H18112B;H1H18113B;H1H18114B;H1H18115B;H1H18116B;H1H18117B;H1H18118B;H1H18119B;H1H18120B;H1H18121B;H1H18122B;H1H18123B;H1H18124B;H1H18125B;H1H18126B;H1H18127B;H1H18128B;H1H18129B;H1H18130B;H1H18131B;H1H18132B;H1H18133B;H1H18134B;H1H18135B;H1H18136B;H1H18137B;H1H18138B;H1H18139B;H1H18140B;H1H18141B;H1H18142B;H1H18143B;H1H18144B;H1H18145B;H1H18146B;H1H18147B;H1H18148B;H1H18149B;H1H18150B;H1H18151B;H1H18152B;H1H18153B;H1H18154B;H1H18155B;H1H18156B;H1H18157B;H1H18158B;H1H18159B;H1H18160B;H1H18161B;H1H18162B;H1H18163B;H1H18164B;H1H18165B;H1H18166B;H1H18167B;H1H18168B;H1H18169B;H1H18170B;H1H18171B;H1H18172B;H1H18173B;H1H18174B;H1H18175B;H1H18176B;H1H18177B;H1H18178B;H1H18179B;H1H18180B;H1H18181B;H1H18182B;H1H18183B;H1H18184B;H1H18185B;H1H18186B;H1H18187B;H1H18188B;H1H18189B;H1H18190B;H1H18191B;H1H18192B;H1H18193B;H1H18194B;H1H18195B;H1H18196B;H1H18197B;H1H18198B;H1H18199B;H1H18200B;H1H18201B;H1H18202B;H1H18203B;H1H18204B;H1H18205B;H1H18206B;H1H18207B;H1H18208B;H1H18209B;H1H18210B;H1H18211B;H 1H18212B;H1H18213B;H1H18214B;H1 H18216B;H1H18217B;H1H18218B;H1H 18219B;H1H18220B;H1H18221B;H1H18 222B;H1H18223B;H1H18224B;H1H182255B;H1H18226B;H1H1827B;H1H1822 8B;H1H18229B;H1H1823B;H1H18231B;H1H18232B;H1H182334B;H1H18234 H1H18235B;H1H18236B;H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1 H18241B;H1H18242B;H1H18243B;H1H 18244B;H1H18245B;H1H18246B;H1H18 247B;H1H18248B;H1H18249B;H1H1825B;H1H18251B;H1H18252B 3B;H1H18254B;H1H18255B;H1H18256 B;H1H18257B;H1H18258B;H1H18259B; H1H18261B;H1H18262B;H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1 H18267B;H1H18268B;H1H18269B;H1H18270B;H1H18271B;H1H18 274B;H1H18275B;H1H18276B;H1H18277B;H1H18278B;H1H1828 0B;H1H18281B;H1H18282B;H1H18283 B;H1H18284B;H1H18285B;H1H18286B; H1H18287B;H1H18288B;H1H18289B;H1H18290B;H1H18291B;H1 H18293B;H1H18294B;H1H18295B;H1H18297B;H1H18298B;H1H18 300B;H1H18301B;H1H18302B;H1H18303B;H1H18304B;H1H18305B;H1H1830 6B;H1H18307B;H1H18308B;H1H18309B;H1H18310B;H1H18312B;H1H18313B; H1H18314B; H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H 1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1 The composition or kit according to claim 21 or 22, comprising an antibody selected from the group consisting of H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B, or an antigen-binding fragment thereof.

24. A container or injection device comprising an antigen-binding protein, polynucleotide, or composition according to any one of claims 1 to 9, 14 to 16, and 19 to 23.

25. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 9 for treating or preventing cancer or infection caused by influenza virus, coronavirus, SARS-Covirus, MERS-Covirus, parainfluenza virus, human metapneumovirus, or hepatitis C virus (HCV), characterized in that a therapeutically effective amount of the pharmaceutical composition is administered to a subject in need thereof.

26. The pharmaceutical composition according to claim 25, for treating or preventing cancer which is prostate cancer, 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.

27. The pharmaceutical composition according to claim 25 or 26, characterized in that the subject is administered one or more further therapeutic agents.

28. The pharmaceutical composition according to claim 27, characterized in that the subject is administered one or more further therapeutic agents, which are antiviral drugs or vaccines.

29. Members selected from the group consisting of ledipasvir, sofosbuvir, a combination of ledipasvir and sofosbuvir, oseltamivir, zanamivir, ribavirin, and interferon-alpha 2b, interferon-alpha 2a, and antibodies or antigen-binding fragments thereof that specifically bind to influenza HA; and / or H1H14611N2; H1H14612N2; H1H11723P; H1H11729P; H1H11820N; H1H11829N; H1H11829N2; H2aM11829N; H2M11830N; H1H11830N 2;H1H11903N;H1H14571N;H2a14571N;H1H11704P;H1H11711P;H1H11714P; H1H11717P; H1H11724P; H1H11727P; H1H11730P2; H1H11731P2; H1H11734P2; H1H11736P2; H1H11742P2; H1H11744P2; H1H11745P2; H1H11747P2; H1H1174 8P2; H1H17952B; H1H17953B; H1H17954B; H1H17955B; H1H17956B; H1H17957B ;H1H17958B;H1H17959B;H1H17960B;H1H17961B;H1H17962B;H1H17963B;H 1H17964B; H1H17965B; H1H17966B; H1H17967B; H1H17968B; H1H17969B; H1H1 7970B; H1H17971B; H1H17972B; H1H17973B; H1H17974B; H1H17975B; H1H179 76B; H1H17977B; H1H17978B; H1H17979B; H1H17980B; H1H17981B; H1H17982B ;H1H17983B;H1H17984B;H1H17985B;H1H17986B;H1H17987B;H1H17988B;H 1H17989B; H1H17990B; H1H17991B; H1H17992B; H1H17993B; H1H17994B; H1H1 7995B; H1H17996B; H1H17997B; H1H17998B; H1H17999B; H1H18000B; H1H1800 1B; H1H18002B; H1H18003B; H1H18004B; H1H18005B; H1H18006B; H1H18007B;H1H18008B;H1H18009B;H1H18010B;H1H18011B;H1H18012B;H1H18013B;H1H18014B;H1H18015B;H1H18016B;H1H18017B;H1H18018B;H1H18019B;H1H18020B;H1H18021B;H1H18022B;H1H18023B;H1H18024B;H1H18025B;H1H18026B;H1H18027B;H1H18028B;H1H18029B;H1H18030B;H1H18031B;H1H18032B;H1H18033B;H1H18034B;H1H18035B;H1H18037B;H1H18038B;H1H18039B;H1H18040B;H1H18041B;H1H18042B;H1H18043B;H1H18044B;H1H18045B;H1H18046B;H1H18047B;H1H18048B;H1H18049B;H1H18051B;H1H18052B;H1H18053B;H1H18054B;H1H18055B;H1H18056B;H1H18057B;H1H18058B;H1H18059B;H1H18060B;H1H18061B;H1H18062B;H1H18063B;H1H18064B;H1H18065B;H1H18066B;H1H18067B;H1H18068B;H1H18069B;H1H18070B;H1H18071B;H1H18072B;H1H18073B;H1H18074B;H1H18075B;H1H18076B;H1H18077B;H1H18078B;H1H18079B;H1H18080B;H1H18081B;H1H18082B;H1H18083B;H1H18084B;H1H18085B;H1H18086B;H1H18087B;H1H18088B;H1H18089B;H1H18090B;H1H18091B;H1H18092B;H1H18093B;H1H18094B;H1H18095B;H1H18096B;H1H18097B;H1H18098B;H1H18099B;H1H18100B;H1H18101B;H1H18102B;H1H18103B;H1H18104B;H1H18105B;H1H18107B;H1H18108B;H1H18109B;H1H18110B;H1H18111B;H1H18112B;H1H18113B;H1H18114B;H1H18115B;H1H18116B;H1H18117B;H1H18118B;H1H18119B;H1H18120B;H1H18121B;H1H18122B;H1H18123B;H1H18124B;H1H18125B;H1H18126B;H1H18127B;H1H18128B;H1H18129B;H1H18130B;H1H18131B;H1H18132B;H1H18133B;H1H18134B;H1H18135B;H1H18136B;H1H18137B;H1H18138B;H1H18139B;H1H18140B;H1H18141B;H1H18142B;H1H18143B;H1H18144B;H1H18145B;H1H18146B;H1H18147B;H1H18148B;H1H18149B;H1H18150B;H1H18151B;H1H18152B;H1H18153B;H1H18154B;H1H18155B;H1H18156B;H1H18157B;H1H18158B;H1H18159B;H1H18160B;H1H18161B;H1H18162B;H1H18163B;H1H18164B;H1H18165B;H1H18166B;H1H18167B;H1H18168B;H1H18169B;H1H18170B;H1H18171B;H1H18172B;H1H18173B;H1H18174B;H1H18175B;H1H18176B;H1H18177B;H1H18178B;H1H18179B;H1H18180B;H1H18181B;H1H18182B;H1H18183B;H1H18184B;H1H18185B;H1H18186B;H1H18187B;H1H18188B;H1H18189B;H1H18190B;H1H18191B;H1H18192B;H1H18193B;H1H18194B;H1H18195B;H1H18196B;H1H18197B;H1H18198B;H1H18199B;H1H18200B;H1H18201B;H1H18202B;H1H18203B;H1H18204B;H1H18205B;H1H18206B;H1H18207B;H1H18208B;H1H18209B;H1H18210B;H1H18211B;H1H18212B;H1H18213B;H 1H18214B;H1H18216B;H1H18217B;H1 H18218B;H1H18219B;H1H18220B;H1H18221B;H1H1822B;H1H18 224B;H1H182255B;H1H182266B;H1H18227B;H1H1822B;H1H1823 0B;H1H18231B;H1H18232B;H1H18233B;H1H18234B;H1H18236B; H1H18237B;H1H18238B;H1H18239B;H1H18240B;H1H18242B;H1 H18243B;H1H18244B;H1H18245B;H1H 18246B;H1H18247B;H1H18248B;H1H18 249B;H1H1825B;H1H18251B;H1H18252B;H1H18253B;H1H18254B;H1H1825 5B;H1H18256B;H1H18257B;H1H18258B;H1H18261B;H1H18262B; H1H18263B;H1H18264B;H1H18265B;H1H18266B;H1 H18269B;H1H18270B;H1H18271B;H1H18272B;H1H18275B;H1H18 276B;H1H18277B;H1H18278B;H1H18279B;H1H1828B;H1H1828B;H1H1828 2B;H1H18283B;H1H182844B;H1H18285B;H1H18286B;H1H18287B;H1H18288B; H1H18289B;H1H18290B;H1H18291B;H1H18292B;H1H18293B;H1H18294B;H1 H18295B;H1H18297B;H1H18298B;H1H18299B;H1H18300B;H1H18 302B;H1H18303B;H1H18304B;H1H18305B;H1H18306B;H1H18307B;H1H1830 8B;H1H18309B;H1H18310B;H1H18311 B;H1H18312B;H1H18313B;H1H18314B;The pharmaceutical composition according to claim 25 or 26, characterized by administering one or more further therapeutic agents, which are antibodies or antigen-binding fragments thereof selected from the group consisting of H1H18315B; H1H18316B; H1H18317B; H1H18318B; H1H18319B; H1H18320B; H1H18321B; H1H18322B; H1H18323B; H1H18324B; H1H18325B; H1H18326B; H1H18327B; H1H18328B; H1H18329B; H1H18330B; H1H18331B; H1H18332B; H1H18333B; H1H18334B; and H1H18335B, to the target of one or more further therapeutic agents. ;

30. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 9 or a polynucleotide according to any one of claims 14 to 16, wherein the pharmaceutical composition is formulated for injection.

31. The pharmaceutical composition according to claim 30, characterized in that the pharmaceutical composition is formulated for subcutaneous, intravenous, or intramuscular injection.

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