EA044791B1 - HUMAN ANTIBODIES TO HEMAGGLUTIININ OF THE INFLUENZA VIRUS - Google Patents

Description

Technical field of the invention

This application claims the benefit of US Provisional Patent Application Serial No. 62/622,480; filed January 26, 2018; which is incorporated herein by reference in its entirety.

The present invention relates to human antibodies and antigen-binding fragments thereof that specifically bind to hemagglutinin (HA) of group 2 influenza A virus, compositions containing these antibodies, as well as therapeutic and diagnostic routes for using these antibodies.

List of sequences

An official copy of the sequence listing was sent along with the description electronically via EFS-Web as a sequence listing in ASCII format called 10201WO01_SEQ_LIST.txt, created January 24, 2019, and approximately 21 kb in size. The sequence listing contained herein in ASCII format is part of the specification and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Influenza is a highly contagious disease that has a long history, characterized by peaks of pandemics, epidemics, repeated outbreaks and relapses. Despite annual vaccination efforts, influenza infections result in significant morbidity and mortality.

Influenza viruses are composed of three types, A, B, and C. In addition, influenza A viruses can be classified into subtypes based on allelic changes in the antigenic regions of two genes that encode the surface glycoproteins, hemagglutinin (HA) and neuraminose (NA), which are essential for virus attachment and entry into the host cell.

Hemagglutinin is a three-dimensional glycoprotein that contains two structural domains: a globular head domain, which consists of a receptor-binding region (which undergoes frequent antigenic drift), and a stalk domain (more conserved among different influenza virus strains). The HA protein is synthesized as a precursor (HA0), which is proteolytically processed to produce two subunits (HA1 and HA2) that associate with each other to form a stalk/globular head structure. The HA1 peptide is responsible for attaching the virus to the cell surface. The HA2 peptide forms a rod-like structure that mediates the fusion of viral and cellular membranes in endosomes, allowing the release of the ribonucleic acid complex into the cytoplasm.

There are currently eighteen subtypes defined by their hemagglutinin proteins (H1-H18). 18 HA can be divided into two groups. Group 1 consists of subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, and group 2 includes subtypes H3, H4, H7, H10, H14 and H15.

New strains of the same subtype can arise as a result of a phenomenon called antigenic drift, or mutations of the HA or NA molecules that create new and different epitopes. The consequence of this is that a new vaccine must be produced every year against the viruses that are predicted to emerge - a method that is not only expensive, but also extremely ineffective. Although technological developments have improved the ability to produce improved influenza antigen(s) for vaccine compositions, there remains a need to provide additional sources of protection to combat emerging influenza subtypes and strains.

Although the idea of creating a vaccine composition containing an antigen of interest (eg, HA and/or NA) to generate broadly neutralizing antibodies in a patient is generally considered a good approach, it is not always desirable to use this approach in certain patient populations. For example, in certain patients, a vaccine composition containing the antigen of interest may not always be effective, such as in the elderly, in the very young, in immunocompromised patients, etc. In these patient populations or in any patient who is unable to establish effective immune response, it may be more preferable to provide a composition already containing broadly neutralizing antibodies that can target epitopes common to a number of strains in group 1 and/or group 2 subtypes.

To date, there has been limited success in identifying antibodies that broadly neutralize or inhibit influenza viruses. Okuno et al. immunized mice with influenza A/Okuda/57 (H2N2) virus and isolated an antibody, designated C179, which binds to a conserved conformational epitope in HA2 and neutralizes group 1 influenza A viruses H2, H1 and H5 subtypes in vitro and in vivo (Okuno et al. (1993) J Virol 67(5):2552–2558). Throsby et al. identified 13 monoclonal antibodies from human B cells that had broad spectrum activity against group 1 subtypes (Throsby et al. (2008), PLOS one 3(2):e3942). Sui et al. identified a human monoclonal antibody (F10) that binds H5 and other group 1 viruses (Sui, et al. (2009), Nat. Struct. Mol. Biol. 16(3):265-273). Tharakaraman et al. identified a broadly neutralizing antibody, designated VIS410, that was effective in neutralizing group 2 influenza virus strains, including H3N2 and H7N9 strains, in vitro and in vivo (Tharakaraman, K, et al., (2015), Proc Natl Acad Science 112 (35 ): 10890-10895).

- 1 044791

However, after decades of research in this area, only a few antibodies are currently in clinical trials to evaluate their ability to neutralize influenza viruses of different subtypes (see, for example, antibodies being developed by Cracell Holland ((US2012/0276115,

US2014/0065156, US8470327, US2014/0120113, EP2731967, US8691223, US2013/0243792,

US2014/0065165, WO2008/028946 and WO2010/130636); Osaka University (US2011/0319600, EP2380976, US2012/0058124, US2012/0058124), Celltrion (US2013/0004505, EP2545074; WO2014/158001); Vanderbilt University (US2013/0289246), SeaLane Biotechnologies (US2012/0128671), Trellis Bioscience, Inc. (US2012/0020971 EP2582721); Visterra, Inc. (US2013/0302349); Burnham Institute/Dana Farber (US2014/011982, EP2222701, WO2010/027818); Temasek (US8444986, US8574581, US 8637644, US8637645, US8383121, US8540996, US8574830, US8540995); HUMABS Biosciences/Institute for Research in Biomedicine (US8871207, US8685402, EP2313433); MedImmune (WO2015/051010); AIMM Therapeutics (WO2013/081463, EP12798020) and Genentech (US2014/0161822), however, there are still no commercially available antibodies that broadly neutralize or suppress influenza A virus infection or attenuate disease caused by various subtypes of the virus. Accordingly, there is still a need in the art to identify new antibodies that neutralize multiple subtypes of influenza A virus that can be used to prevent or treat influenza virus infection.

Brief description of the invention

The present invention provides antibodies and antigen-binding fragments that bind hemagglutinin (HA) of group 2 influenza A virus. The antibodies of the present invention are useful, among other things, for inhibiting or neutralizing the activity of group 2 HA of influenza A virus. In some embodiments, the antibodies are useful for blocking attachment influenza virus to a host cell and/or to prevent influenza virus from entering host cells. In some embodiments, the antibody functions to inhibit the transmission of a virus between cells. In certain embodiments, the antibodies are used in preventing, treating, or reducing the severity of at least one symptom of an influenza virus infection in a subject. In certain embodiments, antibodies can be administered prophylactically or therapeutically to a subject having or susceptible to influenza virus infection. In certain embodiments, compositions containing at least one antibody of the present invention can be administered to a subject for whom the vaccine is contraindicated or for whom the vaccine is less effective, for example, an elderly patient, a very young patient, a patient who may be allergic to one or more components of the vaccine, or an immunocompromised patient who may not respond to the immunogens in the vaccine. In certain embodiments, compositions containing at least one antibody of the present invention can be administered to healthcare personnel, hospitalized patients or nursing home residents, or other high-risk patients during an influenza outbreak. In certain embodiments, compositions containing at least one antibody of the present invention may be administered as a first line of treatment to patients in the event that a predicted annual vaccine is ineffective, or in the event of a pandemic with a strain that has been severely antigenically challenged.

Antibodies of the present invention may be full length (eg an IgG1 or IgG4 antibody) or may contain only an antigen binding portion (eg a Fab, F(ab') ₂ or scFv fragment) and may be modified to affect functionality, e.g. , to increase stability in the host, or to enhance effector function, or eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In some embodiments, the antibody may be bispecific.

In a first aspect, the present invention provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that specifically bind to influenza A virus group 2 hemagglutinin (HA).

In one embodiment, the present invention provides an isolated recombinant antibody or antigen binding fragment thereof that specifically binds to influenza A virus group 2 HA, wherein the antibody has two or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to influenza A virus group 2 HA with a dissociation constant (K _D ) of less than 10 ^-8 M, as measured by surface plasmon resonance;

(c) exhibits a dissociative half-life (t1/ ₂ ) of greater than 75 minutes;

(d) demonstrates neutralization of group 2 influenza A viruses selected from the H3N2 and H7N9 strains, characterized by an IC ₅₀ value of less than 200 nM and 500 nM, respectively;

(e) demonstrates complement-mediated lysis of influenza virus-infected cells having an EC ₅₀ value of less than about 150 nM;

(f) demonstrates antibody-dependent cell-mediated cytotoxicity against virus-infected target cells using a reporter bioassay characterized

- 2044791 having an EC ₅₀ value of less than approximately 0.9 nM;

(g) demonstrates antibody-dependent cell-mediated cytotoxicity to virus-infected target cells in the presence of human peripheral blood mononuclear cells (PBMCs) characterized by an _EC50 value of less than approximately

0.180 nM;

(h) demonstrates an increase in the survival of an animal infected with influenza virus when administered 24, 48, 72 or 96 hours after infection with the virus;

(i) demonstrates increased survival of an animal infected with influenza virus when administered in combination with oseltamivir 96 hours after infection; or (j) wherein the antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within any of the heavy chain variable region (HCVR) sequences listed in Table. 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any of the light chain variable region (LCVR) sequences listed in table. 1.

In certain embodiments, the antibody of the present invention exhibits enhanced protective function when administered to a mammal infected with influenza virus as a single intravenous dose of from about 7 to 15 mg/kg, starting on day 3 postinfection, compared to oral administration of oseltamivir. administered twice daily for 5 days at a dose of approximately 2 mg/kg, starting on day 3 postinfection (and continuing until day 7 postinfection).

In a related embodiment, an antibody of the present invention provides enhanced protective function in a mammal infected with influenza virus when administered either subcutaneously or intravenously, and/or when administered before or after infection with influenza virus.

In one embodiment, an antibody of the present invention exhibits enhanced protection relative to an animal administered an isotype (negative) control antibody when administered to an infected mammal as a single subcutaneous or intravenous dose ranging from about 5 mg/kg to about 15 mg /kg.

In one embodiment, the antibody of the present invention exhibits enhanced protective function when administered to a mammal infected with influenza virus as a single intravenous dose of from about 7 mg/kg to about 15 mg/kg, compared to oral oseltamivir administered over two once a day for 5 days at a dose of approximately 2 mg/kg.

In one embodiment, the antibody of the present invention exhibits a survival rate of approximately 100% in a mammal infected with influenza virus when administered as a single dose of approximately 15 mg/kg, 24 hours or more after infection.

In one embodiment, an antibody of the present invention exhibits a 100% survival rate in a mammal infected with influenza virus when administered as a single intravenous dose of approximately 15 mg/kg at 24, 48, 72, or 96 hours postinfection.

In one embodiment, the antibody of the present invention exhibits a survival rate of approximately 100% in a mammal infected with influenza virus when administered as a single intravenous dose of approximately 15 mg/kg, compared to the 80% survival rate observed with oseltamivir when administered orally. administered twice daily for 5 days at a dose of approximately 2 mg/kg.

In one embodiment, the antibody of the present invention provides additional protective effect in a mammal infected with influenza virus when administered with oseltamivir for more than 48 hours after infection.

In one embodiment, the antibody of the present invention provides additional protective effect in a mammal infected with influenza virus when administered together with oseltamivir 72 hours after infection.

In one embodiment, the antibody of the present invention provides an additional protective effect when used in combination with oseltamivir, where the antibody is administered to a mammal infected with influenza virus as a single intravenous dose ranging from about 7 mg/kg to about 15 mg/kg, and oseltamivir administered orally twice daily for 5 days at a dose of approximately 2 mg/kg.

In a related embodiment, the antibody of the present invention provides additional protective effect when administered in combination with oseltamivir 96 hours after influenza virus infection, where the antibody is administered as a single intravenous dose ranging from about 7 mg/kg to about 15 mg/kg, and Oseltamivir is administered orally twice daily for 5 days at a dose of approximately 2 mg/kg.

In one embodiment, the antibody of the present invention can be administered intravenously, intranasally, subcutaneously, intradermally or intramuscularly, and oseltamivir can be administered orally.

In one embodiment, oseltamivir is administered before, simultaneously with, or after the administration of an antibody of the present invention.

In one embodiment, the antibody and/or oseltamivir can be administered as a single dose or as multiple doses.

Exemplary anti-influenza A virus group 2 HA antibodies of the present invention are listed in Table. 1 and 2 of this document. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary antibodies to ON influenza virus. In table 2 sets forth the nucleic acid sequence identifiers HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of illustrative antibodies to influenza virus HA.

The present invention provides antibodies or antigen-binding fragments thereof that contain an HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereto, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect thereto.

In one embodiment, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind influenza A virus group 2 HA containing HCVR having the amino acid sequence of SEQ ID NO: 2 or 18.

The present invention also provides antibodies or antigen-binding fragments thereof that contain an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

In one embodiment, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind influenza A virus group 2 HA containing LCVR having the amino acid sequence of SEQ ID NO: 10 or 26.

In one embodiment, an isolated antibody or antigen binding fragment that specifically binds influenza A virus group 2 HA comprises the amino acid sequence pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10 or 18/26.

In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 2/10 (eg, H1H14611N2) or 18/26 (eg, H1H14612N2).

In one embodiment, the isolated antibody or antigen binding fragment comprises:

(a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or 20;

(b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 6 or 22;

(c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 8 or 24;

(d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12 or 28;

(e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14 or 30; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16 or 32.

In one embodiment, an isolated antibody or antigen binding fragment that specifically binds influenza A virus group 2 HA comprises (a) HCDR1 of SEQ ID NO: 4, (b) HCDR2 of SEQ ID NO: 6; (c) HCDR3 from SEQ ID NO: 8; (d) LCDR1 from SEQ ID NO: 12; (e) LCDR2 from SEQ ID NO: 14 and (f) LCDR3 from SEQ ID NO: 16.

In one embodiment, an isolated antibody or antigen binding fragment that specifically binds influenza A virus group 2 HA comprises (a) HCDR1 of SEQ ID NO: 20, (b) HCDR2 of SEQ ID NO: 22; (c) HCDR3 from SEQ ID NO: 24; (d) LCDR1 from SEQ ID NO: 28; (e) LCDR2 from SEQ ID NO: 30 and (f) LCDR3 from SEQ ID NO: 32.

The present invention also provides antibodies or antigen-binding fragments thereof that contain a heavy chain CDR1 (HCDR1) containing an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in table. 1, or a substantially similar sequence thereto, which has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to it.

The present invention also provides antibodies or antigen binding fragments thereof that comprise a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof, that is at least 90% characterized , at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides antibodies or antigen binding fragments thereof that comprise a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof, that is at least 90% characterized , at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides antibodies or antigen binding fragments thereof that comprise a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof, that is at least 90% characterized , at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides antibodies or antigen-binding fragments thereof that contain a light chain CDR2 (LCDR2) containing an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in table. 1, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides antibodies or antigen binding fragments thereof that contain a light chain CDR3 (LCDR3) containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in table. 1, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides antibodies or antigen-binding fragments thereof containing a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3), containing any of the amino acid sequences of HCDR3 listed in table. 1, paired with any of the LCDR3 amino acid sequences listed in table. 1. In certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof that comprise the HCDR3/LCDR3 amino acid sequence pair contained in the exemplary influenza A virus group 2 HA antibodies listed in Table. 1. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8/16 (eg, H1H14611N2) or 24/32 (eg, H1H14612N2).

The present invention also provides antibodies or antigen binding fragments thereof that comprise a group of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary influenza A virus group 2 HA antibodies listed in Table 1. In certain embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NO: 4-6-8-1214-16 (for example, H1H14611N2) or SEQ ID NO: 20-22-24-28-30-32 (for example, H1H14612N2).

In a related embodiment, the present invention provides antibodies or antigen binding fragments thereof comprising a group of six CDRs (i.e., HCDR1-HCDR2-HCDR3LCDR1-LCDR2-LCDR3) contained within the amino acid sequence pair HCVR/LCVR, as defined for any of the exemplary antibodies to NA of group 2 influenza A virus listed in table. 1. For example, the present invention provides antibodies or antigen-binding fragments thereof comprising a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3 contained in a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10 ( e.g. H1H14611N2) or SEQ ID NO: 18/26 (e.g. H1H14612N2). Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventional methods that may be used to identify CDR boundaries include, for example, the Kabat determination, the Chothia determination, and the AbM determination. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the loop structural regions, and the

- 5 044791 according to AbM is a compromise solution between the approaches according to Kabat and Chothia. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md.

(1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997) and Martin et al., Proc. Natl. Acad. Sci. USA 56:92689272 (1989). Public databases are also available to identify CDR sequences in antibodies.

The present invention also provides antibodies and antigen binding fragments thereof that compete for specific binding to influenza A virus group 2 HA, an antibody or antigen binding fragment comprising CDRs consisting of a CDR in HCVR and a CDR in LCVR, each having an amino acid sequence, selected from the HCVR and LCVR sequences listed in table. 1.

The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete for binding to influenza A virus group 2 HA or that bind the same epitope on influenza A virus group 2 HA as a reference antibody or antigen-binding fragment thereof, comprising CDRs from HCVR and CDR from LCVR, where each of HCVR and LCVR has an amino acid sequence selected from the sequences of HCVR and LCVR listed in table. 1.

The present invention also provides isolated antibodies and antigen-binding fragments thereof that block influenza A virus group 2 HA from attaching to and/or entering a host cell.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific, comprising a first binding specificity to a first epitope in the influenza A virus group 2 HA and a second binding specificity to a different antigen.

In a second aspect, the present invention provides nucleic acid molecules encoding the influenza A virus group 2 HA antibodies of the present invention or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in table. 2; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR amino acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 amino acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 amino acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 amino acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence,

- 6 044791 selected from any of the amino acid sequences of LCDR1 listed in table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 amino acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in table. 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 amino acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it.

The present invention also provides nucleic acid molecules encoding HCVR, wherein HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the set of amino acid sequences HCDR1-HCDR2-HCDR3 is as determined by any of the exemplary antibodies to the HA of the influenza virus listed in Table. 1.

The present invention also provides nucleic acid molecules encoding LCVR, wherein LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences LCDR1-LCDR2-LCDR3 is as determined by any of the exemplary antibodies to the HA of the influenza virus listed in Table. 1.

The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, where HCVR contains an amino acid sequence that is any of the HCVR amino acid sequences listed in table. 1, and where LCVR contains an amino acid sequence that is any of the LCVR amino acid sequences listed in table. 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to it, and a polynucleotide sequence selected from any of the sequences LCVR nucleic acids listed in table. 2, or a substantially similar sequence thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to it. In certain embodiments, in accordance with this aspect of the present invention, the nucleic acid molecule encodes HCVR and LCVR, where both HCVR and LCVR are derived from the same influenza A virus group 2 HA antibody shown in Table. 1.

The present invention provides nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in table. 1. The present invention also provides nucleic acid molecules encoding any of the light chain amino acid sequences listed in table. 1.

In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-influenza A virus group 2 HA antibody. For example, the present invention includes recombinant expression vectors comprising any of the above-mentioned nucleic acid molecules, i.e. .e. nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences given in table. 1. Also included within the scope of the present invention are host cells into which expression vectors have been introduced, as well as methods for producing antibodies or parts thereof by culturing the host cells under conditions allowing the production of antibodies or antibody fragments, and isolating the antibodies and fragments thus obtained antibodies.

In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen binding fragment thereof that specifically binds influenza A virus group 2 HA, and a pharmaceutically acceptable carrier. In a related aspect, the present invention provides a composition that is a combination of an anti-influenza A virus group 2 HA antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined

- 7 044791 with an anti-influenza A virus group 2 HA antibody. Exemplary agents that may advantageously be combined with an anti-influenza A virus group 2 HA antibody include, without limitation, other agents that bind and/or inhibit the activity of influenza A virus HA (including other antibodies or their antigen-binding fragments, etc.) and/or agents that do not directly bind the HA of the influenza virus, but nevertheless suppress viral activity, including the infectivity of host cells. In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first influenza A virus group 2 HA antibody or antigen binding fragment thereof; (b) a second antibody to influenza A virus group 2 HA or an antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on influenza A virus group 2 HA, and the second antibody binds to a second epitope on influenza A virus group 2 HA, wherein the first and the second epitopes are different and do not overlap; and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first influenza A virus group 2 HA antibody or antigen binding fragment thereof; (b) a second antibody to influenza A virus group 2 HA or an antigen binding fragment thereof, wherein the first antibody does not cross-compete with the second antibody for binding to influenza A virus group 2 HA; and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first influenza A virus group 2 HA antibody or antigen binding fragment thereof; (b) a second influenza virus antibody or antigen-binding fragment thereof that interacts with another viral (non-influenza virus) antigen, where the first antibody binds to the group 2 HA epitope of influenza A virus and the second antibody binds to an epitope on the other viral ( non-influenza virus antigen; and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the present invention provides a pharmaceutical composition comprising: (a) a first influenza A virus group 2 HA antibody or antigen binding fragment thereof; (b) a second antibody or antigen-binding fragment thereof that interacts with another antigen, wherein the first antibody binds to an HA epitope of group 2 influenza A virus and the second antibody binds to an epitope on the other antigen; and (c) a pharmaceutically acceptable carrier or diluent. Additional combination therapeutics and combination formulations comprising the influenza A virus group 2 HA antibodies of the present invention are disclosed elsewhere herein.

In a fourth aspect, the present invention provides therapeutic methods for treating a disease or disorder associated with influenza A virus group 2 HA (such as a viral infection in a subject), or at least one symptom associated with a viral infection, using an antibody to the group 2 HA virus influenza A or an antigen binding portion of an antibody of the present invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen binding portion of an antibody of the present invention to a subject in need thereof. The disorder to be treated is any disease or condition that is reduced, ameliorated, suppressed, or prevented by inhibiting the activity of the influenza virus HA. In certain embodiments, the present invention provides methods for preventing, treating, or alleviating at least one symptom of an influenza A virus infection, the method comprising administering a therapeutically effective amount of an influenza virus HA antibody or antigen binding fragment thereof of the present invention to a subject in need thereof.

In some embodiments, the present invention provides methods for alleviating or reducing the severity, duration, or frequency of at least one symptom of an influenza virus infection in a subject by administering an anti-influenza A virus group 2 HA antibody of the present invention, wherein at least one symptom is selected from the group , consisting of headache, fever, prolonged pain, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis and death.

In certain embodiments, the present invention provides methods for reducing the viral load in a subject, the methods comprising administering to the subject an effective amount of an antibody or fragment thereof of the present invention that binds influenza A virus group 2 HA and blocks influenza virus binding to and/or cell entry -the owner.

In certain embodiments, the antibody or antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject who has, or is at risk of, or predisposed to developing an influenza virus infection. Subjects at risk include, but are not limited to, an immunocompromised person, for example, an immunocompromised person due to an autoimmune disease, or people receiving immunosuppressive therapy (for example, after an organ transplant), or people affected by human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS). with certain forms of anemia that deplete or destroy white blood cells, those people who receive radiation or chemotherapy, or those people who suffer from inflammatory diseases

- 8 044791 violation. Other people at risk for influenza virus infection include older adults (over 65 years of age), children younger than 2 years of age, healthcare workers, and people with underlying medical conditions such as lung infection, heart disease, or forms of diabetes. Also, anyone who comes into physical contact with or is physically close to an infected person has an increased risk of developing an influenza virus infection. In addition, the subject is at risk of contracting influenza virus infection due to proximity to a disease outbreak, such as being in a densely populated city or being in close proximity to subjects with confirmed or suspected influenza virus infections, or choice of place of work, such as a worker hospitals, pharmaceutical researcher, traveler to an infected area or frequent flyer.

In certain embodiments, the antibody or antigen-binding fragment thereof is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of an anti-inflammatory drug (such as corticosteroids and non-steroidal anti-inflammatory drugs), an anti-infective drug, another anti-influenza virus HA antibody, an anti-influenza antigen antibody (for example, neuraminidase), an antiviral drug, a decongestant drug, an antihistamine, an influenza vaccine, a nutritional supplement such as antioxidants, and any other drug or therapeutic agent known in the art useful for reducing the severity of at least one symptom of an influenza virus infection or for reducing a patient's viral load . In certain embodiments, the second therapeutic agent may be an agent that helps neutralize or reduce any potential side effects associated with an antibody or antigen binding fragment thereof of the present invention if such effect(s) are to occur . The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially. In one embodiment, the antibody can be administered through a single intravenous infusion to achieve a maximum concentration of the antibody in the subject's blood serum. The antibody or fragment thereof can be administered at a dose of from about 0.01 mg/kg body weight to about 100 mg/kg body weight of the subject. In certain embodiments, the antibody of the present invention can be administered in one or more doses containing from 50 mg to 5000 mg.

The present invention also includes the use of an anti-influenza A virus group 2 HA antibody or an antigen-binding fragment thereof of the present invention for the treatment of a disease or disorder in which blocking influenza A virus HA binding and/or activity would be beneficial. The present invention also includes the use of an anti-influenza A virus group 2 HA antibody or an antigen-binding fragment thereof of the present invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blocking influenza A virus HA binding and/or activity.

Other embodiments will be apparent from a review of the following detailed description.

Brief description of graphic materials

Fig. 1A, 1B, 1C: A single dose of H1H14611N2 and H1H14612N2 has been shown to effectively neutralize three different strains of group 2 influenza A viruses in vitro. 1A: A/Aichi/02/1968-PR8-X31 (H3N2); IB: A/Philippines/01/1982 (H3N2) and 1C: A/Shanghai/01/2013-PR8 (H7N9).

Fig. 2: H1H14611N2 and H1H14612N2 were shown to mediate complement-dependent cytotoxicity of cells infected with A/Aichi/02/1968-PR8-X31.

Fig. 3: Shows the dose-response curve for group 2 HA antibodies H1H14611N2 and H1H14612N2 in the FcyRIIIA activation assay.

Fig. 4: Shows the dose-response curve for group 2 HA antibodies H1H14611N2 and H1H14612N2 in the ADCC assay using human donor PBMCs.

Fig. 5A, 5B, 5C: A single dose of group 2 HA antibodies H1H14611N2 and H1H14612N2 are shown to demonstrate complete protection against lethal influenza virus when administered as a single dose of 15 mg/kg at 24 (A), 48 (B) or 72 (C) hours after infection with 5 X MLD50 A/Aichi/02/1968-PR8-X31 (H3N2).

Fig. 6: Anti-HA group 2 antibody H1H14611N2 has been shown to have an additive effect in treating influenza virus infection when administered in combination with oseltamivir 96 hours after infection.

Detailed Description of the Invention

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

- 9 044791

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, only the preferred methods and materials are described herein. All publications referred to herein are incorporated herein by reference in their entirety.

Definitions

The term influenza virus hemagglutinin, also called influenza virus HA, is a three-dimensional glycoprotein found on the surface of influenza virions that mediates viral attachment (via binding of HA1 to α-2,3- or α-2,6-sialic acids) and entry ( through a conformational change) into host cells. HA consists of two structural domains: a globular head domain containing a receptor-binding region (subject to a high frequency of antigenic mutations) and a stalk region (more conserved among different influenza virus strains). Influenza virus HA is synthesized as a precursor (HAO) that is proteolytically processed to produce two subunits (HA1 and HA2) that associate with each other to form a stalk/globular head structure. Viral HA is the most variable antigen on the virus (18 subtypes can be divided into two groups), but the stem (HA2) is highly conserved in each group.

The amino acid sequence of the full-length influenza virus HA is illustrated by the amino acid sequence of H3N2A/Wisconsin/67/X-161/2005, submitted to GenBank under accession number ACF41911.1 (also shown herein as SEQ ID NO: 33), or H7N7A/chicken/Netherlands /01/2003, accession number AAR02640.1 (also shown herein as SEQ ID NO: 34). The term influenza virus HA also includes variants of influenza virus HA proteins isolated from different influenza isolates. The term influenza virus HA also includes recombinant HA or a fragment thereof. The term also includes an influenza virus HA or fragment thereof linked, for example, to a histidine tag, a mouse or human Fc, or a signal sequence.

The term influenza virus infection as used herein, also referred to as influenza, refers to a severe acute respiratory illness caused by an influenza virus. The term includes respiratory tract infection and symptoms that include fever, headache, general aches and pains, fatigue and weakness, in some cases severe exhaustion, nasal congestion, sneezing, sore throat, chest discomfort, cough, shortness of breath, bronchitis, pneumonia and death in severe cases.

The term antibody, as used herein, is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds (i.e., complete antibody molecules), and also their multimers (eg IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region (HCVR or VH) AND a heavy chain constant region (consisting of the _CH1 , CH2 and CH3 domains). Each light chain consists of a light chain variable region (LCVR or _VL ) and a light chain constant region ( _CL ). The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FR antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially altered. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

It is also possible to replace one or more CDR residues or remove one or more CDRs. The scientific literature describes antibodies that can dispense with one or two CDRs for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the sites of contact between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan has also discovered many antibodies in which one or two CDRs do not have amino acid residues that come into contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues that do not contact the antigen can be identified based on previous studies (e.g., residues H60-H65 in CDRH2 are often unnecessary) among Kabat CDR regions outside the Chothia CDR, molecular modeling, and/or empirical way. When a CDR or residue(s) thereof is removed, they are typically replaced by an amino acid occupying a corresponding position in the sequence of another human antibody or a consensus sequence of such sequences. The positions to be replaced within the CDR and the amino acid to be replaced can also be selected empirically. Empirical

- 10 044791 logical substitutions can be conservative or non-conservative substitutions.

The fully human anti-influenza HA monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains relative to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and/or CDR regions are mutated to the corresponding residue(s). ki) the germline sequence from which the antibody is derived, or into the corresponding residue(s) of a different germline sequence, or into a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are called collectively referred to herein as germline mutations). One skilled in the art, from the heavy and light chain variable region sequences disclosed herein, can readily obtain numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework residues and/or CDRs in the VH and/or _VL domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework residues and/or CDRs are mutated to the corresponding residue(s) of another germline sequence (i.e., a germline sequence that is different from the germline sequence from which it was originally derived). antibody obtained). In addition, antibodies of the present invention may contain any combination of two or more germline mutations in framework regions and/or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a certain germline sequence, while certain others residues that differ from the original germline sequence are retained or mutated into the corresponding residue of a different germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced biological antagonistic or agonistic properties (in the case of necessary), reduced immunogenicity, etc. The present invention covers antibodies and antigen binding fragments obtained using this general method.

The present invention also includes fully human monoclonal antibodies to influenza virus HA containing variants of any of the amino acid sequences HCVR, LCVR and/or CDR disclosed herein with one or more conservative substitutions. For example, the present invention includes antibodies to influenza virus HA with amino acid sequences HCVR, LCVR and/or CDR, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions with respect to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

The term human antibody as used herein is intended to include antibodies having variable and constant regions derived from human immunoglobulin germline sequences. The human mAbs of the present invention may include amino acid residues not encoded by human immunoglobulin germline sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example in the CDRs, and in particular CDR3. However, the term human antibody as used herein is not intended to include mAbs in which CDR sequences derived from the germ line of another mammalian species (eg, mouse) are grafted onto human FR sequences. The term includes antibodies produced recombinantly in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or derived from a human subject.

The term recombinant as used herein refers to antibodies or antigen binding fragments thereof of the present invention that are generated, expressed, isolated or produced using technologies or methods known in the art, such as recombinant DNA technology, which includes, for example, DNA splicing and transgene expression. The term refers to antibodies expressed in a non-human mammal

- 11 044791 (including in non-human transgenic mammals, for example, transgenic mice), or in a cellular expression system (for example, CHO cells), or they are isolated from a combinatorial library of recombinant human antibodies.

The term specifically binds or specifically binds to or the like means that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10'8 M or less (eg, a lower KD value indicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies were identified using real-time analysis using a marker-free biolayer interferometry on the Octet® HTX biosensor, which specifically binds to the HA of the influenza virus. Moreover, multispecific antibodies that bind to one domain in the influenza virus HA and one or more additional antigens or bispecific antibodies that bind to two different regions of the influenza virus HA are nevertheless considered to be antibodies that specifically bind, as used herein. document.

The term high-affinity antibody refers to those mAbs that have a binding affinity to the HA of the influenza virus, expressed as K _D , of at least ^10'8 M; preferably 10' ⁹ M; more preferably 10' ¹⁰ M, even more preferably 10' ¹¹ M, even more preferably 10' ¹² M, as measured by real-time analysis using marker-free biolayer interferometry, such as the Octet® HTX biosensor, or by surface plasmon resonance , such as BIACORE™, or by solution affinity determination using ELISA.

By the terms slow dissociation rate, Koff, or kd is meant an antibody that dissociates from influenza virus HA with a rate constant of 1x10'3 ^s'1 or less, preferably ^{1x10'4 s'1} or less, as determined by real-time analysis using markerless biolayer interferometry, such as the Octet® HTX biosensor, or surface plasmon resonance, such as BIACORE™.

The terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms antigen binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to bind to the HA of an influenza virus.

In specific embodiments, the antibody or antibody fragments of the present invention may be conjugated to a functional molecule, such as a ligand, or a therapeutic molecule (immunoconjugate), such as an antiviral drug, a second antibody to an influenza virus, or any other therapeutic molecule useful in treating an infection, caused by the influenza virus.

The term isolated antibody, as used herein, is intended to mean an antibody that does not inherently contain other antibodies (Abs) having different antigen specificities (for example, an isolated antibody that specifically binds to the HA of an influenza virus, or a fragment thereof that essentially does not include Abs that specifically bind antigens other than the HA of the influenza virus.

The terms blocking antibody or neutralizing antibody as used herein (or an antibody that neutralizes influenza virus HA activity or an antagonist antibody) are intended to mean an antibody whose binding to an anti-influenza virus HA antibody results in inhibition of at least one biological HA activity of the influenza virus. For example, an antibody of the present invention can prevent or block influenza virus from attaching to or entering a host cell. Moreover, a neutralizing antibody is one that can neutralize, i.e. prevent, suppress, reduce, interfere with or interfere with the ability of a pathogen to initiate and/or establish infection in a host. The terms neutralizing antibody and antibody that neutralizes or antibodies that neutralize are used interchangeably herein. These antibodies can be used alone or in combination as prophylactic or therapeutic agents with other antiviral agents when appropriately formulated, or in connection with active vaccination, or as a diagnostic tool.

The term surface plasmon resonance refers to an optical phenomenon that involves the analysis of biomolecular interactions in real time by detecting changes in protein concentrations in a biosensor array, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey, USA.

- 12 044791

Biolayer interference is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: an immobilized protein layer on the biosensor tip and an internal reference layer. Any change in the number of molecules associated with the biosensor tip causes a shift in the interference pattern, which can be measured in real time (Abdiche, Y.N., et al. Analytical Biochemistry, (2008), 377(2), 209-217). In certain embodiments of the present invention, a biosensor (Octet HTX assay) based on real-time biolayer interferometry was used to evaluate the binding characteristics of certain antibodies to influenza virus HA.

As used herein, the term KD is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. One antigen can have more than one epitope. Thus, different antibodies can bind to different sites on the antigen and can 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 site of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain those residues that directly contribute to the affinity property of the interaction. Epitopes can also be conformational, i.e., consist of nonlinear amino acids. In certain embodiments, epitopes may include determinants, which are reactive surface moieties of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structure characteristics and/or specific charge characteristics.

The term cross-compete as used herein means an antibody or antigen-binding fragment thereof that binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both directions, that is, a first antibody that binds and blocks the binding of a second antibody, and vice versa. In certain embodiments, the first antibody and the second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but non-overlapping epitopes such that binding of one inhibits or blocks binding of the second antibody, for example, through steric mismatch. Cross-competition between antibodies can be measured using methods known in the art, for example, using real-time analysis using marker-free biolayer interferometry. To determine whether the test antibody cross-competes with the influenza virus group 2 protein reference antibody of the present invention, the reference antibody is allowed to bind to the HA or influenza virus peptide under saturation conditions. The ability of the test antibody to bind to the HA of the influenza virus is then assessed. If the test antibody is able to bind to influenza virus HA after saturation of binding by the reference influenza virus HA antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-influenza virus HA antibody. On the other hand, if the test antibody is unable to bind to the influenza virus HA after saturation of binding with the reference anti-influenza virus HA antibody, then the test antibody may bind to the same epitope as the epitope that is bound by the reference anti-influenza virus HA antibody herein. invention.

The term substantially identical or substantially identical to a nucleic acid or fragment thereof indicates that when the corresponding nucleotide insertions or deletions are optimally aligned with another nucleic acid sequence (or its complementary strand), the nucleotide sequence identity is at least about 90% and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by a well-known sequence identity algorithm such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

When applied to polypeptides, the term essentially similar or substantially similar means that two peptide sequences, when optimally aligned, such as by GAP or BESTFIT, using default gap penalty values, have at least 90% sequence identity, and more preferably at least 95%, 98% or 99% sequence identity. Preferably, the positions of the residues that are not identical differ in conservative amino acid substitutions. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by another

- 13 044791 gim amino acid residue containing a side chain (R-group) with similar chemical properties (for example, charge or hydrophobicity). Typically, a conservative amino acid substitution will not essentially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of groups of amino acids that contain side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic side chains with hydroxyl groups: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate and 7) sulfur containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinarginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change with a positive value in the PAM250 log-likelihood function matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, which is incorporated herein by reference. A moderately conservative replacement is any change with a non-negative value in the PAM250 log-likelihood function matrix.

Sequence similarity of polypeptides is usually measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, the GCG software package includes programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutein . See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and calculations of percent sequence identity in regions of greatest overlap between the query and found sequences (Pearson (2000), supra). Another preferred algorithm when comparing a sequence of the present invention to a database containing a large number of sequences from various organisms is the BLAST computer program, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

The phrase therapeutically effective amount means an amount that produces the desired effect for which it is administered.

The exact amount will depend on the purpose of treatment and will be determined by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term subject refers to an animal, preferably a mammal, in need of reducing the severity, preventing and/or treating a disease or disorder, such as a viral infection. The subject may have an influenza virus infection or may be predisposed to developing an influenza virus infection. Subjects predisposed to developing influenza virus infection or patients who may be at increased risk of influenza virus infection include those with a weakened immune system due to an autoimmune disease, people receiving immunosuppressive therapy (eg, after an organ transplant), people affected by immunodeficiency syndrome people with HIV (HIV) or acquired immunodeficiency syndrome (AIDS), with certain forms of anemia that deplete or destroy white blood cells, those people who receive radiation or chemotherapy, or those people who have an inflammatory disorder. In addition, extremely young or older subjects are at increased risk. Anyone who comes into physical contact with or is physically close to an infected person has an increased risk of developing an influenza virus infection. In addition, the subject is at risk of contracting influenza virus infection due to proximity to a disease outbreak, such as being in a densely populated city or being in close proximity to subjects with confirmed or suspected influenza virus infections, or choice of place of work, such as a worker hospitals, pharmaceutical researcher, traveler to an infected area or frequent flyer.

As used herein, the terms treat, administer a treatment, or cure refer to reducing or reducing the severity of at least one symptom or sign of infection.

- 14 044791 infection by influenza virus due to the administration of a therapeutic agent, such as an antibody of the present invention, to a subject in need thereof. Terms include suppression of disease progression or worsening infection. The terms also include a positive prognosis of the disease, i.e. the subject may not have an infection or may have reduced or absent viral titers when administered a therapeutic agent, such as an antibody of the present invention. The therapeutic agent can be administered to a subject at a therapeutic dose.

The terms prevent, warn or warn refer to suppression of the manifestation of an influenza virus infection or any symptoms or signs of an influenza virus infection upon administration of an antibody of the present invention. The term includes preventing the spread of infection in a subject who has been exposed to or is at risk of becoming infected with an influenza virus.

As used herein, a protective effect can be demonstrated using any standard procedure known in the art to determine whether an agent, such as an antiviral agent, or an antibody, such as an anti-influenza virus HA antibody of the present invention, can demonstrate one or more of the following: for example, increasing survival after exposure to an infectious agent, reducing viral load, or reducing the intensity of at least one symptom associated with an infectious agent.

As used herein, the term antiviral drug refers to any antibacterial drug or therapeutic agent used to treat, prevent, or reduce the severity of a viral infection in a subject. The term antiviral drug includes, without limitation, TAMIFLU® (oseltamivir), RELENZA® (zanamivir), ribavirin or interferon-alpha2b. In the present invention, the infection to be treated is caused by an influenza virus.

General description.

Influenza is an infectious disease caused by RNA viruses of the Orthomyxoviridae family (influenza viruses). Influenza viruses are classified based on the core protein into three varieties - A, B and C, which are further subdivided into subtypes defined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). Influenza A virus infects a range of mammalian and avian species, while infections B and C are largely limited to humans. The disease in humans is caused only by types A and B.

High mutation rates and frequent genetic differences in influenza viruses contribute to the high variability of the HA and NA antigens. Small mutations causing small changes (antigenic drift) occur relatively frequently. Antigenic drift allows the virus to evade immune recognition, leading to repeated outbreaks of influenza during interpandemic years. Significant changes in the HA antigen (antigenic drift) are caused by reassortment of genetic material from different subtypes of influenza A virus. Antigenic drift due to new pandemic strains is a rare phenomenon that occurs through reassortment between animal subtypes and human subtypes, for example in coinfected pigs.

The neutralizing antibody response to influenza A virus is typically specific for a given viral subtype. There are 18 subtypes of influenza A virus, defined by their hemagglutinin (HA) proteins. 18 HA, H1-H18, can be divided into two groups. Group 1 consists of subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, and group 2 includes subtypes H3, H4, H7, H10, H14 and H15. For these reasons, it would be highly desirable to have a vaccine that broadly induces neutralizing antibodies capable of neutralizing all influenza A virus subtypes as well as their annual variants. In addition, broadly neutralizing heterosubtypic antibodies could be administered as drugs to prevent or treat influenza A virus infection.

HA is synthesized as the homo-trimeric precursor HA0. Each monomer can be independently cleaved post-translationally to produce two polypeptides, HA1 and HA2, linked by a single disulfide bond. The larger N-terminal fragment (amino acids HAL 320-330) forms a membrane-distal globular domain that contains the receptor-binding site and most of the determinants recognized by virus-neutralizing antibodies. The HA1 HA polypeptide is responsible for the attachment of the virus to the cell surface. The smaller C-terminal portion (HA2, approximately 180 amino acids) forms a rod-like structure that anchors the globular domain to the cellular or viral membrane. The HA2 polypeptide mediates the fusion of viral and cellular membranes in endosomes, ensuring the release of the ribonucleic acid complex into the cytoplasm.

There has been limited success only in identifying antibodies that neutralize more than one subtype of influenza A virus. Additionally, the need for neutralizing antibodies identified to date is narrow and their potency is low. Okuno et al. immunized mice with influenza virus A/Okuda/57 (H2N2) and isolated a monoclonal antibody (C179), which binds to a conserved conformational epitope in HA2 and neutralizes viruses

- 15 044791 influenza A subtypes H2, H1 and H5 group 1 in vitro and in vivo in animal models ((Okuno et al., J. Virol.

67:2552-8, 1993).

Despite a decade of research, there are no commercially available antibodies that significantly neutralize or suppress influenza A virus infection or attenuate disease caused by influenza A virus. Thus, there is a need to identify new antibodies that neutralize multiple subtypes of influenza A virus, and they can be used as drugs to prevent or treat influenza A virus infection.

Passive immunotherapy for the prevention or treatment of infectious diseases has been used for more than a century, typically in the form of convalescent human serum that contains high titers of neutralizing antibodies (Good et al. 1991; Cancer 68: 1415–1421). Currently, a variety of purified monoclonal antibodies are undergoing preclinical and clinical studies for use as antimicrobial agents (Marasco et al. 2007; Nature Biotechnology 25: 1421–1434).

The present inventors have described herein fully human antibodies and antigen-binding fragments thereof that specifically bind to influenza virus hemagglutinin and modulate the interaction of the influenza virus with host cells. Antibodies to influenza A virus group 2 HA can bind to influenza virus HA with high affinity. In certain embodiments, the antibodies of the present invention are blocking antibodies, where the antibodies can bind to the HA of the influenza virus and block the virus from attaching to and/or entering a host cell. In some embodiments, the blocking antibodies of the present invention can block influenza virus binding to cells and, as such, can inhibit or neutralize viral invasion of host cells. In some embodiments, blocking antibodies may be useful for treating a subject suffering from an influenza virus infection. When administered to a subject in need thereof, the antibodies can reduce the subject's infection with a virus, such as influenza. They can be used to reduce a subject's viral loads. They can be used alone or as an adjuvant therapy with other therapeutic molecules or modalities known in the art to treat a viral infection. In certain embodiments, these antibodies can bind to an epitope in the stem region of the viral HA. In addition, the identified antibodies may be used prophylactically (before infection) to protect a mammal from infection, or may be used therapeutically (after infection has developed) to relieve a pre-existing infection or to alleviate at least one symptom associated with an infection.

The full-length amino acid sequences of two exemplary influenza A virus group 2 HAs are shown in GenBank under accession number ACF41911.1 (from A/Wisconsm/67/X-161/2005 (H3N2), see also SEQ ID NO: 33) and accession number AAR02640 .1 (from A/chicken/Netherlands/01/2003 (H7N7) (see also SEQ ID NO:34).

In certain embodiments, the antibodies of the present invention are obtained from mice immunized with a primary immunogen, such as full-length influenza virus HA, or a recombinant form of influenza virus HA, or fragments thereof, followed by immunization with a secondary immunogen or an immunogenetically active influenza virus HA fragment. In certain embodiments, the antibodies are obtained from mice immunized with an influenza virus vaccine composition followed by a booster immunization with one or more recombinantly produced HA peptides.

In certain embodiments, mice are immunized with A/Hong Kong/08/1968 (H3N2), then A/Hong Kong/05/1972-PR8-X36 (H3N2), and then with A/Hong Kong/08/1968 ( H3N2). All mice were boosted with a 1:1 mixture of DNA encoding HA from A/Wisconsin/67/X-161/2005 (H3N2) and A/chicken/Netherlands/01/2003 (H7N7).

The immunogen may be a biologically active and/or immunogenic fragment of HA or influenza virus DNA encoding its active fragment. The fragment can be obtained from the stalk region of the HA protein. (See Sui et. al., Nature Struct, and Mol. Biol., published online February 22, 2009; pages 1-9).

These peptides can be modified to add or replace certain residues for labeling or for conjugation to carrier molecules such as KLH. For example, a cysteine can be added at either the N-terminus or the C-terminus of the peptide, or a linker sequence can be added to produce a peptide for conjugation, such as KLH for immunization.

Certain antibodies to influenza A virus group 2 HA are capable of binding to and neutralizing influenza virus HA activity, as determined by in vitro or in vivo assays. The ability of the antibodies of the present invention to bind to the group 2 HA of influenza A virus and neutralize its activity, and thereby the attachment and/or entry of the virus into a host cell followed by viral infection, can be measured using any standard method known to those skilled in the art. , including binding assays or activity assays as described herein.

- 16 044791

Non-limiting exemplary in vitro assays for measuring binding activity are illustrated in Example 3 herein. In Example 3, the binding affinity and dissociation constants of influenza A virus group 2 HA for influenza A virus group 2 HA were determined by surface plasmon resonance using a Biacore instrument. In Example 4, neutralization assays were used to determine the infectivity of various group 2 influenza virus strains. In Example 5, certain antibodies were shown to mediate complement-dependent cytotoxicity (CDC), or in Example 6, certain antibodies were shown to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of virus-infected cells in vitro. Example 7 demonstrates that certain antibodies of the present invention are capable of neutralizing influenza A virus infection in vivo.

Antibodies specific for influenza A virus group 2 HA may contain no additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the location of the tag (if present) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, then the peptide containing N-terminal biotin will be oriented such that the C-terminal part of the peptide is located distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or an MRI detectable label. In certain embodiments, such labeled antibodies can be used in diagnostic studies, including diagnostic imaging studies.

Antigen-binding fragments of antibodies.

Unless specifically stated otherwise, the term antibody as used herein should be understood to include antibody molecules that contain two immunoglobulin heavy chains and two immunoglobulin light chains (ie, complete antibody molecules), as well as antigen-binding fragments thereof. The terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms antigen binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to specifically bind to influenza A virus group 2 HA. An antibody fragment may include a Fab fragment, an F(ab') ₂ fragment , Fv fragment, dAb fragment, CDR-containing fragment, or isolated CDR. In certain embodiments, the term antigen-binding fragment refers to a fragment of a polypeptide or a multispecific antigen-binding molecule thereof. Antigen-binding antibody fragments can be produced, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering technologies involving manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and chemically manipulated or manipulated using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, insert pisteine residues, modify, add or remove amino acids, and etc.

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 molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR), such as the CDR3 peptide) or the FR3-CDR3-FR4 restricted peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g. monovalent antibodies, bivalent antibodies, etc.), immunopharmaceuticals small modular protein (SMIP) drugs and shark IgNAR variable domains are also included in the expression antigen binding fragment used herein.

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one CDR that is in frame with or adjacent to one or more framework sequences. In antigen binding fragments having a _VH domain linked to a _VL domain, the VH and _VL domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain dimers V _H -V _H , V _H -V _L or V _L -V _L . Alternatively, the antigen-binding antibody fragment may comprise a monomeric V _H or VL domain.

- 17 044791

In certain embodiments, an antigen binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that may be found in an antigen binding fragment of an antibody of the present invention include: (i) Vh-Ch1, (ii) Vh-Ch2, (iii) Vh-Ch3, (iv) Vh-Ch1-Ch2 , (v) Vh-Ch1-Ch2-Ch3, (vi) VhCh2-Ch3, (vii) Vh-C _l , (viii) V _l -Ch1, (ix) Vl-Ch2, (x) Vl-Ch3, ( xi) Vl-Ch1-Ch2, (xii) Vl-Ch1-Ch2-Ch3, (xiii) V _l -C _h 2-C _h 3 and (xiv) V _l -C _l . In any configuration of variable and constant domains, including any of the exemplary configurations set forth above, the variable and constant domains may either be directly linked to each other or may be linked by all or part of a hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that provide flexible or semiflexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, an antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations set forth above in non-covalent association with each other and/or with one or more monomeric VH or _Vl domains ( for example, via disulfide bond(s).

As with full antibody molecules, antigen-binding fragments can be monospecific or polyspecific (eg, bispecific). A polyspecific antigen-binding antibody fragment will typically contain at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using standard techniques available in the art.

Obtaining human antibodies.

Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to produce human antibodies that specifically bind to influenza A virus group 2 HA. An immunogen containing any of the following can be used to produce antibodies to influenza A virus group 2 HA. In certain embodiments, The antibodies of the present invention are derived from mice immunized with full-length native influenza A virus group 2 HA (see, for example, GenBank accession numbers ACF41911.1 or AAR02640.1), or live attenuated or inactivated virus, or DNA encoding a protein or its fragment. Alternatively, influenza A virus group 2 HA or a fragment thereof can be produced using standard biochemical techniques and can be modified and used as an immunogen. In one embodiment, the immunogen may be a recombinantly produced influenza A virus group 2 HA protein or a fragment thereof. In certain embodiments of the present invention, the immunogen may be an influenza virus vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, booster injections may contain one or more influenza viruses or hemagglutinins derived from these strains, for example, A/Hong Kong/08/1968 (H3N2) followed by A/Hong Kong/05/1972-PR8-X36 (H3N2 ), and then with A/Hong Kong/08/1968 (H3N2). All mice were boosted with a 1:1 mixture of DNA encoding HA from A/Wisconsin/67/X-161/2005 (H3N2) and A/chicken/Netherlands/01/2003 (H7N7). In certain embodiments, booster injections may contain a 1:1 mixture of influenza virus strains or a 1:1 mixture of hemagglutinins derived from strains or DNA encoding HA. In certain embodiments, the immunogen may be a recombinant influenza virus HA peptide expressed in E. coli or other eukaryotic or mammalian cells, such as Chinese hamster ovary (CHO) cells or its influenza virus.

Using VELOCIMMUNE™ technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for creating monoclonal antibodies, high-affinity chimeric antibodies to influenza A virus group 2 HA having a human variable region and a murine constant region are initially isolated. VELOCIMMUNE® technology involves the production of a transgenic mouse having a genome containing human heavy and light chain variable regions functionally linked to endogenous mouse constant region loci, whereby the mouse, in response to antigen stimulation, produces an antibody containing a human variable region and a mouse constant region. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and functionally linked to DNA encoding the human constant regions of the heavy and light chains. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is injected with the antigen of interest, and lymphatic cells (such as B cells) are isolated from the mice that express the antibodies. Lymphatic

- 18 044791 cells can be fused with a myeloma cell line to produce immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the constant regions of the desired heavy chain and light chain isotypes. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

Initially, high-affinity chimeric antibodies are isolated that have a human variable region and a murine constant region. As shown in the experimental section below, antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to produce a fully human antibody of the present invention, e.g., wild type or modified IgG1 or IgG4. Although the selected constant region may vary depending on the particular application, the characteristics of high antigen binding affinity or target specificity are inherent in the variable region.

Biological equivalents.

The influenza A virus group 2 HA antibodies and antibody fragments of the present invention encompass proteins with amino acid sequences that differ from those of the described antibodies, but which retain the ability to bind influenza A virus HA. Such antibody and antibody fragment variants contain one or more of the additions deletions or amino acid substitutions when compared with the original sequence, but exhibit biological activity that is almost equivalent to that of the described antibodies. Likewise, DNA sequences encoding an antibody of the present invention include sequences that contain one or more of the additions, deletions or substitutions of nucleotides when compared to the disclosed sequence, but which encode an antibody or antibody fragment that is essentially biological equivalents of the antibody or fragment antibodies of the present invention.

Two antigen-binding proteins or antibodies are considered biological equivalents if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and/or extent of absorption do not differ significantly when the same molar dose is administered under similar experimental conditions, either in a single dose or multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption, but not in the rate of their absorption, and may still be considered biologically equivalent, since such differences in the rate of absorption are intended and reflected at the time of labeling are not essential to achieve effective concentrations of the drug in the body, for example, during long-term use, and are considered medically insignificant for the particular drug product being studied.

In one embodiment, two antigen binding proteins are biological equivalents if there are no clinically significant differences in their safety, purity, or activity.

In one embodiment, two antigen-binding proteins are biological equivalents if switching from the reference product to the biological product can be made one or more times without an expected increase in the risk of side effects, including a clinically significant change in immunogenicity or reduced efficacy, compared to long-term therapy without such a transition.

In one embodiment, two antigen binding proteins are biological equivalents if they both act according to a common mechanism or mechanisms of action with respect to the condition or conditions of use, to the extent such mechanisms are known.

Biological equivalence can be determined using in vivo and/or in vitro methods. Biological equivalence measurements include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum, or other body fluids over time; (b) an in vitro test that correlated with and was sufficiently predictive of in vivo bioavailability data in humans; (c) an in vivo test in humans and other mammals in which the relevant early pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antibody.

Biological equivalents of the antibodies of the present invention can be constructed by, for example, creating various substitutions of residues or sequences or deletion of terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not significant for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or inappropriate intramolecular disulfide bridges upon renaturation. In other situations, biologically equivalent antibodies may include antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, for example, mutations that eliminate or remove glycosylation.

Antibodies to influenza virus NA. Antibodies containing Fc variants.

In accordance with certain embodiments of the present invention, there are provided anti-influenza A virus group 2 HA antibodies comprising an Fc domain that contains one or more mutations that increase or decrease the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. pH. For example, the present invention includes anti-influenza virus HA antibodies containing a mutation in the CH2 or C _H 3 region of the Fc domain, where the mutation(s) provide(s) an increase in the affinity of the Fc domain for FcRn in an acidic environment (for example, in endosome, where the pH varies from about 5.5 to about 6.0). The result of such mutations may be an increase in the half-life of the antibody in the blood serum when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T), or modification at position 428 and/or 433 (for example, H/L/R/S/P/Q or K), and/or 434 (for example, A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]), or modification in position 250 and/or 428, or modification at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (e.g., M428L) and 434S (e.g., N434S), modification 428L, 2591 ( for example, V259I) and 308F (for example, V308F), modification 433К (for example, Н433К) and 434 (for example, 434Y), modification 252, 254 and 256 (for example, 252Y, 254Т and 256Е), modification 250Q and 428L (for example, T250Q and M428L) and modification 307 and/or 308 (for example, 308F or 308P). In yet another embodiment, the modification includes modification 265A (eg, D265A) and/or 297A (eg, N297A).

For example, the present invention includes anti-influenza A virus group 2 HA antibodies containing an Fc domain that contains one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (for example, M252Y, S254T and T256E), 428L and 434S (for example, m428L and N434S), 257I and 311I (for example, P257I and Q311I), 257I and 434H (for example, P257I and N434H), 376V and 434H (for example , D376V and N434H), 307A, 380A and 434A (for example, T307A, E380A and N434A), as well as 433K and 434F (for example, H433K and N434F). All possible combinations of the above-mentioned mutations in the Fc domain and other mutations within the variable domains of the antibodies disclosed herein are contemplated within the scope of the present invention.

The present invention also includes anti-influenza A virus group 2 HA antibodies comprising a chimeric heavy chain constant region (CH), wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the present invention may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule in combination with part or all of a CH3 domain derived from a human IgG1 molecule. human IgG2 or human IgG4. In accordance with certain embodiments, the antibodies of the present invention comprise a chimeric _CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a hinge region of human IgG1, human IgG2 or human IgG4, in combination with a lower hinge sequence (amino acid residues at positions 228-236 according to EU numbering). EUt) derived from the hinge region of human IgG1, human IgG2 or human IgG4. In certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge sequence and amino acid residues derived from a human IgG2 lower hinge sequence. An antibody containing a chimeric _CH region described herein can, in certain embodiments, exhibit the effector functions of the modified Fc without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, US Provisional Patent Application No. 61/759578, filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety.)

Biological characteristics of antibodies.

In general, the antibodies of the present invention function by binding to the group 2 HA of the influenza A virus. For example, the present invention includes antibodies and antigen-binding antibody fragments that bind to the group 2 HA of the influenza A virus (for example, at 25° C. or 37 °C) with a KD of less than 10 nM, as measured by surface plasmon resonance on a Biacore instrument, or by a biosensor based on real-time biolayer interferometry (Octet HTX assay). In certain embodiments, the antibodies or antigen-binding fragments thereof bind influenza A virus group 2 HA with a KD of less than about 5 nM,

- 20 044791 less than about 2 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM, as measured by surface plasmon resonance, for example, using the assay format described herein or substantially similar analysis.

The present invention also includes antibodies and antigen-binding fragments thereof that bind influenza A virus group 2 HA with a dissociative half-life (t ¹ /2) of greater than about 75 minutes, as measured by surface plasmon resonance at 37° C., e.g. using an analysis format as defined herein, or a substantially similar analysis. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind influenza virus HA with a t ¹ /2 of greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, or greater than about 1000 minutes, as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined herein (e.g., mAb trap format or antigen trap) or a substantially similar assay.

The present invention also includes antibodies or antigen-binding fragments thereof that neutralize the infective ability of the influenza virus against its host cells. In some embodiments, the antibodies exhibit neutralizing activity against various representative strains of group 2 influenza A/Aichi/02/1968-PR8-X31 (H3N2) viruses; A/Philippines/01/1982 (H3N2) and A/Shanghai/01/2013-PR8 (H7N9) with IC ₅₀ ranging from about 5.7 Nm to about 405 Nm in a microneutralization assay, for example, as shown in Example 4, or essentially similar analysis.

The present invention also includes antibodies or antigen-binding fragments thereof that mediate complement-dependent cytotoxicity of infected cells characterized by an EC ₅₀ value of 1 to 40 nM with a maximum lysis of 78.3% (see Example 5). In one embodiment, the antibodies or antigen-binding fragments thereof mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of infected cells, as demonstrated by a reporter assay, and the ability to lyse cells having HA using peripheral blood mononuclear cells (PBMCs). In the reporter assay, the EC ₅₀ was 0.8714 nM for H1H14611N2 and 0.6882 nM for H1H14612N2. In the PBMC type assay, the EC ₅₀ was 0.1463 nM for H1H14611N2 and 0.1762 nM for H1H14612N2. (See example 6).

The present invention also includes anti-influenza A virus group 2 HA antibodies that exhibit enhanced protective function or potent neutralization of influenza A virus infection in vivo. Certain antibodies exhibit potent neutralization when administered for therapeutic purposes (following infection; see Example 7). In one embodiment, a single dose of H1H14611N2 at 7 mg/kg or 15 mg/kg administered 96 hours postinfection resulted in 80% and 100% survival in mice (respectively) when administered for therapeutic purposes. One percent survival was also observed when certain exemplary antibodies (H1H14611N2 and H1H14612N2) were administered as a single dose of 15 mg/kg at 24, 48, or 72 hours postinfection. In one embodiment, H1H14611N2 demonstrated an additional protective effect in influenza virus-infected mammals when combined with the antiviral drug oseltamivir.

In one embodiment, the present invention provides an isolated recombinant antibody or antigen binding fragment thereof that specifically binds to influenza A virus group 2 HA, wherein the antibody has two or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to influenza A virus group 2 HA with a dissociation constant (KD) of less than 10 ^-8 M, as measured by surface plasmon resonance; (c) exhibits a dissociative half-life ( ^t1 / ₂ ) of greater than 75 minutes; (d) demonstrates neutralization of group 2 influenza A viruses selected from the H3N2 and H7N9 strains having an IC ₅₀ of less than about 200 nM and 500 nM, respectively; (e) demonstrates complement-mediated lysis of influenza virus-infected cells having an EC ₅₀ value of less than about 150 nM; (f) demonstrates antibody-dependent cell-mediated cytotoxicity to virus-infected target cells using a reporter bioassay, characterized by an EC ₅₀ value of less than about 0.9 nM; (g) exhibits antibody-dependent cell-mediated cytotoxicity to virus-infected target cells in the presence of human peripheral blood mononuclear cells (PBMCs) characterized by an EC ₅₀ value of less than about 0.180 nM; (h) demonstrates an increase in the survival of an animal infected with influenza virus when administered 24, 48, 72 or 96 hours after infection with the virus; (i) demonstrates increased survival of an animal infected with influenza virus when administered in combination with oseltamivir 96 hours after infection; or (j) wherein the antibody or antigen-binding fragment contains three defining

- 21 044791 complementarity of the heavy chain CDR region (HCDR1, HCDR2 and HCDR3) contained in any of the heavy chain variable region (HCVR) sequences listed in table. 1; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in any of the heavy chain variable region (LCVR) sequences listed in table. 1.

Antibodies of the present invention may have two or more of the above biological characteristics or any combinations thereof. Other biological characteristics of the antibodies of the present invention will be apparent to one skilled in the art from a review of the present invention, including the practical examples herein.

Epitope mapping and related techniques.

The present invention includes antibodies to influenza A virus group 2 HA that react with one or more amino acids found in one or more domains of the influenza A virus group 2 HA molecule. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within the viral HA molecule influenza (for example, a linear epitope in the domain). Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the influenza A virus group 2 HA molecule (eg, a conformational epitope).

Various techniques known to those skilled in the art can be used to determine whether an antibody interacts with one or more amino acids within a polypeptide or protein. Exemplary techniques include, for example, standard epitope cross-competition assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide digestion crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids in the polypeptide with which the antibody interacts is hydrogen-deuterium exchange, detected using mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding of the antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water and the exchangeable protons in the amino acids that are protected by the antibody complex undergo reverse deuterium-hydrogen exchange at a lower rate than the exchangeable protons in the amino acids that are not part of the interaction surface. As a result, amino acids that form part of the protein/antibody interaction surface can retain deuterium and thus exhibit relatively higher masses compared to amino acids not included in the interaction surface. After antibody dissociation, the target protein is subjected to protease digestion and mass spectrometric analysis, which identifies deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Chemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73. 256A-265A.

The term epitope refers to a site on an antigen that B and/or T cells react with. B-cell epitopes can be formed from both continuous amino acids and non-continuous amino acids brought together by folding the protein into a tertiary structure. Epitopes formed by continuous amino acids are generally retained when exposed to denaturing solvents, while epitopes formed by folding into a tertiary structure are typically lost when exposed to denaturing solvents. Typically, an epitope includes at least 3, and more often at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-assisted profiling (MAP), also known as antigen structure-modified antibody profiling (ASAP), is a method of categorizing large numbers of monoclonal antibodies (mAbs) directed against the same antigen based on the similarity of each antibody's binding profile to a chemical or enzymatically modified antigen surfaces (see US 2004/0101920, which is specifically incorporated herein by reference in its entirety). Each category may have a unique epitope that is either clearly different from or overlaps with an epitope present in another category. This technology enables the rapid selection of genetically identical antibodies so that characterization can be focused on genetically dissimilar antibodies. When used in relation to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to sort the antibodies of the present invention into groups of antibodies that bind different epitopes.

In certain embodiments, anti-influenza A virus group 2 HA antibodies or antigen-binding fragments thereof bind to an epitope within any one or more regions present in the influenza virus HA, either in a naturally occurring or recombinantly produced form, or with a fragment of it.

The present invention also includes antibodies to influenza A virus group 2 HA that bind to the same epitope or part of an epitope. Likewise, the present invention also includes antibodies to influenza A virus group 2 HA that compete for binding to influenza A virus group 2 HA or a fragment thereof with any of the specific exemplary antibodies described herein. For example, the present invention includes antibodies to influenza A virus group 2 HA that cross-compete for binding to influenza A virus group 2 HA with one or more antibodies derived from the antibodies described in table. 1.

Using standard methods known in the art, one can readily determine whether an antibody binds to the same epitope as or competes with the influenza A virus group 2 HA reference antibody. For example, to determine whether a test antibody binds to the same epitope as an influenza A virus group 2 HA reference antibody of the present invention, allow the reference antibody to bind to influenza A virus group 2 HA or peptide under saturating conditions. The ability of the test antibody to bind to the influenza A virus group 2 HA molecule is then assessed. If the test antibody is able to bind to the influenza A virus group 2 HA molecule after saturating binding to the influenza A virus group 2 HA reference antibody, it can be concluded that the test antibody binds to a different epitope than the epitope to which the influenza A virus group 2 HA reference antibody binds. On the other hand, if the test antibody is unable to bind influenza A virus group 2 HA after saturating binding to the influenza A virus group 2 HA reference antibody A, then the test antibody can bind to the same epitope as the epitope that is bound by the reference anti-influenza A virus group 2 HA antibody of the present invention.

To determine whether there is competition for binding to the reference antibody to the group 2 HA of the influenza A virus, the binding assay described above is carried out in two directions: in the first direction, it is possible to bind the reference antibody to the group 2 HA of the influenza A virus under saturating conditions with subsequent assessment of the binding of the test antibody to the HA molecule of group 2 of the influenza A virus. In the second direction, it is possible to bind the test antibody to the HA of group 2 of the influenza A virus under saturating conditions, followed by assessment of the binding of the reference antibody to the HA molecule of group 2 of the influenza A virus. If in both directions, only the first (saturating conditions) antibody is capable of binding to the group 2 HA molecule of influenza A virus, then it is concluded that the test antibody and the reference antibody compete for binding to the group 2 HA molecule of influenza A virus. One skilled in the art will understand that an antibody that competes for binding to a reference antibody may not necessarily bind to an identical epitope as the reference antibody, but may spatially block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the other antibodies from binding to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of others by at least 50%, but preferably 75%, 90%, or even 99%, according to competitive binding assays (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the antigen that weaken or eliminate the binding of one antibody weaken or eliminate the binding of the other antibody. Two antibodies have an overlapping epitope if some amino acid mutations that weaken or eliminate the binding of one antibody weaken or eliminate the binding of the other antibody.

Additional routine experiments (eg, peptide mutations and binding assays) can be subsequently performed to confirm that the observed lack of binding of the test antibody is due to binding to the same epitope to which the reference antibody also binds, or that steric blocking (or other phenomenon) is responsible for the lack of observed binding. Experiments of this type can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay known in the art.

Immunoconjugates.

The present antibody encompasses a human monoclonal anti-influenza A virus group 2 HA antibody conjugated to a therapeutic moiety (immunoconjugate), such as a toxoid or antiviral drug for the treatment of an influenza virus infection. As used herein, the term immunoconjugate refers to an antibody that is chemically or biologically linked to a radioactive substance, cytokine, interferon, target or reporter fragment, enzyme, peptide or protein, or therapeutic agent. The antibody may be associated with a radioactive substance, a cytokine, an interferon, a target fragment or reporter fragment, an enzyme, a peptide or protein, or a therapeutic agent.

- 23 044791 anywhere along the entire length of a molecule that is so long that it is capable of binding to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second, different anti-influenza virus HA antibody. In certain embodiments, the antibody may be conjugated to an agent that is specific for cells infected with the virus. When selecting the type of therapeutic moiety that can be conjugated to an anti-influenza virus HA antibody, the condition being treated and the therapeutic effect to be achieved will be taken into account. Examples of suitable agents for forming immunoconjugates are known in the art; see for example WO 05/103081.

Polyspecific antibodies.

Antibodies of the present invention may be monospecific, bispecific or polyspecific.

Polyspecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains that are specific for multiple target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

Any of the multispecific antigen binding molecules of the present invention or variants thereof can be constructed using standard molecular biology techniques (eg, recombinant DNA and protein expression techniques) known to one skilled in the art.

In some embodiments, antibodies specific for influenza A virus group 2 HA are produced in a bispecific format (bispecific), in which variable regions that bind to distinct influenza A virus group 2 HA domains are linked together to impart dual-domain specificity to a single binding molecule. Accordingly, the resulting bispecificity properties may enhance the overall inhibitory efficacy against influenza A virus group 2 HA by both increasing binding specificity and avidity. Variable regions that are specific to individual domains (for example, segments of the N-terminal domain), or those that can bind to different regions within the same domain, are paired in a structural framework, allowing each region to simultaneously bind to separate epitopes, or to different areas within the same domain. In one example, to create bispecificity, heavy chain variable regions (VH) from a binding molecule with specificity for one domain are recombined with variable light chain ( _VL ) regions from a number of binding molecules with specificity for a second domain to identify non-cognate _VL partners that can pair with the original VH without disrupting the original VH specificity. In the same way, you can combine a single V _L segment (for example, V _L 1) with two different VH domains (for example, VH1 and VH2) to create a bispecific molecule consisting of two binding arms (VH1 - V _L 1 and VH2 - V _L 1). The use of a separate V _L segment reduces the complexity of the system and thereby simplifies and increases the efficiency of the cloning, expression and purification methods used to create bispecific molecules (see, for example, USSN13/022759 and US2010/0331527).

Alternatively, antibodies that bind multiple domains and a second target, such as, but not limited to, a second distinct anti-influenza A virus group 2 HA antibody, can be produced in a bispecific format using the techniques described herein or other techniques known to one of ordinary skill in the art. technology. Antibody variable regions that bind to different sites can be linked together with variable regions that bind to corresponding sites on, for example, an influenza virus to impart dual antigenic specificity within a single binding molecule. Accordingly designed bispecific molecules with these properties serve as dual function molecules. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for the outer part of the extracellular domain and paired on a structural framework, allowing each variable region to bind to distinct antigens.

An exemplary antibody in a bispecific format that can be used in the context of the present invention includes the use of a C _H 3 domain of a first immunoglobulin (Ig) and a C _H 3 domain of a second immunoglobulin Ig, wherein the C _H 3 domains of the first and second Ig are different from each other. at least one amino acid, and wherein the at least one amino acid difference impairs binding of the bispecific antibody to Protein A compared to a bispecific antibody that lacks the amino acid difference. In one embodiment, the first Ig C _H 3 domain binds Protein A, and the second Ig C _H 3 domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (IMGT exon numbering; H435R EU numbering). The second CH3 may additionally contain the modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that can be found within the second C _H 3: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; EU N384S, K392N and V422I) for IgG2 antibodies; and Q15R, N44S, K52N,

- 24 044791

V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies. The bispecific antibody format options described above are contemplated within the scope of the present invention.

Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, scFv or diabody based bispecific formats, IgG-scFv fusions, double variable domain (DVD)-Ig, quadroma, peak-valve, conventional lung chain (e.g. regular peak-to-trough light chain, etc.), CrossMab, CrossFab, (SEED)-antibody, leucine zipper, DuoBody, IgG1/IgG2, Dual Action Fab (DAF)-IgG and Mab ² bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and the sources cited herein for an overview of the above formats). Bispecies antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where non-naturally occurring amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valence and geometric shape. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic administration and formulations.

The present invention provides therapeutic compositions containing antibodies to group 2 HA of the influenza A virus or antigen-binding fragments thereof according to the present invention. Therapeutic compositions in accordance with the present invention are administered with suitable carriers, excipients and other agents that are included in the formulations to provide improved transport, delivery, tolerability and the like. Many suitable formulations can be found in a reference book well known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-oil emulsions and water-in-oil emulsions. , carbowax emulsions (polyethylene glycols with different molecular weights), semi-liquid gels and semi-liquid mixtures containing carbowax. See also Powell et al. Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238–311.

The dosage of the antibody may vary depending on the age and size of the subject to be administered, the target disease, conditions, route of administration, etc. When using the antibody of the present invention to treat a disease or disorder in an adult patient or to prevent a disease, it is advantageous to administer antibodies of the present invention, typically in a single dose of from about 0.1 to about 60 mg/kg body weight, more preferably from about 5 to about 60, from about 10 to about 50, or from about 20 to about 50 mg /kg body weight. Depending on the severity of the condition, the frequency of administration and duration of treatment can be adjusted. In certain embodiments, an antibody or antigen binding fragment thereof of the present invention may be administered as an initial dose of at least about 0.1 mg to about 5000 mg, about 1 to about 2000 mg, about 5 to about 1000 mg, or from about 10 to about 500 mg, to about 100 mg, or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a secondary dose or multiple subsequent doses of an antibody or antigen-binding fragment thereof in an amount that may be approximately the same as or less than the initial dose, wherein the repeat doses are separated by at least 1 day to 3 days. days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks or at least 14 weeks.

Various delivery systems are known that can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J Biol Chem 262:4429–4432). Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous integuments (for example, oral mucosa, rectal and intestinal mucosa, etc.), and it can be administered together with other biologically active means. Administration may be systemic or local. The pharmaceutical composition can also be delivered in a vial, particularly in a liposome (see, for example, Langer (1990) Science 249:1527-1533).

The use of nanoparticles for delivering the antibody of the present invention is also provided herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods for their preparation and use are described in detail in Arruebo, M., et al. 2009 (Antibody-conjugated nanoparticles for biomedical applications in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. Nanoparticles can be formulated and conjugated to antibodies contained in pharmaceutical compositions to target virus-infected cells. Nanoparticles for drug delivery have also been described in patent documents, for example, US 8257740 or US 8246995, each of which is incorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used. In yet another embodiment, the controlled release system can be placed near the target of the composition, requiring only a fraction of the systemic dose.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, intracranial, intraperitoneal and intramuscular injection, drip infusion, etc. These injectable preparations can be prepared using generally known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium commonly used for injection. The aqueous injection medium is, for example, physiological saline solution, isotonic solution containing glucose and other auxiliaries, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol ( for example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oil medium, for example, sesame oil, soybean oil and the like are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, gasoline alcohol and the like. The injectable composition obtained in this manner is preferably filled into a suitable ampoule.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with regard to subcutaneous delivery, when delivering the pharmaceutical composition of the present invention, a pen-type delivery device can be freely used. This pen-type delivery device can be reusable or disposable. A reusable pen-type delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition from the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and a new cartridge that contains the pharmaceutical composition placed. The pen-type delivery device can then be reused. In the case of a disposable pen-type delivery device, there is no replaceable cartridge. More specifically, the disposable pen-type delivery device is pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition reservoir is empty, the entire device is discarded.

For subcutaneous delivery of the pharmaceutical composition of the present invention, a variety of reusable pen syringes and auto-injector delivery devices are used. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG pen ™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of refillable pen delivery devices used for subcutaneous administration of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTA pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, Illinois), but only a few of them are mentioned.

Advantageously, the pharmaceutical compositions described above for oral or parenteral administration are prepared in dosage forms in a unit dose adapted to correspond to the dose of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is typically from about 5 to about 5000 mg per unit dose dosage form; Preferably, the antibody content in other dosage forms ranges from about 5 to about 500 mg and from about

- 26 044791 up to approximately 250 mg.

Types of use of antibodies in therapy.

The antibodies of the present invention are useful for treating and/or preventing a disease, disorder, or condition associated with an influenza virus infection and/or reducing the severity of at least one symptom associated with such a disease, disorder, or condition.

In certain embodiments, the antibodies of the present invention are useful for treating subjects suffering from severe and acute respiratory infection caused by influenza virus. In some embodiments, the antibodies of the present invention are useful in reducing viral titers or reducing viral load in a host. In one embodiment, an antibody or antigen binding fragment thereof of the present invention can be administered at a therapeutic dose to a patient with an influenza virus infection.

One or more antibodies of the present invention can be administered to alleviate, or prevent, or reduce the severity of one or more symptoms or conditions of a disease or disorder. Antibodies can be used to alleviate or reduce the severity of at least one symptom of an influenza virus infection, including, but not limited to, fever, cough, sore throat, headache, body aches, fatigue, severe exhaustion, shortness of breath, bronchitis, pneumonia and death.

Also provided herein is the prophylactic use of one or more antibodies of the present invention in subjects at risk of developing influenza virus infection, such as immunocompromised individuals, the elderly (over 65 years of age), children under 2 years of age, workers health care providers, family members of a patient with influenza virus infection, and patients with a history of associated disorders (eg, increased risk of pulmonary infection, cardiovascular disease, or diabetes).

In a further embodiment of the present invention, the antibodies of the present invention are used to prepare a pharmaceutical composition for treating patients suffering from an influenza virus infection. In another embodiment of the present invention, the antibodies of the present invention are used as an adjuvant therapy with any other agent or any other therapy known to those skilled in the art useful for treating or reducing the severity of an influenza virus infection.

Types of combination therapy.

Combination therapies may include an anti-influenza A virus group 2 HA antibody of the present invention and any additional therapeutic agent that may advantageously be combined with an antibody of the present invention or a biologically active fragment of an antibody of the present invention. The antibodies of the present invention can be synergistically combined with one or more drugs or agents (eg, antivirals) used to treat influenza.

For example, exemplary antiviral agents include, for example, vaccines, neuraminidase inhibitors, or nucleoside analogues. Other exemplary antiviral agents that may be used in combination with an antibody of the present invention may include, for example, zidovudine, ganciclovir, vidarabine, idoxuridine, trifluridine, foscarnet, acyclovir, ribavirin, amantadine, rimantadine, saquinavir, indinavir, ritonavir, alpha interferons and other interferons, a neuraminidase inhibitor (eg, zanamivir (RELENZA®), oseltamivir (TAMIFLU®), laninamivir, peramivir) or rimantadine.

Other exemplary antiviral drugs include, but are not limited to, an HA inhibitor, a sialic acid inhibitor, and an M2 ion channel inhibitor. In one embodiment, the M2 ion channel inhibitor is amantadine or rimantadine.

In some embodiments, the antibodies of the present invention can be combined with a second therapeutic agent to reduce the viral load in a patient with an influenza virus infection or to reduce the severity of one or more symptoms of the infection.

The antibodies of the present invention can be used in combination with an anti-inflammatory drug (eg, corticosteroids and non-steroidal anti-inflammatory drugs), a decongestant, an antihistamine drug, an antimicrobial drug, another anti-influenza virus antibody, an antiviral drug, an influenza vaccine such as FLUMIST ® or FLUVIRIN®, a dietary supplement such as antioxidants, or any other palliative therapy for the treatment of influenza virus infection.

In certain embodiments, the second therapeutic agent is another influenza virus antibody. In certain embodiments, the second therapeutic agent is another anti-influenza hemagglutinin antibody. In certain variants

- 27 044791 implementation of the second therapeutic agent is another antibody to another influenza virus protein, such as neuraminidase or the tetrameric ectodomain of matrix protein 2 (M2e protein). In certain embodiments, the second therapeutic agent is an antibody to another protein, such as transmembrane serine protease 2 (TMPRSS2). The second antibody may be specific for one or more different influenza virus proteins from different subtypes or strains of the virus. This document presents the use of a combination (cocktail) of antibodies with a broad spectrum of neutralizing or inhibitory activity against the influenza virus. In some embodiments, non-competing antibodies can be combined and administered to a subject in need thereof to reduce the ability of the influenza virus to escape through rapid mutation as a result of selective pressure. In some embodiments, antibodies comprising the combination bind to different non-overlapping epitopes on the HA protein. Antibodies containing the combination can block virus attachment and/or entry into host cells and/or fusion with host cells. The antibodies may react with hemagglutinin selected from any one or more subtypes of influenza A group 2, including subtypes H3, H4, H7, H10, H14 and H15, and when used alone or in combination with any one or more of the agents indicated above, can neutralize any one or more subtypes of group 2 influenza virus, including without limitation the following: H3N2, H7N9.

Also provided herein is the use of a combination of anti-influenza A virus group 2 HA antibodies of the present invention, wherein the combination contains one or more antibodies that do not cross-compete; in some embodiments, the combination includes a first antibody with broad spectrum neutralizing activity with a second antibody with activity against a narrow range of isolates and that does not compete with the first antibody.

As used herein, in combination with means that the additional therapeutically active component(s) may be administered prior to, concurrently with, or subsequent to the administration of the influenza A virus group 2 HA antibody of the present invention. Expression in combination with also includes sequential or co-administration of an anti-influenza virus HA antibody and a second therapeutic agent.

The additional therapeutically active component(s) may be administered to the subject prior to administration of the influenza A virus group 2 HA antibody of the present invention. For example, the first component may be considered administered before the second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before or less than 1 minute before introducing the second component. In other embodiments, additional therapeutically active component(s) may be administered to a subject following administration of the influenza A virus group 2 HA antibody of the present invention. For example, the first component may be considered administered after the second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after or 72 hours after administration second component. In yet other embodiments, additional therapeutically active component(s) may be administered to a subject concomitantly with administration of the influenza A virus group 2 HA antibody of the present invention. Coadministration for purposes of the present invention includes, for example, administering to a subject an anti-HA influenza A virus group 2 antibody and an additional therapeutically active component in a single dosage form or in separate dosage forms administered to the subject within approximately 30 minutes or less of each other. When administered in separate dosage forms, each dosage form is administered via the same route (eg, both influenza A virus group 2 HA antibodies and an additional therapeutically active component can be administered intravenously, etc.); alternatively, each dosage form may be administered by different routes (eg, the anti-influenza A virus group 2 HA antibody may be administered intravenously, and the additional therapeutically active component may be administered orally). Either way, all administrations of components in a single dosage form, in separate dosage forms, or in separate dosage forms by different routes are considered simultaneous administration for the purposes of the present invention. For purposes of this disclosure, administration of an anti-influenza A virus group 2 HA antibody before, concurrently with, or after (as those terms are defined herein above) administration of an additional therapeutically active component is considered to be administration of an anti-influenza A virus group 2 HA antibody in combination with an additional therapeutic agent. active component.

The present invention includes pharmaceutical compositions in which the influenza A virus group 2 HA antibody of the present invention is formulated in conjunction with one or more additional therapeutically active component(s) described elsewhere herein.

- 28 044791

Administration schemes.

In certain embodiments, a single dose of an anti-influenza A virus group 2 HA antibody of the present invention (or a pharmaceutical composition comprising a combination of an anti-influenza A virus group 2 HA antibody and any additional therapeutically active agents mentioned herein) can be administered to the subject in need. In accordance with certain embodiments of the present invention, multiple doses of an anti-influenza A virus group 2 HA antibody of the present invention (or a pharmaceutical composition comprising a combination of an anti-influenza A virus group 2 HA antibody and any additional therapeutically active agents mentioned herein ) can be administered to a subject over a period of time. Methods in accordance with this aspect of the present invention involve sequentially administering to a subject multiple doses of the influenza A virus group 2 HA antibody of the present invention. As used herein, sequential administration means that each dose of influenza A virus group 2 HA antibody is administered to a subject at different points in time, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that comprise sequentially administering to a patient one initial dose of an influenza A virus group 2 HA antibody, then one or more secondary doses of an influenza A virus group 2 HA antibody, and then optionally one or more tertiary doses of an influenza A virus group 2 HA antibody. influenza A virus.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the influenza A virus group 2 HA antibody of the present invention. Thus, the starting dose is the dose that is administered at the beginning of the treatment regimen (also referred to as the initial dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. Each of the initial, secondary and tertiary doses may contain the same amount of anti-influenza A virus HA antibody, but may generally differ in frequency of administration. However, in certain embodiments, the amount of influenza A virus group 2 HA antibody contained in the primary, secondary, and/or tertiary doses differs from each other (eg, adjusted upward or downward as necessary) over the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered at less frequent intervals (eg, maintenance doses).

In certain illustrative embodiments of the present invention, each secondary and/or tertiary dose is administered over a period of 1 to 48 hours (e.g., 1.11/2, 2.21/2, 3, 31/2, 4, 41/2, 5 , 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131 /2, 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2 , 22, 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2 or more) after the immediately previous dose. The phrase immediately preceding dose, as used herein, means, in a sequence of multiple administrations, a dose of influenza A virus group 2 HA antibody administered to a patient prior to administration of the next next dose in the sequence, with no intervening doses.

Methods in accordance with this aspect of the present invention may involve administering to a patient any number of secondary and/or tertiary doses of an influenza A virus group 2 HA antibody antagonist. For example, in certain embodiments, only one secondary dose is administered to a patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In certain embodiments of the present invention, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of the treatment regimen. The frequency of administration may be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical examination.

Types of use of antibodies in diagnostics.

The anti-influenza A virus group 2 HA antibodies of the present invention can be used to detect and/or measure influenza A virus group 2 HA in a sample, for example, for diagnostic purposes. In some embodiments, one or more antibodies of the present invention are used in assays to detect a disease or disorder, such as a viral infection. Exemplary diagnostic assays for influenza A virus group 2 HA may involve, for example, contacting a patient-derived sample with an anti-influenza A virus group 2 HA antibody of the present invention, wherein the anti-influenza A virus group 2 HA antibody is labeled with a detectable label or molecule - reporter or used as a capture ligand to selectively isolate influenza A virus group 2 HA from patient samples. Alternatively, an unlabeled anti-influenza A virus group 2 HA antibody can be used diagnostically in combination with

- 29 044791 with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter molecule may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure influenza A virus group 2 HA in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in diagnostic assays for influenza A virus group 2 HA in accordance with the present invention include a sample of any tissue or fluid obtained from a patient that contains detectable amounts of either influenza A virus group 2 HA or fragments thereof in normal or pathological conditions. states. Typically, influenza A virus group 2 NA levels in a specific sample obtained from a healthy patient (e.g., a patient not suffering from influenza-associated illness) can be measured to initially determine a baseline or standard influenza A virus group 2 NA level. This baseline influenza A virus group 2 NA level can then be compared to influenza A virus group 2 NA levels measured in samples obtained from individuals suspected of having or having an influenza A virus group 2 NA associated condition or symptoms associated with such a condition.

Antibodies specific for influenza A virus group 2 HA may contain no additional tags or fragments, or they may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the location of the tag (if present) can determine the orientation of the peptide relative to the surface to which the peptide is bound. For example, if the surface is coated with avidin, then the peptide containing N-terminal biotin will be oriented such that the C-terminal part of the peptide is located distal to the surface.

Examples

The following examples are set forth to provide those skilled in the art with complete disclosure and description of how to carry out and use the methods and preparation and use of the compositions of the present invention, and are not intended to limit the scope of what the inventors of the present invention consider their invention to be. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantity, temperature, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is approximately 25° C., and pressure is at or near atmospheric.

Example 1. Preparation of human antibodies to hemagglutinin (HA) of group 2 influenza A virus.

Human antibodies to influenza A virus group 2 HA were generated using the VELOCIMMUNE® mouse containing DNA encoding the variable regions of the human immunoglobulin heavy chain and kappa light chain. Mice were immunized with A/Hong Kong/08/1968 (H3N2), then A/Hong Kong/05/1972-PR8-X36 (H3N2), and then with A/Hong Kong/08/1968 (H3N2). All mice were boosted with a 1:1 mixture of DNA encoding HA from A/Wisconsin/67/X-161/2005 (H3N2) and A/chicken/Netherlands/01/2003 (H7N7). The humoral immune response was monitored using an immunoassay specific for influenza A virus group 2 HA. Once the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to maintain their viability and generate hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce antibodies to influenza A virus group 2 HA. Using this technique and the various immunogens described above, several chimeric antibodies (i.e., antibodies having human variable domains and rodent constant domains).

Also, anti-influenza A virus group 2 HA antibodies were isolated directly from antigen-positive murine B cells without fusion with myeloma cells, as described in US Pat. No. 7,582,298, which is specifically incorporated herein by reference in its entirety. Using this method, several fully human antibodies specific for influenza virus HA (ie, antibodies having human variable domains and human constant domains) were obtained.

Two exemplary antibodies described herein are designated H1H14611N2 and H1H14612N2.

The biological properties of illustrative antibodies produced in accordance with the methods of this example are described in detail in the examples below.

Example 2. Nucleotide and amino acid sequences of the variable regions of the heavy and light chains.

- 30 044791

In table 1 shows the amino acid sequence identifiers of the CDRs and variable regions of the heavy and light chains of selected antibodies to group 2 HA of the influenza A virus of the present invention. The corresponding nucleic acid sequence identifiers are given in Table. 2.

Table 1

Amino acid sequence identifiers

SEQ ID NO: Antibody designation HCV R HCD RI HCD R2 HCD R3 LCV R LCD RI LCD R2 LCD R3 H1H14611N2 2 4 6 8 10 12 14 16 H1H14612N2 18 20 22 24 26 28 thirty 32

table 2

Nucleic acid sequence identifiers

SEQ ID NO: Antibody designation HCV R HCD RI HCD R2 HCD R3 LCV R LCD RI LCD R2 LCD R3 H1H14611N2 1 3 5 7 9 eleven 13 15 H1H14612N2 17 19 21 23 25 27 29 31

In this document, antibodies are generally referred to according to the following nomenclature: the Fc prefix (e.g., H1H, H2M, etc.) is followed by a numerical identifier (e.g., 14611, 14612, etc., as shown in Table 1 or 2 ), followed by the suffix P, P2, N, N2, or B. Thus, according to this nomenclature, the antibody may be referred to herein as, for example, H1H14611N2, H1H14612N2, etc. The prefixes NS or H2M in antibody names used herein indicate the specific isotype of the Fc region of the antibody. For example, antibody H1M has a mouse IgG1 Fc, and antibody H2M has a mouse IgG2 Fc (isotype a or b) (all variable regions are fully human, as indicated by the first 'H' in the antibody designation). One skilled in the art will appreciate that an antibody with a specific isotype Fc can be converted into an antibody with a different isotype Fc (e.g., an antibody with a mouse IgG1 Fc can be converted into an antibody with a human IgG4, etc.), but in any case, the variable domains (including CDR) - which are shown by the numeric identifiers shown in the table. 1 and 2 will remain the same and the antigen binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain.

Example 3. Binding affinities and kinetic constants for monoclonal antibodies to group 2 HA of influenza A virus.

Binding affinities and kinetic constants for human monoclonal antibodies to influenza A virus group 2 HA were determined by surface plasmon resonance using an antigen trap format. Measurements were carried out on a Biacore device. In this format, the surface of the Biacore high-density touch sensor was derivatized by amino immobilization with a mouse monoclonal anti-human Fc antibody to capture antibodies to influenza A virus group 2 HA expressed with human Fc constant regions. The association of all HA proteins with each of the captured mAbs was monitored using standard methods. The foldon domain proteins used in this experiment were obtained from BEI Resources or Influenza Reagent Resource (IRR), as shown in Table. 3 below. Equilibrium binding dissociation constants (KD) and dissociation half-periods (t1/ ₂ ) were calculated based on the kinetic rate constants:

ka K _D (M) = - kd and t!/2 (min.) = (2) x kd

The binding kinetics parameters for HA proteins with surface-bound anti-HA antibodies at 37°C are presented in Table. 3.

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Table 3

Binding affinity and kinetic parameters for the binding of H1H14611N2 to foldon domain HA proteins at 37°C

Group 1 Group 2 A/Brisbane/59/2007 (Hl) ΒΕΙ Resources (NR28607) A/California/04/20 09 (Hl) BEI Resource s (NR15749) A/Puerto Rico/08/1 934 (Hl) BEI Resource (NR19240) A/Vietna m/1203/2 004 (H5) IRR (FR-39) A/Indonesi a/05/2005 (H5) IRR (FR-59) A/Turkey/Wis consin/01/196 6(H9) BEI Resources (NR-43782) A/Hiroshi ma/52/200 5 (H3) IRR (FR-63) A/Shangh ai/01/2013 (H7) BEI Resources (NR44365) K _D tl/2 K _D tl/2 K _D tl/2 K _D tl/2 K _D tl/2 K _D tl/2 K _D tl/2 K _D tl/2 shаb mol/l min. mol b/l MIN. mo l/ L MIN. mol b/l MIN. mol/l MIN. mol/L MIN. mol/L MIN. mol/l MIN. Н1Н146 11N2 (group 2, specificity) N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. 8.22E-09 86 ^IC§ IC Buffer N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B. N.B.

IC§: inconclusive result NB: no binding Results H1H14611N2 binds group 2 H3 HA with high affinity (Table 3). The equilibrium dissociation constant (KD value) was in the lower nM range for A/Hiroshima/52/2005 (H3N2; KD = 8.22E ^-09 ). H1H14611N2 was not associated with group 1 strains.

Example 4: H1H14611N2 and H1H14612N2 effectively neutralize a wide range of group 2 influenza A viruses.

Exemplary monoclonal antibodies (mAbs) H1H14611N2 and H1H14612N2 were selected for in vitro microneutralization assays using two different formats to assess antibody spectrum and activity.

Cell viability in microneutralization assay.

Monoclonal antibodies were tested in a microneutralization assay to evaluate spectrum and activity. Briefly, Madin-Darby canine kidney (MDCK) cells were seeded at 6.0 x 10 ³ cells/well in a 96-well plate. Only virus diluent was added to background control wells. Cells were incubated for 4-5 hours at 37°C with 5% CO ₂ . Monoclonal antibodies were diluted at 4 times the final concentration in virus diluent. Antibodies were diluted 1:3 in two parallels. The virus was thawed on ice and diluted to the appropriate predetermined concentration. The diluted virus was added to the diluted mAbs. The virus-mAb mixture was immediately added to MDCK cells and incubated at 37°C with 5% CO ₂ for 72 hours. Wells with virus control and control uninfected cells were also included. On day 4, the plates were centrifuged at 1200 rpm for 3 minutes. Cells were lysed using 100 μl of CelTiter-Glo substrate and ATP release was measured by luminescence (Victor X3, PerkinElmer). The percentage of viability was determined in comparison with an uninfected control. Viability indices were analyzed using nonlinear 4PL regression to determine IC ₅₀ (GraphPad Prism).

NA microneutralization assay.

Briefly, Madin-Darby canine kidney (MDCK) cells were seeded and incubated overnight, achieving 80–100% confluency the next day. Monoclonal antibodies were diluted in viral medium (VIM) to 50 μg/ml and diluted 1:2 in triplicate or quadruple dilutions. H5N1 A/Vietnam/1203/2004 or H1N1 A/California/07/2009 was diluted in VIM and added to the diluted antibodies and incubated for 1 h. The samples were then transferred to MDCK and incubated for 48 h. After incubation, 50 μl of the supernatant was transferred into a new 96-well plate. Diluted turkey or horse erythrocytes were added to the supernatant and incubated at room temperature for 30 or 60 min. The hemagglutination titer was recorded as the reciprocal of the last dilution that completely inhibited hemagglutination.

Results.

The results showed that H1H14611N2 and H1H14612N2 neutralized a range of diverse influenza A virus group 2 isolates in microneutralization assays. IC50 (nM) for H1H14611N2 and H1H14612N2 in

- 32 044791 regarding the neutralization of three different influenza strains (A/Aichi/02/1968-PR8-X31(H3N2), A/Philippines/01/1982 (H3N2) and A/Shanghai/01/2013-PR8 (H7N9)) are shown in table 4, dose-response curves for strains including A/Aichi/02/1968-PR8-X31 (H3N2), A/Phillipines/02/1982 (H3N2) and A/Shanghai/01/2013 (H7N9) are also shown respectively in Fig. 1A, B and C (H1H14611N2 shown by triangles; H1H14612N2 shown by inverted triangles; nonspecific hIgG1 shown by diamonds). The results of additional neutralization studies of influenza virus strain A/Victoria/316/2011 (H3N2) using hemagglutination data are shown in Table. 5. Given in table. Value 5 is the lowest mAb concentration (nM) at which RBC hemagglutination was not detected. A negative control hIgG1 was also included in these studies.

Table 4

Summary information on the neutralizing activity of group 2 anti-HA mAbs against several strains of influenza virus ( _IC50 , nM)

shаb A/Aichi/02/19 68-PR8-X31 (H3N2) A/Philippines/01/1 982 (H3N2) A/Shanghai/01/20 13-PR8 (H7N9) H1H14611N2 7,830 68.37 404.7 H1H14612N2 5,700 37.60 >6666 Non-target/negative control hlgGl >833 >3333 >6666

Data are presented from one of at least three studies showing similar results.

Table 5

Summary of neutralizing activity of group 2 HA mAb against influenza virus strain A/Victoria/316/2011 (H3N2)

mAb Concentration (nM) H1H14611N2 41.7 H1H14612N2 166.7

Example 5 H1H14611N2 and H1H14612N2 mediate the killing of cells infected with group 2 HA virus through complement-dependent cytotoxicity (CDC).

Exemplary monoclonal antibodies (mAbs) H1H14611N2 and H1H14612N2 were selected for in vitro complement-dependent cytotoxicity (CDC) assays. Briefly, Madin-Darby canine kidney (MDCK) cells were infected with A/Aichi/02/1968-PR8-X31 (H3N2) at a multiplicity of infection (MOI) of 3 30 hours before analysis. Target cells were combined with the indicated mAbs at the indicated concentrations. Healthy human serum complement (NHSC) was prepared three times at a final concentration (15%) in CDC assay medium. Percent cytotoxicity was measured using CytoTox-Glo (Promega). To determine the maximum and background lysis values, background controls with lysis detergent and blood serum were used. In fig. 2 shows H1H14611N2 (shown as triangles), H1H14612N2 (shown as inverted triangles) along with nonspecific hlgG1 (shown as diamonds) as a control. The percentage of specific lysis was calculated as ((lysis caused by mAb+NHSC) - (lysis caused by NHSC only))/((maximum lysis caused by detergent) - (lysis caused by NHSC only)) x 100.

Results.

H1H14611N2 and H1H14612N2 exhibited a dose-dependent increase in the ability to specifically lyse virus-infected cells when tested in the CDC assay with A/Aichi/02/1968-PR8-X31(H3N2)-infected MDCKs in the presence of complement protein-retained human serum. The results of one representative CDC analysis of three are shown in FIG. 2. A nonspecific/negative control hlgG1 was also used in this experiment. H1H14611N2 and H1H14612N2 were effective in mediating cytolysis with maximum lysis of 78.3% and 86.3% and EC ₅₀ values of 140 nM and 126 nM, respectively.

Example 6 H1H14611N2 and H1H14612N2 effectively kill cells expressing group 2 HA through antibody-dependent cell-mediated cytotoxicity (ADCC).

Exemplary monoclonal antibodies H1H14611N2 and H1H14612N2 were selected to test their ability to specifically lyse cells surface expressing HA in two antibody-dependent cell-mediated cytotoxicity assays.

A. FcyRIIIA activation assay.

To test the ability of the H1H14611N2 and H1H14612N2 antibodies to specifically lyse cells overexpressing HA, an FcyRIIIA activation assay was initially used using the ADCC-Glo reporter bioassay (Promega) with 3T3 cells overexpressing

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A/Wisconsin/67/200 (H3N2). Target cells were combined with the indicated mAbs at the indicated concentrations. Target and effector cells were then combined at an E:T ratio of 5:1. In fig. Figure 3 shows the results of this analysis using H1H14611N2 (shown as triangles),

H1H14612N2 (shown as inverted triangles) together with nonspecific hlgG1 (shown as diamonds) as a control.

B. ADCC assay using donor human peripheral blood mononuclear cells (PBMCs).

Two exemplary group 2 HA antibodies, H1H14611N2 and H1H14612N2, were further tested in an ADCC assay using human PBMC. PBMCs were isolated from donor human buffy coats and stimulated with IL-2 (5 ng/ml) for 10-12 hours before use. Target 3T3 cells overexpressing A/Wisconsin/67/2005 (H3N2) were combined with the indicated mAbs at the indicated concentrations. Target and effector cells were then combined at an E:T ratio of 30:1. Percent cytotoxicity was measured using CytoTox-Glo (Promega). In fig. Figure 4 shows the results of this assay using H1H14611N2 (shown as circles), H1H14612N2 (shown as inverted triangles) along with nonspecific hlgG1 (shown as diamonds) as a control.

Results.

H1H14611N2 and H1H14612N2 showed a dose-dependent increase in the ability to specifically lyse cells surface expressing HA in both ADCC assays.

H1H14611N2 and H1H14612N2 showed a dose-dependent increase in FcyyyR III A activation in the ADCC-Glo reporter bioassay (Promega) with 3T3 cells overexpressing A/Wisconsin/67/2005 (H3N2) (Fig. 3). The EC ₅₀ value for H1H14611N2 was 0.8714 nM and for H1H14612N2 - 0.6882 nM.

ADCC activity was confirmed using direct PBMC-mediated cytotoxicity of infected and overexpressing cells. The results of using human donor PBMCs in the H1H14611N2- and H1III4612Ni-mediated ADCC assay against 3T3 cells overexpressing A/Wisconsin/67/2005 (H3N2) are shown (Fig. 4). The EC ₅₀ value for H1H14611N2 was 0.1463 nM and for H1H14612N2 - 0.1762 nM.

All experiments using donor PBMCs used an effector to target (E:T) ratio of 30:1, whereas an E:T ratio of 5:1 was used in the reporter bioassay.

Example 7 Selected monoclonal antibodies specific for influenza A virus group 2 hemagglutinin are effective in vivo in the treatment of fatal influenza virus infection.

There is a significant unmet need for improved standards of care to treat or prevent influenza virus infections in humans. Currently, only two classes of drugs are available: adamantanes and neuraminidase inhibitors (NAIs). Adamantanes (amantadine and rimantadine) have been associated with the rapid emergence of drug-resistant strains and are no longer recommended for the treatment of influenza. NAIs, such as oseltamivir (TAMIFLU®), are first-line medications for the treatment and prevention of influenza, but their range of effectiveness is limited: NAIs have been shown to shorten the duration of fever and illness symptoms by approximately one day per therapeutic plan if the antiviral the drug is administered within 48 hours of symptom onset, but has little clinically proven effectiveness when administered after 48 hours.

To evaluate the in vivo effectiveness of H1H14611N2 and H1H14612N2 in the treatment of severe influenza, experiments were carried out taking into account the following objectives:

study 1: evaluate the efficacy of a single dose of H1H14611N2 and H1H14612N2.

Study 2: Determine the effectiveness of H1H14611N2 when administered in combination with oseltamivir.

The strain used in these studies was a mouse-adapted influenza A virus group 2 isolate A/Aichi/02/1968-X31 (H3N2). All experiments were performed on 6-week-old female wild-type mice (BALB/c). Mice were infected with 5 x MLD ₅₀ (10,000 PFU) A/Aichi/02/1968-X31 (H3N2). In treatment models, mice were challenged intranasally (i/n) on day 0 and fixed doses according to the manufacturer's instructions and mice were dosed every 12 h (i.e., twice daily, BID) by gavage for 5 days, at In this case, the first dose was administered on day 4. Mice were weighed and monitored daily until day 14 p.i. and then sacrificed when they had lost 20% of their initial weight. Results are presented as percentage of survival.

The first experiment compared the efficacy of either a single dose of H1H14611N2 (15 mg/kg; shown by triangles) or H1H14612N2 (15 mg/kg; shown by inverted triangles) starting at 24, 48, or 72 hours postinfection using 5 X MLD ₅₀ A/Aichi/02/1968-X31 (H3N2) to evaluate the effect in a mouse model at these time points. All mice receiving 15 mg/kg isotype control at 24 hours postinfection

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Claims (18)

died by day 7 (shown in Fig. 5A only as a solid gray line); in contrast, all mice receiving a single dose (15 mg/kg) of H1H14611N2 or H1H14612N2 survived dosing at 24 (Fig. 5A), 48 (Fig. 5B), or even 72 hours (Fig. 5C) postinfection . In the second experiment, mice received a single subeffective dose of 7 mg/kg (shown by squares, dotted line) or 15 mg/kg H1H14611N2 (shown by circles, dotted line), control IgG (shown by triangles), 2 mg/kg oseltamivir BID for 5 days (shown as diamonds, solid lines) or a combination of a single dose of 7 (shown as squares, solid line) or 15 mg/kg H1H14611N2 (shown as circles, solid line) and oseltamivir for 5 days 96 hours post-infection using 5 x MLD ₅₀ A /Aichi/02/1968-X31 (H3N2) (see Fig. 6). Single doses of 15 mg/kg and 7 mg/kg H1H14611N2 respectively produced 100% and 80% survival, and 80% of mice treated with 2 mg/kg oseltamivir alone survived. The combination of H1H14611N2 with oseltamivir increased survival to 100% in both H1H14611N2 dose groups (15 mg/kg or 7 mg/kg), demonstrating that this combination allows the use of lower doses of H1H14611N2 to achieve sustained treatment effect (Figure 6). In summary, H1H14611N2 and H1H14612N2 demonstrated consistent efficacy in treating mice infected with a historical strain of influenza virus, and furthermore, H1H14611N2 demonstrated efficacy when administered in combination with oseltamivir. CLAIM 1. An isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to hemagglutinin (HA) of group 2 influenza A virus, wherein the antibody or antigen-binding fragment thereof contains three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained in a heavy chain variable region (HCVR) containing the amino acid sequence shown under SEQ ID NO: 2 or 18; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) containing the amino acid sequence shown under SEQ ID NO: 10 or 26. 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in an HCVR containing the amino acid sequence given under SEQ ID NO: 2; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in the LCVR containing the amino acid sequence set forth in SEQ ID NO: 10. 3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in an HCVR containing the amino acid sequence shown under SEQ ID NO: 18; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in the LCVR containing the amino acid sequence set forth in SEQ ID NO: 26. 4. An isolated antibody or an antigen-binding fragment thereof according to claim 1, where the antibody or an antigen-binding fragment thereof: (a) is a fully human monoclonal antibody; (b) binds to influenza A virus group 2 HA with a dissociation constant (K _D ) of less than 10 ^-8 M, as measured by surface plasmon resonance; (c) exhibits a dissociative half-life (t1/ ₂ ) of greater than 75 minutes; (d) demonstrates neutralization of group 2 influenza A viruses selected from the H3N2 and H7N9 strains, characterized by an IC ₅₀ value of less than 200 nM and 500 nM, respectively; (e) demonstrates complement-mediated lysis of influenza virus-infected cells having an EC ₅₀ value of less than about 150 nM; (f) demonstrates antibody-dependent cell-mediated cytotoxicity against virus-infected target cells using a reporter bioassay, characterized by an EC50 of less than about 0.9 nM; (g) exhibits antibody-dependent cell-mediated cytotoxicity to virus-infected target cells in the presence of human peripheral blood mononuclear cells (PBMCs) characterized by an EC50 of less than about 0.180 nM; (h) demonstrates an increase in the survival of an animal infected with influenza virus when administered 24, 48, 72 or 96 hours after infection with the virus; and/or (i) demonstrates increased survival of an animal infected with influenza virus when administered in combination with oseltamivir 96 hours after infection. 5. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein, when administered to a mammal infected with influenza virus as a single intravenous dose of from about 7 to 15 mg/kg, starting on day 3 after infection, the mammal is protected from said influenza virus. infection to a greater extent than a mammal infected with influenza virus - 35 044791 melting patient who was orally administered oseltamivir twice daily for 5 days at a dose of approximately 2 mg/kg, starting on day 3 postinfection and continuing until day 7 postinfection. 6. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein when administered to mammals infected with influenza virus as a single dose of approximately 15 mg/kg, 96 hours after infection, said mammals have a survival rate of approximately 100% . 7. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein when administered to mammals infected with influenza virus as a single dose of approximately 15 mg/kg, said mammals exhibit a survival rate of approximately 100%. 8. The isolated antibody or antigen-binding fragment thereof according to claim 1, where when administered to a mammal infected with influenza virus, an additional protective effect is manifested in the mammal when administered together with oseltamivir 96 hours after infection. 9. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein when administered to a mammal infected with influenza virus as a single intravenous dose ranging from about 7 mg/kg to about 15 mg/kg, there is an additional protective effect in the mammal when administered intravenously. combination with oseltamivir orally twice daily for 5 days at a dose of approximately 2 mg/kg. 10. An isolated antibody or antigen binding fragment thereof according to claim 1, comprising an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 18. 11. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10 or 26. 12. An isolated antibody or its antigen-binding fragment according to claim 1, containing: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 20; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 22; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 24; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 28; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 30; (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 32. 13. An isolated antibody or its antigen-binding fragment according to claim 12, containing: (a) HCDR1 containing the amino acid sequence of SEQ ID NO:4, (b) HCDR2 containing the amino acid sequence of SEQ ID NO:6; (c) HCDR3 containing the amino acid sequence of SEQ ID NO:8; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 12; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 14 and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 16. 14. An isolated antibody or its antigen-binding fragment according to claim 12, containing: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 20, (b) HCDR2 containing the amino acid sequence of SEQ ID NO: 22; (c) HCDR3 containing the amino acid sequence of SEQ ID NO: 24; (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 28; (e) LCDR2 containing the amino acid sequence of SEQ ID NO: 30 and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 32. 15. The isolated antibody or antigen-binding fragment thereof according to claim 1, comprising HCVR, which contains the amino acid sequence shown under SEQ ID NO: 2 and LCVR, which contains the amino acid sequence shown under SEQ ID NO: 10. 16. An isolated antibody or antigen-binding fragment thereof according to claim 1, comprising an HCVR that contains the amino acid sequence set forth in SEQ ID NO: 18 and an LCVR that contains the amino acid sequence set forth in SEQ ID NO: 26. 17. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof prevents the influenza virus from attaching to and/or entering the host cell. 18. A pharmaceutical composition containing an isolated antibody or an antigen-binding fragment thereof according to claim 1, which binds to the HA of an influenza virus, and a pharmaceutically acceptable carrier or diluent. -