HK1237800B - Antibodies useful in passive influenza immunization, and compositions, combinations and methods for use thereof - Google Patents

Description

Antibodies useful for passive influenza immunization and compositions, combinations, and methods of use thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application was filed as a PCT International Application on February 4, 2015, and claims the benefit of priority to U.S. Provisional Application No. 61/935,746, filed on February 4, 2014, and U.S. Provisional Application No. 62/051,630, filed on September 17, 2014, the entire contents of each of which are incorporated herein by reference.

Citations for sequence listings submitted via EFS-WEB

The complete contents of the electronic submission of the following sequence listing via the USPTO-EFS-WEB server (as authorized and set forth in MPEP 1730 II.B.2(a)(C)) are hereby incorporated by reference in their entirety for all purposes. The sequence listing is identified on the electronically submitted text file as follows:

File name Creation Date Size (bytes) 9079004_1.txt February 4, 2015 285.473

Technical Field

Antibodies, compositions, and methods for treating and preventing influenza viruses are provided. Antibodies and antigen-binding fragments that bind to the consensus sequence of influenza hemagglutinin A near the HA ₀ mature cleavage site are provided. Antibody compositions, combinations, and methods for effective passive immunization across influenza A and influenza B virus strains are also provided.

background

Influenza is a major cause of death and illness, affecting the upper and lower respiratory tract. There are three types of influenza viruses, influenza A, B, and C. Influenza A and influenza B viruses cause seasonal epidemics of illness in humans. Influenza C infection causes mild respiratory illness and is not considered to cause epidemics. Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (H) and neuraminidase (N). There are 17 different hemagglutinin subtypes and 10 different neuraminidase subtypes. Influenza A viruses can be further divided into different strains. The current subtypes of influenza A viruses commonly found in humans are influenza A (H1N1) virus and influenza A (H3N2) virus. Influenza B viruses are not divided into subtypes, but are divided into two different lineages: B/Victoria/2/87-like and B/Yamagata/16/88-like. Influenza A viruses (such as H1N1), influenza A viruses (such as H3N2), and influenza B viruses are all included in the annual flu vaccine.

Five types of clinically relevant influenza viruses (three types of influenza A and two types of influenza B) are currently prevalent in the population with varying frequencies, and influenza A viruses are divided into two different phylogenetic groups 1 and 2. Group 1 includes hemagglutinin subtypes H1, H2, H5, H6, H8, H9, H11, H13 and H16. Group 2 includes H3, H4, H7, H10, H15 and H14. The currently relevant popular influenza A viruses of group 1 are H1 subtypes, which are further divided into subtypes in human and pig sources, and the relevant popular viruses of group 2 are H3 subtypes at present. Influenza A viruses are responsible for most seasonal diseases, and H3 viruses dominate 8 influenza seasons (CDC seasonal influenza in the past 12 influenza seasons in the U.S.; U.S. monitoring data). In 1968, H3 viruses caused one of 3 pandemic influenzas in the 20th century, and since then H3 viruses have remained the important material for people's diseases. Except people, H3 influenza viruses also infect birds, pigs and horses conventionally. Influenza B viruses have been circulating in humans for over 100 years, and current strains are divided into two lineages: the Yamagata lineage and the Victoria lineage. Recently, the trivalent influenza vaccine has been expanded to include a quadrivalent antigen-containing vaccine covering both lineages of influenza B, as well as H1 and H3 viruses.

Current prevention and treatment of influenza are inadequate and can be ineffective. Despite widespread vaccination, susceptibility to influenza persists. Factors contributing to susceptibility include: (1) incomplete vaccination coverage, such as for the 2009 H1N1 influenza pandemic, when vaccine shortages were widespread, (2) years when vaccine formulations did not well represent circulating strains, such as in 2008, (3) reduced vaccination efficacy in the elderly, with average efficacy ranging from 40-50% at age 65 to only 15-30% after age 70, and (4) the emergence of pandemic strains that are not represented in seasonal vaccines, with the H5N1 virus being of particular concern. In addition, resistance to currently available antiviral therapies for the treatment of influenza has become a serious problem. Resistance to the adamantanes (amantadine and rimantadine), drugs that act on the M2 protein and inhibit viral integration, increased from 1.9% in mid-2004 to 14.5% during the first six months of the 2004-2005 influenza season and is now over 90% (Sheu, T.G. et al. (2011) J Infect Dis 203:13-17). Resistance to oseltamivir phosphate, an antiviral drug that inhibits the influenza neuraminidase protein, increased dramatically from 1-2% of H1N1 viruses during the 2006-2007 influenza season to 12% in 2007-2008 and exceeded 99% of seasonal H1N1 viruses in 2009. Fortunately, the 2009 pandemic H1N1 strain was susceptible to Tamiflu, which may result in fewer deaths. Therefore, there is an overwhelming need for new influenza prevention/treatment methods.

Unfortunately, determining the diagnosis of influenza virus strains typically requires a report time of 12-24 hours, which leads to unfavorable treatment delays if the strain needs to be determined to select appropriate therapy. Therefore, there is still a need for antibodies that combine with multiple clades and show enhanced affinity therefor. In particular, a passive vaccine comprising antibodies that are effective against influenza A and influenza B and have a wide range of reactivity with multiple strains is desirable to avoid the need for exhaustively characterizing infectious viruses before administering antibodies or antibody mixtures. Broadly reactive antibodies and compositions that are effective against all strains of influenza A and B are desirable, particularly because prior strain diagnosis is not necessary before treatment. High efficacy is further desired to facilitate the production and administration of reagents.

Overview

In some embodiments, recombinant human antibodies or antigen-binding fragments thereof that are reactive with the major influenza B lineages - the Yamagata and Victoria clades - are provided.

In some embodiments, monoclonal antibodies that bind with high affinity to trimers representing influenza B are provided.

In some embodiments, antibodies are provided that are capable of conferring passive immunity in the event of infection, for example, by a previously unidentified strain of influenza B virus or a strain against which seasonal vaccines (which do not include actual strains in circulation approximately every 2-3 years) do not confer protection: Monto, A.A., et al., Vaccine (2009) 27:5043–5053.

In some embodiments, antibodies are provided that bind to many strains (indicating targeting of essential sites), and thus may bind even strains not previously encountered. Such antibodies are also useful for ameliorating or preventing infection in subjects or reducing their toxicity in subjects for whom vaccination does not produce a fully protective response or who are at high risk due to potentially impaired bronchial function, such as in patients with chronic obstructive pulmonary disease or patients with weakened immune systems (e.g., very young patients, elderly patients, transplant patients, and cancer- or HIV-chemotherapy-treated patients).

In some embodiments, compositions comprising a cocktail of mAbs that confer broad passive immunity are provided.

In some embodiments, compositions are provided comprising: (1) one or more binding moieties, monoclonal antibodies, or immunoreactive fragments thereof, which have reactivity with two major influenza B lineages, and (2) one or more binding moieties, monoclonal antibodies, or immunoreactive fragments thereof, which have reactivity with influenza A viruses of Group 1 (including H1, H2, H5, H6, H8, H9, H11, H13, H16) and/or Group 2 (including H3 and H7 as model samples), including those binding moieties, monoclonal antibodies, or immunoreactive fragments thereof that exhibit group cross-reactivity. In some embodiments, the antibodies provided herein specifically bind to an epitope contained in the HA ₀ protein and recognize the native trimeric form of HA, as well as the proteolytically activated form.

In another embodiment, provided are binding portions selected from bispecific and multispecific antibodies and fragments thereof that are capable of expanding the range of virus types and evolutionary branches that can be specifically bound, and compositions comprising two or more portions that react with multiple strains having characteristics of influenza A and influenza B.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided that exhibits a binding affinity (KD) of 10 nM or tighter or 3 nM or tighter to one or more strains of each of the influenza B Yamagata and influenza B Victoria clades. In some embodiments, the antibody or fragment is a recombinant antibody or fragment thereof.

In some embodiments, the antibody or antigen-binding fragment is an engineered monospecific antibody or a multispecific human monoclonal antibody.

In some embodiments, antibodies or antigen-binding fragments are provided that neutralize infection of cells by one or more strains of the influenza B Yamagata and influenza B Victoria clades.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided, comprising the amino acid sequence of (a) a heavy chain complementary determining region 1 (HCDR1), (b) a heavy chain complementary determining region 2 (HCDR2), and (c) a heavy chain complementary determining region 3 (HCDR3), wherein HCDR1/HCDR2/HCDR3 are selected from the group consisting of: SEQ ID NO: NO:31/32/33; 41/42/43; 51/52/53; 61/62/63; 71/72/73; 81/82/83; 91/92/93; 101/102/103; 111/112/113 ;121/122/123;131/132/133;141/142/143;151/152/153;161/162/163;171/172/173;181/182/183;191/1 92/193; 201/202/203; 211/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies or fragments having the property of binding to and/or inhibiting influenza virus.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided, comprising (a) a light chain complementary determining region 1 (LCDR1), (b) a light chain complementary determining region 2 (LCDR2), and (c) a light chain complementary determining region 3 (LCDR3), wherein LCDR1/LCDR2/LCDR3 are selected from the group consisting of: SEQ ID NO: NO:34/35/36; 44/45/46; 54/55/56; 64/65/66; 74/75/76; 84/85/86; 104/105/106; 114/115/16; 124/125 /126;134/135/136;144/145/146;154/155/156;164/165/166;174/175/176;184/185/186;194/195/19 6; 204/205/206; 214/215/216; 224/225/226; 234/235/236; 244/245/246; 254/255/256; 264/265/266; 274/275/276 and 284/285/286, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies or fragments having the property of binding to and/or inhibiting influenza virus.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided, comprising heavy and light chain CDR sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 selected from the group consisting of: SEQ ID NO: 31/32/33/34/35/36; 41/42/43/44/45/46; 51/52/53/54/55/56; 61/62/63/64/65/66; 71/72/73/74/75/76; 81/82/83/84/85/86; 91/92/93/94/95/96; 101/102/103/104/105/106; 111/112/113 13/114/115/116; 121/122/123/124/125/126; 131/132/133/134/135/136; 141/142/143/144/145/146; 151/152/153/154/155/156; 161/162/163/164/165/166; 171/172/173/174/175/176; 181/ 182/183/184/185/186; 191/192/193/194/195/196; 201/202/203/204/205/206; 211/212/213/214/215/216; 221/222/223/224/225/226; 231/232/233/234/235/236; 241/242/243/244/245/246 ; 251/252/253/254/255/256; 261/262/263/264/265/266; 271/272/273/274/275/276; and 281/282/283/284/285/286; or highly homologous variants thereof having 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies or fragments having the property of binding to and/or inhibiting influenza virus.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided that specifically binds to one or more strains of each of the influenza B Yamagata and influenza B Victoria clades and comprises a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159, 169, 179, 189, 199, 209, 219, 229, 239, 249, 259, 269, 279 and 289, or a homologous variant thereof having at least 80% sequence identity to said HCVR; said variant and said antibody or fragment having properties of binding to and/or inhibiting influenza virus.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided, comprising a light chain variable sequence (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, and 290, or a homologous variant thereof having at least 80% sequence identity to said LCVR, said variant and said antibody or fragment having the property of binding to and/or inhibiting influenza virus.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided that comprises a HCVR/LCVR sequence pair selected from the group consisting of: 39/40, 49/50, 59/60, 69/70, 79/80, 89/90, 99/100, 109/110, 119/120, 129/130, 139/140, 149/150, 159/160, 169/170, 179/180, 189/190, 200/2100, 211/220, 229/230, 239/240, 249/250, 259/260, 269/270, 279/280, 289/300, 290/310 9/180, 189/190, 199/200, 209/210, 219/220, 229/230, 239/240, 249/250, 259/260, 269/270, 279/280 and 289/290, or homologous variants thereof having at least 80% sequence identity to said HCVR and/or LCVR; said variants and said antibodies having the property of binding to and/or inhibiting influenza virus.

In some embodiments, an isolated human antibody or antigen-binding fragment thereof is provided, comprising a HCVR/LCVR pair selected from the group consisting of SEQ ID NO: 79/80, 129/130, 219/220, 229/230, 239/240, 249/250, and 269/270, or a homologous variant thereof having at least 80% sequence identity to said HCVR and/or LCVR; said variant and said antibody having the property of binding to and/or inhibiting influenza virus.

In some embodiments, isolated human antibodies or antigen-binding fragments thereof are provided that bind to one or more epitopes selected from the group consisting of SEQ ID NOs: 307, 308, 309, 310, 311, 312, and 313, or discontinuous epitopes thereof.

In some embodiments, isolated human antibodies or antigen-binding fragments thereof are provided that compete with an antibody or fragment as disclosed herein for binding to one or more strains of each of the influenza B Yamagata and influenza B Victoria clades, displacing at least 50% of the latter when present in less than a 100-fold excess.

In some embodiments, isolated codon-optimized nucleic acid molecules encoding the antibodies or fragments of the present disclosure are provided. In some embodiments, expression vectors comprising nucleic acid molecules encoding the antibodies or fragments of the present disclosure are provided.

In some embodiments, a host cell for expressing a recombinant polypeptide comprising an expression vector comprising a nucleic acid molecule encoding an antibody or fragment of the disclosure is provided.

In some embodiments, a method of producing an anti-influenza B antibody or an antigen-binding fragment thereof is provided, the method comprising growing a host cell under conditions that allow production of the antibody or fragment thereof, and recovering the produced antibody or fragment thereof.

In some embodiments, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment that exhibits a binding affinity (KD) of 10 nM or tighter or 3 nM or tighter for one or more strains of each of the influenza B Yamagata and influenza B Victoria clades and a pharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition comprising an antibody or antigen-binding fragment and a pharmaceutically acceptable carrier is provided, wherein the antibody or antigen-binding fragment comprises an amino acid sequence of HCDR1/HCDR2/HCDR3 selected from the group consisting of SEQ ID NO: NO:31/32/33; 41/42/43; 51/52/53; 61/62/63; 71/72/73; 81/82/83; 91/92/93; 101/102/103; 111/112/113; 121/122/12 3;131/132/133;141/142/143;151/152/153;161/162/163;171/172/173;181/182/183;191/192/193;201/202/203;21 1/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies or fragments having the property of binding to and/or inhibiting influenza virus, wherein said antibody or fragment exhibits a first melting temperature (Tm1) greater than or equal to 55°C as measured in PBS.

In some embodiments, compositions are provided comprising one or more human antibodies or antigen-binding fragments having binding specificity for at least one strain of influenza A. In some aspects, the antibodies or antigen-binding fragments having specificity for influenza A include specificity for one or more strains of Group 1 and Group 2.

In some embodiments, a pharmaceutical composition is provided, comprising: (a) a first antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, 13; and a light chain amino acid sequence comprising a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, 16, or a highly homologous variant thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, wherein the variant is capable of binding to and/or inhibiting influenza virus; and (b) a second antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, 23, and a light chain amino acid sequence comprising a light chain amino acid sequence comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 21, 22, 23. NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain variable region (LCVR), or a highly homologous variant thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, the variant being capable of binding to and/or inhibiting influenza virus; and (c) a third antibody or antigen-binding fragment thereof, comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3, and a light chain amino acid sequence comprising a light chain variable region (LCVR) comprising a light chain CDR domain sequence LCDR1/LCDR2/LCDR3, wherein the HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 are respectively selected from:

(i) SEQ ID NO: 71, 72, 73, and SEQ ID NO: 74, 75, 76;

(ii) SEQ ID NO:91, 92, 93, and SEQ ID NO:94, 95, 96;

(iii) SEQ ID NOs: 101, 102, 103, and SEQ ID NOs: 104, 105, 106;

(iv) SEQ ID NOs: 121, 122, 123, and SEQ ID NOs: 124, 125, 126;

(v) SEQ ID NOs: 181, 182, 183, and SEQ ID NOs: 184, 185, 186;

(vi) SEQ ID NOs: 191, 192, 193, and SEQ ID NOs: 194, 195, 196;

(vii) SEQ ID NOs: 201, 202, 203, and SEQ ID NOs: 204, 205, 206;

(viii) SEQ ID NOs: 211, 212, 213, and SEQ ID NOs: 214, 215, 216;

(ix) SEQ ID NO: 221, 222, 223, and SEQ ID NO: 224, 225, 226;

(x) SEQ ID NO: 231, 232, 233, and SEQ ID NO: 234, 235, 236;

(xi) SEQ ID NO: 241, 242, 243, and SEQ ID NO: 244, 245, 246;

(xii) SEQ ID NO: 261; 262, 263, and SEQ ID NO: 264, 265, 266; or

(xiii) SEQ ID NO: 271, 272, 273, and SEQ ID NO: 274, 275, 276,

or a highly homologous variant thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, wherein the variant and the antibody or fragment are capable of binding to and/or inhibiting influenza virus.

In some embodiments, the first antibody or fragment comprises the HCVR/LCVR pair of SEQ ID NO: 19/20, or a highly homologous variant thereof comprising at least 80% sequence identity in one or both of the HCVR or LCVR domain sequences; and the variant and the antibody or fragment have the property of binding to and/or inhibiting influenza virus.

In some embodiments, the second antibody or fragment comprises the HCVR/LCVR pair of SEQ ID NO: 29/30, or a highly homologous variant thereof comprising at least 80% sequence identity in one or both of the HCVR or LCVR domain sequences; and said variant and said antibody or fragment have the property of binding to and/or inhibiting influenza virus.

In some embodiments, the third antibody or fragment comprises a HCVR/LCVR pair selected from the group consisting of SEQ ID NO: 79/80, 99/100, 109/110, 129/130, 189/190, 199/200, 209/210, 219/220, 229/230, 239/240, 249/250, 269/270, and 279/280, or a highly homologous variant thereof comprising at least 80% sequence identity in one or both of the HCVR or LCVR domain sequences; and the variant and the antibody or fragment have properties of binding to and/or inhibiting influenza virus.

In some embodiments, the third antibody or fragment comprises a HCVR/LCVR pair selected from the group consisting of SEQ ID NO: 79/80, 129/130, 219/220, 229/230, 239/240, 249/250, and 269/270, or a highly homologous variant thereof comprising at least 80% sequence identity in one or both of the HCVR or LCVR domain sequences, and the variant and the antibody or fragment have properties of binding to and/or inhibiting influenza virus. In some embodiments, a composition comprising the first, second, and third antibodies or fragments is provided, wherein the antibodies or fragments exhibit an isoelectric point (pi) that is within 2 pi points.

In a specific embodiment, a composition comprising a first antibody, a second antibody, and a third antibody is provided, wherein the first antibody is TRL053/Mab53 comprising the HC/LC amino acid sequence of SEQ ID NO: 17/18, the first antibody is TRL053/Mab53 comprising the HC/LC amino acid sequence of SEQ ID NO: 17/18, NO: 27/28 HC/LC amino acid sequence antibody TRL579/Mab579, the third antibody is selected from the group consisting of: TRL847, TRL845, TRL849, TRL848, TRL846, TRL854, TRL809 and TRL832, respectively comprising HC/LC amino acid sequences selected from the group consisting of 227/228, 207/208, 247/248, 237/238, 217/218, 267/268, 77/78 and 127/128, or highly homologous variants thereof comprising at least 80% sequence identity in one or both of the HC or LC domain sequences; the variants and the antibodies or fragments have the property of binding to and/or inhibiting influenza virus.

In a specific embodiment, a composition is provided comprising each of the first, second, and third antibodies or fragments of the present disclosure, each of which is formulated in a single dose in an amount effective for treating or preventing influenza A and influenza B infection or disease in a subject in need thereof.

In a specific embodiment, a composition comprising the first, second and third antibodies or fragments of the present invention is provided in an amount of 100 mg/kg or less per dose of each of the first, second and third antibodies or fragments; in an amount of 10 mg/kg or less per dose of each of the first, second and third antibodies or fragments; or in an amount of 1 mg/kg or less per dose of each of the first, second and third antibodies or fragments. In some embodiments, each of the first, second and third antibodies or fragments is each present in the composition in an amount of 10 mg/kg or less of the total antibody or antibody fragment per dose.

In specific embodiments, compositions comprising carriers, diluents, and/or excipients for nasal or pulmonary delivery are provided.

In some embodiments, a composition is provided, comprising an antibody or fragment of the present disclosure and further comprising one or more of an antiviral therapeutic, a viral replication inhibitor, a protease inhibitor, a polymerase inhibitor, a hemagglutinin inhibitor, a bronchodilator, or an inhaled corticosteroid. In some embodiments, the immunomodulator is interferon beta 1a; the antiviral therapeutic is a neuraminidase inhibitor selected from the group consisting of oseltamivir, zanamivir, peramivir, and laninamivir; the antiviral therapeutic is an RNA polymerase inhibitor selected from the group consisting of favipiravir (T-705) and VX 787; the antiviral therapeutic is a host cell targeted therapeutic selected from the group consisting of influenza enzymes (Das181) and AB-103 (p2TA); the antiviral therapeutic is an ion channel inhibitor selected from the group consisting of rimantadine and amantadine; and the bronchodilator is selected from albuterol, levalbuterol, or famoterol.

In some embodiments, a composition is provided comprising each of the first, second and third antibodies or fragments of the present disclosure in an amount effective for treating or preventing influenza A and influenza B infection or disease in a subject in need thereof, wherein one or more of the antibodies or antigen-binding fragments thereof is an antibody fragment selected from Fab, Fab' and F(ab') ₂ , scFv, dAb or a multispecific antibody, comprising a HCDR1/HCDR2/HCDR3 amino acid sequence selected from the group consisting of: 11/12/13, 21/22/23, 71/72/73, 74/75/76, 91/92/93, 101/102/103, 121/122/123, 181/182/183, 191/192/19 3, 201/202/203, 211/212/213, 221/222/223, 231/232/233, 241/242/243, 261/262/263 and 271/272/273, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies having the property of binding to and/or inhibiting influenza virus.

In some embodiments, a method for treating or preventing influenza infection in a subject is provided, the method comprising administering to the subject (a) a first antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, 13, and a light chain amino acid sequence comprising a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, 16, or a highly homologous variant thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, wherein the variant and the antibody or fragment are capable of binding to and/or inhibiting influenza virus; and (b) a second antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, 23, and a light chain amino acid sequence comprising a light chain amino acid sequence comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 24, 25, 26. NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain variable region (LCVR), or SEQ ID NO: 74, 75, 76 highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies or fragments being capable of binding to and/or inhibiting influenza virus; and (c) a third antibody or antigen-binding fragment thereof comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3, and a light chain amino acid sequence comprising a light chain variable region (LCVR) comprising light chain CDR domain sequence LCDR1/LCDR2/LCDR3, said HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 being selected from the group consisting of: (i) SEQ ID NO: 71, 72, 73, and SEQ ID NO: 74, 75, 76; (ii) SEQ ID NO: 91, 92, 93, and SEQ ID NO: ID NO:94,95,96; (iii) SEQ ID NO:101,102,103, and SEQ ID NO:104,105,106; (iv) SEQ ID NO:121,122,123, and SEQ ID NO:124,125,126; (v) SEQ ID NO:181,182,183, and SEQ ID NO:184,185,186; (vi) SEQ ID NO:191,192,193, and SEQ ID NO:194,195,196; (vii) SEQ ID NO:201,202,203, and SEQ ID NO:204,205,206; (viii) SEQ ID NO:211,212,213, and SEQ ID NO:214,215,216; (ix) SEQ ID NO: 221, 222, 223, and SEQ ID NO: 224, 225, 226; (x) SEQ ID NO: 231, 232, 233, and SEQ ID NO: 234, 235, 236; (xi) SEQ ID NO: 241, 242, 243, and SEQ ID NO: 244, 245, 246; (xii) SEQ ID NO: 261; 262, 263, and SEQ ID NO: 264, 265, 266 or (xiii) SEQ ID NO: 271, 272, 273, and SEQ ID NO: 274, 275, 276, or a highly homologous variant thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variant and said antibody or fragment being capable of binding to and inhibiting influenza virus. In some embodiments, each of said first, second, and third antibodies or fragments is administered simultaneously or sequentially.

In some embodiments, an anti-influenza composition of the present disclosure is administered to a subject, wherein the influenza infection status of the subject is determined without exhaustive viral strain determination.

In some embodiments, an anti-influenza composition of the present disclosure is administered to a subject, wherein the subject is protected from influenza infection or disease.

As is well known in the art, non-immunoglobulin based proteins can have similar epitope recognition properties as antibodies and may also provide suitable embodiments, including binding agents based on fibronectin, transferrin, and lipocalin. Nucleic acid based moieties, such as aptamers, also have these binding properties.

In other embodiments, the present invention relates to pharmaceutical compositions and formulations comprising the binding moieties of the invention, particularly those compositions and formulations in which the binding moieties are covalently or non-covalently associated with erythrocytes in a manner that promotes distribution from the blood to the lungs, and to methods of using the binding moieties of the invention to passively inhibit viral infection in a subject (as a prophylactic agent in a normal population or in a subject that may have been exposed to the virus, or as a therapeutic agent in an already infected subject). The invention also relates to recombinant materials and methods for producing antibodies or fragments, including the use of these recombinant materials and methods for in situ production of antibodies or fragments in a subject.

In some embodiments, a combination of binding molecules, particularly human monoclonal antibodies or fragments thereof, that bind to influenza virus and are effective against influenza virus are provided, wherein the combination is effective against Group 1 influenza A virus, Group 2 influenza A virus, and influenza virus B. The combination of antibodies is effective in treating or preventing influenza A and B viruses, thereby providing an effective agent against all related and circulating influenza viruses in a single composition or dose.

In some embodiments, compositions comprising monoclonal antibodies are provided, each monoclonal antibody binding to influenza virus with low nM or sub-nM affinity. Combination compositions are provided comprising antibodies or antibody fragments specifically directed against Group 1 influenza A virus, antibodies or binding fragments specifically directed against Group 2 influenza A virus, and antibodies or binding fragments specifically directed against influenza B virus (including Yamagata and Victoria B lineages).

In some embodiments, a combination of antibodies is provided comprising a first antibody directed against Group 1 influenza A, in more specific embodiments, particularly at least H1 influenza virus, a second antibody directed against Group 2 influenza A, in more specific embodiments, particularly at least H3 influenza virus, and a third antibody directed against influenza B virus, in more specific embodiments, particularly the Yamagata and Victoria lineages.

In some embodiments, compositions are provided comprising or consisting of a combination of influenza monoclonal antibodies or fragments thereof that are effective in combination for the treatment or prevention of influenza A and influenza B.

In one embodiment, the invention comprises an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO: 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159, 169, 179, 189, 199, 209, 219, 229, 239, 249, 259, 269, 279 and 289, or a homologous sequence thereof having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In another embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 79, 99, 109, 129, 189, 199, 209, 219, 229, 239, 249, 269 and 279. In another embodiment, the antibody or fragment thereof comprises a HCVR comprising SEQ ID NO: 209, 229, 239, 249, or 269.

In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, and 290, or a substantially homologous sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In another embodiment, the antibody or antigen-binding portion of an antibody comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 80, 100, 110, 130, 190, 200, 210, 220, 230, 240, 250, 270, or 280. In another embodiment, the antibody or fragment thereof comprises a LCVR comprising SEQ ID NO: 210, 230, 240, 250, or 270.

In one embodiment, the invention comprises a composition comprising an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) selected from the group consisting of one or more of SEQ ID NOs: 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159, 169, 179, 189, 199, 209, 219, 229, 239, 249, 259, 269, 279, and 289, or a homologous sequence thereof having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In another embodiment, the composition comprises an antibody, or antigen-binding fragment thereof, comprising a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 99, 109, 129, 189, 199, 209, 219, 229, 239, 249, 269, and 279. In another embodiment, the composition comprises an antibody, or fragment thereof, comprising a HCVR comprising SEQ ID NOs: 209, 229, 239, 249, or 269.

In one embodiment, the invention comprises a composition comprising an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 and 290, or a homologous sequence thereof having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In another embodiment, the composition comprises an antibody, or an antigen-binding portion thereof, comprising a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 80, 100, 110, 130, 190, 200, 210, 220, 230, 240, 250, 270, or 280. In another embodiment, the composition comprises an antibody, or fragment thereof, comprising a LCVR comprising SEQ ID NO: 210, 230, 240, 250, or 270.

In one embodiment, the invention comprises a composition comprising a B antibody (anti-B type) or fragment as provided herein and an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) selected from the group consisting of one or more of SEQ ID NOs: 19 and 29, or a homologous sequence thereof having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In one embodiment, the invention comprises a composition comprising a B antibody or fragment as provided herein and an antibody or antigen-binding fragment of an antibody comprising a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 20 and 30, or a homologous sequence thereof having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In more specific embodiments, the composition comprises at least one B antibody and at least one or at least two A antibodies.

In some embodiments, an antibody or antigen-binding fragment comprising HC and LC variable region amino acid sequences is provided, wherein the HCVR comprises a HCDR1/HCDR2/HCDR3 amino acid sequence selected from any one of the following sequences: SEQ ID NO: 31/32/33; 41/42/43; 51/52/53; 61/62/63; 71/72/73; 81/82/83; 91/92/93; 101/102/103; 111/112/113; 121/122/123; 131/132/133; 141/142/143; 151/152/153; 161/162/163; 171/172/173; 181/182/183; 191/192/193; 2 01/202/203; 211/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, and the LCVR comprises a LCDR1/LCDR2/LCDR3 amino acid sequence selected from any one of the following sequences: SEQ ID NO: NO:34/35/36; 44/45/46; 54/55/56; 64/65/66; 74/75/76; 84/85/86; 104/105/106; 114/115/16; 124/125/126; 134/135/136; 144/145/146; 154/155/156; 164/165/166; 174/175/176; 184/185/ 284/285/286, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences.

In some embodiments, antibodies or antigen-binding fragments comprising Fc modifications to extend serum half-life are provided. Such modifications are disclosed in Xencor Xtend ^™ antibody engineering technology (Xencor, Melbourne, AU). In some aspects, antibodies or fragments comprising Fc modifications show an amino acid sequence having a sequence identity of greater than 80%, greater than 90%, greater than 95%, or greater than 99% to SEQ ID NO: 297, 17, 2737, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, or 287.

In some embodiments, without being bound by theory, because the model Fab is used in an inhaled formulation, an antigen binding fragment that is a scFv is provided. Such engineered antibodies do not exist in nature. In some embodiments, an engineered antibody or binding fragment is provided that comprises a HCDR1/HCDR2/HCDR3 amino acid sequence selected from any one of the following sequences: SEQ ID NO: 31/32/33; 41/42/43; 51/52/53; 61/62/63; 71/72/73; 81/82/83; 91/92/93; 101/102/103; 111/112/113; 121/122/123; 131/132/133; 141/142/143; 151/152/153; 161/162/163; 171/172/173; 181 /182/183; 191/192/193; 201/202/203; 211/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences.

In some embodiments, codon-optimized nucleic acids for expressing antibodies and fragments provided herein are provided. In some embodiments, such non-naturally occurring nucleic acids are used to produce higher expression levels. In some embodiments, codon-optimized nucleic acids are used to produce recombinant antibodies or fragments provided herein.

In some embodiments, an antibody or antigen-binding fragment comprising an HC and LC variable region amino acid sequence is provided, wherein the HCVR comprises a HCDR1/HCDR2/HCDR3 amino acid sequence selected from any one of the following sequences: SEQ ID NO: 31/32/33; 41/42/43; 51/52/53; 61/62/63; 71/72/73; 81/82/83; 91/92/93; 101/102/103; 111/112/113; 121/122/123; 131/132/133; 141/142/143; 151/152/153; 161/162/163; 171/172/173; 181/182/183; 191/192/193; 2 01/202/203; 211/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, and the LCVR comprises a LCDR1/LCDR2/LCDR3 amino acid sequence selected from any one of the following sequences: SEQ ID NO: NO:34/35/36; 44/45/46; 54/55/56; 64/65/66; 74/75/76; 84/85/86; 104/105/106; 114/115/16; 124/125/126; 134/135/136; 144/145/146; 154/155/156; 164/165/166; 174/175/176; 184/185/ 284/285/286, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences.

In some embodiments, antibodies or antigen-binding fragments comprising Fc modifications to extend serum half-life are provided. Such modifications are disclosed in Xencor Xtend ^™ antibody engineering technology (Xencor, Melbourne, AU). In some aspects, antibodies or fragments comprising Fc modifications exhibit an amino acid sequence having greater than 80%, greater than 90%, greater than 95%, or greater than 99% sequence identity to SEQ ID NO: 297, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, or 287.

In some embodiments, an engineered antibody or binding fragment is provided comprising a HCDR1/HCDR2/HCDR3 amino acid sequence selected from any one of the following sequences: SEQ ID NO: 31/32/33; 41/42/43; 51/52/53; 61/62/63; 71/72/73; 81/82/83; 91/92/93; 101/102/103; 111/112/113; 121/122/123; 131/132/133; 141/142/143; 151/152/153; 161/162/163; 171/172/173; 181 /182/183; 191/192/193; 201/202/203; 211/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences.

In some embodiments, codon-optimized nucleic acids for expressing the antibodies and fragments provided herein are provided. In some embodiments, such non-naturally occurring nucleic acids are used to produce higher expression levels. In some embodiments, codon-optimized nucleic acid molecules are used to produce recombinant antibodies and fragments provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the classification of influenza A and B viruses known in the art into groups of important clades, modified from Suzuki, Y. et al., Mol. Biol. Evol. (2002) 19:501–509.

Figure 2 shows representative strains within two influenza B clades, Yamagata and Victoria, from Dreyfus, C. et al., Science (2012) 337:1343–1348. Representative Yamagata clade strains include Mississippi/04/2008, Florida/4/2006, Jilin/20/2003, Houston/B60/1997, Harbin/7/1994, and Nashville/45/1991. Representative Victoria clade strains include Malaysia/2506/2004, Mississippi/07/2008, Brisbane/60/2008, and Ohio/01/2005.

Fig. 3 shows the table 7 of the recombinant HA ELISA data with the reactivity of several anti-influenza B mAbs and two anti-influenza A mAbs to influenza A and B virus strains.Recombinant HA ELISA was repeated in triplicate and the average multiple reactivity (relative fluorescence unit) relative to background was shown. Fig. 3 shows anti-B mAb TRL809, TRL812, TRL813; Each of TRL832, TRL841, TRL842, TRL845, TRL846, TRL847, TRL848, TRL849, TRL854 and TRL856 has a wide cross reactivity against two lineages of influenza B virus (comprising B/Florida/06, B/Mass/12 and B/Wisconsin/10 of the Yamagata clade and B/Brisbane/08, B/Malaysia/04 and B/Victoria/87 of the Victoria clade). Anti-influenza A mAbs CF401 (mAb 53) and CF402 (mAb 579) were reactive with members of the influenza A H1 (A/California/09) and H3 (A/Sydney/97) subtypes but not with influenza B strains.

FIG4 shows Table 8 with neutralization IC50 data for anti-influenza B antibodies TRL845, TRL848, TRL849, TRL854, TRL832, and TRL809 against representative strains B/Yamagata/16/1988 and B/Victoria/2/1987.

Figure 5A shows the in vivo activity of the anti-influenza B antibody 5A7 in mice, expressed as a percentage of body weight, following intranasal (IN) versus intraperitoneal (IP) administration of 5A7 in mice infected with 10xLD50 of B/Florida (Yamagata lineage). Mice were treated with mAb via the IN or IP route 24 hours post-infection (24 hpi), and body weight was recorded daily as an indicator of disease severity, with PBS and no virus used as controls. Animal body weight was monitored daily for 14 days post-infection, and the percentage of body weight relative to the initial day 0 body weight was used to plot the curve. Mice with >30% weight loss were euthanized. 1 mg/kg IN provided complete protection from weight loss, whereas 1 mg/kg via the IP route had some weight loss. This demonstrates enhanced efficacy of the IN route of administration relative to the IP route of administration.

Figure 5B shows the in vivo activity of the anti-influenza B antibody 5A7, expressed as a percentage of body weight, in mice infected with 10xLD50 of B/Malaysia (Victoria lineage) following IN versus IP administration of 5A7. Mice were treated with mAb at 24 hpi via the IN or IP route, and body weight was measured daily as an indicator of disease severity. Mice with >30% weight loss were euthanized. 1 mg/kg IN provided complete protection from weight loss, whereas 1 mg/kg via the IP route resulted in some weight loss. This demonstrates enhanced efficacy of the IN route of administration relative to the IP route.

Figures 6A-D show the in vivo activity of the anti-influenza B mAbs of the present invention against 10xLD50 (50% lethality) of virus in a murine model, following IN administration of 1 mg/kg. Mouse body weights were assessed up to 14 days post-infection, with PBS and no virus used as controls. Animal body weights were monitored daily for 14 days post-infection, and the percentage of body weight relative to the initial day 0 weight was used to plot the curves.

Figure 6A shows that 1 mg/kg of mAbs TRL845, TRL847, TRL 848, TRL849, and 5A7 administered by the IN route provided protection from disease in mice infected with B/Malaysia/2506/04 representing the Victoria clade.

Figure 6B shows that 1 mg/kg of mAbs TRL845, TRL847, TRL 848, TRL849, and 5A7 administered by the IN route provided protection from disease in mice infected with B/Florida/04/2006 representing the Yamagata clade.

Figure 6C shows that mAbs TRL849, TRL846, TRL854, TRL 856, TRL847, and 5A7 administered by the IN route at 1 mg/kg provided protection from disease in mice infected with B/Malaysia/2506/04 representing the Victoria clade.

Figure 6D shows that 1 mg/kg of TRL849, TRL846, TRL854, TRL 856, TRL847, and 5A7 administered by the IN route provided protection from disease in mice infected with B/Florida/04/2006 representing the Yamagata clade.

Figures 7A-7D show that co-administration of three anti-influenza mAbs does not interfere with anti-B mAb efficacy. Mice were infected with 10xLD50 virus and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (Cocktail 1), anti-B TRL847 (Cocktail 2), or anti-B TRL849 (Cocktail 3). PBS and no virus were used as controls. Animal body weights were monitored daily for 14 days post-infection, and the percentage of body weight at the initial day 0 weight was used to plot the curve.

7A-7D together show that the cocktail will provide the expected level of protection without interference with other mabs in the cocktail against representative strains from all seasonal influenza subtypes (H1N1, H3N2, and both lineages of B).

Figure 7A shows in vivo protection of mice infected with 10xLD50 H1N1 and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7B shows in vivo protection of mice infected with 10xLD50 H3N2 and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7C shows in vivo protection of mice infected with 10xLD50 B/Yamagata lineage and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7D shows in vivo protection of mice infected with 10xLD50 B/Victoria lineage and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 8 shows a CLIPS ^™ peptide array (top panel) of the B/Lee/1940/HA protein (SEQ ID NO: 291) manufactured by PEPSCAN Inc. The shaded regions of the B/Lee/1940/HA protein correspond to the residues used to generate the peptide arrays from aa_15-65 (SEQ ID NO: 292), aa_300-359 (SEQ ID NO: 293), and aa_362-481 (SEQ ID NO: 294). Table 9 (bottom panel) shows mAb 5A7 epitope 1 - aa_333-338 (SEQ ID NO: 304), epitope 2 - aa_342-346 (SEQ ID NO: 305), and epitope 3 - aa_457-463 (SEQ ID NO: 306); mAb TRL845 epitope - aa_455-463 (SEQ ID NO: 307); TRL848 epitope 1 - aa_64-71 (SEQ ID NO: 308); epitope 2 - aa_336-348 (SEQ ID NO: 309); epitope 3 - aa_424-428 (SEQ ID NO: 310); mAb 849 epitope 1 - aa_317-323 (SEQ ID NO: NO: 311), epitope 2 - aa_344-349 (SEQ ID NO: 312), epitope 3 - aa_378-383 (SEQ ID NO: 313); mAb 854 epitope 1 - aa_457-463 (SEQ ID NO: 314). The lower right figure shows the region resolved to the stem of HA in dark grey.

Figure 9 illustrates epitopes mapped along the stem of the HA protein by PEPSCAN ^™ analysis. The shaded area depicts the epitope bound by each mAb. mAbs TRL848, TRL845, TRL854, and TRL849 recognize overlapping epitopes along the stem of HA. A similar but unique epitope is generated by the control mAb 5A7, derived from a published sequence.

Figures 10A-10F show melting curve measurements of mAbs TRL845, TRL847, TRL848, TRL849, and TRL854. Each mAb exhibited high thermal stability.

FIG10A shows the melting curve of TRL845, which exhibits two melting temperatures (Tm1, Tm2) at 58.3° C. and 68.7° C., respectively.

FIG. 10B shows Table 10 with the melting temperatures (Tm) of mAbs TRL845, TRL847, TRL848, TRL849, and TRL854, as shown in FIG. 10A and 10C-10F, respectively.

FIG10C shows the melting curve of TRL847, which exhibits a melting temperature (Tm1) of 70.3°C.

FIG10D shows the melting curve of TRL848, which exhibits a melting temperature (Tm1) of 70.1°C.

FIG10E shows the melting curve of TRL849, which exhibits two melting temperatures (Tm1, Tm2) at 70° C. and 81.8° C., respectively.

FIG10F shows the melting curve of TRL854, which exhibits two melting temperatures (Tm1, Tm2) at 59.7° C. and 68.9° C., respectively.

Figure 11A shows the IN and IP administrations performed at comparable doses, and shows that the effectiveness of Mab IN is 10 to 100 times greater than that of the same Mab administered by IP. Animals were inoculated with 10XLD50 and H1 influenza virus (A/Puerto Rico/8/1934) and treated with 10mg/kg, 1mg/kg, and 0.1mg/kg of neutralizing CA6261 Mab at 24hpi by IN or IP, with PBS and virus-free controls. The body weight of the animals was monitored daily, and after 14 days of persistent infection, the percentage body weight relative to the initial day 0 weight was used to plot the curve. Data show that IN delivery of neutralizing antibodies significantly enhances their therapeutic efficacy compared to systemic delivery.

Figure 11B shows the evaluation of IN and IP administration of antibody MAb53 (TRL053) for the therapeutic efficacy of H1 virus. Animals were inoculated with 10XLD50 of H1 influenza A virus/Puerto Rico/8/1934 (PR8), and treated with 10mg/kg, 1mg/kg and 0.1mg/kg of neutralizing antibody TRL053 (MAb53) (administered by IN or IP) at 24hpi, with PBS treatment and virus-free as controls. The body weight of the animals was monitored daily for 14 days after continuous infection, and the percentage body weight of the initial day 0 weight was used to plot the curve. The TRL053 administered by IN showed a greater efficacy than the IP administration. TRL053 was only effective to a certain extent at the IP dosage of 10mg/kg. The IN dosage was effective at 10mg/kg and 1mg/kg.

Figure 12A shows a comparison of comparable doses administered IN and IP, and demonstrates that the potency of Mab CA8020 IN was 10 to 100 times greater than the potency of the same Mab administered IP. Animals were inoculated with 10×LD50 of H3 influenza virus and treated with 10 mg/kg, 1 mg/kg, and 0.1 mg/kg of the neutralizing CA8020 Mab either IN or IP 24 hpi, with PBS and no virus used as controls. Animal body weights were monitored daily for 14 days post-infection, and the percentage of body weight relative to the initial day 0 weight was used to plot the curve.

Figure 12 B shows the evaluation of the IN and IP administration of antibody MAb579 (TRL579) for the therapeutic efficacy of H3 viruses. Animals were inoculated with 10XLD50 of Vic/11H3 virus and treated with 10mg/kg, 1mg/kg and 0.1mg/kg of the neutralizing antibody TRL579 (administered by IN or IP) at 24hpi. The body weight of the animals was monitored daily for 14 days after continuous infection. TRL579 is effective for IP or IN, but the TRL579 administered by IN shows a greater efficacy than IP administration. TRL579 is only effective to a certain extent at an IP dosage of 10mg/kg. In this study, the IN dosage is effective at 10mg/kg and 1mg/kg.

Figure 13 is presented at the preventive IN and IP administration carried out 3 or 4 days before being infected with the virus. Before attacking with 3XLD50 of H1 influenza virus A/Puerto Rico/8/1934 (expressed as PR8) 3 or 4 days, antibody CA6261 was administered by IN or IP. Before infection 3 days (-3dpi) or before infection 4 days (-4dpi), CA6261 antibody was administered by IN (0.1mg/kg) or IP (0.1mg/kg and 1mg/kg), and attacked with H1 influenza virus. The control was virus-free and non-treated. The body weight of the animals was monitored daily, and after 14 days of continuous infection, the percentage body weight relative to the weight at initial day 0 was used to draw a curve. Figure 13 is presented at IN administration (assessed at 0.1mg/kg) for up to 4 days (-4dpi) before infection to protect mice from viral attack. IP administration carried out 3 or 4 days before infection with the same dose (0.1mg/kg) was completely ineffective. IP administration 3 or 4 days before infection was effective at 1 mg/kg. By comparing IN (0.1 mg/kg) with IP (1 mg/kg) administration at -3 dpi and -4 dpi, in both cases, the IN dose was 10 times less effective than the IP.

Figure 14 is shown in the preventive IN and IP administration carried out 5, 6 or 7 days before infection with the virus. Antibody CA6261 was administered by IP (at 1 mg/kg) or IN (at 1 mg/kg) 5, 6 or 7 days before attacking with 3XLD50 of H1 influenza virus PR8. Controls were Tamiflu (10 mg/kg orally administered, twice a day for 5 days), no treatment and no virus. The body weight of the animals was monitored daily and, after 14 days of persistent infection, the percentage body weight relative to the initial day 0 weight was used to plot the curve. In all cases, the antibody was more effective than Tamiflu.

Figure 15 shows preventive IN and IP administration 5, 6 or 7 days before infection with the virus (virus challenge at a higher dose of 10XLD50). Antibody CA6261 was administered by IP (at 1 mg/kg) or IN (at 1 mg/kg) 5, 6 or 7 days before attacking with 10XLD50 of H1 influenza virus PR8. The controls were Tamiflu (10 mg/kg orally administered, twice a day for 5 days), no treatment and no virus. The weight of the animals was monitored daily for 14 days after continuous infection, and the percentage weight relative to the initial weight on day 0 was used to draw the curve. In this study, only animals that were administered (by intranasal administration) antibodies to the airways survived the virus attack completely. Mice treated with 1 mg/kg of the antibody were not fully protected 5, 6 or 7 days before the virus attack, and the mice died of infection. Tamiflu was completely ineffective in protection. Mice treated with 0.1 mg/kg of the antibody administered intranasally 5 or 6 days before viral infection survived viral challenge almost as well as uninfected control animals.

Figure 16 shows table 11 with the assessment of monoclonal anti-influenza B antibodies in hemagglutination inhibition, B/Victoria and B/Yamagata pedigrees in vitro neutralization, predicted binding site (head, stem) and in vivo efficacy. In vivo results are provided as a summary of 3 different studies relative to the percentage of initial body weight of the animal of the specified TRL antibody of evaluation with 10xLD50 virus inoculation and administered 1mg/kg at 24hpi. Observe animal body weight, continue 14 days, indicate the lowest body weight observed. Shown is the result of each of antibody TRL 809, TRL832, TRL846, TRL845, TRL847, TRL848, TRL849 and TRL854, which is effective for maintaining at least 95% initial animal body weight throughout the in vivo efficacy evaluation. Each of TRL845, TRL847, TRL848, TRL849 and TRL854 was effective in maintaining at least 96% of the initial animal body weight throughout the in vivo efficacy evaluation. Although TRL846 did not show neutralization in conventional in vitro assays, it did show egress inhibition and was therefore considered neutralizing.

Figure 17 shows Table 12 with isoelectric points and affinity (K in _nM ) of B antibodies for B/Florida (Yamagata lineage) and B/Malaysia (Victoria lineage) influenza virus strains. The affinity of B antibodies for influenza virus was evaluated to serve as an indicator of relative efficacy. The affinity of each of TRL845, TRL847, TRL848, TRL849, and TRL854 for B/Malaysia was sub-nM (<1.0 nM) in each case (data not shown).

Figure 18 shows an animal efficacy study using B/Florida (Yamagata lineage) virus for various influenza B antibodies used at 24hpi. All antibodies were administered at 1mg/kg via IN at 24hpi for 10XLD50 virus. The antibodies tested were TRL845, TRL847, TRL848, TRL849, 5A7, and CA8033. PBS and no virus were used as controls. The body weight of the animals was monitored daily for 14 days after continuous infection, and the percentage body weight relative to the initial day 0 weight was used to plot the curve. The anti-influenza B antibodies administered via IN showed efficacy against infection with the Yamagata lineage virus using 10XLD50 dosage, with several antibodies showing that the infected animals treated with the antibodies maintained 100% body weight.

Figure 19 shows the animal efficacy study of various influenza B antibodies used at 24hpi using B/Florida (Yamagata lineage) virus. For the virus of 10XLD50, all antibodies were used at 24hpi with 1mg/kg by IN. The antibodies tested were TRL849, TRL846, TRL854, TRL856, TRL847, 5A7, TRL809 and TRL848. PBS and no virus were used as controls. The body weight of the animals was monitored every day, and after 14 days of continuous infection, the percentage body weight relative to the weight at the initial day 0 was used to draw a curve. The anti-influenza B antibodies used by IN showed efficacy against the infection of the Yamagata lineage virus using 10XLD50 dosage, and several antibodies showed that the infected animals treated with the antibodies maintained 100% body weight.

Figure 20 shows the animal efficacy study of various influenza B antibodies used at 24hpi using B/Malaysia (Victoria lineage) virus. For the virus of 10XLD50, all antibodies were used at 24hpi by IN with 1mg/kg. The antibodies tested were TRL845, TRL847, TRL848, TRL849, 5A7 and CA8033. PBS and no virus were used as controls. The body weight of the animals was monitored daily, and after 14 days of continuous infection, the percentage body weight relative to the weight at the initial day 0 was used to draw the curve. The anti-influenza B antibodies used by IN showed efficacy against the infection of the Y Victoria lineage virus using 10XLD50 dosage, and several antibodies showed that the infected animals treated with the antibodies maintained 100% body weight.

Figure 21 shows the animal efficacy study of various influenza B antibodies used at 24hpi using B/Malaysia (Victoria lineage) virus. For the virus of 10XLD50, all antibodies were used at 24hpi with 1mg/kg by IN. The antibodies tested were TRL849, TRL846, TRL854, TRL856, TRL847, 5A7, TRL809 and TRL848. PBS and no virus were used as controls. The body weight of the animals was monitored daily, and after 14 days of continuous infection, the percentage body weight relative to the weight at the initial day 0 was used to draw a curve. The anti-influenza B antibodies used by IN showed efficacy against the infection of the Victoria lineage virus using 10XLD50 dosage, and several antibodies showed that the infected animals treated with antibodies maintained 100% body weight.

Figure 22 A and 22B are presented at the efficacy evaluation of the anti-influenza B antibodies carried out in the animal study utilizing (Figure 22 A) Victoria lineage virus B/Malaysia and (Figure 22 B) Yamagata lineage virus B/Florida. Infect mice with 10xLD50 of virus, and treat mice with the antibody of 1mg/kg dosage by IN at IN 24hpi. Monitor the body weight of animals every day, and after 14 days of continuous infection, the percentage body weight relative to the weight of the initial day 0 is used to draw the curve. The antibodies tested are TRL809, TRL832, antibody 5A7 and TRL579 as specified. TRL579 is an anti-H3 antibody and is used as a negative control. TRL809 and TRL832 neutralize influenza B by blocking virus binding. IN-administered anti-influenza B antibodies TRL809, TRL832, and 5A7 demonstrated efficacy against infection with Victoria lineage viruses at a dose of 10XLD50, showing that antibody-treated infected animals maintained -100% of their body weight.

Figure 23 shows an animal study evaluating the antiviral efficacy of the combination of antibodies TRL053, TRL579 and 5A7 for influenza A or influenza B viruses using IN administration. Animals were infected with H1 virus PR8, H3 virus Victoria/11, influenza B virus B/Malaysia or influenza B virus B/Florida, and then either the animals were not treated or the mixture of the three antibodies was used to treat the animals. Each antibody was included at a dosage of 1 mg/kg, thereby providing a total antibody dose of 3 mg/kg. For mixture-treated animals, the combination antibodies were administered at 24 hpi for 10XLD50 of the specified virus. No virus was described as a control. The body weight of the animals was monitored daily, and after 14 days of continuous infection, the percentage body weight relative to the initial weight on day 0 was used to plot the curve. Figure 23 provides evidence that the antibody cocktail administered as a single mixed dose 24 hours after infection with 10xLD50 of virus exhibits efficacy against infection with each or any of HI, H3, B/Yamagata lineage, and B/Victoria lineage viruses.

Figure 24 shows an animal study in which 100% of mice survived a lethal dose of H1N1 challenge when a single dose of 1 mg/kg of CF-401 was administered 4 days after infection. Administration of standard of care Tamiflu was not protective, and CF-401 mAb therapy also provided additional protection from weight loss. Tamiflu was administered orally at 10 mg/kg twice daily on days 4-8. The data show that standard of care (Tamiflu) plus mAb therapy is effective when administered at late time points.

Figure 25 provides the results of IN versus IP administration of a model antibody fragment Fab of the CA6261 antibody for therapeutic efficacy against H1 virus. Animals were inoculated with 10XLD50 of H1 influenza virus and treated with 10 mg/kg, 1 mg/kg, and 0.1 mg/kg of neutralizing CA6261 Fab administered either IN or IP at 24 hpi. PBS treatment and no virus were used as controls. The body weight of the animals was monitored daily for 14 days post-infection, and the percentage body weight relative to the initial day 0 weight was used to plot the curve. All doses of the Fab CA6261 antibody administered IN showed greater efficacy than any IP dose. Administration of the neutralizing Fab IP did not show detectable efficacy even at the highest dose of 10 mg/kg.

Details

Life-threatening seasonal and pandemic influenza infections remain serious illnesses, killing 40,000 people in the United States alone. The availability of seasonal vaccines has failed to eliminate influenza as a major clinical problem. There is only one approved drug for influenza used in practice, yet influenza has shown a strong tendency to develop resistance to neuraminidase inhibitors and other neuraminidase inhibitors. Furthermore, it is known to lose its effectiveness substantially if administered after 48 hours of symptom onset.

Antibodies targeting influenza offer an alternative to vaccines and can be rapidly effective in treating or preventing infection, whereas vaccines typically require weeks to induce effective antiviral activity. Particularly attractive are monoclonal antibodies that react with the main protein on the surface of the influenza virus, hemagglutinin, in a region known as the hemagglutinin stem, which is genetically stable and does not change significantly or at all between seasons.

Numerous antibodies have been characterized and are being developed as therapeutic antibodies for influenza, including those based on conserved epitopes of the virus. Some cross-reactive antibodies target the hemagglutinin (HA) glycoprotein, which elicits the most potent neutralizing antibodies during vaccination or natural infection. HA comprises two subunits, HA1 and HA2, which are crucial components in viral infection. MAb CR6261 is a well-characterized antibody that is thought to bind to H1 viruses and other subtypes (H5) within group 1, and binds to the HA2 subunit (Throsby M et al. (2008) PL0S ONE 3:e3942; Eckert DC et al. (2009) Science 324:246-251; Friesen RHE et al. (2010) PLoS ONE 5(2):e1906; U.S. Patent 8,192,927). MAb CR8020 is believed to bind to the membrane-proximal region of HA2 on H3 and another subtype (H7) of group 2 viruses (Eckert DC et al. (2011) Science 333:843-850). The antibody FI6v3 from Swiss researchers is believed to have the ability to bind to epitopes present on group 1 (H1) and 2 (H3) viruses, however, FI6 has shown limited efficacy in mice (Corti D et al. (2011) Science 333:850-856). Palese and colleagues have reported the generation of broadly protective monoclonal antibodies against H3 influenza viruses using sequential immunization in mice with different hemagglutinins (Wang TT et al. (2010) PLoS Pathog 6(2):e1000796; US application 20110027270). By using this approach, an H1 antibody that is thought to be broadly reactive was isolated (Tan GS et al. (2012) J Virol 86(11):6179-6188).

Therapeutic treatment of influenza with monoclonal antibodies to HA is dose dependent and requires higher doses if administered later after infection. Common therapeutic doses of HA-specific antibodies with broad reactivity, given IP or IV, require doses in the range of 2 mg/kg to 50 mg/kg to see protection from lethal challenge. Later after infection, the same effect requires administration in the range of >10 mg/kg. The average adult in North America is approximately 80.7 kg, which would require 807 mg of antibody if administered at 10 mg/kg. Two current Phase I studies of influenza monoclonal antibodies CR6261 and CR8020, conducted by Crucell Holland BV, are evaluating the safety and tolerability of single doses escalating from 1 mg/kg to 50 mg/kg (trials NCT01406418 and NCT01756950, respectively; clinicaltrials.gov). In mice, these antibodies require 15 mg/kg to protect mice from death (Friesen, RHE et al. (2010) PLoS ONE 5(2):e1906); Ekiert DC et al. (2011) Science 333:843-850). Based on these IP or IV dosages, a single antibody in the amount of grams or grams per patient would be required based on human body weight (approximately 70 kg). This is compounded by the fact that in any therapy for multiple influenza subtypes, more than one antibody is needed to treat the three different subtypes of influenza (influenza A H3, influenza A H1, and influenza B) present in circulation, potentially requiring a total of about 3 grams of antibody (assuming about 1 gram per antibody). This large amount of antibody becomes costly and difficult to administer, and poses a major obstacle in the development of therapeutic antibodies for influenza.

We have identified a solution that significantly reduces the amount of antibody by more than 10-fold while significantly maintaining or even improving efficacy. We have found that intranasal delivery of antibodies, or more commonly, delivery by inhalation, provides a significant and significant improvement in efficacy compared to IV or IP routes. Effective airway administration of influenza monoclonal antibodies is described in USSN 61/782,661 and PCT/US2014/27939 (incorporated herein by reference).

Remarkably, by using a recognizable and known influenza mouse model, the intranasal (IN) delivery of neutralizing antibodies significantly increases therapeutic efficacy to more than 10 times compared to intraperitoneal (IP) or intravenous (IV) delivery routes. When given by the IN route rather than by IV or IP routes, comparable efficacy can be achieved using the same dose less than 1/10. Current treatment designs for the treatment of influenza utilize intravenous delivery as a standard (ClinicalTrials.gov Identifier: NCT01390025, NCT01756950, NCT01406418). This delivery method is standard in the art because the ability to utilize the neutralization characteristics of antibodies is unknown. The vast majority of research on antibody therapeutics utilizes IV or IP delivery, and it is not recognized that the IN delivery of neutralizing antibodies for influenza will improve efficacy compared to IV or IP delivery.

Previous reports of IN delivery have evaluated polyclonal serum gamma globulin IVIG or antibodies of the IgA class (IgA antibodies are intrinsically common to the lungs) (Akerfeldt S et al. (1973) Biochem Pharmacol 22:2911-2917; Ramisse F et al. (1998) Clin Exp Immunol 111:583-587; Ye J et al. (2010) Clin Vaccine Immunol 17(9):1363). One research group tested an ascites formulation of an antibody (C179) by the IN route and described protective IN delivery (before challenge) comparable to IP (Sakabe S et al. (2010) Antiviral Res 88(3):249-255). C179 exhibited low neutralizing activity against the 2009 pandemic H1N1 virus but reportedly protected mice from infection.

In contrast, the inventors have found that it is important that the enhanced efficacy is not simply accompanied by any cross-reactivity against influenza antibodies regardless of the mode of administration. Generally, non-neutralizing antibodies do not exhibit anti-influenza efficacy when given IN. To our knowledge, earlier studies have failed to recognize that this effect can be more widely applied to antibodies that exhibit in vitro neutralizing activity regardless of their viral epitopes or protein targets. In addition, antibodies do not have to have cross-reactivity against HA, because when administered by IN, neutralizing strain-specific antibodies will exhibit enhanced efficacy. We have found that neutralizing antibodies (rather than simply cross-reactive anti-HA antibodies) are necessary to significantly reduce the amount of antibodies required to achieve comparable efficacy, depending on the route of administration. In fact, we have found that the opposite occurs when cross-reactive anti-HA antibodies without neutralizing ability are used. The therapeutic use of these cross-reactive non-neutralizing anti-HA antibodies results in a significant reduction in therapeutic efficacy when mice are treated intranasally; however, when administered by IP or IV routes, these antibodies exhibit significant efficacy.

Without wishing to be bound by theory, a possible mechanism behind this phenomenon lies in the fact that intranasal delivery achieves certain levels of IgG antibodies in the airway mucosa, where the neutralizing capacity of the antibody-antigen combination site can be exploited, whereas IV or IP delivery of antibodies is Fc-dependent. In the airways, inhibitory mechanisms rely on the neutralizing properties of the antibody, and Fc-dependent effects are severely limited. When IgG antibodies are administered IP or IV, the amount of antibody reaching this space in the airways is too low to exploit the neutralizing effect of the antibody. For example, when neutralizing antibodies are administered IP or IV, the primary observed therapeutic effect is due to the antibody's effector function. We have found comparable efficacy levels for neutralizing and non-neutralizing antibodies when administered IP or IV, but not IN. To further illustrate that this effect is dependent on neutralization, antibodies against the M2 protein do not exhibit in vitro neutralization and are only able to demonstrate Fc-mediated effects. Previous work using antibodies against the M2 ion channel (a molecule that is more genetically conserved than HA) has shown promise in preclinical models and has completed Phase I studies (TCN-032 from Theraclone; NCT01390025, NCT01719874; Grandea AG et al. (2010) Proc Natl Acad Sci USA 107(28):12658-12663). Antibodies against the M2 protein cannot neutralize the virus but may have well-documented therapeutic efficacy mediated by effector functions (Wang, R. et al. (2008) Antiviral Research 80:168-177; Grandea, A.G., 3rd Edition et al. (2010) Proc Natl Acad Sci USA 107(28):12658-12663). We believe that neutralizing and non-neutralizing antibodies act primarily through effector functions similar to those of M2-targeting antibodies when administered IP or IV. The M2 protein is far less abundant than HA and does not protrude from the surface. Antibodies against HA can neutralize the virus, thereby providing the potential for further improving efficacy. In this way, antibodies against HA are generally more effective than anti-M2 antibodies in treatment. However, antibodies that are not neutralizing agents but still target HA show efficacy levels comparable to neutralizing antibodies when administered by IP, which indicates that this delivery route cannot utilize the potent effect that can be utilized when administered by IN. In addition, delivery of neutralizing Fabs by IN but not IP leads to therapeutic efficacy. Non-neutralizing Fabs administered by IN did not show therapeutic efficacy. In short, only neutralizing antibodies given by IN showed this enhanced efficacy. By extending this observation, for neutralizing antibodies targeting other influenza proteins (e.g., neuraminidase) and neutralizing antibodies against other respiratory pathogens (palivizumab for RSV), this phenomenon of enhanced efficacy through pulmonary delivery can occur. The enhanced efficacy, or the level of enhanced efficacy, may depend on the apical replication life cycle of the respiratory pathogens of this subgroup, where these viruses are susceptible to the neutralizing ability of these antibodies in the apical space. Since antibody delivery via the IN and IP/IV routes can achieve effectiveness in their own way, the combined use of the two routes will exploit the maximum therapeutic potential of neutralizing antibodies. This approach will allow for maximum efficacy by utilizing both enhanced neutralizing activity (via the IN route) and enhanced Fc-dependent activity (via the IP/IV route).

This example shows the intranasal efficacy of neutralizing antibodies (including known antibodies and newly separated antibodies) on low dosage. Many different and known antibodies (as exemplary antibodies, including CR6261, CR8020, CR9114, 5A7, mAb53 (TRL053), mAb579 (TRL579), TRL845, TRL846, TRL847, TRL848, TRL849, TRL854, TRL809 and TRL832) are provided. Such activity and efficacy have not been previously demonstrated, regardless of many studies such as CR6261 and CR8020, including preclinical trials. Many different antibodies are assessed herein, including known and newly separated antibodies, and the antibodies are effective for airway administration.

The present invention relates to novel and unique combinations of antibodies, which are effectively directed against influenza A and influenza B viruses, and can be produced and applied together without adverse interactions and with significant formulation efficiency. The antibodies of the combination according to the present invention are directed against different but all clinically relevant subtypes, including subtype H1, H3, H5 of influenza A virus and influenza B Yamagata and Victoria pedigrees. Therefore, the invention provides generally applicable antibody combinations and mixtures, wherein the unique combination or mixture of antibodies can neutralize related circulating influenza viruses, can be applied via the airway (such as intranasal or to the lungs or anti-tracheal mucosal delivery) to administer the antibody combination or mixture of the present invention, for methods and compositions used by the airway and for treating or preventing influenza virus, influenza infection and/or propagation. Due to the unique and useful effect of the antibody combination of the present invention or mixture, clinical evaluation or specific diagnosis of influenza virus or subtype is not necessary or is not required before application or use. Antibody fragments, derivatives or variants are envisioned. According to the present disclosure, antibody fragments, including Fab, are shown to be effective herein. In one aspect of the presently disclosed embodiments, antibody Fab fragments are active and effective when administered via an airway route (e.g., intranasally or via inhalation) and are ineffective when administered by IP or IV. Model Fabs are effective in vivo in an absorbable formulation, as shown in Figure 25. In some embodiments, antigen-binding fragments that are scFvs are provided.

In some embodiments, antibodies, antigen-binding fragments, compositions and methods for passive immunization against influenza are provided. In some embodiments, antibody combinations, compositions and methods for the treatment or prevention of influenza virus are provided. In some aspects, compositions comprising monoclonal antibodies against influenza A and B are provided, which are suitable for systemic administration or direct administration to the respiratory tract, including administration through the airways, such as by intranasal or inhalation administration. Compositions and methods for treating or preventing via airway administration of antibodies or combining intranasal or inhalation administration of antibodies with intraperitoneal or intravenous administration are provided.

Conserved epitopes within the hemagglutinin (HA) molecule have recently been discovered. There are several reports on the isolation and characterization of human monoclonal antibodies (MAbs) that can recognize and neutralize many influenza A virus subtypes. Many of these antibodies target the hemagglutinin (HA) glycoprotein, which can elicit the most potent neutralizing antibodies during vaccination or natural infection. HA consists of two subunits, HA1 and HA2, which are crucial components in viral infection. HA1 is involved in attachment to the host cell receptor sialic acid, while HA2 mediates fusion of the virus with the endosomal membrane.

Current antibody therapy dosages are well established in multiple mg/kg per dose based on research and clinical experience to date using many recombinant antibodies, including more than 20 monoclonal antibodies that have been approved for clinical use in the United States (Newsome BW and Ernstoff MS (2008) Br J Clin Pharmacol 66(1):6-19). For example, panitumumab, an anti-EGFR fully human antibody approved for colorectal cancer, is administered intravenously at 6 mg/kg over 1-1.5 hours once every 2 weeks. Using an average human weight of 70 kg, this amount is 420 mg of antibody per dose.

So far, no monoclonal antibody has been clinically approved for influenza. Reports on studies utilizing influenza antibodies in animals show that the effective dose range of these antibodies for treatment or prevention purposes when given intravenously or intraperitoneally is in the range of 1 mg/kg to 100 mg/kg. Phase I clinical trials utilizing some of these antibodies (CR6261, CR8020, TCN-032) conducted in the U.S. used dose escalation (clinicaltrials.gov; NCT01390025, NCT01406418, NCT01756950) from 2 mg/kg to 50 mg/kg in safety and tolerance studies. Subsequent Phase IIa studies utilizing these antibodies were conducted with the highest Phase I dose (e.g., 30 mg/kg or 50 mg/kg). This large amount of material has set a major obstacle in the development of the therapeutic agent of this new route. Specifically, the systemic dose within this range results in a large amount of material costs, and also brings the time, space and personnel costs associated with infusion. Therefore, there is an urgent need to enhance the efficacy and/or reduce the amount of material required to make antibody therapy or prevention against influenza a viable alternative.

While influenza B is slightly less common than influenza A, it remains a serious health concern. Generally, influenza B viruses are divided into two lineages: B/Victoria/2/87-like and B/Yamagata/16/88-like. Influenza B viruses differ from influenza A viruses in that they lack a protein called basic 1-F2 (PB1-F2), but possess additional proteins not found in influenza A, such as glycoprotein B (NB). Additional differences exist. However, the HA proteins of influenza B strains share the same homology with some influenza A HA proteins as they do with each other.

The hemagglutinin protein (HA) of influenza virus has a globular head domain (which is highly heterogeneous between influenza virus strains) and a stem region containing the fusion site required for entry into cells. HA is a trimer on the viral envelope. The uncleaved form of the hemagglutinin protein (HA ₀ ) is activated by being cleaved into HA ₁ and HA ₂ parts (to allow the fusion site to achieve toxicity) via trypsin. The two cleaved parts use a disulfide bond to maintain coupling, but undergo conformational changes in the low pH environment of the endosomal compartment of the host cell, which leads to fusion of the virus with the host cell membrane.

The cleavage site contains a consensus sequence shared by different strains of influenza A and B. The uncleaved hemagglutinin protein trimer (HA ₀ ) is referred to as the inactive form, whereas when cleaved into the HA ₁ and HA ₂ parts, the hemagglutinin protein is referred to as being in the activated form.

Bianchi, E. et al., J. Virol. (2005) 79:7380-7388 describe a "universal" influenza B vaccine based on a consensus sequence for this cleavage site, wherein a peptide comprising this site, when conjugated to the outer membrane protein complex of Neisseria meningitidis, is able to generate antibodies in mice. Monoclonal antibodies (mAbs) that show binding to the consensus sequence are also described. In addition, successful passive transfer of antiserum was observed in mice. Other prior art vaccines, such as those described in WO 2004/080403 that contain peptides derived from the M2 and/or HA proteins of influenza, include antibodies that have poor efficacy or are ineffective across strains.

Antibodies that bind to the HA stem region described in the art include those developed by Crucell, such as CR6261 and CR8020 described in WO 2008/028946 ('946); Throsby, M. et al., PLoS One (2008) 3:e3942; Ekiert, D.C. et al., Science (2011) 333:843-850; and Sui, J. et al., Nat. Struct. Mol. Biol. (2009) 16:265-273. According to the aforementioned PCT publication '946, these antibodies bind not only to H5N1 but also to H2, H6, H9, and H1. mAbs directed against the conserved M2E antigen have also been developed, such as those described by Grandea, A.G. et al., PNAS USA (2010) 107:12658-12663. M2E is a virally encoded protein that appears on the surface of infected cells and is also the target of amantadine and rimantadine. Resistance to these antiviral agents has become widespread, suggesting that this target is unable to perform essential functions.

Additional prior art antibodies have been described by the Lanzavecchia group: Corti, D. et al., Science (2011) 333: 850-856, which bind to and neutralize group 1 and group 2 strains of influenza A, but are not as potent as those described herein. Additionally, mAbs immunoreactive against the stem regions of influenza A and B were described in Dreyfus, C. et al., Science (2012) 337: 1343-1348, but did not have detectable neutralizing potency. The same authors described two mAbs that bind to the head group of HA, one of which had high variability in affinity and potency for strains from two clades of influenza B, the other of which had an affinity of ~1 nM. These results confirm that broadly neutralizing mAbs for influenza B are difficult to obtain.

PCT Application Publication No. WO2011/160083 (incorporated herein by reference) describes monoclonal antibodies derived from human cells and used in vaccines. The antibodies show high binding affinity to influenza virus clade H1 present in Group 1, and some of the antibodies also show high affinity to H9, also present in Group 1, and/or H7, present in Group 2, and/or H2, also present in Group 1. Some of the disclosed antibodies bind only to the inactive trimeric form (presumably at the common cleavage region), while other antibodies are able to bind to the activated hemagglutinin protein that has been cleaved.

PCT Publication No. WO2013/086052 (incorporated herein by reference) discloses a panel of antibodies, including bispecific antibodies, that bind to an epitope in this consensus region and bind to a wide range of influenza A viruses from Groups 1 and 2, including H1, H2, H5, H6, H8, H9, H11, H13, and H16 in Group 1 and H3 and H7 in Group 2. Furthermore, a recent publication by Yasugi, M. et al., PLoS Pathogens (2013) 9: published online as e1003150, pp. 1-12, describes human mAbs that neutralize influenza B virus, specifically, a mAb designated 5A7 that is believed to have therapeutic efficacy even when administered 72 hours after infection. The _KD of 5A7 against the only strain tested was reported to be 5 nM, and the epitope was identified in the highly conserved C-terminal stem region; the reported efficacy was comparable across all strains of influenza B. This result establishes that high affinity (sub-nM) mAbs against influenza B are difficult to achieve.

In some embodiments, there is provided an antibody or similar binding portion for prevention and treatment. Therefore, they can be used to protect a subject from viral infection and for treating a subject who has been exposed to or has been infected with influenza B. The ultimate goal subject is a human subject, and for the use in a human subject, it is preferred to use a human form or a humanized form of the binding portion of a conventional natural antibody or its immunoreactive fragment. However, the antibody containing the appropriate binding characteristics determined by the complementary determining region (CDR) can retain non-human characteristics when used in the research of laboratory animals. The antibody used in the research of the following examples, although the research is carried out in mice, contains the variable and constant regions for people.

definition

In light of the present disclosure, conventional molecular biology, microbiology, and recombinant DNA techniques within the capabilities of those skilled in the art may be employed. Such techniques have been fully explained in the literature. See, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology," Vols. I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook," Vols. I-III [J. E. Celis, ed. (1994)]; "Current Protocols in Immunology," Vols. I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M. J. Gait, ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J. Higgins, eds. (1985)]; "Transcription And Translation" [B. D. Hames & S. J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, editor (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Accordingly, if appearing herein, the following terms shall have the definitions set out below, unless the context clearly requires otherwise.

As used herein, the term "antibody" or "binding portion" refers to an antibody, a co-immunoreactive fragment or antigen-binding fragment, as well as a monospecific monoclonal antibody, a multispecific antibody such as a bispecific monoclonal antibody or a trispecific monoclonal antibody, an isolated monoclonal antibody, a recombinant monoclonal antibody, and an isolated human or humanized monoclonal antibody, or an antigen-binding fragment thereof. A molecule comprising two heavy immunoglobulin chains and two light immunoglobulin chains, and also includes immunoreactive fragments or antigen-binding fragments of conventional antibodies, even though "fragment" is sometimes referred to redundantly. The antibodies, thereby including Fab fragments, _Fv single-chain antibodies containing essentially only the variable regions, bispecific or trispecific antibodies, and various fragmented forms thereof that still retain immunogenicity, as well as proteins that mimic the activity of "natural" antibodies, typically by comprising amino acid sequences that approximate the activity of the variable regions of more conventional naturally occurring antibodies or modified amino acid sequences (i.e., pseudopeptides). Thus, the term encompasses antibody fragments, derivatives, functional equivalents, and homologs of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or fully or partially synthesized, such as recombinant or otherwise engineered. Thus, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, as well as U.S. Patent Nos. 4,816,397 and 4,816,567.

The term "antibody" also includes immunoglobulins, whether natural or partially or completely synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is an antibody or a binding domain homologous thereto. The term also includes CDR-grafted antibodies. An "antibody" is any immunoglobulin that binds to a specific epitope, including antibodies and fragments thereof. The term includes polyclonal, monoclonal, and chimeric antibodies, the last of which is described in U.S. Patent Nos. 4,816,397 and 4,816,567. The term "antibody" includes wild-type immunoglobulin (Ig) molecules, which typically comprise four full-length polypeptide chains, two heavy (H) chains and two light (L) chains, or equivalent Ig homologs thereof (e.g., camel nanobodies, which comprise only heavy chains); including full-length functional mutants, variants, or derivatives thereof, which retain the basic epitope binding properties of Ig molecules, and include dual-specific, bispecific, multispecific, and dual variable domain antibodies; immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Preferably, the antibody is of the IgG class.

The "antibody combining site" is the structural portion of an antibody molecule composed of the variable and hypervariable regions of the heavy and light chains that specifically binds to an antigen.

As used herein, the phrase "antibody molecule" in its various grammatical forms contemplates both intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies (i.e., comprising a single antibody), except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the monoclonal antibody preparation process, such variants typically existing in small amounts), the population is identical and/or binds to the same epitope (or multiple epitopes). A monoclonal antibody is an antibody having a type of antibody combining site that can immunoreact with a specific antigen. A monoclonal antibody thus typically exhibits a single binding affinity for any antigen that immunoreacts with it. In contrast to polyclonal antibody preparations that typically include multiple antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. However, if a monoclonal antibody contains an antibody molecule with multiple antibody combining sites (each site having immunospecificity for different antigens), it can be multispecific; for example, a bispecific (chimeric) monoclonal antibody. The modifier "monoclonal" indicates that the antibody is obtained from a substantially uniform antibody population and should not be interpreted as requiring the production of antibodies by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure can be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Such methods and other exemplary methods for making monoclonal antibodies are described herein.

The term "antigen-binding fragment," used synonymously with "immunoreactive fragment,""bindingfragment," and "antibody fragment," refers to an enzymatically obtainable, synthetic, or recombinant "engineered" polypeptide or glycoprotein that specifically binds to an antigen to form a complex. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of "engineered" antibody fragments include, but are not limited to, Fv, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In addition, antibody fragments include single-chain polypeptides having the characteristics of a VH domain (i.e., capable of assembling together with a VL domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody) or the characteristics of a VL domain (i.e., capable of assembling together with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody). "Antibody fragments" include molecules that contain at least one polypeptide that is not full-length, including (i) a Fab fragment, which is a monovalent fragment consisting of a variable light (VL) domain, a variable heavy (VH) domain, a constant light (CL) domain, and a constant heavy 1 (CH1) domain; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the heavy chain portion of a Fab (Fd) fragment, which consists of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, and (v) a domain antibody (dAb) fragment, which contains a single variable domain (Ward, ES et al., Nature 341, 544-546 (1989)); (vi) camelid antibodies; (vii) isolated complementarity determining regions (CDRs); (viii) single-chain Fv fragments in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (ix) diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (WO 94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 906444-6448, (1993)); and (x) linear antibodies comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242:193-204 9 (2000)); (xii) minibodies, which are bivalent molecules consisting of scFv fused to constant immunoglobulin domains CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al. (2004) Prot Eng Des Sel 17(4):315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9):1126-1136); and (xiii) other non-full-length portions of the heavy and/or light chains, or mutants, variants or derivatives thereof (alone or in any combination). Single-chain Fabs (scFAbs) are known and described in US20070274985.

Antigen binding fragments include Fab fragments; F(ab') ₂ fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; dAb fragments; and the smallest recognition unit consisting of amino acid residues that mimics the hypervariable region (CDR) of an antibody. Other engineered molecules, such as double-chain antibodies, three-chain antibodies, four-chain antibodies, and miniantibodies are also included in the expression "antigen binding fragment" as used herein.

The antibodies used and mentioned in combination herein, in addition to the antibodies first described herein and constituting new polypeptide entities, may include those having amino acid sequences as reported and publicly known, including antibodies, proteins, polypeptides having modifications to known or disclosed amino acid sequences and retaining or exhibiting substantially equivalent activity (including target neutralization or recognition and binding activity). Thus, proteins exhibiting substantially equivalent or altered activity are also contemplated. These modifications may be intentional, for example, such as those obtained by site-directed mutagenesis, or may be accidental, such as those obtained by mutations in a host that is a producer of the complex or its named subunit. The antibodies are intended to include within their scope the antibodies explicitly cited herein and all substantially homologous analogs and allelic variants, such as sequences having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the explicitly disclosed or publicly reported antibodies.

The amino acid residues described herein are preferably present in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin binding is retained by the polypeptide. _NH2 refers to the free amino group present at the amino terminus of the polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of the polypeptide. In accordance with standard polypeptide nomenclature (J. Biol. Chem., 243:355-259 (1969)), the abbreviations for the amino acid residues are shown in the corresponding Table 1 below:

Table 1 Corresponding amino acid symbols

It should be noted that all amino acid residue sequences are represented herein by formulas with their left and right orientation shown in the conventional direction of amino terminus to carboxyl terminus.Table 1 is provided to correlate three-letter to one-letter symbols that may appear alternately herein.

The following are examples of various groupings of amino acids: amino acids with non-polar R groups: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; amino acids with uncharged polar R groups : glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; amino acids with charged polar R groups (negatively charged at pH 6.0) : aspartic acid, glutamic acid; basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (at pH 6.0); another grouping can be those amino acids with a phenyl group: phenylalanine, tryptophan, tyrosine.

Another grouping can be based on molecular weight (i.e., the size of the R group):

Particularly preferred conservative amino acid substitutions are:

- Substitution of Arg by Lys and vice versa so that the positive charge can be maintained;

- Substitution of Asp by Glu and vice versa so that the negative charge can be maintained;

-Ser substitution for Thr so that a free -OH group is maintained; and

- Substitution of Asn by Gln so that free _NH2 can be maintained.

Amino acid substitutions can also be introduced to replace amino acids with particularly preferred properties. For example, a Cys can be introduced as a potential site for forming a disulfide bridge with another Cys. His can be introduced as a particularly "catalytic" site (i.e., His can act as an acid or a base and is the most commonly used amino acid in biochemical catalysis). Pro can be introduced because of its particularly flat structure, which induces a beta turn in the structure of the protein.

Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid sequences (preferably at least about 80%, in more preferred embodiments, at least about 90 or 95%, at least about 98% or at least about 99% sequence identity) are identical, or represent conservative substitutions. In some embodiments, substantially homologous sequences are provided having one or more amino acid substitutions.

Nucleic acids encoding antibodies for use according to the present disclosure can be used to prepare and/or generate antibodies or active fragments thereof for use in the presently disclosed embodiments. Vectors comprising such nucleic acids can be used to express or isolate antibodies as provided or used herein.

A "replicon" is any genetic element (eg, plasmid, chromosome, virus) that is an autonomous unit of DNA replication in vivo (ie, capable of replicating under its own control).

A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment can be attached so as to enable replication of the attached segment.

"DNA molecule" refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its single-stranded form or double-stranded helical form. The term refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found in, among other things, linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the normal convention of giving the sequence only along the non-transcribed strand of the DNA (i.e., the strand having a sequence homologous to the mRNA) in the 5' to 3' direction.

"Origin of replication" refers to those DNA sequences that participate in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence that is transcribed and translated into protein in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences can include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

A "promoter sequence" or "promoter" is a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For the purposes of defining the presently disclosed embodiments, the promoter sequence is bounded at its 3' terminus by the transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. Within the promoter sequence, a transcription start site (conveniently defined by utilizing the localization of nuclease S1) and a protein binding domain (consensus sequence) responsible for the binding of RNA polymerase are found. Eukaryotic promoters typically, but not always, contain a "TATA" box and a "CAT" box. Prokaryotic promoters contain a Shine-Dalgarno sequence in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a form of regulatory sequence and is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is subsequently translated into the protein encoded by the coding sequence.

When such DNA is introduced into the cell, the cell has been "transformed" by exogenous or heterologous DNA. The transforming DNA may or may not be integrated (covalently linked) into the chromosomal DNA, thereby constituting the genome of the cell. In, for example, prokaryotic cells, yeast cells, and mammalian cells, the transforming DNA may be maintained on an additional element such as a plasmid. With regard to eukaryotic cells, stably transformed cells are cells in which the transforming DNA has been integrated into the chromosome so that it can be inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish a cell line or clone consisting of a colony of daughter cells containing the transforming DNA." Clone" is a colony of cells derived from a single cell or a common ancestor by mitosis. A "cell line" is a clone of primary cells that can stably grow many generations in vitro.

Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, more preferably at least about 90 or 95%) of the nucleotides match over a defined length of the DNA sequence. Substantially homologous sequences can be identified by comparing the sequences using standard software available in sequence databases, or in a Southern hybridization experiment under, for example, stringent conditions (as defined for that particular system). Defining appropriate hybridization conditions is within the capabilities of those skilled in the art. See, for example, Maniatis et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

It should be understood that also within the scope of the present disclosure are DNA sequences encoding or for use in antibodies of the present disclosure that encode antibodies, polypeptides, or active fragments thereof having the same amino acid sequence, but are degenerate to the original or known coding sequence. "Degenerate to" means that different three-letter codons are used to specify a specific amino acid. It is well known in the art that the following codons can be used interchangeably to encode each specific amino acid:

It will be appreciated that the codons specified above are for RNA sequences. The corresponding codons for DNA are substituted with T for U.

Mutation can be produced in antibody or active fragment coding sequence so that specific codon is changed into the codon of encoding different amino acids.Such mutation is usually produced by producing minimum nucleotide changes as far as possible.This type of substitution mutation can be produced in a "non-conservative" manner (that is, by changing the amino acid of different groups of amino acids with a specific size or feature to the amino acid belonging to another grouping) or in a "conservative" manner (that is, by changing the amino acid of the group of amino acids with a specific size or feature to the amino acid belonging to the same grouping). This type of conservative change usually causes less variation in the structure and function of the resulting protein. Non-conservative changes are more likely to change the structure, activity or function of the resulting protein. Some embodiments are considered to be included in the amino acid sequence containing conservative changes or replacement sequences on 1, 2, 3, 4, 5, 6 or 7 amino acid residues, and the conservative changes or replacements can not significantly change the activity or binding characteristics of the resulting protein.

As mentioned above, DNA sequences encoding antibodies, polypeptides, or active fragments thereof can be prepared by synthesis rather than cloning. Appropriate codons can be used to design the DNA sequence of the antibody or fragment amino acid sequence. Generally, if the sequence is used for expression, codons that are preferred for the intended host are selected. Complete sequences are assembled from overlapping oligonucleotides prepared by standard methods, and the sequences are assembled into complete coding sequences. See, for example, Edge, Nature, 292: 756 (1981); Nambair et al., Science, 223: 1299 (1984); Jay et al., J. Biol. Chem., 259: 6311 (1984). Synthetic DNA sequences allow for the convenient construction of genes that will express analogs or "mutants". Alternatively, DNA encoding mutant proteins can be produced by site-directed mutagenesis of natural genes or cDNAs, and conventional polypeptide synthesis can be used to directly produce mutant proteins.

The " heterologous " district of DNA construct is the identifiable section of the DNA in the larger DNA molecule that is not found to be connected with the larger molecule in nature.Therefore, when the heterologous region encodes a mammalian gene, the gene is usually flanked by the DNA of the mammalian genomic DNA in the genome of the biological source.Another example of a heterologous coding sequence is a construct (for example, a cDNA wherein the genomic coding sequence contains introns, or a synthetic sequence with a codon different from the natural gene) in which the coding sequence itself does not exist in nature.Allelic variation or naturally occurring mutation events do not produce heterologous regions of DNA as defined herein.

A DNA sequence is "operably linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of the DNA sequence. The term "operably linked" includes having an appropriate start signal (e.g., ATG) before the DNA sequence to be expressed and maintaining a correct reading frame to allow expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If the gene to be inserted into the recombinant DNA molecule does not contain an appropriate start signal, a start signal can be inserted before the gene.

The term "standard hybridization conditions" refers to salt and temperature conditions that are substantially equivalent to 5xSSC and 65°C for hybridization and washing. However, it will be understood by those skilled in the art that such "standard hybridization conditions" depend on specific conditions, including the concentrations of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatches, percent formamide, and the like. It is also important in the determination of "standard hybridization conditions" whether the two sequences are hybridizing RNA-RNA, DNA-DNA, or RNA-DNA. Such standard hybridization conditions can be readily determined by those skilled in the art according to well-known formulas, wherein hybridization is typically 10-20°C lower than the predicted or determined _Tm , and, if necessary, washing uses higher stringency.

The term "binding affinity" for antibodies and fragments is described herein as KD (equilibrium dissociation constant between an antibody and its antigen), which can be determined by various methods known in the art. In some embodiments, the antibody or antigen-binding fragment exhibits the following binding affinity for influenza A virus, influenza B virus, or HA protein derived therefrom, such as recombinant HA protein. In one embodiment, the binding affinity is between about ^5x10-8 M and about ^5x10-12 M, in some embodiments, the binding affinity is between about ^5x10-9 M and ^5x10-11 M, in some embodiments, the binding affinity is between about ^3x10-9 M and about ^5x10-11 M, and in some embodiments, the binding affinity is between about ^5x10-10 M and about ^5x10-11 M. In some embodiments, antibodies and antigen-binding fragments that exhibit a KD for recombinant HA protein of less than 10 nM, less than 3 nM, or less than 1 nM, or tightly are provided. In some embodiments, antibodies and fragments showing a KD between 10nM and 0.1pM are provided. In some embodiments, antibodies and fragments showing a KD between 3nM and 1pM are provided. As used herein, the term "tight" represents the KD value mentioned or less (lower KD value), because the higher the intensity or tightness of affinity, the lower the KD value. In some embodiments, KD is measured by measuring the oblique incidence reflectivity difference (OI-RD) (e.g., by using a microarray or fluid system, such as (see Landry et al., 2012, Assay and Drug Dev Technol 10 (3), 250-259), or using (i.e., surface plasmon resonance) or competitive binding assay).

Neutralization of antibody-mediated viruses is defined as defined or accepted in this article, and as mentioned and used in this article, can be tested in various conventional or generally recognized neutralization assays. The example of neutralization assay includes a conventional neutralization assay based on the inhibition of the viral cytopathic effect (CPE) of the cells in culture. For example, influenza neutralization can be tested by reducing or blocking the formation of CPE in MDCK cells infected by influenza. Virus and neutralizing reagent can be pre-mixed, subsequently added to cells, and then the blocking of virus entry can be measured. Hemagglutinin inhibition (HI) can be tested in vitro, and the hemagglutinin inhibition can detect the blocking of the ability of virus binding to red blood cells. Exemplary known and accepted neutralization assays are provided in WHO Manual on Animal Influenza (who/cds/csr/ncs/2002.5, 48-54 pages). The antibody that blocks the sialic acid receptor binding site can neutralize the binding of the virus to the cell, thereby blocking infection. Conversely, neutralization assay can detect the blocking of virus discharge, as in the case of neuraminidase inhibitors such as. Recently, neutralizing antibodies that work in a similar manner by preventing viral discharge have been identified, the examples of which include the CR9114 antibody (Dreyfus et al. (2012) Science 337:1343-1348) on influenza B virus. Similarly, a micro-neutralization assay was performed using a microtiter plate composed of ELISA to detect viral nucleoprotein (NP) in infected cells. Quantitative PCR assays have been described to measure viral proteins (Dreyfus C et al. (2012) Emerging Inf Diseases 19 (10:1685-1687). In some embodiments, there is provided an antibody or fragment that is not shown as a neutralizing antibody in another conventional in vitro neutralization assay, but does show neutralization inhibition by going out, thereby being effectively administered by IN. Therefore, as defined herein, "neutralizing" antibodies will refer to antibodies that show neutralization in conventional in vitro neutralization assays, and/or antibodies that suppress going out inhibition. Therefore, in one sense of the term, discharge inhibitors have a neutralizing effect because they inhibit the spread of influenza infection.

"Non-neutralizing" antibodies or non-neutralizing antibodies can bind to the virus but do not inhibit the virus or viral replication in a recognition neutralization assay or in a routine evaluative assessment. Binding to the virus can inhibit the virus by preventing its effective interaction with cells, cell receptors, or cell targets. Non-neutralizing antibodies can bind to conserved proteins in the virus or epitopes on proteins. For example, in clinical trials, the M2 antibody TCN-032 can bind to a wide range of influenza A viruses, but does not show neutralization in conventional neutralization assays. Similarly, antibodies that do not neutralize but bind to HA can be identified.

As used herein, "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising the VL domain and constant domain of a light chain (CL), and a VH domain and the first constant domain (CH1) of a heavy chain. The Fab and F(ab') ₂ portions of an antibody molecule can be prepared by proteolytic reactions of papain and pepsin on substantially intact antibody molecules, respectively, using known methods, or can be synthesized or recombinantly prepared. The Fab' antibody molecule portion is also well known and can be produced from the F(ab') ₂ portion by, for example, reducing the disulfide bond connecting the two heavy chain portions using mercaptoethanol, followed by alkylating the resulting protein thiol with a reagent such as iodoacetamide.

The term "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. For example, in native antibodies, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of antibodies in the IgG, IgA, and IgD isotypes; IgM and IgE Fc domains contain three heavy chain constant domains ( _CH domains 2-4) in each polypeptide chain.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of immunoglobulin molecules that contain the paratope, including those portions known in the art such as Fab, Fab', F(ab') ₂ , and F(v), which are preferred for use in the therapeutic methods described herein.

In some cases, as taught in the present disclosure, for use, particularly for intranasal or inhalation administration, the neutralizing activity and necessary characteristics of the antibody of certain level or amount are needed. Therefore, any fragment, variant, derivative, synthetic antibody or antibody portion for use according to the present disclosure need to retain neutralizing ability and activity for target virus or pathogen (influenza virus in one aspect). On the other hand, the antibody used via selectable systemic route (including intraperitoneal or intravenous) must be combined with or identify target virus or pathogen (influenza virus in one aspect), yet, does not need neutralization. Therefore, as an example, the Fab fragment (multiple fragments) of the antibody retaining neutralization can be used intranasally. Effector functions mediated via Fc are not needed for intranasal efficacy and neutralization. On the contrary, the antibody delivered systemically will obviously mediate its effect by effector functions.

The term "antigen binding domain" refers to the portion of an antigen binding molecule that comprises a region that specifically binds to part or all of an antigen and is complementary to that antigen. When the antigen is large, the antigen binding molecule may only bind to a specific portion of the antigen, which is referred to as an epitope. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a library of complementary VL or VH domains, respectively, screened using VH or VL domains from antibodies that bind to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term "antigen-binding portion of an antibody", when used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody comprises amino acid residues from "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions excluding the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In particular, the heavy chain CDR3 is the region that contributes most to antigen binding and determines the properties of the antibody. Antibodies can be fully defined in terms of their heavy and light chain CDRs in terms of amino acid sequence, and can be described and characterized in particular in terms of their heavy chain variable region CDR1, CDR2, and CDR3 sequences and their light chain variable region CDR1, CDR2, and CDR3 sequences. An antibody can be defined or characterized as an antibody or fragment comprising a heavy chain and a light chain, wherein the heavy chain variable region comprises a specific CDR1, CDR2, and CDR3 sequence and the light chain variable region comprises a specific CDR1, CDR2, and CDR3 sequence. The CDRs and FRs of an antibody can be determined according to standard methods and analyses available to and known to those skilled in the art. Therefore, the CDRs and FRs can be determined according to the standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991) or according to the International Immunogenetics Information System (IMGT) (imgt.org; LeFranc, M-P (1999) Nucl Acids Res 27:209-212; LeFranc, M-P (2005) Nucl Acids Res 33:D539-D579).

By "substantially as listed" is meant that the variable region sequences of the antibodies of the present disclosure, and/or in particular the CDR sequences, will be identical or highly homologous to the designated sequences provided in the sequence listing appended herein. Wherein Xaa appears in the sequence listing and represents any naturally occurring amino acid residue.

So-called "high homology" envisions that only a few substitutions can be produced in the variable region sequence and/or CDR sequence, preferably 1 to 8, preferably 1 to 5, preferably 1 to 4, or 1 to 3 or 1 or 2 substitutions. The term is essentially as listed including particularly conservative amino acid substitutions that do not substantially or significantly affect the specificity and/or activity of the present antibody. It is envisioned that conservative amino acid substitutions are used for CDR region sequences. Exemplary CDR substitutions and variants are envisioned and provided herein. In some aspects, substitutions can be produced in the variable region sequence outside the CDR to retain the CDR sequence. Therefore, changes can be introduced or utilized in the variable region sequence or selectable non-homologous or mosaic variable region sequences so that the CDR sequence is maintained and the remainder of the variable region can be substituted.

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitopic determinants include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

Methods and methodologies for producing monoclonal antibodies by hybridomas or other means and methods are well known. Panels of monoclonal antibodies produced against pathogens, viruses, or influenza peptides can be screened for various properties; that is, neutralization, isotype, epitope, affinity, etc. Particularly attractive are monoclonal antibodies that neutralize the activity of viruses or their subunits. Such monoclones can be easily identified in neutralizing activity assays. High-affinity antibodies are also useful for effective binding and/or neutralization, or when immunoaffinity purification of natural or recombinant viruses is desired or when the purification is of interest.

Monoclonal antibodies for practicing the presently disclosed embodiments can be produced by initiating a monoclonal hybridoma culture containing a nutrient medium containing a hybridoma that secretes an antibody molecule having the appropriate antigen specificity. The culture is maintained under conditions sufficient for the hybridoma to secrete the antibody molecule into the medium, and for a period of time sufficient for the antibody molecule to be secreted into the medium. The antibody-containing medium is then collected. The antibody molecule can then be further isolated by known techniques.

The media used to prepare these compositions are well known in the art and commercially available, including synthetic culture media, inbred mice, and the like. An exemplary synthetic culture medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and 20% fetal bovine serum. An exemplary inbred mouse strain is Balb/c.

Methods for producing monoclonal antiviral antibodies are also well known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983). Typically, a virus, viral protein, or peptide analog is used alone or conjugated to an immunogenic carrier, such as an immunogen for producing monoclonal antibodies. Hybridomas are screened for their ability to produce antibodies that immunoreact with the virus, protein, or peptide analog.

Antibodies can also be bispecific or multispecific, for example, wherein one binding domain of an antibody is a virus neutralizing antibody for the present invention, and another binding domain has different specificities, for example, in conjunction with the apical surface of a cell or associated therewith, in conjunction with airway epithelial cells, etc. The bispecific antibodies of the present invention include wherein one binding domain of an antibody is a neutralizing agent for the present invention, including fragments thereof, and another binding domain is a different antibody or fragment thereof, including fragments of different antiviral specific antibodies (including selectable neutralizing antibodies or non-neutralizing antibodies). Another binding domain can be an antibody that recognizes or targets a specific cell type, such as in lung epithelium, alveolar macrophages, neural or glial cell specific antibodies. In the bispecific antibodies of the present invention, a binding domain of an antibody of the present invention can be combined with other binding domains or molecules, and the other binding domains or molecules recognize specific cell receptors and/or regulate cells in a specific manner, such as, for example, immunomodulators (e.g., interleukins), growth regulators or cytokines or toxins (e.g., ricin) or anti-mitotic or apoptotic agents or factors. Thus, the antibodies of the invention can be used to direct agents, markers, or target other molecules or compounds or antibodies in indications such as infection, inflammation, and the like.

The bispecific antibodies used in the present invention may comprise at least two Fab fragments. In one embodiment, the variable or constant regions of the heavy and light chains of the second Fab fragment are exchanged. Due to the exchange of variable or constant regions, the second Fab fragment is also referred to as a "cross-Fab fragment," "xFab fragment," or "cross-Fab fragment." This type of bispecific is described in US2013006011.

In specific and further aspects, the combined or sequential administration of neutralizing antibodies by IN together with the administration of antibodies by IP or IV provides an effective and enhanced synergistic approach for treating and/or preventing viral infections. The antibodies administered systemically (including IP or IV) can be neutralizing or non-neutralizing, and thus can be the same antibodies as those administered by IN, or can be modified antibodies or different antibodies. Thus, for intranasal delivery, the neutralizing antibodies can be different or distinct antibodies from the antibodies used in combination therewith (for delivery by another method, particularly systemic delivery, including IP or IV delivery).

The present disclosure demonstrates that neutralizing Fc function and the Fc portion of an antibody, and thus effector function, is not required for enhanced efficacy intranasally. Thus, antibodies and fragments such as Fab fragments, or antibodies lacking Fc or effector function, are effective in intranasal administration. Conversely, Fab fragments of antibodies (neutralizing or non-neutralizing) or antibodies lacking Fc or effector function are ineffective in IP or IV administration.

In some embodiments, immunoconjugates or antibody fusion proteins are provided in which antibodies, antibody molecules, or fragments thereof for use in the present invention are conjugated or attached to other molecules or reagents, including, but not limited to, antibodies, molecules, or fragments conjugated to chemical ablative agents, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, antivirals, antimicrobials, or peptides, cell wall and/or cell membrane disrupting agents, or drugs. In one aspect, the immunomodulator or antibody fusion may include antibodies, molecules, or fragments conjugated to antivirals, particularly anti-influenza agents. The anti-influenza agent may be a neuraminidase inhibitor. The anti-influenza agent may be selected from Tamiflu and Relenza. The anti-influenza agent may be an M2 inhibitor, such as amantadine or rimantadine. The anti-influenza agent may be a viral replication inhibitor.

The "subjects" or "subjects" for which antibodies, fragments thereof, and compositions (including the antibodies of the present invention) are used to treat and prevent influenza include, in addition to humans, any subject susceptible to influenza infection. Thus, various mammals, such as cattle, pigs, sheep, and other mammalian subjects, including horses and household pets, and seals, will benefit from the prophylactic and therapeutic use of these binding moieties. In some cases, antibodies are used that are appropriate for the subject species. Additionally, influenza is known to infect avian species, which will also benefit from compositions containing the antibodies of the present invention, again potentially appropriate for the subject species.

A "therapeutically effective amount" or "effective amount" of an antibody or fragment is a predetermined amount that imparts therapeutic efficacy to the subject being treated at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of the effect or the infection effect, or the doctor observes a change). The effective amount of each antibody in the composition can be in the range of about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/Kg to about 1 mg/kg. The effects contemplated herein, as the case may be, include medical therapeutic and/or prophylactic treatments. The specific dosage of the antibody or fragment administered according to the present disclosure to obtain a therapeutic and/or prophylactic effect will of course be determined according to the specific circumstances surrounding the case, including, for example, the compound administered, the route of administration, the co-administration of other active ingredients, the condition to be treated, the specific composition used, the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific compound used and the duration of treatment. The effective amount to be administered can be determined by a physician based on the aforementioned relevant circumstances and the exercise of sound medical judgment. 'Therapeutically effective amount' means the amount of a drug, compound, antimicrobial agent, antibody or pharmaceutical agent that will elicit the biological or medical response in the subject being sought by the medical doctor or other clinician. Specifically, with respect to viral infection and viral proliferation, the term "effective amount" is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful reduction in the amount or extent of viral replication or pathogenesis in a subject, and/or a reduction in the duration of illness (fever, joint pain, malaise), or a reduction in weight loss in an infected individual. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to reduce, preferably prevent, viral load, viral replication, viral spread or other characteristics of pathology such as, for example, fever or increased white blood cell count (which may be accompanied by the presence and activity of the virus).

In certain embodiments, an "effective amount" in the context of administering a therapy to a subject refers to an amount of a therapy sufficient to achieve one, two, three, four, or more of the following effects: (i) a reduction or improvement in the severity of an influenza virus infection, an influenza virus disease, or symptoms associated therewith; (ii) a reduction in the duration of an influenza virus infection, an influenza virus disease, or symptoms associated therewith; (iii) an inhibition of the progression of an influenza virus infection, an influenza virus disease, or symptoms associated therewith; (iv) a regression of an influenza virus infection, an influenza virus disease, or symptoms associated therewith; (v) an inhibition of the development or onset of an influenza virus infection, an influenza virus disease, or symptoms associated therewith; (vi) an inhibition of the recurrence of an influenza virus infection, an influenza virus disease, or symptoms associated therewith; (vii) a reduction or inhibition of the spread of influenza virus from one cell to another, from one tissue to another, or from one organ to another; (viii) an inhibition or reduction of the spread/transmission of influenza virus from one subject to another; (ix) a reduction in organ failure associated with an influenza virus infection or influenza virus disease; (x) a reduction in hospitalizations in subjects; (xi) a reduction in the length of hospital stays. (xii) increase in the survival rate of subjects infected with influenza virus or a disease associated therewith; (xiii) elimination of influenza virus infection or a disease associated therewith; (xiv) inhibition or reduction of influenza virus replication; (xv) inhibition or reduction of influenza virus binding or fusion with host cells; (xvi) inhibition or reduction of influenza virus entry into host cells; (xvii) inhibition or reduction of influenza virus genome replication; (xviii) inhibition or reduction of influenza virus protein synthesis; (xix) inhibition or reduction of influenza virus particle assembly; (xx) inhibition or reduction of influenza virus particles from Inhibition or reduction of the release of host cells; (xxi) reduction of influenza virus titer; (xxii) reduction in the number of symptoms associated with influenza virus infection or influenza virus disease; (xxiii) enhancement, improvement, supplementation or improvement of the preventive or therapeutic effect of another therapy; (xxiv) prevention of the onset or progression of secondary infection associated with influenza virus infection; (xxv) prevention of the onset of bacterial pneumonia secondary to influenza virus infection or reduction of disease severity; and/or (xxvi) changes in the immune response to influenza including cytokines, chemokines, complement, cellular responses, etc. In some embodiments, the "effective amount" of the therapy has a beneficial effect, but does not cure influenza virus infection or a disease associated therewith. In certain embodiments, the "effective amount" of the therapy may include administering multiple doses of therapy at a certain frequency to obtain a certain amount of therapy with a preventive and/or therapeutic effect. In other cases, the "effective amount" of the therapy may include administering a single dose of therapy in a specific amount.

"Symptoms" or "symptoms" associated with a viral infection (including specifically influenza infection), viral illness, or viral exposure may include, but are not limited to, a fever of 100°F or higher, feeling hot, cough and/or sore throat, runny or stuffy nose, headache and/or body aches, chills, fatigue, malaise, nausea, vomiting, and/or diarrhea, aches and pains in the joints and muscles and/or around the eyes.

The terms 'prevent' or 'prevent' refer to the reduction of the risk of acquiring or developing a disease or condition in a subject who may be exposed to a disease-causing agent or who is susceptible to the disease prior to the onset of the disease (i.e., preventing the development of at least one symptom of the clinical symptoms of the disease).

The term "prevention" is related to and encompassed by the term 'prevention' and refers to measures or methods whose purpose is to prevent rather than to treat or cure a disease. The term 'treating/treatment' of any disease or infection refers in one embodiment to ameliorating the disease or infection (i.e., preventing the growth of the disease or infectious agent or virus or reducing the manifestation, extent or severity of at least one of its clinical symptoms). In another embodiment, 'treatment' refers to improving at least one physical parameter that the subject may not be able to discern. In another embodiment, 'treatment' refers to regulating the disease or infection physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In other embodiments, 'treatment' relates to slowing the progression of the disease, the spread of the disease, or reducing infection.

As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "ul" or "μl" means microliter, "ml" means milliliter, and "l" means liter.

Methods for prevention and treatment are conventional and generally well known. Antibodies or other binding moieties are generally provided by injection, but oral vaccines are also known to be effective. Dosage levels and administration times are easily optimized and are within the capabilities of those skilled in the art. In an alternative, recombinant materials for in situ production of antibodies can be administered. Technology can now be used in the cells of animal subjects, including, for example, lymphocytes or myocytes, to express antibody genes (see, for example, Johnson, P.R. et al., Nature Medicine (2009) 15: 901-907). The in situ generation of such antibodies can reduce the cost of manufacturing medicaments and simplifies management. The administration of the antibodies of the present invention carried out by such methods is another aspect of the present invention.

The human cells (or cells from any given species) that secrete useful antibodies can be identified using the CellSpot ^™ method described in particular U.S. Patent No. 7,413,868 (the contents of which are incorporated herein by reference). In short, this method can screen single cells obtained from human (or other species) subjects in a high-throughput assay utilizing labeling (utilizing microparticle markers) and microscopy. In an illustrative embodiment, even the secreted antibodies of a single cell can be analyzed by adsorbing or coupling the secreted antibodies on a surface, followed by treating the surface with the required reagents (each of which is coupled to a unique microparticle marker). The footprint of the cell can therefore be identified by means of a microscope. By using this technology, the desired antibody secretion of millions of cells can be screened, and even rare antibodies can be recovered, such as those antibodies that are intended to be used for cross-strain passive influenza immunization herein. Since human subjects have existing antibodies for at least some influenza strains, and since the antibodies obtained by the method of the present invention bind to conserved sequences, these antibodies are used for addressing new strains and strains encountered by the population.

Methods for obtaining suitable antibodies are not limited to CellSpot ^™ technology, nor are they limited to human subjects. Cells producing suitable antibodies can be identified in various ways, and the cells can be cells from laboratory animals such as mice or other rodents. The nucleic acid sequences encoding these antibodies can be isolated and the various antibody forms produced, including chimeric and humanized forms of antibodies produced by non-human cells. In addition, recombinantly produced antibodies or fragments include single-chain antibodies or their Fab or Fab ₂ regions. Human antibodies can also be obtained using hosts such as those with humanized immune systems. Methods for producing antibodies for screening suitable binding characteristics are well known in the art.

Similarly, means for constructing RNA aptamers with desired binding modes are also known in the art.

As noted above, antibodies or other binding moieties may bind to the activated form, the inactive form, or both forms of the hemagglutinin protein. In some cases, it may be advantageous for the epitope to be present at the cleavage site of the protein, as this is relatively conserved between strains, but it is preferred that the binding moiety bind to both the trimer and the activated form.

The cleavage sites of various strains of influenza A and influenza B are known. For example, the above-cited paper by Bianchi et al. shows in Table 2 the sequences surrounding the cleavage sites of several such strains:

Table 2. Consensus sequences of the solvent-exposed regions of the mature cleavage sites of influenza A and B viruses.

^a The position of the cleavage between HA ₁ and HA ₂ is indicated by an arrow.

^b The consensus sequence is identical for Victoria and Yamagata lineages.

As indicated, the strict consensus sequence begins at an arginine residue upstream of the cleavage site, and thus a preferred consensus sequence for inclusion in the test peptides of the present invention has the sequence RGI/L/FFGAIAGFLE (SEQ ID NO: 57). It is possible to use only a portion of this sequence in a test peptide.

As noted above, once cells secreting the desired antibodies have been identified, the nucleotide sequences encoding them can be directly retrieved and the desired antibodies produced recombinantly on a large scale. This also enables manipulation of antibodies so that they can be produced, for example, as single-chain antibodies or as only their variable regions, or as bispecific antibodies.

The retrieved nucleic acid can be physically stored and recovered for later recombinant production, and/or sequence information about the coding sequence of the antibody can be retrieved and stored to allow for subsequent synthesis of the appropriate nucleic acid. The availability of information contained in the coding sequence and rapid synthesis and cloning techniques, together with known recombinant production methods, allow for the rapid production of desired antibodies in the event of a pandemic or other emergency.

For reference, the sequences of the human constant regions of the two heavy and light chains have been described and are shown herein as SEQ ID NOs: 33-35. In WO 2011/16008 and WO 2013/086052, cited above, various monoclonal antibodies with variable regions having defined amino acid sequences and corresponding nucleotide coding sequences were recovered that bind with varying degrees of affinity to HA proteins of various strains of influenza. These antibodies include mAb53 and mAb579. mAb53 binds to H1 with specific affinity; in addition, mAb53 binds tightly to H5, H7, and H9. mAb579 binds to H3 and H7 with affinities as low as the subnanomolar range. Reactivity with the native trimer of HA was verified using HA expressed on the surface of HEK293 cells. Antibody binding was measured by flow cytometry. The plasmid encoding HA was provided by S. Galloway and D. Steinhauer of Emory University, and the trimers displayed on the cell surface of various clades were recognized by the monoclonal antibodies of the present invention.

There are currently multiple technologies for producing single antibody-like molecules (bispecific antibodies) that integrate the antigen-specific domains from two separate antibodies. Therefore, single antibodies with very broad strain reactivity can be constructed using single antibodies with broad reactivity to group 1 and group 2, respectively, or with Fab domains of antibodies that bind to one of these groups in combination with influenza B. Suitable technologies have been described by Macrogenics (Rockville, MD), Micromet (Bethesda, MD), and Merrimac (Cambridge, MA). (See, e.g., Orcutt, K.D. et al., "A modularIgG-scFv bispecific antibody topology," Protein Eng Des Sel. (2010) 23:221-228; Fitzgerald, J. et al., "Rational engineering of antibody therapeutics targeting multiple oncogene pathways," MAbs. (2011) 1:3(3); and Baeuerle, P.A. et al., "Bispecific T-cell engaging antibodies for cancer therapy," Cancer Res. (2009) 69:4941-4944.)

Therefore, it is particularly useful to provide antibodies or other binding moieties that bind to multiple types of hemagglutinin proteins by constructing bispecific antibodies. Particularly useful combinations are those that combine the binding specificities of mAb53 (H1, H5, and H9) with mAb579 (H3 and H7).

While mAb53 bound to _HA0 with high affinity, it did not bind to _HA1 , suggesting binding to the complementary _HA2 fragment, which was confirmed. Since mAb53 did not bind to _HA0 when tested by immunoblotting, it was assumed that the dominant epitope was at least partially conformational.

Tables 3 and 4 provide the KD and _IC50 values shown by mAbs 53 and 579 for hemagglutinin proteins of various strains of influenza A.

Table 3. KD values of mAbs 53 and 579 for monomeric and trimeric HA of various influenza virus strains.

Table 4. _IC50 values of mAb 53 and 579 against various influenza virus strains

These values were obtained in MDCK monolayer microneutralization assays.

The present invention provides a variety of novel mAbs with specificities complementary to those cited above (e.g., mAb53 and mAb579), including mAbs that recognize influenza B. The availability of these additional mAbs provides the opportunity to prepare passive vaccines that are effective across a wide range of influenza strains, thereby alleviating the need for accurate determination or diagnosis of the infectious agent strain prior to treatment.

For those binding portions of the present disclosure that are indeed mAbs, as is well known, specificity is essentially determined by the complementarity determining regions (CDRs) present in the variable regions of the light and heavy chains. The influence of the heavy chain CDRs is understood to be more important, while the identity of the light chain is more flexible. Therefore, the overall specificity of a mAb or fragment is generally determined by the properties of the heavy chain CDRs, while the CDRs present in the light chain are subject to more variation while maintaining essentially the same specificity of the antibody.

In addition to bispecific antibodies per se, the present disclosure contemplates the use of only heavy chains in constructs for neutralizing viral infection; such antibodies can also be bispecific. Because it is understood in the art that specificity is primarily conferred by the heavy chain variable region, in some cases, heavy chains alone have been successfully and herein successfully used as active ingredients in vaccines. Alternatively, heavy chains with appropriate specificity can be associated with various light chain forms to enhance affinity for viruses or the ability to neutralize viruses.

As noted above, the binding specificity of the binding moieties of the present invention is determined by the CDRs, primarily those of the heavy chain, but also complemented by those of the light chain. Thus, a binding moiety of the present invention may contain the three CDRs of the heavy chain and, optionally, the three CDRs of the light chain that match them. The present invention also encompasses binding moieties that bind to the same epitope as the binding moiety that actually contains these CDRs. Thus, for example, aptamers with the same binding specificity—that is, those that bind to the same epitope as the binding moiety that actually contains the CDRs—are also encompassed. Because binding affinity is also determined by the arrangement of the CDRs within the framework, a binding moiety of the present invention may contain a complete variable region of the heavy chain containing the three relevant CDRs and, optionally, a complete variable region of the light chain containing the three CDRs associated with the light chain that complements the heavy chain. This is also true for binding moieties immunospecific for a single epitope, as well as for bispecific antibodies or binding entities capable of binding to two separate epitopes.

Therefore, for the binding portion of the variable region of an antibody with suitable affinity, the important amino acid sequence is the CDR sequence arranged on the framework, which can be changed without affecting specificity or reducing affinity to an unacceptable level. The definition of these CDRs can be achieved by methods known in the art. Specifically, the most commonly used method for identifying relevant CDRs is the method of Kabat disclosed in Wu, TT et al., J. Exp. Med. (1970) 132: 211-250 and in the book (Kabat, EA, et al. (1983) Sequence of Proteins of Immunological Interest , Bethesda National Institute of Health, page 323). Another similar and commonly used method is the method of Chothia disclosed in Chothia, C. et al., J. Mol. Biol. (1987) 196: 901-917 and Chothia, C. et al., Nature (1989) 342: 877-883. Additional modifications have been suggested by Abhinandan, KR et al., Mol. Immunol. (2008) 45:3832-3839. The present invention includes CDRs as defined by any of these systems.

Some criticisms have been addressed by different workers on the two systems; therefore, it should be understood that the CDRs as specified herein and in the claims may differ slightly. As long as the resulting variable regions retain their binding ability, the precise location of the CDRs is unimportant, and those regions specified in the claims are considered to include the CDRs identified by any accepted system.

Can use standard method and preparation by this antibody of the present invention or other binding portion as passive vaccine administration.Usually, by injection (normally intramuscular or subcutaneous) administer this type of vaccine, but other modes of administration are by no means excluded, comprise intravenous administration.By suitable design, also oral administration vaccine can be used.As noted above, developing technology is expected to allow in situ production mAb in human muscle or lymphocyte, for example, antibody of the present invention is also suitable for this production method.

About preparation, use conventional passive antibody vaccine formulation excipient, or can be used in carrier such as liposome, micelle, nanoparticle etc. in binding moiety.The particularly interesting method that is included in the scope of the present invention is by the adsorption of nanoparticles prepared from mAb by binding moiety attached to erythrocyte, as Anselmo, A.C. etc., described in ACS Nano. and published online as 10.1021/NN404853Z in 2013. According to this technology, by carrier particles or medicament or both being adsorbed on red blood cells, obtain preferential delivery to lung, thereby prevent the shortened half-life caused by the processing in liver and spleen, and provide higher concentration in lung. This is particularly suitable for influenza passive vaccine, and the various methods that binding moiety is attached to erythrocyte can be used. However, according to this paper, all that is required is adsorption. Other methods for strengthening the distribution of monoclonal antibodies to the lung can also be used to enhance the effectiveness of passive vaccines, such as the atomized products delivered by inhalation.

In some embodiments, the present invention provides methods and devices for effectively treating and preventing influenza viruses and influenza virus infections by simultaneous or combined administration of antibodies directed against Group 1 and Group 2 influenza A and influenza B. According to the present invention, a combination of broadly neutralizing antibodies is administered comprising an antibody or active fragment that is broadly directed against Group 1 influenza A virus, an antibody or active fragment that is broadly directed against Group 2 influenza A virus, and an antibody or active fragment that is broadly directed against influenza B virus.

In some embodiments, a passive vaccine, antibody composition or antibody combination is provided, which comprises antibodies that are effective against influenza A and influenza B and have a wide range of reactivity with multiple strains. This will provide an effective anti-influenza agent in a single dose and can be used when initial symptoms develop, or after exposure to influenza. Likewise, this will bypass the need to characterize the infectious virus in detail before administering the antibody or antibody mixture. Determining the diagnosis of influenza strains requires clinical laboratory facilities and generally requires a minimum report time of 12-24 hours, which results in a disadvantageous delay in treatment if the influenza virus strain needs to be determined before selecting the appropriate targeted therapy. By utilizing a broadly reactive composition for influenza A and B strains, prior strain diagnosis before treatment is not necessary.

In some embodiments, novel methods and means are provided for effectively treating and preventing influenza viruses by administering (including intranasal or inhaled administration) a combination of antibodies or a combination antibody composition directly to the respiratory tract or airway. The present invention shows that direct delivery of neutralizing antibodies or a combination of antibodies to the respiratory tract, including airway delivery, such as by inhalation (IH) and/or intranasal (IN) delivery and administration is superior to systemic administration (IV or IP) of the same amount of the same antibody or combination of antibodies at lower doses, more effective and more efficient. Treatment or prevention using antibodies delivered by IN or IH before or even after viral exposure or infection is effective.

In some embodiments, there is provided a composition for airway administration that is effective for the treatment or prevention of influenza virus infection in a mammal, particularly an inhaled or intranasal composition, particularly an antibody combination composition, the composition comprising antibodies or active fragments broadly directed against group 1 influenza A virus, antibodies or active fragments broadly directed against group 2 influenza A virus, and antibodies or active fragments broadly directed against influenza B virus. In a first aspect, the present invention provides an inhaled or intranasal composition that is effective for the treatment or prevention of influenza virus infection in a mammal, comprising a combination of virus neutralizing monoclonal antibodies in a single unit dose, wherein each antibody is included in the dose at an administration amount of 1 mg/kg or less. The present invention provides an inhaled or intranasal composition that is effective for the treatment or prevention of influenza virus infection in a mammal, comprising a combination of neutralizing monoclonal antibodies in a single unit dose, wherein each antibody is administered in an effective amount of 10 mg/kg or less. The present invention provides an inhaled or intranasal composition that is effective for the treatment or prevention of influenza virus in a mammal, comprising a combination of influenza neutralizing monoclonal antibodies (each antibody is less than 1 mg/kg) in a single unit dose. The present invention provides an inhaled or intranasal composition effective for the treatment or prevention of influenza virus in mammals, comprising a combination of influenza neutralizing monoclonal antibodies (less than 0.5 mg/kg of each antibody) in a single unit dose. The present invention provides an inhaled or intranasal composition effective for the treatment or prevention of influenza virus in mammals, comprising a combination of antibodies, wherein each antibody is administered at a dose of less than 0.1 mg/kg.

In other aspects, the present invention provides an inhaled or intranasal composition effective for treating, preventing, or reducing the spread of influenza virus in mammals, comprising a combination of influenza neutralizing antibodies against circulating influenza virus strains. In one aspect, the present invention provides a composition for intranasal administration consisting of a combination of influenza neutralizing antibodies against circulating influenza virus strains, particularly consisting of influenza A anti-H1 antibodies, influenza A anti-H3 antibodies, and anti-influenza B antibodies. In one aspect, the composition includes influenza A antibodies that are effective against or further effective against other influenza strains, including but not limited to H2, H5, and H7 strains.

The composition is suitable for use in and is applicable to the treatment or prevention of influenza virus. In a specific aspect, the composition is suitable for reducing the spread of influenza virus.

In a specific embodiment, a composition is provided comprising a combination of antibodies comprising antibody Mab53 (TRL053), antibody Mab579 (TRL579), an antibody fragment thereof, or a bispecific antibody based thereon, and one or more influenza antibodies against influenza B virus selected from TRL847, TRL845, TRL849, TRL848, TRL846, TRL854, TRL809, and TRL832.

In some embodiments, a pharmaceutical composition is provided, which comprises (a) a first antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence, wherein the heavy chain amino acid sequence comprises a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, and 13, and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, and 16; (b) a second antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence, wherein the heavy chain amino acid sequence comprises a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, and 23, and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain variable region (LCVR); and (c) one or more influenza virus neutralizing antibodies against influenza B, particularly against the Yamagata lineage and/or Victoria lineage, wherein the antibody or fragment thereof is selected from an antibody or fragment thereof comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3) (HCDR1/HCDR2/HCDR3), and the HCDR1/HCDR2/HCDR3 is selected from the group consisting of: SEQ IDNO:31/32/33;41/42/43;51/52/53;61/62/63;71/72/73;81/82/83;91/92/93;101/102/103;111/112/1 13;121/122/123;131/132/133;141/142/143;151/152/153;161/162/163;171/172/173;181/182/183;19 1/192/193; 201/202/203; 211/212/213; 221/222/223; 231/232/233; 241/242/243; 251/252/253; 261/262/263; 271/272/273 and 281/282/283, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants and said antibodies having the property of binding to and inhibiting influenza virus.

In some embodiments, a pharmaceutical composition is provided, which comprises (a) a first antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence, wherein the heavy chain amino acid sequence comprises a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, and 13, and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, and 16; (b) a second antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence, wherein the heavy chain amino acid sequence comprises a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, and 23, and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 24, 25, and 26. NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain variable region (LCVR); and (c) one or more influenza B, particularly influenza virus neutralizing antibodies against the Yamagata lineage and/or Victoria lineage, wherein the antibody or fragment thereof is selected from an antibody or fragment thereof comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain complementary determining region 3 (LCDR3) (LCDR1/LCDR2/LCDR3), wherein the LCDR1/LCDR2/LCDR3 is selected from the group consisting of: SEQ ID NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3; and (c) one or more influenza B, particularly influenza virus neutralizing antibodies against the Yamagata lineage and/or Victoria lineage, wherein the antibody or fragment thereof is selected from an antibody or fragment thereof comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain complementary determining region 3 (LCDR3) (LCDR1/LCDR2/LCDR3), wherein the LCDR1/LCDR2/LCDR3 is selected from the group consisting of: SEQ ID NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 NO:34/35/36; 44/45/46; 54/55/56; 64/65/66; 74/75/76; 84/85/86; 104/105/106; 114/115/16; 124/ 125/126; 134/135/136; 144/145/146; 154/155/156; 164/165/166; 174/175/176; 184/185/186; 194/ 195/196; 204/205/206; 214/215/216; 224/225/226; 234/235/236; 244/245/246; 254/255/256; 264/265/266; 274/275/276 and 284/285/286, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, which variants are capable of binding to and inhibiting influenza virus.

In some embodiments, a composition is provided that comprises (a) a first antibody or an antigen-binding fragment thereof, comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, and 13, and a light chain amino acid sequence comprising a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, and 16; (b) a second antibody or an antigen-binding fragment thereof, comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, and 23, and a light chain amino acid sequence comprising a light chain amino acid sequence comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 24, 25, and 26. NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain variable region (LCVR); and (c) one or more influenza B, particularly against the Yamagata lineage and/or Victoria lineage influenza virus neutralizing antibodies or fragments, wherein the antibody or fragment thereof is selected from an antibody or fragment thereof comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises heavy and light chain CDR sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3, which are selected from the group consisting of: SEQ ID NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3; and (c) one or more influenza B, particularly against the Yamagata lineage and/or Victoria lineage influenza virus neutralizing antibodies or fragments, wherein the antibody or fragment thereof is selected from an antibody or fragment thereof comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises heavy and light chain CDR sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3, which are selected from the group consisting of: SEQ ID NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 NO:31/32/33/34/35/36;41/42/43/44/45/46;51/52/53/54/55/56;61/62/63/64/65/66;71/7 2/73/74/75/76; 81/82/83/84/85/86; 91/92/93/94/95/96; 101/102/103/104/105/106; 111/11 2/113/114/115/116; 121/122/123/124/125/126; 131/132/133/134/135/136; 141/142/143/144/145/146; 151/152/153/154/155/156; 161/162/163/164/165/166; 171/172/173/174/175/1 76; 181/182/183/184/185/186; 191/192/193/194/195/196; 201/202/203/204/205/206; 211/212/213/214/215/216; 221/222/223/224/225/226; 231/232/233/234/235/236; 241/242/243/ 244/245/246; 251/252/253/254/255/256; 261/262/263/264/265/266; 271/272/273/274/275/276 and 281/282/283/284/285/286, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, which variants are capable of binding to and inhibiting influenza virus.

In some embodiments, a pharmaceutical composition is provided, which comprises (a) a first antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence, wherein the heavy chain amino acid sequence comprises a heavy chain variable region (HCVR) of an antibody or fragment thereof comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, and 13, and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, and 16; (b) a second antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence, wherein the heavy chain amino acid sequence comprises a heavy chain variable region (HCVR) comprising HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, and 23, and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a light chain variable region (LCVR) comprising LCDR1/LCDR2/LCDR3 of SEQ ID NOs: NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain variable region (LCVR); and (c) one or more influenza B, particularly influenza virus neutralizing antibodies or fragments thereof against the Yamagata lineage and/or Victoria lineage, wherein the antibody or fragment thereof is selected from an antibody or fragment thereof comprising a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the light chain amino acid sequence comprises a HCVR/LCVR sequence pair selected from the group consisting of: 39/40, 49 /50, 59/60, 69/70, 79/80, 89/90, 99/100, 109/110, 119/120, 129/130, 139/140, 149/150, 159/160, 169/170, 179/180, 189/190, 199/200, 209/210, 219/220, 229/230, 239/240, 249/250, 259/260, 269/270, 279/280 and 289/290.

In some embodiments, neutralizing antibodies for airway delivery and administration can be combined with non-neutralizing antibodies. The application shows that IN administration can be combined with other selectable routes of administration (including IP or IV administration) to give overall and combined enhanced efficacy. As provided herein, the IN and IP administration of the combination of antibodies provides enhanced synergistic activity and efficacy compared to separate IN or IP administration. In addition to providing replacement or alternative administration or treatment methods, the present invention also provides enhanced combination methods for antibody-mediated therapy and prevention, wherein pulmonary administration is combined with systemic administration to obtain the highest efficacy.

In some embodiments, an alternative mode of antibody administration via pulmonary administration allows for lower dosing, lower dosage formulations, and effective broad-spectrum anti-influenza antibody combination compositions.

In some embodiments, a combination of binding molecules, particularly human monoclonal antibodies or fragments thereof, that neutralize influenza viruses and are effective against influenza viruses, wherein the combination is effective against Group 1 influenza A virus, Group 2 influenza A virus, and influenza B virus. The combination of antibodies is effective in the treatment or prevention of influenza disease against both type A and type B, thereby providing an effective agent against all related and circulating influenza viruses in a single composition or dose.

The present invention provides compositions comprising or consisting of a combination of influenza monoclonal antibodies or fragments thereof, which are effective in treating or preventing influenza A and influenza B in combination.

The combination composition can be administered to effectively target uncharacterized and undefined influenza infections. In a specific aspect of the invention, the combination composition is administered directly to the respiratory tract, including by intranasal or inhalation administration. Specifically, the antibody combination or mixture is administered in a single dose, or can be administered in multiple rounds of administration, or in sequential administration of each antibody. Thus, the antibody mixture is as effective against any unknown or unidentified circulating influenza virus as a single specifically defined antibody against a defined target influenza. For example, administration of the mixture is as effective against any given group 1H1 virus as a single specific anti-H1 virus antibody.

In some embodiments, the antibodies of the combination have certain properties and aspects that make them effective and particularly suitable and useful in combination.In specific aspects, the antibodies of the present combination each and all exhibit significant binding and affinity to influenza virus.

In some embodiments, the combined antibodies can be co-formulated, mixed, or administered sequentially with other antibodies to treat a wide range of influenza-like illnesses, including pathogens such as RSV, PAIV, or MPV.

In some embodiments, each antibody in the effective combination exhibits nanomolar or subnanomolar affinities for a variety of influenza strains. This is a different and particularly useful and applicable aspect compared to other known or existing antibodies. For example, Mab53 and Mab579 exhibit nanomolar or subnanomolar affinities for various H1 (and H5) or H3 (and H7) strains, respectively, showing significantly greater binding affinity relative to CR6261 and CR8020 (WO2013/086052). The anti-influenza B antibodies of this combination similarly exhibit nanomolar or subnanomolar binding affinities for influenza B virus strains of the Yamagata and Victoria clades.

In some embodiments, the antibodies of the combination aspects of the present disclosure are designed and selected to have similar biophysical properties, including isoelectric points (pi). In some embodiments, the antibodies selected for combination into the composition each exhibit a pi that is within ±2 pi points of each other, within ±1.5 pi points of each other, within ±1.0 pi points of each other, or within ±0.5 pi points of each other.

In some embodiments, the antibodies of the combination aspects of the present disclosure are designed and selected to have similar biophysical properties, such as robust thermal stability. In some aspects, antibodies are provided that exhibit a first melting temperature (Tm1) in a melting curve assay when performed in PBS at ≥50°C, ≥55°C, ≥60°C, ≥65°C, or ≥70°C.

In some embodiments, it is preferred to design and express antibodies with similar or comparable constant region sequences, and the antibodies are preferably the same IgG selected from IgG1, IgG2, IgG2, IgG3 or IgG4. Modified Fc sequences that provide longer half-life in circulation are also known in the art.

In some embodiments, anti-influenza B antibodies are provided that comprise a human IgG1 constant region amino acid sequence. In some embodiments, anti-influenza B antibodies are provided that comprise a human IgG1 constant region amino acid sequence of SEQ ID NO: 297. In some embodiments, anti-influenza B antibodies are provided that comprise a human light chain kappa constant region of SEQ ID NO: 295 or a human light chain lambda constant region of SEQ ID NO: 296.

In some embodiments, an antibody composition is provided that comprises an anti-influenza B antibody comprising a human IgG1 constant region comprising SEQ ID NO: 297. In some embodiments, an antibody composition is provided that comprises an anti-influenza B antibody comprising a human light chain kappa constant region comprising SEQ ID NO: 295 or a human light chain lambda constant region comprising SEQ ID NO: 296.

In some embodiments, compositions comprising a combination of one or more anti-influenza A antibodies and one or more anti-influenza B antibodies are provided. In some aspects, the antibodies in the combination exhibit one or more properties selected from low to no antibody aggregation, absence of intermolecular association, and/or absence of competitive binding. These aspects are demonstrated and exemplified in the antibodies of the present combination.

The present invention relates to novel methods, regimens and methods for effectively treating and preventing influenza virus infection by administering the present antibody mixture to the airway or respiratory tract (such as by intranasal or inhalation administration of one or more neutralizing antibodies). Intranasal or inhalation administration of influenza virus neutralizing antibodies is more effective in therapeutically or prophylactically treating or blocking the virus than alternative modes of administration such as IP administration. Inhalation and/or intranasal delivery and administration are superior to systemic administration (IV or IP) of the same antibody or combination of antibodies in the same amount at lower doses, are more effective and more efficient. Treatment or prevention using one or more antibodies delivered by IN before or even after viral exposure or infection is effective.

Methods or protocols combining a lung dose of an antibody with a systemic dose of the antibody are particularly effective therapeutically or prophylactically for influenza virus. Such methods or protocols include combining an intranasal or inhaled dose of one or more antibodies with an IP or IV dose of one or more antibodies. The intranasal or inhaled dose can be administered before, after, simultaneously with, or sequentially with the IP or IV dose. One or more intranasal, inhaled, IP or IV doses or multiple doses can be administered. The antibody administered intranasally can be an antibody fragment that does not have Fc or effector functions, such as Fab, whereas the antibody administered IP can have effector functions or enhanced effector functions.

According to the present disclosure, neutralizing antibodies are administered to the airway or respiratory tract to enhance efficacy against viruses (particularly influenza viruses). Administration to the airway or respiratory tract can be by any recognized or known method, and can include inhalation administration or intranasal administration. In order to enhance efficacy, the antibody is delivered to one or more of the upper and lower respiratory tracts, and the respiratory tract may include the nasal cavity, nose, sinus, throat, pharynx, larynx, trachea, bronchi, and lungs.

"Inhalation" refers to the ingestion, particularly in the context of ingestion or administration/administration of an agent or compound, including an antibody or its active fragment, or a composition comprising such an antibody or its active fragment, wherein the agent, compound, antibody, fragment (including as contained in the composition) is delivered to all or part of the respiratory tract. The respiratory tract may include the upper and/or lower respiratory tract. The upper respiratory tract includes the nose, nasal cavity, sinuses, larynx, trachea. The lower respiratory tract includes the lungs, airways (bronchi and bronchioles) and air sacs (alveoli). Inhalation can occur via the nose or mouth, or via direct delivery to the lower respiratory tract (as in intratracheal administration). Thus, inhalation can include only or primarily nasal, intranasal, inhalation via the mouth, oral inhalation, intratracheal inhalation, intratracheal instillation. Thus, inhalation provides and contemplates any mode of administration whereby a drug, agent, composition, antibody, fragment exclusively, specifically or preferentially reaches or is deposited on or in the respiratory tract (including the upper and/or lower respiratory tract).

As used herein, the term "intranasal" includes, but is not limited to, administration, application, or delivery occurring within or via the nose or nasal structures or airways (e.g., by inhalation). The term intranasal as used herein and as exemplified by embodiments in the Examples is not intended to be limited or implied to be limited to administration directly or exclusively or solely via the nose or nasal cavity, and is specifically intended to exclude other modes of administration whereby the drug, agent, antibody, fragment, composition is delivered to or otherwise provided to the respiratory tract, deposited in or on the respiratory tract, or otherwise distributed to the respiratory tract.

Devices for administration or delivery to the respiratory tract or airways are known and recognized by those skilled in the art and in clinical or medical practice and can be adapted for use with the methods, regimens, and compositions of the present invention. Devices include metered dose inhalers, metered spray pumps, manual bulb nebulizers, small or large volume nebulizers, ultrasonic nebulizers, and dry powder inhalers.

The embodiments disclosed herein have been applied and used to particularly treat or prevent targeting, infection or factors or pathogens that affect the respiratory tract. These viruses can exhibit apical replication, thereby allowing susceptibility to be neutralized by mAbs or fragments thereof delivered to the lungs, which can subsequently result in improved efficacy compared to systemic delivery. Therefore, the present embodiment has been applied and used to treat or prevent respiratory infections, particularly respiratory viruses, and factors associated with respiratory diseases or causally related thereto. Common viral respiratory diseases are diseases caused by a variety of viruses with similar properties and affecting the upper respiratory tract. The viruses involved can be influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, and respiratory adenovirus. Parainfluenza virus is the main cause of croup in young children and can cause bronchitis, pneumonia, and bronchiolitis. Adenoviruses mainly invade the respiratory and gastrointestinal tracts and the conjunctiva of the eyes. Adenoviruses can cause a variety of diseases from pharyngitis to pneumonia, conjunctivitis, and diarrhea. Symptoms can appear 1-10 days after exposure to the virus.

The clinical administration of antibodies for treating or alleviating the patient's condition (cancer, inflammatory conditions, antiviral, anti-infective) has been specifically used systemic administration, usually IV administration, which requires a large amount of expensive antibodies, the assistance of medical staff and a large amount of time for administration (common IV doses last for 2 hours). However, other modes of administration can be mentioned, such as intranasal, particularly in patents or applications covering these antibodies, and intranasal administration is at best considered to be an equivalent alternative, completely ignored, or not pursued, probably because it is not well understood, is considered to lack attractiveness or is not very effective, and is considered to indirectly or not as effective as IP or IV administration routes to directly activate the immune system. However, the present invention provided herein and unusual studies show that intranasal administration is indeed preferred, a more effective alternative, particularly for neutralizing antibodies. Specifically, neutralizing antibodies that can act on the site or position of initial pathogen damage or exposure are more effective than alternative modes of administration. Therefore, when administered by pulmonary route compared to systemic route, the neutralizing antibodies targeting influenza show significant differences in the efficacy brought in part by different mechanisms of action.

Airway administration provides a unique opportunity for delivering an effective low-dose and low-cost therapy that does not require confirmation by diagnostic assays. The manifestation of symptoms during the influenza season will be sufficient for the doctor to administer the low-dose mixture, for example as a dry powder inhaler or as a nebulizer or via other airway delivery methods. This standard of care without diagnosis is the current practice of Tamiflu and Relenza, but it is not possible to use expensive intravenous antibody therapy, which is impractical and cost-prohibitive. Following follow-up diagnosis, administration of high-dose antibodies can be administered systemically, such as by intravenous routes or by airway administration, which can be composed of a mixture or of separate specific antibodies that are specific for the influenza type.

Therefore, according to the present invention, the pulmonary delivery of antibodies provides a surprising and significant improvement in efficacy compared to systemic routes such as IV or IP routes. In addition, the enhanced intranasal efficacy is demonstrated by antibodies with neutralizing effects. Non-neutralizing antibodies, particularly antibodies that do not show direct inhibition or blocking of influenza virus by using accepted or known neutralization assays or virus blocking, show impaired efficacy when administered intranasally or by IP. This study shows that by using a recognized and known influenza mouse model, the intranasal (IN) delivery of neutralizing antibodies can enhance therapeutic and preventive efficacy to more than 10 times compared to intraperitoneal (IP) or intravenous (IV) administration routes. When administered by IN rather than by IV or IP routes, comparable efficacy can be obtained using the same dose less than 1/10. Neutralizing antibodies administered to the airways (such as intranasally) can enhance therapeutic efficacy by 10 to 100 times or at least 10 to 100 times at a data level. Compared to intraperitoneal (IP) administration of the same antibody under similar conditions, intranasal administration of neutralizing antibodies can significantly enhance the therapeutic efficacy by at least 10 times, at least 50 times, more than 10 times, more than 50 times, more than 100 times, and up to 100 times. Intranasal administration of neutralizing antibodies provides a new and unexpected method for preventing and treating infections, including influenza infections in particular. IN administration can now be effectively supplemented and combined with other forms of administration to provide more effective and less costly methods for treatment and prevention. Airway administration, including IN administration, enables a combination or mixture of antibodies in a single dose to have efficacy against any expected influenza virus.

Composition

According to the present disclosure, there is provided an antibody combination composition with the purposes and effectiveness for influenza. When applied directly to the airway (such as intranasally), the antibody combination composition is effective at low doses. The composition particularly comprises antibodies, particularly neutralizing antibodies, particularly monoclonal antibodies or their active fragments, particularly antiviral antibodies, particularly combinations of influenza antibodies. One or more neutralizing antibodies that can neutralize more than one type or subtype of influenza are combined with antibodies that neutralize different types or groups of influenza. The composition of the present invention particularly comprises a combination of influenza neutralizing antibodies for circulating influenza virus strains. The composition particularly comprises influenza neutralizing antibodies for circulating influenza virus strains, particularly a combination of anti-influenza A and anti-influenza B antibodies. The composition particularly comprises a combination of influenza neutralizing antibodies, the combination being directed against appropriate and relevant circulating influenza virus strains, particularly against influenza A H1 and H3 subtypes and against influenza B of Yamagata and Victoria lineages. The one or more compositions may comprise three or more neutralizing antibodies, as long as influenza A and influenza B viruses are neutralized by the combination or antibodies.

The composition may particularly comprise neutralizing antibodies against circulating influenza strains of influenza, in particular a combination of influenza A anti-H1 antibodies, influenza A anti-H3 antibodies and anti-influenza B antibodies. The composition may comprise influenza A antibodies that are effective against or further effective against influenza H5 and H7 strains. The influenza antibodies may be strain-specific or non-specific and may neutralize influenza A, including H1 subtypes and/or H3 subtypes and/or H5 and/or H7 or other influenza A strains or subtypes, and/or may neutralize influenza B, including Yamagata and/or Victoria lineages. The composition may have the same components or different components or additive components as an alternative administration composition (such as IV or IP) of the antibodies.

In some embodiments, compositions comprising neutralizing antibodies or fragments thereof, including Fab fragments, are provided. The compositions of the present invention may include influenza virus neutralizing antibodies, particularly group 1 antibody TRL053/Mab53, group 2 antibody TRL579/Mab579, and B antibodies selected from TRL847, TRL845, TRL849, TRL848, TRL846, TRL854, TRL809, and TRL832, fragments thereof, synthetic or recombinant derivatives thereof, humanized or chimeric forms thereof, and combinations of antibodies with their heavy and light chain CDRs.

The virus neutralizing antibody may be, in particular, an antibody fragment capable of neutralizing. In one aspect, the antibody fragment lacks Fc and/or lacks or has reduced effector function. The antibody fragment may be selected from Fab, Fab', and F(ab') _2. The virus neutralizing antibody or fragment may be derived from a recombinant protein, may be recombinantly expressed (including as an active fragment), or may be derived or produced by other means or methods, including means or methods for providing the neutralizing antibody or fragment in the airway or respiratory tract, including expression by genetic material or DNA or DNA vectors, such as by delivering DNA or RNA encoding the neutralizing antibody or fragment.

The compositions of the present invention may further comprise a pharmaceutically acceptable excipient, carrier, or diluent. The compositions may comprise excipients, carriers, diluents, or additives that are suitable or adapted for nasal or pulmonary delivery and for intranasal or inhaled administration. The compositions may comprise excipients, carriers, diluents, or additives that are suitable or adapted for stimulating or enhancing an immune response and/or antibody-mediated cellular or systemic effects. The compositions may comprise immunological mediators or stimulants of an immune response.

The invention provides a method for treating, preventing or reducing or suppressing the propagation of viruses, especially influenza viruses. The invention provides a method for treating or preventing exposure to influenza viruses, contacting influenza viruses or suffering from the viral infection of the mammals of influenza viruses, the method comprising administering (such as intranasal (IN) or via inhalation to the mammals) to the airways of mammals as provided in the present invention. In specific aspects, the combination comprises group 1 antibody TRL053/Mab53, group 2 antibody TRL579/Mab579 and the B antibody selected from TRL847, TRL845, TRL849, TRL848, TRL846, TRL854, TRL809 and TRL832, its fragment, its synthesis or recombinant derivative, its humanization or chimeric form and the antibody with its heavy chain and light chain CDR.

In one aspect of the method, the monoclonal antibodies in the composition are all of the same IgG subtype and have identical or nearly identical constant region sequences. In a specific aspect, all antibodies in the composition are IgG1 antibodies.

According to this method, can after infection or supposition infection, after exposure or clinical symptom manifestation, use described antibody combination.In one aspect thereof, can in the period of reaching 8 hours after infection, use described antibody combination.Or, in the period of reaching 24 hours after infection, use described antibody combination.In other alternatives, in the period of reaching 48 hours after infection, use described antibody combination.In other alternatives, in the period of reaching 72 hours after infection, use described antibody combination.Can be by antibody combination, comprised as single dose, or in multiple sequential dosage, reach infection after 8 hours (8hpi), 12hpi, 18hpi, 24hpi, 36hpi, 48hpi, 72hpi, infection after 1 day, infection after 2 days, infection after 3 days, infection after 4 days, infection after 5 days, infection after 6 days, infection after 7 days, infection after 1 week, infection after 10 days, infection after 2 weeks, infection after 3 weeks, infection after 4 weeks, infection after 1 month, infection after several months and use.

The combination of antibodies is administered in a single dose or in three separate doses administered simultaneously or nearly simultaneously to ensure that any one antibody in the combination, as necessary, is effective at the time of administration.

In some embodiments, there is provided a composition comprising a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antibodies in any proportion. In some embodiments, there is provided a composition comprising 2-10 or 3-5 antibodies or fragments at a weight of about 10-80wt%, 20-50wt%, or 25-40wt% of the total antibody or fragment weight per composition. In a specific embodiment, there is provided a composition comprising each of the first, second, and third antibodies or fragments at a weight of about 33wt% ± 3wt% of the total weight of the antibodies or fragments per composition, comprising substantially equal doses or proportions of the first, second, and third antibodies or fragments. In particularly preferred aspects, the antibodies in the combination are administered in substantially equal dose ratios, with the same dosage amounts, or in a weight ratio or equal proportions of 1:1:1.

In some embodiments, a composition comprising 2-20 antibodies is provided, wherein the effective amount of a single dose of each antibody in the combination may be less than 10 mg/kg body weight, less than 5 mg/kg body weight, less than 2 mg/kg body weight, less than 1 mg/kg body weight or less. The single dose amount of each antibody in the combination may be less than 1 mg/kg body weight, less than 0.5 mg/kg, less than 0.1 mg/kg, less than 0.05 mg/kg. Multiple doses or antibody combinations may be administered. Each combination may be the same or the dosage may be different, such as an initial higher dose, followed by a lower dose, or an initial lower dose, followed by a higher dose. A single dose or multiple doses or any dose may be less than 1 mg/kg body weight, less than 0.5 mg/kg, less than 0.1 mg/kg, less than 0.05 mg/kg. The initial dose may be greater than 1 mg/kg, and further or subsequent doses may be lower or less than 1 mg/kg.

The antibodies can be administered (such as intranasally or via inhalation) to the respiratory tract in multiple doses, wherein each antibody in each single combined dose is less than 1 mg/kg/dose. In such aspects, multiple doses can be administered at intervals of at least 2 hours and up to 72 hours or later after the assumed infection, exposure, or clinical symptoms. Thus, the antibody doses can be administered at intervals of several minutes, hours, or days. After infection or after assumed infection or exposure, the antibody doses can be administered at intervals of several hours or days. The antibody doses can be administered after infection or after assumed infection or exposure and up to 2, 4, 6, 8, 12, 24, 36, 48, or 72 hours.

The administration scheme or method of the present invention may particularly include a first administration (particularly by inhalation or intranasal administration) of an antibody to the airways or respiratory tract in combination with a second or one or more additional administrations (the administration is not by inhalation or intranasal route, for example systemic administration, such as IP or IV administration), or the second or one or more additional administrations may be performed after the first administration. Therefore, the method may include additional IP or IV administration of a virus-specific monoclonal antibody, wherein the additionally administered antibody is a neutralizing or non-neutralizing antibody. In this case, the combination of the antibodies of the invention may be used as an initial dose, followed by administration of a single antibody directed against the infected virus subtype (including as determined by clinical or diagnostic analysis). Therefore, the first dose of the combination of antibodies is preliminary and effective, regardless of the type of influenza virus. Once the virus type is determined, a combination of a second or additional dose, a combination that has changed the ratio, or a single directional antibody may be administered. The second or additional dose may be administered (such as IH or IN) to the airways, or the second or additional dose may be administered systemically. The antibody additionally administered systemically (such as IP or IV) may be an antibody identical to or altered from the antibody administered by IN or via inhalation. The antibody administered additionally (e.g., via IP or IV) may be administered simultaneously with, sequentially with, or after the antibody administered IN or by inhalation. Any such subsequent administration may be several hours later, may be 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, or up to 24 hours later. The subsequent administration may be several days later, may be 1 day, 2 days, or 3 days later. The subsequent administration may be several days later, may be up to 7 days, 1 week later, or several weeks later. The subsequent administration may be performed in a single dose or multiple doses over several hours and/or days and/or weeks.

In other aspects, the present invention provides a regimen for administering a combination of antibodies against influenza A and influenza B viruses, comprising administering a first airway dose (such as an intranasal or inhaled dose) of a combination of antibodies provided herein, followed by or concurrently administering a second dose, or one or more additional doses of antibodies (the antibodies are not administered to the airways or respiratory tract and can be administered intraperitoneally or intravenously), wherein the second dose or additional dose of antibodies is the same or different antibody as the antibody in the combination of the first dose. The second dose or additional dose of antibodies can be altered or modified to become more effective or more pronounced IV or IP altered or modified antibodies. In one aspect, the first dose of antibodies can lack effector functions, such as Fab antibodies, and the second dose of antibodies can have effector functions, have Fc, or can be modified to have enhanced effector functions.

The regimen may include multiple doses of the antibody combination of the invention via inhaled or intranasal routes, and may include multiple doses of the same combination, one or more antibodies of the combination, or alternative antibodies via IP or IV routes. In one aspect of the regimen, the subject or patient to whom the antibody is administered may be monitored for clinical manifestations such as disease or viral infection, and the dose or doses may be altered, reduced, or increased, or administered at closer or less frequent intervals, depending on the patient or subject and the status of the infection or disease.

In one aspect of the regimen, the influenza virus may be influenza A or influenza B or an unknown or unidentified influenza virus. The second dose of antibody that is not administered to the respiratory tract may be a neutralizing or non-neutralizing antibody and may have effector function or enhanced effector function.

In one aspect of the regimen, the first intranasal or inhaled dose may comprise less than 1 mg/kg, less than 0.5 mg/kg, less than 0.1 mg/kg of the combination antibody of the invention. The second or additional IP or IV dose is particularly administered at a dose higher than the first intranasal or inhaled dose. The second or additional IP or IV dose is particularly administered at a dose that is at least 10 times the amount of each or any antibody as compared to the first intranasal or inhaled dose. The second or additional IP or IV dose may be at least 1 mg/kg, at least 5 mg/kg, at least 10 mg/kg, at least 15 mg/kg, or greater than 10 mg/kg, or greater than 20 mg/kg, or greater than 50 mg/kg.

In other aspects of the regimen, the first intranasal or inhaled dose may be less than 1 mg/kg of each antibody in the combination, and the second IP or IV dose may be at least 10 times the first intranasal dose in mg/kg. In other aspects of the regimen, the first intranasal or inhaled dose may be less than 1 mg/kg of each antibody in the combination, and the second IP or IV dose may be at least 50 times the first intranasal dose in mg/kg. In further aspects, the first intranasal or inhaled dose may be less than 0.5 mg/kg of each antibody in the combination, and the second IP or IV dose is at least 5 mg/kg.

The dose of each antibody in the combination in the first intranasal or inhaled dose may be 10 mg/kg or less and is administered within 24 hours of the presumed infection, exposure, or manifestation of clinical symptoms. The dose of each antibody in the combination in the first intranasal or inhaled dose may be 10 mg/kg or less and is administered within 48 hours of the presumed infection, exposure, or manifestation of clinical symptoms. The dose of each antibody in the combination in the first intranasal or inhaled dose may be 10 mg/kg or less and is administered within 72 hours of the presumed infection, exposure, or manifestation of clinical symptoms.

Another aspect of the invention is a method for inhibiting the spread of respiratory viruses, the method comprising administering a combination of antibodies of the invention to the respiratory tract (such as intranasally or via inhalation), administering a combination of antibodies to a subject exposed to, at risk of exposure to, or showing clinical signs of an influenza virus infection, wherein each antibody is administered at a unit dose of 1 mg/kg or less. The unit dose of each antibody in the combination may be less than 10 mg/kg or less than 1 mg/kg. The unit dose of the method may be less than 0.5 mg/kg or less than 0.1 mg/kg or less than 0.05 mg/kg.

The invention provides the antibody combination composition that is suitable for or is selected for pulmonary administration, or the composition of the combination of antibody (particularly influenza antibody, especially monoclonal influenza antibody), wherein the combination of the antibody comprises, including the antibody for the strain consisting of seasonal and pandemic subtype, or is composed of the antibody.For example, because seasonal influenza circulating virus strain is currently influenza B (Yamagata pedigree), influenza B (Victoria pedigree), influenza A H1 subtype and influenza A H3 subtype, therefore provide with or comprise for influenza B (Yamagata), influenza B (Victoria), influenza A H1 subtype and influenza A H3 subtype each one or more antibodies of the present invention joint composition.The present invention includes other antibodies being mixed into the mixture to provide the extra specific coverage to other subtypes such as H7 subtype.

In some embodiments, a composition is provided that comprises a combination of Group 1 antibody TRL053/Mab53, Group 2 antibody TRL579/Mab579, and one or more anti-influenza B antibodies selected from TRL784, TRL794, TRL798, TRL799, TRL809, TRL811, TRL812, TRL813, TRL823, TRL832, TRL833, TRL834, TRL835, TRL837, TRL839, TRL841, TRL842, TRL845, TRL846, TRL847, TRL848, TRL849, TRL851, TRL854, TRL856, and TRL858, immunoreactive fragments thereof, synthetic or recombinant derivatives thereof, humanized or chimeric forms thereof, and antibodies having heavy and light chain CDRs thereof.

In some embodiments, a pharmaceutical composition is provided comprising a combination of Group 1 antibody TRL053/Mab53, Group 2 antibody TRL579/Mab579, and a B antibody selected from TRL845, TRL846, TRL847, TRL848, TRL849, and TRL854, or immunoreactive fragments thereof, synthetic or recombinant derivatives thereof, humanized or chimeric forms thereof, and antibodies having the heavy and light chain CDRs thereof.

In some embodiments, a pharmaceutical composition is provided that comprises a combination of Group 1 antibody TRL053/Mab53, Group 2 antibody TRL579/Mab579, and a B antibody selected from TRL845, TRL847, and TRL849, or an immunoreactive fragment thereof, a synthetic or recombinant derivative thereof, a humanized or chimeric form thereof, and an antibody having the heavy and light chain CDRs thereof. In some embodiments, an anti-influenza composition is provided, which comprises a combination of influenza monoclonal antibodies or binding fragments thereof, wherein the antibodies or fragments thereof comprise: (a) an antibody or fragment thereof comprising a heavy chain amino acid sequence comprising a CDR domain sequence of HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 11, 12, and 13, and a light chain amino acid sequence comprising a CDR domain sequence LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 14, 15, and 16; (b) an antibody or fragment thereof comprising a heavy chain amino acid sequence comprising a CDR domain sequence HCDR1/HCDR2/HCDR3 of SEQ ID NOs: 21, 22, and 23, and a light chain amino acid sequence comprising a CDR domain sequence LCDR1/LCDR2/LCDR3 of SEQ ID NOs: NO:24, 25, 26 CDR domain sequence LCDR1/LCDR2/LCDR3 light chain amino acid sequence; and (c) an antibody or fragment thereof, comprising a heavy chain amino acid sequence and a light chain amino acid sequence comprising the heavy chain and light chain CDR domain sequences HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3, respectively, wherein the HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 are selected from:

(i) SEQ ID NO: 71, 72, 73, and SEQ ID NO: 74, 75, 76;

(ii) SEQ ID NO:91, 92, 93, and SEQ ID NO:94, 95, 96;

(iii) SEQ ID NOs: 101, 102, 103, and SEQ ID NOs: 104, 105, 106;

(iv) SEQ ID NOs: 121, 122, 123, and SEQ ID NOs: 124, 125, 126;

(v) SEQ ID NOs: 181, 182, 183, and SEQ ID NOs: 184, 185, 186;

(vi) SEQ ID NOs: 191, 192, 193, and SEQ ID NOs: 194, 195, 196;

(vii) SEQ ID NOs: 201, 202, 203, and SEQ ID NOs: 204, 205, 206;

(viii) SEQ ID NOs: 211, 212, 213, and SEQ ID NOs: 214, 215, 216;

(ix) SEQ ID NO: 221, 222, 223, and SEQ ID NO: 224, 225, 226;

(x) SEQ ID NO: 231, 232, 233, and SEQ ID NO: 234, 235, 236;

(xi) SEQ ID NO: 241, 242, 243, and SEQ ID NO: 244, 245, 246;

(xii) SEQ ID NO: 261; 262, 263, and SEQ ID NO: 264, 265, 266; and

(xiii) SEQ ID NO:271, 272, 273, and SEQ ID NO:274, 275, 276.

The present invention contemplates and exemplifies highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR domain sequences, said variants being capable of binding to and inhibiting influenza viruses, wherein said compositions are effective against Group 1 and 2 influenza A viruses as well as influenza B viruses.

In a specific embodiment, a composition is provided that comprises a combination of antibodies selected from the group consisting of: (a) an antibody comprising heavy and light chain CDR sequences, HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 11, 12, 13 and 14, 15, 16, respectively, or a fragment thereof; an antibody comprising HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 21, 22, 23 and 24, 25, 26, respectively, or a fragment thereof, and an antibody comprising HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 201, 202, 203 and 204, 205, 206, respectively, or a fragment thereof; (b) an antibody comprising HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 11, 12, 13 and SEQ ID NOs: 14, 15, 16, respectively, or a fragment thereof. NO: 14, 15, 16 of the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof; comprising SEQ ID NO: 21, 22, 23 and SEQ ID NO: 24, 25, 26 of the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof, and comprising SEQ ID NO: 221, 222, 223 and SEQ ID NO: 224, 225, 226 of the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof; (c) comprising the heavy and light chain CDR sequences, HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 of SEQ ID NO: 11, 12, 13 and SEQ ID NO: 14, 15, 16 of the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof; comprising SEQ ID NO: 21, 22, 23 and SEQ ID NO: 24, 25, 26 of the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof, and comprising SEQ ID NO: 221, 222, 223 and SEQ ID NO: 224, 225, 226 of the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof. NO: 24, 25, 26 of HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof, and SEQ ID NO: 231, 232, 233 and SEQ ID NO: 234, 235, 236 of HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof; (d) comprising the heavy and light chain CDR sequences, HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 of SEQ ID NO: 11, 12, 13 and SEQ ID NO: 14, 15, 16, respectively; antibodies or fragments thereof comprising the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 of SEQ ID NO: 21, 22, 23 and SEQ ID NO: 24, 25, 26 of HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 antibodies or fragments thereof, and comprising the HCDR1 / HCDR2 / HCDR3 and LCDR1 / LCDR2 / LCDR3 of SEQ ID NO: 241, 242, 243 and SEQ ID NO: or (e) an antibody or fragment thereof comprising the heavy and light chain CDR sequences, HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 11, 12, 13 and SEQ ID NOs: 14, 15, 16, respectively; an antibody or fragment thereof comprising the HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 21, 22, 23 and SEQ ID NOs: 24, 25, 26, respectively; and an antibody or fragment thereof comprising the HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NOs: 261, 262, 263 and SEQ ID NOs: 264, 265, 266, respectively.

In some embodiments, the antibodies in the combination may be directed against more than one influenza strain or subtype, such as indicated in Figure 3 and shown herein. Antibody Mab53 is effective against influenza A H1, H9, H7, and H5 subtypes of Groups 1 and 2. Antibody Mab579 is effective against H3 and H7 subtypes. Thus, while currently circulating influenza strains are H1, H3, and B subtypes, combinations with efficacy against additional strains and subtypes, including subtypes that may occur and emerge in new or single flu seasons, can be generated and provided herein.

The compositions may be specifically formulated or contain a lower dose or amount of antibody than any alternative dose or form of administration, such as IP or IV. Thus, the compositions for use in the present invention may comprise a 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, greater than 10-fold, greater than 100-fold less neutralizing antibody than a composition thereof for alternative administration, particularly IP or IV administration.

In some embodiments, a composition is provided, which is intended to be used for pulmonary administration, especially each antibody administered intranasally, in an amount less than 1 mg/kg by the body weight of a mammal. In some embodiments, a composition is provided, which includes an antibody totaling less than 10 mg/kg, less than 5 mg/kg or less than 1 mg/kg administered by a person. The composition of the present invention can be particularly less than 1 mg/kg, less than 0.5 mg/kg, less than 0.1 mg/kg, less than 0.05 mg/kg, less than 0.01 mg/kg, less than 0.005 mg/kg, less than 0.0025 mg/kg, less than 0.001 mg/kg by the weight of a mammal (including clinically relevant mammals, such as pigs, dogs, horses, cats or people) and includes an antibody intended for administration (especially intranasal) in an amount of a certain dose. In some embodiments, the therapeutically effective dose is selected from about 100 mg/kg, 50 mg/kg, 10 mg/kg, 3 mg/kg, 1 mg/kg or 1 mg/kg. In some aspects, the effective prophylactic dose or post-exposure prophylactic dose is selected from about 1 mg/kg/0.1 mg/kg or about 0.01 mg/kg.

Those skilled in the art may include determining viral load and viral transmission rate, appropriate and effective dosage in mammals (including humans) based on efficacy in animal models and taking into account clinical and physiological responses. Therefore, the present invention and administration parameters are not limited to the examples provided herein or the specific doses exemplified. The present invention shows that inhalation or intranasal administration is a preferred alternative in terms of efficacy and in reducing, limiting or blocking the effects of the clinical manifestations of viral infection (including influenza virus infection). Inhalation or intranasal administration of neutralizing antibodies provides improved and enhanced efficacy that was not expected or predicted compared to other routes of administration, including IP or IV. The enhanced efficacy produced by pulmonary delivery allows a variety of mAbs to be incorporated into a mixture, making it possible to develop a mixture for all influenza types/subtypes. The amount and time schedule of administration by IN or inhalation route can be further evaluated and determined by those skilled in the art. Studies provided herein show that IN or inhalation administration is more effective than IP or IV at lower doses, and administration can be performed a few days after infection and still maintain efficacy.

The dosage and dosage range used and shown herein may be determined as appropriate and may be converted or applied by those skilled in the art or by clinical or medical professionals using parameters known in the art. Therefore, the mg/kg administration in mice can be extrapolated to comparable or reasonably equivalent administration to humans or other animals. For example, the average weight of laboratory mice is 20 g, whereas the average weight of humans is 70 kg.

It is a routine practice in clinical studies to convert animal doses into human doses, and those skilled in the art would strongly expect that the doses of such conversions would be successful in humans. Interspecies scaling and predicting pharmacokinetic parameters in humans have been described (e.g., Mahmood et al. (2003) J Clin Pharmacol 43:692-697; Mordenti (1986) Journal of Pharmaceutical Sciences, 75:1028-1040). For example, therapeutic levels are generally assumed to be parallel to toxicity, so the conversion factor for converting animal toxicity into human toxicity is generally used to convert the minimum effective dose in animals into the minimum effective dose in humans. In addition, the FDA provides "Industry Guidance" which provides conversion factors for evaluating the maximum safe starting dose of therapeutic agents in clinical trials, including factors for converting animal (mouse) doses into human doses (such as multiplying the mouse dose by 0.08 in one case).

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions, such as gastric upset, dizziness, and the like, when administered to a human.

The present invention also contemplates therapeutic compositions for implementing the therapeutic methods of the present invention. The subject therapeutic compositions comprise, in a mixture, a pharmaceutically acceptable excipient (carrier) and one or more antibodies or active fragments thereof, particularly neutralizing antibodies, peptide analogs thereof, or fragments thereof (as described herein), as active ingredients. In a preferred embodiment, the composition comprises antibodies or fragments capable of neutralizing viruses, particularly influenza viruses, within target cells or in a subject or patient.

The preparation of therapeutic compositions containing antibodies, polypeptides, analogs or active fragments as active ingredients is well known in the art. Typically, such compositions are prepared as liquid solutions or suspensions for administration, but solid forms suitable for forming solutions or suspensions in liquids before administration can also be prepared. Emulsified formulations are also possible. Typically, the active therapeutic ingredient is mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are, for example, water, saline, glucose, glycerol, ethanol, etc. or a combination thereof. In addition, if desired, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, which enhance the efficacy of the active ingredient.

Antibodies, polypeptides, analogs or active fragments can be formulated into therapeutic compositions as neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptide or antibody molecules), which can be formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The compositions containing the therapeutic antibodies, polypeptides, analogs or active fragments are conventionally administered (e.g., by administration of unit doses). The term "unit dose" when used to refer to the therapeutic compositions of the present invention refers to physically discrete units suitable as unitary dosages for human use, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.

In some embodiments, the carrier is selected from any carrier known in the art suitable for pulmonary administration. In some embodiments, the carrier is a suitable carrier selected for intranasal administration, for example, as disclosed in Csaba et al., Adv Drug Deliv Rev. 2009, 61(2): 140-157 (incorporated herein by reference). In some embodiments, the carrier is a nano- or microparticle system. In some embodiments, the carrier is selected from, or comprises one or more of, the following substances: degradable starch, soluble starch, polystyrene, dextran, chitosan, microcrystalline cellulose (MCC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), carbomer, 974P, maltodextrin waxy corn starch, alginate, poly(vinyl alcohol), gelatin polymer, polylactic acid nanoparticles coated with a hydrophilic polyethylene glycol coating (PEG-PLA nanoparticles) (Vila et al., J Aerosol Med 2004; 17(2): 174-185); low molecular weight chitosan nanoparticles (Vila et al., Eur J Pharm Biopharm 2004 Jan; 67(1): 123-131); particles based on polyacrylate polymers (Zaman et al., Curr Drug Deliv 2010 Apr; 7(2): 118-124). In some embodiments, the carrier is phosphate buffered saline (PBS). Intranasal delivery systems are described in Ozsoy et al., Molecules 2009, 14, 3754-3779 (incorporated herein by reference).

In some embodiments, the nanoparticle is delivered to the lung while being adsorbed on red blood cells and avoiding the liver and spleen by the technology of Anselmo et al., ACS Nano 2013, 10.1021/nn404853z disclosed online. In this method, by hatching with different particle/RBC ratios up to 100/1, nanoparticles (e.g., spherical nanoparticles, e.g., 200nm or 500nm in diameter) are for example attached to RBC.

In some embodiments, the potency and activity of the antibody or fragment are formulated in a solution, powder or suspension that can be stabilized without a stimulant or stabilized with an excipient that does not affect the potency of the active ingredient and is non-toxic to the lungs. The antibody can be denatured into an oxidized or hydrolyzed state. In some embodiments, aqueous formulations for delivering the antibody by nasal spray or by injection are provided, which have a chelating agent, or a complexing agent, such as caffeine, dextran or cyclodextrin, or other substances as provided herein, to stabilize the antibody or fragment in solution. Further details of such excipients and aerosol or spray compositions can be found in, for example, WO 2005/025506; Maillet, A. et al. 2008 Phar, Res. 25(6): 1318-1326; Hatcha, J et al., Am. J. Respir. Cell. Mol. Biol. 2012 47(5): 709-717 (all incorporated by reference).

Some stabilizers are incompatible with pulmonary delivery because they cause local inflammation or are acutely toxic. In some embodiments, to further inhibit degradation of the active ingredient solution, the antibody or fragment formulation is sealed in a dark glass vial that must be opened with a special bottle opener and, immediately before use, filtered to remove glass fragments before being transferred to a syringe or sprayer.

In other embodiments, the active ingredient solution is prepared by mixing the active ingredient powder with an injection solution, such as in a biphasic automatic syringe format (mixing the powder portion with the liquid in a glass vial, syringe, or blister pack (such as Pozen MT300)). Such extemporaneous formulations can be attempted to produce solutions for pulmonary delivery by spraying or ultrasonic atomization. However, any known atomization process for producing an inhalation aerosol from an aqueous solution exposes the active ingredient to sufficient heat and oxygen concentration to cause immediate, variable changes in efficacy and activity. Due to these inherent difficulties in obtaining or atomizing stable formulations, the active ingredient has not been suitable for delivery by pulmonary inhalation. Another method of aerosol delivery uses a pressurized metered dose inhaler (pMDI), in which a halogenated hydrocarbon propellant forces a solution or suspension of the antibody or fragment through a small hole, thereby producing a fine, inhalable mist consisting of the antibody or fragment in the propellant droplets. In order to produce stable pMDI preparations, the antibody or fragment must be able to form a solution or particle suspension that is stable and physicochemically compatible with it in propellant and pMDI valve means. The solution stability and pulmonary toxicity problems described above for nasal or injection solutions are also applicable to pMDI preparations, and the additional requirements of propellant compatibility prohibit the use of accepted lung compatibility agents such as water or alcohol. For suspensions, it is necessary to be less than about 5.8 microns (mass median aerodynamic diameter necessary for deep lung penetration), and the particles must be stable in the suspension. By attrition methods such as grinding, micronization, grinding, or by multiphase precipitation methods such as spray drying, solution precipitation or freeze drying, to produce a powder that can be dispersed in a propellant to produce such particles from a large number of antibodies or fragments. These methods directly change the physicochemical properties of the antibody or fragment by heat or chemical interaction. Because some active ingredients can be unstable, these methods have not proven suitable for producing powders that can be redispersed in propellants, or if the powder is initially dispersible, the particles grow in size over time or change their chemical composition over time when exposed to the formulation. This instability can cause changes in potency, activity, or increase the size of particles greater than 3.0 microns, making pMDI suspension formulations unsuitable for aerosol delivery of active ingredients. Another method for producing inhalable aerosols is to use a dry powder inhaler, in which a powder formulation of the antibody or fragment is dispersed in the user's exhaled breath and then inhaled into the lungs. The difficulties described above for pMDI suspension formulations also apply to producing stable dry powder formulations. Clearly, there is a lack of suitable formulations for inhaled delivery of active ingredients in the art. This disclosure describes novel stable formulations of active ingredients or pharmaceutically acceptable salts thereof for dry powder and propellant suspensions administered by inhalation via pulmonary aerosol or nasal spray. Such formulations can be used to treat various disease states and conditions, including, but not limited to, migraine. Additionally, methods for producing novel formulations of active ingredients or pharmaceutically acceptable salts thereof are described.

Active ingredients administered by inhalation must penetrate deeply into the lungs to exhibit local, or alternatively, systemic effects. To achieve this goal, particles of the active antibody or fragment must have a diameter of no more than about 0.5-5.8 μm mass mean aerodynamic diameter (MMAD). Particles in this optimal size range are rarely produced during the crystallization step, and thus secondary treatment is required to produce particles in the range of 0.5-5.8 μm. Such secondary treatments include, but are not limited to, attrition methods (by jet milling, micronization, and mechanical milling), heterogeneous precipitation (such as solution precipitation, spray drying, freeze drying, or lyophilization). Such secondary treatments include large thermal and mechanical gradients, which can directly reduce the potency and activity of the active antibody or fragment, or lead to topological defects or chemical instabilities that change the size, shape, or chemical composition of the particles upon further processing or storage. These secondary treatments also impart a significant amount of free energy to the particles, which is typically stored on the surface of the particles. This free energy stored by the particles generates cohesive forces that cause the particles to aggregate to reduce the free energy stored.

The aggregation process can be so extensive that the inhalable active antibody or fragment particles are no longer present in the microgranule formulation, or can no longer be produced from the microgranule formulation because of the high-intensity cohesion interaction. This method aggravates in the case of inhalation delivery because the particles must be stored in a form suitable for delivery by an inhalation device. Since the particles are stored for a relatively long period, the aggregation process can be increased during storage. The aggregation of particles interferes with the redispersion of the particles by the inhaler device, thereby making it impossible to produce the inhalable particles required for pulmonary delivery and nasal delivery. In addition, most of the conventional pharmaceutical methods that are used to overcome the aggregation effect, such as the use of carriers and/or excipients, can not be used for inhalation in pharmaceutical form because the pulmonary toxicology characteristics of these materials are undesirable.

The present disclosure describes a novel stable formulation of an active ingredient or a pharmaceutically acceptable salt thereof (referred to herein as DHE) for administration as a dry powder and propellant suspension for administration by pulmonary aerosol inhalation or nasal spray inhalation. In one embodiment, DHE is used as a mesylate salt. DHE powders are produced using a supercritical fluid method. The supercritical fluid method provides significant advantages in producing DHE particles for inhalation delivery. Importantly, the supercritical fluid method produces inhalable particles of the desired size in a single step, eliminating the need for secondary processing to reduce particle size. Therefore, the inhalable particles produced using the supercritical fluid method have a reduced surface free energy, which results in reduced cohesion, thereby reducing agglomeration. The particles produced also exhibit a uniform particle size distribution. In addition, the particles produced have a smooth surface and a reproducible crystal structure, which also tends to reduce aggregation. Such supercritical fluid methods may include rapid expansion (RES), solution enhanced diffusion (SEDS), gas antisolvent (GAS), supercritical antisolvent (SAS), gas saturated solution precipitation (PGSS), compressed antisolvent precipitation (PCA), aerosol solvent extraction system (ASES) or any combination of the above methods. The basic technology of each of these supercritical fluid methods is well known in the art and will not be repeated in this disclosure. In a specific embodiment, the supercritical fluid method used is the SEDS method described in U.S. application 20030109421 by Palakodaty et al. The supercritical fluid method produces dry particles that can be used directly by pre-dosing into a dry powder inhaler (DPI) format, or the particles can be directly suspended/dispersed in a suspension medium, such as a pharmaceutically acceptable propellant, in a metered dose inhaler (MDI) format. The particles produced can be crystalline or amorphous, depending on the supercritical fluid method used and the conditions adopted (for example, the SEDS method can produce amorphous particles). As discussed above, the resulting particles possess superior properties compared to particles produced by conventional methods, including, but not limited to, a smooth, uniform surface, low energy, a uniform particle size distribution, and high purity. These characteristics enhance the physicochemical stability of the particles and facilitate particle dispersion when used in a DPI or MDI format. The particle size should be such as to allow the DHE particles to be inhaled into the lungs following aerosol particle administration. In one embodiment, the particle size distribution is less than 20 microns. In an alternative embodiment, the particle size distribution ranges from approximately 0.050 microns to 10.000 microns MMAD, as measured by a cascade impactor; in another alternative embodiment, the particle size distribution ranges from approximately 0.400 to 3.000 microns MMAD, preferably between 0.400 and 3.000 microns MMAD, as measured by a cascade impactor. The supercritical fluid method discussed above produces particle sizes at the lower end of these ranges. In the DPI format, the DHE particles can be metered into the dosage form by electrostatic, cryogenic, or conventional means, as is known in the art. The DHE particles can be used alone (neat) or in combination with one or more pharmaceutically acceptable excipients, such as a carrier or dispersing powder (including, but not limited to, lactose, mannose, maltose, etc., or a surfactant coating). In a preferred formulation, the DHE particles are used without additional excipients. A convenient dosage form commonly used in the art is a foil blister pack. In this embodiment, the DHE particles are metered into a foil blister pack without the need for additional excipients for use with a DPI. Typical metered doses may range from about 0.050 mg to 2.000 mg, or from about 0.250 mg to 0.500 mg. The blister pack is ruptured, and the particles can be dispersed into the inhaled air by electrostatic, aerodynamic, or mechanical forces, or any of these, as is known in the art. In one embodiment, more than 25% of the intended dose is delivered to the lungs upon inhalation; in an alternative embodiment, more than 50% is delivered to the lungs upon inhalation; and in another alternative embodiment, more than 80% is delivered to the lungs upon inhalation. The respirable fraction of DHE particles resulting from delivery in the DPI format (as determined according to USP 601) ranges from 25% to 90%, and the residual particles in the blister pack range from 5% to 55% of the intended dose. In the MDI format, the particles can be suspended/dispersed directly into a suspending medium, such as a pharmaceutically acceptable propellant. In specific embodiments, the suspending medium is a propellant. Ideally, the propellant does not serve as a solvent for the DHE particles. Suitable propellants include C1-4 hydrofluoroalkanes, such as, but not limited to, 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), alone or in any combination. Carbon dioxide and alkanes, such as pentane, isopentane, butane, isobutane, propane, and ethane, can also be used as propellants or mixed with the C1-4 hydrofluoroalkanes discussed above. In the case of a blend, the propellant may contain 0-25% of such carbon dioxide and 0-50% of an alkane. In one embodiment, DHE particle dispersion can be achieved without a surfactant. In an alternative embodiment, if desired, the DHE particles may contain a surfactant, present in a mass ratio of 0.001 to 10 to the DHE. Common surfactants include oleates, stearates, myristates, alkyl ethers, alkylaryl ethers, sorbates, and other surfactants used by those skilled in the art to formulate antibodies or fragments for delivery by inhalation, or any combination of the foregoing. Specific surfactants include, but are not limited to, sorbitan monooleate (SPAN-80) and isopropyl myristate. When used, the DHE microparticle dispersion may also contain a small amount of a polar solvent to aid in solubilizing the surfactant. Suitable polar solvents include C2-6 alcohols and polyols, such as ethanol, isopropyl alcohol, polypropylene glycol, and any combination of the foregoing. Polar antibodies or fragments may be added in a mass ratio of 0.0001% to 4% to the propellant. Amounts of polar solvent exceeding 4% may react with or dissolve DHE. In a specific embodiment, the polar antibody or fragment is ethanol used at a mass ratio of 0.0001% to 1% to the propellant. No additional water or hydroxyl-containing antibody or fragment is added to the DHE particles except for balancing with a pharmaceutically acceptable propellant and surfactant. The propellant and surfactant (if used) may be exposed to water or hydroxyl-containing antibody or fragment prior to their use so that the water and hydroxyl-containing antibody or fragment are at their equilibrium point. Standard metering valves (such as from Neotechnics, Valois or Bespak) and canisters (such as from PressPart or Gemi) may be utilized, which is also applicable to the propellant/surfactant combination. Canister fill volumes of 2.0 ml to 17 ml may be used to achieve dose counts from one (1) to hundreds of actuations. A dose counter with a locking mechanism may optionally be provided to limit a specific dose count regardless of the fill volume. The total mass of DHE in the propellant suspension is typically in the range of 0.100 mg to 2.000 mg of DHE per 100 ml of propellant. By using a standard MDI metering valve in the range of 50 to 100 ml, administration will result in a metered dose in the range of 0.050 μg to 1.000 μg per actuation. An actuator with breath actuation may be preferred for maximizing inhalation coordination, but is not mandatory for achieving therapeutic efficacy. The respirable fraction of such MDIs is in the range of 25% to 75% of the metered dose (as measured according to USP Chapter 601).

As provided herein, for the treatment or prevention of virus, particularly influenza virus, it is effective and useful for the unit dose of the neutralizing antibody for intranasal administration, compared to the unit dose specified or required for alternative administration, such as the unit dose required for IP or IV, relatively reduced. Therefore, in one aspect thereof, there is provided for administration, especially the antibody composition of intranasal administration, wherein the unit dose contrast is for alternative administration or the unit dose specified or required, such as the unit dose required for IP or IV administration is reduced by orders of magnitude, especially reduced by several or multiple quantities. Therefore in one aspect thereof, there is provided for administration, especially the antibody composition of intranasal administration, wherein the unit dose reduces by at least 10 times, 10 times, 20 times, 25 times, 50 times, at least 100 times, 100 times, 500 times, up to 1000 times. Particularly, the composition is thereby reduced compared to administration for IP or IV, especially for identical or comparable indication or effect and/or active equivalent unit dose. IN unit dose can be combined with IP or IV dosage for the efficacy of improvement.

In a manner compatible with the dosage form and with a therapeutically effective amount, the composition is used. The amount to be used depends on the ability of the subject to be treated, the subject's immune system utilizing the active ingredient and the suppression of the desired virus or the degree of neutralization. The precise amount of the active ingredient that needs to be used depends on the doctor's judgment and is unique for each individual. However, for intranasal administration, suitable dosage can be about 0.001 milligram to 10 milligrams per kilogram of individual body weight of each dosage, preferably in the scope of about 0.005 milligram to about 1 milligram, less than 1 milligram, less than 0.5 milligram, less than 0.1 milligram, less than 0.05 milligram, less than 0.01 milligram, more preferably less than 1 milligram, less than 0.5 milligram, less than 0.1 milligram. The suitable scheme for initial administration and subsequent administration is also variable. In one scheme, after initial administration, by subsequently injecting or other administration, the subsequent dosage, single or multiple subsequent dosages, repeated at intervals of 1 hour or many hours are repeated.

Higher doses of the antibody may be administered after an initial IN administration by IP or IV or by other suitable routes. In one aspect of the present disclosure, novel dosing methods or parameters are provided wherein a neutralizing antibody is administered intranasally to a patient or subject, and simultaneously therewith, subsequently or later, a neutralizing or non-neutralizing antibody is administered by IP or IV administration.

Other combinations

In aspects of this embodiment, a virus-binding antibody or binding fragment thereof (particularly where the antibody or fragment is neutralizing) can be combined with an agent or drug to form an antibody-drug or antibody-agent conjugate for respiratory or airway administration (including inhalation or intranasal administration) for use in the embodiments disclosed herein. The drug or agent combined with or conjugated to the antibody or fragment can be a virus-neutralizing drug or agent.

Alternatively, the antibody or combination can be used together with an antiviral agent, an antiviral therapeutic agent, an anti-influenza drug or a reagent (particularly comprising oral anti-influenza drugs). The anti-influenza agent can be a neuraminidase inhibitor. The anti-influenza agent can be selected from Tamiflu and Relenza. The anti-influenza agent can be an M2 inhibitor, such as amantadine or rimantadine. The other reagent for combination can be selected from an antiviral therapeutic agent, a viral replication inhibitor, a protease inhibitor, a polymerase inhibitor, a hemagglutinin inhibitor, a bronchodilator (for example, albuterol, levosalbuterol, salmeterol), or an inhaled glucocorticoid. The antiviral agent can be a virus binding agent or a virus binding inhibitor. In one aspect thereof, the virus binding inhibitor can be an antibody that can bind to influenza virus and suppress influenza by combining rather than a more conventional neutralization mode. TRL809 and TRL832 representatives can be suitable for or effectively combined with a mixture to provide an exemplary binding antibody for enhancing efficacy or synergistic effect (particularly by airway administration). Other agents that are effective for airway administration, particularly in combination with the antibody mixtures disclosed herein, may include surfactants or airway lining modulators, such as surfactant nanoemulsions and cationic airway lining modulators. Agents that modulate or alleviate airway inflammation may also be effective or useful in combination with the antibody mixtures of the invention.

In some embodiments, the therapeutic composition, particularly the pulmonary composition, may further comprise an effective amount of a neutralizing antibody or fragment thereof, and one or more of the following active ingredients: an antibiotic, an antiviral agent, a steroid, an anti-inflammatory agent. In a specific aspect, the composition further comprises an antiviral therapeutic agent. The composition may comprise an anti-influenza agent. In some embodiments, methods and compositions for improving efficacy are provided by combining the mAb therapy of the present invention with other antiviral treatments, such as antiviral therapeutics such as neuraminidase inhibitors (e.g., oseltamivir [Tamiflu ^TM ], zanamivir [Relenza ^TM ]), RNA polymerase inhibitors (e.g., favipiravir, VX-787), immunomodulators (e.g., inhaled interferon β1a), host cell targeting agents (e.g., Fludase ^TM , Radavirsen ^TM ), ion channel inhibitors (e.g., amantadine), or other antiviral agents.

Any such anti-influenza agent may also be compounded or combined as part of the compositions provided herein, or administered in conjunction with the antibody or its active fragment or separately. The anti-influenza agent may be administered in the same or alternative manner as the antibody or its fragment or the inhalation or intranasal composition of the present invention, such as via inhalation or via oral (e.g., pill) administration. Thus, the antibody combinations of the present disclosure and the inhalation or intranasal compositions of the present invention may also comprise an anti-influenza agent or antiviral agent, such as, for example, a neuraminidase inhibitor (including an agent selected from Tamiflu and Relenza), or the antibody combinations and the compositions may be administered in combination with the anti-influenza agent or antiviral agent, or sequentially administered after or before the anti-influenza agent or antiviral agent.

As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "ul" or "μl" means microliter, "ml" means milliliter, and "l" means liter.

The compositions can be formulated in nasal sprays or inhalation solutions or suspensions using methods known and acceptable in the art and in medical science and clinical practice. The FDA provides guidelines and guidance for such sprays, solutions and suspensions, and aerosol pharmaceuticals, including industry guidance documents available at fda.gov. An exemplary industry guidance document from July 2002, entitled Nasal Sprays and Inhalation Solutions, Suspensions, and Aerosol Pharmaceuticals—Chemistry, Manufacturing, and Controls, includes details on formulation components and compositions, their specifications, manufacturing, and closed container systems.

Nasal spray is a medicine containing an active ingredient dissolved or suspended in a preparation (usually water-based), which may contain other excipients and is intended to be used by nasal inhalation. The container closed system for nasal spray includes a container and all components responsible for metering, atomization, and delivery to the patient of the preparation. Nasal spray medicine contains a therapeutically active ingredient (drug substance) dissolved or suspended in a mixture of a solution or excipient (e.g., preservative, viscosity modifier, emulsifier, buffer) in a non-pressurized dispenser, and the non-pressurized dispenser delivers the spray containing the active ingredient of a metered dose. The dose may be measured by a spray pump, or the dose has been measured during the manufacturing process. Nasal spray units may be designed for unit administration or may release many metered sprays containing the preparation of a drug substance. Nasal spray is applied to the nasal cavity to obtain local and/or systemic effects.

Inhalation solutions and suspensions are typically water-based formulations that contain therapeutically active ingredients and also contain additional excipients. Water-based oral inhalation solutions and suspensions must be sterile (21CFR 200.51). Inhalation solutions and suspensions are intended to be delivered to the lungs by oral inhalation to obtain local and/or systemic effects and will be used with a specified nebulizer. Inhalation spray medicines are composed of a preparation and a container closure system. Preparations are typically water-based and do not contain any propellant.

Current container closure system designs for inhalation spray medicines include pre-measured and device-metered presentations using mechanical or electrical assistance and/or energy from the patient's inhalation to produce a spray plume. Pre-measured presentations contain previously measured doses or dose fractions in some type of unit (e.g., single or multiple blisters or other cavities), which are then inserted into the device during the manufacturing process or inserted into the device by the patient before use. Commonly used device-metered units have a reservoir containing a formulation sufficient for multiple doses, which, when activated by the patient, is delivered as a metered spray by the device itself.

Prolonged residence time in the nasal cavity can also be achieved by using bioadhesive polymers, microspheres, chitosan or by increasing the viscosity of the formulation. Drugs, excipients, preservatives and/or absorption enhancers can also be used to stimulate or inhibit nasal mucociliary clearance, thereby achieving delivery of the drug to the absorption site.

Microsphere technology is one of the specialized systems used to design nasal products. Microspheres can provide longer contact with the nasal mucosa, thereby enhancing absorption or efficacy. Microspheres for nasal application have always been prepared using biocompatible materials such as starch, albumin, dextran, and gelatin (Bjork E, Edman P (1990) Int J Pharm 62:187-192).

The aqueous solubility of the drug can be a relevant parameter limitation for nasal drug delivery in solution. Conventional solvents or cosolvents such as glycols, small amounts of alcohol, Transcutol (diethylene glycol monoethyl ether), medium-chain glycerides, and capric glyceride ( _C8 - _C10 glycerides of saturated polysaccharide decomposition) can be used to improve the solubility of the drug. Other options include the use of surfactants or cyclodextrins such as HP-β-cyclodextrin, which act as biocompatible solubilizers and stabilizers in combination with lipophilic absorption enhancers. In this case, their effect on nasal irritants should be considered.

Most nasal preparations are water-based and require preservatives to prevent microbial growth. Parabens, benzalkonium chloride, phenylethyl alcohol, EDTA, and benzoyl alcohol are some of the preservatives commonly used in nasal preparations. Mercury-containing preservatives have rapid and irreversible effects on ciliary motility and are not recommended for use in the nasal system.

Small amounts of antioxidants may be required to prevent drug oxidation. Common antioxidants include sodium metabisulfite, sodium bisulfite, butylated hydroxytoluene, and tocopherol. Generally, antioxidants do not affect drug absorption or cause nasal irritation. Chemical/physical interactions of antioxidants and preservatives with the drug, excipients, manufacturing equipment, and packaging components should be considered as part of the formulation development plan.

Many allergic and chronic diseases are often associated with crusting and drying of the mucous membranes. In addition to excipients, certain preservatives/antioxidants can also cause nasal irritation, especially when used in higher amounts. Adequate nasal moisture is essential to prevent dehydration. Therefore, humectants may be added, especially in gel-based nasal products. Humectants prevent nasal irritation and are less likely to affect drug absorption. Common examples include glycerin, sorbitol, and mannitol.

The choice of delivery system depends on the drug to be used, the indication proposed, the patient population and last but not least, market preferences. Some of these delivery systems include nasal drops, nasal sprays, nasal gels and nasal powders.

In some embodiments, compositions for intranasal and inhalation delivery are provided for administration via a nebulizer. A nebulizer is a drug delivery device for administering a medicament in the form of a mist that is inhaled into the respiratory tract. A nebulizer can be used for intranasal and inhalation delivery of mAbs through the mouth and nasal passages and is an effective device for delivering mAbs to the upper and/or respiratory tract. A nebulizer uses oxygen, compressed air, or ultrasonic power to separate drug solutions and suspensions into small aerosol droplets that can be directly inhaled from the mouthpiece of the device. An aerosol is a mixture of gas and liquid particles, and the best example of a naturally occurring aerosol is the mist formed when small evaporated water particles mixed with hot ambient air are cooled and condensed into a fine cloud of visible airborne water droplets. A metered dose inhaler (MDI) is a device that delivers a specific amount of medicament to the lungs in the form of short pulses of aerosolized medication that is usually self-administered by the patient through inhalation. A dry powder inhaler contains micronized powder that is usually packaged in blisters or gel capsules (containing powdered medication that is inhaled into the lungs by the user's own breath) in a single dose. A major new innovation in the nebulizer market is the introduction of ultrasonic vibrating mesh technology (VMT). With this technology, a mesh/membrane with 1,000-7,000 laser-drilled holes vibrates at the top of a liquid reservoir, forcing a mist of very fine droplets through the holes. This technology is more efficient than having a vibrating piezoelectric element at the bottom of the liquid reservoir, thus also achieving shorter treatment times. Available VMT nebulizers include the Pari eFlow Respironics i-Neb, Omron MicroAir, Beurer Nebulizer IH50, and Aerogen Aeroneb.

In another embodiment, the composition is prepared as a lyophilized powder for reconstruction, or for the preservation or non-preserved sterile liquid composition of the intranasal administration by a mucosal atomization device (MAD). The mucosal atomization device (MAD) is attached to a syringe comprising an antiviral composition via a Luer lock. The syringe plunger is compressed briskly to produce a quick intranasal mist spray of about 0.01mL, 0.05mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL or 1mL per dose. In some cases, the entire dose is applied to a single nostril, or the dose is divided into two nostrils with one or more sprays per dose.

The pH of the nasal formulation is very important for the stability of the antibody and to avoid irritation of the nasal mucosa, allowing the antibody to be in optimal form for absorption, preventing the growth of pathogenic bacteria in the nasal passages, maintaining the functionality of excipients such as preservatives, and supporting normal physiological ciliary motility. In some embodiments, compositions are prepared having a pH within the range of 4.5-8.2, 5.2-7.9, or 4.5 to 6.5, keeping in mind the physicochemical properties of the antibody mixture, drug, or active ingredient. Nasal formulations are typically administered in small volumes ranging from 25 to 200 μL (100 μL is the most commonly used dose volume).

Application

It is again pointed out that normal and reasonably expected antibody therapy dosages are well established as IV or IP dosages in the mg range. This is based on research and clinical experience obtained using numerous recombinant antibodies to date. To date, more than twenty (20) monoclonal antibodies have been clinically approved in the United States (see, for example, Newsome BW and Ernstoff MS (2008) Br J Clin Pharmacol 66 (1): 6-19). Currently used clinically approved antibodies are all in use and are administered by IP or IV in the mg/kg range.

No influenza monoclonal antibody has been clinically approved so far. All trials currently underway or reported utilize intravenous delivery as standard. Specifically, TheraCloneSciences antibody TCN-032 (NCT01390025, clinicaltrails.gov) was evaluated with a single dose escalation within the range of 1-40 mg/kg. The TCN-032 antibody is a human antibody (Grandea AG et al. (2010) PNAS USA 107 (28): 12658-12663) that binds to a conserved epitope of the amino-terminal extracellular domain (M2e) of the influenza matrix protein (M2). The antibody CR6261 and CR8020 (respectively Crucell Holland BV clinical trials NCT01406418 and NCT01756950) were similarly evaluated in safety and tolerability studies using an increasing dose of 2 mg/kg to 50 mg/kg administered by IV within 2 hours.

Flu vaccines are administered by injection. An exception to the flu vaccine is the FluMist live flu vaccine (MedImmune) administered intranasally. FluMist is a combination of three live influenza virus strains – A/H1N1, A/H3N2, and B strains, and is administered in a 0.2 ml dose using a suspension provided in a single dose pre-filled intranasal sprayer. In addition to the virus strains, each dose also contains sodium glutamate, hydrolyzed porcine gelatin, arginine, sucrose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and no preservatives (FluMist Highlights of Prescribing Information, 2012-2013 Formula, MedImmune, RAL-FLUV12, Component No.: 11294).

The present disclosure provides a mode of administration and an antibody administration regimen for treating and preventing viral infections, particularly viruses infected or transmitted by the respiratory route, including novel and effective antibody combinations of influenza viruses. Thus, the present disclosure provides treatment, prevention, or relief of viral infections, particularly influenza viruses, by pulmonary administration of an antibody combination capable of neutralizing any related or circulating influenza virus. The antibody combination can be administered by a single IN dose, or can be administered simultaneously or substantially simultaneously with multiple single doses. Additional combinations or single doses may be subsequently administered, with each administration spaced a few minutes, hours, or days apart.

In a specific aspect, the present disclosure provides treatment, prevention or alleviation of viral infections, particularly influenza viruses, by pulmonary administration of a combination of antibodies to circulating strains of influenza. Thus, according to the present disclosure, treatment, prevention or alleviation of infections, particularly influenza viruses, is provided and achieved by pulmonary administration of a combination of antibodies to influenza B and circulating influenza A viruses (particularly a combination of anti-influenza B antibodies, anti-group 1 influenza A antibodies such as anti-H1 antibodies and anti-group 2 influenza A antibodies such as anti-H3 antibodies in one aspect thereof). According to the present disclosure, intranasal administration of a combination of anti-influenza B antibodies, anti-group 1 influenza A antibodies such as anti-H1 antibodies and anti-group 2 influenza A antibodies such as anti-H3 antibodies is effective in preventing infection or treating influenza infection caused by influenza B or influenza A viruses. To the extent that antibodies are available and have been tested and demonstrated herein to be effective against more than one subtype or strain of a virus, the combinations provided and contemplated herein serve as universal cocktails or combinations effective against many strains and/or subtypes of viruses, particularly influenza virus, including known and circulating strains or subtypes, newly emerging strains or subtypes, and unknown, unexpected, and variant strains or subtypes.

The antibodies used in the embodiments disclosed herein can be administered intranasally or by inhalation, followed by or together with (including simultaneously, in combination, or sequentially or separately) systemic administration of another antibody or the same antibody, particularly IP or IV administration. Thus, combined administrations or methods are contemplated and provided herein in which intranasal and IP (or IV) administration are combined to obtain enhanced efficacy against factors, particularly viruses, particularly influenza viruses. In fact, studies provided herein indicate that by using a combination of intranasal administration with an alternative administration (IP or IV), the combined efficacy is synergistic, and low doses of IN and IP (as an example) can be utilized.

Airway administration provides a unique opportunity to deliver an effective low-dose and low-cost therapy that does not require confirmation by diagnostic assays. The onset of symptoms during flu season is sufficient for a physician to administer the low-dose mixture, for example, with a dry powder inhaler or with a nebulizer or other airway delivery method. This no-diagnosis standard of care is current practice for Tamiflu and Relenza, but is not possible with expensive intravenous antibody therapies, which are impractical and costly. Following a follow-up diagnosis, administration of high-dose antibodies can be administered by the intravenous route or through the airways, which can be composed of a mixture or of separate, specific antibodies specific for the influenza type.

The present disclosure provides a method for treating or preventing viral infection in a mammal exposed to, at risk of exposure to, in contact with, clinically symptomatic for, or suffering from a respiratory virus, the method comprising administering to the mammal (intranasally (IN) or via inhalation) a combination or mixture of antibodies as provided herein. The mixture or combination of antibodies may specifically all be IgG antibodies.

Can be after infection or after the infection of supposition, antibody combination is administered.In one aspect thereof, reach 8 hours (hpi) after infection, comprise 2hpi, 4hpi, 6hpi, 8hpi period in the period of administering antibody combination.Or, reach 24 hours after infection, comprise 4hpi, 8hpi, 12hpi, 18hpi, 24hpi period in the period of administering antibody combination.In other alternatives, reach 48 hours after infection, comprise 12hpi, 24hpi, 36hpi, 48hpi period in the period of administering antibody.In other alternatives, reach 72 hours after infection, comprise 24hpi, 36hpi, 48hpi, 60hpi, 72hpi period in the period of administering antibody.Can be after infection, or after the infection of supposition, or present clinical symptoms such as heating, pain, arthralgia, lethargy after a few days, administer antibody. The antibodies can be administered 1 day after infection, 2 days after infection, 3 days after infection, 4 days after infection, 5 days after infection, 6 days after infection, 7 days after infection, 10 days after infection, 12 days after infection, or 14 days after infection. The antibodies can be administered several weeks after infection or presumed infection, including 1 week, 2 weeks, 3 weeks, 4 weeks, or 1 month.

The antibody combination can be administered before infection or to reduce or prevent spread, or before any clinical indication for a patient, disease or infection. In one aspect thereof, the antibodies can be administered as a prophylactic over a period of several days before infection or a possible or assumed exposure or risk of exposure. The antibody combination can be administered 1 day before or 1 day before, 2 days before or 2 days before, 3 days before or 3, 4 days before or 4 days before, 5 days before or 5 days before, 6 days before or 6 days before, 7 days before or 7 days before, 1 week before or 1 week before, more than 7 days before or more than 7 days before, more than 1 week before or more than 1 week before, up to 9 days before or 9 days before, up to 10 days before or 10 days before. The antibodies can be administered once or multiple times before or before one or more doses at intervals of several hours, days or weeks.

The antibody combination can be administered in a single dose or in a plurality of combined doses repeated. In any preferred aspect, each antibody in the combination or mixture is administered in the same relative amount. Each dosage is the same in units or mg/kg, or can be different in amount. For example, the initial dose can be a higher relative dose, such as, but not limited to, about 1 mg/kg, greater than 1 mg/kg, less than 1 mg/kg or about the maximum or near-maximum tolerated dose, or half the maximum tolerated dose (for mammals to be administered). The subsequent dosage can be the same as the initial dose or can be less than or greater than the initial dose, and can depend on the reaction or response or alleviation or program of clinical symptoms in the subject or patient.

Multiple doses of the same or different amounts of each or any dose may be administered at intervals of hours, minutes, days, or weeks. The timing may vary and may be shortened or extended depending on the response and symptoms. Doses, for example, but not limited to, may be administered at intervals of at least 2 hours, at intervals of at least 4 hours, at intervals of at least 6 hours, at intervals of at least 8 hours, at intervals of at least 24 hours, at intervals of at least 48 hours, at intervals of at least 72 hours. One or more antibody doses may be administered after infection or postulated infection and up to 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours later, for up to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days later, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, or longer.

The method may include additional IP or IV administration of a virus-specific monoclonal antibody, wherein the additional antibody is a neutralizing or non-neutralizing antibody. The antibody additionally administered by IP or IV may be the same antibody as that administered by IN or via inhalation. Simultaneously with the antibody administered by IN or inhalation, sequentially or thereafter, administer the antibody additionally administered by IP or IV. Any such subsequent administration may be after several hours, such as 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or more. Subsequent administration may be after several days, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Subsequent administration may be after several weeks, such as 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks.

Inhaled or intranasal dosing can be used to enhance response or efficacy in patients or subjects who are particularly ill or who continue to show symptoms of infection or disease after an initial IN or IP or IV or combination dose.

In other aspects, the present disclosure provides a regimen for administering a mixture or combination of monoclonal antibodies against influenza virus, the regimen comprising administering a first intranasal or inhaled dose of the antibody combination, followed by or simultaneously administering a second dose of the antibody intraperitoneally or intravenously, or again intranasally or by inhalation, wherein the second dose of the antibody combination is the same or different from the first dose of the antibody combination. The second dose or any additional dose of the antibody can be a neutralizing or non-neutralizing antibody.

The embodiments disclosed herein may be better understood by the following non-limiting examples, which are provided as examples of the present invention. The following examples are provided to more fully illustrate preferred or specific embodiments, which, however, should not be construed as limiting the scope of the present invention.

Antibody structure

Certain heavy and light chain amino acid sequences of the mAbs of the invention are provided below and in the accompanying sequence listing.

TRL 784

Example

The following materials and methods were used in the examples provided herein.

Antibodies: In some cases, Mabs were isolated using phage display as described below. Mabs CR8020 and CR6261 are well-characterized broadly reactive antibodies against group 2 and group 1 viruses, respectively (Throsby M et al. (2008) PLOS ONE 3:e3942; Eckert DC et al. (2009) Science 324:246-251; Friesen RHE et al. (2010) PLoS ONE 5 (2):e1906; U.S. Patent No. 8,192,927; Eckert DC et al. (2011) Science 333:843-850). Antibody CR9114 binds to a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses when administered IV (Dreyfus C et al. (2012) Science Express 2011 Aug 9 10.1126/science.1222908). These neutralizing antibodies were cloned in our hands by synthesizing variable regions and subcloned into mouse IgG2a expression vectors. The variable regions of CR8020 were cloned using disclosed heavy chain GI: 339779688 and light chain GI: 339832448. The variable regions of CR6261 were cloned using disclosed heavy chain GI: 313742594 and light chain GI: 313742595. The variable regions of CR9114 were cloned using gene bank sequence heavy chain accession number JX213639 and light chain accession number JX213640. The CR6261, CR8020 and CR9114 antibodies used for these studies were cloned into IgG expression vectors containing human variable regions fused to mouse IgG2a. The chimeric antibodies of mouse antibodies CR6261, CR8020 and CR9114 are referred to herein as CA6261, CA8020 and CA9114, respectively. Mab 5A7 binds to a common epitope on B virus HA and neutralizes the virus, as well as protecting mice from lethal challenge when given IP (Yasugi M et al. (2013) PLoS Pathog 9(2):e1003150, doi: 10.1371/journal.ppat.1003150).

The human antibody Mab53 (also denoted TRL053) is described in US2012/0020971 and WO2011/160083 (each of which is incorporated herein by reference) and is effective in neutralizing Group 1 and 2 H1, H9, H7, and H5 subtypes.

Antibody Mab579 (also denoted TRL579) is described in WO 2013/086052 (which is incorporated herein by reference) and is effective at neutralizing H3 and H7.

Published sequences including antibody heavy and light chain variable region sequences, particularly the heavy and light chain CDR domain (CDR1, CDR2 and CDR3) sequences of the antibodies described and exemplified herein above (including particularly CR6261, CR8020, CR9114, 5A7, Mab53 and Mab579), are known and may be included in the references cited above and incorporated herein by reference.

Fab Verification: Fab-encoding phage lysis was screened by ELISA against recombinant HA. Individual colonies were picked into 384-well plates containing 2XYT/Cam/Glc medium and grown overnight at 30°C. TG1 cells from the 384-well plates were replicated using Qpix into 384-well expression plates containing 2XYT/Cam with low glucose. The plates were grown at 30°C and 400 rpm for 2-4 hours. Fab expression was induced with 0.5 mM IPTG and grown overnight at 22°C and 400 rpm. Fab-containing cells were lysed with BEL buffer containing Benzonase at 22°C and 400 rpm for 1 hour. Fab-containing lysates were blocked with 12.5% MPBST at 400 rpm and 22°C for 30 minutes. The lysates were added to HA-coated ELISA plates for 1 hour at room temperature. The plates were washed five times with PBST and then incubated with anti-Fab IgG conjugated to alkaline phosphatase for 1 hour at room temperature. Plates were washed five times with TBST and subsequently developed using an AutoPhos (Roche, New Jersey). Plates were read using an Infinite Pro F200. Positive phage lysates were sequenced, and unique Fabs were subcloned into Fab expression vectors containing c-myc and his tags for further characterization.

Fab Expression: Fab expression plasmids were electroporated into TG1F cells, which were plated on LB/Cam agar plates. Plates were incubated overnight at 37°C. A single colony was used to inoculate 5 ml of 2XYT/Cam/Glc and grown overnight at 30°C and 350 rpm. 2 ml of the overnight culture was used to inoculate 500 ml of 2XYT/Cam/low Glc and shaken at 30°C and 180 rpm until an OD600nm of 0.5 was reached. Fab expression was induced by adding IPTG to a final concentration of 0.75 mM. The culture was shaken overnight at 30°C and 160 rpm. The culture was centrifuged at 5,000 g for 30 minutes at 4°C. The bacterial pellet was frozen at -80°C for at least 2 hours. The cells were lysed and filtered overnight on a 0.22 μm filter, followed by IMAC purification and size exclusion steps.

Antibody Cloning and Expression: Fab-encoding phage were sequenced and subcloned into IgG expression plasmids for the respective heavy and light chains. IgG was produced in shake flasks in Invitrogen 293F or Invitrogen 293Expi cells. Cells were transfected with the heavy and light chain expression plasmids. Culture supernatants were collected 6 days after transfection and purified using Protein A affinity chromatography and a buffer exchange step.

Therapeutic Efficacy Studies in Mice: Female BALB/c mice aged 6-7 weeks were used for the experiments. All mice were acclimated and maintained for at least 3 days before the start of the experiment. Mice were weighed on the day of viral challenge and daily thereafter for 2 weeks. A clinical scoring system was used as the clinical endpoint and criteria for exclusion from the study. Clinical weight was scored as follows: hunched posture = 3, piloerection = 3, no food or water intake = 2, weight loss ≥30% = 10, neurological symptoms = 10. Mice were excluded from the study and euthanized when a score of 16 or more was reached. Animal studies were performed according to protocols approved by the Animal Care and Use Committee. Therapeutic treatment of mice was performed on the designated days after infection. Mice were first anesthetized with a ketamine/xylazine mixture before intranasal administration of virus, Mab, or Fab in a volume of 50 μl per mouse. Intraperitoneal administration of Mab or Fab was given in a volume of 100 μl. Average body weights were determined daily over the course of the 14-day study and are shown relative to the average body weight on day 0.

Viruses: According to Cottey, Rowe, and Bender (Current Protocols in Immunology, 2001), influenza virus strains (including A/California/7/09, A/Victoria/11, B/Malaysia/2506/2004, and B/Florida/04/2006) are mouse-adapted. Three rounds of mouse adaptation were performed, followed by one round of virus propagation in embryonated chicken eggs. Briefly, three 6-8 week old mice were anesthetized and infected intranasally with 20 μl of virus. Three days after infection, the mice were euthanized and the lungs removed. The lungs were mechanically homogenized, filtered, and centrifuged to remove large debris. 20 μl of the lung homogenate was additionally passaged in naive mice for three rounds.

References

Huber VC, Lynch JM, Bucher DJ, Le J, Metzger DW: Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virusinfections. J Immunol 2001.166:7381-7388.

Jegerlehner A, Schmitz N, Storni T, Bachmann MF: Influenza A vaccine based on the extracellular domain of M2: weak protection mediated viaantibody-dependent NK cell activity. J Imunol 2004, 172: 5598-5605.

Feng J, Mozdzanowska K, Gerhard W: Complement component Clq enhances thebiological sctivity of influenza virus hemagglutinin-specific antibodies depending on their fine antigen specificity and heavy chain isotype. J Virol2002, 76: 1369-1378.

Mozdzanowska K, Feng J, Eid M, Zharikova D, Gerhard W: Enhancement of neutralizing activity of influenza virus-specific antibodies by serum components. Virology 2006, 352: 418-426.

The following examples are provided to illustrate the present invention but not to limit the present invention.

Example 1. Affinity of monoclonal antibodies.

The initial screening using CellSpot ^™ technology is inherently biased towards finding high-affinity mAbs because if the antigen-antibody interaction is too weak, the antigen binds to the beads that are not retained during the washing process. The sensor instrument used to measure the affinity of the cloned mAbs is the FortéBio ^™ Octet. When the dissociation rate becomes very low, measuring affinity as the ratio of association rate to dissociation rate is less accurate. In order to obtain a better assessment, different concentrations of antibodies were flowed over the antigen fixed to the sensor surface. Table 5 shows a group of influenza B mAbs together with their KD determined in this way.

Table 5. KD of mAbs against influenza B.

The biosensor (Biacore ^™ ) determined affinity (KD) of 5A7 for HA described by Yasugi et al., (paragraph 0008) is ~5 nM. mAb TRL 835 of the present invention competes for binding to 5A7, but has a significantly tighter KD at 0.6 nM, as shown.

Example 2. Binding of mAbs to recombinant influenza B HA.

Antibody binding was determined by ELISA. 384-well ELISA plates were coated with protein rHA in DPBS, pH 7.4 at 20 ng/well. After the plates were coated overnight at 4 °C, the plates were washed twice with PBS and 0.01% Tween 20 (PBS-T). The plates were then blocked with 5% skim milk (M-PBS-T) in PBS-T for 1 hour, and the plates were then washed twice in PBS-T. Purified antibodies were diluted in PBS and subsequently probed for each antigen at 20 ng/well. The plates were washed 5 times with PBS-T and subsequently probed with a secondary antibody conjugated to alkaline phosphatase. The plates were subsequently washed 5 times with PBS-T and developed with AttoPhos AP fluorescent substrate (Promega S1000). Plates were read continuously until the background signal from the negative control wells began to provide a fluorescent signal.

The results of some ELISA assays are shown in Figure 3. Recombinant HA ELISA data illustrate the reactivity of several anti-influenza B mAbs and two anti-influenza A mAbs with influenza A and B strains. Recombinant HA ELISAs were performed in triplicate and the average fold reactivity relative to background is shown. Figure 3 shows that each of anti-B mAbs TRL809, TRL812, TRL813; TRL832, TRL841, TRL842, TRL845, TRL846, TRL847, TRL848, TRL849, TRL854, and TRL856 has extensive cross-reactivity against two lineages of influenza B viruses, including B/Florida/06, B/Mass/12, and B/Wisconsin/10 of the Yamagata clade and B/Brisbane/08, B/Malaysia/04, and B/Victoria/87 of the Victoria clade. Anti-influenza A mAbs CF401 (=mAb 53, TRL053) and CF402 (=mAb 579, TRL579) were reactive with influenza A H1 (A/California/09) and H3 (A/Sydney/97) subtypes, but not with influenza B strains.

Example 3. Neutralization of influenza B virus by mAbs.

The antibodies were incubated with 200 PFU of virus at 37°C for 1 hour. Subsequently, MDCK cells were adsorbed with the mixture at 37°C for 1 hour. MDCK cells were covered with Avicel-containing medium for plaque assay development. Plaques were measured after 2 days of incubation. The identified influenza B mAb with broad reactivity was able to neutralize the Yamagata and Victoria lineages with high efficacy. After 12 hours of incubation, the cells were fixed and subjected to immunofluorescence to detect infected cells. The control anti-B mAb5A7 showed neutralization of the Yamagata and Malaysia lineage strains, data not shown. However, the mAb of the present invention exhibited greater efficacy, as shown in Table 8 of Figure 4.

The results of the neutralization assays for selected anti-influenza B antibodies (anti-influenza B antibodies TRL845, TRL848, TRL849, TRL854, TRL832, and TRL809) are shown in Figure 4. IC50 is the concentration that inhibits the virus in this specific neutralization assay. The mAbs each neutralize a representative member of each of the Yamagata and Victoria pedigrees. The determined strains of these pedigrees are used for this experiment, but the mAbs also neutralize all B viruses tested.

Example 4. In vivo efficacy in a mouse influenza model.

In general, except where otherwise noted, the enhanced efficacy of mAbs was evaluated in a pulmonary delivery model. Mice were infected with 10 x LD50 and treated at 24 hpi by pulmonary delivery via the intranasal route at 1 mg/kg. Body weight and survival were monitored to assess relative efficacy.

Figures 6A-D show the in vivo activity of anti-influenza B mAbs of the present invention against 10xLD50 of virus in a murine model at 1 day post-infection following IN administration at 1 mg/kg. Mouse body weights were assessed up to 14 days post-infection, with PBS and no virus used as controls. Animal body weights were monitored daily for 14 days post-infection, and the percentage of body weight relative to initial day 0 weight was used to plot the curves.

Figure 6A shows that 1 mg/kg of mAbs TRL845, TRL847, TRL 848, TRL849 and 5A7 by the IN route provided protection from disease in mice infected with B/Malaysia/2506/04 representing the Victoria clade.

Figure 6B shows that mAbs TRL845, TRL847, TRL 848, TRL849, and 5A7 provided protection from disease by IN at 1 mg/kg in mice infected with B/Florida/04/2006 representing the Yamagata clade.

Figure 6C shows that 1 mg/kg of mAbs TRL849, TRL846, TRL854, TRL 856, TRL847, and 5A7 by the IN route provided protection from disease in mice infected with B/Malaysia/2506/04 representing the Victoria clade.

Figure 6D shows that 1 mg/kg of mAbs TRL849, TRL846, TRL854, TRL 856, TRL847, and 5A7 by the IN route provided protection from disease in mice infected with B/Florida/04/2006 representing the Yamagata clade.

Example 5. Enhanced in vivo efficacy via pulmonary delivery.

In order to assess the potential of the efficacy enhancement produced by the alternative delivery method, the in vivo efficacy of mAb was tested. Infect mice with 10xLD50, use the conventional systemic delivery via intraperitoneal administration at 10, 1 and 0.1 mg/kg at 24 hpi, compared to the mAb of 1.0 and 0.1 mg/kg used by pulmonary delivery via intranasal route, use the conventional systemic delivery via intraperitoneal administration at 10, 1 and 0.1 mg/kg to process mice. Compare survival rate and body weight. Survival rate and weight loss (measurement of the severity of infection) are used to assess relative activity. Fig. 5 A and 5B show that mAb 5A7 is shown as an example of illustrating the efficacy enhancement produced by pulmonary delivery.

Figure 5A shows the in vivo activity of the anti-influenza B antibody 5A7 in mice as a percentage of body weight following intranasal (IN) versus intraperitoneal (IP) administration of 5A7 in mice infected with 10xLD50 of B/Florida (Yamagata lineage). Mice were treated with mAb at 24 hpi via the IN or IN route, and body weight was measured daily as an indicator of disease severity, with PBS and no virus used as controls. Animal body weight was monitored daily for 14 days post-infection, and the percentage of body weight relative to the initial day 0 weight was used to plot the curve. Mice with >30% weight loss were euthanized. 1 mg/kg IN provided protection from weight loss, whereas 1 mg/kg via the IP route resulted in some weight loss. Enhanced efficacy of the IN route of administration relative to the IP route was demonstrated.

Figure 5B shows the in vivo activity of the anti-influenza B antibody 5A7 in mice, expressed as a percentage of body weight following IN versus IP administration of 5A7 in mice infected with 10xLD50 of B/Malaysia (Victoria lineage). Mice were treated with the mAb at 24 hpi via the IN or IP route, and body weight was measured daily as an indicator of disease severity. Mice with >30% weight loss were euthanized. 1 mg/kg IN provided complete protection from weight loss, whereas 1 mg/kg IP resulted in some weight loss. Enhanced efficacy of the IN route of administration relative to the IP route was demonstrated.

Example 6. In vivo efficacy is not affected by the presence of anti-influenza Group A mAbs.

Utilize and replace influenza antibody assessment and use intranasal administration to the efficacy of applying to the airway.Previously separated for group 1 and group 2 influenza A viruses and neutralize and have the monoclonal antibody for the efficacy of the virus.Human antibody Mab53 (also expressed as TRL053) is described in US2012/0020971 and WO2011/160083, and is effective in neutralizing group 1 and 2H1, H9, H7 and H5 subtypes.Antibody Mab579 (also expressed as TRL579) is described in WO2013/086052, and is effective in neutralizing H3 and H7.As provided herein, prepare these mAbs, optimize to provide anti-H1CF-401 (=mAb 53, TRL053), anti-H3CF-402 (=mAb 579, TRL579) for this research.

The therapeutic efficacy of Mab579 and Mab53 antibodies for influenza A virus infection was tested in a mouse model. Mab579 was tested for H3 influenza, and Mab53 was tested for H1 influenza. IN and IP administration were compared, with 1 mg/kg for IN administration, and 10 mg/kg for IP administration was performed at a 10-fold higher dose. Antibody Mab579 was administered 24 hours after infection (24 hpi) to obtain the therapeutic efficacy (data not shown and Figure 12 B) of the H3 influenza virus Vic11 for 10 × LD50. Antibody Mab53 was administered 24 hours after infection (24 hpi) to obtain the therapeutic efficacy (data not shown and Figure 11 B) of the H1 influenza virus Cal09 and PR8 for 10 × LD50. IN administration was more effective than IP administration, even if IP administration was used with a dosage as high as 10 times in the same experiment.

Comparable survival and weight loss data were obtained for both individual anti-B mAbs and for mixtures of the same mAb with anti-H1 and anti-H3 mAbs in a cocktail, establishing that the mAbs do not interfere with each other's activity.

Figures 7A-7D show that co-administration of three anti-influenza mAbs does not interfere with the efficacy of anti-B mAbs. Mice were infected with 10xLD50 of virus and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401 (=mAb 53, TRL053), anti-H3CF-402 (=mAb 579, TRL579), and anti-BTRL845 (Mixture 1), anti-B TRL847 (Mixture 2), or anti-B TRL849 (Mixture 3). PBS and no virus were used as controls. Animal body weights were monitored daily for 14 days post-infection, and body weight was plotted as a percentage of the initial day 0 weight.

Figures 7A-7D together demonstrate that the cocktail will provide the expected level of protection without interference from another mab in the cocktail against representative strains from all seasonal influenza subtypes (H1N1, H3N2, and both lineages of B).

Figure 7A shows in vivo protection of mice infected with 10xLD50 H1N1 and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-BTRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7B shows in vivo protection of mice infected with 10xLD50 H3N2 and treated at 24 hpi with 3 mg/kg of a triple null mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-BTRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7C shows in vivo protection of mice infected with 10xLD50 B/Yamagata lineage and treated at 24 hpi with 3 mg/kg of a triple-null mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7D shows in vivo protection of mice infected with 10xLD50 B/Victoria lineage and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Example 7. Epitope mapping of anti-B mAbs using PepScan ^™ .

Epitope mapping of anti-B mAbs was performed using Pepscan CLIPS ^™ technology, where various fixed linear and constrained peptides corresponding to segments of the stem region of influenza B hemagglutinin were scored for binding to different anti-B mAbs. The stem sequence of influenza B strain B Lee was chosen because it is ancestral to the Victoria and Yamagata lineages. Peptides that bind to the mAbs were considered epitopes. A unique pattern of discontinuous epitopes was depicted for each mAb tested. The results are summarized in Figures 8 and 9. Figure 8 shows a peptide array of the B/Lee/1940/HA protein (SEQ ID NO: 291) generated by PEPSCAN ^™ (top panel). The shaded regions of the B/Lee/1940/HA protein correspond to the residues used to generate the peptide arrays from aa_15-65 (SEQ ID NO: 292), aa_300-359 (SEQ ID NO: 293), and aa_362-481 (SEQ ID NO: 294). The table (bottom) shows mAb 5A7 epitope 1 - aa_333-338 (SEQ ID NO: 304), epitope 2 - aa_342-346 (SEQ ID NO: 305), and epitope 3 - aa_457-463 (SEQ ID NO: 306); mAb TRL845 epitope - aa_455-463 (SEQ ID NO: 307); TRL848 epitope 1 - aa_64-71 (SEQ ID NO: 308); epitope 2 - aa_336-348 (SEQ ID NO: 309); epitope 3 - aa_424-428 (SEQ ID NO: 310); mAb 849 epitope 1 - aa_317-323 (SEQ ID NO: NO: 311), epitope 2 - aa_344-349 (SEQ ID NO: 312), epitope 3 - aa_378-383 (SEQ ID NO: 313); mAb 854 epitope 1 - aa_457-463 (SEQ ID NO: 314). The lower right panel shows the region resolved to the stem of HA in dark grey.

Figure 9 illustrates epitope mapping along the stem of the HA protein by PEPSCAN ^™ analysis. The shaded area depicts the epitope bound by each mAb. mAbs TRL848, TRL845, TRL854, and TRL849 recognize overlapping, discontinuous epitopes along the stem of HA. The control mAb 5A7, derived from a published sequence, generates a similar but unique epitope.

Example 8. In vivo expression of mAbs.

In some embodiments, the mAbs of the present invention are targeted for in vivo expression. Several delivery systems for transferring the genetic information of mAbs into host cells for in situ production of mAbs have been described. In one embodiment, the encoding DNA is encapsulated into lentiviral particles containing fusogenic proteins on their surface in combination with tissue-targeting antibodies, as described by David Baltimore's laboratory: Proc. Nat'l Acad. Sci. USA 103 (31): 11479–11484 (2006). The targeting antibody can bind to CD20, for example, thereby achieving preferential delivery of the vector to B cells for optimal antibody production. Alternatively, an AAV vector (adeno-associated virus) is used, as described by Johnson, P.R. et al., Nature Medicine (2009) 15: 901-906. Other methods include encapsulating the encoding mRNA into liposomes or lipid particles to facilitate cellular uptake.

Example 9. Melting curve determination.

By in PBS with the concentration of 2ug/mL, utilizing PCR StepOne Plus ^TM instrument, with 1.58 ℃/minute being heated to 99 ℃ from 15 ℃, continuous measurement fluorescence carries out melting curve determination.Figure 10 A-10F shows the melting curve determination of mAb TRL845, TRL847, TRL848, TRL849 and TRL854.Each mAb shows high thermal stability.Figure 10 A shows the melting curve of TRL845, and it shows two melting temperatures (Tm1, Tm2) at 58.3 ℃ and 68.7 ℃ respectively.Figure 10 B shows the table of the melting temperature (Tm) (as shown in Figure 10 A and 10C-10F respectively) of mAb TRL845, TRL847, TRL848, TRL849 and TRL854.The display of two melting temperatures (Tm1, Tm2) may be owing to the Fc of these mAbs and the independent denaturation of Fab domain. Figure 10C shows the melting curve of TRL847, which shows a melting temperature (Tm1) of 70.3°C. Figure 10D shows the melting curve of TRL848, which shows a melting temperature (Tm1) of 70.1°C. Figure 10E shows the melting curve of TRL849, which shows two melting temperatures (Tm1, Tm2) at 70°C and 81.8°C, respectively. Figure 10F shows the melting curve of TRL854, which shows two melting temperatures (Tm1, Tm2) at 59.7°C and 68.9°C. Antibodies TRL053 comprising the HC/LC amino acid sequence of SEQ ID NO: 17/18 and TRL597 comprising the HC/LC amino acid sequence of SEQ ID NO: 27/28 each show melting temperatures as shown in Table 6.

Table 6. Melting temperatures of mAbs.

mAbs Tm1 Tm2 TRL053 70.1 76.5 TRL579 70.5

The display of two melting temperatures (Tm1, Tm2) from some antibodies may be due to the separate denaturation of the Fc and Fab domains of these antibodies. Thus, the antibodies used in certain combinations and compositions provided herein are stable and display melting temperatures greater than 55°C, or in some cases greater than 65°C.

Example 10. Anti-influenza mAbs provide protection when delivered via the pulmonary route.

In order to study the effect of combining CF-401 (mAb 053) and the neuraminidase inhibitor oseltamivir for treating H1N1 infection, mice were challenged with 3xLD50 of H1N1 virus and treated with a single 1mg/kg mAb CF-401 intranasal dose, 10mg/kg oral oseltamivir twice daily (for 4 days) or a combination of the two treatments on the 4th day. As shown in Figure 24, when treated with a single administration of 1mg/kg CF-401, 100% of mice survived the attack. When administered at these late time points after infection, standard of care oseltamivir did not have a protective effect (0% survival rate), but when combined with CF-401mAb therapy, it provided additional protection from weight loss compared to a single mAb therapy. The results are shown in Figure 24, where 100% of mice survived the H1N1 attack from a lethal dose when treated with a single administration of 1mg/kg CF-401 4 days after infection. Administration of standard of care Tamiflu was not protective, but combined with CF-401 mAb therapy, provided additional protection from weight loss. Tamiflu was administered orally at 10 mg/kg twice daily on days 4-8. The data showed that standard of care (Tamiflu) plus mAb therapy was effective when administered at later time points.

Anti-influenza mAbs provide protection when delivered via the pulmonary route. In some embodiments, efficacy can be improved by combining the mAb therapy of the present invention with other antiviral treatments, such as neuraminidase inhibitors (e.g., oseltamivir [Tamiflu ^™ ], zanamivir [Relenza ^™ ]), RNA polymerase inhibitors (e.g., favipiravir, VX-787), immunomodulators (e.g., inhaled interferon beta 1a), host cell targeting agents (e.g., Fludase ^™ , Radavirsen ^™ ), ion channel inhibitors (e.g., amantadine), or other antiviral agents.

Example 11. Neutralizing antibodies are intranasally effective.

The therapeutic efficacy of the antibody of systemic delivery does not only rely on neutralization ability, because the neutralizing and non-neutralizing antibodies given by IP route show similar effects in treating and preventing fatal infections. When administered by IP, neutralizing and non-neutralizing antibodies have similar effectiveness. This brings the question: whether neutralization makes a significant contribution to therapeutic efficacy in systemic delivery. The delivery of non-neutralizing antibodies by IV or IP route did not result in significant efficacy differences (data not shown).

On the contrary, the IN delivery of neutralizing antibodies significantly enhances their therapeutic efficacy compared to systemic delivery (Figure 11A). The enhancement in this therapeutic efficacy is specific for neutralizing antibodies, because the non-neutralizing antibodies established do not show similar enhanced therapeutic efficacy. Different from IP delivery, the IN delivery of neutralizing antibodies provides significant therapeutic benefits compared to non-neutralizing antibodies. The enhanced efficacy delivered by IN is related to the ability of antibody neutralization, because the non-neutralizing antibodies delivered by IN do not show improved therapeutic efficacy. The efficacy enhanced by IN is demonstrated (Figure 11A) by the extensive recognition antibodies (IgG2a antibodies in conjunction with the short α-helix of HA2 subunit) for H1 virus CA6261. In order to further confirm the effect of IN efficacy, we have evaluated another cross-protective antibody CR9114, and show that it is highly effective IN (data not shown, and Figure 24). CR9114 binds to an epitope in the stem of HA and protects against lethal challenge with influenza A and B viruses when administered IV (Dreyfus C et al. (2012) Science Express 2012 Aug 9 10.1126/science.1222908).

We have not observed significant differences in IN efficacy for antibodies with different antibody isotypes. Isotype differences have been observed with IP administration, suggesting that effector function may be relevant. Similarly, single neutralizing antibodies were effective in blocking infection against multiple strains of their target H1 or H3 viruses, suggesting that efficacy is not strain-specific or limited. Therefore, IN administration provides a viable and indeed more effective alternative for neutralizing antibodies against influenza virus.

Example 12. Neutralizing Fab is intranasally effective

A study was conducted to evaluate whether the removal of Fc would eliminate the therapeutic efficacy of neutralizing and non-neutralizing Fabs administered IP or IN. As seen in Figure 25, IP administration of a model Fab (CA6261 antibody Fab) did not provide therapeutic efficacy against H1 viruses at 10 mg/kg or lower. Mice treated with Fab IP died of infection similar to mice treated with PBS. In contrast, mice treated with neutralizing Fab by IN at doses of 10 mg/kg and 1 mg/kg were able to survive lethal infection (Figure 25). All doses administered by IN (even to 0.1 mg/kg) showed greater efficacy than any IP dose administered. By comparing CA6261 Fab IN versus IP or IV in the same experiment, comparable results were observed, wherein Fab CA6261 did not have a protective effect or effectiveness when administered by IP or IV, but when the same dose (5 mg/kg) was administered by IN, significant efficacy (animal retention and 95% or greater body weight) was shown (data not shown). This data suggests that for neutralizing antibodies, Fab effectively blocks or treats viral infection by the intranasal route. The data further suggest that systemic Mab delivery systems require Fc effector function for therapeutic efficacy, as Fab is ineffective.

When administered by IN or IP route, the model Fab from non-neutralizing antibodies does not retain therapeutic efficacy. Mice infected with H3 viruses were treated by IN delivery of purified Fabs of exemplary prior art antibodies CA8020 (neutralizing) and non-neutralizing antibodies (data not shown). Although neutralizing Fab can show therapeutic efficacy, non-neutralizing Fab cannot protect mice from lethal attack. Antibody fragments from non-neutralizing antibodies, particularly Fab, did not show therapeutic efficacy when administered by IN.

Example 13. IN delivery is 10-100 times more effective than IP delivery

The effect of the intranasal (IN) delivery of neutralizing antibodies is 10-100 times that of intraperitoneal (IP) delivery. With 10xLD50 PR8 virus (H1 virus) infection mice, and at 24hpi with antibody treatment mice (Figure 11A). Neutralizing antibody CA6261 is carried out 10 times of serial dilutions, and the antibody is applied by IN or IP approach (Figure 11A). The mice processed by IN approach show lighter disease severity, as indicated by weight loss, and are protected from lethal infection on all dilutions. In comparison, the mice processed by IP only show short-term weight loss and protection from lethal infection at the highest dose (10mg/kg). When applied by IP, all lower dilutions do not protect mice. On the contrary, the IN treatment of the dosage of 0.1mg/kg causes short-term weight loss, and all mice survive. The mice processed by IN utilizing the dosage of 10mg/kg and 1mg/kg are protected from detectable weight loss at all time points after infection. Antibodies administered IP exhibited some degree of weight loss at all doses, and only mice treated at the highest dose of 10 mg/kg survived infection.

We confirm that intranasal delivery of neutralizing antibodies similarly leads to enhanced therapeutic efficacy against H3 viruses. Mice were infected with H3 viruses and treated at 24 hpi (Figure 12A). Neutralizing antibody CA8020 was serially diluted 10 times and administered by IN or IP. As observed in our study for H1 viruses, antibodies administered by IN route provided 100% survival against H3 viruses at all dilutions and showed less weight loss compared to antibodies administered by IP route.

These data together show that neutralization is necessary to obtain enhanced therapeutic efficacy when delivered by IN. In addition, the therapeutic efficacy of systemically delivered antibodies does not depend on neutralization, because similar levels of efficacy are observed for neutralizing and non-neutralizing antibodies. Supporting this observation, the therapeutic efficacy of neutralizing Fab is eliminated when administered by IP, but neutralizing Fab shows efficacy when delivered by IN. Neutralizing Fab administered by IN shows an enhanced efficacy similar to that of IN delivery of their complete Mab counterparts compared to IP administration.

Example 14. Prevention studies using IN antibodies

In view of the significant efficacy of intranasal administration of neutralizing antibodies after infection, studies were conducted to evaluate the efficacy of prophylactic intranasal administration prior to infection with the virus. These studies were used to evaluate and demonstrate the applicability of intranasal administration in situations where individuals are exposed to influenza virus, as well as its applicability as an effective method for preventing and reducing transmission within exposed or high-risk populations, or in patients where infection or disease would be a greater overall health risk clinically. The administration of group 1 (H1) antibody CA6261 several days prior to influenza virus infection was evaluated in a mouse animal model. The administration of CA6261 3, 4, 5, 6, and 7 days prior to infection challenge was evaluated, and different doses of IN and IP administration were directly compared.

In the first study, antibody CA6261 was administered by IN or IP, followed by an H1PR8 virus attack on mice at a dose of 3XLD50. Figure 13 depicts a study of preventive IN and IP administration 3 or 4 days before viral infection. Antibody CA6261 was administered by IN or IP 3 or 4 days before 3XLD50PR attack. CA6261 antibody was administered by IN (0.1 mg/kg) or IP (0.1 mg/kg and 1 mg/kg). IN administration (assessed at 0.1 mg/kg) up to 4 days before infection (-4 dpi) protected mice from viral attack. IP administration at the same dose (0.1 mg/kg) 3 or 4 days before infection was completely ineffective. IP administration 3 or 4 days before infection was effective at 1 mg/kg. By comparing IN (0.1 mg/kg) and IP (1 mg/kg) administered at -3 dpi and -4 dpi, in both cases, an IN dose as low as 1/10 was more effective than IP.

The preventive efficacy of 5, 6 and 7 days before viral infection was subsequently evaluated. The IP of up to 10 times the dose was evaluated by contrast IN administration. Antibody CA6261 was administered by IP (with 1 mg/kg) or IN (with 0.1 mg/kg) 5, 6 or 7 days before attacking with 3XLD50 of H1 influenza virus PR8 (data not shown). Tamiflu administration (oral 10 mg/kg, 2 times a day, for 5 days) was also assessed for comparison. The administration performed at -5dpi showed efficacy on 0.1 mg/kg IN. For the 0.1 mg/kg IN administration performed 6 or 7 days before viral attack, not all mice survived. Up to 10 times the IP dose (10 mg/kg) was effective 5, 6 or 7 days before attack. The antibody IN administration performed with 0.1 mg/kg 5 days before attack was at least as effective as the administration of 10 times the 1 mg/kg dose 7 days before attack.

The higher IN dose of 1 mg/kg 5, 6 and 7 days before the viral attack was subsequently evaluated. Figure 14 depicts a study of the IN versus IP administration of the antibody CA6261 administered at 1 mg/kg 5, 6 or 7 days before the viral PR8 attack utilizing 3XLD50. IN administration of the 1 mg/kg antibody administered 7 days before the viral attack was effective in prevention, and in each case, IN was more effective than the same amount of IP administration. In fact, IN administration at any time (5, 6 or 7 days before the attack) was more effective than any IP administration, even if IP was administered closer to the viral attack. In all cases, the antibody was more effective than Tamiflu.

The above studies show that IN administration is in fact superior to IP administration in terms of preventive protection. IN administration of 0.1 mg/kg antibody up to 5 days before infection (-5 dpi) has a protective effect against attack (3xLD50). The same dose of 0.1 mg/kg administered by IP on any day 3-7 days before viral infection does not protect animals (against the virus at the same 3xLD50 dose). At higher doses of the antibody administered by IN (evaluation of 1 mg/kg), the antibody administered by IN can protect against attack if administered at least 7 days in advance. IN administration of more than 7 days in advance has not been evaluated, but the administration may be effective.

Repeated dosing after infection was effective, and lower intranasal doses were effective when given multiple times at intervals of several hours (8 hours, 32 hours, 52 hours) (data not shown). Similarly, repeated dosing before viral infection or exposure is predicted to be effective and may allow for lower IN prophylactic dosing.

The prophylactic efficacy of CA2621 antibodies, particularly administered several days prior to a 10xLD50 H1 virus PR8 attack, was evaluated under a higher dose of viral challenge. 3 and 4 days prior to a 10xLD50 PR8H1 subtype virus attack, animals were administered 0.1 mg/kg CA6261 antibodies by IN or IP (data not shown). IP administration of 0.1 mg/kg antibody 3 or 4 days prior to viral attack was completely ineffective, with IP-treated animals succumbing to viral infection similarly to untreated animals. In contrast, animals administered 0.1 mg/kg antibody intranasally 3 or 4 days prior to a high titer viral attack were protected from infection.

Antibody administration 5, 6, and 7 days prior to high titer challenge was evaluated, with the antibody administered at 1 mg/kg either IN or IP ( FIG. 15 ). In this study, only animals to which the antibody was administered intranasally (via intranasal administration) fully survived the viral challenge. Mice treated with 1 mg/kg of the antibody (administered 5, 6, or 7 days prior to viral challenge) were not fully protected and succumbed to infection. Tamiflu was completely ineffective in protecting. Mice treated intranasally with 0.1 mg/kg of the antibody 5 or 6 days prior to viral challenge survived the viral challenge almost as well as uninfected control animals.

Therefore, the intranasal administration of anti-influenza antibodies is an effective scheme and method for preventing viral infection. Before viral infection, the influenza neutralizing antibodies administered intranasally for at least 7 days have the protection use of anti-viral attack. For high titer viruses (even higher than what may be reasonably expected for representative human exposure to the virus), protection by intranasal administration of at least 7 days in advance is shown. The protection levels observed in these studies show that intranasal antibody administration is effective in human subjects for protecting from viral attack and blocking or reducing viral transmission. The antibody administered by the lungs has a protective effect under conditions and circumstances where systemic administration is invalid.

Example 16. Combinations for the treatment or prevention of influenza.

The combination of antibodies that cross-react with 3 major strains (H1, H3 and B) of influenza is desirable so that any expected circulating influenza can be treated or prevented with a single dose or a combination of dosages. This can bypass the need for exhaustively characterizing infectious viruses before administering the antibody or antibody mixture. Determining the diagnosis of influenza strains requires clinical laboratory facilities, and by requiring the shortest reporting time of 12-24 hours, if it is necessary to determine the influenza virus strain before selecting appropriate directed therapy, this results in a disadvantageous delay in treatment. By using a broad reactive composition for influenza A and B strains, previous strain diagnosis is not necessary before treatment. In addition, antibodies, particularly neutralizing antibodies, have an immediate therapeutic effect that can not be obtained by inoculation, and this effect only acts as a preventive measure, becoming effective by requiring several weeks.

We seek to evaluate and identify therapeutic combinations of monoclonal antibodies (mAbs) covering influenza A and influenza B. Specifically, suitable and valuable combinations include antibodies that are effective against influenza A group 1, influenza A group 2, and influenza B viruses. This provides a combination that is effective against related and circulating influenza, and the combination can be administered before or without the need for a diagnostic evaluation or assessment of the virus type. The criteria for therapeutic combinations include: effective neutralizing antibodies; antibodies with combined efficacy against related circulating influenza viruses, particularly influenza A H1 and H3 and influenza B; combined efficacy against influenza A or influenza B attacks; the absence of significant interactions or competition between antibodies; antibodies that are effective for IP, IV, and IN routes or via the airway and systemic routes. Preferably, the mAbs contained in the mixture have compatible biophysical properties so that they are easily co-formulated. Matching of pI values can be desirable. The therapeutic combination can be effective against any related circulating influenza virus without the need for diagnostic evaluation or characterization of the patient or subject's influenza. Human monoclonal antibodies, or antibodies comprising human variable regions, particularly at least human complementarity determining regions (CDRs), in at least the heavy and light chains, are preferred and can be utilized.

Evaluate suitable anti-influenza A antibodies. Anti-H1 antibody TRL053 (MAb53) is effective in neutralizing group 1 influenza viruses (including H1) in vitro and in vivo. TRL-053 is isolated from a human antibody phage library, described in US2012/0020971 and WO2011/160083, and is effective in neutralizing group 1 and 2 H1, H9, H7 and H5 subtypes. TRL053 exhibits a binding affinity Kd to influenza H1 and H5 strains in the nanomolar or subnanomolar range, and exhibits an isoelectric point (pI) of 8.54. The heavy and light chain variable region CDR sequences of TRL053 are provided in the accompanying sequence table, and the complete variable region amino acid sequence is indicated above. The efficacy of TRL053 against H1 virus PR8 was evaluated in vivo (Figure 11B). Animals were inoculated with 10 x LD50 of H1 influenza virus PR8 and treated with 10 mg/kg, 1 mg/kg, and 0.1 mg/kg of the antibody TRL053 (MAb53) administered either IN or IP at 24 hpi. The body weights of the animals were monitored daily for 14 days post-infection. TRL053 was effective for both IP and IN administration, however, the TRL053 administered IN showed greater efficacy than the IP administration. TRL053 was only slightly effective at the 10 mg/kg IP dose. The IN dose in this study was effective at 10 mg/kg and 1 mg/kg.

Anti-H1 antibody TRL579 (MAb579) is effective in neutralizing group 2 influenza strains in vitro and in vivo. TRL-579 is isolated from a human antibody phage library, described in WO2013/086-52, and is effective in neutralizing H3 and H7. TRL579 shows a binding affinity Kd for influenza H3 and H7 in the nanomolar and sub-nanomolar range, and shows an isoelectric point (pI) of 8.60. The heavy chain and light chain variable region CDR sequences of TRL-579 are provided in the accompanying sequence table, and the complete variable region amino acid sequence is indicated above. The efficacy of TRL579 for H3 virus Vic/11 was assessed in vivo (Figure 12B). Animals were inoculated with 10XLD50 of virus and treated with 10mg/kg, 1mg/kg and 0.1mg/kg of antibody TRL579 administered by IN or IP at 24hpi. Animal body weights were monitored daily for 14 days post-infection. TRL579 was effective for either IP or IN administration, however, the TRL579 administered by IN showed greater efficacy than the IP administration. TRL579 was only slightly effective at an IP dose of 10 mg/kg. The IN dose in this study was effective at 10 mg/kg and 1 mg/kg.

In order to provide suitable anti-B antibodies in an effective combination, antibodies against influenza B were isolated. Human antibodies that bind to the hemagglutinin protein (HA) of influenza and are effective in neutralizing influenza B viruses of the B/Yamagata and B/Victoria clades were identified using CellSpot (U.S. Patent No. 7,413,868) to identify high-affinity binding antibodies, as initially described in USSN 61/935,746 filed February 4, 2014. A list of B-specific antibodies and their sequences, particularly the monoclonal antibody heavy and light chain CDR sequences of various B antibodies, is described in the sequence listing. The CDR1, CDR2, and CDR3 of the heavy and light chains are indicated, as determined by the IMGT standard (International ImMunoGeneTics Database imgt; Ehrenmann F., Kaas Q., and Lefranc M.-P. (2010) Nucleic Acids Res., 38, D301-307).

The B antibodies are constructed and isolated to have the same heavy chain constant region sequence. The constant region is codon optimized for expression in humans. The heavy chain IgG1 constant region sequence (SEQ ID NO: 297) is provided above. The light chain constant region (SEQ ID NO: 295) of the kappa light chain antibodies (TRL845, 846, 847, 809, 849 and 854) provided above. Antibodies TRL848 and 832 have the lambda light chain constant region (SEQ ID NO: 296) as indicated below.

The heavy and light chain variable region sequences of Antibody B are indicated above and in the accompanying Sequence Listing.

Example 17.B Characterization of Antibodies.

In order to identify and characterize monoclonal antibodies suitable for a wide range of effective antibody combinations, the efficacy of human antibodies against influenza B virus was evaluated. Figure 16 provides a tabulation of the evaluative studies evaluating hemagglutination inhibition, classical in vitro neutralization, and in vivo administration of various anti-B antibodies. Antibodies were evaluated for resistance to B lineages Yamagata and Victoria. In vivo results were provided with a summary of 3 different studies in each case, which evaluated the relative initial body weight percentage of animals inoculated with 10xLD50 of virus and administered the indicated TRL antibodies at 1 mg/kg via IN at 24hpi. Animal weight was observed for 14 days, indicating the lowest weight observed. Each of TRL845, TRL847, TRL848, TRL849, and TRL854 effectively maintained at least 96% of the initial body weight throughout the in vivo efficacy evaluation process.

Evaluate the affinity of B antibody to influenza virus as the index aspect of relative efficacy. Some data are provided in Figure 17. The isoelectric point of the record antibody. The isoelectric point (pi) of TRL053 is 8.54, and the pI of TRL579 is 8.60. The affinity for B/Florida (Yamagata pedigree) and B/Malaysia (Victoria pedigree) influenza virus strains is evaluated, and the K _D list represented by nM is used. The B antibody shows nanometer or subnanometer affinity for B/Florida strain. In each case, the affinity of each of TRL845, TRL847, TRL848, TRL849 and TRL854 for B/Malaysia is subnanomolar (<1.0nM) (data not shown).

The effect of monoclonal influenza B antibodies is tested in animals infected with B/Florida or B/Malaysia viruses representing Yamagata and Victoria pedigrees respectively. Antibodies were evaluated 24 hours after utilizing 10XLD50 virus infection by carrying out IN administration with 1mg/kg. The efficacy results of B/Florida virus are provided in Figure 18, Figure 19 and Figure 22B. The efficacy results of B/Malaysia virus are provided in Figure 20, Figure 21 and Figure 22A. The anti-influenza B antibodies used by IN show the efficacy of the infection of any B pedigree carried out with 10XLD50 dosage, and the animals of the infection treated with several antibodies show that the body weight of 100% is retained. Based on efficacy and novelty research in vivo, antibody TRL847, TRL845, TRL849, TRL848, TRL84, TRL854 and TRL809 and TRL832 are selected as preferred antibodies.

These studies provide for being used in combination with anti-H1 and anti-H3 antibodies, to provide the combination of antibodies with cross-reactivity and effectiveness for the main influenza strains in the crowd particularly effective several B antibodies. The combination of anti-H1 antibody TRL53, anti-H3 antibody TRL579 and anti-B antibodies was tested in a mixture form. B antibody 5A7 is described in the above previous embodiment, and is effective (Fig. 5) for treating B/Florida or B/Malaysia by IP or IN administration at 24hpi. Pulmonary administration shows greater efficacy, is effective at least on 1mg/kg or 0.1mg/kg, yet needs higher dosage to obtain comparable efficacy for systemic delivery.

Example 18. Mixtures, Combinations and Compositions.

The administration of the multiple antibodies (each 10mg/kg) of combination is very large protein antibody dosage load, and may not be well tolerated or adjusted, particularly in patients who are ill or at risk. It is feasible to comprise the antibody combination or the mixture of antibody with lower dosage (particularly in the scope of 1mg/kg). The IN administration of antibody is demonstrated to be effective at 1mg/kg or less herein, even if provided in a preventive manner. Comprise the anti-influenza A antibodies effectively for group 1 and group 2 viruses, particularly for the mixture of H1 and anti-H3 antibodies and anti-influenza B antibodies, with single dose or combination, provide the combination of all same influenza strains in the anti-crowd and the antibody of type. The protective effect of the mixture (each 1mg/kg, 3mg/kg antibody altogether) of evaluation antibody TRL053, TRL579 and 5A7 from the weight loss caused by viral infection in vivo. After 24 hours with 10xLD50 virus infection, the antibody mixture was administered with the dosage of single mixing. The mixture showed efficacy against infection by each or any of H1 virus, H3 virus, B/Yamagata lineage virus, and B/Victoria lineage virus ( FIG. 23 ).

The combination of anti-H1 antibody TRL053, anti-H3 antibody TRL579 and anti-B TRL antibody is evaluated with antibody mixture form.These combinations comprise effectively for relevant influenza type or strain, and are compatible and effective antibody when combined.Utilize the skeleton of same subtype to build them, they have similar pI, do not interact or compete, and neutralize their target influenza virus and the situation that one or more other antibodies exist does not interfere or concentration effect. The protection that is avoided because of the weight loss caused by viral infection of the mixture (each kind 1mg/kg, 3mg/kg antibody altogether) of TRL053, TRL579 and each candidate B TRL antibody used after infecting in vivo evaluation.Shown the effect of each or any infection of H1 virus, H3 virus, B/Yamagata pedigree virus and B/Victoria pedigree virus. Fig. 7 A-D depicts the result of the research that the IN of various mixtures uses.Utilize the combination of antibody to realize the complete protection that infects for H1 virus, H3 virus and B/Yamagata and B/Victoria pedigree virus. 7A-7D provide results from three exemplary mixtures - TRL053, TRL579, and TRL845(c) (Mixture 1), TRL053, TRL579, and TRL847 (Mixture 2), and TRL053, TRL579, and TRL849 (Mixture 3).

Figures 7A-7D show that co-administration of three anti-influenza mAbs does not interfere with the efficacy of anti-B mAbs. Mice were infected with 10xLD50 of virus and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (Cocktail 1), anti-BTRL847 (Cocktail 2), or anti-B TRL849 (Cocktail 3). PBS and no virus were used as controls. Animal body weights were monitored daily for 14 days post-infection, and the percentage of body weight relative to the initial day 0 weight was used to plot the curves.

7A-7D together demonstrate that the cocktail will provide the expected level of protection without interference from other mabs in the cocktail against representative strains from all seasonal influenza subtypes (H1N1, H3N2, and both lineages of B).

Figure 7A shows in vivo protection of mice infected with 10xLD50 H1N1 and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7B shows in vivo protection of mice infected with 10xLD50 H3N2 and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7C shows in vivo protection of mice infected with 10xLD50 B/Yamagata lineage and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

Figure 7D shows in vivo protection of mice infected with 10xLD50 B/Victoria lineage and treated at 24 hpi with 3 mg/kg of a ternary mAb cocktail consisting of anti-H1CF-401, anti-H3CF-402, and anti-B TRL845 (cocktail 1), anti-B TRL847 (cocktail 2), or anti-B TRL849 (cocktail 3).

The data show that the mixed composition of the combination of 3 kinds of novel and unique antibodies is effective for influenza infection caused by any relevant or circulating virus strain or type. The combined antibodies are effectively for group 1 and group 2 influenza A viruses and influenza B viruses. The combination of antibodies administered in a single dose has achieved protection against any attack. In order to make the single dose combination effective and tolerated, and without the need for expensive or excessive antibody dosages, low doses can be achieved in the combination of being administered directly to the airways as a mixture (such as by intranasal administration). The efficacy of the combination of antibodies within the dosage range of 1 mg/kg or less has not previously been achieved by the combination of antibodies for all relevant influenza A and B viruses. The combination of antibodies described and provided herein neutralizes all relevant influenza viruses together and is designed to be particularly capable of combination. The antibodies have compatible biophysical properties. The antibodies in the combination cannot compete or significantly interfere with each other, and the activity of each in the combination is comparable to the activity when they exist alone. The antibodies have similar isoelectric points and are similarly expressed in cell culture. In one aspect herein, the antibodies are built on the same IgG backbone and share a common constant region sequence, with each recombinantly expressed antibody having the same heavy and light chain constant region sequence or related sequences.

The present invention may be embodied in other forms or carried out in other ways without departing from its spirit or essential characteristics. This disclosure is therefore to be interpreted as illustrative in all aspects and not restrictive, the scope of the invention being represented by the appended claims, and all changes falling within the meaning and scope of equivalents are intended to be included herein.

Throughout this specification, various references are cited, each of which is incorporated herein by reference in its entirety.

Claims (32)

1. An isolated human antibody comprising: (1) A heavy chain amino acid sequence containing a heavy chain variable region (HCVR), wherein the HCVR comprises: Heavy chain complementarity-determining region 1 (HCDR1) composed of the amino acid sequence of SEQ ID NO: 241, heavy chain complementarity-determining region 2 (HCDR2) composed of the amino acid sequence of SEQ ID NO: 242, and heavy chain complementarity-determining region 3 (HCDR3) composed of the amino acid sequence of SEQ ID NO: 243; and (2) A light chain amino acid sequence comprising a light chain variable region (LCVR), wherein the LCVR comprises: Light chain complementarity-determining region 1 (LCDR1) composed of the amino acid sequence of SEQ ID NO: 244, light chain complementarity-determining region 2 (LCDR2) composed of the amino acid sequence of SEQ ID NO: 245, and light chain complementarity-determining region 3 (LCDR3) composed of the amino acid sequence of SEQ ID NO: 246; The antibody has the property of binding to and/or inhibiting influenza viruses, wherein the influenza viruses are selected from influenza B strains B/Florida/06, B/Mass/12, B/Wisconsin/10, B/Brisbane/08, B/Malaysia/04 and/or B/Victoria/87. 2. An isolated antibody that specifically binds to one or more strains of each of the influenza B Yamagata and influenza B Victoria clades, said antibody comprising: The heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 249 and the light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 250. The isolated antibody has the property of binding to and/or inhibiting influenza B virus strains from Victoria and Yamagata, selected from B/Florida/06, B/Mass/12, B/Wisconsin/10, B/Brisbane/08, B/Malaysia/04 and/or B/Victoria/87. 3. The isolated human antibody according to claim 1, wherein it binds to one or more epitopes selected from SEQ ID NO: 311, 312 and 313, or discontinuous epitopes thereof. 4. A separated codon-optimized nucleic acid molecule encoding the separated human antibody of claim 1. 5. An expression vector comprising the nucleic acid molecule of claim 4. 6. A host cell for expressing a recombinant polypeptide, comprising the expression vector of claim 5. 7. A method for generating antibodies against influenza B, comprising growing the host cells of claim 6 under conditions that allow the antibody to be generated, and recovering the generated antibodies. 8. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier. 9. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier, wherein the antibody exhibits a first melting temperature (Tm1) greater than or equal to 55°C as measured in PBS. 10. The pharmaceutical composition of claim 9, further comprising one or more human antibodies having binding specificity for at least one strain of influenza A. 11. The pharmaceutical composition of claim 10, wherein the antibody specific for influenza A includes specificity for one or more strains of group 1 and group 2. 12. A pharmaceutical composition comprising: (a) A first antibody comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) containing HCDR1 composed of SEQ ID NO: 11, HCDR2 composed of SEQ ID NO: 12, and HCDR3 composed of SEQ ID NO: 13; and a light chain amino acid sequence comprising a light chain variable region (LCVR) containing LCDR1 composed of SEQ ID NO: 14, LCDR2 composed of SEQ ID NO: 15, and LCDR3 composed of SEQ ID NO: 16, wherein the first antibody is capable of binding to and/or inhibiting influenza A virus subtypes H1, H9, H7, and H5; (b) A second antibody comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) containing HCDR1 (SEQ ID NO: 21), HCDR2 (SEQ ID NO: 22), and HCDR3 (SEQ ID NO: 23); and a light chain amino acid sequence comprising a light chain variable region (LCVR) containing LCDR1 (SEQ ID NO: 24), LCDR2 (SEQ ID NO: 25), and LCDR3 (SEQ ID NO: 26), wherein the second antibody is capable of binding to and/or inhibiting influenza A virus subtypes H3 and H7; and (c) A third antibody comprising a heavy chain amino acid sequence comprising a heavy chain variable region (HCVR) containing HCDR1 composed of SEQ ID NO: 241, HCDR2 composed of SEQ ID NO: 242, and HCDR3 composed of SEQ ID NO: 243; and a light chain amino acid sequence comprising a light chain variable region (LCVR) containing LCDR1 composed of SEQ ID NO: 244, LCDR2 composed of SEQ ID NO: 245, and LCDR3 composed of SEQ ID NO: 246, wherein the third antibody is capable of binding to and/or inhibiting influenza virus, wherein the influenza virus is selected from influenza B strains B/Florida/06, B/Mass/12, B/Wisconsin/10, B/Brisbane/08, B/Malaysia/04, and/or B/Victoria/87. 13. The pharmaceutical composition of claim 12, wherein the first antibody comprises the HCVR/LCVR pair of SEQ ID NO: 19 and 20. 14. The pharmaceutical composition of claim 12, wherein the second antibody comprises the HCVR/LCVR pair of SEQ ID NO:29 and 30. 15. The pharmaceutical composition of claim 12, wherein the third antibody comprises the HCVR/LCVR pair of SEQ ID NO: 249 and 250. 16. The pharmaceutical composition of claim 12, wherein each of the first, second and third antibodies exhibits an isoelectric point (pI) in the range of 2 pI. 17. The pharmaceutical composition of claim 12, wherein the first antibody is TRL053/Mab53 comprising the heavy chain (HC)/light chain (LC) amino acid sequence of SEQ ID NO: 17 and 18, the second antibody is antibody TRL579/Mab579 comprising the HC/LC amino acid sequence of SEQ ID NO: 27 and 28, and the third antibody is antibody TRL849 comprising the HC/LC amino acid sequence of SEQ ID NO: 247 and 248. 18. The pharmaceutical composition of claim 17, wherein each of the first, second, and third antibodies is formulated in a single dose in an amount effective for treating or preventing influenza A and influenza B infection or disease in a subject in need of such treatment. 19. The pharmaceutical composition of claim 18, wherein each of the first, second, and third antibodies is present in the composition in an amount of 100 mg/kg or less per dose of each of the first, second, and third antibodies. 20. The pharmaceutical composition of claim 18, wherein each of the first, second, and third antibodies is present in the composition in an amount of 10 mg/kg or less per dose of each of the first, second, and third antibodies. 21. The pharmaceutical composition of claim 18, wherein each of the first, second, and third antibodies is present in the composition in an amount of 1 mg/kg or less per dose of the first, second, and third antibody. 22. The pharmaceutical composition of claim 18, wherein each of the first, second and third antibodies is present in the composition in an amount of total antibody of 10 mg/kg or less per dose. 23. The pharmaceutical composition of claim 18, wherein the carrier comprises a diluent and/or excipient for nasal or pulmonary delivery. 24. The pharmaceutical composition of claim 18, further comprising one or more of an immunomodulator, an antiviral therapeutic agent, a viral replication inhibitor, a protease inhibitor, a polymerase inhibitor, a hemagglutinin inhibitor, a bronchodilator, or an inhaled corticosteroid. 25. Use of the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating or preventing influenza infection in a subject. 26. The pharmaceutical composition according to claim 24, wherein the immunomodulator is interferon β1a. 27. The pharmaceutical composition of claim 24, wherein the antiviral therapeutic agent is a neuraminidase inhibitor selected from oseltamivir, zanamivir, peramivir, and lanimivir. 28. The pharmaceutical composition of claim 24, wherein the antiviral therapeutic agent is an RNA polymerase inhibitor selected from favipiravir (T-705) and VX 787. 29. The pharmaceutical composition of claim 24, wherein the antiviral therapeutic agent is a host cell-targeting therapeutic agent selected from influenza enzyme (Das181) and AB-103 ( p2TA ). 30. The pharmaceutical composition of claim 24, wherein the antiviral therapeutic agent is an ion channel inhibitor selected from rimantadine and amantadine. 31. The pharmaceutical composition according to claim 24, wherein the bronchodilator is selected from salbutamol, levosalbutamol, or salmeterol. 32. The isolated antibody according to claim 1 is a recombinant antibody.