Monoclonal antibodies against hemagglutinin (HA) and neuraminidase (NA) of influenza H3N2 virus

Abstract

The present disclosure relates to neutralizing anti-influenza monoclonal antibodies. The present disclosure further relates to therapeutic uses of the isolated antibodies. The antibodies are directed against either the hemagglutinin (HA) or neuraminidase (NA) of influenza H3N2.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 003,471, filed on April 1, 2020. The contents of that application are incorporated herein by reference in their entirety.

[0002] Government interests This invention was made with government support under AI116285 and AI145332, granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] This invention relates to broadly neutralizing anti-influenza HA or NA monoclonal antibodies (mAbs) or their antigen-binding fragments. The invention further relates to therapeutic applications of the antibodies or antigen-binding fragments. [Background technology]

[0004] Influenza, commonly known as "flu," is an infectious disease caused by the influenza virus. There are four types of influenza viruses: A, B, C, and D. Human influenza A and B viruses cause seasonal outbreaks of this disease. The first and most important step in preventing influenza is to get vaccinated annually. While approved influenza vaccines have been available for over 70 years, influenza infection remains a major public health concern. In the United States, influenza causes approximately 30,000 deaths and 200,000 hospitalizations annually, while worldwide it accounts for approximately 3 to 5 million severe cases and 200,000 to 500,000 deaths per year. A major vulnerability is the need to select seasonal influenza vaccine compositions each year to properly match the strains expected to be most prevalent during the following season. If a seasonal vaccine does not match the circulating strain, the vaccine may be ineffective. Due to the tendency of influenza antigens to drift and shift, and their tendency to primarily induce specific antibodies, humanity remains vulnerable to waves of new strains with the potential for pandemics, such as the 1918 "Spanish flu" which killed approximately 30 to 50 million people, where immunity may be limited or nonexistent. Seasonal vaccines include influenza A H1, H3, and B strains, but recent pandemics, including the most recent 2009 novel H1N1 pandemic, highlight the need to develop new vaccine strategies and therapies that provide broad protection against a diverse range of influenza strains. [Overview of the project]

[0005] This invention addresses the need by providing broadly neutralizing anti-influenza HA or NA monoclonal antibodies or their antigen-binding fragments.

[0006] In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to hemagglutinin (HA) of an influenza virus, comprising: (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, each containing the sequence of an HCDR set selected from the group consisting of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, and SEQ ID NOs: 7, 8, and 12; and (ii) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, each containing the sequence of an LCDR set selected from the group consisting of SEQ ID NOs: 4-6, and SEQ ID NOs: 10, 5, and 11. In some embodiments, the heavy chain variable region comprises the sequence selected from the group consisting of SEQ ID NOs: 26, 30, and 34, and the light chain variable region comprises the sequence selected from the group consisting of SEQ ID NOs: 28, 32, and 36. In one embodiment, the heavy chain variable region comprises the sequence of SEQ ID NOs: 1-3, and the light chain variable region comprises the sequence of SEQ ID NOs: 4-6. In another embodiment, the heavy chain variable region includes the sequences of sequence numbers 7, 8, and 9, and the light chain variable region includes the sequences of sequence numbers 10, 5, and 11. In yet another embodiment, the heavy chain variable region includes the sequences of sequence numbers 7, 8, and 12, and the light chain variable region includes the sequences of sequence numbers 10, 5, and 11.

[0007] In another embodiment, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds to influenza virus neuraminidase (NA), comprising: (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, each comprising sequences of an HCDR set selected from the group consisting of SEQ ID NOs: 13-15 and SEQ ID NOs: 19-21; and (ii) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, each comprising sequences of an LCDR set selected from the group consisting of SEQ ID NOs: 16-18 and SEQ ID NOs: 22-24. In some embodiments, the heavy chain variable region comprises sequences selected from the group consisting of SEQ ID NOs: 38 and 42, and the light chain variable region comprises sequences selected from the group consisting of SEQ ID NOs: 40 and 44. In one embodiment, the heavy chain variable region comprises sequences of SEQ ID NOs: 13-15, and the light chain variable region comprises sequences of SEQ ID NOs: 16-18. In another embodiment, the heavy chain variable region comprises sequences of SEQ ID NOs: 19-21, and the light chain variable region comprises sequences of SEQ ID NOs: 22-24.

[0008] Furthermore, isolated antibodies or antigen-binding fragments that compete for binding to the HA or NA of the influenza virus in a cross-blocking assay with one or more of the antibodies or antigen-binding fragments described above are provided.

[0009] Each of the aforementioned antibody or antigen-binding fragments may contain a variant Fc constant region. The antibody or antigen-binding fragment may be a chimeric antibody, a humanized antibody, or a human antibody. The antibody or fragment can be conjugated to a therapeutic agent, a polymer, a detectable label, or an enzyme. An example of a polymer is polyethylene glycol (PEG). An example of a therapeutic agent is a cytotoxic agent.

[0010] In another aspect, the present invention provides isolated nucleic acids or sets of nucleic acids encoding one or more of the CDRs, heavy chain or light chain variable regions, or antigen-binding moieties of any of the antibodies or antigen-binding fragments described above. The nucleic acids(s) can be used to express one or more sets of HCDRs or LCDRS, chains of antibodies or antigen-binding fragments, or polypeptides having the antibodies or fragments described above. For this purpose, the nucleic acids(s) can be operably linked to suitable control sequences to generate expression vectors. Thus, within the scope of the present invention are cultured host cells containing vectors, and methods for producing polypeptides, antibodies, or their antigen-binding moieties. The method comprises obtaining cultured host cells containing a vector comprising nucleic acids(s) encoding one or more of the CDRs, polypeptides, heavy chain variable regions, or light chain variable regions of the antibodies or their antigen-binding moieties described above; culturing the cells in a medium under conditions that enable the expression of polypeptides encoded by the vector and assemble antibodies or fragments thereof; and purifying the antibodies or fragments from the cultured cells or the cell medium.

[0011] The antibodies or fragments described above may be used in methods for neutralizing influenza viruses or in methods for treating, preventing, or controlling influenza virus infections. The method comprises administering a therapeutically effective amount of the antibody or fragment to a subject in need. The method may further comprise administering a therapeutically effective amount of a second antibody or antigen-binding fragment to the subject. Accordingly, the present invention also provides a pharmaceutical composition comprising (i) one or more antibodies or antigen-binding fragments thereof, and (ii) a pharmaceutically acceptable carrier.

[0012] Details of one or more embodiments of the present invention are described below. Other features, purposes, and advantages of the present invention will become apparent from the description and claims. [Brief explanation of the drawing]

[0013] [Figure 1A-1B]This is a series of figures showing the binding profiles of H3 and N2 anti-influenza hmAbs. For ELISA, the binding of hmAbs to influenza HA protein (1A) and NA protein (1B) (negative control) was tested at three different dilutions (10–0.01 μg / ml), and the area under the curve (AUC) values ​​are presented. Isotype control hmAbs (ISO) were included as negative controls. [Figure 2] These are a series of photographs showing the binding profiles of hmAbs to H3N2 influenza-infected cells. MDCK cells were pseudo-infected with the indicated virus, fixed after 17 hours, stained with the indicated hmAbs, and binding was evaluated by immunofluorescence assay. Nuclear protein (NP) mAbs are used as an internal control to confirm infection. [Figure 3A-3C] This figure shows that H3 and N2 hmAbs protect mice from H3N2 influenza infection. Female mice aged 5–7 weeks were treated with ip (infusion) at 20 mg / kg of the indicated H3 and N2 hmAbs, or with an isotype control (IgG) or PBS, 24 hours prior to infection. Mice were then challenged with 10 MLD50 of H3N2 X31 virus and monitored daily for weight loss (A) and survival (B). Mice that lost 25% of their body weight were euthanized. Data represent mean + / - SD (n=5). To assess viral replication in the lungs (C), mice were euthanized at 2 (n=3) and 4 (n=3) days post-infection, whole lungs were collected, and viral titers were determined by immunofocus assay (FFU / ml). Symbols represent data from individual mice. Bars: geometric mean lung viral titer; dotted line: limit of detection (200 FFU / ml). The virus was detected in only one out of three mice. *, p<0.05 using Student's t-test. [Figure 4A-4C]Figure showing that 1092E4 and 1122A11 hmAbs have potent therapeutic activity in vivo. Female mice, 5 - 7 weeks old, were infected with 10 MLD50 of H3N2 X31 virus. Twenty-four hours later, they were treated with the indicated hmAbs or PBS at 1 mg / kg or 10 mg / kg, and body weight loss (A) and survival (B) were monitored daily. Mice that had lost 25% of their body weight were euthanized. Data are represented as mean + / − SD (n = 5). To evaluate viral replication (C) in the lungs, mice were euthanized at 2 (n = 3) and 4 (n = 3) days post-infection, whole lungs were harvested, and viral titers were determined by immunofocus assay (FFU / ml).

Mode for Carrying Out the Invention

[0014] The present invention is based, at least in part, on the unexpectedly broad neutralizing anti-influenza activity of certain monoclonal antibodies or antigen-binding fragments thereof. These antibodies and antigen-binding fragments constitute a novel therapeutic strategy for the defense against influenza infections.

[0015] Current anti-viral treatments for influenza (e.g., oseltamivir / Tamiflu, amantadine / rimantadine) are sub-optimal because of the increasing incidence of resistance and the restricted therapeutic window (must be initiated within 48 hours of symptom onset). Continued search for new prophylactic and therapeutic interventions against influenza is ongoing. Monoclonal antibodies (mAbs) continue to be a growing class of drugs, in part because of their high specificity, limited off-target effects, and excellent safety profiles. In addition to their use in the treatment of cancer and autoimmunity, several mAbs are already approved or are being used in clinical trials for the treatment and prevention of various infectious diseases.

[0016] A small number of human monoclonal antibodies (hmAbs) capable of neutralizing a variety of influenza strains have been isolated. These all include hmAbs such as 1F1 (PMC3516549) and CH65 (PMC3161572) that target hemagglutinin (HA) protein expressed on the surface of virions and bind to multiple H1 isolates, hmAbs such as F10 (PMC2692245) and CR6261 (PMC2758658) that recognize all group 1 viruses, hmAb3I14 (PMC5027281), FI6 / MEDI8852 (PMID:21798894, PMC4967455), and VS140 (PMC4568252) that recognize both group 1 (e.g., H1, H2, H5) and group 2 (e.g., H3, H7) viruses, or CR9114 (PMC3538841) that recognize both type A and type B viruses. Some of these hmAbs are currently in clinical trials, and their characterization is leading to the identification of conserved epitopes in influenza HA that may be useful as targets for the development of universal influenza vaccines and therapeutics.

[0017] In a broad sense, antiviral hMAb is an excellent option for effective immunotherapeutic agents to prevent and treat influenza virus infections where vaccine-induced immunity has not yet been achieved (representing vaccine deficiency [e.g., pandemic], suboptimal vaccines, and / or unvaccinated populations). For example, see WO2018 / 213097, WO2019 / 213384, Park JG et al. A Broad and Potent H1-Specific Human Monoclonal Antibody Produced in Plants Prevents Influenza Virus Infection and Transmission in Guinea Pigs.Viruses. 2020 Feb 2;12(2), Piepenbrink MS et al. Broad and Protective Influenza B Virus Neuraminidase Antibodies in Humans after Vaccination and their Clonal Persistence as Plasma Cells.MBio. 2019 Mar 12;10(2), and Nogales A et al. A highly potent and broadly neutralizing H1 influenza-specific human monoclonal antibody. Sci Rep. 2018 Mar 12;8(1):4374. The content of these documents is incorporated by reference. Several HA-specific hMAbs exhibit antiviral activity against a variety of influenza strains and are in clinical trials for the treatment of hospitalized patients and those without associated infections, highlighting the clinical feasibility and potential of influenza-specific hMAbs.

[0018] The broadly neutralizing anti-influenza human monoclonal antibodies or fragments described above can be used to treat, prevent, or control influenza virus infections. Antibodies or fragments may be administered prophylactically to subjects at high risk of influenza infection to potentially prevent the development of the infection. Antibodies or fragments can also be administered to subjects during an influenza infection to reduce the severity and duration of the infection, thereby treating the infection.

[0019] Antibodies or fragments can also be used for the prevention and treatment of pandemic influenza infections. Due to the broad reactivity of Abs, they may be uniquely suited for use against pandemic influenza strains where seasonal influenza vaccines may be ineffective or where existing immunity may be limited. Alternative forms of Abs may be used as disclosed herein. Abs may be used as whole human IgG proteins conjugated with functional labels, their subunits, or as Abs. Within the scope of the invention is the production of vaccines or drugs based on epitopes recognized by Abs. Immunogens or drugs targeting conserved epitopes recognized by hmAbs may confer universal protection from influenza virus infections. Also within the scope of the invention is combination therapy of one or more of the Abs disclosed herein with other antiviral / Abs for the prevention and / or treatment of influenza infections.

[0020] antibody The present invention, as disclosed herein, involves the broad neutralization of anti-influenza monoclonal antibodies or their antigen-binding fragments. These antibodies refer to a class of neutralizing antibodies that neutralize multiple influenza virus strains. The antibodies can preventively and therapeutically protect subjects (e.g., mice as shown in the following examples) from lethal challenge by influenza viruses, as listed in Table 2 below.

[0021] Below are the amino acid sequences of the heavy chain CDR1-3 (HCDR1, HCDR2, and HCDR3), light chain CDR1-3 (LCDR1, LCDR2, and LCDR3), heavy chain (HC) variable region, and light chain (LC) variable region of several exemplary antibodies. The corresponding nucleic acid sequences are also listed. TIFF2023519930000001.tif1401701086G8-heavy chain gaggtgcagctgttggagtctgggggaggcttggttcagcctggggggtccctgagactctcctgtacagcctctggattcacgtttgccagcgatgc catgaactgggtccgccaggctccagggaagggcctggagtgggtctcagctattagtggtaatggtggtatcacatacttcgcagactccgtgaaggg ccggttcaccatctcccgagacaattccaaggacacgctctatctgcaaaatggacagcctgaggggccgaggacacggccgtatattactgtgcaaaaggggtcgcaccctcacatttcaatcttttgactggttattatgcgggacactactactttgacttctggggccagggaaccctggtcaccgtctcctcag (Sequence number 25) / translation EVQLLESGGGLVQPGGSLRLSCTASGFTFASDAMNWVRQAPGKGLEWVSAISGNGGITYFADSVKGRFTISRDNSKDTLYLQMDSLRAEDTAVYYCAKGVAPSHFNLLTGYYAGHYYFDFWGQGTLVTVSS (Sequence ID 26) 1086G8-Kappa gaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttcgtcacaacttagcctggtaccagcacaaacctggccagcctcccaggctcctcatctatggtgcatccactagggccgctagtgtcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataatcactggcctccgtacacttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 27) / Translation EIVMTQSPATLSVSPGERATLSCRASQSVRHNLAWYQHKPGQPPRLLIYGASTRAASVPARFSGSGSGTDFTLTISSLQSEDFAVYYCQQYNHWPPYTFGQGTKLEIK (SEQ ID NO: 28) 1092C4 - Heavy chain gaggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcctctggttacagttttaccagatatggtattagctgggtgcgacaggcccctggacaaggccttgagtggatgggatggatcagcgcttacactggtaacacagactatgcacagaagtttcagggcagaatcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtttattactgtgcgagagatctccctcagggagtagttatattaggctcctattactacggtatggacgtctggggccaagggaacacggtcaccgtctcctca (SEQ ID NO: 29) / Translation EVQLVQSGAEVKKPGASVKVSCKASGYSFTRYGISWVRQAPGQGLEWMGWISAYTGNTDYAQKFQGRITMTTDTSTSTAYMELRSLRSDDTAVYYCARDLPQGVVILGSYYYGMDVWGQGNTVTVSS (Sequence ID 30) 1092C4-Kappa gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttaccagtaggtacttagcctggtaccagcaaaaacttggccaggctcccaggctcctcatctatggtgcatccag cagggccactggcatcccagacaggtttcagtggcagtgggtctgggacagacttcactctcaccatcagcagtctggagcctgaagatcttgcagtttattactgtcagcagtctggtagcccacggacgttcggccaagggaccaaggtggaaatcaaac (Sequence number 31) / translation EIVLTQSPGTLSLSPGERATLSCRASQSVTSRYLAWYQQKLGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDLAVYYCQQSGSPRTFGQGTKVEIK (Sequence ID 32) 1092E4 - Heavy Chain caggtgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcctctggttacagctttaccagatatggtataagctgggtgcgacaggcccctggacaaggccttgagtggatgggatggatcagcgcttacactggtaacacagactatgcacagaaatttcagggcagaatcaccatgaccacagacacatccacgagcacagtctacatggagctgaggagcctaagatctgacgacacggccgtgtattactgtgcgagagatcaccctcagggagtagttatattaggctcctattactacggtatggacgtctggggccaagggaacacggtcaccgtctcctca (SEQ ID NO: 33) / Translation QVQLVQSGAEVKKPGASVKVSCKASGYSFTRYGISWVRQAPGQGLEWMGWISAYTGNTDYAQKFQGRITMTTDTSTSTVYMELRSLRSDDTAVYYCARDHPQGVVILGSYYYGMDVWGQGNTVTVSS (SEQ ID NO: 34) 1092E4-kappa gaaattgtgttgacgcagtctccgggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttaccagtaggtacttagcctggtaccagcaaaaacttggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagtctggagcctgaagattttgcagtgtattactgtcagcagtctggtagcccacggacgttcggccaagggaccaaggtggaaatcaaac (SEQ ID NO: 35) / Translation EIVLTQSPGTLSLSPGERATLSCRASQSVTSRYLAWYQQKLGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQSGSPRTFGQGTKVEIK (Sequence ID 36) 1122A11 - Heavy Chain caggtgcagctggtgcagtctggggctgaggtgagcaagcctggggcctcagtgaaggtctcctgcaaggcatctggatacagcttcaccagccagtctctaggctgggtgcggcaggcccctggacaagggcttgagtggatgggaataatcaaccctagtggtggtatcacaaactacgcacacaagttcc agggcagagtcaccatgaccagggacacgtccacgagcacggtctacatggagctgagcagcctgagatctgaggacacggccctgtattactgtgtgagagatttgagtcattacaatgaagtgggacatgacagggcctactacggtatggacatctggggccaagggaccacggtcaccgtctcctca (Sequence number 37) / translation QVQLVQSGAEVSKPGASVKVSCKASGYSFTSQSLGWVRQAPGQGLEWMGIINPSGGITNYAHKFQGRVTMTRDTSTSTVYMELSSLRSEDTALYYCVRDLSHYNEVGHDRAYYYGMDIWGQGTTVTVSS (Sequence ID 38) 1122A11-Lambda tcctatgagctgattcagccaccctcagtgtccgtgtccccaggacagacagccagcatcacctgttctggagataaattggggaaaaaatatacttgctggtatcagcagaagccaggccagtcccctgtgctggtcatctatcaggataacaagcggccctcagggatccctgagcggttctctggctccaactctgggaacacagccactctgaccatcagcgggacccaggctatggatgaggctgactattactgtcaggcgtgggacagcagcgctgtggtattcggcggagggaccaagctgaccgtcctgg (SEQ ID NO: 39) / Translation SYELIQPPSVSVSPGQTASITCSGDKLGKKYTCWYQQKPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSAVVFGGGTKLTVL (SEQ ID NO: 40) 1122B9 - Heavy chain gaggtgcagctggtggagtctgggggaggcttggtccagccgggggggtccctgagactctcctgtgcagcctctggattcacctttagcggctatgccatgagctgggtccgccaggctccagggaaggggctggagtgcgtctcaggtattattggtagtggtggaagcacatactccgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctggatctggaaatgaacagcctgagagccgaggacacggccgtatattattgtgcgaaacataccaaatcccactactattccggaatgggcgtctggggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 41) / Translation EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLECVSGIIGSGGSTYSADSVKGRFTISRDNSKNTLDLEMNSLRAEDTAVYYCAKHTKSHYYSGMGVWGQGTTVTVSS (Sequence ID 42) 1122B9-Kappa gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagagaccagggaaagcccctaaactcctgatctacgatgcagccaattt ggaaacaggggtcccatcaaggttcagcggaagtggatctgcgacacagtttactttcaccatcagcggcctgcagcctgaagattttgcaacatattactgtcaacagtatgataatctccctctcactttcggcggcgggaccaaggtggaaatcaaac (Sequence number 43) / translation DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQRPGKAPKLLIYDAANLETGVPSRFSGSGSATQFTFTISGLQPEDFATYYCQQYDNLPLTFGGGTKVEIK (Sequence ID 44)

[0022] piece In certain embodiments, the antibodies provided herein are antibody fragments. These include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and single-stranded Fv(scFv) fragments, as well as other fragments described below, such as diabodies, triabodies, tetrabodies, and single-domain antibodies. For an overview of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); also see WO93 / 16185, and U.S. Patents 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and exhibit increased in vivo half-lives, please refer to U.S. Patent No. 5,869,046.

[0023] A diabody is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific. See, for example, EP404,097, WO1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). For triabodies and tetrabodies, see also Hudson et al., Nat. Med. 9:129-134 (2003).

[0024] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see, for example, U.S. Patent No. 6,248,516; DOMANTIS, Inc., Waltham, Mass.).

[0025] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as described herein, and production by recombinant host cells (e.g., E. coli or phages).

[0026] Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. For example, see U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one embodiment, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another embodiment, the chimeric antibody comprises a human variable region and a non-human constant region (e.g., a constant region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey). In further embodiments, the chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from the class or subclass of the parent antibody. The chimeric antibody comprises its antigen-binding fragment.

[0027] In certain embodiments, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains in which HVR, e.g., CDR, (or a portion thereof) is derived from the non-human antibody and FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody also optionally contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to repair or improve antibody specificity or affinity, for example.

[0028] Humanized antibodies and methods for their production are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods Further details can be found in 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol.Immunol.28:489-498 (1991) (describes "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describes "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (describes the "inducible selection" approach to FR shuffling).

[0029] Human framework regions that can be used for humanization include framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (e.g., Baca et al. See al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996). Examples include, but are not limited to, these.

[0030] Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art or using techniques described herein. Human antibodies are generally described in van Dijk and van de Winkel, Curr.Opin.Pharmacol. 5:368-74 (2001) and Lonberg, Curr.Opin.Immunol. 20:450-459 (2008).

[0031] Human antibodies may be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, either replacing endogenous immunoglobulin loci, or located extrachromosomally or randomly incorporated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing the Xenomouse technology, U.S. Patent No. 5,770,429 describing the HUMAB technology, U.S. Patent No. 7,041,870 describing the KM mouse technology, and U.S. Patent Application Publication 2007 / 0061900 describing the Velocimouse technology. Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0032] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human xenomyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). For human antibodies produced via human B-cell hybridoma technology, see Li et al. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (production of monoclonal human IgM antibody from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0033] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0034] The antibodies of the present invention can be isolated by screening a combination library for antibodies having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding properties. Such methods are outlined, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed. Human Press, Totowa, NJ, 2001), and furthermore, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol.222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J.Mol.Biol.338(2):299-310 (2004); Lee et al. This is described in al., J.Mol.Biol.340(5):1073-1093(2004);Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004).

[0035] In certain phage display methods, the repertoire of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically display antibody fragments as either scFv or Fab fragments. Libraries from immunization sources provide high-affinity antibodies to immunogens without requiring the construction of hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self-antigens without any immunization, as described in Griffiths et al. EMBO, J, 12:725-734 (1993). Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), naive libraries can be synthetically constructed by cloning an unrearranged V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving rearrangement in vitro. Examples of patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments in this specification.

[0036] variant In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, the deletion of residues from the amino acid sequence of the antibody, and / or the insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be prepared to reach a final construct, provided that the final construct has desired characteristics, such as antigen binding.

[0037] Substitution, insertion, and deletion variants In certain embodiments, antibody mutants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are defined herein. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC.

[0038] Accordingly, the antibodies of the present invention may contain one or more conservative modifications among the CDRs, heavy chain variable regions, or light variable regions described herein. Conservative modifications or functional equivalents of peptides, polypeptides, or proteins disclosed herein refer to polypeptide derivatives of peptides, polypeptides, or proteins, e.g., proteins having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. They substantially retain activity toward the parent peptide, polypeptide, or protein (such as those disclosed herein). Generally, the conservative modifications or functional equivalents are at least 60% (e.g., any number between 60% and 100% (including both ends), e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to the parent (e.g., one of the amino acid sequences described above). Therefore, within the scope of the present invention are heavy chain variable regions or light variable regions having one or more point mutations, insertions, deletions, cleavages, fusion proteins, or combinations thereof, as well as antibodies having mutant regions.

[0039] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology % = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms, as described in the non-limiting embodiments below.

[0040] The percentage of identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput.Appl.Biosci.4:11-17(1988)), incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using either the Blossum 62 matrix or the PAM250 matrix, and the algorithm of Nedleman and Wunsch (J.Mol.Biol.48:444-453(1970)), incorporated into the gap program of the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, as well as length weights of 1, 2, 3, 4, 5, or 6.

[0041] Additionally or alternatively, the protein sequences of the present invention can be further used, for example, as "query sequences" for performing searches against public databases to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J.Mol.Biol.215:403-10. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecule of the present invention. To obtain gap alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see www.ncbi.nlm.nih.gov).

[0042] As used herein, the term “conservative modification” refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include: Amino acids with basic side chains (e.g., lysine, arginine, histidine), Acidic side chains (e.g., aspartic acid, glutamic acid), Uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), Nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), Beta branched side chains (e.g., threonine, valine, isoleucine) and Aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) are examples.

[0043] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0044] For example substitutional mutations, see, for instance, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). Amino acid insertions include amino-terminus fusions and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion mutations of antibody molecules include fusion of the antibody to the N-terminus or C-terminus of an enzyme (e.g., against ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0045] Glycosylated mutant In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0046] For example, non-glycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. Glycosylation can be modified, for example, to increase the affinity of the antibody to an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the exclusion of glycosylation sites in one or more variable region frameworks, thereby eliminating glycosylation at those sites. Such nonglycosylation can increase the affinity of the antibody to an antigen. Such approaches are described in more detail in U.S. Patents 5,714,350 and 6,350,861 by Co et al.

[0047] Glycosylation of the constant region on N297 may be prevented by mutating the N297 residue to another residue, for example, N297A, and / or by mutating an adjacent amino acid, for example, 298, thereby reducing glycosylation on N297.

[0048] Additionally or alternatively, antibodies with modified types of glycosylation can be produced, such as hypofucosylated antibodies with a reduced amount of fucosyl residues, or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells having an altered glycosylation mechanism. Cells having an altered glycosylation mechanism are described in the Art and can be used as host cells to express recombinant antibodies described herein, thereby producing antibodies with altered glycosylation. For example, Hanai et al. (EP1,176,195) describe a cell line having a functionally disrupted FUT8 gene encoding a fucosyltransferase, in which antibodies expressed in such cell lines exhibit hypofucosylation. Presta's PCT Publication No. 03 / 035835 describes Led 3 cells, a mutant Chinese hamster ovary cell line that exhibits reduced ability to attach fucose to Asn(297)-binding carbohydrates, resulting in hypofucosylation of antibodies expressed in its host cells (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). Umana et al., in PCT Publication No. 99 / 54342, describe cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that the antibodies expressed in the engineered cell lines exhibit an increased bisected GlcNac structure, resulting in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0049] Fc region variant The variable regions of the antibodies described herein may be linked to Fc, for example, IgGl, IgG2, IgG3, or IgG4 (for example, covalently or fused), and these may be any allotype or isoallotype, for example, for IgGl: Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); for IgG2: G2m, G2m23(n For IgG3: G3m, G3m21(gl), G3m28(g5), G3mll(b0), G3m5(bl), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Kml, Km2, Km3 (see, for example, Jefferies et al. (2009) mAbs 1:1). In certain embodiments, the antibody variable region described herein may be linked to Fc that binds to one or more activated Fc receptors (FcγI, FcγIIa, or FcγIIIa), thereby stimulating ADCC and causing T cell depletion. In certain embodiments, the antibody variable region described herein is linked to an Fc that causes depletion.

[0050] In certain embodiments, the antibody variable region described herein may be linked to an Fc containing one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or their glycosylation may be altered to change one or more functional properties of the antibody. The numbering of residues in the Fc region is according to the Kabat EU index.

[0051] The Fc region comprises a domain derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, and includes fragments, analogs, variants, or derivatives of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes, such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide homologous to the C-terminal region of the immunoglobulin, and may include a CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain, individually or in combination. In some embodiments, the antibodies of the present invention have an Fc region other than the region of wild-type IgA1. The antibodies may have an Fc region from IgG (e.g., IgG1, IgG2, IgG3, and IgG4) or from other classes such as IgA2, IgD, IgE, and IgM. The Fc may be a variant of IgA1.

[0052] The constant region of immunoglobulins is involved in important antibody functions such as Fc receptor (FcR) binding and complement fixation. The heavy chain constant region has five major classes, classified as IgA, IgG, IgD, IgE, and IgM, each possessing characteristic effector functions designated by its isotype. For example, IgG is separated into four subclasses known as IgGl, IgG2, IgG3, and IgG4.

[0053] Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific to the IgG class of an antibody: FcγRI, FcγRII, and FcγRIIL. The key sequences for IgG binding to FcγR receptors have been reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to FcR.

[0054] In certain embodiments, the Fc region is a mutant Fc region modified (e.g., by amino acid substitutions, deletions, and / or insertions) relative to the parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to produce a mutant) in order to provide desirable structural features and / or biological activity. For example, modifications may be made in the Fc region to produce an Fc mutant in which (a) ADCC is increased or decreased, (b) complement-mediated cytotoxicity (CDC) is increased or decreased, (c) affinity for Clq is increased or decreased, and / or (d) affinity for the Fc receptor is increased or decreased compared to the parent Fc. Such Fc region mutants generally contain at least one amino acid modification within the Fc region. Combinations of amino acid modifications are considered particularly desirable. For example, the mutant Fc region may contain substitutions of, for example, two, three, four, five, or more of the specific Fc region positions identified herein.

[0055] The mutant Fc region may also include sequence modifications in which an amino acid involved in disulfide bond formation is removed or replaced with another amino acid. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even with the removal of cysteine ​​residues, the single-stranded Fc domain can still form a dimeric Fc domain that is held together non-covalently. In other embodiments, the Fc region may be modified to better suit it with a selected host cell. For example, a PA sequence near the N-terminus of a typical native Fc region may be removed, which may be recognized by digestive enzymes in E. coli, such as proline imino peptidase. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Typically, a glycosylated residue (e.g., asparagine) may confer a cell-lysating reaction. Such a residue may be deleted or replaced with a non-glycosylated residue (e.g., alanine). In other embodiments, a site involved in complement interaction, such as a Clq binding site, may be removed from the Fc region. For example, the EKK sequence of human IgGl may be deleted or substituted. In certain embodiments, a site affecting binding to the Fc receptor may be removed, preferably a site other than the salvage receptor binding site. In other embodiments, the Fc region may be modified to remove the ADCC site. The ADCC site is known in the art; for example, with respect to the ADCC site in IgGl, see Molec.Immunol.29(5):633-9(1992). Specific examples of mutant Fc domains are disclosed, for example, in WO97 / 34631 and WO96 / 32478.

[0056] In one embodiment, the hinge region of Fc is modified, for example, by increasing or decreasing the number of cysteine ​​residues within the hinge region. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of Fc is modified, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody's binding to Staphylococcus protein A(SpA) is impaired compared to the natural Fc-hinge domain SpA binding. This approach is further described in U.S. Patent No. 6,165,745 by Ward et al.

[0057] In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be replaced with a different amino acid residue such that the antibody has a modified affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the CI component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260 by Winter et al.

[0058] In another embodiment, one or more amino acids selected from amino acid residues 329, 331, and 322 may be replaced with different amino acid residues, such that the antibody alters the Clq bond and / or reduces or eliminates the CDC. This approach is described in more detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0059] In another embodiment, one or more amino acid residues within amino acid positions 231 and 239 are modified, thereby altering the antibody's ability to fix complement. This approach is further described by Bodmer et al. in PCT Publication 94 / 29351.

[0060] In yet another embodiment, the Fc region may be modified to increase ADCC and / or increase affinity to the Fcγ receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 2 85, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications to enhance FcγR and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0061] Fc modifications that enhance binding to the Fcγ receptor are located at amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, This includes amino acid modifications at one or more of the following locations: 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, where the numbering of residues in the Fc region is the same as the EU index in abat (WO00 / 42072).

[0062] Other Fc modifications that can be performed on Fc are those that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating the function of Fc-mediated effectors such as ADCC, ADCP, and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, and numbering follows the EU index. Exemplary substitutions include, but are not limited to, 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, and numbering follows the EU index. Fc variants may include 236R / 328R. Other modifications to reduce FcγR and complement interactions include substitutions at 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutation, enzymatic means, or production in organisms such as non-glycosylating bacteria. These and other modifications are outlined in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0063] Optionally, the Fc region may contain non-natural amino acid residues at additional and / or alternative positions known to those skilled in the art (e.g., U.S. Patents No. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720; WO00 / 42072, W See O01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925 and WO06 / 020114.

[0064] Fc mutants that enhance affinity for the inhibitory receptor FcγRIIb may also be used. Such mutants may provide an Fc fusion protein having immunomodulatory activity associated with FcγRIIb cells, for example, B cells and monocytes. In one embodiment, the Fc mutant provides selectively enhanced affinity for FcγRIIb compared to one or more activating receptors. Modifications to alter binding to FcγRIIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index. Exemplary substitutions for improving FcγRIIb affinity include, but are not limited to, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include, but are not limited to, 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc mutants that enhance binding to FcγRllb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0065] The affinity and binding characteristics of the Fc region to the ligand can be determined by equilibrium methods (e.g., ELISA, or radioimmunoassay), kinetic methods (e.g., BIACORE analysis), and various in vitro assay methods (biochemical or immunological assays) known in the art, including but not limited to indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labeling of one or more of the components being examined, and / or various detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0066] In certain embodiments, antibodies are modified to increase their biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn. For example, one or more of the following residues may be mutated, as described in U.S. Patent No. 6,277,375: 252, 254, 256, 433, 435, 436. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, antibodies may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope derived from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M.Other mutants that increase Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al, 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, and 434A (Shields et al, Journal of Biology). Chemistry,2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S(Dall Acqua et al.Journal of Immunology,2002,169:5171-5180,Dall'Acqua et al.,2006,Journal of Biological Chemistry Examples include 281:23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671. In certain embodiments, hybrid IgG isotypes having specific biological characteristics may be used. For example, an IgG1 / IgG3 hybrid mutant may be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids derived from IgG3 at different positions for the two isotypes. Thus, hybrid mutant IgG antibodies containing one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F, may be constructed.In other embodiments described herein, IgGl / IgG2 hybrid mutants may be constructed by substituting IgG2 positions within the CH2 and / or CH3 regions with amino acids from IgGl at different positions for the two isotypes. Thus, hybrid mutant IgG antibodies may be constructed by CHATs containing one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to the insertion of glycine at position 236), and 321h.

[0067] Furthermore, the binding sites of FcγRl, FcγRII, FcγRIII, and FcRn on human IgGl have been mapped, and mutants with improved binding have been described (see Shields, R. Let al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. In addition, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A (these have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity) (Shields et al., 2001). Other IgG1 mutants with strongly enhanced binding to FcγRIIIa have been identified, including mutants with the S239D / I332E and S239D / I332E / A330L mutations that showed the greatest increase in affinity for FcγRIIIa, decreased FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). The introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) resulted in significantly enhanced ADCC activity in vitro, with the S239D / I332E mutant showing enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). In addition, in models of B-cell malignancies and breast cancer, IgG1 mutants containing L235V, F243L, R292P, Y300L, and P396L mutations have been identified in transgenic mice expressing human FcγRIIIa that showed enhanced binding to FcγRIIIa and simultaneously enhanced ADCC activity (Stavenhagen et al., 2007; Nordstrom et al., 2011).Other Fc variants that may be used include: S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0068] In certain embodiments, Fc with reduced binding to FcγR is selected. An exemplary Fc with reduced FcγR binding, e.g., IgGlFc, includes the following three amino acid substitutions: L234A, L235E, and G237A.

[0069] In certain embodiments, Fc with reduced complement fixation is selected. An exemplary Fc with reduced complement fixation, for example, IgGl Fc, has the following two amino acid substitutions: A330S and P331S.

[0070] In certain embodiments, Fc molecules are selected that are essentially devoid of effector function, i.e., have reduced binding to FcγR and reduced complement fixation. Exemplary effector-less Fc molecules, such as IgGl Fc, include the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0071] When using the IgG4 constant domain, it is usually preferable to include the substituted S228P, which mimics the hinge sequence within IgG1 and thereby stabilizes the IgG4 molecule.

[0072] antibody derivative The antibodies provided herein may be further modified to include additional non-proteinoid moieties that are known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.

[0073] Non-limiting examples of water-soluble polymers include, but are not limited to, PEG, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody varies, and if two or more polymers are bound, they may be the same or different molecules. Generally, the number and / or types of polymers used in derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used therapeutically under defined conditions.

[0074] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (see Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength and may not harm normal cells, but may include, but is not limited to, wavelengths that heat the non-proteinaceous moiety to a temperature that kills cells adjacent to the antibody-non-proteinaceous moiety.

[0075] Another modification of antibodies described herein is pegylation. Antibodies can be pegylated to increase, for example, the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or a fragment thereof, is reacted with a PEG, such as a reactive ester or aldehyde derivative of PEG, typically under conditions where one or more PEG groups bind to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any form of PEG used to derivatize other proteins, such as mono(CI-CIO)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody being pegylated is a non-glycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0076] The present invention also includes therapeutic agents, polymers, and human monoclonal antibodies described herein that are conjugated to detectable labels or enzymes. In one embodiment, the therapeutic agent is a cytotoxic agent. In one embodiment, the polymer is PEG.

[0077] Method of production Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence including VL and / or an amino acid sequence including VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, host cells containing such nucleic acid are provided. In one such embodiment, the host cells include (e.g., transformed with them): (1) a vector containing nucleic acid encoding an amino acid sequence including VL of the antibody and an amino acid sequence including VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence including VL of the antibody, and a second vector containing nucleic acid encoding an amino acid sequence including VH of the antibody. In one embodiment, the host cells are eukaryotic cells, for example, Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an antibody is provided, which includes culturing host cells containing nucleic acids encoding an antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0078] For recombinant antibody production, for example, as described above, the nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0079] Suitable host cells for cloning or expressing antibody-coding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (Also see Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed. Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies may be isolated from the bacterial cell paste in the soluble fraction and further purified.

[0080] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-coding vectors, including fungal and yeast strains, in which the glycosylation pathway is "humanized" and antibodies with a partially or completely human glycosylation pattern are produced. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0081] Host cells suitable for the expression of glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. In particular, numerous baculovirus strains have been identified that can be used in conjunction with insect cells for transfection of Spodoptera frugiperda cells.

[0082] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES technology for antibody production in transgenic plants).

[0083] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 cell line transformed with SV40 (COS-7); human embryonic kidney cells (e.g., 293 or 293 cells as described in Graham et al. J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); and TRI cells (e.g., Mather et al., Ananals). These include MRC5 cells and FS4 cells, as described in NYAcad.Sci.383:44-68(1982). Other useful mammalian host cell lines include DHFR - Examples include CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), as well as myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0084] Compositions and formulations The antibodies of the present invention represent an excellent method for developing antiviral therapies for the treatment of human influenza infection in humans, either alone or in an antibody cocktail with additional anti-influenza virus antibodies.

[0085] In another embodiment, the present invention provides a pharmaceutical composition comprising the antibody of the present invention as described herein, formulated with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or therapeutic agent. The pharmaceutical composition of the present invention may also be administered in combination therapy with, for example, another immunostimulant, antiviral agent, or vaccine. In certain embodiments, the composition contains the antibody of the present invention at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1 to 300 mg / ml, or 100 to 300 mg / ml.

[0086] A pharmaceutical composition may contain any number of excipients. Available excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspension aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0087] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the term “parenteral administration” usually means a mode of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the antibodies of the present invention as described herein may be administered via parenteral routes, e.g., topical, epidermal, or mucosal administration routes, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

[0088] The pharmaceutical compositions of the present invention may be in the form of pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxic effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphoric acid, as well as those derived from non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0089] The pharmaceutical composition of the present invention may be in the form of a sterile aqueous solution or dispersion. It can also be formulated in the form of a microemulsion, liposome, or other ordered structure suitable for high drug concentrations.

[0090] The antibodies of the present invention described herein can be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-lives, following humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low doses are administered at relatively infrequent intervals over a long period. Some patients continue treatment for life. In therapeutic use, relatively high doses at relatively short intervals may be required until the progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete remission of the symptoms of the disease. Thereafter, a prophylactic regime can be administered to the patient.

[0091] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the target being treated and the specific mode of administration, and is generally the amount of the composition that produces the therapeutic effect. Generally, out of 100 percent, this amount, when combined with a pharmaceutically acceptable carrier, ranges from about 0.01% to about 99%, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient.

[0092] The dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to a physically distinct unit appropriate as a unit dosage for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. With regard to antibody administration, the dosage is in the range of approximately 0.0001 to 100 mg / kg of host body weight, more typically in the range of 0.01 to 5 mg / kg. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferred dosage regimens for the antibody of the present invention include 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, and the antibody is administered using one of the following administration schedules: (i) for six doses, every four weeks, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once, then 1 mg / kg body weight every three weeks. In some cases, the dose is adjusted to achieve a plasma antibody concentration of about 1 to 1000 μg / ml, and in some cases, a plasma antibody concentration of about 25 to 300 μg / ml. The "therapeutably effective dosage" of the antibody of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or disability caused by the suffering of the disease.For example, for the treatment of influenza infection in a subject, a "therapeutably effective dose" preferably inhibits influenza virus replication or uptake by host cells by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to an untreated subject. A therapeutically effective dose of the therapeutic compound can neutralize the influenza virus or, by other means, improve symptoms in a subject that may typically be human or another mammal. The pharmaceutical composition may be a controlled-release formulation comprising grafts, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0093] Therapeutic compositions can be administered via medical devices such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, and 4,596,556), (2) microinjection pumps (U.S. Patent No. 4,487,603), (3) transdermal devices (U.S. Patent No. 4,486,194), (4) injectors (U.S. Patents No. 4,447,233 and 4,447,224), and (5) osmotic devices (U.S. Patents No. 4,439,196 and 4,475,196), the disclosures thereof being incorporated herein by reference.

[0094] In certain embodiments, the human monoclonal antibodies of the present invention described herein can be formulated to ensure appropriate distribution in vivo. For example, to ensure that the therapeutic compounds of the present invention cross the blood-brain barrier, they can be formulated in liposomes which may additionally contain a target moiety to enhance selective transport to specific cells or organs. For example, U.S. Patent Nos. 4,522,811, 5,374,548, 5,416,016, and 5,399,331; al,(1988)Biochem.Biophys.Res.Commun.153:1038;Bloeman et al.(1995)FEBS Lett.357:140;M.Owais et al.(1995)Antimicrob.Agents Chemother.39:180;Briscoe et al. al.(1995)Am.Physiol.1233:134;Schreier et al.(1994).Biol.Chem.269:9090;Keinanen and Laukkanen(1994)FEBS See Lett.346:123 and Killion and Fidler (1994) Immunomethods 4:273.

[0095] Instructions and methods of use Current antiviral treatments for influenza (e.g., oseltamivir / tamiflu, amantadine / rimantadine) are suboptimal due to increased rates of resistance and limited treatment windows (must be initiated within 48 hours of symptom onset) (Beigel J, et al. 2008. Antiviral Res 78:91-102; Garcia-Sastre A. 2006. Emerg Infect Dis 12:44-47; and Marathe BM, et al. 2016. Sci Rep 6:26742). Monoclonal antibodies continue to be a partially growing class of drugs due to their high specificity, limited off-target effects, and excellent safety profile. The antibodies, compositions, and formulations described herein may be used to neutralize the influenza virus and thereby treat influenza infection.

[0096] Therefore, in one embodiment, the antibodies described herein may be used to neutralize influenza viruses. Neutralization of influenza viruses can be achieved by (i) inhibiting influenza viruses that bind to target cells, (ii) inhibiting the uptake of influenza viruses by target cells, (iii) inhibiting the replication of influenza viruses, and (iv) inhibiting the release of influenza virus particles from infected cells. Those skilled in the art will be able to perform any assay to evaluate the neutralization of influenza viruses. Notably, the neutralizing properties of an antibody may be evaluated by a variety of tests, all of which can evaluate the results of (i) inhibition of influenza viruses that bind to target cells, (ii) inhibition of influenza virus uptake by target cells, (iii) inhibition of influenza virus replication, and (iv) inhibition of influenza virus particles released from infected cells. In other words, performing different tests may lead to the same result, namely, the observation of loss of infectivity of the influenza virus. Therefore, in one embodiment, the present invention provides a method for neutralizing influenza viruses in a subject, comprising administering a therapeutic effect dose of the antibodies of the present invention described herein.

[0097] Another aspect of the present invention provides methods for treating influenza-related illnesses. Such methods include therapeutic (post-influenza infection) and prophylactic (pre-influenza exposure, infection, or pathology) methods. For example, therapeutic and prophylactic methods for treating an individual for influenza infection include methods for treating an individual who has or is at risk of having influenza infection, treating an individual who has influenza infection, and protecting an individual from influenza infection, methods for reducing or mitigating the likelihood of influenza infection in an individual, methods for reducing or mitigating an individual's susceptibility to influenza infection, or methods for inhibiting or preventing influenza infection in an individual, as well as methods for reducing, mitigating, inhibiting, or suppressing the transmission of influenza from an infected individual to an uninfected individual. Such methods include treating (vaccinating or immunizing) an individual who has or is at risk of having influenza infection or pathology therapeutically or prophylactically with an antibody of the present invention or a composition containing an antibody disclosed herein. Thus, the methods can treat influenza infection or pathology, or provide an individual with protection from infection (e.g., prophylactic protection).

[0098] In one embodiment, a method for treating an influenza-related illness comprises administering to an individual in need an antibody or therapeutic composition disclosed herein in an amount sufficient to reduce one or more physiological conditions or symptoms associated with influenza infection or pathology, thereby treating the influenza-related illness.

[0099] In one embodiment, the antibodies or therapeutic compositions disclosed herein are used to treat influenza-related illnesses. The use of the antibodies or therapeutic compositions disclosed herein treats influenza-related illnesses by reducing one or more physiological conditions or symptoms associated with influenza infection or pathology. In aspects of this embodiment, the administration of the antibodies or therapeutic compositions disclosed herein is sufficient to reduce one or more physiological conditions or symptoms associated with influenza infection or pathology, thereby treating the influenza-based illness. In other embodiments of this embodiment, the administration of the antibodies or therapeutic compositions disclosed herein is sufficient to increase, induce, enhance, enhance, promote, or stimulate influenza clearance or elimination, or to reduce, reduce, inhibit, suppress, prevent, control, or limit the transmission of influenza to another individual.

[0100] One or more physiological conditions or symptoms associated with influenza infection or pathology will respond to the therapeutic methods disclosed herein. The symptoms or pathology of influenza infection vary depending on the stage of infection.

[0101] In another aspect of the present invention, the antibodies described herein can be used in various detection methods, for example, to monitor the progression of an influenza infection, or to monitor a patient's response to treatment for such an infection. This disclosure provides a method for detecting HA or NA polypeptides in biological samples obtained from an individual. The method generally comprises a) contacting the biological sample with a target anti-HA or anti-NA antibody, and b) detecting the binding of the antibody to an epitope present in the sample (if any). In some cases, the antibody includes a detectable label. The level of HA or NA polypeptide detected in a biological sample can provide an indicator of the stage, extent, or severity of an influenza infection. The level of HA or NA polypeptide detected in a biological sample can provide an indicator of an individual's response to treatment for an influenza infection.

[0102] The antibodies described herein can be used in combination with one or more other anti-influenza virus antibodies to neutralize influenza virus and thereby treat influenza infections.

[0103] Definitions As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment or single chain thereof. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region comprises three domains, C H 1, C H 2, and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as V L ), and a light chain constant region. The light chain constant region comprises one domain, C L . The V H and V L regions are further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each V H and V L consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFRl, HCDRl, HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs and FRs are LFRl, LCDRl, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigen. The constant regions of the antibody can mediate binding to host tissues or factors of the immune globulin, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (CIq).

[0104] As used herein, the term “antibody” is used in its broadest sense and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), and antibody fragments, any immunoglobulin produced naturally, partially, or completely by synthesis.

[0105] The term "bispecific antibody" refers to an artificial immunoglobulin construct consisting of fragments of two different monoclonal antibodies that bind to two different antigens. Examples include, but are not limited to, trifunctional antibodies and chemically linked Fabs. Several different types of bispecific antibodies exist. The antibodies described herein may include bispecific antibodies, comprising one or more fragments of one or more different anti-HA or NA antibodies, including one or more different antibodies described herein or known anti-influenza virus antibodies. Methods for the preparation and use of such bispecific antibodies are described, for example, in PCT / US16 / 64713, which is incorporated herein by reference.

[0106] As used herein, the term “antigen-binding fragment or portion” of an antibody (or simply “antibody fragment or portion”) refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., the HA or NA of influenza A or B virus). It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment or portion” of an antibody include (i) Fab fragments, V L , V H , C L and C H (ii) a monovalent fragment consisting of domain I, (ii) a bivalent fragment containing an F(ab')2 fragment and two Fab fragments linked by disulfide bridges in the hinge region, and (iii) a Fab' fragment which is essentially a Fab having part of the hinge region (FUNDAMENTAL IMMUNOLOGY (Paul ed., 3 rd See ed.1993), (iv)V H and C HFd fragment consisting of domain I, (v) single arm of antibody V L (vi) Fv fragments consisting of the VH domain, (vi) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546), (vii) isolated CDRs, and (viii) heavy chain variable regions containing nanobodies, a single variable domain, and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by synthetic linkers that allow the VL and VH regions to pair up to form a single protein chain that forms a monovalent molecule (known as single-stranded Fv or scFv) using recombination methods; see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment or portion" of an antibody. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0107] As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to a particular antigen such as the HA or NA of influenza A or B virus substantially does not contain antibodies that specifically bind to antigens other than that particular antigen). Isolated antibodies may substantially not contain other cellular material and / or chemical substances.

[0108] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to a preparation of an antibody molecule in a single-molecule composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0109] The term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence.

[0110] The term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity, in which both the framework and CDR region have variable regions derived from human germline immunoglobulin sequences. In one embodiment, a human monoclonal antibody may be produced by a hybridoma having a genome containing a human heavy chain transgene and a light chain transgene fused to an immortalized cell, including B cells obtained from a transgenic non-human animal, such as a transgenic mouse.

[0111] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomes for human immunoglobulin genes, or hybridomas prepared therefrom (as further described below), (b) antibodies isolated from host cells transformed to express human antibodies, for example, from transfectomas, (c) recombinant antibodies isolated from recombinant human antibody libraries, and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR region are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may undergo in vitro mutagenesis (or, if an animal transgenic to the human Ig sequence is used, in vivo somatic mutagenesis), and thus the V of recombinant antibodies H and V L The amino acid sequence of the region is human germline V H and V L This sequence is derived from and related to other sequences, but it is a sequence that cannot naturally exist in vivo within the human antibody germline repertoire.

[0112] The term "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The terms "antigen-recognizing antibody" and "antigen-specific antibody" are used herein as synonymous with the term "antigen-specific antibody."

[0113] The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody with another drug or antibody. The term "humanized antibody" is intended to refer to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted into a human framework sequence. Additional framework region modifications can be made within the human framework sequence.

[0114] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody. The term can also refer to an antibody in which its variable region sequence or CDR originates from one source (e.g., an IgA1 antibody) and its constant region sequence or Fc originates from a different source (e.g., a different antibody, e.g., an IgG, IgA2, IgD, IgE, or IgM antibody).

[0115] As used herein, the term "affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0116] As used herein, an antibody that "specifically binds to the HA of the influenza virus" refers to an antibody that binds to the HA of the influenza virus but substantially does not bind to the HA of non-influenza viruses. Similarly, an antibody that "specifically binds to the NA of the influenza virus" refers to an antibody that binds to the NA of the influenza virus but substantially does not bind to the NA of non-influenza viruses.

[0117] Preferably, the antibody is "high affinity," i.e., 1 × 10⁻⁶. -7 M or less, more convenient 5×10 -8 M or less, more preferably 3 × 10 -8 M or less, more preferably 1 × 10 -8 M or less, more convenient 5×10 -9 M or less, or more preferably 1 × 10 -9It binds to HA or NA with a KD of M or less. As used herein, the term “substantially unbound” means not binding to or not binding to a protein or cell with high affinity, i.e., 1 × 10 -6 M or higher, comfort 1x10 -5 M or higher, comfort 1x10 -4 M or higher, comfort 1x10 -3 M or more, even more comfortable 1×10 -2 Binds to proteins or cells with a KD of M or higher.

[0118] As used herein, the terms "Kassoc" or "Ka" are intended to refer to the association rate of a particular antibody-antigen interaction, while the terms "Kdis" or "Kd" are intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is derived from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is to use surface plasmon resonance, preferably using a biosensor system such as the Biacore® system.

[0119] As used herein, the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in a variable region of an antibody molecule, known as a paratope. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to the region of the antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also consist of a three-dimensional structure, i.e., nonlinear amino acids. In some embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in some embodiments may have specific three-dimensional structural properties and / or specific charge properties. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in their unique spatial conformation. Methods for determining which epitopes are bound to a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which duplicate or sequential peptides from HA or NA proteins are tested for reactivity with a given antibody. Methods for determining the spatial conformation of epitopes include techniques described in the art and herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0120] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0121] The terms "binding to an epitope" or "recognizing an epitope" refer to a continuous or discontinuous segment of amino acids within an antigen, with respect to an antibody or antibody fragment. Those skilled in the art will understand that the terms do not necessarily imply that the antibody or antibody fragment is in direct contact with all amino acids within the epitope sequence.

[0122] The term “binding to the same epitope” means, with respect to two or more antibodies, that the antibodies bind to encompass the same, overlapping, or continuous or discontinuous segments of an amino acid. Those skilled in the art will understand that the phrase “binding to the same epitope” does not necessarily mean that the antibodies bind to or contact the exact same amino acid. The exact amino acids that the antibodies contact may be different. For example, a first antibody may bind to a segment of amino acid that is completely encompassed by the segment of amino acid bound by the second antibody. In another embodiment, a first antibody may bind to one or more segments of amino acids that significantly overlap with one or more segments bound by the second antibody. For the purposes of this specification, such antibodies are considered to “binding to the same epitope.”

[0123] An antibody that "competes with other antibodies for binding to a target" refers to an antibody that (partially or completely) inhibits the binding of another antibody to its target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the other antibody's binding to the target, can be determined using known competition experiments. In some embodiments, an antibody competes with and inhibits the binding of another antibody to its target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is a "blocking antibody" (i.e., a cold antibody that is initially incubated with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harbor Protoc; 2006; doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competitive antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as demonstrated by steric hindrance).Other competitive binding assays include solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays (Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137:3614 (1986)); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using labels 1-125 (see Morel et al., Mol.Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (see Cheung et al., Virology Examples include 176:546(1990)) and directly labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77(1990)).

[0124] As used herein, the term “immune response” refers to the biological response in vertebrates to an exogenous agent, which protects the organism from these agents and the diseases they cause. The immune response is mediated by the action of soluble macromolecules produced by cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils), and either these cells or the liver (including antibodies, cytokines, and complement), resulting in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invasive pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Examples of immune responses include the activation or inhibition of T cells, e.g., effector T cells or Th cells, e.g., CD4+ or CD8+ T cells, or the inhibition of Treg cells.

[0125] As used herein, the term “detectable label” refers to a detectable molecule, including but not limited to radioisotopes, phosphors, chemiluminescent materials, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, streptavidin, or hapten), and intercalating dyes. The term “phosphor” refers to a substance or part thereof that can exhibit fluorescence to a detectable extent.

[0126] As used herein, the term “subject” refers to an animal. Preferably, the animal is a mammal. The subject also refers to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the subject is a human.

[0127] As used herein, the term “therapeutic dose” of a compound of the present invention refers to an amount of the compound of the present invention that induces a biological or medical response to a target, improves symptoms, delays or slows the progression of a disease, or prevents a disease. In one embodiment, the term refers to an amount that inhibits or reduces microbial colonization or infection. In one embodiment, the term refers to an amount that inhibits or reduces infection or prevents or destroys the formation of a bacterial biofilm. When applied to individual active ingredients, when administered alone, the term refers to that ingredient alone. When applied to combinations, the term refers to the combined amount of active ingredients that produce a therapeutic effect, whether administered together, sequentially, or simultaneously.

[0128] As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium or excipient that does not interfere with the efficacy of the biological activity of the active ingredient of the composition and is not excessively toxic to the host at the concentration administered. In the context of the present invention, pharmaceutically acceptable carriers or excipients are preferably suitable for topical formulations. This term includes, but is not limited to, solvents, stabilizers, solubilizers, isotonic agents, structure-forming agents, suspending agents, dispersants, chelating agents, emulsifiers, antifoaming agents, ointment bases, emollients, skin protectants, gel-forming agents, thickeners, pH adjusters, preservatives, penetration enhancers, complexing agents, lubricants, viscous agents, thickeners, bioadhesive polymers, or combinations thereof. (See, for example, “Remington's Pharmaceutical Sciences,” EW Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety.)

[0129] As used herein, the terms “to treat” or “to cure” any disease or disorder mean, in one embodiment, to alleviate the disease or disorder (i.e., to prevent or reduce the onset of at least one of the disease or its clinical symptoms). In another embodiment, “to treat” or “to cure” means to improve at least one physical parameter that is not identifiable by the patient. In yet another embodiment, “to treat” or “to cure” means to modulate the disease or disorder physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, “to treat” or “to cure” means to prevent or delay the onset, development, or progression of the disease or disorder.

[0130] When used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the invention (especially in the context of the claims) should be interpreted to encompass both singular and plural forms, unless otherwise suggested herein or otherwise clearly contradict the context. Enumerations of value ranges herein are intended merely as abbreviated expressions to refer individually to each distinct value that falls within that range. Unless otherwise indicated herein, each distinct value is incorporated herein as if it were individually listed herein. All methods described herein may be performed in any preferred order, unless otherwise indicated herein or otherwise clearly contradict the context. The use of any and all examples or exemplary language provided herein (e.g., “such as”) is intended only to better illustrate the invention and does not imply any limitation to the scope of the invention as otherwise claimed. No language herein should be interpreted as indicating any non-claimed element essential to the practice of the invention.

[0131] The term "approximately" means within 10%, preferably within 5%, and more preferably within 1%, of a given value or range. Alternatively, the term "approximately" means within the acceptable standard error of the mean, as considered by those skilled in the art. [Examples]

[0132] Example 1: Generation of hmAb. Peripheral blood plasma cells (CD19+IgD-CD38+CD27++) were immunized with the 2014-2015 seasonal inactivated tetravalent influenza vaccine, and single cells were selected from the subjects approximately 7 days later. The immunoglobulin heavy chain (VH) and light chain (Vk or Vl) variable regions were sequenced using single-cell PCR. The heavy and light chain variable regions were expressed from these single-cell selected plasmablasts using the immunoglobulin expression cassette process described in Liao et al. J Virol Methods. 2009 Jun;158(1-2):171-9 (PMC2805188) and the corresponding plasmid obtained by Dr. Kevin Saunders (Duke University). The immunoglobulins were screened for binding to and neutralization of H3 protein, N2 protein, and H3N2 virus, respectively. Next, the immunoglobulin heavy and light chain pairs with the greatest width and activity were cloned as complete IgG1 human monoclonal antibodies, and their gene usage is detailed in Table 1. TIFF2023519930000002.tif48170

[0133] Example 2: In vitro activity of hmAb. Five hmAbs were characterized by ELISA for binding to various HA and NA proteins (Figure 1). 1092C4, 1092E4, and 1086G8 hmAbs exhibit broad reactivity to H3 and H7 proteins. They have minimal reactivity to H1 protein. 1122A11 and 1122B9 exhibit broad reactivity to N2 protein but no reactivity to N1 protein. The hmAbs recognize cells infected with a wide range of H3N2 influenza viruses (Figure 2). The hmAbs were tested for their ability to neutralize a wide range of H3N2 influenza viruses (Table 2). TIFF2023519930000003.tif74170

[0134] Example 3: In vivo activity of hmAb. To evaluate the protective activity of H3 and N2 hmAbs, mice received 20 mg / kg of the indicated hmAb prior to a lethal intranasal challenge dose (10 MLD50) of H3N2 X31 influenza virus. All mice treated with PBS or isotype control hmAbs exhibited severe weight loss and died from infection within 7 days (Figure 3A and B). All mice treated with 1092E4 hmAb maintained their body weight and survived the infection. 80% of mice treated with 1086G8, 1092B6, or 1122A11 survived the infection and maintained their body weight. Consistent with increased survival rates, mice treated with H3 or N2 hmAb had a significant decrease in viral titer in the lungs on post-infection (pi) days 2 and 4, including the absence of detectable virus at d4 in any of the mice treated with 1122A11 hmAb (Figure 3C).

[0135] Having demonstrated the superior protective activity of 1092E4H3-specific and 1122A11N2-specific hmAbs, the inventors then evaluated the therapeutic activity of the antibodies. Mice were challenged with a lethal dose (10MLD50) of H3N2 X31 influenza virus and then treated with 1 or 10 mg / kg of hmAb for 24 hours post-infection. All mice treated with PBS or isotype control hmAb exhibited severe weight loss and died from the infection within 6 days (Figures 4A and 4B). All mice treated with 10 mg / kg of 1092E4 survived the infection and had no detectable virus in the lungs at D4 post-infection (Figure 4C). Treatment with 1 mg / kg of 1122A11 conferred a 40% survival rate. These results, overall, demonstrate that 1092E4 and 1122A11 possess both potent protective and therapeutic activity against H3N2 influenza virus in vivo.

[0136] The above-mentioned examples and descriptions of preferred embodiments should be understood as illustrative and not as limiting the invention as defined by the claims. For ease of understanding, numerous variations and combinations of the above features can be utilized without departing from the invention as described in the claims. Such modifications are not considered to deviate from the scope of the invention, and all such modifications are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to influenza virus hemagglutinin (HA), (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, comprising the sequence of SEQ ID NO: 7-9; (ii) a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the sequences of SEQ ID NOs: 10, 5, and 11.

2. An isolated antibody or antigen-binding fragment thereof that specifically binds to influenza virus hemagglutinin (HA), comprising: (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, comprising the sequences of SEQ ID NOs: 7, 8, and 12; (ii) a light chain variable region comprising LCDR1, LCDR2, and LCDR3 comprising the sequences of SEQ ID NOs: 10, 5, and 11.

3. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 30 and the light chain variable region comprises the sequence of SEQ ID NO:

32.

4. An isolated antibody or antigen-binding fragment thereof described in claim 2, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 34 and the light chain variable region comprises the sequence of SEQ ID NO:

36.

5. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to influenza virus neuraminidase (NA), (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, comprising the sequence of SEQ ID NO: 13-15; (ii) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, the light chain variable region comprising the sequence of SEQ ID NO:16-18.

6. 6. The isolated antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 38 and the light chain variable region comprises the sequence of SEQ ID NO:

40.

7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, further comprising a variant Fc constant region.

8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody is a chimeric antibody or a human antibody.

9. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody or fragment is conjugated to a therapeutic agent, a polymer, a detectable label, or an enzyme.

10. 10. The isolated antibody or antigen-binding fragment thereof of claim 9, wherein the polymer is polyethylene glycol (PEG).

11. The isolated antibody or antigen-binding fragment thereof of claim 9 , wherein the therapeutic agent is a cytotoxic agent.

12. An isolated nucleic acid encoding the HCDR, LCDR, heavy chain variable region, or light chain variable region of the antibody, or antigen-binding portion thereof, of any one of claims 1 to 11.

13. An expression vector comprising the nucleic acid of claim 12.

14. 14. A cultured host cell comprising a nucleic acid according to claim 12 or an expression vector according to claim 13.

15. 1. A method for preparing an antibody, or an antigen-binding portion thereof, comprising: Obtaining a cultured host cell comprising a vector comprising a nucleic acid sequence encoding the HCDR, LCDR, heavy chain variable region, or light chain variable region of the antibody, or antigen-binding portion thereof, of any one of claims 1 to 11; culturing the cells in a culture medium under conditions that allow expression of a polypeptide encoded by the vector and assembly of an antibody or fragment thereof; and purifying said antibody or fragment from said cultured cells or from said medium of said cells.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

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