JP2022061986A - Neutralizing anti-influenza a virus antibodies and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel antibodies that neutralize multiple subtypes of influenza A virus and can be used as a drug for preventing or treating influenza A infection.

SOLUTION: Provided are an antibody and antigen-binding fragments thereof that specifically bind to an epitope present in the stem region of an influenza A hemagglutinin trimer and neutralize group 1 and group 2 subtypes of influenza A virus. Provided is a nucleic acid encoding immortalized B cells and single plasma cells, which produces such antibody and antibody fragments, as well as epitopes that bind to such antibody and antibody fragments. Additionally, provided is a screening method as well as an influenza A virus infection diagnosis, and use of antibodies, antibody fragments, and epitopes in treatment and prevention.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

Typically, the anti-influenza virus neutralizing antibody is specific for a given viral subtype. Based on the hemagglutinin (“HA”) protein, 16 influenza A subtypes have been defined. The 16 types of HA, namely H1 to H16, can be classified into two groups. The first group consists of H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 subtypes, and the second group contains H3, H4, H7, H10, H14 and H15 subtypes. Be included. In birds, all subtypes are present, whereas in humans, the H1, H2 and H3 subtypes cause the disease. The H5, H7 and H9 subtypes cause sporadic severe infections in humans and may lead to new global epidemics. The H1 and H3 viruses are continuously evolving to generate new mutants, a phenomenon called "antigenic drift". As a result, antibodies produced in response to conventional viruses have poor or no protection against the new type of drift virus. As a result, new vaccines are produced each year against the expected emergence of H1 and H3 viruses, but this approach is very costly and not always effective. The same is true for the production of influenza H5 vaccines. Furthermore, it is unclear whether current H5 vaccines based on influenza A isolated in Vietnam or Indonesia will prevent future outbreaks of H5 virus.

For these reasons, it is highly desirable to obtain a vaccine that induces a variety of neutralizing antibodies capable of neutralizing all influenza A virus subtypes, as well as their annually occurring variants. al., 2006). In addition, the broadly neutralizing hetero-subtype antibody can be administered as a prophylactic or therapeutic agent for influenza A infection. In order to reduce the production cost, it is important to select an antibody produced with a high titer when producing such a drug.

So far, the characteristics of antibodies that recognize influenza A virus have been clarified. Antibodies to M2, an invariant small protein that is expressed in infected cells but not on infectious viruses, have some protective effect in the body, probably NK cells or cytotoxic T cells. By targeting infected cells with respect to cell destruction. HA proteins with neutralizing antibodies can also be targeted. HA is synthesized as the homotrimer precursor polypeptide HA0. Each monomer undergoes independent cleavage after translation to form two polypeptides, namely HA1 and HA2, one of which is linked by a disulfide bond. The larger N-terminal fragment (HA1, 320-330 amino acid residues) forms a spherical domain distal to the membrane, which is the receptor binding site and most of the determinants recognized by virus neutralizing antibodies. And contains. The HA1 polypeptide of HA is involved in the attachment of the virus to the cell surface. The smaller C-terminal portion (HA2, about 180 amino acids) forms a stem structure and anchors the globular domain to the cell or viral membrane. The HA2 polypeptide mediates fusion of the viral and cell membranes in the endosome, causing the cytoplasm to release the ribonucleoprotein complex.

Sequence homology between subtypes is smaller for HA1 polypeptides (homology between subtypes: 34% to 59%) than for HA2 polypeptides (homology: 51% to 80%). The most conserved region is the sequence around the cleavage site, in particular 11 N-terminal amino acid residues of HA2 (called fusion peptides) are conserved among all influenza A virus subtypes. Part of this region is exposed as a surface loop in the HA precursor molecule (HA0), but is isolated when HA0 is cleaved into HA1 / HA2. In summary, there are conservative regions between the different HA subtypes, especially in the HA1-HA2 linking sites and the HA2 region. However, these regions may have poor contact with neutralizing antibodies.

So far, there have been very few successful cases in identifying antibodies that neutralize more than one subtype of influenza A virus. Furthermore, the range of neutralization by the antibodies identified so far is narrow and the titer is low. Okuno et al immunizes mice with influenza virus A / Okuda / 57 (H2N2) and binds an isolated monoclonal antibody (C179) to an epitope of HA2 whose conformation is conserved, in vitro and in animal models. In vivo, the H2, H1 and H5 subtypes of the first group of influenza A viruses were neutralized (Okuno et al., 1993; Smirnov et al., 1999; Smirnov et al., 2000).

Gioia et al. Reported the presence of antibodies that neutralize H5N1 virus in the sera of some patients who received the conventional seasonal influenza vaccine (Gioia et al., 2008). They suggest that neutralizing activity is obtained by anti-neuraminidase (N1) antibody. However, no monoclonal antibody was isolated and the target epitope was not characterized. Similarly, it is unclear whether serum antibodies neutralize other subtypes of influenza A virus.

So far, immune donor memory B cells and plasma cells have bound to epitopes present in the stem region of HA and neutralized some of the influenza A subtypes of either Group 1 or Group 2. Heterogeneous subtypes of human antibodies that can be used have been isolated. However, it is influenza A-specific, capable of targeting epitopes of HA trimers conserved in all 16 subtypes and neutralizing both Group 1 and Group 2 subtype viruses. Neutralizing antibodies have not been found so far, and isolation of them remains a major goal in therapeutic approaches and vaccine design.

Despite decades of research, no antibody is commercially available that broadly neutralizes or inhibits influenza A infection or attenuates the disease caused by influenza A virus. Therefore, there is a need to identify novel antibodies that can be used as agents to neutralize multiple subtypes of influenza A virus and to prevent or treat influenza A infection. It is also needed to identify antibodies produced at high titers in order to reduce production costs.

The present invention isolates from individuals vaccinated with the seasonal influenza vaccine a natural human monoclonal antibody that binds to HA and neutralizes infections of more than one subtype of influenza A virus, as well as the present invention. It is based in part on the novel epitope to which the antibody of influenza binds. Accordingly, in one aspect of the invention, the invention neutralizes infection of more than one subtype of influenza A virus selected from group 1 and group 2 subtypes, antibodies and antigens thereof. Contains bound fragments.

In one embodiment of the invention, the invention comprises isolated antibodies or antigen-binding fragments thereof that neutralize infections of influenza A virus type 1 and group 2 subtypes. In another embodiment of the invention, the invention neutralizes the infection of influenza A virus type 1 and group 2 subtypes and stems the influenza A hemagglutination (HA) trimer. An isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope present in the region is contained, and the heavy and light chains of the antibody or the antigen-binding fragment thereof are the first proximal monomers of the HA trimer. And contact the amino acids in the second distal right monomer.

In yet another embodiment of the invention, the invention neutralizes influenza A virus infections of group 1 and group 2 subtypes and in the stem region of the HA trimer of influenza A. An isolated antibody or an antigen-binding fragment thereof that specifically binds to an existing epitope is contained, and the heavy and light chains of the antibody or the antigen-binding fragment thereof are the first proximal monomer of the HA trimer and the antigen-binding fragment thereof. Upon contact with the amino acids in the second distal right monomer, the antibody or antigen-binding fragment thereof is produced in the transgenic cells at least three times higher than when produced by FI6 variant 2. To.

In yet another embodiment of the invention, the invention neutralizes the infection of Group A and Group 2 subtypes of influenza A virus and contaminates the stem region of the HA trimer of influenza A. An isolated antibody or antigen-binding fragment thereof that specifically binds to an existing epitope is contained, and the heavy and light chains of the antibody or the antigen-binding fragment thereof are the first proximal monomer and the first proximal monomer of the HA trimer. Upon contact with the amino acids in the second distal right-side monomer, the antibody or antigen-binding fragment thereof is: (i) as set forth in SEQ ID NOs: 1, 41 and 43, respectively, or set forth in SEQ ID NOs: 1, 41 and 42, respectively. Heavy chain CDR1, CDR2 and CDR3 sequences; and (ii) Light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, or as shown in SEQ ID NOs: 44, 5 and 6, respectively. include.

In another embodiment of the invention, the invention comprises complementary determining region (CDR) sequences having at least 95% sequence identity with any one of SEQ ID NOs: 1-6, 17-22 or 41-44. Includes at least one isolated antibody or antigen-binding fragment thereof, the antibody neutralizing influenza A virus.

In another embodiment of the invention, the invention neutralizes the infection of Group A and Group 2 subtypes of influenza A virus and: (i) to SEQ ID NOs: 1, 41 and 43, respectively. Heavy chain CDR1, CDR2 and CDR3 sequences as shown or as shown in SEQ ID NOs: 1, 41 and 42, respectively; and (ii) as shown in SEQ ID NOs: 4, 5 and 6, respectively, or SEQ ID NOs: 44, 5 respectively. Includes isolated antibodies or antigen-binding fragments thereof, comprising the light chain CDR1, CDR2 and CDR3 sequences as shown in and 6.

In yet another embodiment of the invention, the invention is heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17; heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18 or SEQ ID NO: 41; And an isolated antibody comprising a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 42 or SEQ ID NO: 43 or an antigen-binding fragment thereof, the antibody neutralizing the influenza A virus. In yet another embodiment of the invention, the invention is a light chain CDR1 having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 20 or SEQ ID NO: 44; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 21; And an isolated antibody comprising a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 22 or an antigen-binding fragment thereof, the antibody neutralizes influenza A virus.

In yet another embodiment of the invention, the invention comprises an isolated antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising the amino acid sequence of SEQ ID NO: 14. Chain variable region; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33 and light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and the amino acid of SEQ ID NO: 30. Light chain variable region containing the sequence; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35 and light chain variable region containing the amino acid sequence of SEQ ID NO: 30; or heavy chain variable region and sequence containing the amino acid sequence of SEQ ID NO: 59. Light chain variable region containing the amino acid sequence of SEQ ID NO: 57; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59 and light chain variable region containing the amino acid sequence of SEQ ID NO: 61; or heavy chain containing the amino acid sequence of SEQ ID NO: 55. The antibody comprises a variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61, and the antibody was of type A. Neutralize group 1 and group 2 subtypes of influenza virus. The present invention further comprises an antibody or an antigen-binding fragment thereof, wherein the antibody is FI6 variant 1, FI6 variant 2, FI6 variant 3, FI6 variant 4 or FI6 variant 5.

In yet another embodiment of the invention, the invention comprises antibodies or antigen-binding fragments thereof that neutralize infections of influenza A virus type 1 and group 2 subtypes of the antibodies or theirs. Fragments are expressed by immortalized B cell clones.

In another aspect, the invention comprises a nucleic acid molecule comprising a polynucleotide encoding an antibody or antibody fragment of the invention. In yet another aspect, the invention comprises cells expressing a vector containing the nucleic acid molecule of the invention or an antibody of the invention or an antigen-binding fragment thereof. In yet another embodiment, the invention comprises cells comprising the vector of the invention. In yet another aspect, the invention comprises an immunogenic isolated or purified polypeptide comprising an epitope that binds to an antibody or antigen binding fragment of the invention.

The present invention further relates to an antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule of the present invention, a vector containing the nucleic acid molecule of the present invention, an antibody of the present invention or a cell expressing an antibody fragment thereof, a vector of the present invention. Includes a pharmaceutical composition comprising cells comprising, or the immunogenic polypeptide of the invention, and a pharmaceutically acceptable diluent or carrier. The present invention also includes a pharmaceutical composition comprising a first antibody or an antigen-binding fragment thereof, and a second antibody or an antigen-binding fragment thereof, wherein the first antibody is an antibody of the present invention and a second. An antibody is any antibody or antigen-binding fragment thereof capable of neutralizing an infection with influenza A or influenza B virus.

(I) Antibodies of the present invention or antigen-binding fragments thereof in the manufacture of agents for treating influenza A virus infection, (ii) vaccines, or (iii) infection diagnosis of influenza A virus, the present invention. An immunogenic isolated or purified polypeptide containing a nucleic acid, a vector containing the nucleic acid of the invention, a cell expressing the vector of the invention, an epitope that binds to an antibody or antibody fragment of the invention, or a pharmaceutical of the invention. The use of the composition is also intended to be within the scope of the present invention. In addition, the antibodies of the invention or them for monitoring the quality of anti-influenza A virus vaccines by ensuring that the vaccine antigen contains specific epitopes of appropriate conformation. The use of the antigen-binding fragment of is also intended to be within the scope of the present invention.

In another aspect, the invention provides a method of preventing, treating or reducing influenza A virus infection, or reducing the risk of influenza A virus infection, in a therapeutically effective amount of the invention. It comprises the step of administering an antibody or an antigen-binding antibody fragment to a subject in need of them.

In a further aspect, the invention comprises (i) in the treatment, (ii) in the manufacture of a therapeutic agent for influenza A virus infection, (iii) as a vaccine, or (iv) infecting influenza A virus. Includes an epitope that specifically binds to an antibody of the invention or an antigen-binding fragment thereof for use in the selection of a harmonious ligand.

The antibody-producing titers of 293F cells transiently transfected with a vector expressing a gene encoding the FI6 mutant 2, 3, 4 or 5 are shown. 2A and 2B each represent the surface of the F subdomain of H1 HA forming a complex with FI6 variant 3 (shown as FI6 in the figure), respectively. In HA1 and HA2, side chains that contribute to the preservation of hydrophobic grooves are selected and indicated by arrows. The approximate boundary of this groove is indicated by a black line. Thr (40) and Thr (318) are present in HA1 and Ile (45), Trp (21), Thr (41), Leu (38) and turns 18-21 are present in HA2. FI6 mutant 3 (indicated as FI6 in the figure) bound to the F subdomain of the HA trimer is shown. FIG. 3A shows the H3 HA trimer bound to the three FI6 mutant 3 antibodies by ribbon display. Of the HA monomers, HA1 was colored black and HA2 was colored dark gray, while the other two HA monomers were colored light gray. 3B and 3C show enlarged views of the interaction of the LCDR1 loop with the fusion peptide of the HA monomer flanking the H1 / FI6 mutant 3 and H3 / FI6 mutant 3 complexes, respectively. FIGS. 3D and 3E form a complex with CR6261 (pdb ID: 2GBM) (D) and F10 (pdb: 3FKU) (E), in a region similar to that of FI6 variant 3. However, the structure of the monomer derived from H5 HA, in which the VH domain is located 5 to 10 Å lower than HA and bound to the domain, is shown. The interaction of FI6 variant 3 (see FI6-v3 in the figure) with the H1 and H3 HAF subdomains is shown. A surface view of the F subdomains of H1 HA and H3 HA with selected side chains and HCDR3 and LCDR1 loops of FI6 variant 3 is shown. Proximal HA monomers are shown in light gray, distal right monomers are shown in light gray, and the binding of glycans to N38 in H3 HA1 is shown as dark gray spheres. Shows group-specific differences in cross-reactive antibody binding sites. 5A and 5B show the arrangement of Asn-38 in the sugar side chain in the H3 HA apo structure (A) and the FI6 mutant 3 binding structure (B). 5C and 5D show the orientation of HA1 Trp-21 in different antibody complexes. Panel C shows Ph-100D (see FI6 in the figure) of the HCDR3 loop of FI6 variant 3 interacting with the F subdomain of H1 or H3 HA. Panel D shows the HCDR2 loop of F10 and CR6261 antibodies. Phe-55 and Phe-54, respectively, are shown towards Trp-21 of H5 HA. The contact surface of FI6 mutant 3 with respect to HA is shown. The four panels show the footprint of FI6 mutant 3, CR6261 and F10 antibodies against HA (outlined by black lines). The three HA monomers are shown in white, light or dark gray. FIG. 6A is a contact footprint of uncut FI6 mutant 3 / H1. FIG. 6B is a contact footprint of the truncated FI6 mutant 3 / H3. FIG. 6C is a contact footprint of CR6261 / H5. And FIG. 6D is a contact footprint of F10 / H5. Unlike CR6261 and F10, FI6 variant 3 contacts two HA monomers. For the FI6 variant 3 complex, the glycosylation site at the antibody / HA interface is labeled. Residues in contact with HA in the VH and VK chains of FI6 variant 3 (in bold; numbered in Kabat form) are shown. FI6 variant 3 that provides prophylaxis in a mouse model for influenza A virus infection is shown. FIG. 8A shows BALB / c intravenously administered with different doses of FI6 variant 3 3 hours prior to intranasal infection with 10 MLD50 (50% lethal dose in mice) H1N1 A / PR / 8/34 virus. The survival curve of the mice is shown, and FIG. 8B shows the weight loss (5 animals per condition). FIG. 8C shows weight loss in mice intravenously administered with FI6 variant 3 at different doses 3 hours prior to infection with 3 × 10 ⁵ pfu H3N2 HK-x31 virus (10 per condition). The representative one of the three experiments is shown. Mice intravenously administered 15 mg / kg FI6 variant 3 on day 0 (3 hours before infection) or 1, 2 and 3 days after infection with 10 MLD50 A / PR / 8/34 virus (1). A survival curve (FIG. 8D) and weight loss (FIG. 8E) of 5 animals per condition are also shown. The representative one of the two experiments is shown. The displayed value is the average value ± SD. 8F and 8G show 10 mg / kg (F) or 3 mg / kg (G) FI6 variant 2 (FI6-v2), FI6-v2 KA (FI6-v2) one day prior to infection with the A / PR / 8/34 virus. The survival curve (10 animals per condition) of mice treated with complement fixation test), FI6-v2 LALA (complement and FcR bond loss) or control antibody is shown. The viral titers of the lungs of mice treated with FI6 mutant 3 after administration of a lethal dose of H1N1 PR / 8/34 are shown. In BALB / c mice (4 per condition), 15 mg / kg FI6 mutant 3 hours before (day 0) or 1, 2 or 3 days after intranasal infection with 10 MLD50 H1N1 PR / 8/34. 3 or a control antibody (HJ16, HIV-1-specific antibody) was administered intravenously. Virus titers in the lungs were measured 4 days after infection. The virus was undetected in the brain. Display the data in a box plot. The box extends over the 25th to 75th percentiles and has a median horizon. The whiskers shown above and below the box mean extrema. For Student's t-test analysis, ^* is described as p <0.05 and ^*** is described as <0.001. It is shown that the binding of FI6 mutant 3 to the HA stem region interferes with protease-mediated cleavage of HA0. Recombinant HA from the H1 NC / 99 isolate was incubated with FI6 mutant 3, FE17 (a human antibody that recognizes the Ca2 site of the HA spherical head) or a control antibody. The TPCK-treated trypsin was then exposed to the HA-antibody mixture at 37 ° C. for 5, 10 or 20 minutes. The sample was then run on a polyacrylamide gel, Western blotted and detected by biotinylated human mAb (FO32) that recognizes HA2 and HA0 of all influenza A strains under denaturing conditions. Shown is the HA0 band. The representative one of the three experiments is shown.

The present invention detects and isolates a natural human monoclonal antibody that broadly neutralizes different subtypes of influenza A virus from individuals vaccinated with the seasonal influenza A vaccine, and the antibody of the present invention binds to it. Partially based on new epitopes. Such antibodies are desirable because only one or several antibodies are needed to neutralize different subtypes of influenza A virus. Furthermore, a hetero-subtype antibody that neutralizes widely is produced at a high titer, and the production cost of a drug containing the antibody is reduced. In addition, the epitopes recognized by such antibodies can induce diverse defenses against both different seasonal subtype isolates and subtype isolates that can cause a pandemic. Can be part.

Accordingly, in one aspect, the invention provides isolated antibodies and antigen-binding fragments thereof that neutralize at least two influenza A viruses contained in group 1 and group 2 subtypes. In one embodiment, the invention provides isolated antibodies or antigen-binding fragments thereof that neutralize infections of influenza A virus type 1 and group 2 subtypes.

In another embodiment, the invention neutralizes infections of influenza A virus type 1 and group 2 subtypes and to epitopes present in the stem region of the HA trimer of influenza A. Provided are isolated antibodies or antigen-binding fragments thereof that specifically bind, and the heavy and light chains of the antibody or antigen-binding fragments thereof are the first proximal monomer and the second distant monomer of the HA trimer. Contact with amino acids in the monomer on the right side of the position.

As mentioned above, the HA protein is synthesized as a trimer precursor polypeptide HA0 (homotrimer) containing three identical monomers. Each monomer may or may not be cleaved independently of the other two monomers. When cleaved after translation, each monomer forms two polypeptides, HA1 and HA2, which are otherwise linked by a single disulfide bond. The heavy and light chains of the antibodies of the invention are in contact with two of the three monomers of the HA trimer. The monomers contacted by the antibodies of the invention may or may not be cleaved. For clarity of purpose and for the purpose of understanding the schematics in the figure, we refer to the two monomers contacted by the antibodies of the invention as the proximal monomer and the distal right monomer.

As used herein, the terms "antigen-binding fragment," "fragment," and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the invention that retains the antigen-binding activity of the antibody. To. Examples of antibody fragments include, but are not limited to, single chain antibodies, Fab, Fab', F (ab') 2, Fv or scFv. Further, as used herein, the term "antibody" includes both antibodies and antigen-binding fragments thereof.

As used herein, a "neutralizing antibody" is capable of neutralizing, i.e., preventing, inhibiting, reducing, or delaying the ability of a pathogen to initiate and / or maintain infection of a host. Or it can interfere. The terms "neutralizing antibody" and "neutralizing antibody (s)" are used interchangeably herein. These antibodies may be used alone or in combination as a prophylactic or therapeutic agent in association with an active vaccine, as a diagnostic tool, or as a manufacturing tool as described herein. Can be done.

X-ray crystallography of group 1 specific heterosubtype antibodies co-crystallized with H1 and H5 HA known in the art revealed that the antibody is only one monomer of the HA trimer. It is shown to interact with. In addition, tests show that the antibody contacts HA only with the CDR residues of the heavy chain, not the light chain. In contrast, the antibody or antigen binding fragment of the invention contacts two of the three HA monomers instead of one. In addition, the antibodies of the invention contact HA with CDR residues of both heavy and light chains. In addition, the nature of the interaction between the antibody or antigen binding fragment of the invention and HA is significantly different from that made by other antibodies, CR6261 and F10. The most striking difference is that the interaction of the antibodies of the invention with the hydrophobic groove on HA is mediated solely by heavy chain CDR3 (HCDR3), whereas in CR6261 and F10 all three HCDRs bind. It means being involved.

In one embodiment, the heavy chain of the antibody or antigen-binding fragment of the invention contacts an amino acid residue in a proximal monomer, and the light chain of the antibody or antigen-binding fragment of the invention is close to the HA trimer. Contact with amino acid residues of either the position monomer or the distal right monomer. The monomers contacted by the antibodies of the invention, i.e., the proximal and distal right-side monomers, may be uncleaved or may be cleaved to produce HA1 and HA2 polypeptides. In one embodiment, the proximal monomer and the distal right monomer are cleaved. In other embodiments, the proximal monomer and the distal right monomer are uncut.

The antibodies and antigen-binding fragments of the present invention specifically bind to epitopes conserved between 16 different HAs of 16 subtypes of influenza A virus. In one embodiment, the antibody or antigen binding fragment of the invention binds to an epitope comprising the 329th amino acid residue of HA1 and the 1st, 2nd, 3rd and 4th amino acid residues of HA2, and HA1 and HA2 is present in the uncut monomer of the HA trimer.

In other embodiments, the heavy chain of the antibody or antigen binding fragment of the invention comprises the 318th amino acid residue of HA1 of the proximal or distal right monomer, as well as the 18, 19, 20, 21, 38, of HA2. Contact with amino acid residues 41, 42, 45, 49, 53, and 57. The monomer may be uncut or may be cleaved.

In yet another embodiment, the light chain of the antibody or antigen binding fragment of the invention is the amino acid residues 38, 39, and 43 of HA2 of the uncut proximal monomer, as well as the uncut distal right side. It contacts the amino acid residues 327, 328 and 329 of HA1 of the monomer and the amino acid residues 1, 2, 3 and 4 of HA2.

In yet another embodiment, the antibodies and antigen binding fragments of the invention are the 318th amino acid residue of HA1 of the uncleaved proximal monomer, as well as the 18, 19, 20, 21, 38, 39 of HA2. , 41, 42, 43, 45, 48, 49, 53, 56 and 57 specifically binds to an epitope containing amino acid residues. In addition, the antibody is specific for an epitope containing the amino acid residues 327, 328, 329 of HA1 and the amino acid residues 1, 2, 3, and 4 of HA2 in the uncleaved distal right monomer. Combine to.

In another embodiment, the light chain of the antibody or antigen binding fragment of the invention is the amino acid residues 38, 39, 42, and 46 of HA2 of the proximal monomer, as well as the HA1 of the distal right monomer. Contact with amino acid residues 321 and 323, and amino acid residues 7 and 11 of HA2. In this embodiment, the proximal and distal right side monomers are cleaved.

In yet another embodiment, the antibodies and antigen-binding fragments of the invention are the 318th amino acid residue of HA1 of the truncated proximal monomer, and the 18, 19, 20, 21, 38, 39, of HA2. Amino acid residues 41, 42, 45, 46, 49, 52, 53, and 57, and amino acid residues 321 and 323 of HA1 of the truncated distal right monomer, and amino acids 7 and 11 of HA2. It specifically binds to an epitope containing an amino acid residue of.

As shown herein, the antibody or antigen binding fragment of the invention can specifically bind to all 16 subtypes of influenza A virus. In one embodiment, the antibodies of the invention are the HA subtypes of influenza A H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and. It specifically binds to H16.

In another embodiment of the invention, the invention provides an antibody having a high production titer. For example, when two very similar antibodies of the invention, FI6 variant 1 and FI6 variant 2, are isolated, multiple variants of these antibodies (specifically, variants of FI6 variant 2) are isolated. The body) was synthesized to improve the productivity of the mutated cells. In one embodiment, the antibody or antigen binding fragment of the invention is produced in the transfected cells at a titer at least 1.5 times higher than the titer at the time of production of FI6 mutant 2. In other embodiments, the antibodies of the invention are at least 1.8, 2, 2.2, 2.5, 2.7, 3, 3.2 compared to the titers at the time of production of FI6 variant 2. It is produced with a titer of 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.3, 5.6 or 6 times higher. .. In some embodiments, the antibodies or antigen-binding fragments of the invention have at least 3, at least 4, or at least 4.5-fold higher titers in the transfected cells compared to the titers at the time of production of FI6 variant 2. Produced by titer.

Thus, in one embodiment, the invention neutralizes influenza A virus infections of group 1 and group 2 subtypes and is an epitope present in the stem region of the HA trimer of influenza A. Provided are isolated antibodies or antigen-binding fragments thereof that specifically bind to, and the heavy and light chains of the antibody or antigen-binding fragments thereof are the first proximal monomer of the HA trimer and the second. Upon contact with the amino acids in the distal right monomer, the antibody or antigen-binding fragment thereof is produced in the transgenic cells, for example, at least three times higher than the titer at the time of production of FI6 variant 2. ..

As described herein, a transfected cell is a cell for expressing a nucleic acid sequence encoding an antibody of the invention, which is currently known to or will be discovered by one of ordinary skill in the art. Can be a cell of. Examples of such cells include, but are not limited to, CHO, HEK293T, PER. Mammalian host cells such as C6, NS0, myeloma or hybridoma cells can be mentioned. In addition, cells can be transfected either transiently or stably. Suitable transfection methods and cell types for use in transfection are within the scope of those of skill in the art.

In another embodiment, the antibody or antigen binding fragment of the invention specifically binds to a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 37, 38, 39, or 40.

Human monoclonal antibodies, immortalized B cell clones, or transfected host cells that secrete the antibodies of the invention, and nucleic acids encoding the antibodies of the invention are also included within the scope of the invention. As used herein, the term "broadly specific" binds and / or binds to one or more of the first group subtypes and one or more of the second group subtypes of influenza A virus. Used to refer to an antibody or antigen binding fragment of the invention that can be neutralized.

The antibody or antigen-binding fragment of the present invention is among the first group of influenza A virus subtypes (H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, and variants thereof). Neutralize one or more and one or more of the second group of influenza A virus subtypes (H3, H4, H7, H10, H14 and H15, and variants thereof). In one embodiment, representative subtypes of the first group include H1, H2, H5, H6, and H9, and representative subtypes of the second group include H3, and H7. ..

The antibodies and antibody fragments of the present invention can neutralize various combinations of influenza A virus subtypes. In one embodiment, the antibody is H1 and H3 subtypes of influenza A virus, or H2 and H3 subtypes, or H3 and H5 subtypes, or H3 and H9 subtypes, or H1 and H7 subtypes, or H2 and H7 subtypes, or H5 and H7 subtypes, or H7 and H9 subtypes can be neutralized.

In other embodiments, the antibodies and antibody fragments of the invention are H1, H2 and H3 subtypes of influenza A virus, or H1, H3 and H5 subtypes, or H1, H3 and H9 subtypes, or H2, H3. And H5 subtypes, or H2, H3 and H9 subtypes, or H3, H5 and H9 subtypes, or H1, H2 and H7 subtypes, or H1, H5 and H7 subtypes, or H1, H7 and H9 subtypes. Or H2, H5 and H7 subtypes, or H2, H7 and H9 subtypes, or H5, H7 and H9 subtypes, or H1, H3 and H7 subtypes, or H2, H3 and H7 subtypes, or H3, H5 and H7 subtypes, or H3, H7 and H9 subtypes can be neutralized.

In yet another embodiment, the antibody is H1, H2, H3 and H7 subtypes of influenza A virus, or H1, H3, H5 and H7 subtypes, or H1, H3, H7 and H9 subtypes, or H2. H3, H5 and H7 subtypes, or H2, H3, H7 and H9 subtypes, or H3, H5, H7 and H9 subtypes or H1, H2, H3 and H5 subtypes, or H1, H2, H3 and H9 subtypes. , Or H1, H3, H5 and H9 subtypes, or H2, H3, H5 and H9 subtypes, or H1, H2, H5 and H7 subtypes, or H1, H2, H7 and H9 subtypes, or H1, H5, H7 and H9 subtypes, or H2, H5, H7 and H9 subtypes can be neutralized.

In yet another embodiment, the antibodies of the invention are H1, H2, H3, H5 and H7 subtypes of influenza A virus, or H1, H2, H3, H7 and H9 subtypes, or H1, H3, H5,. H7 and H9 subtypes, or H2, H3, H5, H7 and H9 subtypes, or H1, H2, H3, H5 and H9 subtypes, or H1, H2, H5, H7 and H9 subtypes, or H1, H2, H3, H5, H7 and H9 subtypes can be neutralized. In yet another embodiment, the antibodies and antigen binding fragments of the invention neutralize one or more of the above combinations in addition to neutralizing the H6 subtype of influenza A virus.

The antibody and antigen-binding fragment of the present invention have high neutralizing titers. The concentration of the antibody of the invention required to neutralize 50% of influenza A virus can be, for example, about 50 μg / mL or less. In one embodiment, the concentration of the antibody of the invention required to neutralize 50% of influenza A virus is about 50, 45, 40, 35, 30, 25, 20, 17.5, 15, 12.5, 11, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or about 1 μg / mL or less. In another embodiment, the concentration of the antibody of the invention required to neutralize 50% of influenza A virus is about 0.9, 0.8, 0.75, 0.7, 0. .65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.075 , 0.05, 0.04, 0.03, 0.02, 0.01, 0.008, 0.006, 0.004, 0.003, 0.002 or about 0.001 μg / mL or less. .. This means that the antibody concentration required to neutralize 50% of influenza A virus is very low. Specificity and titer can be measured using standard neutralization assays known to those of skill in the art.

The present invention has a very broad specificity for HA and neutralizes one or more influenza A virus subtypes in group 1 and one or more influenza A virus subtypes in group 2. Provide an antibody. Antibodies of the invention bind to epitopes within the HA region that are conserved between two or more influenza A virus subtypes selected from Group 1 or Group 2.

In one embodiment, the invention is an antibody that specifically binds to a conserved epitope in the stem region of the HA of influenza A virus subtypes of groups 1 and 2 and interferes with virus replication or spread. (For example, isolated antibody or purified antibody) is provided. The invention also specifically binds to epitopes conserved in the stem region of subtypes HA of groups 1 and 2 and inhibits viral entry into cells (eg, isolated antibodies or or isolated antibodies or Purified antibody) is provided. Without being bound by theory, in a exemplary embodiment, the antibody or antigen binding fragment of the invention binds to an epitope conserved in the stem region of influenza A virus and interferes with the fusion process. Inhibits the invasion of the virus into the cells. In one embodiment, the antibodies or antigen-binding fragments of the invention limit the spread of the virus by supplementing complement and FcR-expressing killer cells and intervening antibody-dependent cellular cytotoxicity (ADCC). An epitope or antigenic determinant of a protein corresponds to a site specifically recognized by an antibody binding site (or paratope) in the molecule. Thus, epitopes are correlated sites that require complementary paratopes for recognition in use. When the epitope is conserved among different variants of the protein, it means that the same paratope can contact the same site of the molecule to specifically recognize these different variants.

The antibodies of the invention may be monoclonal (eg, human monoclonal antibodies) or recombinant antibodies. The present invention also provides a fragment of the antibody of the invention, in particular a fragment that retains the antigen-binding activity of the antibody. Including the scope of the patent claim, the present specification expressly in some parts an antigen-binding fragment (one or more), an antibody fragment (one or more), a variant (one or more) and / or an antibody. Although described as a derivative (s), the term "antibody" or "antibody of the invention" includes all that are classified as antibodies, i.e., an antigen-binding fragment (s). , Antibody fragments (s), variants (s) and antibody derivatives (s) are included.

In one embodiment, the antibodies and antibody fragments of the invention neutralize a combination of two or more of the influenza A virus subtypes of groups 1 and 2. Typical influenza A virus subtypes are, but are not limited to, H5N1 (A / Vietnam / 1203/04), H1N1 (A / New Caledonia / 20/99), and H1N1 (A / Salomon Island / 3). / 2006), H3N2 (A / Wioming / 3/03), and H9N2 (A / ticken / Hong Kong / G9 / 97). In other embodiments, the antibody neutralizes, or to, 3, 4, 5, 6, or a combination of 7 or more of the first and second groups of influenza A virus subtypes. It is unique.

In a representative embodiment, the invention is specific for influenza A virus subtypes H1 and H3 (eg, H1N1 and H3N2); or H1, H3, H5 and H9 (eg, H1N1, H3N2, H5N1, and H9N2). Influenza or their antibody fragments. In yet another embodiment, the antibody or antibody fragment thereof is specific for H1, H3, H5, H7 and H9 (eg, H1N1, H3N2, H5N1, H7N1, H7N7, H9N2). Other exemplary combinations of influenza A virus subtypes are described above in the present disclosure.

Table 1 shows the amino acid sequence numbers of the heavy chain variable region (VH) and the light chain variable region (VL) of the representative antibody of the present invention, and the sequence numbers of the nucleic acid sequences encoding them.

In one embodiment, the antibody or antibody fragment of the invention is approximately 70%, 75%, 80%, 85 with the sequence cited in any one of SEQ ID NOs: 13, 33, 55, 59, 29, or 35. Includes heavy chain variable regions with amino acid sequences that are%, 90%, 95%, 97%, 98%, 99%, or 100% identical. In other embodiments, the antibodies or antibody fragments of the invention are about 70%, 75%, 80%, 85%, 90%, 95%, with the sequences cited in SEQ ID NO: 14, 57, 61, or 30. Includes a light chain variable region with an amino acid sequence that is 97%, 98%, 99%, or 100% identical.

In yet another embodiment, the heavy chain variable region of the antibody of the invention is about 70%, 75%, with the sequence cited in any one of SEQ ID NOs: 15, 34, 56, 60, 31, or 36. It can be encoded by nucleic acids having sequences that are 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical. In yet another embodiment, the light chain variable region of the antibody of the invention is approximately 70%, 75%, 80%, 85%, 90%, with the sequence cited in SEQ ID NO: 16, 58, 62, or 32. It can be encoded by nucleic acids having sequences that are 95%, 97%, 98%, 99%, or 100% identical.

The CDRs of the antibody heavy chain are referred to as CDRH1 (or HCDR1), CDRH2 (or HCDR2) and CDRH3 (or HCDR3), respectively. Similarly, the CDRs of the antibody light chain are referred to as CDRL1 (or LCDR1), CDRL2 (or LCDR1) and CDRL3 (or LCDR1), respectively. The positions of the CDR amino acids are determined according to the IMGT numbering method: the positions by CDR1-IMGT are 27-38, the positions by CDR2-IMGT are 56-65, and the positions by CDR3-IMGT are 105-117. be.

Table 2 provides the SEQ ID NOs of the six CDR amino acid sequences of the heavy and light chains of the representative antibodies of the invention, respectively.

In one embodiment, the antibody or antibody fragment of the invention comprises at least one CDR having a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 1-6, 41-44, or 17-22. ..

In another embodiment, the invention is one or more derived from FI6 variant 1, FI6 variant 2, FI6 variant 3, FI6 variant 4, FI6 variant 5, FI28 variant 1, or FI28 variant 2. (Ie, one, two or all three) heavy chain comprising an antibody comprising a heavy chain CDR. In a representative embodiment, the antibody or antigen-binding fragment of the present invention is a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a heavy chain having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 41 or SEQ ID NO: 18. Includes chain CDR2; and heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 19. In certain embodiments, the antibodies or antibody fragments provided herein are heavy chains comprising (i) SEQ ID NO: 1 in CDRH1, SEQ ID NO: 2 in CDRH2 and SEQ ID NO: 3 in CDRH3, and (ii) CDRH1. SEQ ID NO: 1, CDRH2 contains SEQ ID NO: 41 and CDRH3 contains SEQ ID NO: 42, (iii) CDRH1 contains SEQ ID NO: 1, CDRH2 contains SEQ ID NO: 41 and CDRH3 contains SEQ ID NO: 43, or (iv) CDRH1. Includes SEQ ID NO: 17, CDRH2 with SEQ ID NO: 18, and CDRH3 with SEQ ID NO: 19.

One or more (ie, one, two, or) derived from FI6 variant 1, FI6 variant 2, FI6 variant 3, FI6 variant 4, FI6 variant 5, FI28 variant 1 or FI28 variant 2 Antibodies containing light chains, including all three) light chain CDRs, are also provided. In a representative embodiment, the antibody or antigen binding fragment of the invention is a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 44 or SEQ ID NO: 20; light having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 21. Includes chain CDR2; and light chain CDR3 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 22. In certain embodiments, the antibodies provided herein are (i) a light chain comprising SEQ ID NO: 4 in CDRL1, SEQ ID NO: 5 in CDRL2 and SEQ ID NO: 6 in CDRL3, and (ii) SEQ ID NO: 44 in CDRL1. , CDRL2 comprises a light chain comprising SEQ ID NO: 5 and CDRL3 comprising SEQ ID NO: 6, or (iii) CDRL1 comprising SEQ ID NO: 20, CDRL2 comprising SEQ ID NO: 21, and CDRL3 comprising a light chain comprising SEQ ID NO: 22.

In one embodiment, the antibodies of the invention or antigen-binding fragments thereof include all CDRs of FI6 variant 1 of the antibody and neutralize influenza A virus infection, as listed in Table 2. In other embodiments, the antibodies of the invention or antigen-binding fragments thereof contain all CDRs of FI6 variant 2 of the antibody and neutralize influenza A virus infection, as listed in Table 2. .. In other embodiments, the antibodies of the invention or antigen-binding fragments thereof contain all CDRs of FI6 variant 3 of the antibody and neutralize influenza A virus infection, as listed in Table 2. .. In other embodiments, the antibodies of the invention or antigen-binding fragments thereof contain all CDRs of FI6 variant 4 of the antibody and neutralize influenza A virus infection, as listed in Table 2. .. In other embodiments, the antibodies of the invention or antigen-binding fragments thereof contain all CDRs of FI6 variant 5 of the antibody and neutralize influenza A virus infection, as listed in Table 2. ..

In yet another embodiment, the antibodies of the invention or antigen-binding fragments thereof contain all CDRs of FI28 variant 1 of the antibody and neutralize influenza A virus infection, as listed in Table 2. do. In yet another embodiment, the antibodies of the invention or antigen-binding fragments thereof contain all CDRs of FI28 variant 2 of the antibody and neutralize influenza A virus infection, as listed in Table 2. do.

Examples of the antibody of the present invention are, but are not limited to, FI6 mutant 1, FI6 mutant 2, FI6 mutant 3, FI6 mutant 4, FI6 mutant 5, FI28 mutant 1, and FI28 mutant. 2 is mentioned.

The invention further comprises an antibody or fragment thereof that binds to the same epitope as that of the antibody of the invention, or an antibody that competes with the antibody or antigen binding fragment of the invention.

Antibodies of the invention also include hybrid antibody molecules comprising one or more CDRs derived from the antibodies of the invention and one or more CDRs derived from other antibodies to the same epitope. In one embodiment, such a hybrid antibody comprises three CDRs derived from the antibody of the invention and three CDRs derived from another antibody against the same epitope. Typical hybrid antibodies are i) three light chain CDRs derived from the antibody of the present invention and three heavy chain CDRs derived from another antibody against the same epitope, or ii) three heavy chain CDRs derived from the antibody of the present invention. And three light chain CDRs from different antibodies against the same epitope.

Mutant antibodies are also included within the scope of the invention. Therefore, variants of the sequences described in this application are also included within the scope of the invention. Such variants include natural variants produced by somatic mutations in vivo during immune response and in vitro during culture of immortalized B cell clones. Alternatively, as described above, the mutant may be caused by the degeneracy of the genetic code, or may be generated by an error during transcription or translation.

Other variants with improved affinity and / or titer antibody sequences can be obtained by methods known in the art and such variants are also included within the scope of the invention. For example, amino acid substitutions can be used to obtain antibodies with even higher affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve translation efficiency in expression systems for producing antibodies. Further, a polynucleotide containing a sequence optimized for antibody specificity or neutralizing activity by performing a directed evolution method on any nucleic acid sequence of the present invention is also within the scope of the present invention.

In one embodiment, the mutant antibody sequence is 70% or more (ie, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99) with the sequence described in this application. % Or more) share the amino acid sequence identity. In some embodiments, such sequence identity is calculated for the full length of the reference sequence (ie, the sequence described in this application). In some further embodiments, the identity (%) referred to herein is determined by BLAST (ver. 2.1.3) using the default parameters specified by NCBI. (The National Center for Biotechnology Information; http: //www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty = 11 and gap extension penalty = 1].

In other aspects, the invention also includes the light and heavy chains of the antibodies of the invention and the nucleic acid sequences encoding some or all of the CDRs. Provided herein are the light and heavy chains of the representative antibodies of the invention and the nucleic acid sequences encoding some or all of the CDRs. Table 1 provides the SEQ ID NOs of the nucleic acid sequences encoding the heavy and light chain variable regions of the representative antibodies of the invention. For example, the nucleic acid sequences provided herein include SEQ ID NO: 15 (encoding the FI6 mutant single heavy chain variable region), SEQ ID NO: 16 (FI6 mutant 1 and FI6 mutant 2 light chain variable region). (Decoding), SEQ ID NO: 34 (encoding the FI6 mutant double chain variable region), SEQ ID NO: 56 (encoding the FI6 mutant triple chain variable region), SEQ ID NO: 58 (FI6). Mutant 3 and FI6 variant 4 encoding the light chain variable region), SEQ ID NO: 60 (encoding the FI6 variant 4 and FI6 variant quintuple chain variable region), SEQ ID NO: 62 (FI6 variant). 5 encoding the light chain variable region), SEQ ID NO: 31 (encoding the FI28 mutant double chain variable region), SEQ ID NO: 36 (encoding the FI28 mutant double chain variable region) and SEQ ID NO: 32 (encoding the FI28 mutant 1 and mutant 2 light chain variable regions).

Table 3 provides the SEQ ID NOs of the nucleic acid sequences encoding the representative antibody CDRs of the invention. Due to duplication of the genetic code, there may be variants encoding the same amino acid sequences as these sequences.

In one embodiment, the nucleic acid sequence according to the invention comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of the nucleic acid encoding the heavy or light chain of the antibody of the invention. Nucleic acid sequences having at least 95%, at least 98%, or at least 99% identity can be mentioned. In another embodiment, the nucleic acid sequence of the invention has a nucleic acid sequence encoding the heavy or light chain CDR of the antibody of the invention. For example, the nucleic acid sequence according to the invention is at least 75% with the nucleic acid sequence of SEQ ID NOs: 7-12, 15, 16, 34, 23-28, 31, 32, 36, 45-54, 56, 58, 60, or 62. Contains sequences that are identical.

Further, a vector containing a nucleic acid sequence according to the present invention (for example, an expression vector) is included within the scope of the present invention. Cells transfected with such vectors are also included within the scope of the invention. Examples of such cells include, but are not limited to, eukaryotic cells such as yeast cells, animal cells or plant cells. In one embodiment, the cells are of mammalian origin, eg, human, CHO, HEK293T, PER. C6, NS0, myeloma or hybridoma cells.

The present invention also relates to monoclonal antibodies that bind to epitopes capable of binding the antibodies of the present invention, including, but not limited to, FI6 mutant 1, FI6 mutant 2, FI6 mutant 3, and FI6 mutant. 4. Monoclonal antibodies selected from the group consisting of FI6 mutant 5, FI28 mutant 1 and FI28 mutant 2 can be mentioned.

Monoclonal and recombinant antibodies are particularly useful for the identification and purification of individual polypeptides or other antigens that will be detected. The antibody of the present invention has further usefulness in that it can be used as a reagent for immunoassay, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). In these applications, the antibody can be labeled with reagents that can be detected during analysis, such as radioisotopes, fluorescent molecules or enzymes. Antibodies can also be used for identification and characterization of antigen molecules (epitope mapping).

Antibodies of the invention can be linked to a drug for delivery to a therapeutic site, or to facilitate imaging of sites containing cells of interest, such as cells infected with influenza A virus. , Can be linked with a detectable label. Methods of coupling an antibody to a drug and a detectable label, such as techniques for imaging using a detectable label, are well known in the art. Labeled antibodies can be used in various assays that use different labels. Detection of the antigen-antibody complex formed by the antibody of the present invention and the epitope of interest (influenza A virus epitope) can be facilitated by attaching a detectable substance to the antibody. Suitable detection methods include radioactive nuclei, enzymes, coenzymes, fluorescent substances, chemical luminescent substances, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, and dyes. The use of markers is included. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholineresterase; examples of suitable complementary molecular complex are streptavidin / biotin and avidin / biotin; suitable fluorescence. Examples of substances include umveriferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylloride or phylicoleitrin; examples of luminescent substances include luminol; bioluminescent substances. Examples include luciferase, luciferin, and equolin; and examples of suitable radioactive substances include 125I, 131I, 35S, or 3H. Agents labeled in this way can be used in a variety of well-known assays such as radioimmunoassays, enzyme immunoassays such as ELISA and fluorescence immunoassays. (See, for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402;).

Antibodies according to the invention can be combined with therapeutic components such as cytotoxicities, therapeutic agents, or radioactive metal ions or radioisotopes. Examples of radioisotopes are, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212. , Bi-213, Pd-109, Tc-99, In-111 and the like. Such antibody complexes can be used to modify a given biological response and the drug components are not construed as being limited to classical chemotherapeutic agents. For example, the drug component may be a protein or polypeptide that processes the desired biological activity. Examples of such proteins include toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for combining such therapeutic components with antibodies are well known. For example, Arnon et al. (1985) Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy (Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss,). Inc.), pp. 243 to 256); Hellstrom et al. Ed. (1987) "Antibodies for Drug Delivery" (Control Drug Delivery, Robinson et al. (2nd edition; Marcel Decker, Inc.), pp. 623-653); Thorpe (1985). ) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy" ("Monoclonal Antibodies '84: Biological and Clinical Applications) Ed., Pinchera et al., Pp. 475-506 (Editure Curtis, Milan, Italy, 1985); "Analysis, Results, and Future of Radiolabeled Antibodies for Treatment in Cancer Treatment". Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy ”(Monocular Antibodies for Cancer Detection and Therapy, Baldwin et al. (Academic Press, New York, 1985), pp. 303-316). 1982) See Immunol. Rev. 62: 119-158.

Alternatively, as described in US Pat. No. 4,676,980, an antibody or antibody fragment thereof can be combined with a secondary antibody or antibody fragment thereof to form an antibody heterocomplex. In addition, a linker can be used between the label and the antibody of the invention (eg, US Pat. No. 4,831,175). Antibodies or antigen-binding fragments thereof may be labeled directly with radioactive iodine, indium, yttrium, or other radioactive particles known in the art (eg, US Pat. No. 5,595,721). Treatment may consist of a combination of treatments by simultaneous or continuous administration of combined or non-combined antibodies (eg, WO 00/52031; 00/52473).

The antibodies of the invention may also be attached to a solid support. In addition, the antibodies of the invention or functional antibody fragments thereof can be covalently bound to form a complex with the polymer for chemical modification, eg, to prolong the circulating half-life. Examples of polymers and methods for binding peptides are described in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. ing. In some embodiments, the polymer can be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is water soluble at room temperature and has the general formula: R (O-CH2-CH2) nOR, where R may be hydrogen or a protecting group such as an alkyl or alkanol group. In one embodiment, the protecting group may have 1-8 carbon atoms. In a further embodiment, the protecting group is methyl. The symbol n is a positive integer. In one embodiment, n is 1 to 1,000. In other embodiments, n is 2 to 500. In one embodiment, PEG has an average molecular weight of 1,000-40,000. In a further embodiment, PEG has a molecular weight of 2,000-20,000. In a further additional embodiment, the PEG has a molecular weight of 3,000 to 12,000. In one embodiment, the PEG has at least one hydroxy group. In other embodiments, the PEG has a terminal hydroxy group. In yet another embodiment, it is the terminal hydroxy group that is activated to react with the free amino group on the inhibitor. However, it will be appreciated that the type and amount of reactive groups may vary in the complexing of PEG with the antibodies of the invention by covalent bonding.

Water-soluble polyoxyethylated polyols are also useful in the present invention. Useful water-soluble polyoxyethylated polyols include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG) and the like. In one embodiment, POG is used. Without being bound by any theory, the glycerol backbone of polyoxyethylated glycerol is, for example, the same backbone naturally produced as monoglycerides, diglycerides, triglycerides in animals and humans. Therefore, it is not necessary to be recognized as a foreign molecule in the living body by this branch. In some embodiments, POG has a molecular weight in the same range as PEG. Another drug delivery system that can be used to prolong the circulating half-life is liposomes. A method for preparing a liposome-based delivery system is described in Gabizon et al. (1982), Caffiso (1981) and Szoka (1980). Other drug delivery systems are also known in the art and are described, for example, in Poznansky et al. (1980) and Poznansky (1984) are mentioned.

The antibody of the present invention can also be provided in a purified state. Typically, the antibody is present in a composition that is almost free of other polypeptides, eg, in such cases, less than 90% (weight) of the composition, generally less than 60%. And more generally less than 50% is composed of other polypeptides.

Antibodies of the invention may be immunogenic in a non-human (or heterologous) host, such as a mouse. Specifically, an antibody may have an idiotope that is immunogenic in a non-human host but not in a human host. Antibodies of the invention for use in humans cannot be easily isolated from hosts such as mice, goats, rabbits, rats, mammals other than primates, and are generally by humanized or xeno mice. It includes things that cannot be obtained.

The antibodies of the invention are generally IgG, although they may be of any isotype (eg, IgA, IgG, IgM, ie α, γ or μ heavy chains). Antibodies included in the IgG isotype include those of the IgG1, IgG2, IgG3, or IgG4 subclass. The antibodies of the invention may have a κ or λ light chain.

Antibody production The antibody according to the present invention can be produced by any method known in the art. For example, the general technique of producing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C, .1975; Kozbar et al. 1983). In one embodiment, the EBV immortalization method disclosed in WO 2004/076677 is used instead.

The method described in WO 2004/076677 can be used to transform B cells producing the antibodies of the invention with EBV in the presence of polyclonal B cell activating factor. Transformation with EBV is a common technique and can be easily configured to contain polyclonal B cell activating factor.

If desired, other stimulants involved in cell growth and differentiation can be added during the transformation step to further increase efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve immortalization efficiency, but the use of IL-2 is not essential. Immortalized B cells produced using these techniques can then be cultured by techniques known in the art and antibodies can be isolated from the culture.

A single plasma cell can be cultured in a microwell culture plate using the technique described in UK Patent Application No. 0819376.5. Antibodies can be isolated from a single plasma cell culture. In addition, RNA can be extracted from a single plasma cell culture and single cell PCR can be performed using techniques known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR, sequenced, cloned into an expression vector, and then transfected into HEK293T cells or other host cells. Cloning of nucleic acids in expression vectors, transfection of host cells, culturing of transfected host cells, and isolation of produced antibodies can be performed using any technique known to those of skill in the art.

If desired, the antibody may be further purified using various chromatographic methods such as filtration, centrifugal sedimentation and HPLC or affinity chromatography. Methods for purifying antibodies (eg, monoclonal antibodies), including techniques for producing pharmaceutical grade antibodies, are well known in the art.

The antibody fragments of the invention can be obtained from the antibody by techniques involving digestion with enzymes such as pepsin or papain and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, the antibody fragment can be obtained by cloning and expressing a portion of the heavy or light chain sequence. Antibody "fragments" include Fab, Fab', F (ab') 2 and Fv fragments. The present invention also includes single chain Fv fragments (scFv) derived from the heavy and light chains of the antibody of the present invention, for example, the present invention includes scFv containing a CDR derived from the antibody of the present invention. .. Similarly, heavy or light chain monomers and dimers, heavy chain single domain antibodies, light chain single domain antibodies, and single chain antibodies such as heavy and light chain variable regions are linked by peptide linkers. The single chain Fv that has been used is also included.

The antibody fragment of the present invention can be imparted with a monovalent or multivalent interaction and can contain various structures as described above. For example, a scFv molecule can be synthesized to produce a trivalent "trimer antibody" or a tetravalent "tetramer antibody". The scFv molecule can contain an Fc region domain obtained from a divalent mini-antibody. In addition, the sequences of the invention may be multispecific molecular components, the sequences of the invention target the epitopes of the invention and other regions associated with other targets to which the molecule binds. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and bispecific antibodies (Holliger and Hoodson, 2005, Nature Biotechnology 9). 1126 to 1136).

Common techniques of molecular biology can also be used to prepare DNA sequences encoding the antibodies or antibody fragments of the invention. Oligonucleotide synthesis methods can also be used to synthesize the desired DNA sequence in full or in part. If desired, site-specific mutagenesis and polymerase chain reaction (PCR) methods may be used.

Any suitable host cell / vector system can be used to express the DNA sequences encoding the antibody molecules of the invention or fragments thereof. Some bacteria (eg, E. coli (eg, E. coli)) are used to express antibody fragments such as Fab and F (ab') 2 fragments, and in particular Fv fragments, as well as single chain antibody fragments such as, for example, single chain Fvs. coli)) and other microbial systems can be used. Expression systems by eukaryotic (eg, mammalian) host cells can also be used to produce larger antibody molecules, such as complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER. Examples include C6, NS0, myeloma or hybridoma cells.

The present invention also provides a method for producing an antibody molecule according to the present invention, wherein the production method encodes a nucleic acid of the present invention under conditions suitable for expressing a protein from the DNA encoding the antibody molecule of the present invention. It comprises a step of culturing a host cell containing the vector, and a step of isolating the antibody molecule.

If only heavy or light chain polypeptide coding sequences need to be used for transfection into host cells, the antibody molecule may contain only heavy or light chain polypeptides. When producing products containing both heavy and light chains, cell lines can be transfected with two types of vectors. The first vector encodes a light chain polypeptide and the second vector encodes a heavy chain polypeptide. Alternatively, a single vector may be used, which contains the sequences encoding the light and heavy chain polypeptides.

Alternatively, the antibody according to the present invention can be produced by i) the step of expressing the nucleic acid sequence according to the present invention in a host cell, and ii) the step of isolating the expressed antibody product. In addition, the method may include iii) the step of purifying the antibody.

Screening of Transformed B Cells, Cultured Single Plasma Cell and Transfected HEK293T Cell Transformed B Cell and Cultured Single Plasma Cell Screen for Production of Antibodies with Desired Specificity or Function can do.

The screening step is the technique for identifying the specificity or function desired, by any immunoassay (eg, ELISA), by tissue or cell staining (including transfected cells), or by neutralization assay. It can be carried out by any of a number of other techniques known in the art. Assays can be selected based on the simple recognition of one or more antigens, or even the desired function, eg, the function desired to select a neutralizing antibody rather than simply an antibody that binds to the antigen. , Desirable functions for selecting antibodies that can alter the properties of the target cell, eg, signal cascade, cell shape, growth rate, ability to affect other cells, by other cells, or by other reagents. Alternatively, it can be selected based on the response to the influence of changes in conditions, differentiation state, and the like.

The transformed positive B cell culture can then be cloned into individual transformed B cells. Cloning steps to isolate individual clones from a positive cell mixture should be performed using limiting dilution, micromanipulation, single cell deposition by cell selection, or any other method known in the art. Can be done.

Nucleic acid can be isolated from a single cultured plasma cell, cloned and expressed in HEK293T cells, or expressed in other host cells by techniques known in the art.

The immortalized B cell clones or transfected HEK293T cells of the present invention are nucleic acids (DNA) encoding the monoclonal antibody of interest in various techniques for research, for example, as a source of the monoclonal antibody. Or it can be used as a source of mRNA).

The invention includes immortalized B memory cells or transfected host cells that produce antibodies that neutralize at least two different subtypes of influenza A virus selected from group 1 and group 2 subtypes. The composition is provided.

Epitopes As described above, the antibodies of the invention can be used to map their binding epitopes. We have discovered that the antibody that neutralizes influenza A virus infection is against an epitope found on HA. In one embodiment, the antibody is against one or more epitopes in the stem region of HA that are conserved between one or more of the first and second subtypes of influenza A virus. be. The epitope to which the antibody of the present invention binds may be a linear (continuous) type or a three-dimensional structure (discontinuous) type. In one embodiment, the antibodies and antibody fragments of the invention bind to a polypeptide region comprising SEQ ID NO: 37, 38, 39 or 40 as disclosed herein.

In another embodiment, the epitope to which the antibody of the invention binds comprises amino acid residues in HA1 and HA2 polypeptides consisting of one or two HA monomers as described above. The HA monomer may be uncut or may be cleaved.

The epitopes recognized by the antibodies of the invention can have numerous uses. Purified or synthesized forms of epitopes and their mimotopes can be used to generate an immune response (ie, an immune response to produce antibodies related to vaccines or other uses), or epitopes or Serums can be screened for antibodies that immunoreact with those mimotopes. In one embodiment, such epitopes or mimotopes, or antigens containing such epitopes or mimotopes, can also be used as vaccines to elicit an immune response. The antibodies and antibody fragments of the present invention can also be used in methods for monitoring the quality of vaccines. In particular, antibodies can be used to confirm that the antigen in the vaccine contains the appropriate immunogenic epitope with the appropriate conformation.

Epitopes may also be useful in screening for ligands that bind to such epitopes. Such ligands include, but are not limited to, antibodies (including those derived from camels, sharks, and other species), antibody fragments, peptides, phage display products, aptamers, or adnectins. Or other fragments of viral or cellular proteins that can block epitopes and thus prevent infection. Such ligands are included within the scope of the invention.

Recombinant expression The immortalized B cell clone or cultured plasma cell of the present invention can also be used as a nucleic acid resource for cloning an antibody gene for recombinant expression thereafter. In pharmaceutical applications, for example, for reasons of stability, reproducibility, ease of culture, etc., it is more common to express from recombinant resources than to express from B cells or hybridomas.

Accordingly, the present invention is: (i) obtaining one or more nucleic acids (eg, heavy and / or light chain mRNA) from a B cell clone or a single vector culture encoding the antibody of interest. And (ii) a step of inserting this nucleic acid into an expression vector and (iii) a step of transfecting the vector into the host cell in order to express the antibody of interest in the host cell. Provide a manufacturing method.

Similarly, the invention comprises (i) sequencing the nucleic acid (s) encoding the antibody of interest from a B cell clone or a single plasma cell culture, and (ii). ) Recombination comprising the step of preparing the nucleic acid (s) to be inserted into the host cell using the sequence information obtained from step (i) in order to express the antibody of interest in the host cell. Provided is a method for producing cells. Although not required, the codons used can be changed by performing an operation to introduce a restriction site into the nucleic acid between steps (i) and (ii), and / or transcription and / or translation control sequences. Can also be optimized.

The present invention also provides a method for producing a transfected host cell, comprising the step of transfecting one or more nucleic acids encoding the antibody of interest into the host cell, wherein the nucleic acid is immortalized by the present invention. A nucleic acid derived from a B cell clone or a single plasma cell culture. Therefore, the procedure for first preparing the nucleic acid (s) and then using this nucleic acid to transfect host cells is performed by different people at different locations (eg, in different countries). It can be performed different times.

These recombinant cells of the invention can then be used for expression and culture. These are particularly useful for expressing antibodies during large-scale pharmaceutical manufacturing. These can also be used as active ingredients in pharmaceutical compositions. Any suitable culture method, such as, but not limited to, static culture, roller bottle culture, ascites, hollow fiber bioreactor cartridge, small modular fermenter, stirring tank, microcarrier culture, ceramic core perfusion, etc. Can be used.

Methods for obtaining and sequencing B cell or plasma cell-derived immunoglobulin genes are well known in the art (eg, Kuby Immunology, 4th Edition, Chapter 4, 2000). Please refer to).

Host cells to be transfected are yeast and animal cells, especially mammalian cells (eg, CHO cells, NS0 cells, PER. C6 (Jones et al 2003) or HKB-11 (Cho et al. 2001; Cho et al. 2003). ) Human cells such as cells, myeloma cells (US Pat. No. 5,807,715; No. 6,300,104, etc.)), and eukaryotic cells such as plant cells. Preferred expression hosts are capable of glycosylating the antibodies of the invention, especially by a sugar chain structure that is not immunogenic in itself in humans. In one embodiment, the transfected host cells can be grown in serum-free medium. In a further embodiment, the transfected host cells can also be grown by culture in the absence of animal-derived components. Cell lines can also be obtained by culturing the transfected host cells.

The present invention encodes (i) a step of preparing an immortalized B cell clone according to the present invention, or a step of culturing plasma cells, and (ii) an antibody of interest from a B cell clone or a single plasma cell culture. Provided are a method for producing one or more nucleic acid molecules (eg, heavy chain and light chain genes) encoding an antibody of interest, comprising the step of obtaining the nucleic acid of interest. The present invention comprises (i) a step of preparing an immortalized B cell clone according to the present invention, or a step of culturing a single plasma cell, and (ii) a B cell clone or plasma cell encoding the antibody of interest. Also provided is a method of obtaining a nucleic acid sequence encoding a antibody of interest, comprising the steps of sequencing the nucleic acid from the culture.

The present invention also comprises a method for producing a nucleic acid molecule (s) encoding a antibody of interest, comprising the step of obtaining a nucleic acid obtained from a transformed B cell clone or plasma cell of the present invention. offer. Therefore, the procedure for first obtaining a B cell clone or plasma cell culture and then obtaining nucleic acid (s) from a B cell clone or plasma cell culture may be in different locations (eg, different countries). ) Can be performed differently by different people.

The present invention is: (i) obtaining one or more nucleic acids (eg, heavy and light chain genes) from a selected B cell clone or plasma cell culture expressing the antibody of interest and / or. A step of sequencing and (ii) a step of inserting a nucleic acid (s) or using a nucleic acid (s) sequence (s) to prepare an expression vector (s). iii) A step of transfecting a host cell capable of expressing the target antibody, and (iv) a step of culturing or subculturing the transfected host cell under conditions in which the target antibody is expressed, as desired. (V) Provided is a method for producing an antibody (for example, for pharmaceutical use), which comprises a step of purifying the target antibody.

The present invention comprises a step of culturing or subculturing the transfected host cell population under conditions in which the antibody of interest is expressed, and optionally purifying the antibody of interest. Methods for producing antibodies are also provided and such transfected host cell populations encode (i) selected antibodies of interest produced by B cell clones or plasma cell cultures prepared as described above. A step of preparing the nucleic acid (s) to be prepared, (ii) a step of inserting the nucleic acid (s) into an expression vector, and (iii) a vector in a host cell capable of expressing the target antibody. It is produced by a step of transfecting and (iv) a step of culturing or subculturing the transfected host cell containing the inserted nucleic acid to produce a target antibody. Therefore, the procedure for first preparing the recombinant host cell and then culturing the cell to express the antibody should be performed in different places (eg, in different countries) different times by different people. Can be done.

Pharmaceutical Compositions The present invention comprises pharmaceutical compositions containing the antibodies and / or antibody fragments of the invention and / or the nucleic acids encoding such antibodies and / or the epitopes recognized by the antibodies of the invention. offer. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier to allow administration. The carrier itself should not induce antibody production to the extent that it is detrimental to each individual receiving the composition, and should be non-toxic. Suitable carriers are large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, high molecular weight amino acids, amino acid copolymers and inert viral particles. It's okay.

For example, pharmaceutically acceptable mineral salts (such as hydrochlorides, hydrobromates, phosphates and sulfates) or organic acid salts (such as acetates, propionates, malonates and benzoates). Salt can be used.

The pharmaceutically acceptable carrier in the therapeutic composition may further contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary components such as wetting agents or emulsifiers or pH buffering substances may be present in such compositions. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions upon ingestion by the subject.

Examples of the administration form included within the scope of the present invention include a form suitable for parenteral administration, and examples thereof include injection or infusion (for example, bolus administration or continuous infusion). If the product is for injection or infusion, the product may be in the form of suspensions, solutions or emulsions with oily or aqueous excipients and pharmaceutical additives such as suspending agents, preservatives, stables. It may contain an agent and / or a dispersant. Alternatively, the antibody molecule may be in a dry form in which it is dissolved in a suitable sterile solution prior to use.

Once prescribed, the compositions of the invention can be administered directly to the subject. In one embodiment, the composition is tailored to the administration of the target human.

The pharmaceutical composition of the present invention is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous. It can be administered by any route such as topical, subcutaneous, nasal, enteral, sublingual, intravaginal or rectal route. A subcutaneous injector can also be used for administration of the pharmaceutical composition of the present invention. Typically, the therapeutic composition can be prepared as an injection in either a solution or suspension state. It can also be prepared in solid form suitable for dissolution or suspension in liquid excipients prior to injection.

Direct delivery of the composition is generally performed by subcutaneous, intraperitoneal, intravenous or intramuscular injection, or delivered to the interstitial space of the tissue. The composition can also be administered intralesional. The dosing procedure may be a single dose schedule or a multiple dose schedule. For known antibody drugs, guidance on dosing frequency is provided, for example, whether the drug should be delivered daily, weekly, monthly, or the like. Frequency and dosage also depend on the severity of the symptoms.

The composition of the present invention can be prepared in various forms. For example, the composition can be prepared as an injection in either the form of a solution or a suspension. Solids in a form suitable for dissolution or suspension in liquid excipients may be prepared prior to injection (eg, Synagis ™ and Synagis ™ for preparation by dissolving in sterile water containing preservatives. Freeze-dried composition such as Herceptin ™). The composition can be prepared for topical administration, eg, as an ointment, cream or powder. The composition can be prepared for oral administration, eg, as a tablet or capsule, as a spray, or as a syrup (which is optionally flavored). The composition can be prepared as an inhaler for transpulmonary administration, eg, using a fine powder or spray. The composition can be prepared as a suppository or vaginal suppository. The composition can be prepared, for example, as a droplet for administration to the nose, ear or eyeball. The composition may be in the form of a kit, such as preparing a solution by combining the compositions immediately before administration to the subject. For example, the lyophilized antibody can be provided in the form of a kit containing sterile water or sterile buffer.

As a matter of course, the active ingredient in the composition is an antibody molecule, an antibody fragment or a variant or derivative thereof. Therefore, the active ingredient is easily decomposed in the gastrointestinal tract. Therefore, when the composition is administered by a route using the gastrointestinal tract, the composition must contain an agent that protects the antibody from degradation but releases the antibody when it is absorbed by the gastrointestinal tract. ..

A thorough discussion of pharmaceutically acceptable carriers is available from Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

The pharmaceutical composition of the present invention generally has a pH of 5.5 to 8.5, may be 6 to 8 in some embodiments, and may be about 7 in further embodiments. The pH may be maintained by the use of a buffer. The composition can be sterile and / or free of pyrogens. The composition may be isotonic to humans. In one embodiment, the pharmaceutical composition of the invention is provided in a closed container.

The pharmaceutical composition is sufficient to provide an effective amount of a polypeptide comprising one or more antibodies of the invention and / or an epitope that binds to the antibodies of the invention, i.e., for treatment, remission or prevention of the desired disease or condition. , Or, in an amount sufficient to exhibit a detectable therapeutic effect. The therapeutic effect also includes reduction of physical symptoms. The exact effective amount for any particular subject will depend on the subject's physical constitution and health, the nature and extent of symptoms, and the treatment or combination of treatments selected for administration. The effective amount for a given situation is determined by a given experimental method and determined by the clinician. For the purposes of the invention, the effective dose is generally from about 0.01 mg / kg to about 50 mg / kg, or about 0.05 mg / kg to about 10 mg of the composition of the invention in the subject receiving the administration. It is like / kg. Known antibody drugs provide guidance in this regard, for example, Herceptin ™ is administered by intravenous injection of a 21 mg / mL solution, with an initial dose of 4 mg / kg per body weight per week. The maintenance dose per body weight is 2 mg / kg, whereas Rituxan ™ is administered at 375 mg / m2 per week.

In one embodiment, the composition may contain one or more antibodies of the invention (eg, two, three, etc.) to provide additional or synergistic therapeutic effects. In other embodiments, the composition comprises one or more antibodies of the invention (eg, two, three, etc.) and one or more against influenza A or B (eg, two, three). Seeds, etc.) may contain additional antibodies. For example, one antibody may bind to an epitope on HA while another antibody may bind to a different epitope on HA or to an epitope on neuraminidase and / or matrix protein. Furthermore, those that administer the antibody of the present invention together with the influenza A vaccine or with an antibody having specificity other than the influenza A virus (for example, influenza B) are within the scope of the present invention. The antibodies of the invention can be administered either in combination with / simultaneously with an antibody having specificity other than the influenza vaccine or influenza A virus, or at another time point.

In another embodiment, the invention provides a pharmaceutical composition comprising two or more antibodies, wherein the first antibody is the antibody of the invention and is specific for an HA epitope. The second antibody is specific for a neuraminidase epitope, a second HA epitope, and / or a matrix epitope. For example, the invention provides a pharmaceutical composition comprising two or more antibodies, the first antibody being specific for an epitope in the HA stem of influenza A virus and the second antibody. Is specific for a neuraminidase epitope, a second HA epitope (eg, an epitope in the spherical head of HA, a second epitope in the stem of HA), and / or a matrix epitope. The second epitope in the stem, or epitope in the spherical head of influenza A virus HA, can be conserved among more than one influenza A virus subtype, but must be conserved. There is no.

In yet another embodiment, the invention provides a pharmaceutical composition comprising two or more antibodies, the first antibody being specific for a neuraminidase epitope and the second antibody being a second antibody. It is specific to 2 neuraminidase epitopes, HA epitopes, and / or matrix epitopes.

In yet another embodiment, the invention provides a pharmaceutical composition comprising two or more antibodies, the first antibody being specific for a matrix epitope and the second antibody being a second antibody. It is specific to 2 matrix epitopes, HA and / or epitopes on the matrix.

Representative antibodies of the present invention specific to the target protein of influenza A virus are, but are not limited to, FI6 mutant 1, FI6 mutant 2, FI6 mutant 3, FI6 mutant 4, and FI6. Examples thereof include mutant 5, FI28 mutant 1 or FI28 mutant 2.

In one embodiment, the invention provides a pharmaceutical composition comprising antibody FI6 variant 1 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the invention provides a pharmaceutical composition comprising antibody FI6 variant 2 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the invention provides a pharmaceutical composition comprising antibody FI6 variant 3 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the invention provides a pharmaceutical composition comprising antibody FI6 variant 4 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the invention provides a pharmaceutical composition comprising antibody FI6 variant 5 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In yet another embodiment, the invention provides a pharmaceutical composition comprising antibody FI28 variant 1 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In yet another embodiment, the invention provides a pharmaceutical composition comprising antibody FI28 variant 2 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier.

The antibodies of the invention may be administered (in combination or separately) with other therapeutic agents such as chemotherapeutic agents, radiotherapy agents and the like. In one embodiment, therapeutic compounds include antiviral compounds such as Tamiflu ™. Such combination therapy results in additive or synergistic improvements in therapeutic efficacy compared to administration of each therapeutic agent alone. The term "synergistic effect" is used to describe that the combination of the effects of two or more activators is greater than the sum of the individual effects of each activator. Therefore, if the combined effect of two or more agents results in "synergistic inhibition" of the activity or process, the inhibition of the activity or process is greater than the sum of the inhibitory effects of the individual activators. Intended to be. The term "synergistic therapeutic effect" refers to the therapeutic effect observed when two or more treatments are combined, in which case the therapeutic effect (measured by a number of parameters) is the individual treatment. Greater than the combined individual therapeutic effects observed for.

Antibodies can be administered to subjects who have not previously responded to treatment for influenza A virus infection, i.e., subjects who are known to respond to anti-influenza treatment. Such treatments include existing treatments with antiviral agents. Such a condition can be caused, for example, by infection with an antiviral drug-resistant strain of influenza A virus.

In one embodiment, the composition of the invention may contain an antibody of the invention, wherein the antibody is at least 50% by weight (eg, 60%, 70%, 75%, 80%) of the total protein of the composition. , 85%, 90%, 95%, 97%, 98%, 99% or more). In such compositions, the antibody is in a purified state.

The present invention provides a method for producing a pharmaceutical product, comprising (i) a step of preparing the antibody of the present invention and (ii) a step of mixing the purified antibody with one or more pharmaceutically acceptable carriers.

The invention also provides a method of producing a pharmaceutical product comprising mixing the antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is from transformed B cells or plasma cell cultures of the invention. The obtained monoclonal antibody. Therefore, the procedure for first obtaining a monoclonal antibody and then for producing a drug can be performed different times by different people in different places (eg, in different countries).

As an alternative to delivering antibodies or B cells for therapeutic purposes, nucleic acids (typically DNA) encoding the monoclonal antibodies of interest (or active fragments thereof) from B cells or plasma cell cultures are used. It can also be delivered to the subject to express the nucleic acid in situ in the patient to provide the desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

The composition of the invention may be an immunogenic composition and, in some embodiments, a vaccine composition comprising an antigen comprising an epitope recognized by the antibody of the invention. The vaccine according to the invention may be either prophylactic (ie, to prevent infection) or therapeutic (ie, to treat infection). In one embodiment, the invention provides a vaccine comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 37, 38, 39 or 40. In another embodiment, the invention provides a vaccine comprising a polypeptide comprising amino acid residues in HA1 and HA2 polypeptides consisting of one or two HA monomers as described above. The HA monomer may be uncut or may be cleaved.

The composition may contain an antimicrobial agent, especially when packaged in a multi-dose format. The composition may contain, for example, a surfactant such as Tween (polysorbate) such as Tween 80. Surfactants are generally present at low concentrations, eg less than 0.01%. The composition may contain sodium salts (eg, sodium chloride) to adjust the tonicity. The NaCl concentration is generally 10 + 2 mg / mL.

Further, in particular, if the composition is a lyophilized product, or if it contains components prepared by dissolving the lyophilized material in a solution (has been reconstituted), the composition may contain a sugar alcohol (eg, mannitol) or. Disaccharides (eg, sucrose or trehalose) may be contained, for example, from about 15-30 mg / mL (eg, 25 mg / ml). For lyophilization, the pH of the composition can be adjusted to about 6.1 prior to lyophilization.

The composition of the present invention may contain one or more immunomodulators. In one embodiment, the one or more immunomodulators contain an adjuvant (s).

For the purpose of effectively coping with influenza A virus infection, the epitope compositions of the present invention can elicit both a cell-mediated immune response as well as a humoral immune response. This immune response can induce an antibody- and cell-mediated, long-term immune response that can respond rapidly to exposure to influenza A virus.

Medical Treatment and Usage The antibodies and antibody fragments or derivatives and variants thereof of the present invention are used for treatment of influenza A virus infection, prevention of influenza A virus infection, or diagnosis of influenza A virus infection. can do.

The diagnostic method comprises contacting the antibody or antibody fragment with the sample. Such samples were taken from, for example, the nasal passages, nasal passages, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart, ovaries, pituitary glands, adrenal glands, thyroid glands, brain or skin. It may be a tissue sample. Diagnostic methods also include detection of antigen / antibody complexes.

Thus, the invention is (i) an antibody, antibody fragment, or variant or derivative thereof according to the invention, (ii) an immortalized B cell clone according to the invention, (iii) an epitope capable of binding to an antibody of the invention, or (Iv) provides a ligand, preferably an antibody capable of binding to an epitope that binds to a therapeutic antibody of the invention.

The present invention comprises a step of administering to a subject an antibody capable of binding an antibody of the present invention, an antibody fragment, or a variant and derivative thereof, or a ligand, preferably an epitope that binds to the antibody of the present invention. Treatment methods are also provided. In one embodiment, the method reduces influenza A virus infection in a subject. In other embodiments, the method prevents infection of influenza A virus in a subject, reduces the risk of infection, or delays infection.

Further, the present invention relates to (i) an antibody, an antibody fragment, or a variant and a derivative thereof, and (ii) immortalization according to the present invention during the production of a drug for preventing or treating influenza A virus infection. Also provided are B cell clones, (iii) epitopes capable of binding to antibodies of the invention, or (iv) ligands, preferably antibodies that bind to epitopes capable of binding antibodies of the invention.

The present invention provides the compositions of the present invention for use as agents for the prevention or treatment of influenza A virus infection. Also provided is the use of the antibodies and / or proteins of the invention that contain epitopes that bind to the antibodies during the treatment and / or production of diagnostic agents of the subject. Also provided are methods for treating a subject, comprising the step of administering the composition of the invention to the subject. In some embodiments, the subject may be human. Some of the methods for confirming the effectiveness of a therapeutic procedure include the step of monitoring the symptoms of the disease after administration of the composition of the present invention. Treatment may be a single dose schedule or a multiple dose schedule.

In one embodiment, an antibody, antibody fragment, immortalized B cell clone, epitope or composition according to the invention is administered to a subject in need of treatment. Such subjects include, but are not limited to, those who have a high risk of infection with influenza A virus or those who are susceptible to infection, for example, those who are susceptible to infection. Antibodies or antibody fragments of the invention can also be used for activation by passive immunization or vaccination.

Antibodies and fragments thereof as described in the present invention can also be used in an infection diagnostic kit for influenza A virus. In addition, the epitopes capable of binding the antibodies of the invention can also be used in kits for monitoring the efficacy of vaccination by detecting the presence of prophylactic anti-influenza A virus antibodies. Antibodies, antibody fragments, or variants and derivatives thereof as described in the present invention can also be used in kits for monitoring the production of vaccines with the desired immunogenicity.

The present invention also provides a method for producing a pharmaceutical product, which comprises mixing a monoclonal antibody with one or more pharmaceutically acceptable carriers, wherein the monoclonal antibody is obtained from the transfected host cells of the present invention. It is a monoclonal antibody. Therefore, the procedures for first obtaining a monoclonal antibody (eg, expressing and / or purifying the monoclonal antibody) and then mixing the resulting monoclonal antibody with a pharmaceutical carrier are at different locations (eg, eg). It can be performed different times by different people in different countries).

Steps such as various cultures, subcultures, cloning, subcloning, sequencing, and nucleic acid preparation initiated by the transformed B cells or plasma cell cultures of the present invention are the transformed B cells or plasma cell cultures. In order to perpetuate the antibody expressed by the substance, it can be optimized and carried out as desired in each step. In a preferred embodiment, the method further comprises the technique of optimizing the nucleic acid encoding the antibody (eg, affinity maturation or optimization). The present invention includes all of the cells, nucleic acids, vectors, sequences, antibodies, etc. used and prepared during such steps.

In all of these methods, the nucleic acid used in the expression host can be engineered for insertion, deletion or modification. Changes by such an operation include, but are not limited to, introduction of restriction sites, modification of codons used, addition or optimization of transcription and / or translation control sequences, and the like. By changing the nucleic acid, the encoded amino acid can also be changed. For example, such alterations may result in one or more amino acid substitutions, deletions and / or insertions into the amino acid sequence of the antibody (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). Can be useful for introducing. Such point mutations that can alter effector function, antigen binding affinity, post-translational modifications, immunogenicity, etc. can introduce amino acids for binding covalent groups (eg, labels) or tags. Can be introduced (eg, for purification purposes). Mutations can be introduced at specific sites or can be sorted after introduction at random locations (eg, molecular evolution). For example, to introduce different properties into the encoded amino acids, randomly into one or more nucleic acids encoding any of the CDR regions, heavy chain variable regions or light chain variable regions of the antibodies of the invention. Alternatively, the mutation can be introduced directly. Such changes can occur through an iterative process, retaining the initial changes and introducing new changes at other nucleotide positions. Furthermore, the changes may be combined with those carried out in an independent process. Amino Acids Encoded Different properties introduced include, but are not limited to, improved affinity.

The general term "comprising" includes "including" as well as "consisting", for example, a composition "comprising" containing X is approximately composed of X. Or may contain some other additional component, such as XY + Y.

The term "substantially" does not exclude "completely", for example, if the composition is "substantially free" of Y, it may be completely free of Y. If necessary, the term "substantially" may be omitted from the description of the present invention.

The term "about" with respect to the numerical value x means, for example, x + 10%.

As used herein, the term "disease" is generally synonymous with the terms "disease" and "symptoms" (assuming they are medical symptoms) and are used interchangeably with these terms. Reflects all abnormalities in the condition of the body or part of the body of a human or animal whose normal function is impaired, typically distinguished from signs and symptoms, and the lifespan or quality of life of the human or animal. Intended to reduce.

As used herein, reference "treatment" with respect to a subject or patient is intended to include prevention, prophylaxis and treatment. The terms "subject" or "patient" are used interchangeably herein to mean all mammals, including humans. Examples of subjects include humans, dairy cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In one embodiment, the patient is a human.

Representative embodiments of the present invention are provided in subsequent examples. Subsequent examples are shown for illustrative purposes only and are intended to assist one of ordinary skill in the art in using the present invention. The examples are by no means meant to limit the scope of the invention thereby.

Example 1. Generation and characterization of plasma cell-derived antibodies that broadly neutralize influenza A virus To identify individuals capable of producing heterosubtype antibodies in response to seasonal influenza vaccines (containing H1 and H3 HA). We screened circulating plasma cells by ELISPOT 7 days after vaccination to amplify the ability to secrete antibodies that bind to the vaccine or negative control H5 HA (A / VN / 1203/04). Notably, in one donor, 14% of IgG-secreting plasma cells antibody to H5, whereas four of the five donors tested for H5-specific plasma cells were undetected. Produced, 57% produced antibodies to the vaccine. CD138 + plasma cells were isolated from peripheral blood mononuclear cells (PBMCs) collected 7 days after vaccination by cell sorting using magnetic microbeads followed by FACSAria. A limited number of plasma cells were seeded on microwell culture plates. Recombinant H5 or H9 HA was used as the antigen, and tetanus toxoid was used as the negative control, three ELISAs were performed in parallel and the culture supernatant was tested. Of the 4,928 culture supernatants screened, 12 were H5 bound but not H9 HA, 25 were H9 bound but not H5 HA, 54 were H5 and It bound to both of H9. Of the 54 types of cultures, RT-PCR was performed on some of the cultures having the highest OD signal, and two pairs of VH and VL genes were recovered.

The VH and VL genes were cloned into expression vectors and transfected into HEK293T cells to produce recombinant antibodies. The two monoclonal antibodies, FI6 mutant 2 and FI28, shared most of the V, D and J gene fragments (IGHV3-30 ^* 01, IGHD3-9 ^* 01, IGHJ4 ^* 02 and IGKV4-1 ^* 01). However, there were differences in the N region, the use of IGKJ and the pattern of somatic mutations, and therefore were not relevant as clones.

The specificity of the recombinant antibody was investigated by ELISA using a series of HAs belonging to different subtypes. Notably, FI6 binds to all influenza A subtypes tested, including groups 1 (H1, H5 and H9) and groups 2 (H3 and H7), and HA from influenza B. Did not bind to. In contrast, FI28 bound only to 3 HAs of the first group (H1, H5 and H9).

Considering the homology of VH and VL sequences of the two antibodies, H of FI6 mutant 2, FI28 mutant 1 and hCMV-specific antibody 7I13 (antibody using the same V, D and J sequences as the H chain) And L chains were used to perform shuffling experiments. Binding to H7 required FI6 mutant 2 H and L chain pairs, whereas binding to H5 was maintained when the L chains of FI6 mutant 2 and FI28 mutant 1 were shuffled. rice field. Furthermore, binding to H5 was also observed when the H chain of FI6 mutant 2 was combined with the L chain of unmodified 7I13. In contrast, no binding to H5 was observed when the homologous 7I13 H chain was combined with the L chain of FI6 variant 2. Without being bound by any particular theory, these results indicate that binding to H5 is primarily due to the H chain, whereas binding to H7 is between the H and L chains of FI6 variant 2. It was suggested that accurate pairing is required.

Next, using pseudoviruses (Table 5) and infectious viruses (Table 6), FI6 mutant 2 and FI28 mutant 1 were used for the ability to neutralize the subtypes of influenza A type 1 and group 2. Was tested. Notably, FI6 variant 2 includes 6 H5 isolates belonging to the intrinsically divergent viruses 0, 1, 2.1, 2.2 and 2.3, and 2 tri-H7 isolates. Neutralized all pseudoviruses tested. In addition, FI6 variant 2 compared all tested infectious viruses, including two H3N2 viruses and four H1N1 viruses, with the recent H1N1 infectious explosive isolate A / CA / 04/09. It was neutralized up to several tens of times (Table 6). FI28 mutant 1 neutralized all H5 pseudoviruses, but not H7 pseudoviruses as well as the infectious virus tested. The neutralizing titer against pseudovirus was higher than the titer against infectious virus.

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nd, not implemented

Example 2. Antigenic site of FI6 variant 2 and FI28 variant 1 To identify the antigenic site to which antibody FI6 variant 2 and FI28 variant 1 bind, we first conserved the mouse monoclonal antibody C179 (conservation of the HA stem region). These antibodies were tested for their ability to inhibit binding to (mapped to a sequence) (Y. Okuno, et al., J Virol 67, 2552 (1993)). Both FI6 mutant 2 and FI28 mutant 1 completely inhibited the binding of C179 to the recombinant H5 VN / 1203/04 HA, indicating that the epitopes recognized by these antibodies overlap. It was suggested. In contrast, FI6 mutant 2 and FI28 mutant 1 do not compete with the set of H5-specific antibodies isolated from the H5N1 immune donor and recognize different epitopes in the spherical head of HA (C.I. P. Simmons et al., PLoS Med 4, e178 (2007); S. Khurana et al., PLoS Med 6, e1000049 (2009)). Screening for defective escape variants and attempting to map the FI6 variant 2 epitope suggests that this epitope cannot be easily mutated without impairing virus adaptability.

We then performed peptide mapping using a library of linear and cyclized peptides HA A / VN / 1194/04, and performed helix scans with the Pepscan Presto BV system (Lelystad, The Netherlands). By this analysis, HA2 fusion peptide FGAIAG (3rd to 8th amino acids according to H3 numbering; SEQ ID NO: 37), HA2 helix A peptide DGVTNKVNS (46th to 54th amino acids; SEQ ID NO: 38), HA2 helix B peptide MENULDFWDSNVK. The binding region of FI6 variant 2 was identified, including (amino acids 102-116; SEQ ID NO: 39) as well as the HA1 C-terminal peptide LVLATGLRNSP (amino acids 315-325; SEQ ID NO: 40). The binding region of FI28 mutant 1 was different from that of FI6 because the antibody did not react with the HA1 C-terminal peptide and the HA2 helix B peptide.

Example 3. Generation and characterization of improved productive FI6 variants 3, 4 and 5 to improve productivity in mammalian cells and eliminate unwanted somatic mutations and unwanted properties. Several variants of VL were synthesized. The VH and VL genes were cloned with the constant portion of IgG1 and the expression vector encoding Cκ, respectively. The IMGT database was referenced to identify germline sequences associated with FI6 mutant 2. By synthesis (Genscript, Piscatawy, NJ) or site-specific mutagenesis (Promega), a single or multiple germline mutation produced an antibody variant in which the germline was restored and sequenced. To optimize expression in human cells, GenScript's OptimumGene ™ system was used to codon-optimize the sequences of all mutants. Freestyle 293 cells (Invitrogen) cultured in suspension were transiently transfected with PEI to produce a monoclonal antibody. After 7 days from the start of culture, the supernatant derived from the transfected cells was collected, IgG was affinity purified by protein A chromatography (GE Healthcare), and desalted by PBS. Productivity in a transient expression system was evaluated by multiple independent studies. The median is shown in FIG. As shown in FIG. 1, FI6 mutant 2 is produced at a titer of 13.5 μg / mL, FI6 mutant 3 is produced at a titer of 46.3 μg / mL, and FI6 mutant 4 is produced at a titer of 60.7 μg / mL. The FI6 variant 5 is produced at a titer of 61.6 μg / mL. Therefore, we were able to increase the production titers of FI6 mutants 3, 4 and 5 by 3.4-fold, 4.5-fold, and 4.6-fold, respectively, compared to FI6 mutant 2. ..

In addition, the recombinant antibody was characterized for its binding to H5 and H7 HA by ELISA, and also for the neutralization of H5 and H7 pseudoviruses (Table 7), and the original FI6 mutant 2 IgG was evaluated. Compared with. Half of the area of the ELISA plate (Corning) was coated with 5 μl of 1 μg / mL baculovirus-derived recombinant HA (Protein Sciences) PBS solution. After blocking with 1% PBS / BSA, antibodies were added and binding was revealed using alkaline-phosphatase labeled F (ab') 2 goat anti-human IgG (Southern Biotech). The plate was then washed, substrate (p-NPP, sigma) was added and the plate was read at 405 nm. The antibody concentration required to obtain 50% of maximum binding (EC50) was measured by ELISA and the relative affinity of the antibody binding to HA was quantified. For the pseudovirus neutralization assay, serially diluted antibodies were incubated with medium supernatant containing a constant concentration of pseudovirus at 37 ° C. for 1 hour. HEK 293T / 17 cells were then added to the mixture and incubated at 37 ° C. for 3 days. The cells were then solubilized with a Britelite reagent (PerkinElmer) and the relative luminescence (RLU) of the cell solubilizer was measured with a luminometer microplate reader (Veritas, Turner Biosystems). The decrease in infectivity was measured by comparing RLUs in the presence and absence of the antibody and expressed as the neutralization rate. The 50% inhibition concentration (IC50) was defined as the sample concentration at which the RLU was reduced by 50% compared to the virus control well after subtracting the background RLU in the cell-only control well. Table 7 shows FI6 mutants 3-5 selected from which the binding activity was preserved or improved based on the improvement in sequence characteristics.

FI6 mutant 2 and FI6 mutant 3 belong to the first group (H1, H2, H5, H6, H8 and H9) and the second group (H3, H4, H7 and H10) and range from 10 to 270 ng / mL. Bound to all recombinant or purified HA tested with an EC50 value of (Table 8). Furthermore, FI6 mutant 2 and FI6 mutant 3 stain cells transfected with HA genes belonging to the first group (H11, H12, H13 and H16) and the second group (H4, H10, H14 and H15). (Table 8).

（１）ＥＣ５０値（ｎｇ／ｍＬ）はＥＬＩＳＡにより測定されたものである
（２）「＋」は、ＨＡを形質移入された２９３細胞が染色に陽性だったことを意味する。

(1) EC50 value (ng / mL) was measured by ELISA. (2) "+" means that 293 cells transfected with HA were positive for staining.

Example 4. Structural evaluation of FI6 variant 3 epitopes on H1 and H3 HA FI6 variant 3 identifies epitopes recognized on HA in groups 1 and 2, and molecular interactions between antibodies and their target antigens. To illustrate, we crystallized a complex consisting of the FI6 variant 3 Fab fragment and the H1 (group 1) and H3 (group 2) HA homotrimers. To crystallize the FI6 mutant 3 / H1 homotrimer HA complex, the external domain of H1 HA0 was expressed in Sf9 insect cells. The cDNAs corresponding to residues 11 to 329 (HA1) and 1 to 176 (HA2) (according to H3 numbering) were cloned into the BioFocus expression vector incorporating the GP67 secretion signal, and the expressed protein was secreted into the culture medium. .. The cloned cDNA was fused with the C-terminal trimer-forming foldon sequence to form the H1 HA0 trimer. In order to remove the foldon tag before crystallization, a thrombin cleavage sequence is introduced between the foldon sequence of HA2 and the C-terminal, and the 6-His tag used for affinity purification is expressed in the C of the polypeptide sequence. Incorporated on the outermost side of the terminal.

Sf9 insect cells are infected with recombinant baculovirus, Ni-NTA resin (Qiagen) is passed through the culture medium, and gel filtration (S200 column) is performed. HA0 was recovered. Of the eluted proteins, the protein corresponding to the trimer H1 HA0 was concentrated to 1 mg / mL and then treated with thrombin (5 U / mg with respect to HA0) at 20 ° C. for 2 hours to remove the C-terminal tag. Finally, the purified cleavage protein H1 HA0 was fractionated on a Mono Q anion exchange column. To form a complex with Fab-FI6 variant 3, 0.5-1 mg / mL purified H1 HA0 was mixed with a 2-fold molar excess of purified Fab-FI6 variant 3. After incubating at 4 ° C. for 3 hours to form a complex, fractionation was performed on an S200 gel filtration column to separate excess Fab-FI6 mutant 3.

The purified complex of Fab-FI6 mutant 3 and the uncleaved H1 HA0 trimer was concentrated to 12 mg / mL for use in crystallization. Vapors are diffused from a well solution consisting of 0.1 M bistris propane (pH 7.0) and 2.2 M ammonium sulfate to grow a complex crystal of Fab-FI6 mutant 3 and H1 HA0 by the hanging drop method. rice field. The crystals were grown at 20 ° C. for 4 weeks and recovered from the drops into a 1: 1 mixture of well solution and 3.4 M sodium malonate (pH 7.0) for freeze protection and then flash frozen in liquid nitrogen. Data was collected at beamline IO3 by Diamond Light Source, indexed, integrated and scaled by MOSFLM and SCALA, respectively.

Statistical analysis of unit cell parameters and protein molecular weight suggested that one hemagglutinin monomer and one Fab fragment were included per unit of asymmetry, so molecular replacement was used using a study model in the monomeric state. Was carried out. Initial phases were obtained using the coordinates of the H1 HA (PDB ID: 1RD8) monomer as a search model by the CCP4 program PHASER. The automated model building program FFFEAR successfully densified the variable domains of the heavy chain, followed by the placement of the light chain variable domains compared to the HA-antibody structures 3FKU and 3GBN. Models were constructed and fine-tuned alternately by COOT and REFMAC5, respectively. This process was repeated until the electron density map was filled as fully as possible and the R-work and R-free values were flat.

Amino acids in the final PDB file were numbered according to Kabat's convention. The final model contains all of H1 HA and heavy and light chain variable domains. For crystallization of the FI6 mutant 3 / H3 heterotrimer HA complex, HA (BHA) released by the X-31 (H3N2) virus and bromelain was purified and a Fab fragment was prepared by papain digestion. FI6 variant 3 Fab was purified by Protein A Sepharose Affinity Chromatography (HiTrap Protein A HP, 1 mL) followed by an S-200 size exclusion column. 3.5 mg of Fab was mixed with 3 mg of X-31 BHA and incubated overnight at 4 ° C. to form a complex, which was purified by a superose 6 SEC column. For crystallization, the peak fraction corresponding to the Fab-HA complex was pooled and concentrated.

A sitting drop vapor diffusion method was performed by an Oryx-6 crystallization robot (Douglas Instruments) to grow FI6 mutant 3-H3 complex crystals. 25% glycerol was added to the reservoir solution to freeze protect the crystals. Data were collected at beamline IO3 by Diamond Light Source, indexed, integrated and scaled by Denzo and Scalepack, respectively. Molecular replacement was performed by Amore and crystals containing an H3 HA trimeric complex with three FI6 mutants 3 Fabs in an asymmetric unit were analyzed. The molecular replacement calculation is performed on the H3 HA trimer, the heavy chain and light chain variable domains of the FI6 mutant 3 of the FI6 mutant 3 / H1 complex, and the constant domain (PDB ID: 3HC0.pdb) as independent search targets. This was done using the coordinates of the 2Å structure. The solutions obtained by molecular replacement were refined by Refmac5 and Pheonix using Coot, interwoven with manual adjustments. The electron density map was significantly improved with non-crystallographic averaging using DM.

X-ray crystallography showed that FI6 mutant 3 was bound to a conserved epitope of the F subdomain. The two HAs were phylogenetically and structurally different, and although the complex was crystallized in different packing arrangements, the interacting surfaces were found to be very similar (FIGS. 2, A and B). .. In each case, each monomer of the HA trimer binds to one molecule of FI6 variant 3 (FIG. 3). The HCDR3 loop of FI6 variant 3 binds to a shallow groove on the F subdomain of HA. The surface of this groove is formed by residues from the A helix of HA2 and some of the two strands of HA1 (38-42 and 318-320), whereas the bottom is residues 18-21. Formed by HA2 turns comprising (FIGS. 2, A and B).

The HCDR3 loop crosses Helix A at an angle of approximately 45 °, allowing Leu-100A, Tyr-100C, Phe-100D and Trp-100F to make hydrophobic contact with residues in the groove (FIG. 4). Tyr-100C and Trp-100F also form hydrogen bond potentials with Thr-318 in the side chain of HA1 and the main chain carbon of the 19th residue of HA1. At two other locations, polar interactions are formed by residues 98 and 99 of the backbone carbonyl of HCDR3 and Asn-53 and Thr-49 on helix A. Based on these, the interaction between HCDR3 and HA (H1 and H3) fills the surface of about 750 Å2 of the antibody, and about two-thirds of this interaction is due to the interaction with the HA2 chain. Is considered to be.

Finally, the interaction between FI6 variant 3 and the hydrophobic grooves of H1 and H3 is very similar. The LCDR1 loop of FI6 variant 3 provides two contacts on the helix A side opposite to the side contributing to the hydrophobic groove; Ph-27D makes hydrophobic contact with fatty Lys-39. And since Asn-28 hydrogen bonds with Asn-43, it is considered that both H1 and H3 fill a surface area of about 190 Å2 in consideration of these. For H1 HA co-crystallized in the uncut state, LCDR1 is also in extensive contact with the uncut "fusion peptide" of the adjacent distal right HA monomer (FIGS. 3, B and C and FIG. 4). , A and B), whereby the packed surface of FI6 variant 3 is further 320 Å2.

Residues 28 and 29 of LCDR1 form a backbone amide hydrogen bond with the backbone carbonyl of residue 329 of HA1 and the adjacent residue Leu-2 at one of HA2, thus the bond is at the cleavage site. It extends to. The Phe-27D of LCDR1 is hydrophobically in contact with Leu-2 of the adjacent distal right HA2, whereas the side chain hydroxyl Tyr-29 is a residue of the backbone carbonyl of the adjacent distal right HA1 chain. Hydrogen bond with 325. In contrast to the very similar interactions between H1 and H3 HA and HCDR3, the interaction between LCDR1 and the "fusion peptide" derived from the adjacent truncated distal right H3 HA monomer. The action was very weak compared to the interaction formed with the uncleaved H1 HA (FIG. 3, Insert B). Although Phe-27D interacts again with Lys-39, the fatty site of HA2, Tyr-29 is a residue in the backbone carbonyl of the adjacent distal right HA2, Ala-7 (uncleaved H1 HA). (Not 329) and may come into contact with hydrogen bonds.

In contrast, considering that the contact area is smaller than 114 Å (compared to 320 Å 2 in H1 HA), there is no backbone contact between the LCDR1 loop and the cleaved H3 HA “fusion peptide”. It was also observed that FI6 mutant 3 orients slightly differently with respect to HA in the FI6 mutant 3 / H3 and FI6 mutant 3 / H1 complexes due to the production of H3 HA by cleavage of the HA precursor. Will be done. Table 9 reports the residues that make contact at the interface between the FI6 mutant 3 VH and the VL chain and the cleaved H3 homotrimer HA. Table 10 reports the residues that make contact at the interface between the FI6 mutant 3 VH and the VL chain and the uncleaved H1 homotrimer HA.

The structures of the two cross-reactive antibodies CR6261 and F10 (specific to Group 1) have been previously reported as a complex of H5 and H1 HA. Binding to HA by CR6261 and F10 antibodies is mediated only by the VH domain and orients to HA in approximately the same manner as each other, but both antibodies are significantly rotated with respect to FI6 variant 3 and HA. It was 5-10 Å closer to the proximal end of the membrane (FIGS. 3, D and E).

From the FI6 mutant 3 / H1 and FI6 mutant 3 / H3 represented here, the HA binding sites of the three antibodies overlap widely, but the nature of the interaction formed by the FI6 mutant 3 is It is clear that they are significantly different from those made with CR6261 and F10 antibodies. The interaction between the FI6 mutant 3 and the hydrophobic groove of HA is mediated only by the long-chain HCDR3, whereas for CR6261 and F10, a total of three HCDRs are most involved in the binding. It's a big difference.

Like H7, H10 and H15 HA in the second group, H3 HA is glycosylated with Asn-38 (HA1), whereas H1 common to all HAs in the first group is not glycosylated. The point is the important difference between the FI6 mutant 3 / H1 complex and the FI6 mutant 3 / H3 complex. In the unbound H3, this sugar side chain projects from the β chain of HA1 containing the Asn-38 residue towards the helix A of HA2 of the same HA subunit, and the footprint of FI6 variant 3. (Fig. 5A). Since sugar side chains are known to affect the antigenicity of viral glycoproteins, this overlap results in the cross-reactivity of the other first group targeting regions proximal to the HA membrane. The antibody suggested that it was due to the lack of binding to HA in group 2. However, the FI6 mutant 3 is bound to H3 HA by reorienting the oligosaccharides, rotating them about 80 ° away from the HA surface and making new contacts with ASP-53 and Asn-55 of the HCDR2 loop. Can be done (Fig. 5B).

Given that FI6 variant 3 binds to H3 HA due to the flexibility of the sugar side chain at Asn-38, we consider that the reason why H3 HA does not bind to CR6261 or F10 is not at the glycosylation site. I had a question about it. Simple modeling suggests that by making similar changes in the orientation of the sugar side chains, the cross-reactive antibody exhibits binding to CR6261 but not to F10. The β-turn containing the VH residues 73-77 of F10 is sterically hindered by the Asn-38 linked sugar chain in the orientation adopted for the FI6 mutant 3 / H3 complex. In this case, it is unclear whether the sugar chain can rotate further away from the binding site to which it is bound by F10. However, neither CR6261 nor F10 from which the glycosylation site (Asn-38) was removed could neutralize the H7 pseudovirus (A / ticken / Italy / 99) (IC50: over 50 μg / ml). It was suggested that the glycan-related steric hindrance is not limited to structural constraints that prevent binding of CR6261 and F10 antibodies to Group 2 HA.

The most notable difference is that, in addition to the glycosylation of Asn-38, the F subdomain structure between the HAs of groups 1 and 2 is involved in the environment and orientation of HA2 Trp-21, which is unique to the group. In group 1 HA, Trp-21 is approximately parallel to the surface of the F subdomain, whereas group 2 HA is oriented approximately perpendicular to the surface (FIGS. 5, C). And D). All three antibodies (FI6 mutant 3, CR6261 and F10) contact Trp-21 primarily with the phenylalanine side chain (Phe-100D for FI6 mutant 3, Phe-54 for CR6261 and Phe-55 for F10). (FIGS. 5, C and D). For FI6 variant 3, due to local rearrangement in the HCDR3 loop, Phe-100D is located approximately 2 Å deeper in the hydrophobic groove of the H1 complex than in the case of the H3 complex. Thus, in either case, it means maintaining a more similar contact distance to Trp-21.

In complex formation with H1 HA, the positions of the two group-specific antibodies Phe-54 (CR6261) and Phe-55 (F10) were similar to those of FI6 variant 3. However, when Ph-54 (CR6261) and Phe-55 (F10) are placed on a short loop HCDR2 connecting two adjacent antiparallel strands, they are separated from the hydrophobic groove and the second group of Trp-21. The flexibility with respect to the transfer of phenylalanine to accommodate FI6 variants appears to be reduced compared to FI6 variant 3. Therefore, the binding of CR6261 and F10 to Group 2 HA appears to be blocked by steric hindrance between HCDR2 phenylalanine and Trp-21.

In summary, the structural data obtained suggest that the core epitope of helix A is similar to that recognized by CR6261 and F10, but the FI6 variant 3 binds at different angles and is further 5-10 Å away from the membrane. It is shown to be in contact with a larger region that is located and extends to the cleaved and uncut form of the fusion peptide of the adjacent distal right monomer containing the helix A (FIG. 6). Binding of FI6 variant 3 is mediated by both VH and VL CDRs, in particular by long chain HCDR3 with different conformation of group-specific Trp-21 loops and severely mutated LCDR1. To. The use of both VH and VK chains and long-chain HCDR3 is characteristic of naturally selected antibodies and is possessed by phage-derived antibodies such as CR6261 and F10 that bind using only the VH chain. In contrast to the characteristics. FI6 mutants 3 Contact residues in VH and VK are shown in FIG.

Example 5. In vivo preventive effect of FI6 mutant 3 An in vivo test was conducted on the preventive effect of FI6 mutant 3 using a mouse model for influenza A virus infection. 6-week-old and 8-week-old female BALB / c mice were injected intravenously (iv) with purified antibodies at varying concentrations of 1-16 mg / kg. After 3 hours, the mice were comatose and 10 MLD50 (50% lethal dose of mice) H1N1 A / PR / 8/34 was intranasally administered (in). As a treatment setting, mice were administered the antibody one, two or three days after intranasal administration. Mice survival and weight loss were monitored daily until day 14 after infection (pi). Mice that lost more than 25% of their body weight from the start of the experiment were euthanized in compliance with the animal experiment protocol.

To assess the effect of FI6 variant 3 on viral replication, mice administered 10 MLD50 H1N1 A / PR / 8/34 were administered antibodies at different time points and sacrificed 4 days later to lung and brain. Was collected. Tissues were homogenized in Leibovitz L-15 medium (Invitrogen) supplemented with an antibiotic-antifungal agent solution (Invitrogen) to prepare a 10% (w / v) tissue suspension. The dose of the tissue homogenic solution was set for MDCK cells, and the virus titer was measured. In a prophylactic setting, FI6 variant 3 is completely prophylactic, somewhat prophylactic when administered at 4 mg / kg (survival rate 80%), and when administered at 2 mg / kg. Mice were infected with Group 1 H1N1 A / PR / 8/34 virus (FIG. 8). In the mice treated with FI6 mutant 3, the lung virus titer 4 days after infection was reduced to about 1/100 of that on the day or 1 day after infection (FIG. 9). In addition, FI6 mutant 3 also prevented weight loss in mice infected with Group 2 H3N2 HK-x31 virus (FIG. 8).

Example 6. Mechanism of virus neutralization by FI6 mutant 3 In an in vivo test aimed at evaluating the protective effect of FI6 antibody, we found complement fixation (FI6-v2 KA) or complement and FcR binding (FI6). FI6 mutant 2 Fc mutant lacking -v2 LALA) was prepared. These antibodies exhibited similar binding and in vitro neutralization properties as FI6 variant 2 and half lethal periods comparable to in vivo tests (mean half of FI6-v2, FI6-v2 KA and FI6-v2 LALA). The lethal period is 3.3, 3.4 and 3.5 days, respectively). The prophylactic effects of these antibodies were tested in mice fatally infected with the A / Puerto Rico / 8/34 (H1N1) virus. FI6. v2 completely prevented mouse mortality when administered at 4 mg / kg and prevented 80% of mouse mortality when administered at 2 mg / kg (FIG. 8F). When administered at 10 mg / kg, FI6-v2 and FI6-v2 KA were completely prevented, whereas the activity of FI6-v2 LALA was significantly reduced and could only be prevented in 40% of mice (Fig.). 8F). This reduction in effect was particularly pronounced when the antibody variant was administered at a critical concentration of 3 mg / kg (FIG. 8G).

To investigate the mechanism that contributes to the neutralization activity of FI6 mutant 3, NC / 99 in PBS solution containing FI6 mutant 3, FE17, non-specific mAb (HBD85) or mAb at a molar concentration of 15 times. Baculovirus-derived HA (Protein Sciences) was incubated at 37 ° C. for 40 minutes. Trypsin treated with TPCK was added to each sample to a final concentration of 2.5 μg / mL and digested at 37 ° C. for 5, 10 and 20 minutes. At each time point, a buffer containing SDS and DTT was added and boiled at 95 ° C. for 5 minutes to stop digestion. The sample was then packed in a 12% Tris-glycine polyacrylamide gel. The protein was transferred to the PVDF membrane by the iBlot blotting system (Invitrogen). The PVDF membrane was blocked for 30 minutes with a TBS-Tween solution of 10% fat-free dry milk. The primary antibody against HA0 (FO32 biotinylated in the laboratory) was incubated overnight at 4 ° C. with a TBS-Tween solution at a concentration of 0.5 μg / mL. PVDF membranes were washed 3 times with TBS-Tween and incubated with HRP-labeled streptavidin (Sigma) for 1 hour at room temperature.

The PVDF membrane was washed 3 times with TBS-Tween and positive bands were detected by ECL Plus ™ Western Blotting Detection Reagent (GE Healthcare) and the LAS4000 CCD camera system. The data in FIG. 10 show that FI6 variant 3 inhibits cleavage of HA0 by TPCK-trypsin. This cleavage suggests that at least for these viruses, the antibody light chain binds to uncleaved HA0 to block infection where extracellular cleavage occurs.

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Claims (35)

An isolated antibody or antigen-binding fragment thereof, which neutralizes the infection of influenza A virus type 1 and group 2 subtypes, and is a trimeric of influenza A hemagglutinin (HA). It specifically binds to an epitope present in the stem region, where the heavy and light chains of the antibody or antigen-binding fragments thereof are the first proximal monomer and the second distant of the HA trimer. Upon contact with the amino acid in the monomer on the right side of the position, the antibody or its antigen-binding fragment is produced in the transgenic cells at least three times higher than that produced by the FI6 mutant strain 2. Antibodies or antigen-binding fragments thereof. The heavy chain of the antibody or an antigen-binding fragment thereof contacts an amino acid residue in the proximal monomer, wherein the light chain of the antibody or an antigen-binding fragment thereof is the said of the HA trimer. The antibody according to claim 1 or an antigen-binding fragment thereof, which contacts an amino acid residue in the proximal monomer and the distal right-side monomer. The first or second monomer in the HA trimer is uncleaved or cleaved by specifically binding to an epitope present in the stem region of influenza A influenza HA trimer. , The antibody according to claim 1 or an antigen-binding fragment thereof. The heavy chain of the antibody or an antigen-binding fragment thereof is the 318th amino acid of HA1 of the first or second monomer, and the 18, 19, 20, 21, 38, 41, 42, 45, of HA2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the monomer is uncleaved or cleaved by contacting the amino acid residues 49, 53, and 57. The light chain of the antibody or an antigen-binding fragment thereof is the amino acid residues 38, 39 and 43 of HA2 of the proximal monomer and the amino acids 327, 328 and 329 of HA1 of the distal right monomer. The antibody or antigen thereof according to claim 1, which is in contact with the residue and the first, second, third and fourth amino acid residues of HA2, and the proximal and distal right-side monomers are uncut. Bonded fragment. The light chain of the antibody or an antigen-binding fragment thereof is the amino acid residues 38, 39, 42 and 46 of HA2 of the proximal monomer and the amino acids 321 and 323 of HA1 of the distal right monomer. The antibody or antigen-binding fragment thereof according to claim 1, wherein the residue and the 7th and 11th amino acid residues of HA2 are in contact with each other and the proximal and distal right-side monomers are cleaved. The 318th amino acid of HA1 and the remaining 18th, 19th, 20th, 21st, 38th, 39th, 41st, 42nd, 43rd, 45th, 48th, 49th, 53rd, 56th and 57th amino acids of HA2 of the proximal monomer. It specifically binds to an epitope containing the group and the amino acid residues 327, 328, 329 of HA1 and the amino acid residues 1, 2, 3 and 4 of the HA2 polypeptide of the distal right monomer. The antibody or antigen-binding fragment thereof according to claim 1, wherein the proximal and distal right-side monomers are uncut. The 318th amino acid of HA1 and the 18th, 19th, 20th, 21st, 38th, 39th, 41st, 42nd, 45th, 46th, 49th, 52nd, 53rd and 57th amino acid residues of HA2 of the proximal monomer. In addition, the proximal and distal right side monomers specifically bind to an epitope containing the 321st and 323rd amino acid residues of HA1 and the 7th and 11th amino acid residues of HA2 of the distal right side monomer. The antibody according to claim 1 or an amino acid-binding fragment thereof, which is cleaved. It specifically binds to an epitope containing the 329th amino acid of HA1 and the 1st, 2nd, 3rd and 4th amino acid residues of HA2, and the HA1 and HA2 are contained in the uncleaved monomer of the HA trimer. The antibody according to claim 1 or an antigen-binding fragment thereof, which is present. The antibody or antigen-binding fragment thereof is associated with H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 of the HA subtype of influenza A. The antibody according to claim 1 or an antigen-binding fragment thereof, which specifically binds to the antibody. An isolated antibody or an isolated antibody that neutralizes the infection of group 1 and group 2 subtypes of influenza A virus and specifically binds to an epitope present in the stem region of the HA trimer of influenza A. The antigen-binding fragment of the antibody, the heavy chain and the light chain of the antibody, or the antigen-binding fragment thereof, is an amino acid in the first proximal monomer and the second distal right monomer of the HA trimer. Upon contact, the antibody or antigen-binding fragment thereof is: (i) heavy chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 1, 41 and 43, respectively, or as shown in SEQ ID NOs: 1, 41 and 42, respectively. And (ii) isolated antibodies or antigen bindings thereof, comprising the light chain CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, or as shown in SEQ ID NOs: 44, 5 and 6, respectively. piece. The heavy chain of the antibody or an antigen-binding fragment thereof contacts an amino acid residue in the proximal monomer, wherein the light chain of the antibody or an antigen-binding fragment thereof is the said of the HA trimer. The antibody according to claim 11 or an antigen-binding fragment thereof, which contacts an amino acid residue in the proximal monomer and the distal right-side monomer. The first or second monomer in the HA trimer is uncleaved or cleaved by specifically binding to an epitope present in the stem region of influenza A influenza HA trimer. , The antibody according to claim 11 or an antigen-binding fragment thereof. The heavy chain of the antibody or an antigen-binding fragment thereof is the 318th amino acid of HA1 of the first or second monomer, and the 18, 19, 20, 21, 38, 41, 42, 45, of HA2. The antibody according to claim 11, or an antigen-binding fragment thereof, which is in contact with the amino acid residues 49, 53, and 57 and the monomer is uncleaved or cleaved. The light chain of the antibody or an antigen-binding fragment thereof is the amino acid residues 38, 39 and 43 of HA2 of the proximal monomer and the amino acids 327, 328 and 329 of HA1 of the distal right monomer. The antibody or antigen thereof according to claim 11, which is in contact with the residue and the first, second, third and fourth amino acid residues of HA2, and the proximal and distal right-side monomers are uncut. Bonded fragment. The light chain of the antibody or an antigen-binding fragment thereof is the amino acid residues 38, 39, 42 and 46 of HA2 of the proximal monomer and the amino acids 321 and 323 of HA1 of the distal right monomer. The antibody or antigen-binding fragment thereof according to claim 11, wherein the residue and the 7th and 11th amino acid residues of HA2 are in contact with each other and the proximal and distal right-side monomers are cleaved. The 318th amino acid of HA1 and the remaining 18th, 19th, 20th, 21st, 38th, 39th, 41st, 42nd, 43rd, 45th, 48th, 49th, 53rd, 56th and 57th amino acids of HA2 of the proximal monomer. It specifically binds to an epitope containing the group and the amino acid residues 327, 328, 329 of HA1 and the amino acid residues 1, 2, 3 and 4 of HA2 of the distal right monomer, and is described above. The antibody or antigen-binding fragment thereof according to claim 11, wherein the position and the distal right-side monomer are uncleaved. The 318th amino acid of HA1 and the 18th, 19th, 20th, 21st, 38th, 39th, 41st, 42nd, 45th, 46th, 49th, 52nd, 53rd and 57th amino acid residues of HA2 of the proximal monomer. In addition, the proximal and distal right side monomers specifically bind to an epitope containing the 321st and 323rd amino acid residues of HA1 and the 7th and 11th amino acid residues of HA2 of the distal right side monomer. The antibody according to claim 11 or an amino acid-binding fragment thereof, which is cleaved. It specifically binds to an epitope containing the 329th amino acid of HA1 and the 1st, 2nd, 3rd and 4th amino acid residues of HA2, and the HA1 and HA2 are contained in the uncleaved monomer of the HA trimer. The antibody according to claim 11 or an antigen-binding fragment thereof, which is present. The antibody or antigen-binding fragment thereof is associated with H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 of the HA subtype of influenza A. The antibody according to claim 11 or an antigen-binding fragment thereof, which specifically binds to the antibody. It neutralizes the infection of Group A and Group 2 subtypes of influenza A virus and: (i) as shown in SEQ ID NOs: 1, 41 and 43, respectively, or as SEQ ID NOs: 1, 41 and 42, respectively. Heavy chain CDR1, CDR2 and CDR3 sequences as shown in; and (ii) Light chain CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively, or as shown in SEQ ID NOs: 44, 5 and 6, respectively. Isolated antibodies or antigen-binding fragments thereof, including sequences. A heavy chain variable region having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 59 or 55 and a light chain having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 57 or 61. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, which comprises a variable region. Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59 and light chain variable region containing the amino acid sequence of SEQ ID NO: 57; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59 and light chain containing the amino acid sequence of SEQ ID NO: 61. Variable region; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 55 and light chain variable region containing the amino acid sequence of SEQ ID NO: 57; or heavy chain variable region containing the amino acid sequence of SEQ ID NO: 55 and the amino acid sequence of SEQ ID NO: 61. An isolated antibody or an antigen-binding fragment thereof comprising a light chain variable region containing, wherein the antibody neutralizes the first group subtype and the second group subtype of influenza A virus. Those antigen-binding fragments. The antibody according to any one of claims 1 to 23, wherein the antibody is a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, Fab, Fab', F (ab') 2, Fv or scFv. Or their antigen-binding fragments. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 24 for treating an infection with influenza A virus. A nucleic acid molecule containing a polynucleotide encoding the antibody according to any one of claims 1 to 25 or an antigen-binding fragment thereof. 26. The nucleic acid molecule of claim 26, wherein the polynucleotide comprises a sequence that is at least 75% identical to the nucleic acid sequence as set forth in any of SEQ ID NOs: 45-54, 56, 58, 60 or 62. 26. The nucleic acid molecule of claim 26, wherein the polynucleotide comprises the sequences set forth in SEQ ID NOs: 60 and 58; or SEQ ID NOs: 60 and 62; or SEQ ID NOs: 56 and 58; or SEQ ID NOs: 56 and 62. A vector comprising the nucleic acid molecule of claim 26, 27, or 28. A cell expressing the antibody according to any one of claims 1 to 25 or an antigen-binding fragment thereof, or comprising the vector according to claim 29. An immunogenic isolated or purified polypeptide comprising the epitope according to any one of claims 1 to 25, or an epitope that binds to an antigen-binding fragment thereof. The antibody according to any one of claims 1 to 25 or an antigen-binding fragment thereof, the nucleic acid according to any one of claims 26 to 28, the vector according to claim 29, and the invention according to claim 30. A pharmaceutical composition comprising cells, or the immunogenic polypeptide of claim 31, and a pharmaceutically acceptable diluent or carrier. The antibody according to any one of claims 1 to 25 in (i) production of a drug for treating influenza A virus infection, (ii) vaccine, or (iii) diagnosis of influenza A virus infection. Or an antigen-binding fragment thereof, the nucleic acid according to any one of claims 26 to 28, the vector according to claim 29, the cell according to claim 30, or the immunogenic polypeptide according to claim 31. , Or the use of the pharmaceutical composition according to claim 32. Any of claims 1-25 for monitoring the quality of an anti-influenza A virus vaccine by ensuring that the antigen of the vaccine contains a specific epitope having an appropriate three-dimensional structure. Use of the antibody according to paragraph 1 or an antigen-binding fragment thereof. The antibody according to any one of claims 1 to 25, which is a method for reducing the infection of influenza A virus or the risk of infection with influenza A virus and effective for prevention or treatment. A method comprising the step of administering an antigen-binding fragment to a subject in need thereof.

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