HK1191974A

HK1191974A - Influenza virus neutralizing antibody and method for screening same

Info

Publication number: HK1191974A
Application number: HK14105188.5A
Authority: HK
Inventors: 黒泽良和; 伊庭善孝; 大岛信子; 奥野良信
Original assignee: Perseus Proteomics Inc.
Priority date: 2010-09-03
Filing date: 2011-09-02
Publication date: 2014-08-08

Description

Anti-influenza virus neutralizing antibody and screening method thereof

[ technical field ]

The present invention relates to an anti-influenza virus antibody which surpasses the barrier between subtypes and shows a general neutralizing activity against all influenza viruses, a method for producing the same, and a method for detecting the same in a subject.

[ background art ]

Influenza virus is an RNA enveloped virus with particles about 100nm in diameter and belongs to the orthomyxoviridae family. They can be classified into type 3 according to their intrinsic protein antigenicity: type a (type a), type B (type B) and type C (type C). Influenza viruses are composed of an inner nucleocapsid surrounded by a viral envelope having a lipid bilayer structure or a ribonucleic acid (RNA) core that meets with nucleoproteins, and an outer glycoprotein. The inner layer of the viral envelope is formed primarily by matrix proteins, while the outer layer is mostly formed by host-derived lipids. Influenza virus RNA exhibits a segmented structure. Influenza spreading worldwide is caused by influenza a viruses. The type a virus HAs two envelope glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA). HA is classified into 16 subtypes and NA is classified into 9 subtypes according to antigenicity.

In recent years, highly pathogenic H5N1 type avian influenza virus has spread fiercely worldwide; such a situation is not strange even when a new virus that infects humans appears and causes a pandemic. In order to cope with this situation, the global virus detection system is being enhanced, therapeutic drugs such as Tamiflu (Tamiflu) are being stocked in large quantities, and development, production and mass stocking of vaccines are also being performed. However, the situation is still much unexplained, including when and in what form such viruses appear, when tamiflu et al have a therapeutic effect, whether the vaccine developed is effective, when and to whom it was vaccinated, when to start a high warning state and when to declare the state to be released. This is because we have come to a situation that has never been experienced by humans in which vaccines and therapeutic drugs are prepared against pathogens and viruses that have not yet emerged. In particular, vaccine development is problematic due to the fact that many mutations are introduced into the hemagglutinin gene every year in the influenza virus genome, resulting in antigenic drift (antigenic change), which is considered to be the cause of epidemic epidemics. Therefore, vaccination with a vaccine that does not match the prevailing type does not have the expected prophylactic effect. The reason why no attempt is made to develop an antibody therapeutic drug (prophylactic drug) for influenza virus is that there is a fear that viruses that could be neutralized during development change their characteristics by antigenic drift, and thus drugs developed by great efforts are no longer useful.

The present inventors screened a phage-displayed human antibody library made from a large number of B lymphocytes collected from a single individual against 12 influenza H3N2 virus strains isolated during 1968-2004, and as a result, found that most of the clones showing neutralizing activity were anti-hemagglutinin antibodies, and they could be roughly classified into three groups: an antibody that specifically neutralizes a virus strain isolated in the years 1968-. Although this finding breaks the general understanding that the conventional development of antibody therapeutic drugs (prophylactic drugs) for influenza viruses is not meaningful, phage antibody libraries have not been studied seriously so far because the combination of heavy and light chains does not necessarily reflect the in vivo situation, and the like.

Under such circumstances, human monoclonal antibodies capable of neutralizing influenza virus H5 were successfully isolated independently by three groups (patent documents 1 and 2, non-patent documents 2 to 4). These antibodies have neutralizing activity not only against influenza virus type H5 but also against other subtypes (e.g., type H1, etc.). However, although 16 subtypes of hemagglutinin (H1-H16) can be divided into two large groups (groups 1 and 2) according to epitopes, these antibodies show neutralizing activity only to group 1 (e.g., H1, H2, H5, H6, H8, H9 type, etc.) among them, and do not show neutralizing activity to influenza viruses belonging to group 2 (e.g., H3, H7 type, etc.). That is, an anti-influenza virus antibody which can exert a wide neutralizing activity beyond the obstacle between two groups classified based on the sequence of hemagglutinin has not been isolated and reported so far.

X-ray structural analysis showed the binding pattern of these antibodies to hemagglutinin, and it was apparent that amino acid 38 of hemagglutinin was changed to asparagine in H3 and H7 forms of group 2, and underwent N-type sugar chain modification (non-patent documents 4 and 5). Furthermore, it has been reported that introduction of an N-type sugar chain modification site at position 38 of H5 reduces the binding ability of a neutralizing antibody by 70% (non-patent document 5). It is also known that when influenza viruses having mutations in hemagglutinin escape neutralizing antibodies, these mutations are mainly concentrated in 5 regions (A, B, C, D and E region) within the hemagglutinin gene which reportedly contain neutralizing epitopes (non-patent documents 6 and 7). These findings suggest that it is difficult to obtain neutralizing antibodies that can go beyond the barrier between the two groups.

[ Prior art documents ]

[ patent document ]

[ patent document 1] WO2007/134327

[ patent document 2] WO2008/028946

[ non-patent document ]

[ non-patent document 1] Virology Vol.397, pp.322-330,2010

[ non-patent document 2] Proc. Natl.Acad.Sci.USA., Vol.105, pp.5986-5991,2008

[ non-patent document 3] PLoS ONE, Vol.3, pp.5986-5991, e3942,2008

[ non-patent document 4] Nature Structural & Molecular Biology, Vol.16, pp.265-273,2009

[ non-patent document 5] Science, Vol.324, pp.246-251,2009

[ non-patent document 6] Nature, Vol.289, pp.373-378,1981

[ non-patent document 7] J.Gen.Virol., Vol.62, pp.153-169,1982

[ summary of the invention ]

[ problems to be solved by the invention ]

In preparation for coping with a pandemic of influenza type H5N1 predicted to occur in the near future and a pandemic by viruses of H7 and H9 that are likely to occur later, it is highly necessary to design a vaccine instead of predicting a change in antigenicity as a conventional preventive concept against influenza, and a preventive means based on a more general, more reliable new concept is developed.

Accordingly, it is an object of the present invention to provide an anti-influenza virus antibody which exerts neutralizing activity exceeding the obstacles between groups 2 classified according to the conservation of the amino acid sequence of the hemagglutinin protein, preferably an antibody having neutralizing activity against influenza viruses of all subtypes of H1-H16 types, and a method for producing the same. Further, it is another object of the present invention to provide a detection method capable of simply and relatively inexpensively investigating whether a subject carries the above-mentioned general-purpose neutralizing antibody.

[ means for solving problems ]

The inventors used apheresis (apheresis)(isolation of one component of the collected blood), 10 samples were taken from a single individual⁹A phage-displayed human antibody library reflecting almost the entire antibody spectrum was prepared from a large number of such B lymphocytes, and antibody clones that bind to the antigen were screened in a comprehensive manner by panning method using inactivated H3N2 influenza virus as the antigen. Antibodies were recovered from the selected phages and tested for their neutralizing activity against influenza viruses type H3 belonging to group 2 and influenza viruses types H1, H2 and H5 belonging to group 1. As a result, it was surprisingly found that an antibody of 40 or more clones showing neutralizing activity not only to H3 type used as a screening antigen but also to influenza viruses of H1 type, H2 type and H5 type belonging to a different group from that of H3 type was successfully obtained. These clones had 6 different heavy chain variable domain (VH) amino acid sequences, but all of these sequences were subjected to IgBLAST search and were judged to belong all to VH1-69 germline (germline). Moreover, IgBLAST searches showed that these 6 clones had light chain variable domains (VL) limited to 3 germline types.

Considering that the previously reported neutralizing antibodies against the H5 type were based on VH1-69 or similar VH1-e, and the binding pattern of the neutralizing antibodies with hemagglutinin as shown by X-ray structural analysis, it is a very surprising matter that the antibodies obtained by the present inventors are capable of neutralizing the H3 type influenza virus. However, the present inventors have thought that, when searching for an antibody capable of neutralizing a virus subtype belonging to a group different from the antigen, it is possible to obtain an antibody capable of neutralizing all influenza virus subtypes beyond the barrier between groups by thoroughly screening an antibody that interacts with a certain subtype as an antigen. Thus, the present inventors collected 10 from one individual⁹B lymphocytes, two orders of magnitude larger than conventional practice, and generated a human antibody library that almost completely reflected the donor antibody profile, screened antibody clones that could bind influenza virus H3 comprehensively, and examined for their neutralizing activity against influenza viruses H1, H2 and H5, thereby successfully isolating antibodies capable of neutralizing influenza viruses in both group 1 and group 2 for the first time.

Analysis of the amino acid sequences of the resulting neutralizing antibodies showed that all neutralizing antibodies used the VH1-69 gene as the heavy chain variable domain V region. Notably, clones with higher neutralizing activity against influenza virus in group 1 than other clones were found to have one amino acid deletion in the heavy chain variable domain V region compared to other clones. Because the process of transformation (antibody maturation) of antibody-producing cells under growth and differentiation stimuli is accompanied by cleavage of one of the DNA double strands encoding the heavy and light chains, and mutations are introduced during the process that often results in repair of the cleavage by the wrong DNA polymerase, the resulting mutations are almost all based on amino acid substitutions of single base substitutions. Therefore, the frequency of deletion of one amino acid (3 bases) is extremely low; even if such deletion occurs, since it has almost bad influence, it stimulates B cells producing the antibody, and further the possibility of the mechanism that exists in the body as a memory cell for a long time is further reduced. In view of the common technical knowledge in the related art, it was surprising to find that an antibody which essentially neutralizes influenza viruses of group 2 acquires an activity which can also strongly neutralize influenza viruses belonging to group 1 due to the deletion of one amino acid in the heavy chain variable domain other than CDR 3.

The present inventors have further studied based on these findings and have formed the present invention.

Accordingly, the present invention provides:

[1] an isolated antibody which neutralizes at least one influenza virus selected from group 1 and at least one influenza virus selected from group 2, wherein group 1 consists of influenza viruses of H1 type, H2 type, H5 type, H6 type, H8 type, H9 type, H11 type, H12 type, H13 type, and H16 type, and group 2 consists of influenza viruses of H3 type, H4 type, H7 type, H10 type, H14 type, and H15 type;

[2] the antibody according to [1] above, wherein the antibody neutralizes at least influenza virus of H1 type and/or H5 type, and neutralizes influenza virus of H3 type;

[3] the antibody according to [1] above, wherein the antibody neutralizes influenza virus types H1-H16;

[4] the antibody according to [1] above, wherein the heavy chain variable domain V region utilizes VH1-69 or VH1-e gene;

[5] the antibody according to [1] above, wherein the heavy chain variable domain V region has an amino acid deletion;

[6] the antibody according to [5] above, wherein the heavy chain variable domain V region encoded by VH1-69 or VH1-e gene has at least a mutation deleting glycine at position 27;

[7] the antibody according to [1] above, wherein the light chain variable domain V region utilizes VL1-44, VL1-47 or VL1-51 gene;

[8]according to [1] above]The antibody of (1), wherein the minimum inhibitory concentration in a focus formation inhibition test (focus formation inhibition test) of the antibody at the time of conversion into an IgG type is 10^-11-10^-12Magnitude of M;

[9] the antibody according to [1] above, wherein the antibody is a human antibody;

[10] the antibody according to [1] above, wherein the complementarity determining region 1 of the heavy chain variable domain consists of the amino acid sequence represented by SEQ ID NO.1, and the complementarity determining region 2 consists of the amino acid sequence represented by SEQ ID NO. 2;

[11] the antibody according to [10] above, wherein the framework region 1 of the heavy chain variable domain consists of the amino acid sequence represented by SEQ ID NO. 3;

[12] the antibody according to [1] above, wherein the heavy chain variable domain V region consists of the amino acid sequence shown in any one of SEQ ID Nos. 4 to 9;

[13] the antibody according to [1] above, wherein the heavy chain variable domain consists of an amino acid sequence represented by any one of SEQ ID Nos. 10 to 15;

[14] the antibody according to [1] above, wherein the heavy chain variable domain and the light chain variable domain are each composed of an amino acid sequence shown in any one of the following (a) to (l):

(a) serial number 10 and serial number 16;

(b) sequence number 10 and sequence number 17;

(c) serial number 10 and serial number 18;

(d) serial number 10 and serial number 19;

(e) serial number 10 and serial number 20;

(f) serial number 10 and serial number 21;

(g) serial number 10 and serial number 22;

(h) serial No. 11 and serial No. 23;

(i) sequence number 13 and sequence number 24;

(j) sequence number 14 and sequence number 25;

(k) serial number 15 and serial number 26, and

(l) Serial number 12 and serial number 70.

[15] A passive immunotherapeutic agent for influenza, which comprises an antibody according to [1] above;

[16] a passive immunotherapy method for influenza, which comprises administering an effective amount of an antibody according to [1] above to a subject mammal or bird that has been infected with influenza virus or has a possibility of infection;

[17] the method according to [16] above, wherein the subject is a human;

[18] a method for producing an antibody according to [1] above, comprising the steps of:

(1) providing an antibody library comprising antibody clones from about 10 collected from an individual⁸More than one B cell is selected from the group consisting of,

(2) an influenza virus selected from H1 to H16, or a hemagglutinin protein of the virus, or an extracellular domain thereof, is contacted with an antibody library (1) as an antigen, and antibody clones reactive with the antigen are screened in a comprehensive manner,

(3) recovering antibody molecules from each of the antibody clones screened in step (2),

(4) detecting the neutralizing activity of the antibody against at least one influenza virus selected from group 1 and at least one influenza virus selected from group 2 for each antibody obtained in step (3), and

(5) producing an antibody that neutralizes both influenza viruses belonging to group 1 and influenza viruses belonging to group 2 using a clone that produces the antibody, and recovering the antibody;

[19] the method according to [18] above, wherein the antibody is a human antibody;

[20] the method according to [18] above, wherein the antibody library is a phage display library;

[21]according to [20] above]The method of (1), wherein the number of antibody clones is 10¹⁰-10¹¹；

[22] The method according to [18] above, wherein the B cells are collected by apheresis;

[23] the method according to [18] above, wherein, in the step (2) above, an influenza virus isolate from which the individual from which the B cells have been collected has no history of infection, or a hemagglutinin protein thereof, or an extracellular domain thereof, is used as the antigen;

[24] the method according to [23] above, wherein the influenza virus isolate belongs to the H1, H2 or H3 type;

[25] the method according to [23] above, wherein the influenza virus isolate belongs to a hemagglutinin subtype with no history of infection in the B cell-collected individual;

[26] the method according to [25] above, wherein the influenza virus isolate belongs to the H5, H7 or H9 type;

[27] the method according to [18] above, comprising detecting neutralizing activity against at least influenza virus type H1 and/or H5 and influenza virus type H3 in step (4) above;

[28] the method according to [20] above, further comprising a step of converting the antibody into an IgG type;

[29] a method for detecting an antibody according to [1] above in a subject, comprising the steps of:

(1) inoculating a subject with hemagglutinin of a subtype of any one of types H1 to H16,

(2) after inoculation, when the antibody-producing cells have sufficiently expanded, collecting blood from the subject, and

(3) examining the blood for the presence of antibody-producing cells presenting an antibody that binds to both hemagglutinin of a subtype selected from group 1 and hemagglutinin of a subtype selected from group 2 and has a heavy chain variable domain V region encoded by VH1-69 or VH1-e gene;

[30] a method for detecting an antibody according to [1] above in a subject, comprising the steps of:

(1) inoculating the subject with hemagglutinin from a subtype selected from group 1 and hemagglutinin from a subtype selected from group 2, respectively,

(2) after each hemagglutinin inoculation, when the antibody-producing cells have sufficiently expanded, collecting blood from the subject, and

(3) examining each blood for the presence or absence of antibody-producing cells presenting an antibody that binds to hemagglutinin of a subtype selected from the group different from the inoculated hemagglutinin and has a heavy chain variable domain V region encoded by VH1-69 or VH1-e gene,

and the like.

[ Effect of the invention ]

According to the present invention, a human antibody having a neutralizing ability against all influenza virus hemagglutinin subtypes can be obtained. Passive immunization with the neutralizing antibody is effective in preventing or treating influenza, even in the event of antigenic drift and antigenic shift. The invention also enables determining whether a subject has memory B cells that can produce antibodies that exhibit neutralizing activity against influenza viruses beyond interclass barriers.

[ brief description of the drawings ]

FIG. 1-1 shows the results of ELISA determination of the binding activity of antibodies screened against a phage-displayed human antibody library with the H3N2 strain against all 12H 3N2 strains and one H1N1 strain.

FIGS. 1-2 show the results of ELISA determination of the binding activity of antibodies screened against a phage-displayed human antibody library with the H3N2 strain against all 12H 3N2 strains and one H1N1 strain.

Fig. 2 shows which influenza H3 virus strain was used as an antigen when screening clones classified in groups 11 and 22.

Figure 3 shows the influenza strain used to screen each antibody clone in group 11, as well as the number of clones isolated.

Fig. 4 shows the amino acid sequences of the cloned VH and VL in group 11, wherein the dot indicated in FR045-092 FR1 region ". indicates the missing amino acid.

Fig. 5 shows the results of comparing the clones in group 11 with the amino acids of the heavy chain variable domain of the antibody that has been reported to show neutralizing activity against both the H1 strain and the H5 strain, wherein the dot shown in the FR1 region of FR045-092 "", indicates the missing amino acid.

Fig. 6 shows the results of ELISA determination of the binding activity of the clones in group 11 against the H3N2 influenza strain.

FIG. 7 provides a Western blot showing that the clones in group 11 recognize HA of strains H3 and H1.

FIG. 8 shows the results of the study of the hemagglutination inhibitory activity of the clones in group 11 against influenza viruses H3 and H1.

Fig. 9 shows the results of the range formation inhibitory activity study of the clones in group 11 against influenza viruses H3 and H1.

FIG. 10 shows the results of ELISA determination as to whether the clones in group 11 competitively inhibited the binding activity of mouse monoclonal antibody C179 to influenza virus H3. ELISA experiments were performed with influenza A New Caslidonian strain of C179 in the presence of Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092, F005-126) and in the absence (no Fab-p 3), wherein F005-126, which was non-reactive with influenza A New Caslidonian strain HA, was used as a negative control.

FIG. 11 shows the results of ELISA determination as to whether mouse monoclonal antibody C179 competitively inhibits the binding activity of the clones in group 11 to influenza virus H3. The effect of Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092) on influenza A New Caslidonia Soviet-type strains was determined in the presence (C179(+)) and absence (C179(-)) of C179.

Fig. 12 shows the results of ELISA determination of whether antibody clone F005-1269 shown in fig. 2, which has broad strain specificity for influenza H3 virus strain, and antibody clones recognizing both influenza H3 and H1 viruses in group 11 share epitopes. The effect of F005-126 of Fab-PP type on Airtight strains of influenza A hong Kong type was determined by performing an ELISA experiment in the presence of antibodies to Fab-p3 (F022-360, F026-146, F026-427, F045-092, F005-126, F019-102) and in the absence (no Fab-p 3), wherein F005-126 of Fab-p3 type was used as a positive control for competitive inhibition and F019-102 was used as a negative control for non-reaction with Airtight strains of influenza A hong Kong type.

FIG. 13 shows the results of FACS analysis of the binding capacity of the clones in group 11 to cells expressing hemagglutinin from influenza virus type H3, with all grey peaks from the negative control F008-038 and the solid line peaks from a) F026-427, b) F045-092 and c) F49, respectively.

Fig. 14 shows the measurement results of the neutralizing activity of each IgG antibody against influenza viruses H3, H5, H2 and H1, in which the vertical axis represents the infection inhibition rate (%), and the horizontal axis represents the antibody concentration (μ g/mL).

FIG. 15-1 shows a graphical representation of the reactivity of Fab clones to influenza A/H3N2 Airy strains HA0 and HA1, where the gray peak indicates mock transfection results, the green peak indicates pDisp-Aic68HA0 transfection results, the pink peak indicates pDisp-Aic68HA1 transfection results, the blue peak indicates pDisp-Fuk85HA0 transfection results, and the orange peak indicates pDisp-Fuk85HA1 transfection results.

FIG. 15-2 shows a graphical representation of the reactivity of Fab clones to influenza A/H3N2 Airy strains HA0 and HA1, where the gray peak indicates mock transfection results, the green peak indicates pDisp-Aic68HA0 transfection results, the pink peak indicates pDisp-Aic68HA1 transfection results, the blue peak indicates pDisp-Fuk85HA0 transfection results, and the orange peak indicates pDisp-Fuk85HA1 transfection results.

FIG. 16 shows the results of ELISA determination of whether antibody F004-104 recognizing epitope B of the HA molecule shares an epitope with the antibody clones in group 11. The effect of monoclonal antibodies F026-427p3, F045-092p3, F004-104p3 of Fab-p3 and of the mouse-derived anti-influenza A/H3N2 antibody F49 on the strain influenza A/H3N2 Panama was determined by ELISA in the presence (F026-427IgG, F045-092IgG, F004-104IgG) and in the absence (no IgG) of IgG antibodies.

Fig. 17-1 shows the amino acid sequences of HA1 and HA2 of influenza virus subtypes and each strain.

Fig. 17-2 shows the amino acid sequences of HA1 and HA2 for influenza virus subtypes and each strain.

Fig. 18 shows the results of FACS analysis of the binding ability of various HA antibodies to cells expressing hemagglutinin produced by replacing serine residue 136 of hemagglutinin from an Aic68 influenza strain with threonine or alanine, where the gray peak indicates the results of mock transfection, the green peak indicates the results of HA of wild-type Aic68, the blue peak indicates the results of S136T of HA of Aic68, and the pink peak indicates the results of S136A of HA of Aic 68.

FIG. 19 shows the results of FACS analysis of the binding capacity of F045-092 to cells expressing mutant HA1 produced by mutual replacement of amino acids at positions 142-146 or 133-137 from HA1 of various H3N2 influenza viruses, wherein the gray peak indicates the result of mock transfection, the green peak indicates the reactivity to wild type, the pink peak indicates the reactivity to the chimera 142A grafted with the amino acid sequence at position 142-146, and the blue peak indicates the reactivity to the chimera 133A grafted with the amino acid sequence at position 133-137.

FIG. 20 shows three-dimensional structures of amino acid portions at positions 91-260 of a mutant HA1 region generated by mutually replacing amino acids at positions 142-146 and 133-137 of HA1 from various H3N2 influenza viruses, wherein the receptor binding region shows orange, the amino acids at positions 133-137 of Aic68_ wild type show blue, the amino acids at positions 142-146 show brilliant blue, the amino acids at positions 133-137 of Wyo03_ wild type show red, the amino acids at positions 142-146 show pink, the amino acids at positions 133-137 of Fuk85_ wild type show green, and the amino acids at positions 142-146 show yellow-green.

Fig. 21 shows the three-dimensional structure of the amino acid portions from positions 91 to 260 of the HA1 region of H3N2 influenza virus, the site of the HA1 region recognized by various antibodies, and the name of a virus strain used for determining the site by the EMAC method, in which the receptor binding region shows orange color, the site recognized by each anti-HA antibody shows pink color, the amino acid number within the receptor binding region shows black color, the amino acid number of the antigen recognition site shows blue color, and the amino acid contained in the receptor binding region as the antigen recognition site shows blue color.

FIG. 22 is a graphical representation of the results of competition experiments between various anti-HA antibodies that bind to the A, B, C, D and E sites in HA1 and F045-092 antibodies, wherein the left panel was generated with F045-092 as the competitor and the right panel was generated with a cp3 type anti-HA antibody as the competitor (+: with cp3 antibody, —: without cp3 antibody). The virus strains used are shown in the lower left of the figure. Fab-pp free by using PBS instead of pp-type antibody; no Ab PBS was used instead of all antibodies.

[ embodiments of the invention ]

Herein, influenza virus includes all currently known subtypes, even subtypes that may be isolated and identified in the future. The currently known influenza virus subtypes include a combination of subtypes selected from the group consisting of hemagglutinin type selected from H1-H16 and neuraminidase type selected from N1-N9.

Influenza viruses can be roughly divided into two groups based on the amino acid sequence similarity of hemagglutinin. Here, a group consisting of influenza viruses of H1 type, H2 type, H5 type, H6 type, H8 type, H9 type, H11 type, H12 type, H13 type, and H16 type is referred to as group 1, and a group consisting of influenza viruses of H3 type, H4 type, H7 type, H10 type, H14 type, and H15 type is referred to as group 2. The ceramidase subtypes are not considered when selecting the classes in these groups. The new subtypes isolated and identified in the future will be classified into group 1 or group 2 based on the amino acid sequence similarity of hemagglutinin.

The present invention provides an isolated antibody, which neutralizes at least one influenza virus selected from group 1 (H1 type, H2 type, H5 type, H6 type, H8 type, H9 type, H11 type, H12 type, H13 type, and H16 type) and at least one influenza virus selected from group 2 (H3 type, H4 type, H7 type, H10 type, H14 type, and H15 type). Preferably, the neutralizing antibody of the present invention neutralizes at least influenza viruses H1 and/or H5 in group 1 and at least influenza virus H3 in group 2. More preferably, the neutralizing antibody of the present invention further neutralizes influenza virus H9 in group 1 and further neutralizes influenza virus H7 in group 2. The neutralizing antibody of the present invention particularly preferably neutralizes all influenza viruses of types H1-H16, most preferably even novel influenza viruses of the hemagglutinin subtype which will be isolated and identified in the future.

The neutralizing antibody of the present invention can be produced by a method comprising the steps of:

(5) the antibody is produced using a clone that produces an antibody that neutralizes both influenza viruses belonging to group 1 and influenza viruses belonging to group 2, and the antibody is recovered.

The collection of B cells as donors of antibody-producing cells for the production of antibody libraries may be any mammal (e.g., human, pig, horse, etc.) or bird (chicken, duck, etc.) that has been infected with influenza virus; the same animal species as the subject to be passively immunized with the neutralizing antibody of the present invention can be appropriately selected, for example, a human being. In the case of selecting a human, the age, sex, vaccination status (vaccinated or unvaccinated), and the like of the donor are not limited; however, since the donor desirably has as many influenza virus infection experiences as possible, the donor is preferably over 20 years old, preferably over 30 years old, further preferably over 40 years old, particularly preferably over 50 years old, however, this is not a limitation. Antibodies that exhibit neutralizing activity beyond the interclass barrier, because of their ability to neutralize all viral isolates within the group and within the subtype, donors with cells that can produce such neutralizing antibodies are considered to be less susceptible to annual seasonal influenza infection. Thus, people who have no history of influenza a virus infection in a given period of time in the past are more desirable.

The amount of blood collected for collecting B cells sufficient for preparing a general antibody library is about 20-30mL, and the number of B cells contained in the blood is about 10⁷. All groups that isolated human neutralizing antibodies against highly pathogenic H5N1 avian influenza virus produced approximately 10% of their total serum content by collecting a common volume of blood from multiple donors¹⁰Antibody libraries of individual clones. But originally send outAttempts have been made to generate antibody libraries of a size that reflects the entire antibody spectrum in order to obtain antibodies that bind to a hemagglutinin subtype in a thorough (comprehensive) manner. Typically, the limit of blood volume collected from one person in a single operation is about 200-; thus, the number of B cells that can be harvested using this method is about 10⁸. Therefore, the present inventors collected more than the above amount of B cells contained in blood from one individual using apheresis. Preferably, the antibody library for the purposes of the present invention is composed of 10⁹More than one B cell. For example, the isolation of B cells from about 3L of blood by apheresis may enable the collection of about 10 from a human individual⁹And (4) B cells.

So long as it contains about 10 from one individual⁸One or more, preferably about 10⁹The one or more B cells, the antibody-producing cells used to generate the antibody library may further comprise antibody-producing cells from another individual. Specifically, for example, the number of mononuclear cells corresponding to about 3L of blood is recovered by apheresis, after which B cells are separated and recovered by a method such as Ficoll-Paque density gradient centrifugation.

Antibody libraries include, but are not limited to, for example, phage display libraries, libraries obtained by immortalizing B cells with EB virus, hybridoma libraries obtained by fusing B cells with myeloma cells, and the like. Preferably, a phage display library can be used.

Examples of methods for generating phage-displayed human antibody libraries as referred to herein include, but are not limited to, the following.

Although the selection of the phage used is not particularly limited, filamentous phage (Ff phage) is generally preferably used. Methods for presenting a foreign protein onto a phage surface include, a method of expressing a foreign protein and presenting it onto an envelope protein as a fusion protein with one of the envelope proteins g3p (cp3) and g6p (cp6) -g9p (cp9), and a method generally used in which a foreign protein is fused to the N-terminus of cp3 or cp 8. The phage display vector includes 1) a method of introducing a foreign gene into a phage genome envelope protein gene in a fused form so that all envelope proteins are presented on the surface of a phage as a fusion protein with the foreign protein, and 2) a method of inserting a gene encoding a fusion protein separately from a wild-type envelope protein gene so that the fusion protein and the wild-type envelope protein are expressed simultaneously, and 3) a method of infecting Escherichia coli carrying a phagemid vector carrying the gene encoding the fusion protein with a helper phage having the wild-type envelope protein gene and producing phage particles expressing the fusion protein and the wild-type envelope protein simultaneously. In the example of 1), fusion with a large foreign protein results in loss of infectivity; thus, in this case, an antibody library is generated using a type 2) or 3) method.

Specifically, useful vectors include those described by Holt et al (Curr. Opin. Biotechnol.,11: 445-. For example, pCES1 (see J.biol.chem.,274:18218-18230,1999) is a Fab expressing phagemid vector carrying DNA encoding the kappa L chain constant region placed downstream of the cp3 signal peptide, encoding C_H3And the cp3 coding sequence was placed downstream of the cp3 signal peptide by a His-TAG, a c-myc TAG and an amber stop codon (TAG), under the control of a lactose promoter. This vector, when introduced into E.coli with an amber mutation, presents Fab onto the cp3 envelope protein. However, when expressed in HB2151 strain without the amber mutation or the like, this strain produced soluble Fab antibody. Useful scFv-expressing phagemid vectors include, for example, pHEN1(J.mol.biol.,222:581-597,1991), and the like.

Meanwhile, helper phages include, for example, M13-KO7, VCSM13, and the like.

Other phage display vectors include those designed to link a sequence containing a codon encoding cysteine to the 3 'end of an antibody gene and the 5' end of an envelope protein gene, thereby expressing both genes simultaneously and separately (not as fusion proteins), and to present an antibody to the envelope protein on the surface of phage through an S-S bond between introduced cysteine residues (CysDisplayTM technology by Morphosis), and the like.

The types of antibody libraries generated in the present invention include natural (naive)/non-immune libraries, synthetic libraries, immune libraries, and the like.

Natural (naive)/non-immune libraries were obtained by RT-PCR to obtain VH and VL genes retained by normal animals and randomly cloning them into one of the phage display vectors described above. Generally, mRNA or the like derived from lymphocytes (preferably peripheral blood lymphocytes) of peripheral blood, bone marrow, tonsil of a normal animal is used as a template. Libraries generated by amplifying only mRNA from IgM that has not undergone a species shift due to antigen sensitization, thereby avoiding bias associated with the V gene, are specifically referred to as natural libraries.

Representative natural/non-immune libraries include libraries from CAT (see J.mol.biol.,222: 581-; 597,1991; nat.Biotechnol.,14: 309-; 314,1996), libraries from MRC (see Annu.Rev.Immunol.,12: 433-; 455,1994), libraries from Dyax (see J.biol.Chem.,1999(ibid.); Proc.Natl.Acad.Sci.USA,14: 7969-; 7974,2000), and the like.

Synthetic libraries are generated by selecting specific antibody genes that function in human B cells and replacing a portion of the V gene fragment, e.g., the antigen-binding portion of CDR3, with DNA encoding a random amino acid sequence of appropriate length. Synthetic libraries are considered to be excellent in terms of antibody expression efficiency and stability because they can be constructed with a combination of VH and VL genes that can produce scFv and Fab that are functional from the outset. Representative examples include the Morphosys HuCAL library (see J.mol.Biol.,296:57-86,2000), the BioInvent library (see Nat.Biotechnol.,18:852,2000), the Crucell library (see Proc.Natl.Acad.Sci.USA,92:3938,1995; J.Immunol.methods,272:219-233,2003), and the like. When synthetic libraries are used, it is desirable to use VH1-69 and VH1-e gene segments as the V gene segments for the heavy chain variable domains.

The immune library is generated by preparing mRNA from lymphocytes collected from a human having a high blood antibody titer against a target antigen, such as a recipient for vaccination, or from lymphocytes collected from a human artificially immunized with a target antigen by external immunization or the like, in the same manner as the above-described natural/non-immune library, and amplifying VH and VL genes by RT-PCR. Since the desired antibody gene is already present in the library from the beginning, the desired antibody can be obtained even when the library volume is relatively small. However, in the human example, because the antibody specific to the virus subtype injected by vaccination is amplified, when vaccination is performed with an influenza virus one of the H1-H3 hemagglutinin subtypes for which a number of antibodies already exist in the body, leads to amplification of antibodies with a narrow range of neutralizing activity, so that only specific isolates among the subtypes are neutralized; this is likely to mask the desired neutralizing antibodies. Therefore, at the time of inoculation, it is preferable to inoculate influenza viruses of a subtype whose widespread infection has not been reported so far (for example, in the human example, H5, H7, H9, etc.).

The greater the diversity of the library, the better; in practice, however, the number of phages (10) that can be processed in a subsequent panning operation (panning) is taken into account¹¹-10¹³One phage) and the number of phage required for clonal isolation and amplification in ordinary panning (100-1,000 phage/clone), a suitable library size is about 10⁸-10¹¹And (4) cloning. Preferably, for V_HAnd V_LGenes, size 10 respectively⁹And 10⁶The number of individual clones, Fab or scFv clones, was 10¹⁰-10¹¹And (4) cloning.

Methods for generating antibody libraries by immortalizing cells with EB virus include, but are not limited to, for example, the methods described in PLos Medicine4(5): e1780928-9936 (2007). Most people are immunized against the epstein barr virus because they have been infected with the virus in an asymptomatic infection with infectious mononucleosis; however, when using the common epstein barr virus, virosomes (virion) are still produced and therefore appropriate purification has to be performed. It is also preferred to use recombinant EB viruses that retain the ability to immortalize B lymphocytes, but lack virion replication capacity (e.g., lack of a switch gene for transition from a late stage infection state to a lytic infection state, etc.) as EB systems that are never contaminated with viruses.

Since the marmoset-derived B95-8 cells secrete EB virus, B lymphocytes can be easily transformed using the culture supernatant thereof. The antibody-producing B cell line can be obtained by, for example, culturing cells in a medium (e.g., RPMI 1640) supplemented with serum and penicillin/streptomycin (P/S) or a serum-free medium supplemented with a cell proliferation factor, followed by separating the culture by filtration, centrifugation or the like at an appropriate concentration (e.g., about 10)⁷Individual cells/mL) of antibody-producing B lymphocytes suspended therein and the suspension is incubated, usually for about 0.5-2 hours, at 20-40 ℃, preferably 30-37 ℃. When the human antibody-producing cells are provided as mixed lymphocytes, it is preferable to first remove the T lymphocytes by allowing them to form E rosettes with, for example, sheep red blood cells, etc., thereby increasing the transformation frequency, since most people have T lymphocytes toxic to cells infected with EB virus. It is also possible to select lymphocytes specific for the target antigen by mixing sheep red blood cells previously coupled with soluble antigen with antibody-producing B lymphocytes and separating rosettes using a Percoll density gradient or the like. Further, only antigen-non-specific B lymphocytes will form rosettes when mixed with sheep red blood cells previously coupled with anti-IgG antibodies, because they are covered by a large excess of added antigen, resulting in their no longer presenting IgG to the surface. Therefore, it is possible to select antigen-specific B lymphocytes by collecting a cell layer in which rosettes are not formed from the mixture with a Percoll density gradient or the like.

The antibody-secreting cells that have acquired an immortal ability due to transformation can be fused back with mouse or human myeloma cells (back-fused) so as to stably maintain the antibody-secreting ability. Examples of myeloma cells include mouse myeloma cells such as NS-1, P3U1, SP2/0 and AP-1, and human myeloma cells such as SKO-007, GM1500-6TG-2, LICR-LON-HMy2 and UC 729-6.

The generation of antibody libraries by cell fusion can be accomplished according to the general procedures used for hybridoma preparation for the production of monoclonal antibodies. Specifically, an antibody-producing hybridoma can be prepared by fusing a B cell collected from a donor with one of the myeloma cells described above.

The fusion operation can be performed according to known methods, for example, the method of Koehler and Milstein [ Nature, vol.256, p.495(1975) ]. The fusion promoter includes polyethylene glycol (PEG), Sendai virus, etc., and PEG, etc. is preferably used. Although the molecular weight of PEG is not particularly limited, PEG1000-PEG6000, which has low toxicity and relatively low viscosity, is preferable. Examples of PEG concentrations include about 10-80%, preferably about 30-50%. Useful solutions for diluting PEG include various buffered solutions, such as serum-free media (e.g., RPMI 1640), complete media containing about 5-20% serum, Phosphate Buffered Saline (PBS), and Tris buffer. DMSO (e.g., about 10-20%) can be added as desired. Examples of the pH of the fusion solution include about 4 to 10, preferably about 6 to 8.

The ratio of B cells to myeloma cells is typically about 1:1-20: 1; efficient cell fusion can be achieved by incubation at normal 20-40 ℃, preferably 30-37 ℃ for typically 1-10 minutes.

Hybridoma screening and propagation are typically performed in a medium suitable for animal cells containing 5-20% FCS (e.g., RPMI 1640) or serum-free medium supplemented with cell proliferation factors, and supplemented with HAT (hypoxanthine, aminopterin, thymidine). Examples of concentrations of hypoxanthine, aminopterin and thymidine include about 0.1mM, about 0.4. mu.M and about 0.016mM, respectively, and the like. To screen for human-mouse hybridomas, ouabain resistance may be used. Since the human cell line is more sensitive to ouabain than the mouse cell line, it is possible to add about 10 to the medium^-7-10^-3M ouabain scavenges unfused human cells.

In selecting hybridomas, it is preferable to use a culture supernatant of feeder cells or specific cells. As the feeder cell, an allogeneic cell type having a life limit so as to be dead after the occurrence of the hybridoma is used, a cell capable of producing a large amount of growth factors favorable for the occurrence of the hybridoma and having a proliferation potential reduced by radioactive irradiation or the like is used. For example, mouse trophoblasts include spleen cells, macrophages, blood, thymocytes, and the like; the human feeder cells include peripheral blood mononuclear cells and the like. Cell culture supernatants include, for example, primary culture supernatants of the various cells described above and culture supernatants of various established cell lines.

The step of selecting antibodies against the target antigen by the phage display method is called panning. Specifically, the phages presenting antigen-specific antibodies are concentrated by repeating the following series of operations approximately 2 to 4 times: a carrier (carrier) having immobilized thereon an influenza virus of any one of subtypes H1-H16 or a viral hemagglutinin protein or an extracellular domain thereof and a phage library are contacted with each other, unbound phages are washed away, and then the bound phages are eluted from the carrier and Escherichia coli is infected with the phages to amplify the phages. In the present invention, the influenza virus used as an antigen can be inactivated by formalin treatment. Preferably, the influenza virus isolate used as an antigen is never infected by the individual from which the B-cells were collected. This is because, when a virus isolate that an individual has been infected with is used as an antigen, it is feared that an antibody clone showing only a narrow-range neutralizing activity is dominant, thereby masking the neutralizing antibody desired in the present invention. Therefore, preferably, influenza viruses belonging to hemagglutinin subtypes, which have never been infected by individuals from which B cells were collected, or hemagglutinin thereof, can be used as antigens. Examples include influenza viruses of type H5, H7 and H9. Alternatively, when an influenza virus isolate belonging to any one of H1-H3 types is used as the antigen, it is desirable, for example, to use a subtype isolate that has been prevalent before the birth of an individual as a source of B cell collection.

The cDNA sequences encoding various subtypes of hemagglutinin are well known; recombinant hemagglutinin of any desired subtype can be produced using common genetic recombination techniques. Furthermore, a trimeric ectodomain structure of hemagglutinin can be produced according to the method described in the above non-patent document 6.

Useful carriers for immobilizing antigens include various carriers used in ordinary antigen-antibody reactions or affinity chromatography, for example, insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide and silicon; microplates, tubes, membranes, columns, beads, etc., comprising glass, metal, or the like; surface Plasmon Resonance (SPR) sensor chips, and the like. For antigen immobilization, physical adsorption may be used, and it is also acceptable to use a chemical bonding method, which is used for insolubilization and immobilization of proteins, enzymes, or the like. For example, the biotin- (strept) avidin system or the like is preferably used. For washing of unbound phage, blocking solutions such as BSA solution (once or twice), PBS containing a surfactant such as Tween (3-5 times), and the like can be used sequentially. One report mentions that the use of a citrate buffer (pH5) or the like is preferable. For elution of specific phage, an acid (e.g., 0.1M hydrochloric acid or the like) is generally used; it is also possible to cleave with a specific protease (for example, a gene sequence coding for a trypsin cleavage site can be introduced into the junction site between the antibody gene and the envelope protein gene; in this case, even when all envelope proteins are expressed as a fusion protein, it is possible to effect infection and amplification of Escherichia coli because of the presence of the wild-type envelope protein on the surface of the eluted phage), to competitively elute with a soluble antigen, or to elute by reducing the S-S bond (for example, in the aforementioned CysDisplay)^TMIn (e), after panning, the antigen-specific phage may be recovered by desorbing the antibody and envelope protein using a suitable reducing agent). When elution is performed with an acid, the eluate is neutralized with Tris or the like, and then Escherichia coli is infected with the eluted phage, followed by culturing, after which the phage is recovered by a conventional method.

In the antigen fixed to the carrier, it is possible to use yeast display on the cell membrane surface expression of hemagglutinin trimer.

After the phage presenting antigen-specific antibodies are concentrated by panning, Escherichia coli is infected with the phage, and cells are seeded on a plate and cell cloning is performed. The phage are recovered again, and the antigen binding activity is confirmed by antibody titer determination (e.g., ELISA, RIA, FIA, etc.) or measurement using Fluorescence Activated Cell Sorting (FACS) or Surface Plasmon Resonance (SPR).

The step of infecting Escherichia coli with the phage antibody clone obtained above and recovering the antibody from the culture supernatant can be performed, for example, when using a vector containing an amber stop codon at the junction site between the antibody gene and the envelope protein gene, infecting an Escherichia coli strain (e.g., HB2151 strain) having no amber mutation with a phage, producing and secreting a soluble antibody molecule in the periplasm or the medium, lysing the cell wall with lysozyme or the like, recovering an extracellular fraction, and purifying the fraction using the same purification technique as that used above for the phage display vector. If a His-tag or a c-myc tag has been previously introduced, the antibody can be easily purified using IMAC, an anti-c-myc antibody column, or the like. When a specific protease is used for cleavage in panning, antibody molecules can be separated from the phage surface by acting on it with the protease, so that the desired antibody can be purified by performing the same purification operation. In the present invention, since the neutralizing activity of the antibody is about 100-fold higher than that of the Fab type when the antibody is an IgG type competitive antibody, plasmid DNA is recovered from the resulting Fc phage clone, a sequence corresponding to the domain linked to the Fc of IgG is added by genetic manipulation, Escherichia coli is transformed therewith, and the transformant is cultured, the manipulation of which is described in the following examples. The antibody recovered from the culture supernatant was purified using an IgG agarose column, and then tested for neutralizing activity.

The desired antibody can also be selected from antibody-producing cell lines immortalized by EB virus or cell fusion, for example, by reacting the above-mentioned antigen previously labeled with a fluorescent substance with immortalized cells or fused cells and then isolating the cells bound to the antigen by Fluorescence Activated Cell Sorting (FACS). In this case, hybridomas or immortal B cells producing antibodies against the target antigen can be directly selected, thereby significantly reducing the labor for cloning.

Hybridomas that produce monoclonal antibodies against a target antigen can be cloned using various methods. Because aminopterin inhibits many cellular functions, it is preferred to remove it from the culture medium as soon as possible. However, human hybridomas are typically maintained in aminopterin-containing medium for about 4-6 weeks after fusion. Desirably, hypoxanthine and thymidine are removed after 1 week or more after the removal of aminopterin. When clones appear and reach approximately 1mm in diameter, the antibody content in the culture supernatant can be measured.

The amount of the antibody can be measured, for example, by a method in which a hybridoma culture is added to a solid phase (e.g., a microplate) having adsorbed thereon a target antigen or a derivative or partial peptide thereof, the target antigen or the like can be adsorbed alone or together with a carrier, an anti-immunoglobulin (IgG) antibody (an antibody against IgG from the same species as the animal from which the original antibody-producing cell was used) or protein A, the carrier or the like is labeled with a radioactive substance (e.g., 125I,131I,3H,14C), an enzyme (e.g., β -galactosidase, β -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase), a fluorescent substance (e.g., fluorescamine, isothiocyanatofluorescin), a chemiluminescent substance (e.g., luminol derivative, luciferin, lucigenin) or the like in advance, detecting antibodies directed against the target antigen bound to the solid phase; or a method in which a hybridoma culture supernatant is added to a solid phase having an anti-IgG antibody or protein A adsorbed thereon, a target antigen labeled with the same labeling substance as above or a derivative or partial peptide thereof is added, and an antibody against the target antigen bound to the immobilization is detected, and the like.

Although limiting dilution is commonly used as a cloning method, it is also possible to use soft agar cloning or to use FACS cloning (as described above). Cloning by limiting dilution may be performed, for example, by the following procedure, which should not be construed as limiting.

The amount of antibody was measured as described above and positive wells were selected. Previously, appropriate feeder cells were selected and added to 96-well plates. Cells were aspirated from antibody positive wells and suspended in complete medium [ e.g., RMPI1640 supplemented with 10% FCS (fetal calf serum) and P/S ] at a density of 30 cells/mL; add 0.1mL of suspension (3 cells/well) to a 96-well plate with feeder cells added; part of the remaining cell suspension was diluted to 10 cells/mL and seeded into the other wells (1 cell/well) in the same way; the remaining cell suspension was diluted to 3 cells/mL and seeded into additional wells (0.3 cells/well). Culturing the cells for 2-3 weeks until macroscopic colonies appear; antibody amounts were measured, positive wells were selected, and recloned. In the case of human cells, cloning is relatively difficult, so plates containing 10 cells per well are also prepared. Although hybridomas producing monoclonal antibodies can be usually obtained by two subcloning, recloning is ideally repeated regularly for more months to confirm their stability.

The hybridoma can be cultured in vitro or in vivo.

The in vitro culture method comprises gradually enlarging the monoclonal antibody-producing hybridomas obtained as described above from a well plate while maintaining the cell density at, for example, about 10⁵-10⁶Individual cells/mL, and gradually decreasing FCS concentration.

The in vivo culture method comprises, for example, intraperitoneal injection of mineral oil into a mouse (the mouse is histocompatible with the parent strain of hybridoma) to induce plasma cell Myeloma (MOPC), and intraperitoneal injection of about 10 days after 5-10 days⁵-10⁷Individual hybridoma cells, and ascites were collected under anesthesia 2 to 5 weeks later.

The separation and purification of monoclonal antibodies are performed according to the separation and purification method of immunoglobulin [ e.g., salting out, ethanol precipitation, isoelectric point precipitation, electrophoresis, adsorption-desorption with ion exchangers (e.g., DEAE, qae), ultracentrifugation, gel filtration, specific purification by selective collection of antibodies by antigen-coupled solid phase or active adsorbents such as protein a or protein G, and decrosslinked adsorption to obtain antibodies, etc. ], in the same manner as in the preparation and purification of ordinary polyclonal antibodies.

As described above, by culturing the hybridoma in vivo or in vitro in a warm-blooded animal and collecting the antibody from the body fluid or the culture thereof, it is possible to screen for a monoclonal antibody that binds to an influenza virus of a specific hemagglutinin subtype.

Whether the thus obtained monoclonal antibody can neutralize influenza virus beyond the barriers among groups can be determined by examining the neutralizing activity against at least one influenza virus selected from group 1 and at least one influenza virus selected from group 2.

In general, studies on the neutralizing activity of influenza viruses are often performed by a Hemagglutination Inhibition (HI) assay. Influenza viruses bind to erythrocytes via the hemagglutinin head region with sialic acid-containing sugar chains (sialic acid chains) presented on the surface of erythrocytes as influenza virus receptors. As a result, influenza viruses cause agglutination of erythrocytes. Since antibodies having influenza virus neutralizing activity recognize and bind to hemagglutinin, the hemagglutination properties of influenza virus are inhibited by neutralizing antibodies. Thus, whether to inhibit hemagglutination can be used as an indication of whether neutralizing activity is present. Although the region involved in the coagulation of hemagglutinin may undergo antigenic drift, the amino acids involved in the sialic acid bonding of this region tend to be highly conserved among influenza virus subtypes, suggesting that the neutralizing antibody of the present invention having a broad neutralizing activity can recognize this amino acid as an epitope. Alternatively, the neutralization activity test method of the present invention includes, for example, a range formation inhibition test [ J.Clin.Microbiol.Vol.28, pp.1308-1313(1990) ]. Specifically, the influenza virus and the host cell are contacted with each other in the presence and absence of the test antibody, and the presence or absence of the neutralizing activity and the level thereof are determined according to whether the test antibody significantly inhibits the formation of foci due to viral infection of the host cell.

Although the subtype of influenza virus to be tested for neutralizing activity is not particularly limited, it preferably includes at least influenza virus type H1 and/or H5 in group 1 and at least influenza virus type H3 in group 2. Alternatively, it is also preferable to further test the neutralizing activity against influenza virus H9 in group 1 and influenza virus H7 in group 2.

For an antibody that has been confirmed to neutralize at least one influenza virus selected from group 1 and at least one influenza virus selected from group 2 in an antibody molecule neutralizing activity test, the antibody molecule can be produced in a large amount using a clone that produces the antibody molecule. When the antibody library used is a phage display library, Escherichia coli can be infected with phage clones presenting the desired neutralizing antibody Fab or scFv, which can be cultured to produce Fab-type or scFv-type antibodies, which are preferably converted to IgG-type antibodies to significantly improve the neutralizing activity thereof. For example, Fab to IgG conversion can be achieved by excising fragments encoding VHCH1 and VLCL from phage DNA, inserting the fragments into a plasmid containing a fragment encoding the Fc region, constructing a plasmid comprising DNA encoding the heavy and light chains, transfecting animal cells such as CHO cells with it, and culturing the cells to secrete IgG-type antibodies in the culture supernatant. The obtained antibody can be purified and recovered by a known method.

When the antibody library used is a hybridoma prepared by B cells immortalized by EB virus or cell fusion, it is possible to produce antibody molecules in vitro or in vivo by the hybridoma as described above, purify the antibody by a conventional method, and recover the antibody.

In order to neutralize the resulting antibody, it is possible to mimic the steps (somatic mutation and selection) adopted by the immune system in vitro, in order to increase its affinity for the antigen. Methods of mutation in antibody genes include chain rearrangement, random mutation using Escherichia coli which is likely to lack a repair system so as to cause mutation, or error-prone polymerase chain reaction (error-prone PCR), CDR walking, and the like. Screening for neutralizing antibodies with higher affinity for the antigen can be achieved by selecting high affinity neutralizing antibodies from a library of mutants generated by mutation. For example, 1) a method of recovering antibody phages having high affinity with a low concentration of antigen for screening, 2) a method of recovering antibody phages that do not easily leave the antigen with strong washing conditions, 3) a method of using an antagonistic reaction, and the like can be used.

The types of neutralizing antibodies of the present invention obtained as described above mostly use VH1-69 or VH1-e gene as the heavy chain variable domain V region. This feature is common to a variety of antibodies that have been reported to date to neutralize a wide variety of influenza subtype viruses in group 1; it is interesting to note that although the same V gene fragment was used, there was a difference in the range of neutralizing activity shown, and it is a feature of the neutralizing antibody of the present invention that only VL1-44, VL1-47 or VL1-51 gene was used as the light chain variable domain V region. In addition, the neutralizing antibody of the present invention has 10 in the cell foci formation inhibition test of IgG type antibody^-11-10^-12The minimum inhibitory concentration of the order of M shows a higher level of neutralizing activity than all antibodies reported so far that can neutralize influenza viruses of various subtypes in group 1.

The antibody undergoes immunoglobulin gene rearrangement, i.e., recombination of the heavy chain variable domain V, D and the J region or recombination of the light chain variable domain V and J regions during B cell differentiation, followed by introduction of somatic mutations in the base sequence of the variable region. As a result, an antibody having a variable region with high antigen affinity can be produced. Therefore, the species of neutralizing antibodies of the present invention, which are B cell-derived antibody clones, may even include neutralizing antibodies having amino acid sequences generated by somatic mutation in the original immunoglobulin genes. All points of contact with the hemagglutinin molecule are present within the heavy chain variable domain when the neutralizing antibody forms an antigen-antibody conjugate with the influenza virus antigen; the heavy chain variable domain is therefore considered to be the site that has the most substantial contribution to the affinity of influenza virus neutralizing antibodies; however, convergence was also observed in the mutation of the light chain variable domain (convergence), suggesting that the light chain variable domain also plays a role in the neutralizing antibodies of the present invention. Thus, in the neutralizing antibody of the present invention, the contribution of complementarity determining region 3(CDR3) to antigen binding is small, so complementarity determining regions 1 and 2 present in the heavy chain variable domain V region are more important. Here, the heavy chain variable domain V region (light chain variable domain V region) refers to a V region constituting a heavy chain (light chain) variable region after rearrangement, and may be a region including, for example, framework regions 1,2, 3 and complementarity determining regions 1, 2. The heavy chain (light chain) variable region refers to a portion of an antibody that is not a constant region of a Fab region, and may be a region comprising, for example, framework regions 1,2, 3 and complementarity determining regions 1,2, 3. Therefore, the neutralizing antibody of the present invention is preferably, for example, a neutralizing antibody having the amino acid sequence of SEQ ID NO.1 as the heavy chain variable domain complementarity determining region 1 and the amino acid sequence of SEQ ID NO. 2 as the complementarity determining region 2.

The present inventors also obtained clones having significant neutralizing activity against H1, H2, and H5 types belonging to group 1, while having neutralizing activity against influenza H3 type belonging to group 2, as compared with other clones. This clone differs from the other clones in that the structure lacks one amino acid in framework region 1 of the heavy chain variable domain (glycine 27 of SEQ ID NO: 27). Therefore, a neutralizing antibody in which the framework region 1 of the heavy chain variable domain is composed of the amino acid sequence shown in SEQ ID NO. 3 is also preferable as the neutralizing antibody of the present invention.

Another specific example includes a neutralizing antibody in which the V region of the heavy chain variable domain is composed of an amino acid sequence shown in any one of SEQ ID Nos. 4 to 9, a neutralizing antibody in which the heavy chain variable domain is composed of an amino acid sequence shown in any one of SEQ ID Nos. 10 to 15, and a neutralizing antibody in which the heavy chain variable domain (SEQ ID Nos. 10 to 15) and the light chain variable domain (SEQ ID Nos. 16 to 26 and 70) are composed of one of the amino acid sequence combinations shown below.

(a) Serial number 10, serial number 16;

(b) serial number 10, serial number 17;

(c) serial number 10, serial number 18;

(d) serial number 10, serial number 19;

(e) serial number 10, serial number 20;

(f) serial number 10, serial number 21;

(g) serial number 10, serial number 22;

(h) serial No. 11, serial No. 23;

(i) serial No.13, serial No. 24;

(j) sequence number 14, sequence number 25;

(k) serial number 15, serial number 26, or

(l) Serial No. 12, serial No. 70

Examples thereof further include the nucleotide sequences represented by SEQ ID Nos. 71 to 76 as the nucleotide sequences encoding the heavy chain variable domain amino acid sequences of the aforementioned antibodies (SEQ ID Nos. 10 to 15), and the nucleotide sequences represented by SEQ ID Nos. 77 to 88 as the nucleotide sequences encoding the light chain variable domain amino acid sequences (SEQ ID Nos. 16 to 26 and 70). Thus, the neutralizing antibody of the present invention can be exemplified by a neutralizing antibody wherein the heavy chain variable domain of the neutralizing antibody is composed of an amino acid sequence encoded by the base sequence shown in any one of SEQ ID Nos. 71 to 76, and the heavy chain variable domain and the light chain variable domain of the neutralizing antibody are composed of an amino acid sequence encoded by one of the combinations of the base sequences shown below.

(a) Serial No. 71, serial No. 77;

(b) serial number 71, serial number 78;

(c) sequence No. 71, sequence No. 79;

(d) serial No. 71, serial No. 80;

(e) serial No. 71, serial No. 81;

(f) serial number 71, serial number 82;

(g) serial No. 71, serial No. 83;

(h) serial number 72, serial number 84;

(i) sequence number 74, sequence number 85;

(j) sequence number 75, sequence number 86;

(k) sequence No. 76, sequence No. 87, or

(l) Serial number 73, serial number 88

These findings demonstrate that the method of the present invention is very useful because it not only provides an antibody that can exhibit a wider range of neutralizing activity than that obtained by conventional methods, i.e., surpasses the interclass barrier, but also provides an antibody that has higher neutralizing activity than that obtained by conventional methods.

Due to the broad nature of their neutralizing activity, it is contemplated that the recognition sites of influenza virus neutralizing antibodies obtained by the methods of the invention will differ from the epitopes recognized by conventional neutralizing antibodies. If the epitope recognized by the neutralizing antibody in the present invention is definite, a peptide comprising the amino acid sequence of the epitope (antigen amino acid sequence) can be used as a vaccine for influenza virus, and a nucleic acid (gene) comprising a base sequence encoding the antigen peptide can be used as an influenza test agent and a test agent kit. Immunoreactive epitopes can be identified by known methods; examples include 1) a method of examining reactivity between a limited degradation product prepared by enzymatically or chemically treating hemagglutinin and an IgG type neutralizing antibody obtained in the present invention, 2) a method of examining reactivity between an overlapping peptide synthesized by referring to an amino acid sequence database and an IgG type neutralizing antibody obtained in the present invention, and the like.

After transcription and translation, hemagglutinin as a precursor is glycosylated; glycosylated hemagglutinin is known to be cleaved into two subunits (subbunit) HA1 and HA 2. Table 1 shows the correspondence between the amino acid sequences and the sequence numbers of the various influenza virus HA1 and HA2 subunits.

[ Table 1]

Reported antibodies reactive with H1 and H5 types identified by the three groups share the common feature with the antibodies of the invention of utilizing VH1-69 in the heavy chain variable domain; suggesting that they may share epitopes. Thus, it is expected that this epitope is present in the HA2 subunit (the region involved in membrane fusion). However, surprisingly, the inventors demonstrated that the neutralizing antibody of the invention does not compete with antibody (C179), and that C179 competes with the above reported antibody for epitopes (Nature Structural & molecular biology, Vol.16, pp.265-273,2009). This supports the fact that although VH1-69 was used in each case, these antibodies do not share an epitope. Furthermore, the neutralizing antibody of the present invention showed Hemagglutination Inhibition (HI) activity, suggesting that it recognizes and binds HA1 subunit (cell receptor binding region).

Isoleucine (amino acid 54 in the amino acid sequence of SEQ ID NO: 27) and phenylalanine (amino acid 55 in the amino acid sequence of SEQ ID NO: 27) present in the CDR2 region of VH1-69 are consecutive hydrophobic amino acid residues, are known to form hydrophobic end portions (hydrophobic tips), and interact with hydrophobic clusters. The aforementioned isoleucine in the neutralizing antibodies of the present invention was replaced by phenylalanine, suggesting that there was some effect on the binding ability to the hydrophobic cluster. Hemagglutinin contains a hydrophobic pocket with highly conserved amino acids, the sialic acid binding site, which is classified as an epitope candidate (Nature, Vol.333, pp.426-431,1988). The amino acids forming the sialic acid binding site include, for example, tyrosine 98, tryptophan 153, threonine 155, histidine 183, glutamic acid 190, lysine 194, amino acid 134 and 138, and amino acid 224 and 228 (corresponding amino acids in the case of influenza viruses of different subtypes or strains) of influenza A/Aizhi/2/68 HA1 and the like. Thus, the region comprising these amino acids may be an epitope. HA1 from influenza virus HAs been reported to contain 5 sites [ A, B (B1, B2), C (C1, C2), D, and E regions ] that are susceptible to accumulating mutations (P.A. Underwood, J.Gen.Virol.vol.62,153-169,1982; Wiley et al, Nature, vol.289,366-378,1981). In the present invention, the A region refers to the amino acids at position 121-146 of SEQ ID NO. 28 or a region corresponding to the amino acid region; the B1 region refers to amino acids 155 and 163 of SEQ ID NO. 28 or a region corresponding to the amino acid region; the B2 region indicates amino acids 155 and 163 in SEQ ID NO. 28 or a region corresponding to the amino acid region; the C1 region is amino acids 50 to 57 of SEQ ID NO. 28 or a region corresponding to the amino acid region; the C2 region refers to the 275 th and 279 th amino acids of the sequence No. 28 or the region corresponding to the amino acid region; the D region refers to the 207-229 th amino acid of the sequence No. 28 or the region corresponding to the amino acid region; the E region refers to amino acids 62 to 83 of SEQ ID NO. 28 or a region corresponding to the amino acid region. Since the species of the antibody of the present invention, particularly F045-092, competes with an antibody recognizing the vicinities of the A-region, the B1-region and the B2-region present in HA1, it is suggested that the vicinities of the A-and B-regions are epitopes.

Because the neutralizing antibodies of the present invention are capable of neutralizing influenza viruses of all hemagglutinin subtypes beyond the barriers of the group, they are effective preventive and/or therapeutic means not only against seasonal influenza caused by antigen drift but also against pandemics due to antigen shift. Thus, by applying the neutralizing antibody, passive immunization against all influenza virus subtypes can be performed, which are expected to have a therapeutic effect on patients who have been infected with influenza due to any influenza virus, and a prophylactic effect on subjects who are feared to be infected with influenza virus or will be infected. In addition, the neutralizing antibody of the present invention is considered to be less likely to cause side effects because it is an antibody already present in the human body.

The neutralizing antibody of the present invention can be used as a passive immunotherapeutic agent for influenza, either directly or after preparing a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier.

Here, as the pharmaceutically acceptable carrier, various organic or inorganic carrier substances generally used as a pharmaceutical material can be used, and they can be formulated into excipients, solvents (dispersants), solubilizing agents, suspending agents, stabilizers, isotonic agents, buffers, pH adjusting agents, analgesics, and the like. Pharmaceutical additives such as preservatives and antioxidants may also be used as required.

Examples of suitable excipients include lactose, sucrose, D-mannose, D-sorbose, starch, alpha starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light silicic anhydride (light silicic anhydride), synthetic aluminum silicate, magnesium metasilicate (magnesium metasilicate), and the like.

Examples of suitable solvents include water for injection, physiological saline, ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, and the like.

Examples of suitable co-solvents include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, tris, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, and the like.

Examples of suitable suspending agents include surfactants such as triethanolamine stearate (stearyltriethanolamine), sodium lauryl sulfate, dodecylpropionic acid (laurylaminoproprionic acid), lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose; polysorbate, polyoxyethylene hardened castor oil (polyoxyyethylene hardened castor oil), and the like.

Examples of suitable stabilizing agents include Human Serum Albumin (HSA), sodium metabisulfite, sodium hydrosulfite (Rongalite), sodium metabisulfite (sodium hydrosulfite), and the like.

Examples of suitable isotonicity agents include sodium chloride, glycerol, D-mannitol, D-sorbitol, glucose, and the like.

Examples of suitable buffers include buffers such as phosphate buffers, acetate buffers, carbonate buffers, citrate buffers, and the like.

Examples of suitable pH adjusters include acids or bases, such as hydrochloric acid and sodium hydroxide.

Examples of suitable analgesics include benzyl alcohol and the like.

Examples of suitable preservatives include p-hydroxybenzoate, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.

Examples of suitable antioxidants include sulfites, ascorbates, and the like.

Examples of the dosage form of the aforementioned pharmaceutical composition include injectable preparations such as injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intraarterial injection, etc.), instillations, and the like.

These pharmaceutical compositions can be produced by a method generally used in the field of pharmaceutical technology, for example, a method described in Japanese pharmacopoeia and the like. Specific methods for preparing pharmaceutical preparations are described in detail below. The amount of antibody in the pharmaceutical composition may vary depending on the dosage form, dosage, etc., e.g., from about 0.1% to 100% by weight.

For example, an injection can be produced by dissolving, suspending or emulsifying the antibody together with a dispersant (e.g., polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), a preservative (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol, etc.), an isotonizing agent (e.g., sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.) and the like in an aqueous solvent (e.g., distilled water, physiological saline, ringer's solution, etc.) or an oily solvent (e.g., vegetable oils such as olive oil, sesame oil, cottonseed oil, and corn oil, propylene glycol, etc.). Additives such as co-solvents (e.g., sodium salicylate, sodium acetate, etc.), stabilizers (e.g., human serum albumin, etc.), and analgesics (e.g., benzyl alcohol, etc.) may be used if desired. These injections may be subjected to sterilization treatment such as filtration sterilization using a membrane filter or the like as required, and are usually filled in a suitable container such as an ampoule.

The injection can also be used by dissolving (dispersing) a powder prepared by treating the above liquid by a method such as vacuum drying, and the like, to obtain a fresh supply. Examples of the method of vacuum drying include freeze drying and a method using a Speedback concentrator (SAVANT Company). When freeze-drying is performed, the sample is preferably lyophilized by cooling to below-10 ℃ using a laboratory flask or a tray or vial in an industrial setting. When a Speedback concentrator is used, lyophilization is performed at about 0-30 ℃ under a vacuum of about 20mmHg or less, preferably about 10mmHg or less. Preferably, a buffer, such as phosphate, is added to the liquid to be dried to a pH of about 3-10. The powder preparation obtained by lyophilization is a long-term stable preparation which can be prepared into a fresh injection by dissolving in water for injection, physiological saline, ringer's solution, or the like, or by dispersing in olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol, or the like, before use.

The above antibody may be used in combination with another therapeutic agent, as required. Examples of therapeutic agents include tamiflu, leqing (Relenza), amantadine, and the like.

Alternatively, the above antibody may be conjugated with another therapeutic agent, as desired. The antibody transports the drug to or near the site where the influenza virus is present and inhibits the virus from entering the cell, where the drug kills the virus or treats, alleviates or alleviates the symptoms of influenza. Examples of the drug include all drugs which have been used or will be used as a drug for treating influenza. These drugs are, for example, synthetic or naturally occurring, low or high molecular weight, proteinaceous, non-proteinaceous, nucleic acid or nucleotide substances. The coupling of the antibody to the drug is preferably achieved through a linker. One example of the linker is a linker comprising a substituted or unsubstituted aliphatic alkylene chain having functional groups capable of binding to an antibody or a drug, such as N-hydroxysuccinimide group, ester group, thiol group, iminocarbonate group, aldehyde group, etc., at both ends thereof (Koutai Kogaku Nyuumon, Chijin Shoka, 1994).

The antibodies may also be encapsulated in liposomes to deliver drugs into cells as needed. Preferred liposomes include positively charged liposomes, positively charged cholesterol, transmembrane peptide-bound liposomes, and the like (Mamoru Nakanishi et al, Protein, Nucleic Acid and Enzyme,44: 1590-.

The neutralizing antibodies of the present invention are administered by a non-oral route, such as intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, and the like. Examples of the content of the antibody as the active ingredient are, but not limited to, 2,500. mu.g/mL per dose or 1.0 to 10mg per kg of body weight for adult patients. The frequency of dosage is, for example, once every 1-2 weeks, administered once to several times, or once every 2-3 weeks for about 2 months.

The neutralizing antibodies of the present invention are useful not only for the prevention and/or treatment of human influenza, but also for the prevention and/or treatment of avian influenza, such as chickens, and non-human mammals, such as pigs and horses, to which the risk of human infection can be reduced in advance. When the neutralizing antibody of the present invention is applied to an animal, the same technique for preparing a pharmaceutical preparation as above can be used.

The neutralizing antibody of the present invention is isolated by screening for antibodies originally present in humans. It is reasonable to assume that these antibodies have been carried by humans at a certain frequency, rather than occurring very infrequently. Thus, individuals who are able to produce neutralizing antibodies of the invention may be considered to be resistant to the novel influenza as well. At the same time, individuals lacking the ability to produce such antibodies may be said to be threatened by the potential for infection by new influenza types. If it can be determined whether the neutralizing antibody is carried or not by a relatively convenient procedure, it can be judged that passive immunoprophylaxis is desirably preferred for non-neutralizing antibody carriers.

Accordingly, the present invention also provides a method of detecting a neutralizing antibody of the present invention in a subject, comprising the steps of:

(3) investigating the presence or absence in the blood of antibody-producing cells presenting an antibody that binds to both hemagglutinin of a subtype selected from group 1 and hemagglutinin of a subtype selected from group 2, and has a heavy chain variable domain V region encoded by VH1-69 or VH1-e gene, or

A method comprising the steps of:

(3) each blood was examined for the presence of antibody-producing cells presenting an antibody that binds to hemagglutinin of a subtype selected from the group different from the inoculated hemagglutinin and has a heavy chain variable domain V region encoded by VH1-69 or VH1-e gene.

In order to detect the objective neutralizing antibody with a small amount of collected blood, it is necessary to proliferate and concentrate the B lymphocytes producing the desired antibody. Therefore, the present invention detects the presence of neutralizing antibodies of the present invention by introducing hemagglutinin inoculation, using the binding activity to hemagglutinin belonging to different groups as an index, and using the V gene fragment common to most species of the neutralizing antibodies.

The present invention is explained in more detail below with reference to examples, which are only illustrative and not intended to limit the present invention.

[ examples ]

Blood sampling

Mononuclear cells corresponding to a blood volume of 3L were collected by apheresis from a pediatrician born in 1974. Blood collection was performed 5 months in 2004.

Preparation of human phage antibody library

Human phage antibody libraries were prepared by phage display methods. About 10% of the blood components obtained from the blood collection were recovered by Ficoll-Paque⁹And (4) separating RNA from lymphocytes. From the RNA amplified cDNA libraries of heavy (VH) and light (VL) chains of antibodies were constructed, respectively. The number of clones for the heavy and light chains was about 10, respectively⁹An (10)⁶And (4) respectively. Then, the heavy chain and the light chain were combined to construct a Fab-cp3 type human phage antibody library, which is a library containing about 10¹⁰Library of individual clones.

Influenza virus strains used

The following influenza virus strains were used in this example. Unless otherwise indicated, the abbreviations in the following examples represent the following influenza virus strains.

(H3N2 type)

Aic68: A/Aizhi/2/68, Fuk70: A/Fugang/1/70, Tok73: A/Tokyo/6/73, Yam77: A/sorb/2/77, Nii81: A/New Ejection/102/81, Fuk85: A/Fugang/C29/85, Gui89: A/Guizhou/54/89, Kit93: A/North Jiuzhou/159/93, Syd97: A/Sydney/5/97, Pan99: A/Panama/2007/99, Wyo03: A/wyoming/3/2003, NY04: A/New York/55/2004

(H1N1 type)

NC 99A/New Cardonia/20/99, SI 06A/Solomon island/3/2006

Screening

Screening was performed by the panning method. Influenza virus strains inactivated by formalin treatment were coated on immune tubes, followed by antigen-antibody reaction between the virus strains coated in the tubes and a phage antibody library. After washing the tubes with PBS, the phage bound to the antigen were eluted with acid, and then immediately neutralized and recovered. The recovered phage was used to infect Escherichia coli, the recovery rate was calculated, and phage antibodies were prepared. Using the phage, the above operation was repeated. This operation was performed three times, and Escherichia coli was infected with the eluted phage, which was then cultured overnight on a LBGA plate to give a single colony. The single colony was isolated, Fab-cp3 antibody was prepared, and the binding activity of the antibody to the strain used for screening was confirmed by ELISA. Further analysis was performed using clones showing binding activity as positive clones.

Preparation of Fab-cp3 type antibody

Escherichia coli infected with the phage obtained in the screening was inoculated into YT containing 0.05% glucose, 100. mu.g/ml ampicillin and 1mM IPTG, and cultured overnight at 30 ℃ with shaking. Culture supernatants containing Fab-cp3 type antibodies secreted from Escherichia coli were recovered by centrifugation and used for ELISA, competitive ELISA, Western blotting, flow cytometry, and the like.

ELISA

The virus strain solution inactivated by formalin treatment was added to a 96-well Maxisorp plate and coated at 37 ℃ for 1 hour. The virus solution was removed from the wells and blocked for 1 hour by the addition of 5% BSA/PBS. After removing the BSA solution, the E.coli culture supernatant containing the Fab-cp3 type antibody was added and reacted for 1 hour. After washing with pbs (pbst) supplemented with 0.05% Tween20, rabbit anti-cp 3 antibody was added and reacted for 1 hour. After further washing with PBST, goat anti-rabbit IgG (H + L) -HRP was added and reacted for 1 hour. After washing with PBST, an OPD solution, which is a substrate for HRP, was added and reacted at room temperature, and then the reaction was terminated by 2N sulfuric acid, after which OD was measured at a wavelength of 492 nm. All reactions were carried out at 37 ℃ unless otherwise indicated.

Sequence analysis of isolated antibody clones

The nucleotide sequences of the heavy chain (VH) and light chain (VL) of clones positive for the viral antigen were confirmed by sequencing reaction.

Grouping of isolated clones

After the nucleotide sequence of the cloned VH was confirmed, the sequence was converted into an amino acid sequence, and the sequence was compared between isolated clones. Clones were grouped centering on the similarity of the amino acid sequences of VH, particularly in the CDR3 sequence.

Western blotting

The formalin-treated virus strain was subjected to SDS-PAGE in a non-reducing state to separate fractions, which were transferred onto a PVDF membrane. PVDF membranes were blocked with PBST supplemented with 2.5% skim milk for 1 hour, then washed with PBST, and reacted with Fab-cp3 type antibody in culture supernatant for 1 hour. After washing with PBST, the membrane was reacted with rabbit anti-cp 3 antibody for 1 hour, further washed with PBST, and reacted with goat anti-rabbit IgG (H + L) -HRP for 1 hour. After washing with PBST, the membrane was reacted with ECL solution for 4-5 minutes and the bands were detected with a CCD camera. All reactions were carried out at room temperature.

Preparation of Fab-pp-type antibodies

Plasmid DNA of the Fab-cp3 type antibody was genetically transformed into Fab-PP type (PP is the Fc binding domain of protein A) and then transformed into Escherichia coli. Escherichia coli was inoculated into YT supplemented with 0.05% glucose, 100. mu.g/ml ampicillin and 1mM IPTG, and cultured overnight with shaking at 30 ℃. The culture supernatant containing the Fab-pp type antibody secreted from Escherichia coli was recovered by centrifugation, precipitated with ammonium sulfate, dissolved in PBS, and purified with an IgG-agarose column for HI activity, virus-neutralizing activity, and the like.

HI Activity measurement

The purified Fab-pp type antibodies were serially diluted with PBS, mixed with 4 HA/unit virus solution per well, and reacted at room temperature for 1 hour. Adding red blood cells, mixing, and reacting at room temperature for 30min-1 h. The results are expressed as the dilution ratio of the antibody.

Isolation from N libraryBinding Activity of clones of (4) against influenza virus strain H3N2

The binding activity of clones of antibodies isolated from the N library screening, against the 12H 3N2 virus strains used for the screening, and against the 1H 1N1 virus strain, is shown as ELISA results. Is an experiment to determine what cross-reactivity the screened isolated clones show to the virus strain. The results are shown in FIG. 1.

Isolated clones were grouped according to the degree of binding activity against each virus. The leftmost indicates the group number. The column "isolated clone number" on the right side of the clone name indicates the number of clones showing the same VH amino acid sequence among the screened isolated clones, and the "isolated virus strain" on the further right side indicates which virus strain these clones were screened for isolation.

The ELISA was numerically processed as follows:

greater than or equal to 1: red colour

Less than 1, not less than 0.5: orange color

Less than 0.5, not less than 0.1: yellow colour

Less than 0.1: white colour

The rightmost Western Blotting (WB) column shows only the results of the clones tested. Clones with detectable bands at HA position are denoted "HA", clones with detectable bands at other positions are denoted "? ", as it is currently not known what they recognize. The blank column indicates that no experiment was performed.

Results of N library screening

The number of clones picked up in each screen of the H3N2 strain (before confirmation by ELISA) and among them the clones belonging to groups 11 and 22 (fig. 1) were isolated by which strain of virus was screened (fig. 2) are shown.

Group 11 is a group of antibody clones recognizing two strains H3 to H1 as antigens, and group 22 is a group of antibody clones recognizing all 12 strains H3 but not H1.

Isolation of clones belonging to group 11

The screened virus strains when individual antibody clones belonging to group 11 were isolated, and the numbers isolated in the screen are shown (FIG. 3).

The number of isolates of clones having the same VH sequence based on the VH sequence of each clone (VL sequence not considered) is shown.

Amino acid sequence of a clone belonging to group 11

FIG. 4 shows the amino acid sequences of VH and VL of clones belonging to group 11.

(1) VH amino acid sequence

From germ line searches with IgBlast from NCBI, clones belonging to group 11 were found to have the highest degree of identity with IGHV1-69 x 01. Therefore, IGHV1-69 × 01 was judged to be germline.

From this result, comparison of VH amino acid sequences was performed between IGHV1-69 × 01 and each clone. "the degree of identity (%) with the germline FR1-FR 3" is the result of calculation of the degree of identity with the FR1-FR3 amino acid sequence of IGHV1-69 x 01. Furthermore, as a result of comparison with the amino acid sequence of IGHV1-69 x 01, portions having different amino acids were highlighted with different colors. However, as for CDR3 and FR4, amino acids that differ between the cloned sequences are highlighted in different colors.

(2) VL amino acid sequence

To date, 15 clones with the same VH amino acid sequence as F022-360 have been isolated. Sequence analysis of VL sequences from 12 of these clones resulted in the identification of 7 VLs. As a result, F022-360 was found to have 7 VH-VL combinations among the VH sequences. Of these 7 combinations, the VL sequence highlighted in grey was the combination F022-360. For clones with other VH sequences, only one type of combination has been identified so far, since only the VL sequences of the clones that are represented are identified. The VL sequence of F026-245 has not been confirmed.

Similar to VH, the line with the highest degree of identity was used as the line for the amino acid sequence of each VL according to the results of the Igblast search at NCBI. Since some, approximately 3, germ lines were detected, the sequences of each clone were compared based on IGLV1-44 x 01. Different amino acids are highlighted with other colors.

Antibody clones previously reported to neutralize H1 and H5 and clone lines 1-69, and comparison of VH amino acid sequences between clones belonging to group 11

Three prior publications (precisely, 4) reported human antibodies that could neutralize two strains of H1-H5, all of which were cloned in IGHV 1-69. Therefore, these clones were compared to the amino acid sequences of IGHV1-69 x 01 germline and clones belonging to group 11 (fig. 5).

The same amino acids as IGHV1-69 x 01 are shown by bars (bars).

The volumes and pages of each document are as follows:

2009Nat.Struct.Mol.Biol.:Vol.16265-273

2009Science Vol.324246-251(2008PLoS One: Vol.3e3942 is a literature related to the isolation of this clone)

2008PNAS:Vol.1055986-5991

Binding Activity of clones belonging to group 11 against the H3N2 influenza Virus Strain

The binding activity of clones belonging to group 11 was confirmed by ELISA for 12H 3 virus strains and H1 virus strains used in the screening. Assays were performed in duplicate for each strain and mean and standard deviation were calculated (figure 6).

Western blotting of clones belonging to group 11

These experiments are experiments showing that clones belonging to group 11 recognized HA of the H3 and H1 virus strains (fig. 7).

All samples were subjected to SDS-PAGE in a non-reduced state.

The upper half of FIG. 7 shows data for F022-360 and F045-092, and the lower half of FIG. 7 shows data for F026-146 and F026-427. The difference between the left and right is the difference in exposure time for data capture.

F032-093 was a positive control against HA from H3 strain A/North Kyushu/159/93, and F078-155 was a positive control against HA from H1 strain/New Carlonia/20/99.

HI Activity of clones belonging to group 11

It was confirmed whether or not the clone belonging to group 11 had HI activity (fig. 8).

HIU is expressed as the dilution rate at which the antibody concentration is diluted in 2-fold increments from 100. mu.g/ml.

Neutralizing Activity of Fab-pp type antibodies

Whether clones belonging to group 11 neutralized the H3 and H1 influenza virus strains was confirmed (fig. 9).

The inhibition rate of foci (focus) formation was expressed as% inhibition when 250 and 100. mu.g/ml of Fab-pp-type antibody were added.

Inhibition of C179ELISA Activity by Fab-p3

A mouse monoclonal antibody C179 capable of neutralizing Influenza A SulLing-type neo-Kalidonia strain was reacted with an immunoplate to which the vaccine had been adsorbed, in the presence or absence of Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092, F005-126; F005-126 was a negative control that did not react with Influenza A SulLing-type (Sobiet Union type Influenza A) neo-Kalidonia strain HA). Then, the immunoplates were reacted with HRP-labeled anti-mouse IgG (manufactured by MBL corporation) and developed by OPD to detect C179 bound to the vaccine.

There was no significant difference in the observed ELISA values in the presence and absence of the Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092, F005-126) (FIG. 10). Thus, the response of C179 to the vaccine was found not to be inhibited by F022-360, F026-146, F026-427 or F045-092. This suggests that C179 is different from the recognition epitope of F022-360, F026-146, F026-427 or F045-092.

Inhibition of ELISA Activity of C179 against Fab-p3

Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092, F005-126) were reacted with immunoplates to which a vaccine of influenza A Soviet New Carlonia strain had been adsorbed, in the presence or absence of C179. The immunoplates were then reacted with rabbit anti-p 3 polyclonal antibody, followed by reaction with HRP-labeled anti-rabbit IgG (manufactured by MBL Co.) and color development by OPD to detect Fab-p3 antibody bound to the vaccine.

There was no significant difference in the observed ELISA values in the presence and absence of C179 (fig. 11). Thus, it was found that the reaction of F022-360, F026-146, F026-427 or F045-092 with the vaccine was not inhibited by C179. This suggests that F022-360, F026-146, F026-427 or F045-092 recognizes a different epitope from C179.

Inhibition of ELISA Activity by Fab-p3 on F005-126

The Fab-PP type monoclonal antibody F005-126 was reacted with an immunoplate of a vaccine that had adsorbed Airy virus strain of influenza A hong Kong type in the presence or absence of Fab-p3 antibody (F022-360, F026-146, F026-427, F045-092, F005-126, F019-102; F019-102 is a negative control that did not react with Airy virus strain of influenza A hong Kong type). The immunoplates were then reacted with HRP-labeled rabbit IgG and developed by OPD to detect vaccine-bound Fab-PP type F005-126.

F005-126 is an antibody that does not react with the New Kalidoni strain of influenza A SulAN, but broadly reacts with and neutralizes many hong Kong strains of influenza A. Since F022-360, F026-146, F026-427 and F045-092 also react extensively with a variety of hong Kong influenza A strains, it is envisaged that similar recognition epitopes are possible. For this reason, competitive inhibition experiments with F005-126 were performed. There was no significant difference in the observed ELISA values when F005-126 of Fab-PP type was reacted with a vaccine of Airy virus strain of hong Kong influenza A in the presence (F022-360, F026-146, F026-427, F045-092, F019-102) and in the absence of Fab-p3 antibody (FIG. 12). In the presence of F005-126, type Fab-p3, as a positive control, the ELISA values were significantly reduced. Thus, it was found that the reactivity of F005-126 with the vaccine was not inhibited by F022-360, F026-146, F026-427 or F045-092. This suggests that F005-126 recognizes a different epitope than F022-360, F026-146, F026-427 or F045-092.

Reactivity of F026-427 and F045-092 to HA expressed on 293T cells

The HA gene of the hong Kong sorb virus influenza A strain was inserted into the cloning site of an expression vector pNOW to prepare pNOW-Yam77 HA. pNOW-Yam77HA was mixed with lipofectamine LTX and added to 93T cells for transfection. After 24 hours of culture, transfected cells were recovered and subsequently treated with 2.5% BSA-PBS-0.05% Na-N₃Blocking at 4 ℃ for 30min, and reacting with Fab-p3 antibody (F026-427, F045-092, F008-038) at 4 ℃ for 30min (negative control that did not react with the sorbic virus strain HA at F008-038). Then, the cells were reacted with a rabbit anti-p 3 polyclonal antibody, and then reacted with Alexa 488-labeled anti-rabbit IgG (produced by Pierce), and FACS analysis was performed. Similarly, as a positive control, the cells were reacted with the hong Kong mouse monoclonal antibody F49 for influenza A, and then reacted with Alexa 488-labeled anti-mouse IgG (produced by Pierce), and FACS analysis was performed.

The FACS of F026-427 and F045-092 showed peak shifts compared to the negative control F008-038, which did not react with HA of hong Kong sorbic virus strain influenza A (FIG. 13). Thus, F026-427 and F045-092 are considered antibodies against sorb strains.

Measurement of neutralizing Activity of fully human IgG against influenza Virus

[ samples and reagents ]

1. Purified fully human IgG antibodies

F026-427 (batch No. 100614), F045-092 (batch No. 100614)

2. Virus

The following virus strains were used.

Human H3N2, A/Aizhi/2/1968 strain, A/Beiyuzhou/159/1993 strain

Avian H3N8, A/budgerigar/Aizhi/1/1977 strain

Pandemic H1N1, A/Suita/1/2009pdm strain

Pig H1N1, A/pig/Hokkaido/2/1981 strain

Human H1N1, A/New Cardonia/20/1999 Strain

Human H2N2, A/Okuda/1957 strain

Bird H2N2, A/duck/hong Kong/273/1978 strain

Avian reassortment H5N1;

A/Duck/Mongolia/54/2001 (H5N2) strain HA x A/Duck/Mongolia/47/2001 (H7N1) strain NA x A/Duck/Hokkaido/49/98 (H9N2) internal (internal)

Human H5N1, strain A/Vietnam/1194/2004

Human H5N1, A/Anhui/1/2005 strain

Human H5N1, A/Indonesia/5/2005 strain

3. Nuclear media

MDCK cells were sub-cultured in MEM with 10% FCS, and for the neutralization test and culture after viral infection, MEM with 0.4% BSA without FCS was used.

4. Reagents for PAP staining

Mouse monoclonal antibody against influenza A NP (C43)

Rabbit antiserum cappel55436 against mouse IgG (whole molecule)

Goat antiserum cappel55602 against rabbit IgG (whole molecule)

Rabbit Peroxidase Antiperoxidase (PAP) cappel55968

3, 3' -diaminobenzidine tetrahydrochloride Sigma D5637

Hydrogen peroxide special reagent Sigma-Aldrich13-1910-5

[ Experimental method ]

Human IgG antibodies having the VH and VL amino acid sequences of clones F026-427 and F045-092, respectively, were prepared and used for the neutralization assay. Each purified human IgG antibody solution was diluted to 250. mu.g/mL and 100. mu.g/mL with MEM containing 0.4% BSA, and 4-fold serial dilutions were made using 100. mu.g/mL as stock solutions. For each of the resulting diluted antibody solutions, an equal amount of an influenza virus solution adjusted to 100FFU for each subtype was added, followed by 1 hour neutralization reaction at 37 ℃. MDCK cells initially sub-cultured in MEM containing 10% FCS were monolayer-cultured in 96-well plates, washed with PBS (-), and adsorbed to viruses at 37 ℃ for 1 hour using 30. mu.L/well of MEM containing 0.4% BSA supplemented with the reaction solution after the neutralization reaction. After removing the neutralizing solution and washing it once with PBS (-), MEM containing 0.4% BSA was added at 50. mu.L/well and the mixture was washed with CO₂Incubate at 37 ℃ for 16 hours in the presence. After removing the culture solution, the cells were fixed with 100% ethanol and dried. Subsequently, the infected cells were stained by the enzyme antibody technique (PAP method), and the infection inhibition rate was calculated by counting the number of infected cells under a microscope. The results are shown in FIG. 14.

F026-427 showed neutralizing activity against human H3N3 strain, avian H3N8 strain, human H1N1 strain and human H2N2 strain, and had weak neutralizing activity against human H5N1 strain. On the other hand, F045-092 showed neutralizing activity against human H3N2 strain, avian H3N8 strain, human H1N1 strain, human and avian H2N2 strain, and had weak neutralizing activity against human and avian H5N1 strain. The above results suggest that F026-427 and F045-092 show reactivity to human-and avian-derived virus strains, and, with a few exceptions, to porcine-derived virus strains.

Reactivity of F026-427 and F045-092 to HA0 and HA1 expressed on 293T cells

Genes HA0 and HA1 of an Airy virus strain of influenza A/H3N2 were inserted into the cloning site of an expression vector pDisplay to prepare pDisp-Aic68HA0 and pDisp-Aic68HA 1. In the same manner, HA0 and HA1 genes of influenza A/H3N2 type Fugang virus strain were inserted into the cloning site of expression vector pDisplay to prepare pDisp-Fuk85HA0 and pDisp-Fuk85HA 1. Each of these four plasmids was mixed with lipofectamine LTX and added to 293T cells for transfection. Samples with lipofectamine LTX added only to 293T without plasmid were also prepared as negative controls (mock transfection). After 24 hours of culture, transfected cells were recovered and washed with 2.5% BSA-PBS-0.05% Na-N₃Blocking at 4 ℃ for 30min, followed by contact with Fab-PP antibodies (F026-427PP, F045-092PP),

Anti-influenza a/H3N2 type antibody F49 or rabbit anti-V5 tag antibody reacted at 4 ℃ for 30 min. For the positive control, Fab-PP antibody F003-137PP against an Airy virus strain of influenza A/H3N2 type reacted at 4 ℃ for 30min with cells transfected with pDIsp-Aic68HA0 and pDIsp-Aic68HA1 and mock-transfected cells. Similarly, for the positive control, Fab-PP antibody F019-102PP from influenza A/H3N2 Fukan county strain reacted at 4 ℃ for 30min with cells transfected with pDIsp-Fuk85HA0 and pDIsp-Fuk85HA1 and mock transfected cells. Then, the cells reacted with the Fab-PP type antibody were reacted with Alexa 488-labeled anti-human IgG (produced by Pierce), the cells reacted with F49 were reacted with Alexa 488-labeled anti-mouse IgG (produced by Pierce), the cells reacted with the rabbit anti-V5-tag antibody were reacted with Alexa 488-labeled anti-rabbit IgG (produced by Pierce), and FACS analysis was performed, respectively.

The reaction of F045-092PP with cells expressing HA0 and HA1 of the influenza a/H3N2 type erichian strain and HA0 and HA1 of the influenza a/H3N2 type foxan county strain caused peak shifts in FACS compared to control mock-transfected cells that did not express HA (fig. 15-1). Compared to control mock transfected cells that did not express HA, F026-427PP reacted weakly with cells expressing HA0 and HA1 of the Airy virus strain influenza A H3N2, causing peak shifts in FACS (FIGS. 15-2). The V5-tag antibody, which is an antibody for confirming the expression of HA0 and HA1, was shown to be sufficiently expressed in any of the HA0 and HA 1-expressing cells. In addition, F004-137PP and F019-102PP were sufficient to react with HA0 and HA1, respectively. F49 reacted with HA0 but not HA 1.

Since F026-427 and F045-092 reacted with HA1, it is believed that the epitopes recognized by these antibodies were present on the HA1 molecule and were different from F49, where F49 had as broad a strain specificity as F026-427 and F045-092. Antibodies from VH1-69 germline have recently been reported to have broad virus strain specificity, with the epitope recognized being located mainly in the HA2 region and believed to exert neutralizing activity by inhibiting fusion activity. However, the recognition epitopes of said F026-427 and F045-092 from the VH1-69 germline are in the HA1 region and are apparent from FIG. 8, and thus, it is considered that they show neutralizing activity due to their HI activity. There is no precedent for antibodies with this property, and therefore, it is an antibody with entirely new properties. Furthermore, as is apparent from FIG. 16, the recognition epitopes of F026-427 and F045-092 are considered to be in the vicinity of epitope B.

Inhibition of ELISA Activity by F004-104

Monoclonal antibodies F026-427p3, F045-092p3 and F004-104p3 of Fab-p3 type, and an antibody F49 of mouse origin against influenza A/H3N2 type, were reacted with immunoplates to which an influenza A/H3N2 type panama strain vaccine had been adsorbed, in the presence of IgG antibodies (F026-427IgG, F045-092IgG, F004-104IgG) and in the absence (no IgG). Then, in order to detect the Fab-p3 antibody bound to the vaccine, it was reacted with a rabbit-derived anti-p 3 antibody, followed by reaction with an HRP-labeled anti-rabbit IgG antibody, and developed by OPD. In addition, to detect F49, the reaction was performed with HRP-labeled anti-rabbit IgG antibody, and color was developed by OPD. The results are shown in FIG. 16.

Significant inhibition of ELSIA activity was fully observed between the same type of antibodies. F045-092IgG and F004-104IgG inhibited the ELISA activity of any of F026-427p3, F045-092p3 and F004-104p 3. In addition, F026-427IgG inhibited the ELISA activity of F004-104p 3. On the other hand, F026-427IgG did not significantly inhibit F045-092p3, which is believed to be due to the much stronger binding activity of F045-092 compared to F026-427. For F49, no significant inhibition of its ELISA activity was observed by IgG antibodies.

By escape mutation analysis, F004-104 was an antibody recognizing an epitope located near the 159 th and 190 th amino acid sequences of HA1 molecule (FIG. 17-1, FIG. 17-2). The recognition epitopes of F026-427 and F045-092 were considered to be in the vicinity of the recognition epitope of F004-104 by competition ELISA. Thus, the recognition epitopes of F026-427 and F045-092 are presumed to be located near the 159 and 190 amino acid sequences of the HA1 molecule.

Reactivity of F045-092 and F026-427 to mutation of amino acid at position 136

Mutant HA was prepared by replacing the serine residue 136 of the Aic68 strain HA with either threonine (mutant Aic68S136T) or alanine (mutant Aic68S 136A). These mutant and wild-type Aic68 strains Aic68wt were expressed on 293T cells and reacted with F026-427, F045-092, F003-137, F035-015 or F033-038 before reactivity was detected by flow cytometry analysis. The results are shown in FIG. 18.

As a result, the reactivity of F035-015 and F033-038 against these two mutants was not changed. F045-092 was slightly less reactive to Aic68S136T than to Aic68S 68 wt. F045-092 is less reactive to Aic68S 136A. Since mutation of serine residue 136 affects the recognition of HA by F045-092, residue 136 is presumed to be the HA recognition epitope of F045-092 or an amino acid in the vicinity thereof.

FCM analysis of chimeric HA133A and 142A by F045-092

The amino acid sequence at position 142-146 of the Wyo03 strain HA was grafted into the amino acid sequence at position 142-146 of the Aic68 strain HA to prepare a chimeric HA (Aic68_ 142A). In contrast, the amino acid sequence at position 142-146 of the Aic68 strain HA was grafted into the amino acid sequence at position 142-146 of the Wyo03 strain HA to prepare a chimeric HA (Wyo03_ 142A). On the other hand, the amino acid sequence at position 142-146 of the Aic68 strain HA was grafted into the amino acid sequence at position 142-146 of the Fuk85 strain HA to prepare a chimeric HA (Fuk85_ 142A). Further, the amino acid sequence at positions 133-137 of the Wyo03 strain HA was grafted into the amino acid sequence at positions 133-137 of the Fuk85 strain HA to prepare a chimeric HA (Fuk 85-133A). These mutants and the Wild-type Aic68 strain HA (Aic68_ Wild), the Wild-type Wyo03 strain HA (Wyo03_ Wild), the Wild-type Fuk85 strain HA (Fuk85_ Wild) were expressed on 293T cells and subsequently reacted with F045-092 before checking the reactivity by flow cytometry analysis (EMAC method [ epitope mapping by chimerism analysis ]; Okada et al, Journal of general Virology, vol.92,326-335,2011). The results are shown in FIGS. 19 and 20.

F045-092 was sufficiently reactive with Aic68_ Wild, but weakly reactive with chimeric Aic68_142A grafted with the amino acid sequence from position 142 to 146 of the Wyo03 strain HA. This demonstrates that the amino acid sequence at position 142-146 of the Wyo03 strain inhibits HA recognition by F045-092. The amino acid sequence at position 142-146 of the Wyo03 strain had many amino acid residues with relatively high molecular weights, suggesting the possibility of causing steric hindrance when antibodies were bound to HA. On the other hand, F045-092 reacted very weakly with Fuk85_ Wild but strongly with chimeric Fuk85_133A grafted with the amino acid sequence at position 133-137 of the Wyo03 virus strain HA. In order to influence the recognition of HA by F045-092, it was considered that the amino acid sequence at position 133-137 was preferably of the Wyo03 strain type, rather than of the Fuk85 strain type. Thus, the amino acid sequence at position 133-137 was presumed to be the recognition epitope of F045-092, or to be located in the vicinity thereof.

HA1 antigen recognition site of anti-HA antibodies used in competition studies

An HA X-ray crystal structure analysis file (1HA0) of H3 was downloaded from a Protein database (Protein Data Bank) and the three-dimensional structure of the amino acid part from position 91 to 260 in the HA1 region was constructed using Rasmol2.7.5 software (FIG. 21). According to the EMAC method, the antigen recognition site of each H3N2 antibody of each H3N2 influenza virus was predicted in advance. For F033-038, for example, regions A and B are estimated to be the antigen recognition sites for HA, the Aic68 strain.

Competition between anti-HA antibodies binding to HA1A, B, C, D and E sites and F045-092 antibody War research

Competitive ELISA was performed by the EMAC method between anti-HA antibodies (F041-342, F041-360, F019-102, F004-111, F033-038, F010-073, F010-014, F004-136, F010-077, F008-055, F008-038, F008-046, F010-032, F035-015, F037-115, F004-104, F003-137) and F045-092 antibodies, for which recognition sites on the antigen have been identified. The results are shown in FIG. 22.

Fab-pp and Fab-cp3 types of each anti-HA antibody were prepared, and pp-type and cp3 type antibodies were added as competitors to compete for the antigen, after which the binding activity of the pp-type antibody to the antigen was measured. Specifically, a formalin inactivated strain of H3N2 was plated on immunoplates and blocked with 5% BSA. Each 50. mu.l of the optimum concentration of Fab-pp antibody, and 20. mu.g/ml of the antibody F045-092 of the cp3 type or the antibody F3 of Fab-cp prepared by 20-fold dilution of the culture supernatant of Escherichia coli were added to the immunoplates after blocking was complete, and incubated at 37 ℃ for 1 hour. After washing with PBST, rabbit anti-streptavidin-HRP antibody was added to detect pp-type antibody bound to the antigen and further incubated at 37 ℃ for 1 hour. After washing the plate, an HRP substrate OPD was added and reacted for 20min, and then the reaction was terminated with 2N sulfuric acid, after which the OD of the sample was measured at a wavelength of 492 nm.

The H3N2 type virus strains used were as follows:

aic 68A/Aizhi/2/68

Yam 77A/sorb/2/77

Syd 97A/Sydney/5/97

Pan 99A/Panama/2007/99

The F045-092 antibody does not compete at all with F041-342 and F041-360 recognizing the C site, F019-102 recognizing the E site, or F004-111 recognizing both the C and E sites, and is considered not to bind to the C and E sites. On the other hand, anti-HA antibodies (F033-038, F010-073, F010-014, F004-136, F010-077) that recognize both A and B sites are highly competitive with F045-092. As for the antibody recognizing the A site around the receptor binding region (F035-015), the antibody recognizing only the B site (F008-055, F008-038, F008-046, F010-032, F037-115, F004-104) and the antibody recognizing the B2/D site (F003-137), although they compete with the F045-092 antibody, the antibodies other than F008-038 did not provide a competition result comparable to that of the antibody recognizing both the A and B sites. The possibility was suggested that the F045-092 antibody recognizes the A and B sites, as well as the receptor binding region located between them and having a high degree of amino acid conservation in the virus type. This is consistent with F045-092 showing HI activity.

[ Industrial Applicability ]

According to the present invention, human antibodies showing neutralizing activity against all influenza virus subtypes can be screened. It is also possible to determine whether a subject carries an antibody against influenza virus. An important task for humans is to develop methods for preventing the pandemic of new viral strains, methods for checking the spread of infection if it occurs, and methods for developing truly effective vaccines to gain time for vaccinating many people. Measures currently being taken include the implementation of a global virus surveillance system, the mass stocking of therapeutic drugs such as tamiflu, and the development, production and mass stocking of vaccines, but no one can tell what form of novel virus will be present before it actually appears. As a new promising solution, the present invention will make a great contribution to public health and medical care.

This patent application is based on provisional patent application Nos.61/380,051 and 61/452,785, filed in the United states, and non-provisional patent application No.13/198,147, filed in the United states, the entire contents of which are incorporated herein by reference.

Claims

1. An isolated antibody that neutralizes at least one influenza virus selected from group 1 consisting of influenza viruses of H1 type, H2 type, H5 type, H6 type, H8 type, H9 type, H11 type, H12 type, H13 type, and H16 type, and at least one influenza virus selected from group 2 consisting of influenza viruses of H3 type, H4 type, H7 type, H10 type, H14 type, and H15 type.

2. The antibody according to claim 1, wherein the antibody neutralizes at least influenza virus type H1 and/or H5 and influenza virus type H3.

3. The antibody of claim 1, wherein the antibody neutralizes influenza viruses types H1-H16.

4. The antibody of claim 1, wherein the heavy chain variable domain V region utilizes VH1-69 or VH1-e genes.

5. The antibody according to claim 4, wherein the heavy chain variable domain V region has a deletion of amino acids.

6. The antibody of claim 5, wherein the heavy chain variable domain V region encoded by VH1-69 or VH1-e gene has at least a mutation lacking glycine 27.

7. The antibody according to claim 1, wherein the light chain variable domain V region utilizes VL1-44, VL1-47 or VL1-51 genes.

8. The antibody of claim 1, wherein the antibody has a minimum inhibitory concentration in a range formation inhibition assay of 10 when converted to an IgG-type antibody^-11-10^-12Of the order of M.

9. The antibody according to claim 1, which is a human antibody.

10. The antibody according to claim 1, wherein complementarity determining region 1 of the heavy chain variable domain consists of the amino acid sequence represented by SEQ ID NO.1, and complementarity determining region 2 consists of the amino acid sequence represented by SEQ ID NO. 2.

11. The antibody according to claim 10, wherein the framework region 1 of the heavy chain variable domain consists of the amino acid sequence shown in SEQ ID NO. 3.

12. The antibody according to claim 1, wherein the heavy chain variable domain V region consists of an amino acid sequence represented by any one of SEQ ID Nos. 4 to 9.

13. The antibody according to claim 1, wherein the heavy chain variable domain consists of the amino acid sequence shown in any one of SEQ ID Nos. 10 to 15.

14. The antibody according to claim 1, wherein the heavy chain variable domain and the light chain variable domain are each composed of an amino acid sequence represented by any one of the following (a) to (l):

(a) serial number 10 and serial number 16;

(b) sequence number 10 and sequence number 17;

(c) serial number 10 and serial number 18;

(d) serial number 10 and serial number 19;

(e) serial number 10 and serial number 20;

(f) serial number 10 and serial number 21;

(g) serial number 10 and serial number 22;

(h) serial No. 11 and serial No. 23;

(i) sequence number 13 and sequence number 24;

(j) sequence number 14 and sequence number 25;

(k) serial number 15 and serial number 26, and

(l) Serial number 12 and serial number 70.

15. A passive immunotherapeutic agent for influenza, comprising an antibody according to claim 1.

16. A method for passive immunotherapy of influenza, which comprises administering to a mammalian or avian subject already infected with, or having a potential to be infected with, influenza virus an effective amount of an antibody according to claim 1.

17. The method according to claim 16, wherein the subject is a human.

18. A method of producing an antibody according to claim 1, the method comprising the steps of:

19. The method of claim 18, wherein the antibody is a human antibody.

20. The method of claim 18, wherein the antibody library is a phage display library.

21. The method of claim 20, wherein the number of antibody clones is 10¹⁰～10¹¹。

22. The method of claim 18, wherein said B cells are collected by apheresis.

23. The method according to claim 18, wherein, in the step (2), the influenza virus isolate from which the B-cell-collected individual has no history of infection, or the hemagglutinin protein thereof, or the extracellular domain thereof, is used as the antigen.

24. The method of claim 23, wherein the influenza virus isolate is of type H1, H2, or H3.

25. The method of claim 23, wherein said influenza virus isolate belongs to a hemagglutinin subtype with no history of infection in said B cell harvested individual.

26. The method of claim 25, wherein the influenza virus isolate is of type H5, H7, or H9.

27. The method according to claim 18, wherein in the step (4), the neutralizing activity against at least influenza virus type H1 and/or H5 and against influenza virus type H3 is detected.

28. The method according to claim 20, further comprising the step of converting the antibody to an IgG class.

29. A method of detecting an antibody according to claim 1 in a subject, the method comprising the steps of:

(3) the blood was examined for the presence of antibody-producing cells presenting an antibody that binds to both hemagglutinin of a subtype selected from group 1 and hemagglutinin of a subtype selected from group 2 and has a heavy chain variable domain V region encoded by VH1-69 or VH1-e gene.

30. A method of detecting an antibody according to claim 1 in a subject, the method comprising the steps of: