Antibody or fragment thereof, and Anti-influenza vaccine

Abstract

The present invention addresses the problem of providing an anti-idiotype antibody against a broadly neutralizing antibody and an antigen that is produced using the anti-idiotype antibody and can induce reactivity to a wide range of subtypes of influenza viruses.　The present invention provides an antibody or a fragment thereof which specifically binds to a "MEDI8852 antibody" or a "FluA-20 antibody" that is a specific anti-influenza HA broadly neutralizing antibody.

Description

Antibody or fragment thereof, and anti-influenza vaccine

[0001] The present invention relates to an antibody or a fragment thereof, and an anti-influenza vaccine.

[0002] Many pathogenic viruses, such as influenza viruses, can evade the host immune system by altering the immunogenicity of their surface proteins. As a result of these changes, various subtypes of pathogenic viruses are produced.

[0003] Because the subtype of a pathogenic virus can weaken the effectiveness of a vaccine, there is a need for a vaccine that can protect against infection with a single inoculation, regardless of the subtype (known as a "universal vaccine").

[0004] A known method for developing a universal vaccine is to use antibodies (referred to as broadly neutralizing antibodies, or "bnAbs") that can broadly cross-react with and neutralize viral antigens with different amino acid sequences (e.g., Non-Patent Document 1).

[0005] Front Immunol 2021 Oct 19:12:708227. doi: 10.3389 / fimmu. 2021.708227.

[0006] However, antigens and immunization methods capable of inducing broadly neutralizing antibodies that protect against a wide range of influenza virus subtypes have not yet been fully established. Under these circumstances, the present inventors aimed to develop anti-idiotype antibodies (hereinafter also referred to as "anti-Id antibodies") that are directed against broadly neutralizing antibodies. Such anti-idiotype antibodies are expected to function as effective vaccines against influenza viruses.

[0007] The present invention has been made in view of the above circumstances, and aims to provide an anti-idiotype antibody against a broadly neutralizing antibody, and an antigen that can induce reactivity against a wide range of influenza virus subtypes using the anti-idiotype antibody.

[0008] As a result of investigations, the present inventors have newly discovered that the above-mentioned problems can be solved by using an antibody or a fragment thereof composed of a predetermined sequence, and have thus completed the present invention. More specifically, the present invention provides the following.

[0009] (1) An antibody or fragment thereof that specifically binds to the "MEDI8852 antibody," an anti-influenza HA broadly neutralizing antibody, and that satisfies all of the following requirement A. [Requirement A] - The heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 2. - The heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 3. - The 27th amino acid of heavy chain FR1, as defined by Kabat, is an amino acid selected from the group consisting of Tyr, Phe, Leu, Ile, and Glu. - The 29th amino acid of heavy chain FR1, as defined by Kabat, is an amino acid selected from the group consisting of Phe, Leu, Ile, Val, and Ala. - The 73rd amino acid of heavy chain FR3, as defined by Kabat, is Lys. - The 76th amino acid of heavy chain FR3, as defined by Kabat, is Ser.

[0010] (2) The antibody or fragment thereof according to (1), wherein the 27th amino acid of the heavy chain FR1 is Tyr and the 29th amino acid of the heavy chain FR1 is Phe.

[0011] (3) The antibody or fragment thereof according to (1) or (2), wherein the amino acid sequences of CDR1 to CDR3 of the heavy and light chains further satisfy all of the following [Requirement B]. [Requirement B] The heavy chain CDR3 is the amino acid sequence represented by SEQ ID NO: 4. The light chain CDR1 is the amino acid sequence represented by SEQ ID NO: 6. The light chain CDR2 is the amino acid sequence represented by SEQ ID NO: 7. The light chain CDR3 is the amino acid sequence represented by SEQ ID NO: 8.

[0012] (4) The antibody or fragment thereof according to any one of (1) to (3), further satisfying all of the following [Requirement C]: [Requirement C] The heavy chain variable region has the amino acid sequence represented by SEQ ID NO: 1. The light chain variable region has the amino acid sequence represented by SEQ ID NO: 5.

[0013] (5) The antibody or fragment thereof according to any one of (1) to (3), further satisfying all of the following [Requirement D]: [Requirement D] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 17 or 18. The light chain variable region is the amino acid sequence shown in SEQ ID NO: 19 or 20.

[0014] (6) An antibody or fragment thereof that specifically binds to the "FluA-20 antibody," an anti-influenza HA broadly neutralizing antibody, wherein the amino acid sequences of CDR1 to CDR3 of the heavy and light chains satisfy all of the following [Requirement E]. [Requirement E] Heavy chain CDR1 is the amino acid sequence represented by SEQ ID NO: 10. Heavy chain CDR2 is the amino acid sequence represented by SEQ ID NO: 11. Heavy chain CDR3 is the amino acid sequence represented by SEQ ID NO: 12. Light chain CDR1 is the amino acid sequence represented by SEQ ID NO: 14. Light chain CDR2 is the amino acid sequence represented by SEQ ID NO: 15. Light chain CDR3 is the amino acid sequence represented by SEQ ID NO: 16.

[0015] (7) The antibody or fragment thereof according to (6), further satisfying all of the following [Requirement F]: [Requirement F] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9. The light chain variable region has the amino acid sequence shown in SEQ ID NO: 13.

[0016] (8) An anti-influenza vaccine comprising the antibody or fragment thereof according to any one of (1) to (7), or a nucleic acid encoding the antibody or fragment thereof.

[0017] According to the present invention, there are provided anti-idiotype antibodies against broadly neutralizing antibodies, and antigens that can induce reactivity against a wide range of influenza virus subtypes using the anti-idiotype antibodies.

[0018] 1 is a diagram showing the reactivity of mouse serum immunized with "#2-911" against HA in an example. 2 is a conceptual diagram showing the relationship between a template antibody and an anti-Id antibody induced from that antibody, and the mechanism by which an antibody similar to the template antibody is induced by the anti-Id antibody. 3 is a diagram showing the competitiveness of mouse serum immunized with "#2-911" with "MEDI8852 antibody" in an example. 4 is a diagram showing the reactivity of mouse serum immunized with "#2-911 Fab nanoparticles" against HA in an example. 5 is a diagram showing the reactivity of mouse serum immunized with "#2-911 Fab nanoparticles" against HA in an example. 6 is a diagram showing the reactivity of mouse serum immunized with "#2-911 Fab nanoparticles" against HA in an example. 7 is a diagram showing the reactivity of mouse serum immunized with "#2-911" against HA in an example. 1 is a diagram showing the reactivity of serum from rats immunized with "#2-911 Fab nanoparticles" to HA in an example. FIG. 2 is a diagram showing the reactivity of serum from rabbits immunized with "#2-911 Fab nanoparticles" to HA in an example. FIG. 3 is a diagram showing the reactivity of "Hu #2-911 mouse chimera" to "MEDI8852 antibody" in an example. FIG. 4 is a diagram showing the reactivity of serum from mice immunized with "R2-150 Fab nanoparticles" to HA in an example. FIG. 5 is a diagram showing the reactivity of serum from mice immunized with "R2-150 Fab nanoparticles" to HA in an example. FIG. 6 is a diagram showing the analysis results of the interaction between "#2-911" and "MEDI8852 antibody". FIG. 7 is a diagram showing the analysis results of the interaction between "#2-911" and "MEDI8852 antibody". 1 shows the binding affinity between alanine-substituted mutants of "#2-911" and "MEDI8852 antibody." 2 shows the reactivity of monoclonal antibodies obtained from mice administered with "#2-911" as an antigen to various HAs.

[0019] Hereinafter, an embodiment of the present invention will be described, but the present invention is not limited to this.

[0020] <Antibody of the Present Invention or Fragment Thereof> The antibody of the present invention or a fragment thereof specifically binds to a predetermined anti-influenza HA broadly neutralizing antibody, i.e., the "MEDI8852 antibody" or "FluA-20 antibody" described below. The antibody of the present invention or a fragment thereof corresponds to the anti-idiotype antibody of the "MEDI8852 antibody" or "FluA-20 antibody."

[0021] Influenza viruses are classified into multiple types (types A, B, etc.) based on differences in the antigenicity of their surface proteins. Furthermore, 18 subtypes of influenza A are known due to sequence polymorphisms in hemagglutinin (HA). Such polymorphisms make it difficult to produce antibodies that are broadly cross-reactive with influenza virus proteins, hindering the development of vaccines against influenza virus infections.

[0022] In view of these circumstances, the present inventors focused on the development of anti-idiotype antibodies against the known broadly neutralizing anti-influenza HA antibodies "MEDI8852 antibody" and "FluA-20 antibody." An "anti-idiotype antibody" refers to an antibody that specifically binds to an idiotope formed in the antigen-binding region of a specific antibody molecule ("MEDI8852 antibody" or "FluA-20 antibody" in the present invention).

[0023] Furthermore, the present inventors believed that the following results could be expected by inoculating any subject with an anti-idiotype antibody of the "MEDI8852 antibody" or "FluA-20 antibody" as an antigen: - Antibodies and the like having functions or cross-reactivity equivalent to or similar to those of the target antibody (in this invention, the "MEDI8852 antibody" or "FluA-20 antibody") could be produced in the body of the vaccinated subject. - Antibodies and the like having functions that are more extensive than those of the target antibody (in particular, broader cross-reactivity than the target antibody) could be produced in the body of the vaccinated subject. - Antibodies and the like having reactivity to a wide range of influenza HA subtypes could be produced in the body of the vaccinated subject. - Furthermore, the produced antibodies and the like could be cloned.

[0024] Therefore, the present inventors have conducted extensive research and discovered certain anti-idiotype antibodies ("MEDI8852 antibody-anti-Id antibody" and "FluA-20 antibody-anti-Id antibody" which will be described later), thereby completing the present invention.

[0025] The present inventors confirmed that the sera obtained by inoculating a living body with "MEDI8852 antibody-anti-Id antibody" and "FluA-20 antibody-anti-Id antibody" react with a wide range of influenza A subtypes.

[0026] Furthermore, the present inventors have surprisingly confirmed that the serum obtained by inoculating a living body with "MEDI8852 antibody-anti-Id antibody" and "FluA-20 antibody-anti-Id antibody" also reacts with influenza B virus HA. Neither "MEDI8852 antibody" nor "FluA-20 antibody" binds to influenza B virus. Therefore, it was a highly unexpected result to obtain an anti-idiotype antibody that induces the production of antibodies that bind to antigens that are not bound by the template antibodies ("MEDI8852 antibody" and "FluA-20 antibody"). Therefore, the antibody of the present invention or a fragment thereof functions as an antigen that can induce the production of broadly neutralizing antibody-like antibodies (or serum containing such antibodies) that bind to a wide range of known subtypes of influenza A and B viruses.

[0027] In the present invention, the term "influenza virus" includes any influenza virus, and particularly includes influenza A virus and influenza B virus.

[0028] Inoculation with the antibody or fragment thereof of the present invention can produce antibodies or serum reactive to many subtypes of influenza A virus and influenza B virus.

[0029] In a preferred embodiment of the present invention, the antibody or fragment thereof of the present invention includes the following: - An antibody capable of inducing antibodies (or sera) reactive to influenza A virus HA subtypes (preferably H1 to H8, H10 to H18). - An antibody capable of inducing antibodies (or sera) reactive to influenza B virus (particularly Victoria strain and Yamagata strain) HA. - An antibody capable of inducing antibodies (or sera) reactive to all of the above HAs.

[0030] In the present invention, the term "antibody" includes proteins with a Y-shaped four-arm structure, also known as immunoglobulins.

[0031] In the present invention, the term "antibody fragment" refers to a protein or peptide having the structure of the antibody's complementarity determining region (hereinafter also referred to as "CDR") or the structure of the variable region of the antibody of the present invention. Examples of such proteins or peptides include Fab, Fab', F(ab') and the like. ２ Examples of such antibodies include Fv (variable fragment of antibody), single-chain antibodies (H chain, L chain, H chain V region, L chain V region, etc.) including scFv, diabodies (scFv dimers), dsFv (disulfide-stabilized V region), and VHH (variable domain of heavy chain of heavy chain antibody), as well as proteins or peptides comprising at least a portion of the CDR.

[0032] "Fab" is an antibody fragment obtained by treating an antibody molecule with the protease papain, in which approximately the N-terminal half of the H chain and the entire L chain are linked by a disulfide bond, and has a molecular weight of approximately 50,000 and has antigen-binding activity. Fab can be produced by inserting DNA encoding the Fab of an antibody into a prokaryotic or eukaryotic expression vector, and then introducing the vector into a prokaryotic or eukaryotic organism to express the Fab.

[0033] "F(ab') ２" refers to an antibody fragment having antigen-binding activity and a molecular weight of approximately 100,000, which is slightly larger than that of Fab fragments linked via disulfide bonds in the hinge region, among fragments obtained by treating an antibody molecule with the protease pepsin. F(ab') ２ The method for producing Fab is not particularly limited, but examples thereof include a method in which Fab is produced by bonding Fab to a thioether bond or a disulfide bond.

[0034] "Fab'" means "F(ab') ２ Fab' is an antibody fragment having antigen-binding activity and a molecular weight of approximately 50,000, obtained by cleaving the disulfide bond in the hinge region of Fab'. There are no particular limitations on the method for producing Fab', but examples include a method in which DNA encoding the Fab' fragment of an antibody is inserted into an expression vector for prokaryotes or eukaryotes, and the vector is introduced into a prokaryote or eukaryote to express Fab'.

[0035] "Fv" is the minimum unit of an antibody fragment having antigen-binding activity, consisting of one heavy chain variable region (VH) and one light chain variable region (VL). Methods for producing Fv are not particularly limited, but include constructing cDNA encoding VH and VL, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryote or eukaryote to express Fv. However, since it is generally difficult to obtain Fv, scFv, which will be described later, is widely used.

[0036] An "scFv" is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker (P). Methods for producing scFv are not particularly limited, but include a method in which cDNA encoding the VH and VL of an antibody is obtained, DNA encoding the scFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and the expression vector is introduced into a prokaryote or eukaryote to express the scFv.

[0037] A "diabody" is an antibody fragment formed by dimerization of scFv and has bivalent antigen-binding activity. The bivalent antigen-binding activities may be the same, or one of the two may have a different antigen-binding activity. Methods for producing diabodies are not particularly limited, but include obtaining cDNA encoding the VH and VL of an antibody, constructing DNA encoding the scFv so that the amino acid sequence of the peptide linker (P) is 8 residues or less in length, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryote or eukaryote to express the diabody.

[0038] A "dsFv" is a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine ​​residue, and the polypeptides are linked via a disulfide bond between the cysteine ​​residues. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method described by Reiter et al. (Protein Engineering, 7, 697-704, 1994). Methods for producing dsFv are not particularly limited, but include a method in which cDNA encoding the VH and VL of an antibody is obtained, DNA encoding the dsFv is constructed, the DNA is inserted into a prokaryotic or eukaryotic expression vector, and the expression vector is introduced into a prokaryote or eukaryote to express the dsFv.

[0039] A "VHH" is a protein or peptide that consists only of an antibody heavy chain region and is capable of specifically binding to an antigen. Methods for producing VHHs are not particularly limited, but include constructing DNA encoding the VHH, inserting the DNA into an expression vector for prokaryotes or eukaryotes, and introducing the expression vector into a prokaryote or eukaryote to express the VHH.

[0040] A CDR-containing peptide comprises at least two regions of the three heavy chain CDRs (heavy chain CDR1 to CDR3 or HCDR1 to HCDR3) and the three light chain CDRs (light chain CDR1 to CDR3 or LCDR1 to LCDR3). In a preferred embodiment of the present invention, the CDR-containing peptide comprises two heavy chain CDRs (heavy chain CDR1 and CDR2).

[0041] In one aspect of the present invention, the antibody of the present invention may have specific amino acids not only in the heavy chain CDRs and / or light chain CDRs, but also in positions other than the CDRs of the heavy chain variable region and / or light chain variable region.

[0042] Hereinafter, when a number relating to the position of a specific amino acid is referred to (for example, "the 27th amino acid in the heavy chain FR1 is an amino acid selected from the group consisting of Tyr, Phe, Leu, Ile, and Glu"), the number refers to the number defined by Kabat. For the Kabat definition, see "Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991." According to the Kabat definition, amino acids appearing at the same position in the three-dimensional structure of an antibody molecule can be uniquely designated, particularly for amino acids constituting a framework region.

[0043] In the case of "#2-911" (an example of an antibody that satisfies requirements A, B, and C, described below; see Table 6), specific amino acids present in locations other than the CDRs of the heavy chain variable region and / or light chain variable region include the amino acids Tyr27 (tyrosine at position 27) and Phe29 (phenylalanine at position 29) located in the N-terminal framework (also referred to as FR1) of heavy chain CDR1, and further include the amino acids Lys73 (lysine at position 73) and Ser76 (serine at position 76) located in the framework (also referred to as FR3) located between heavy chain CDR2 and heavy chain CDR3 of "#2-911." Note that the antibody function is not lost even if Tyr27 is replaced with Phe (phenylalanine), Leu (leucine), Ile (isoleucine), or Glu (glutamic acid) instead of tyrosine. The above Phe29 may be substituted with Leu (leucine), Ile (isoleucine), Ala (alanine), or Val (valine) instead of phenylalanine without impairing the function of the antibody.

[0044] In one aspect of the present invention, the antibody of the present invention may have a region containing specific amino acids in place of one or more heavy chain CDRs as defined in Requirement A, described below. In the case of "#2-911," such amino acids include the following: His35 (histidine at position 35) contained in the heavy chain CDR1 sequence region and Phe53 (phenylalanine at position 53) contained in the heavy chain CDR2 sequence region; amino acids Tyr27 (tyrosine at position 27) and Phe29 (phenylalanine at position 29) located in the N-terminal framework of heavy chain CDR1; and amino acids Lys73 (lysine at position 73) and Ser76 (serine at position 76) located in the framework (also referred to as FR3) located between heavy chain CDR2 and heavy chain CDR3. Note that antibody function is not lost even if Tyr27 is replaced with Phe, Leu, Ile, or Glu instead of tyrosine. Furthermore, even if Phe29 is substituted with Leu, Ile, Ala, or Val instead of phenylalanine, the antibody function is not lost.

[0045] The antibody or fragment thereof of the present invention will be described in detail below.

[0046] (1) Antibodies or fragments thereof that specifically bind to the "MEDI8852 antibody" The present invention encompasses antibodies or fragments thereof that specifically bind to the "MEDI8852 antibody" (hereinafter, these antibodies or fragments thereof are also collectively referred to as "MEDI8852 antibody-anti-Id antibodies").

[0047] The "MEDI8852 antibody" is a broadly neutralizing antibody whose epitope is the stalk region of HA of influenza A virus, as reported in WO2017 / 123685 and Kallewaard et al. (2016), Cell, 166, 596-608.

[0048] In the present invention, the phrase "specifically binds to MEDI8852 antibody" includes the case where the antibody of the present invention or a fragment thereof binds to the "MEDI8852 antibody" but hardly binds or does not bind at all to other proteins (antibodies, etc.). Whether an antibody or a fragment thereof specifically binds to the "MEDI8852 antibody" is determined by a method such as enzyme-linked immunosorbent assay (ELISA).

[0049] (1-1) Aspect A In one aspect of the present invention, the "MEDI8852 antibody - anti-Id antibody" satisfies all of the following requirement A. [Requirement A] - The heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 2. - The heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 3. - The amino acid at position 27 of heavy chain FR1 according to the Kabat definition is one amino acid selected from the group consisting of Tyr, Phe, Leu, Ile, and Glu. - The amino acid at position 29 of heavy chain FR1 according to the Kabat definition is one amino acid selected from the group consisting of Phe, Leu, Ile, Val, and Ala. - The amino acid at position 73 of heavy chain FR3 according to the Kabat definition is Lys. - The amino acid at position 76 of heavy chain FR3 according to the Kabat definition is Ser.

[0050] In the above embodiment, the heavy chain CDR1 and CDR2, and four amino acids in the FRs (i.e., the 27th amino acid in heavy chain FR1, the 29th amino acid in heavy chain FR1, the 73rd amino acid in heavy chain FR3, and the 76th amino acid in heavy chain FR3 based on the above-mentioned Kabat definition) directly interact with the "MEDI8852 antibody" and contribute to the binding between the "MEDI8852 antibody-anti-Id antibody" and the "MEDI8852 antibody." The four amino acids in the FRs are exposed to the "MEDI8852 antibody" during binding with the "MEDI8852 antibody," and thereby participate in the binding between the antibodies.

[0051] From the viewpoint of facilitating the production of antibodies equivalent to those antibodies that were found to have good reactivity in the Examples described below, in a preferred embodiment of the present invention, the 27th amino acid in heavy chain FR1 based on the Kabat definition is Tyr and the 29th amino acid in heavy chain FR1 based on the Kabat definition is Phe.

[0052] (1-2) Aspect B In one aspect of the present invention, the "MEDI8852 antibody - anti-Id antibody" has heavy and light chain CDR1 to CDR3 amino acid sequences that satisfy all of the following [Requirement B] in addition to the requirements of "Aspect A" above. [Requirement B] The heavy chain CDR3 is the amino acid sequence represented by SEQ ID NO: 4. The light chain CDR1 is the amino acid sequence represented by SEQ ID NO: 6. The light chain CDR2 is the amino acid sequence represented by SEQ ID NO: 7. The light chain CDR3 is the amino acid sequence represented by SEQ ID NO: 8.

[0053]

[0054] However, the present invention also encompasses antibodies or fragments thereof that specifically bind to the "MEDI8852 antibody," an anti-influenza HA broadly neutralizing antibody having the two CDRs of [Requirement A]. For example, the present invention encompasses antibodies or fragments thereof that specifically bind to the "MEDI8852 antibody," whose heavy chain CDR1 is the amino acid sequence represented by SEQ ID NO: 2 and whose heavy chain CDR2 is the amino acid sequence represented by SEQ ID NO: 3. For example, the present invention encompasses antibodies or fragments thereof that specifically bind to the "MEDI8852 antibody," whose heavy chain CDR1 is the amino acid sequence represented by SEQ ID NO: 2, whose heavy chain CDR2 is the amino acid sequence represented by SEQ ID NO: 3, and whose heavy chain CDR3 is the amino acid sequence represented by SEQ ID NO: 4.

[0055] From the viewpoint of more easily achieving the effects of the present invention, the "MEDI8852 antibody-anti-Id antibody" preferably satisfies all of the following [Requirement C]: [Requirement C] The heavy chain variable region is the amino acid sequence represented by SEQ ID NO: 1. The light chain variable region is the amino acid sequence represented by SEQ ID NO: 5.

[0056]

[0057] From the viewpoint of more easily achieving the effects of the present invention, the "MEDI8852 antibody-anti-Id antibody" preferably satisfies all of the following [Requirement D]: [Requirement D] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 17 or 18. The light chain variable region is the amino acid sequence shown in SEQ ID NO: 19 or 20.

[0058]

[0059] The "MEDI8852 antibody-anti-Id antibody" satisfies at least requirement A. Furthermore, the "MEDI8852 antibody-anti-Id antibody" preferably satisfies requirement B, and more preferably satisfies requirement C or requirement D.

[0060] The "MEDI8852 antibody-anti-Id antibody" according to a preferred embodiment of the present invention includes the antibodies referred to in the Examples as "#2-911 (#2-911 antibody)" and "Hu #2-911" (an antibody obtained by humanizing "#2-911," humanized #2-911 antibody).

[0061] (2) Antibodies or fragments thereof that specifically bind to "FluA-20 antibody" The present invention encompasses anti-idiotype antibodies or fragments thereof that specifically bind to "FluA-20 antibody" (hereinafter, these antibodies or fragments thereof are also collectively referred to as "FluA-20 antibody-anti-Id antibodies").

[0062] The "FluA-20 antibody" is a broadly neutralizing antibody reported in WO2020 / 232426 and Bangaru et al. (2019), Cell, 177, 1136-1152, whose epitope is the trimer-forming surface of the head region of HA of influenza A virus (HA-head trimer interface).

[0063] In the present invention, the phrase "specifically binds to Flu A-20 antibody" includes the case where the antibody of the present invention or a fragment thereof binds to "Flu A-20 antibody" but hardly binds or does not bind at all to other proteins (antibodies, etc.). Whether an antibody or a fragment thereof specifically binds to "Flu A-20 antibody" can be determined by a method such as enzyme-linked immunosorbent assay (ELISA).

[0064] In one embodiment of the present invention, the "FluA-20 antibody-anti-Id antibody" has heavy and light chain CDR1 to CDR3 amino acid sequences that satisfy all of the following [Requirement E]. [Requirement E] Heavy chain CDR1 is the amino acid sequence represented by SEQ ID NO: 10. Heavy chain CDR2 is the amino acid sequence represented by SEQ ID NO: 11. Heavy chain CDR3 is the amino acid sequence represented by SEQ ID NO: 12. Light chain CDR1 is the amino acid sequence represented by SEQ ID NO: 14. Light chain CDR2 is the amino acid sequence represented by SEQ ID NO: 15. Light chain CDR3 is the amino acid sequence represented by SEQ ID NO: 16.

[0065]

[0066] From the viewpoint of more easily achieving the effects of the present invention, the "FluA-20 antibody-anti-Id antibody" preferably satisfies all of the following [Requirement F]: [Requirement F] The heavy chain variable region is the amino acid sequence represented by SEQ ID NO: 9. The light chain variable region is the amino acid sequence represented by SEQ ID NO: 13.

[0067]

[0068] The "FluA-20 antibody-anti-Id antibody" according to a preferred embodiment of the present invention includes each antibody referred to as "R2-150 (R2-150 antibody)" in the examples. The "FluA-20 antibody-anti-Id antibody" satisfies at least requirement E. Furthermore, the "FluA-20 antibody-anti-Id antibody" preferably satisfies requirement F.

[0069] <Method for Producing the Antibody of the Present Invention or a Fragment Thereof> The method for producing the antibody of the present invention or a fragment thereof is not particularly limited, and conventional methods for producing various antibodies (monoclonal antibodies, polyclonal antibodies, etc.) and various polypeptides can be used.

[0070] The antibody of the present invention is preferably produced as a monoclonal antibody or a fusion protein with other proteins (ferritin protein, hemocyanin, serum albumin, linker protein, etc.).

[0071] The antibody or fragment thereof of the present invention may be purified as needed.

[0072] Whether an antibody or a fragment thereof is an antibody or a fragment thereof of the present invention can be determined by any method for identifying an amino acid sequence (such as N-terminal amino acid sequence analysis).

[0073] The present invention encompasses genes encoding the antibodies of the present invention or fragments thereof, expression vectors containing the genes, etc. These genes and expression vectors can be prepared by conventionally known methods.

[0074] <Uses of the antibody of the present invention or a fragment thereof> The antibody of the present invention or a fragment thereof functions as an anti-idiotype antibody against the anti-influenza HA broadly neutralizing antibodies "MEDI8852 antibody" and "FluA-20 antibody." Therefore, the antibody of the present invention or a fragment thereof can be used for any application that utilizes this function.

[0075] The antibody or fragment thereof of the present invention can be used in an anti-influenza vaccine. Accordingly, the present invention encompasses an anti-influenza vaccine comprising the antibody or fragment thereof of the present invention.

[0076] The present invention also encompasses methods for immunizing against influenza viruses and methods for preventing influenza virus infections, which comprise the step of administering the antibody or fragment thereof of the present invention to any animal (human or non-human animal).

[0077] <Anti-influenza vaccine> The present invention encompasses vaccines (anti-influenza vaccines) comprising the antibody or a fragment thereof of the present invention, or a nucleic acid encoding the antibody or a fragment thereof.

[0078] The antibody or fragment thereof of the present invention functions as an antigen and can produce antibodies or serum that are broadly cross-reactive with known type A or type B influenza virus HAs that infect any organism. Therefore, the antibody or fragment thereof of the present invention, or nucleic acids encoding them, can be preferably used as a universal vaccine against influenza viruses. In the present invention, "nucleic acid" includes DNA, mRNA, etc.

[0079] The vaccine of the present invention can be effective in preventing, for example, human seasonal influenza virus infections, novel infectious diseases caused by influenza viruses that have newly acquired infectivity in humans, and influenza virus infections that infect non-humans.

[0080] The form of the vaccine of the present invention is not particularly limited, and it may be prepared as a protein vaccine (such as a vaccine comprising the antibody of the present invention or a fragment thereof), a nucleic acid vaccine (such as a vaccine comprising a nucleic acid encoding the antibody of the present invention or a fragment thereof), etc.

[0081] The subjects to which the vaccine of the present invention is administered are not particularly limited, and examples include mammals (humans and non-human animals), birds, etc. Non-human animals include rats, mice, rabbits, monkeys, pigs, etc.

[0082] The dosage and frequency of administration of the vaccine of the present invention can be appropriately determined depending on the condition (age, body weight, symptoms, etc.) of the subject to be administered.

[0083] The method of administering the vaccine of the present invention can be appropriately selected depending on the form of the vaccine, and examples include subcutaneous administration, nasal administration, and transdermal administration.

[0084] The vaccine of the present invention may contain the antibody of the present invention or a fragment thereof, or a nucleic acid encoding the antibody or the fragment thereof, as well as any pharmaceutically acceptable carrier or adjuvant, if necessary.

[0085] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0086] In this example, the following abbreviations are used where appropriate: Anti-Id antibody: anti-idiotype antibody CDR: complementarity determining region VH: heavy chain variable region HCDR: heavy chain CDR VL: light chain variable region LCDR: light chain CDR HA: hemagglutinin

[0087] The CDRs are defined according to the method of Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991).

[0088] <Test 1: Establishment of anti-Id antibody-producing cells against "MEDI8852 antibody"> Hybridoma clones producing anti-Id antibodies against "MEDI8852 antibody," a known broadly neutralizing anti-influenza HA antibody, were obtained according to the following method.

[0089] (1) Preparation of "MEDI8852 Antibody" Based on the amino acid sequence of "MEDI8852" (Kallewaard et al., Cell, 166, 596-608, 2016, and WO2017 / 123685), DNA was synthesized with a Kozak translation initiation sequence added to the 5' end of each of the heavy chain variable region and light chain variable region. The resulting heavy chain variable region was cloned into the "pFUSE-CHIg-hG1" vector (InvivoGen) to obtain a "MEDI8852 heavy chain expression plasmid." The resulting light chain variable region was cloned into the "pFUSE2-CLIg-hk" vector (InVivogen) to obtain a "MEDI8852 light chain expression plasmid." Azenta's services were used for cloning.

[0090] Next, transient expression was carried out using the "MEDI8852 heavy chain expression plasmid" and the "MEDI8852 light chain expression plasmid" as the introduction plasmids, using the "Expi293 Expression System" (Thermo Fisher Scientific). After transient expression, the culture supernatant was collected and affinity purified using a "HiTrap Protein A HP column" (Cytiva). The resulting eluate was concentrated and subjected to gel filtration purification using a "HiLoad 26 / 600 Superdex 200pg column" (Cytiva). The eluted fractions of the gel filtration purification were confirmed by SDS-PAGE, and fractions containing the target protein were collected and subjected to the following tests as "MEDI8852 antibody".

[0091] (2) Preparation of MEDI8852 Mouse Fc Chimeric Antibody Based on the amino acid sequence of the heavy chain Fab component region of the "MEDI8852 antibody," DNA was synthesized with a Kozak translation initiation sequence added to the 5' end. The resulting DNA was cloned into the "pFUSE-CHIg-mG2a" vector (InvivoGen) to obtain the "MEDI8852 Fab-mouse Fc chimeric heavy chain expression plasmid." The cloning was performed using the services of Azenta.

[0092] Next, a MEDI8852 Fab-mouse Fc chimeric antibody (hereinafter also referred to as "MEDI8852 mouse Fc chimeric antibody") was prepared in the same manner as in "(1) Preparation of 'MEDI8852 antibody'" above, using the "MEDI8852 Fab-mouse Fc chimeric heavy chain expression plasmid" and the "MEDI8852 light chain expression plasmid" as introduction plasmids.

[0093] (3) Preparation of Hybridoma Clones Producing Anti-Id Antibodies Against "MEDI8852 Antibody" Hybridoma clones producing anti-Id antibodies specific to "MEDI8852 antibody" were obtained according to the following method.

[0094] (3-1) Immunization of Mice "MEDI8852 mouse Fc chimeric antibody" was used as the antigen, which was mixed with the adjuvant "TiterMax Gold, cat. G-1" (TiterMax) and administered to the soles of BALB / c mice. The antigen dose was set to 50 μg per mouse. Booster immunizations were performed 3, 10, and 12 days after administration. However, the antigen dose for booster immunization was set to 5 μg per mouse, and PBS buffer was used instead of the adjuvant.

[0095] After immunization (2 days after the final administration), popliteal lymph nodes were collected from the mice and a cell suspension was prepared. The resulting cell suspension was mixed with SP2 / 0-Ag14 myeloma cells and subjected to electrofusion using an electrofusion device "ECFG21" (Neppa Gene). After fusion, the resulting cells were suspended in "ClonaCell-HY Medium D, cat. ST-03804" (STEMCELL Technologies) and seeded onto plastic dishes. Colonies formed 10 days after seeding were isolated into 96-well plastic plates containing hybridoma medium, and the culture supernatant was used for the following evaluations. The hybridoma medium used was "RPMI1640, cat. A1049101" (Thermo Fisher Scientific) supplemented with 1 / 10 of the amount of "Doma-Drive, cat. T31-1003SF" (Immune Systems) and 1 / 50 of the amount of "HAT Supplement, cat. 21-60017" (Thermo Fisher Scientific).

[0096] (3-2) Selection of clones producing anti-Id antibodies specific to "MEDI8852 antibody" Using each culture supernatant obtained in "(3-1) Immunization of mice" above, hybridoma clones producing anti-Id antibodies specific to "MEDI8852 antibody" were screened. Specifically, clones producing antibodies that meet all of the following criteria were selected by ELISA. (Criterion 1) React (bind) to "MEDI8852 antibody". (Criterion 2) Do not react with a control antibody "human IgG1 kappa-UNLB, cat. 0151K-01" (Southern Biotech) that has the same isotype as "MEDI8852 antibody".

[0097] ELISA was performed using an antigen solid-phase format in which "MEDI8852 antibody" or "human IgG1-kappa-UNLB" was immobilized. The secondary antibody used for detection was "anti-mouse IgG-Fc fragment, HRP conjugated, cat. A90-131" (Bethyl Laboratories). For color development, "ELISA POD Substrate TMB Kit (popular), cat. 05298-80" (Nacalai Tesque) was used, and absorbance at a wavelength of 450 nm was measured using an "Infinite M1000 pro microplate reader" (Tecan).

[0098] <Test 2: Selection of anti-Id antibodies that induce antibodies reactive to HA> Anti-Id antibodies against the "MEDI8852 antibody" were obtained according to the following method. This antibody can induce antibodies reactive to HA when administered as an antigen.

[0099] (1) Sequence analysis of anti-Id antibodies Hybridoma clones were expanded and cultured, and RNA was extracted from the resulting culture using "RNeasy mini Kit, cat. 74104" (QIAGEN). Next, cDNA synthesis was performed using "SuperScript III First-Strand Synthesis Supermix, cat. 18080400" (Thermo Fisher Scientific).

[0100] The synthesized cDNA was used as a template to amplify the antibody variable region sequences (heavy chain variable region and light chain variable region) by PCR. Both the heavy chain variable region and the light chain variable region were amplified using primers that recognize the upstream of the variable region and the downstream of the constant region. The resulting DNA fragments were cloned using "Zero Blunt TOPO PCR Cloning Kit, cat. 450159" (Thermo Fisher Scientific), and DNA sequence analysis of the antibody variable region was performed. Azenta's services were used for the analysis.

[0101] The antibody sequence identified as a result of DNA sequence analysis of the anti-Id antibody and its CDR region sequence are shown in Table 6. The anti-Id antibody having these sequences is also referred to as "#2-911" hereinafter. In addition, in the VH sequence shown in Table 6, the 27th amino acid of the heavy chain framework is Tyr, the 29th amino acid is Phe, the 73rd amino acid is Lys, and the 76th amino acid is Ser, based on the Kabat definition.

[0102]

[0103] (2) Preparation of Recombinant Anti-Id Antibody Based on the sequence of the variable region of the anti-Id antibody (#2-911), DNA was genetically synthesized with a Kozak translation initiation sequence added to the 5' end of the heavy chain variable region and light chain variable region. The obtained heavy chain variable region was cloned into the "pFUSE-CHIg-mG2a" vector (InvivoGen) to obtain an "anti-Id antibody heavy chain expression plasmid." The obtained light chain variable region was cloned into the "pFUSE2-CLIg-ml1" vector (InvivoGen) to obtain an "anti-Id antibody light chain expression plasmid." The cloning was performed using the services of Azenta.

[0104] Next, transient expression was carried out using the "anti-Id antibody heavy chain expression plasmid" and the "anti-Id antibody light chain expression plasmid" as the introduction plasmids using the "Expi293 Expression System" (Thermo Fisher Scientific). After transient expression, the culture supernatant was collected and affinity purified using a "HiTrap Protein G HP column" (Cytiva). The resulting eluate was concentrated and purified by gel filtration using a "HiLoad 26 / 600 Superdex 200 pg column" (Cytiva). The eluted fractions from the gel filtration purification were confirmed by SDS-PAGE, and the fractions containing the target protein were collected and subjected to the following tests as recombinant antibodies (hereinafter also referred to as "recombinant anti-Id antibodies").

[0105] (3) Verification of reactivity to HA Based on the following method, the "recombinant anti-Id antibody" (#2-911) was verified from the viewpoint of whether it functions as an antigen capable of inducing antibodies that react with HA in mouse serum.

[0106] (3-1) Immunization of Mice A recombinant anti-Id antibody was used as the antigen, mixed with the adjuvant TiterMax Gold (TiterMax), and intraperitoneally administered to BALB / c mice (3 mice per group). The antigen dose was set to 200 μg per mouse. After administration, booster immunizations were administered twice at 2-week intervals from the start of administration. However, the antigen dose for booster immunizations was set to 20 μg per mouse, and Imject Alum Adjuvant, cat. 77161 (Thermo Fischer Scientific) was used as the adjuvant.

[0107] (3-2) Verification of reactivity with HA One week after the first booster immunization, partial blood was collected from mice immunized with the "recombinant anti-Id antibody," and the generation of antibodies against the anti-Id antibody was confirmed by ELISA. Therefore, it was found that this anti-Id antibody functions as an antigen. Therefore, one week after the second booster immunization, whole blood was collected from the mice, and serum was prepared (immune serum), which was then used to verify reactivity with HA. As a control, serum from a non-immunized mouse was also prepared (naive serum).

[0108] Reactivity with HA was verified by ELISA using an antigen solid-phase format in which an antigen panel consisting of various HAs was immobilized. These HAs correspond to H1-H18, which represent the antigenicity of circulating influenza A viruses. As a "positive control," to compare the immune induction ability of anti-Id antibodies, serum from mice immunized with Ab2 antibody, an anti-Id antibody specific to A / BK79 (H3N2) HA, as described in a previous paper (Betakova et al. J. Gen. Virol. 1998), was reacted with immobilized A / BK79 HA. Each serum was diluted 300-fold with PBS buffer containing 1% BSA (hereinafter also referred to as "1% BSA / PBS") and used as a sample. The secondary antibody used for detection was "anti-mouse IgG-Fc fragment, HRP conjugated" (Bethyl Laboratories). The color-developing reagent used was "ELISA POD substrate TMB kit" (popular) (Nacalai Tesque). After the ELISA reaction, absorbance at a wavelength of 450 nm was measured using an "Infinite M1000 pro microplate reader" (Tecan). The ELISA was performed in duplicate at each point, and the average value was calculated.

[0109] The results based on the above-mentioned ELISA are shown in Figure 1. This figure shows the reactivity of naive or immune serum with a panel of HA antigens. These results demonstrate that the anti-Id antibody (#2-911) functions as an antigen capable of inducing antibodies against all known influenza A virus HA subtypes (except H9).

[0110] (4) Verification of binding competition between "#2-911" and "MEDI8852 antibody" From the results of (3) above, it was found that "#2-911," obtained using the "MEDI8852 antibody" as a template, is an anti-Id antibody of the "MEDI8852 antibody," and further functions as an antigen to induce antibodies that react with HA. Here, when mice are immunized with "#2-911" as an antigen, the antibodies contained in the resulting serum may contain antibodies similar to the "MEDI8852 antibody" (Figure 2). In such cases, such "MEDI8852 antibody"-like antibodies should compete with the "MEDI8852 antibody" for binding to HA. Therefore, the competition between mouse serum obtained by inoculation with "#2-911" and the "MEDI8852 antibody" was verified by competitive ELISA.

[0111] As the HA antigen in the competitive ELISA, one of the following was used: NC99 / H1N1: a recombinant protein of the ectodomain of human influenza A / New Caledonia / 20 / 99 (H1N1); Vic361 / H3N2: a recombinant protein of the ectodomain of A / Victoria / 361 / 2011 (H3N2).

[0112] Each serum (diluted 100-, 300-, or 900-fold) was reacted with the antigen-immobilized wells at room temperature for 0.5 hours, and then washed with PBS buffer (hereinafter also referred to as "PBST") containing 0.05% Tween 20. Next, the "MEDI8852 antibody" (presumably more than three times the EC50 of 13.5 ng / ml for NC99 / H1N1) prepared at 45 ng / ml using 1% BSA / PBS was reacted with the "NC99 / H1N1"-immobilized wells at room temperature for 1 hour, and then washed with PBST. On the other hand, the wells containing Vic361 / H3N2 were incubated with MEDI8852 antibody (90 ng / ml, more than three times the EC50 of 26.7 ng / ml for Vic361 / H3N2) in 1% BSA / PBS at room temperature for 1 hour, followed by washing with PBST. As a control, a 20 μg / ml mouse isotype control antibody, mouse IgG2a isotype control (clone C1.18.4), cat. I-118 (Leinco Technologies), was used instead of serum.

[0113] After the above reaction, the "MEDI8852 antibody" bound to HA was detected using "anti-human IgG-Fc fragment, HRP conjugated, cat. A80-104P" (Bethyl Laboratories) as a secondary antibody. ELISA was performed in duplicate for each point, and the average value was calculated.

[0114] The results based on the above-mentioned ELISA are shown in Figure 3. As shown in Figure 3, the binding of the "MEDI8852 antibody" to HA decreased in a concentration-dependent manner in the mouse serum obtained by inoculation with "#2-911". This indicates that the mouse serum obtained by inoculation with "#2-911" contains an antibody that competes with the "MEDI8852 antibody" in binding to HA, that is, it contains an antibody similar to the "MEDI8852 antibody".

[0115] Furthermore, analysis of the complex formed by binding of the above-mentioned "#2-911" and MEDI8852 suggested that the heavy chain CDR1 (amino acid sequence represented by SEQ ID NO: 2) and heavy chain CDR2 (amino acid sequence represented by SEQ ID NO: 3) of "#2-911" are directly involved in the binding.

[0116] <Test 3: Evaluation of anti-Id antibody (#2-911) against "MEDI8852 antibody" - 1> As in Test 2, it was found that mouse serum obtained by inoculation with "#2-911" competes with "MEDI8852 antibody" in binding to HA, that is, antibodies similar to "MEDI8852 antibody" were induced. Therefore, nanoparticles using a self-associating protein designed to enable multivalent antigen presentation of the Fab region of "#2-911" (hereinafter also referred to as "#2-911Fab nanoparticles") were prepared. Note that nanoparticles using self-associating proteins are known as a form of vaccine antigen. Furthermore, serum from animals immunized with the nanoparticles as an antigen was obtained, and their binding to HA molecules was evaluated.

[0117] (1) Preparation of #2-911 Fab Nanoparticles. DNA encoding a fusion protein sequence containing a ferritin protein sequence was ligated downstream of the heavy chain Fab region sequence of the #2-911 expression plasmid via a linker sequence containing repeats (3, 5, or 7 times) of the four amino acid sequence serine-glycine-glycine-glycine. This fusion protein was designed according to Cell, 162, 1090-1100, 2015, and contained a Kozak translation initiation sequence at the 5' end and a stop codon at the 3' end. The resulting gene was cloned into the pcDNA3.4 vector (Thermo Fisher Scientific) to obtain the #2-911 Fab-nanoparticle expression plasmid (heavy chain).

[0118] The light chain Fab component region of the "#2-911" expression plasmid was also cloned into the "pcDNA3.4" vector (Thermo Fisher Scientific) to obtain an expression plasmid (light chain) for "#2-911Fab-nanoparticles."

[0119] For the cloning, the services of Azenta were used.

[0120] (2) Preparation of "#2-911" immunized mouse serum The resulting "#2-911Fab-nanoparticle" expression plasmids (heavy chain and light chain) were introduced using the "Expi293 Expression System" (Thermo Fisher Scientific) for transient expression. After transient expression, the culture supernatant was collected and affinity purified using a "CaptureSelect LC-lambda (mouse) affinity matrix, cat. 194323010" (Thermo Fisher Scientific). The resulting eluate was concentrated and purified by gel filtration using a "HiPrep 26 / 60 Sephacryl S-500HR column" (Cytiva). The eluted fractions from the gel filtration purification were confirmed by SDS-PAGE, and the fractions containing the target protein were collected and used hereinafter as "#2-911Fab nanoparticles."

[0121] (3) Immunization of Mice "#2-911Fab nanoparticles" were used as the antigen, mixed with the adjuvant "TiterMax Gold" (TiterMax), and intraperitoneally administered to BALB / c mice (6 mice per group). The antigen dose was set to 200 μg per mouse. After administration, booster immunizations were performed twice at 2-week intervals from the start of administration. However, the antigen dose for booster immunizations was set to 20 μg per mouse, and "Imject Alum Adjuvant, cat. 77161" (Thermo Fischer Scientific) was used as the adjuvant. One week after the final immunization, whole blood was collected, and serum was prepared and used for the following evaluation.

[0122] (4) Verification of reactivity with HA Based on the same method as in "(3-2) Verification of reactivity with HA" in Test 2, the reactivity of antibodies contained in serum obtained using "#2-911 Fab nanoparticles" as an antigen with a panel of HA antigens was evaluated by ELISA using a solid-phase antigen format.

[0123] The results of the ELISA are shown in Figure 4. This figure shows the reactivity of naive serum or immune serum with a panel of HA antigens. These results confirmed that mouse serum obtained using "#2-911 Fab nanoparticles" as an antigen was capable of inducing antibodies that exhibit broad cross-reactivity against various HAs, similar to the anti-Id antibody (#2-911).

[0124] Furthermore, it was confirmed that the "#2-911 Fab nanoparticles" induced a more stable immune response than immunization using the "#2-911" antibody as an antigen, and that the serum of mice immunized with this antigen showed stronger reactivity to HAs from H4 to H15.

[0125] Furthermore, it was confirmed that the antibodies contained in the immune serum reacted with both the HA of influenza B Victoria strain and the HA of Yamagata strain, which cannot be reacted with the template antibody for "#2-911," "MEDI8852 antibody" (Figure 5).

[0126] (5) Evaluation of binding to HA molecules expressed on the cell surface Using serum obtained using "#2-911 Fab nanoparticles" as an antigen, binding to HA molecules expressed on cells was evaluated according to the following method.

[0127] (5-1) Preparation of HA-Expressing Cells The full-length HA gene of "A / Indonesia / 5 / 2005 (H5N1)" (hereinafter also referred to as "Indo05 / H5N1") including the signal sequence was cloned into the "pCMVbeta" vector (Takara Bio Inc.). The obtained full-length HA expression plasmid was used as a transfer plasmid and transiently expressed in Expi293 cells using the "Expi293 Expression System" (Thermo Fisher Scientific). Hereinafter, the obtained cells are also referred to as "HA-expressing cells."

[0128] (5-2) Verification by flow cytometry 100,000 HA-expressing cells and 100,000 Expi 293 cells (hereinafter also referred to as "mock cells") transfected with a plasmid not containing the HA gene were prepared. These cells were reacted at 4°C in the presence of one of the following antibodies, washed with PBS, and then stained using the detection antibody "BD Pharmingen PE goat anti-mouse Ig (multiple adsorption), cat. 550589" (BD Biosciences). - Immune serum of "#2-911 Fab nanoparticles" - Negative control: mouse IgG2a (mIg2a) isotype control (clone C1.18.4) (Leinco Technologies) - Positive control: MEDI8852 mouse Fc chimera

[0129] After staining, flow cytometry analysis was performed using a "FACS Canto II" (BD Biosciences). Figure 6 shows the histogram obtained by flow cytometry. In the negative control group, no change was observed between HA-expressing cells and mock cells. In the positive control, a peak shift was observed in HA-expressing cells compared to mock cells, confirming that HA molecules reacting with MEDI8852 mouse Fc chimera were expressed on the cell surface. On the other hand, a reaction against HA-expressing cells was also observed in the immune serum group of "#2-911 Fab nanoparticles." This confirmed that the immune serum contained antibodies that react with HA expressed on the cell surface.

[0130] <Test 4: Evaluation of anti-Id antibody (#2-911) against "MEDI8852 antibody" - 2> In the above Test 3, it was confirmed that when mice were immunized with nanoparticles containing a multivalent Fab region of "#2-911," antibodies reactive with HA were contained. Therefore, it was examined whether antibodies reactive with HA were similarly induced in animals other than mice.

[0131] (1) Immunization of Animals Rats and rabbits were used instead of mice and were immunized as follows.

[0132] (1-1) Immunization of rats "#2-911Fab nanoparticles" were used as the antigen, mixed with the adjuvant "TiterMax Gold" (TiterMax), and intraperitoneally administered to Jcl:Wistar rats (3 rats per group). The antigen dose was set to 400 μg per rat. After administration, booster immunizations were administered twice at 2-week intervals from the start of administration. However, the antigen dose for booster immunizations was set to 40 μg per rat, and "Imject Alum Adjuvant, cat. 77161" (Thermo Fischer Scientific) was used as the adjuvant. One week after the final immunization, whole blood was collected, and serum was prepared and used for the following verification.

[0133] (1-2) Immunization of Rabbits "#2-911 Fab nanoparticles" were used as the antigen, mixed with the adjuvant "TiterMax Gold" (TiterMax), and intradermally inoculated into Scl:NZW rabbits (2 rabbits per group). The antigen dose was set at 300 μg per rabbit. After administration, booster immunizations were administered twice, at two-week intervals from the start of administration. However, the booster antigen dose was set at 300 μg per rabbit, and "Imject Alum Adjuvant, cat. 77161" (Thermo Fischer Scientific) was used as the adjuvant. Japan Bioserum's services were used for immunization and blood collection. One week after the final immunization, whole blood was collected, and serum was prepared and used for the following verification.

[0134] (2) Verification of reactivity with HA Verification of reactivity with HA was performed by ELISA using an antigen solid-phase format in which an antigen panel consisting of various HAs was immobilized, as in Test 2, "(3-2) Verification of reactivity with HA."

[0135] Figures 7 and 8 show the results based on the above ELISA. Figure 7 shows the results using rats, and Figure 8 shows the results using rabbits. The results below confirmed that "#2-911" is able to induce antibodies in immune serum that react broadly against various subtypes of influenza A not only in mice but also in rats and rabbits.

[0136] Furthermore, it was confirmed that the antibodies contained in each immune serum reacted with both HA of influenza B Victoria strain and Yamagata strain, which the template antibody for "#2-911," "MEDI8852 antibody," could not react with (Figures 9 and 10). These results suggest that by using "#2-911" as an antigen, for example, by immunizing animals to obtain antibodies, it is possible to obtain new antibodies that are similar in function and morphology to the template antibody (MEDI8852 antibody), or that have functions that exceed those of the template antibody.

[0137] <Test 5: Preparation of humanized antibody> Based on the following method, an antibody (hereinafter referred to as a humanized antibody) was obtained that had the CDR sequences of "#2-911" and had the remaining sequences converted to human antibody sequences. Humanized antibodies can be a form of more effective vaccine antigen. Therefore, the humanized antibody was examined to determine whether it exhibited reactivity equivalent to that of the original antibody "#2-911."

[0138] (1) Design of humanized sequence of "#2-911" The humanized sequence was designed using the CDR grafting method with reference to the method of Kuramochi et al. (Human monoclonal antibodies, Methods and protocols, Methods in Molecular Biology 1904, ISBN 978:1-4939-8957-7, Chapter 9).

[0139] First, known structures of mouse antibodies with high amino acid sequence homology to "#2-911" were searched for, and the positions of amino acids thought to be important for CDR structure formation and reaction with antigen were predicted. In parallel with this prediction work, cDNA databases for human antibody heavy chain variable regions and human antibody light chain variable regions were searched for sequences with high homology to the framework regions of the heavy chain variable region and light chain variable region of "#2-911".

[0140] A sequence was designed by linking the framework sequence of the human antibody sequence identified as a result of the search with the CDR sequence of "#2-911." Amino acid sequences at positions thought to be important for CDR structural formation and antigen reactivity were grafted into this sequence, resulting in the design of the humanized antibody sequence "Hu #2-911" shown in Table 7. The CDR sequences in this humanized antibody sequence are identical to those in the original mouse antibody. Furthermore, in this humanized antibody sequence, as in the original mouse antibody sequence, the 27th amino acid in the heavy chain framework is Tyr, the 29th amino acid is Phe, the 73rd amino acid is Lys, and the 76th amino acid is Ser, based on the Kabat definition.

[0141]

[0142] (2) Evaluation of the antigenic reactivity of "Hu #2-911" Whether "Hu #2-911" has the same antigenic reactivity as "#2-911" was evaluated based on the following method.

[0143] (2-1) Preparation of "Hu #2-911" Mouse Chimera DNA was synthesized by adding a Kozak translation initiation sequence to the 5' end of the heavy chain variable region and light chain variable region of "Hu #2-911." The resulting heavy chain variable region was cloned into the "pFUSE-CHIg-mG2a" vector (InvivoGen). The resulting light chain variable region was cloned into the "pFUSE2-CLIg-ml1" vector (InvivoGen). Genscript's services were used for cloning. These procedures resulted in a chimeric antibody expression plasmid containing the variable region of a humanized anti-Id antibody and the constant region of a mouse antibody.

[0144] Next, the resulting chimeric antibody expression plasmid was used as an introduction plasmid and transient expression was carried out using the "Expi293 Expression System" (Thermo Fisher Scientific). After transient expression, the culture supernatant was collected and affinity purified using "rProtein A Sepharose fast flow, cat. 17127903" (Cytiva). The resulting eluate was concentrated and buffer exchanged into PBS using a "PD-10 column, cat. 17085101" (Cytiva). The eluted fractions of the buffer-exchanged eluate were confirmed by SDS-PAGE, and the fractions containing the target protein were collected and subjected to the following tests as "Hu #2-911" mouse chimeric antibody (hereinafter also referred to as "Hu #2-911 mouse chimera").

[0145] (2-2) Verification of reactivity with "MEDI8852" The "MEDI8852" antibody corresponds to the antigen of "Hu #2-911." Therefore, the reactivity of "Hu #2-911 mouse chimera" with "MEDI8852" was verified based on the following method.

[0146] The verification was carried out using ELISA in an antigen solid-phase format in which "MEDI8852" was immobilized. Note that "MEDI8852" is a human antibody, and "#2-911" is a mouse antibody. In this example, "Hu #2-911" was prepared as a "Hu #2-911 mouse chimera" so that the reactivity of "Hu #2-911" to "MEDI8852" and the reactivity of "#2-911" to "MEDI8852" could be compared using the same secondary antibody.

[0147] For each of "#2-911" and "Hu #2-911 Mouse Chimera," a seven-stage dilution series was prepared using 1% BSA / PBS, starting from 10 μg / ml at a 3-fold common ratio. The resulting dilution series was reacted with the antigen and detected using the secondary antibody "anti-mouse IgG-Fc fragment, HRP conjugated" (Bethyl Laboratories). For color development, an "ELISA POD Substrate TMB Kit" (popular) (Nacalai Tesque) was used, and the absorbance at a wavelength of 450 nm was measured using an "Infinite M1000 pro microplate reader" (Tecan). ELISA was performed in duplicate at each point, and the average value was calculated.

[0148] The results based on the above ELISA are shown in Figure 11. This figure shows the reactivity between "Hu #2-911 mouse chimera" and "MEDI8852." The EC50 of each antibody calculated from these results is as follows: - "#2-911": 1.67 μg / mL - "Hu #2-911 VH1 / VL1" (combination of SEQ ID NOs: 17 and 19): 1.71 μg / mL - "Hu #2-911 VH1 / VL2" (combination of SEQ ID NOs: 17 and 20): 2.34 μg / mL - "Hu #2-911 VH2 / VL1" (combination of SEQ ID NOs: 18 and 19): 1.43 μg / mL - "Hu #2-911 VH2 / VL2" (combination of SEQ ID NOs: 18 and 20): 0.9 μg / mL Therefore, it was found that "Hu #2-911 mouse chimera" exhibits reactivity to the antigen "MEDI8852" equivalent to that of the original antibody "#2-911".

[0149] Although the framework sequence was replaced with a different sequence due to humanization of the antibody sequence, the antibody showed reactivity equivalent to that of "#2-911," suggesting that the antigen reactivity of "#2-911" is determined by the CDR sequence.

[0150] <Test 6: Establishment of anti-Id antibody-producing cells against "FluA-20 antibody"> Hybridoma clones producing anti-Id antibodies against "FluA-20 antibody," a known broadly neutralizing anti-influenza HA antibody, were obtained according to the following method.

[0151] (1) Preparation of "FluA-20 Antibody" Based on the amino acid sequence of "FluA-20" (Bangaru, et al., Cell, 177, 1136-1152, 2019, WO2020 / 232426), DNA was synthesized with a Kozak translation initiation sequence added to the 5' end of each of the heavy chain variable region and light chain variable region. The obtained heavy chain variable region was cloned into the "pFUSE-CHIg-hG1" vector (InvivoGen) to obtain a "FluA-20 heavy chain expression plasmid." The obtained light chain variable region was cloned into the "pFUSE2-CLIg-hk" vector (InvivoGen) to obtain a "FluA-20 light chain expression plasmid." Azenta's services were used for cloning.

[0152] Next, transient expression was carried out using the "FluA-20 heavy chain expression plasmid" and the "FluA-20 light chain expression plasmid" as the introduction plasmids using the "Expi293 Expression System" (Thermo Fisher Scientific). After transient expression, the culture supernatant was collected and affinity purified using a "HiTrap Protein A HP column" (Cytiva). The resulting eluate was concentrated and subjected to gel filtration purification using a "HiLoad 26 / 600 Superdex 200 pg column" (Cytiva). The eluted fractions from the gel filtration purification were confirmed by SDS-PAGE, and fractions containing the target protein were collected and subjected to the following tests as "FluA-20 antibody."

[0153] (2) Preparation of Hybridoma Clones Producing Anti-Id Antibodies Against "FluA-20 Antibody" Hybridoma clones producing anti-Id antibodies specific to "FluA-20 antibody" were obtained according to the following method.

[0154] (2-1) Immunization of Mice "FluA-20 antibody" was used as the antigen, which was mixed with the adjuvant "TiterMax Gold" (TiterMax) and administered to the soles of BALB / c mice. The antigen dose was set to 50 μg per mouse. Booster immunizations were performed 3, 10, and 12 days after administration. However, the antigen dose for booster immunization was set to 5 μg per mouse, and PBS buffer was used instead of the adjuvant.

[0155] After immunization (2 days after the final administration), popliteal lymph nodes were collected from the mice and a cell suspension was prepared. The resulting cell suspension was mixed with SP2 / 0-Ag14 myeloma cells and subjected to electrical cell fusion using an electrical cell fusion device "ECFG21" (Neppa Gene). After fusion, the resulting cells were suspended in "ClonaCell-HY Medium D" (STEMCELL Technologies) and seeded onto plastic dishes. Colonies formed 10 days after seeding were isolated into 96-well plastic plates containing hybridoma medium, and the culture supernatant was used for the following evaluations. The hybridoma medium used was "RPMI1640" (Thermo Fisher Scientific) to which 1 / 50 of "Nutridoma-CS, cat. 1136374001" (Merck Millipore) and "HAT Supplement" (Thermo Fisher Scientific) were added.

[0156] (2-2) Selection of clones producing anti-Id antibodies specific to "Flu A-20 antibody" Using each culture supernatant obtained in "(2-1) Immunization of mice" above, hybridoma clones producing anti-Id antibodies specific to "Flu A-20 antibody" were screened. Specifically, clones producing antibodies that meet all of the following criteria were selected by ELISA. (Criterion 1) React (bind) to "Flu A-20 antibody". (Criterion 2) Do not react with a control antibody "human IgG1 kappa-UNLB" (Southern Biotech) that has the same isotype as "Flu A-20 antibody".

[0157] ELISA was performed in an antigen solid-phase format using "FluA-20 antibody" or "human IgG1-kappa-UNLB (Southern Biotech)." The secondary antibody used for detection was "anti-mouse IgG-Fc fragment, HRP conjugated" (Bethyl Laboratories). For color development, an "ELISA POD Substrate TMB Kit" (popular) (Nacalai Tesque) was used, and absorbance at a wavelength of 450 nm was measured using an "Infinite M1000 pro microplate reader" (Tecan).

[0158] <Test 7: Selection of anti-Id antibodies that induce antibodies reactive to HA> Anti-Id antibodies against "FluA-20 antibody" were obtained according to the following method. This antibody can induce antibodies reactive to HA when administered as an antigen.

[0159] (1) Sequence analysis of anti-Id antibody Hybridoma cells were expanded and RNA was extracted from the resulting culture using "RNeasy mini Kit, cat. 74104" (QIAGEN). The DNA sequence of the antibody variable region was then analyzed by an external contractor (Azenta).

[0160] The antibody sequence identified as a result of DNA sequence analysis of the anti-Id antibody and its CDR region sequence are shown in Table 8. The anti-Id antibody having these sequences is hereinafter also referred to as "R2-150."

[0161]

[0162] (2) Preparation of anti-Id antibody Fab nanoparticles "R2-150 Fab nanoparticles" DNA encoding the sequence of a fusion protein bound to ferritin protein was linked downstream of the sequence of the heavy chain Fab component of "R2-150" via a linker sequence containing five repeats of a sequence consisting of the four amino acids serine-glycine-glycine-glycine. This fusion protein was designed according to Cell, 162, 1090-1100, 2015, and contained a Kozak translation initiation sequence added to the 5' end and a stop codon added to the 3' end. The resulting gene was cloned into the "pcDNA3.4" (Thermo Fisher Scientific) vector to obtain an anti-Id antibody Fab-nanoparticle expression plasmid (heavy chain).

[0163] The light chain Fab constituent region of "R2-150" was also cloned into a "pcDNA3.4" (Thermo Fisher Scientific) vector in the same manner as above to obtain an anti-Id antibody Fab-nanoparticle expression plasmid (light chain).

[0164] Next, the obtained expression plasmids (heavy chain and light chain) were used as introduction plasmids and transient expression was carried out using the "Expi293 Expression System" (Thermo Fisher Scientific). After transient expression, the culture supernatant was collected and affinity purified using a "CaptureSelect LC-kappa (mur) affinity matrix, cat. 191315005" (Thermo Fisher Scientific). The obtained eluate was concentrated and subjected to gel filtration purification using a "HiPrep 26 / 60 Sephacryl S-500HR column" (Cytiva). The eluted fractions from the gel filtration purification were confirmed by SDS-PAGE, and the fractions containing the target protein were collected and used hereinafter as "R2-150Fab nanoparticles."

[0165] (3) Immunization of Mice "R2-150Fab nanoparticles" were used as the antigen, mixed with the adjuvant "TiterMax Gold" (TiterMax), and intraperitoneally administered to BALB / c mice. The antigen dose was set to 200 μg per mouse. After administration, two booster immunizations were performed at two-week intervals from the start of administration. However, the antigen dose for booster immunizations was set to 20 μg per mouse, and "Imject Alum Adjuvant, cat. 77161" (Thermo Fischer Scientific) was used as the adjuvant. One week after the first booster immunization, partial blood was collected from the mice, and the production of antibodies against the anti-Id antibody used as the antigen was confirmed by ELISA. One week after the final immunization, whole blood was collected, and serum was prepared and used for the following verification.

[0166] (4) Verification of reactivity with HA Verification of reactivity with HA was performed by ELISA using an antigen solid-phase format in which an antigen panel consisting of various HAs was immobilized, as in Test 2, "(3-2) Verification of reactivity with HA."

[0167] The results based on the above-mentioned ELISA are shown in Figure 12. This figure shows the reactivity of the serum from mice immunized with "R2-150 Fab nanoparticles" with a panel of recombinant influenza A HA antigens. These results demonstrate that the anti-Id antibody (R2-150) functions as an antigen capable of inducing antibodies against all known influenza A virus HA subtypes (except H9).

[0168] Furthermore, it was confirmed that the antibodies contained in the immune serum reacted with both HA of influenza B Victoria and Yamagata strains, to which the template antibody for "R2-150," the "FluA-20 antibody," could not react (Figure 13). These results suggest that new antibodies similar in function and morphology to, or even superior in function to, the template antibody (FluA-20 antibody) can be obtained by using "R2-150" as an antigen, for example, by immunizing animals to obtain antibodies.

[0169] <Test 8: Analysis of the interaction between "#2-911" and template antibody> In this example, a complex of the Fab of the anti-Id antibody "#2-911" and its template Fab of the "MEDI8852 antibody" was crystallized and its three-dimensional structure was analyzed. This analysis identified detailed structural coordinates, such as the positions of the amino acid side chains that make up the Fab and the hydration water located on the surface of the protein molecule.

[0170] The analysis results are shown in Figures 14, 15, and 16. These figures show the relationship of the interaction (binding) between "#2-911" and "MEDI8852 antibody" from three perspectives. The numbers assigned to each amino acid shown in Figures 14, 15, and 16 are numbers according to Kabat's definition (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991).

[0171] FIG. 14 shows the crystal structure of the complex between Fab #2-911 and Fab #MEDI8852, with the interaction region between the two displayed and analyzed using the software "Pymol" (Schrodinger). The presence or absence of contact between amino acids between Fab #2-911 and Fab #MEDI8852 was objectively determined by calculation from the structural coordinates using the "InterfaceResidues" script (https: / / pymolwiki.org / index.php / InterfaceResidues) on the software. More specifically, amino acids with a contact area of ​​1.0 Å^2 or greater between Fab #2-911 and Fab #MEDI8852 were determined to have a direct interaction. FIG. 14 illustrates the main chain structures of the "#2-911" Fab and the "MEDI8852 antibody" Fab, which showed direct binding to it, in a "ribbon model." For each Fab, the CDR regions are shown in dark color. To facilitate understanding of the interaction, a "space-filling model" including the side chains is shown for HCDR1 and HCDR3 of the "MEDI8852 antibody." In addition, the side chains of the amino acids constituting the "#2-911" HCDR1 and HCDR2 are shown in a "stick model." In the crystal structure, direct binding to the "MEDI8852 antibody" was observed other than those in the "#2-911" HCDR1 and HCDR2. For amino acids on the framework that were considered to be particularly important, their side chains are shown in a "stick model," and the observed hydrogen bonds are illustrated. In the figure, the CDR numbers and residue numbers of particularly noteworthy amino acids are shown (those of the "MEDI8852 antibody" are shown in italics).

[0172] Like Figure 14, Figures 15 and 16 show the interaction between "#2-911" Fab and "MEDI8852 antibody" Fab, observed from a different direction than Figure 14. Of these, Figure 16 more clearly shows the three-dimensional positional relationship between "#2-911" Fab (shown as a "cartoon model") and "MEDI8852 antibody" Fab (shown as a "space-filling model") that constitute the complex. The three CDRs of the "#2-911" heavy chain and the "#2-911" light chain are each shown in dark colors in the figures.

[0173] As can be seen from these figures, the crystal structure of the complex revealed that the "MEDI8852 antibody" directly interacts with the region composed of HCDR1 and HCDR2 of "#2-911" and the surrounding framework regions. Specifically, the crystal structure of the complex revealed that HCDR1 and HCDR2 of "#2-911" have a large binding area (area of ​​direct interaction), particularly with HCDR3, which is composed of highly hydrophobic amino acids of the "MEDI8852 antibody," and are responsible for the primary interaction with the antigen (FIGS. 14 and 16). It was revealed that not only HCDR1 and 2, but also specific amino acids described below are particularly involved in binding. On the other hand, no amino acids that directly interact with the "MEDI8852 antibody" were detected in the HCDR3 and light chain of "#2-911," and the HCDR3 and light chain did not bind to the "MEDI8852 antibody." This was particularly evident visually in Figure 16, where a clear distance was observed between these HCDR3 and light chains and the molecules that make up the "MEDI8852 antibody."

[0174] The side chains of amino acids Tyr27 and Phe29 on the N-terminal framework (also referred to as FR1) of HCDR1 of "#2-911" each showed direct interaction with heavy chain amino acids of the "MEDI8852 antibody." Here, depending on the antibody, the 27th amino acid may be substituted for Tyr with, for example, Phe, Leu, Ile, or Glu. Furthermore, the 29th amino acid may be substituted for Phe with, for example, Leu, Ile, Val, or Ala. Using the structural coordinates identified in this example, Tyr27 and Phe29 were substituted with the respective amino acids listed above in the "Pymol" program, and it was confirmed that, like Tyr27 and Phe29, these amino acids also showed direct interaction with heavy chain amino acids of the "MEDI8852 antibody."

[0175] Furthermore, amino acid Lys73 on the framework (also referred to as FR3) between HCDR2 and HCDR3 of "#2-911" showed direct interaction with the side chain of Ser53 on the light chain of "MEDI8852 antibody." Lys73 formed hydrogen bonds with the side chains of amino acids Ser30 and Ser31 on the light chain of "MEDI8852 antibody" (hydrogen bonds are indicated by dotted lines extending from Lys73 in Figures 14 and 15). Furthermore, the side chain of amino acid Ser76 on the heavy chain FR3 of "#2-911" formed a hydrogen bond with the main chain of heavy chain Phe100a of "MEDI8852 antibody" (hydrogen bonds are indicated by dotted lines extending from Ser76 in Figure 15).

[0176] These results demonstrate that Tyr27, Phe29, Lys73, and Ser76 present on the heavy chain framework of "#2-911," together with HCDR1 and HCDR2 of "#2-911," are particularly involved in the binding between "#2-911" and the "MEDI8852 antibody."

[0177] The importance of the binding of HCDR1 and HCDR2 of "#2-911" to the "MEDI8852 antibody" deduced from the above-mentioned three-dimensional structure, and the importance of specific amino acids in the FR region to the "MEDI8852 antibody," were demonstrated by the change in binding affinity of the alanine-substituted mutants of "#2-911" to the "MEDI8852 antibody" in Figure 17. That is, when His35 contained in the HCDR1 sequence of "#2-911," Phe53 contained in the HCDR2 sequence, Tyr27 in FR1, and Lys73 in FR3 are substituted with alanine, the binding affinity with the "MEDI8852 antibody" is significantly reduced or lost. It was also found that the binding affinity between "#2-911" and the "MEDI8852 antibody" is strengthened when Phe29 in FR1 is substituted with alanine.

[0178] <Test 9: Obtaining new antibodies that exceed the function of template antibodies using anti-Id antibodies> By using an anti-Id antibody as an antigen, it is expected that it will be possible to obtain antibodies similar in function or morphology to the template antibody, or new antibodies that exceed the function of the template antibody. Therefore, to verify this point, five new monoclonal antibodies (clones A to E) were obtained from hybridomas expressing antibodies that use the anti-Id antibody "#2-911" as an antigen. Specifically, the "MEDI8852 antibody," which is the template antibody for "#2-911," broadly cross-reacts with influenza A HA but does not bind to influenza B HA. Therefore, according to the following procedure, an attempt was made to obtain clones expressing monoclonal antibodies that cross-react with both influenza A HA and influenza B HA from hybridomas expressing antibodies against "#2-911."

[0179] (1) Immunization of Mice The antigen used was the "#2-911 Fab nanoparticles" used in Experiments 3 and 4, mixed with the adjuvant "TiterMax Gold" (TiterMax), and intraperitoneally administered to BALB / c mice (13 mice). The antigen dose was set at 200 μg per mouse. After administration, booster immunizations were administered twice, at two-week intervals from the start of administration. However, the antigen dose for booster immunizations was set at 20 μg per mouse, and "Imject Alum Adjuvant, cat. 77161" (Thermo Fischer Scientific) was used as the adjuvant. One week after the final immunization, popliteal lymph nodes and spleens were collected from the mice, and cell suspensions were prepared from parts of the spleens.

[0180] The resulting cell suspension was mixed with SP2 / 0-Ag14 myeloma cells and subjected to electrical cell fusion using an electrical cell fusion device "ECFG21" (Neppa Gene Co., Ltd.). After fusion, the resulting cells were suspended in "ClonaCell-HY Medium D, cat. ST-03804" (STEMCELL Technologies) and seeded onto plastic petri dishes.

[0181] Colonies formed 10 days after seeding were isolated into 96-well plastic plates containing hybridoma medium, and the culture supernatant was used for the following evaluation. The hybridoma medium used was "RPMI1640, cat. A1049101" (Thermo Fisher Scientific) supplemented with 1 / 10 of the amount of "Doma-Drive, cat. T31-1003SF" (Immune Systems) and 1 / 50 of the amount of "HAT Supplement, cat. 21-60017" (Thermo Fisher Scientific).

[0182] (2) Screening of monoclonal antibodies cross-reactive with influenza A and B HAs Using each culture supernatant obtained in "(1) Immunization of mice" above, hybridoma clones producing monoclonal antibodies cross-reactive with both influenza A HA and influenza B were screened. Specifically, clones producing antibodies that met all of the following criteria were selected by ELISA. (Criterion 1) React with (bind to) "#2-911 Fab nanoparticles." (Criterion 2) Do not react with mouse polyclonal Fab "mouse IgG Fab fragment, cat. 010-0105" (Rockland). (Criterion 3) React with a mixed antigen (influenza A HA mixed antigen) consisting of influenza A HAs (8 types shown in Table 9). (Criterion 4) Reacts to a mixed antigen (mixed influenza B HA antigen) consisting of influenza B HA (8 types shown in Table 5).

[0183] ELISA was performed in an antigen solid-phase format in which each of the above antigens was immobilized. The secondary antibody used for detection was "anti-mouse IgG-Fc fragment, HRP conjugated, cat. A90-131" (Bethyl Laboratories). For color development, "ELISA POD Substrate TMB Kit (popular), cat. 05298-80" (Nacalai Tesque) was used, and absorbance at a wavelength of 450 nm was measured using an "Infinite M200 pro microplate reader" (Tecan).

[0184] The following three types of controls were set: "#2-911Fab-nps" is the "#2-911Fab nanoparticles" antigen used for immunization (positive antigen); "mIgG polyclonal Fab" is mouse polyclonal Fab "mouse IgG Fab fragment, cat. 010-0105" (Rockland) (negative antigen); and "SARS-CoV-2 Spike" is the spike protein of the SARS-CoV-2 virus (Wuhan strain) prepared by adding the same sequence as the purification peptide tag added to the C-terminus of each HA protein during the preparation of the HA antigen panel (negative antigen).

[0185]

[0186] The five clones, clones A to E, selected as hybridoma clones that satisfy all of the above-mentioned (Criteria-1) to (Criteria-4), were tested for their individual reactivities with 14 types of influenza A and two types of influenza B HA, and the results are shown in Figure 18. It was confirmed that multiple clones were obtained that produce antibodies that show cross-reactivity with various subtypes of influenza A HA and also cross-react with HA of the Yamagata and Victoria lineages (known as influenza B HA).

[0187] The template antigen for "#2-911," "MEDI8852 antibody," is an antibody that specifically reacts with influenza A HA and strongly reacts with all known subtypes, from H1 to H18 (Kallewaard et al., Cell, 166, 596-608, 2016, and WO2017 / 123685). These 18 types of influenza A HA are classified into "Group 1" and "Group 2" due to differences in amino acid sequences resulting from differences in their common ancestors (see Table 9), and it is known that antigenicity differs significantly between the two groups. However, all antibodies obtained from mice immunized with "#2-911" as an antigen not only broadly cross-react with HAs belonging to both groups, like the "MEDI8852 antibody," but also cross-react with influenza B HA, which the "MEDI8852 antibody" cannot bind to. In other words, the approach of this example yielded an antibody that is similar to the template antibody but has better functionality.

Claims

1. An antibody or fragment thereof that specifically binds to the "MEDI8852 antibody," an anti-influenza HA broadly neutralizing antibody, and that satisfies all of the following requirement A. [Requirement A] - The heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO:

2. - The heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO:

3. - The 27th amino acid of heavy chain FR1, as defined by Kabat, is an amino acid selected from the group consisting of Tyr, Phe, Leu, Ile, and Glu. - The 29th amino acid of heavy chain FR1, as defined by Kabat, is an amino acid selected from the group consisting of Phe, Leu, Ile, Val, and Ala. - The 73rd amino acid of heavy chain FR3, as defined by Kabat, is Lys. - The 76th amino acid of heavy chain FR3, as defined by Kabat, is Ser.

2. The antibody or fragment thereof according to claim 1, wherein the 27th amino acid of the heavy chain FR1 is Tyr and the 29th amino acid of the heavy chain FR1 is Phe.

3. The antibody or fragment thereof according to claim 1 or 2, wherein the amino acid sequences of CDR1 to CDR3 of the heavy and light chains further satisfy all of the following [Requirement B]: [Requirement B] Heavy chain CDR3 is the amino acid sequence shown in SEQ ID NO:

4. The light chain CDR1 has the amino acid sequence shown in SEQ ID NO:

6. The light chain CDR2 has the amino acid sequence shown in SEQ ID NO:

7. The light chain CDR3 has the amino acid sequence shown in SEQ ID NO:

8.

4. The antibody or fragment thereof according to any one of claims 1 to 3, further satisfying all of the following [Requirement C]: [Requirement C] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

1. The light chain variable region has the amino acid sequence shown in SEQ ID NO:

5.

5. The antibody or fragment thereof according to any one of claims 1 to 3, further satisfying all of the following [Requirement D]: [Requirement D] The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 17 or 18. The light chain variable region is the amino acid sequence shown in SEQ ID NO: 19 or 20.

6. An antibody or fragment thereof that specifically binds to the "FluA-20 antibody," an anti-influenza HA broadly neutralizing antibody, wherein the amino acid sequences of CDR1 to CDR3 of the heavy and light chains satisfy all of the following [Requirement E]. [Requirement E] Heavy chain CDR1 is the amino acid sequence represented by SEQ ID NO:

10. Heavy chain CDR2 is the amino acid sequence represented by SEQ ID NO:

11. Heavy chain CDR3 is the amino acid sequence represented by SEQ ID NO:

12. The light chain CDR1 has the amino acid sequence shown in SEQ ID NO:

14. The light chain CDR2 has the amino acid sequence shown in SEQ ID NO:

15. The light chain CDR3 has the amino acid sequence shown in SEQ ID NO:

16.

7. The antibody or fragment thereof according to claim 6, further satisfying all of the following [Requirement F]: [Requirement F] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

9. The light chain variable region has the amino acid sequence shown in SEQ ID NO:

13.

8. An anti-influenza vaccine comprising the antibody or fragment thereof according to any one of claims 1 to 7, or a nucleic acid encoding the antibody or fragment thereof.

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