JP2006347922A - Antibody enzyme against hemagglutinin of influenza virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody enzyme capable of recognizing surface glycoprotein hemagglutinin and cleaving and / or decomposing hemagglutinin regardless of continuous or discontinuous mutation of influenza virus.
An antibody or fragment thereof prepared by using an antigen peptide containing an amino acid sequence present in the highly conserved region of hemagglutinin HA1 and / or an amino acid sequence present in the highly conserved region of hemagglutinin HA2 as a surface glycoprotein hemagglutinin And is an antibody enzyme capable of cleaving and / or degrading the hemagglutinin.
[Selection figure] None

Description

  The present invention relates to an antibody enzyme against hemagglutinin of influenza virus.

  Influenza viruses are classified based on the antigenicity of core proteins (nuclear protein (NP protein) and membrane protein (M1 protein)). In 1940, a virus that was an influenza virus but antigenically different from the previous virus was isolated. The previous virus was designated as type A, and the new virus was designated as type B. If newer viruses were classified thereafter, they would be C, D, E ... types. The influenza C virus was discovered by Taylor in 1949, and it is now clear that there are three types of influenza A, B and C.

  Influenza A virus is further abbreviated as hemagglutinin (Hemagglutinin (HA), hereinafter appropriately abbreviated as HA) and neuraminidase (NA), hereinafter abbreviated as NA. And subtypes of H1 to H15 in HA and N1 to N9 in NA. HA plays an important role when influenza virus particles are taken into cells by binding to sialic acid on the cell surface when adsorbed / invaded into cells such as humans. On the other hand, NA has a function of cleaving sialic acid when virus particles leave the cell surface in the late stage of infection, and is useful for acquiring infectivity. As for HA, since 1993, three subtypes have been identified for humans, two for pigs, two for horses, and fifteen for waterfowl. Influenza B virus has the same surface glycoprotein as influenza A virus, but only one subtype. In addition, influenza C virus has only hemagglutinin esterase (HE). Influenza B virus is only prevalent in humans and influenza C virus is prevalent only in humans and swine.

  Influenza A and B viruses, which are both large and small, are recurrent almost every year. The antigen structure is slightly different, and this shift in antigen structure causes the epidemic. Antigen mutations are observed in hemagglutinin (HA) and neuraminidase (NA), which are surface proteins of virus particles, and there are two types of mutations, discontinuous mutation (antigenic shift) and continuous mutation (antigenic drift). Discontinuous mutation refers to a mechanism in which cells infected by two different strains suddenly appear a new serotype by various recombination of RNA genome components, and is found only in influenza A virus. Discontinuous mutations are often the cause of outbreaks. To date, humans have repeated a pandemic worldwide with Spanish cold (H1N1) in 1918, Asian cold (H2N2) in 1957, Hong Kong cold (H3N2) in 1968, and Russian cold (H1N1) in 1977. . Continuous mutations are those observed in influenza A, B, and C influenza viruses, which are not the exchange of genomic RNA segments but maintain the same genome, but are the mechanisms that arise as a result of mutations in the HA and NA genes. is there. The origin of type A virus is single, and from there, a new type of virus develops and spreads due to discontinuous and continuous mutations, and it shows a single lineage system that can replace other strains. However, the C virus exhibits a multi-lineage pattern in which various virus strains are simultaneously prevalent. B type shows a mode between A type and C type.

  Due to such mutations of hemagglutinin (HA) and neuraminidase (NA), it is difficult to produce a vaccine suitable for the antigen type in the currently used HA vaccine, and its preventive effect is a problem.

As a technique for solving such a problem, a report has been made on an antibody that recognizes an antigen site that is common to the subtypes of the HA molecule and hardly causes an antigenic mutation (see, for example, Patent Document 1). Patent Document 1 discloses an antibody prepared using influenza viruses H1N1 and H2N2 as antigens and recognizing polypeptide sequences TGLRN and GITNKVNSVIEK in a hemagglutinin molecule.
Japanese Patent Laid-Open No. 6-100594 (published on April 12, 1994)

  However, it not only functions as an antibody having a virus neutralizing activity by recognizing a highly conserved region (highly conserved region) without mutating to hemagglutinin (HA) in which antigenic variation is observed, but also cleaving HA and If an antibody enzyme that also has a function as an enzyme capable of degrading can be obtained, it recognizes HA by the antibody enzyme and cleaves and / or degrades the recognized HA in spite of antigenic mutation that occurs every year. can do. That is, compared to an antibody that binds to one (or two) HA molecules per molecule, the antibody enzyme can destroy HA molecules one after the other with one molecule. Therefore, if such an antibody enzyme can be obtained, the infectivity of influenza virus can be destroyed, and it can be expected to contribute to the prevention and treatment of influenza.

  The present invention has been made in view of the above problems, and its object is to recognize the surface glycoprotein hemagglutinin and to cleave and / or cleave the hemagglutinin regardless of continuous or discontinuous mutation of influenza virus. Another object is to provide an antibody enzyme that can be degraded.

  In order to solve the above-mentioned problems, the inventors of the present application have a highly conserved region TGLRN (318-) of the HA1 segment of hemagglutinin (hereinafter abbreviated as HA1 as appropriate) conserved between the H1 and H2 types of influenza virus. 322) and the highly conserved region GITNKVNSVIEK (47 to 58) of the HA2 segment of hemagglutinin (hereinafter abbreviated as HA2 as appropriate) conserved in the same manner, and intensively studied to obtain an antibody enzyme. As a result, it recognizes the surface glycoprotein hemagglutinin and cleaves and / or degrades the hemagglutinin among antibodies obtained by using antigen peptides containing these highly conserved regions TGLRN and GITNKVNSVIEK as immune antigens. As a result, the inventors have found that there is a substance that exhibits the function as an antibody enzyme capable of producing the present invention, and completed the present invention.

  In order to solve the above problems, an antibody enzyme according to the present invention is an antibody against an influenza virus having hemagglutinin, and is characterized by recognizing hemagglutinin and having an activity of degrading the hemagglutinin.

  The antibody enzyme is an antibody enzyme that recognizes the amino acid sequence shown in SEQ ID NO: 1 present in the highly conserved region of hemagglutinin HA1 and / or the amino acid sequence shown in SEQ ID NO: 2 present in the highly conserved region of hemagglutinin HA2. preferable.

  In addition, the above antibody enzyme uses an antigen peptide containing the amino acid sequence shown in SEQ ID NO: 1 present in the highly conserved region of hemagglutinin HA1 and / or the amino acid sequence shown in SEQ ID NO: 2 present in the highly conserved region of hemagglutinin HA2 as an antigen. The produced antibody enzyme is preferable. Here, the antigen peptide used as the antigen may be one obtained by covalently binding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to a protein.

  In the antibody enzyme, for example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4, wherein one or several amino acids are substituted, deleted, inserted and The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5, wherein one or several amino acids are substituted, deleted, inserted, And / or consisting of an added amino acid sequence.

  In the above antibody enzyme, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 6, or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 6, substitution, deletion, insertion, and / or The light chain variable region is composed of an added amino acid sequence, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 7 or the amino acid sequence shown in SEQ ID NO: 7, wherein one or several amino acids are substituted, deleted, inserted, and / or Or it may consist of an added amino acid sequence.

  The present invention also includes an antibody enzyme fragment containing the variable region of the antibody enzyme.

  As described above, the antibody enzyme according to the present invention is an antibody against an influenza virus having hemagglutinin, and has a configuration of recognizing hemagglutinin and having an activity of degrading the hemagglutinin. Regardless of continuous or discontinuous mutation, the surface glycoprotein hemagglutinin can be recognized and the hemagglutinin can be cleaved and / or degraded. Therefore, the antibody enzyme according to the present invention can be expected to contribute to the prevention and treatment of infection with influenza virus having hemagglutinin.

  The present invention will be described more specifically below, but the present invention is not limited to this description.

(I) Antibody enzyme according to the present invention The antibody enzyme according to the present invention may be an antibody against an influenza virus having hemagglutinin, as long as it is an antibody enzyme that recognizes hemagglutinin and has an activity of degrading hemagglutinin.

  Here, the antibody against the influenza virus having hemagglutinin may be a polyclonal antibody against these influenza viruses, but more preferably a monoclonal antibody against these influenza viruses. This makes it possible to select an antibody with excellent specificity. The antibody recognizes hemagglutinin. Therefore, it can be said that the antibody is an antibody against hemagglutinin of influenza virus.

  Here, the influenza virus recognized by the antibody enzyme of the present invention is not particularly limited as long as it has hemagglutinin as a surface glycoprotein. For example, influenza virus H1, H2, and H3 types Etc. Among these, influenza virus H1 type and H2 type are more preferable. Examples of such influenza virus H1 include (H1N1), and examples of influenza virus H2 include (H2N2). Moreover, since the antibody enzyme of the present invention can recognize a highly conserved region (highly conserved region) without mutating to hemagglutinin (HA) in which antigenic variation is observed, it is present at present (H1N1), Not only type A influenza viruses such as (H2N2), but also mutated H1 type and H2 type influenza viruses can be recognized in the future by continuous and discontinuous mutations.

  The antibody enzyme according to the present invention has an activity of recognizing hemagglutinin and decomposing the hemagglutinin. That is, the antibody enzyme according to the present invention has both properties as an antibody that specifically recognizes hemagglutinin and properties as an enzyme that cleaves and / or degrades the recognized hemagglutinin. Here, the “antibody enzyme” refers to an antibody that has an enzymatic action and exhibits degradation activity targeting its antigenic protein. Therefore, the antibody enzyme of the present invention can recognize HA by the antibody enzyme and cleave and / or degrade the recognized HA, regardless of antigenic mutations that occur every year. That is, compared to an antibody that binds to one (or two) HA molecules per molecule, the antibody enzyme can destroy HA molecules one after the other with one molecule. Therefore, the effect reaches several hundred times or several thousand times that of a normal antibody. Therefore, if such an antibody enzyme can be obtained, the infectivity of influenza virus can be destroyed, and it can be expected to contribute to the prevention and treatment of influenza. In addition, since it is an enzyme, an enzyme sensor can be constructed, and its application range is wider than that of an antibody. The enzyme activity of the antibody enzyme according to the present invention may be one that cleaves and / or degrades other types of HA protein in addition to the recognized hemagglutinin.

  The antibody enzyme of the present invention may be any one that recognizes hemagglutinin, but recognizes an amino acid sequence present in the highly conserved region of hemagglutinin HA1 and / or an amino acid sequence present in the highly conserved region of hemagglutinin HA2. Is preferred. Here, the amino acid sequence present in the highly conserved region of hemagglutinin HA1 is not particularly limited as long as it is a region that is highly conserved without mutating due to continuous mutation or discontinuous mutation in HA1. For example, the amino acid sequence shown in SEQ ID NO: 1 can be mentioned. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the highly conserved region TGLRN (318 to 322) of hemagglutinin HA1. Further, the amino acid sequence present in the highly conserved region of hemagglutinin HA2 is not particularly limited as long as it is a region that is highly conserved without mutating due to continuous mutation or discontinuous mutation in HA2. The amino acid sequence shown in No. 2 can be mentioned. The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of the highly conserved region GITNKVNSVIEK (47 to 58) of hemagglutinin HA2.

  In addition, the antibody enzyme of the present invention is preferably prepared using an antigen peptide containing an amino acid sequence present in the highly conserved region of hemagglutinin HA1 and / or an amino acid sequence present in the highly conserved region of hemagglutinin HA2 as an antigen. .

  That is, an antibody enzyme that recognizes hemagglutinin HA1 or its highly conserved region and has the activity of degrading hemagglutinin containing hemagglutinin HA1 is prepared using an antigen peptide containing an amino acid sequence present in the highly conserved region of HA1 as an antigen. It is preferable that Such an antigenic peptide may be a peptide consisting of an amino acid sequence present in the highly conserved region of HA1, or may be a peptide obtained by binding another amino acid sequence to the amino acid sequence present in the highly conserved region of HA1. The amino acid sequence present in the highly conserved region of HA1 may be bound to other proteins.

  An antibody enzyme that recognizes hemagglutinin HA2 or a highly conserved region thereof and has an activity of degrading hemagglutinin including hemagglutinin HA2 is prepared using an antigen peptide containing an amino acid sequence present in the highly conserved region of HA2 as an antigen. It is preferable that Such an antigenic peptide may be a peptide consisting of an amino acid sequence present in the highly conserved region of HA2, or may be a peptide obtained by binding another amino acid sequence to the amino acid sequence present in the highly conserved region of HA2. The amino acid sequence present in the highly conserved region of HA2 may be bound to other proteins.

  An antibody enzyme that recognizes hemagglutinins HA1 and HA2 or their highly conserved regions and has an activity of degrading hemagglutinin containing hemagglutinins HA1 and HA2 is an amino acid sequence present in the highly conserved region of HA1 and the high degree of HA2. It is preferable that the antigen peptide containing the amino acid sequence present in the conserved region is prepared as an antigen. Such an antigenic peptide may be a fusion peptide in which an amino acid sequence present in the highly conserved region of hemagglutinin HA1 and an amino acid sequence present in the highly conserved region of HA2 may be combined, or another amino acid sequence may be bound to such a fusion peptide. It may be a peptide obtained by binding another protein to such a fusion peptide. Here, the order in which the amino acid sequence present in the highly conserved region of hemagglutinin HA1 and the amino acid sequence present in the highly conserved region of HA2 are not particularly limited, and either may be on the C-terminal side. .

  Here, the amino acid sequence present in the highly conserved region of hemagglutinin HA1 is not particularly limited as long as it is a region that is highly conserved without mutating due to continuous mutation or discontinuous mutation. The amino acid sequence shown in 1 can be mentioned. Further, the amino acid sequence present in the highly conserved region of hemagglutinin HA2 is not particularly limited as long as it is a region that is highly conserved without being mutated due to continuous mutation or discontinuous mutation. The amino acid sequence shown can be mentioned. Further, the “other amino acid sequence” is not particularly limited, and examples thereof include alanine two molecules (AA). By adding AA to the C-terminal, it is possible to adopt a structure close to the natural hemagglutinin helix structure. The “other amino acid sequence” may be an amino acid sequence composed of, for example, 2 to 3 amino acids for improving the immune response. The position at which these “other amino acid sequences” are added is not particularly limited, and the amino acid sequence present in the highly conserved region of hemagglutinin HA1 and / or the N-terminal of the amino acid sequence present in the highly conserved region of hemagglutinin HA2 Or may be added to the C-terminal.

  The “other protein” is not particularly limited as long as it is a protein that can be usually used to bind to a low molecule to obtain an antigen. For example, Human IgG, BSA (bovine serum albumin) ), HSA (human serum albumin), KLH (keyhole limpet hemocyanin) and the like. The above-mentioned various antigen peptides can be used as the above-mentioned antigen peptide. For example, a preferred example is an antigen peptide obtained by covalently binding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to the above-mentioned other protein. be able to. The peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 connects the highly conserved region TGLRN (318 to 322) of influenza virus hemagglutinin HA1 to the highly conserved region GITNKVNSVIEK (47 to 58) of hemagglutinin HA2 of influenza virus, and further AA It is a 19mer peptide (TGLRNGITNKVNSVIEKAA).

  The antibody enzyme of the present invention is an antibody against influenza virus having hemagglutinin.

  Here, the structure and origin of the antibody are not particularly limited. Therefore, the antibody may be a monoclonal antibody such as a mouse obtained by cell fusion, an antibody produced in E. coli, animal cells, etc. using genetic recombination techniques, or hemagglutinin. The antibody may be a chimeric antibody such as a humanized antibody in which the variable region of an antibody against an influenza virus having an antibody is grafted to an antibody of a human or other animal.

  The antibody enzyme of the present invention is not particularly limited as long as it is the above-mentioned antibody enzyme. As a specific example, for example, the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain An antibody enzyme whose variable region consists of the amino acid sequence shown in SEQ ID NO: 5 can be mentioned. The amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of the heavy chain variable region of antibody enzyme HA1-1 produced in the Examples described later, and the amino acid sequence shown in SEQ ID NO: 5 is the same as that of antibody enzyme HA1-1. Amino acid sequence of the light chain variable region. FIG. 26 (a) shows the amino acid sequence of the heavy chain variable region of HA1-1 (SEQ ID NO: 4), and FIG. 26 (b) shows the amino acid sequence of the light chain variable region of HA1-1 (SEQ ID NO: 5). As shown in FIG. 26 (a), the heavy chain variable region of HA1-1 has complementarity determining regions CDR1, CDR2, and CDR3 indicated by underlining. That is, the heavy chain variable region of HA1-1 consists of CDR1 consisting of the 31st to 35th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 4, and CDR2 consisting of the 50th to 66th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 4. And CDR3 consisting of the 99th to 102nd amino acid sequences of the amino acid sequence shown in SEQ ID NO: 4. Further, as shown in FIG. 26 (b), the light chain variable region of HA1-1 has CDR1, CDR2, and CDR3 indicated by underlining. That is, the heavy chain variable region of HA1-1 consists of CDR1 consisting of the 24th to 40th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 5, and CDR2 consisting of the 56th to 62nd amino acid sequence of the amino acid sequence shown in SEQ ID NO: 5. And CDR3 consisting of the 95th to 103rd amino acid sequences of the amino acid sequence shown in SEQ ID NO: 5.

  In the antibody enzyme according to the present invention, the heavy chain variable region comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 4. And / or the light chain variable region consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 5, and hemagglutinin May be recognized and have an activity of degrading the hemagglutinin. Such an antibody enzyme is a variant of HA1-1 and has an activity of recognizing hemagglutinin and decomposing the hemagglutinin. The range of “one or several” in the above “amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 4 (5)” Is not particularly limited, but means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably 1 to 3.

  Another example is an antibody enzyme in which the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 6 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 6 is the amino acid sequence of the heavy chain variable region of antibody enzyme HA1-2 produced in the examples described later, and the amino acid sequence shown in SEQ ID NO: 7 was produced in the examples described later. It is an amino acid sequence of the light chain variable region of antibody enzyme HA1-2. FIG. 27A shows the amino acid sequence of the heavy chain variable region of HA1-2 (SEQ ID NO: 6), and FIG. 27B shows the amino acid sequence of the light chain variable region of HA1-2 (SEQ ID NO: 7). As shown in FIG. 27 (a), the heavy chain variable region of HA1-2 has CDR1, CDR2, and CDR3 indicated by underlining. That is, the heavy chain variable region of HA1-2 consists of CDR1 consisting of the 31st to 35th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6, and CDR2 consisting of the 50th to 66th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6. And CDR3 consisting of the 99th to 102nd amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6. As shown in FIG. 27 (b), the light chain variable region of HA1-2 has CDR1, CDR2, and CDR3 indicated by underlining. That is, the heavy chain variable region of HA1-1 consists of CDR1 consisting of the 24th to 40th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 7, and CDR2 consisting of the 56th to 62nd amino acid sequence of the amino acid sequence shown in SEQ ID NO: 7. And CDR3 consisting of the 95th to 103rd amino acid sequences of the amino acid sequence shown in SEQ ID NO: 7.

  In the antibody enzyme according to the present invention, the heavy chain variable region comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 6. And / or the light chain variable region consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 7, and hemagglutinin May be recognized and have an activity of degrading the hemagglutinin. Such an antibody enzyme is a variant of HA1-2, which recognizes hemagglutinin and has an activity of decomposing hemagglutinin.

  The present invention also includes an antibody fragment that is a fragment of the antibody enzyme of the present invention and includes the variable region of the antibody enzyme. Such an antibody fragment is not particularly limited as long as it is an antibody fragment containing the variable region of the above antibody enzyme and recognizes hemagglutinin and has an activity of degrading hemagglutinin. Such an antibody fragment only needs to contain the variable region of the antibody enzyme. Therefore, it may be the heavy chain (H chain) of the antibody enzyme, the light chain (L chain) of the antibody enzyme, the variable region of the heavy chain of the antibody enzyme, It may be the variable region of the light chain of an antibody enzyme, or any antibody fragment containing these. Among them, the variable region of the heavy chain or light chain of the above antibody enzyme can be suitably used for preparing a chimeric antibody by transplanting such a variable region to an antibody of a human or other animal.

  Examples of such antibody fragments include an antibody fragment consisting of the amino acid sequence shown in SEQ ID NO: 4, 5, 6 or 7, and an antibody fragment containing these amino acid sequences. The amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of the heavy chain variable region of antibody enzyme HA1-1 produced in the examples described later, and the amino acid sequence shown in SEQ ID NO: 5 was produced in the examples described later. The amino acid sequence of the light chain variable region of the antibody enzyme HA1-1. The amino acid sequence shown in SEQ ID NO: 6 is the amino acid sequence of the heavy chain variable region of the antibody enzyme HA1-2 produced in the Examples described later. The amino acid sequence shown in 7 is the amino acid sequence of the light chain variable region of antibody enzyme HA1-2 produced in the examples described later.

  Further, the above antibody fragment is an antibody fragment consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 4, 5, 6 or 7. An antibody fragment comprising such an amino acid sequence may be one that recognizes hemagglutinin and has an activity of degrading the hemagglutinin. Such antibody fragments include the heavy chain (HA1-1-H) of the antibody enzyme HA1-1, the light chain (HA1-1-L) of the antibody enzyme HA1-1, and the heavy chain (HA1-) of the antibody enzyme HA1-2, respectively. 2-H), or a variable region variant of the light chain (HA1-2-L) of antibody enzyme HA1-2 or an antibody fragment comprising such variant, which recognizes hemagglutinin and degrades hemagglutinin It has activity to do.

  The present invention also includes a gene encoding the above antibody enzyme, or a gene encoding a fragment of the above antibody enzyme and a fragment of the antibody enzyme containing the variable region of the above antibody enzyme. By introducing such a gene so that it can be expressed in a suitable host, the antibody enzyme of the present invention or a fragment thereof can be expressed in the host. In addition, the variable region of the antibody enzyme of the present invention or the variable region incorporating the CDR thereof may be expressed as it is, but it can also be expressed as a chimeric antibody enzyme linked to a gene encoding the constant region.

  The “gene” includes not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand. Further, the “gene” includes sequences such as a sequence of an untranslated region (UTR) and a vector sequence (including an expression vector sequence) in addition to the sequence encoding the antibody enzyme of the present invention or a fragment thereof. There may be.

  Specific examples of the gene of the present invention include a gene consisting of the base sequence shown in SEQ ID NO: 12, 13, 14 or 15, or a gene containing these. The gene consisting of the base sequence shown in SEQ ID NO: 12 is an example of a gene encoding the heavy chain variable region of the antibody enzyme HA1-1 produced in the examples described later, and the amino acid sequence shown in SEQ ID NO: 13 is an implementation described later. It is an example of the gene which codes the light chain variable region of the antibody enzyme HA1-1 manufactured in the example, The amino acid sequence shown in SEQ ID NO: 14 is the heavy chain variable region of the antibody enzyme HA1-2 manufactured in the Examples described later. It is an example of the gene to encode, The amino acid sequence shown to sequence number 15 is an example of the gene which codes the light chain variable region of the antibody enzyme HA1-2 manufactured in the Example mentioned later.

  The gene is not necessarily the same as the nucleotide sequence shown in SEQ ID NO: 12, 13, 14 or 15, but an antibody enzyme or a fragment thereof that recognizes hemagglutinin and has an activity of degrading hemagglutinin. If it is a gene that encodes, its variants are also included. Examples of such mutants include mutants in which one or more bases are deleted, substituted, or added in the base sequence of the gene encoding the antibody enzyme or fragment thereof.

(2) Method for producing antibody enzyme according to the present invention The antibody enzyme according to the present invention includes, for example, an antigen comprising an amino acid sequence present in the highly conserved region of hemagglutinin HA1 and / or an amino acid sequence present in the highly conserved region of hemagglutinin HA2. The antibody can be produced by producing a monoclonal antibody using a hybridoma obtained by fusing a spleen cell of an immunized animal such as a mouse immunized with the peptide and a fusion partner such as a mouse myeloma cell. When obtaining a heavy chain and a light chain, the obtained monoclonal antibody may be separated into a heavy chain and a light chain. In addition, when obtaining the antibody fragment of the present invention, first, the relevant monoclonal antibody is obtained, and then the above monoclonal antibody may be cleaved using a suitable protease so that the desired antibody fragment can be obtained. .

  Monoclonal antibodies can be obtained by the usual hybridoma method (Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor, Immunology Today 4, 72 ( 1983)), EBV-hybridoma method (Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., 77-96 (1985)).

  Examples of the antigen peptide include peptides composed of amino acid sequences present in the highly conserved region of HA1, peptides obtained by linking other amino acid sequences to amino acid sequences present in the highly conserved region of HA1, and amino acid sequences present in the highly conserved region of HA1. , A peptide consisting of an amino acid sequence that exists in the highly conserved region of HA2, a peptide that binds another amino acid sequence to the amino acid sequence that exists in the highly conserved region of HA2, and a highly conserved region of HA2 A fusion peptide in which an amino acid sequence is combined with another protein, an amino acid sequence present in the highly conserved region of hemagglutinin HA1 and an amino acid sequence present in the highly conserved region of HA2, and the other amino acid sequence is combined with such a fusion peptide. Bound peptides, other tampering with such fusion peptides And the like can be mentioned those that combines the quality. Here, the order in which the amino acid sequence present in the highly conserved region of hemagglutinin HA1 and the amino acid sequence present in the highly conserved region of HA2 are not particularly limited, and either may be on the C-terminal side. .

  Here, the amino acid sequence present in the highly conserved region of hemagglutinin HA1 is not particularly limited as long as it is a region that is highly conserved without mutating due to continuous mutation or discontinuous mutation. The amino acid sequence shown in 1 can be mentioned. Further, the amino acid sequence present in the highly conserved region of hemagglutinin HA2 is not particularly limited as long as it is a region that is highly conserved without being mutated due to continuous mutation or discontinuous mutation. The amino acid sequence shown can be mentioned. Further, the “other amino acid sequence” is not particularly limited, and examples thereof include alanine two molecules (AA). By adding AA to the C-terminal, it is possible to adopt a structure close to the natural hemagglutinin helix structure. Moreover, in order to improve an immune response, you may add 2-3 amino acids. The “other protein” is not particularly limited as long as it is a protein that can be usually used to bind to a low molecule to form an antigen. For example, Human IgG, BSA, HSA, KLH Etc. The above-mentioned various antigen peptides can be used as the above-mentioned antigen peptide. For example, a preferred example is an antigen peptide obtained by covalently binding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to the above-mentioned other protein. be able to. The peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 connects the highly conserved region TGLRN (318 to 322) of influenza virus hemagglutinin HA1 to the highly conserved region GITNKVNSVIEK (47 to 58) of hemagglutinin HA2 of influenza virus, and further AA It is a 19mer peptide (TGLRNGITNKVNSVIEKAA).

  As for the antibody enzyme whose amino acid sequence has been clarified, the antibody enzyme of the present invention can be produced using a conventionally known genetic recombination technique or the like. In this case, after the gene encoding the amino acid sequence of the above antibody enzyme is incorporated into a vector or the like, it can be introduced into an appropriate host cell so that it can be expressed, and a peptide translated in the cell can be purified. . It should be noted that the target antibody enzyme can be efficiently produced by incorporating a gene encoding the above antibody enzyme together with an appropriate promoter capable of mass expression.

[Example 1: Production of monoclonal antibody against influenza virus hemagglutinin]
A peptide of 19mer (TGLRNGITNKVNSVIEKAA) was obtained by further binding two alanine molecules to the C-terminal of the peptide in which GITNKVNSVIEK, which is a highly conserved region of HA2, was bound to TGLRN, which is a highly conserved region of the influenza virus surface protein hemagglutinin HA1. Hereinafter, IAH (Influenza A type Hemagglutinin) peptide) was synthesized as appropriate. The reason for adding two alanine molecules is to make the helix structure close to the structure of natural hemagglutinin. Human IgG was bound to the obtained IAH peptide to obtain an antigen peptide. A Blb / C mouse (female) was immunized with this antigenic peptide, and a monoclonal antibody against influenza virus hemagglutinin was prepared by an ordinary cell fusion method using polyethylene glycol.

<Immunity to mice>
Prepare two glass syringes in the clean bench, attach a 19G needle to one glass syringe, and dilute antigen peptide diluted to 1 mg / mL with sterile PBS (phosphate buffered saline). Collected. The needle was then removed and a three-way stopcock was attached. Another glass syringe was prepared, and a complete Freund's adjuvant of the same amount as a 1 mg / mL PBS solution of the antigen peptide was collected with a 19G needle, and both glass syringes were attached to a three-way stopcock. By alternately pushing glass syringes connected by three-way stopcocks, the antigenic peptide and adjuvant were mixed well until a white emulsion was formed to obtain a W / O (water-in-oil) state.

  The abdomen of the mouse was wiped with alcohol cotton and sterilized, and 100 μL (per mouse) was administered to two subcutaneous sites of the mouse. This means that 100 μg was administered per mouse.

  The second and third immunizations were performed using complete Freund's adjuvant. Incomplete Freund adjuvant was used for the fourth immunization.

  For final immunization, 1 μg / mL PBS solution of antigen peptide was administered to each mouse's tail vein without adjuvant. Cell fusion was performed 3 days after the final immunization.

  Antisera of mice 7 to 10 days after each immunization injection were collected, and titer measurement was performed by the usual ELIZA method. When the first immunization was taken as day 0, the second immunization was performed on the 20th day, the third immunization was performed on the 36th day, the fourth immunization was performed on the 50th day, and the final immunization was performed on the 65th day.

  IAH peptide and human-IgG were prepared to 4 μg / mL using PBS. 50 μL each of these IAH peptides and human-IgG were injected into 96-well immunoplates in separate series. After coating at 4 ° C. overnight, the plate was washed three times with PBS containing 0.05% Tween 20 (PBS-T) using an immunowasher, and 150 μL of 2% gelatin PBS was added to all wells. It was. Incubation for 1 hour at room temperature completes blocking.

  The collected antiserum was first diluted 1/100, and serial dilution from 1/100 to 4 times was repeated 7 times to prepare 8 types of antiserum dilutions per sample. The serum of untreated mice was also diluted as antiserum and used as a control. 50 μL of serially diluted antiserum was added to each well after blocking, and incubated at room temperature for 1 hour (primary reaction). After washing, 50 μL of alkaline phosphatase (ALP) rabbit labeled anti-mouse antibody diluted 1/1000 in PBS-T was added to each well. Incubated for 1 hour at room temperature (secondary reaction). After washing the plate, 100 μL of ALP substrate (p-nitrophenyl phosphate) was added to each well (substrate reaction), and after 30 minutes, the absorbance was measured with an immunoreader (wavelength 405 nm).

  FIG. 1 shows the results of titer measurement after the first immunization (14 days after the first immunization). As shown in FIG. 1, the titer was sufficiently increased against human-IgG, but the titer was not increased at all against IAH peptide.

  FIG. 2 shows the results of titer measurement after the second immunization. As shown in FIG. 2, it can be seen that the titer against the IAH peptide has increased after the second immunization.

  As the third immunization (FIG. 3) and the fourth immunization (FIG. 4) proceeded, the titer against the IAH peptide further increased. The titer after the final immunization increased to several hundred times (FIG. 5).

<Cell fusion>
Cell fusion was performed 3 days after the final immunization. The mice were anesthetized with diethyl ether, and the tip of the semi-sharp scissors was brought to about 80% of the neck of the mouse, and the carotid artery was cut. The blood was collected in a blood collection tube, allowed to stand at room temperature until the end of the experiment, then allowed to stand overnight at 4 ° C., centrifuged the next day (3000 rpm, 10 minutes), antiserum was collected, and titer measurement was performed. FIG. 5 shows the results of titer measurement after the final immunization. Table 1 shows the titer against IAH peptide in each mouse after the final immunization. The titer is O.D. D. _1/2 was taken, and the dilution factor at that time was taken as the titer.

  The mice were sterilized by thoroughly spraying ethanol for disinfection. The sterilized mouse was placed in a clean bench, and the spleen was removed so as not to damage other organs. The removed spleen was transferred to a petri dish containing 5 mL of a washing MEM medium, washed well after removing fat and the like, and transferred to a petri dish containing 10 mL of a washing MEM medium. The spleen was divided into three equal parts with two ground glass plates. The spleen divided into three equal parts was slid one by one in order, and the spleen cells being crushed were taken out while being rubbed with another slick glass plate. A screen mesh was placed on a 50 mL centrifuge tube, and splenocytes taken out in a petri dish were poured into the screen mesh and centrifuged (1400 rpm, 6 minutes). Myeloma cells (NS-1) previously cultured in a T-flask during centrifugation were collected, placed in a 50 mL centrifuge tube, and centrifuged. The supernatant of the centrifuge tube after the first washing was aspirated with a Pasteur pipette, and 10 mL MEM medium was added again while pipetting. The supernatant of myeloma cells after centrifugation was aspirated. It was again suspended in MEM medium and centrifuged again with myeloma cells. After centrifugation, each supernatant was aspirated, again suspended in 10 mL of MEM medium and centrifuged, and then the supernatant was aspirated, and 10 mL of MEM medium was added and suspended uniformly. Spleen cells and myeloma cells were aligned so that myeloma cells: spleen cells = 1: 5. These two cells were mixed, and cell fusion was carried out by slowly adding 1 mL of PEG over 1 minute. After washing with MEM, HAT selection medium was added, and culture was started in a 37 ° C. incubator. Three days later, HAT medium was further added, and after seven days, the medium was changed. Table 2 shows the number of each of myeloma cells and spleen cells, the ratio of myeloma cells to spleen cells, and the number of sown wells in the cell fusion of each mouse used.

<Cloning and screening>
Screening was performed on wells in which a medium exchange colony was confirmed on the seventh day after cell fusion. Screening was performed in the same manner as the above-described ELISA method for titration, and the supernatant of a well in which colonies were confirmed was placed in a 96-well U-bottom plate, diluted 1/2 with PBS-T, and used for the primary reaction. It was. Antisera at the time of cell fusion was used as a positive control for the primary reaction, and untreated mouse serum was diluted 1/100 as a negative control. Strains that were positive in the primary screening were cloned. After the secondary screening, the plate that reacted most strongly with the IAH peptide in each plate was cloned by the limiting dilution method. The screening was repeated until all wells in which colonies were confirmed became positive. A strain in which 100% appears twice in succession and specifically reacts with HA was established as a monoclonal antibody against influenza virus type A hemagglutinin. Table 3 shows the cell fusion efficiency and positive appearance rate in each mouse.

  As shown in Table 3, the fusion efficiency by cell fusion was as low as 32% for mouse I, but as high as 96% and 94% for mouse II and mouse III, respectively. On the other hand, the number of positive clones that appeared was very small, with 4 strains for mouseII and 1 strain for mouseI and mouseIII. Table 4 shows the finally established monoclonal antibodies (HA1-1 and HA1-2).

<Determination of class / subclass>
The class and subclass of established monoclonal antibodies (HA1-1 and HA1-2) were determined using Iso Strip ™ mouse monoclonal antibody isotyping kit.

  150 μL of the culture supernatant diluted 10-fold with PBS was placed in a development tube and placed at room temperature for 30 seconds. Vortexed to dissolve the colored latex beads. The strip was placed in the development tube with the black part down, and allowed to stand for 1-5 minutes until a positive control band was detected. As a result, as shown in Table 4, both HA1-1 and HA1-2 were IgG (κ).

[Example 2: Cross-reactivity test of monoclonal antibodies HA1-1 and HA1-2 against influenza virus type A hemagglutinin]
<Cross-reactivity test with peptide>
Using the obtained culture supernatants of 2 clones, cross-reactivity with several peptides was examined by ELISA. As peptides, TP41-1, ECL2A, HIV V3 loop, and RT-1 were used. TP-41 is a highly conserved region peptide of the HIV surface protein gp41, ECL2A is a peptide at the deletion site of the second extracellular region of the chemokine receptor CCR5Δ32, which is an HIV co-receptor, and the HIV V3 loop is HIV with CCR5. RT-1 is a partial peptide of HIV reverse transcriptase, which is a peptide synthesized based on the conserved region of the site that binds to the co-receptor. TP41-1 consists of the amino acid sequence shown in SEQ ID NO: 8, ECL2A consists of the amino acid sequence shown in SEQ ID NO: 9, HIV V3 loop consists of the amino acid sequence shown in SEQ ID NO: 10, RT-1 is the amino acid shown in SEQ ID NO: 11. Consists of an array. The respective amino acid sequences are shown in Table 5 below.

  TP41-1, ECL2A, HIV V3 loop, and RT-1 were each adjusted to 5 μg / mL with PBS, and 200 μL was placed in a 96-well immunoplate and used for coating. In addition, in order to compare with the target IAH peptide, the IAH peptide was prepared to 5 μg / mL with PBS in the same manner as the other peptides and coated. The culture supernatant was diluted 1/2 with PBS-T and used for the primary reaction. Other operations were performed in the same manner as the titer measurement described above.

  FIG. 6 shows the results of the cross-reactivity test. As shown in FIG. 6, neither HA1-1 nor HA1-2 reacted with peptides other than IAH peptide.

<Cross-reactivity test with influenza A virus and other proteins>
Using the obtained culture supernatants of 2 clones, cross-reactivity with influenza A viruses (H1N1, H2N2, H3N2) and other proteins was examined by ELISA. As other proteins, human IgG, BSA, HSA, KLH, and human-hemoglobin were used.

  Influenza A virus H1N1, influenza A virus H2N2, influenza A virus H3N2, human IgG, BSA, HSA, KLH, and human-hemoglobin were each adjusted to 5 μg / mL with PBS and 200 μL on a 96-well immunoplate. One by one was coated. For comparison with the IAH peptide, the IAH peptide was prepared to 5 μg / mL with PBS in the same manner as the other proteins and coated. The culture supernatant was diluted 1/2 with PBS-T and used for the primary reaction. Other operations were performed in the same manner as the titer measurement described above.

  As a result of the cross-reactivity test, neither HA1-1 nor HA1-2 antibody reacted with Human IgG, BSA, HSA, KLH, and Human-hemoglobin. FIG. 7 shows the results of a cross reaction test with influenza A virus H1N1, influenza A virus H2N2, and influenza A virus H3N2. As shown in FIG. 7, both HA1-1 and HA1-2 naturally reacted strongly with the IAH peptide, but showed a high response to H1N1. It turns out that it reacts also to H2N2. On the other hand, H3N2 appears weak even in negative control (Negative control), so it is considered that it does not react to this. From this, it can be seen that HA1-1 and HA-2 are antibodies that recognize the conserved region of hemagglutinin targeted this time.

[Example 3: Purification and separation of monoclonal antibodies HA1-1 and HA1-2 against hemagglutinin of influenza virus>
<Large acquisition and purification of HA1-1 and HA1-2>
In order to obtain high-purity HA1-1 and HA1-2, salting out with 50% ammonium sulfate was performed twice before purification to remove albumin, and then the Affigel Protein A MAPS-II kit (BIO-RAD) was used. Used and purified.

Hybridomas (1 × 10 ⁶ mice / mouse) obtained by the cell fusion method using an antigen peptide obtained by binding human IgG to IAH peptide as an immunogen are intraperitoneally administered to mice, and HA1-1 (or HA1-2) is obtained. ) Ascites. About 8 mL of this ascites was filtered with filter paper to remove suspended matter in the ascites. Thereafter, the solution was diluted with an equal volume of PBS to the filtered amount. Next, ascites was equally divided into two centrifuge tubes (made of glass), and each equivalent amount of saturated ammonium sulfate solution was added dropwise while stirring by vortex. This was left to stand in ice for 30 minutes and centrifuged at 4 ° C. and 5000 rpm for 10 minutes. The supernatant was decanted and the pellet was dissolved in 1 mL each of PBS. Once again, an equal amount of saturated ammonium sulfate solution was added dropwise while stirring by vortexing, and salting out was performed in the same manner. When salting out was completed, the two tubes were combined and dialyzed twice against 2 L of PBS.

  After completion of dialysis, HA1-1 (or HA1-2) was purified. The operation was performed at 4 ° C. in a cold room according to the instructions of the Affigel Protein A MAPS-II kit. The column was packed with gel with a Pasteur pipette so that it would not be bubbled, and a UV monitor, recorder, etc. were set in the cold room. Monitoring was performed at UV 280 nm, and the binding buffer was spontaneously dropped until the baseline fell, and the gel was equilibrated. The flow rate was adjusted to 0.2 to 0.4 mL / min, and the sample was applied when the Binding buffer reached the gel surface. When the sample reached the gel surface, a binding buffer was applied, and washing was performed by natural dropping until the peak settled. Next, Elution buffer was applied and a sample was collected. At this time, since the antibody was contained in the Elution buffer (acidic), it was immediately neutralized with 2M Tris-HCl pH 8.0. Each fraction of the collected sample was dialyzed twice against 3 L PBS. The protein concentration was determined by DC protein standard assay (BIO-RAD).

<Separation and purification of heavy and light chains>
The antibody HA1-1 (or HA1-2) (5 mg) purified with the Affigel Protein A MAPS-II kit was subjected to ultrafiltration concentration with Centriprep-10 (MILLIPORE) until the antibody solution was about 1 mL. . Further, 5 mL of 50 mM Tris-HCl and 0.15 M NaCl buffer (pH 8.0) was added, and the mixture was concentrated again by centrifugation until it reached about 1 mL. This operation was repeated, and after ultrafiltration, the solution was adjusted to 2.7 mL with 50 mM Tris-HCl, 0.15 M NaCl buffer (pH 8.0), and stored in a brown bottle.

To 2.7 mL of the antibody solution, 0.3 mL of 2M 2-mercaptoethanol was added and stirred gently. After adjusting to pH 8.0 with 1M Tris solution, N ₂ was enclosed, and a reduction reaction was performed with stirring at 15 ° C. for 3 hours. During this period, 0.6 M iodoacetamide was prepared, adjusted with 50 mM Tris-HCl, 0.15 M NaCl buffer (pH 8.0), and deaerated with an aspirator. After completion of the reduction reaction, 3 mL of 0.6 M iodoacetamide was added to the reaction solution and stirred gently. After adjusting the pH to 8.0 with 1M Tris solution, N ₂ was enclosed, and an alkylation reaction was performed with stirring at 15 ° C. for 15 minutes. Using a disk filter (MILLIPORE sample prep LCR13-LH, φ0.5 μm), particles of the reaction solution were removed. Centriprep-10 (MILLIPORE) was used, the reaction solution was centrifuged at 4 ° C. and 2800 rpm for 10 minutes using a high-speed cooling centrifuge, and concentrated by ultrafiltration to about 0.5 mL. The concentrated sample was then filtered through ultrafree C3 (0.2 μm).

  The heavy chain and light chain were purified by size exclusion chromatography (column: Protein-Pak 300, 7.8 × 300 mm, manufactured by Nippon Waters) using 6M guanidine hydrochloride as the mobile phase. 6M guanidine hydrochloride was prepared with HCl to pH 6.5. The separated antibody solution was divided into 3 portions (injection amount of 0.2 mL or less per injection) and injected into HPLC to separate heavy and light chains. Subsequently, dialysis was performed a total of 8 times against PBS, and further twice against 15 nM PB pH 6.5. Purity was confirmed by SDS-PAGE (12% separation gel, 3% concentrated gel, silver stain). Finally, the heavy chain and light chain concentrations were determined by DC protein assay (BIO-RAD) and stored at 4 ° C. Thereafter, it was handled aseptically.

[Example 4: Sequencing of heavy chain and light chain of HA1-1, HA1-2]
MRNA encoding HA1-1 and HA-1-2 was extracted from the hybridoma using QuickPrep mRNA Purification Kit (Amersham Bioscience). Thereafter, cDNA was synthesized from the obtained mRNA using AMV RT cDNA Synthesis Kit (manufactured by Life Science Inc.). Genes encoding the desired HA1-1 and HA-1-2 were amplified using Ig-Prime Kits (Novagen) using the synthesized cDNA as a template and TOPO TA Cloning Kits for Sequencing (Invitrogen). After subcloning, sequencing was performed from the M13 forward (-20) and reverse priming sites. A fully automated DNA sequencer was used for sequencing. The amino acid sequences of the heavy and light chains of HA1-1 and HA-1-2 were deduced based on the determined DNA sequences.

  The amino acid sequence of the heavy chain variable region of HA1-1 is shown in SEQ ID NO: 4, the nucleotide sequence of the gene encoding the heavy chain variable region of HA1-1 is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of HA1-1 is shown. In SEQ ID NO: 5, the nucleotide sequence of the gene encoding the light chain variable region of HA1-1 is shown in SEQ ID NO: 13, the amino acid sequence of the heavy chain variable region of HA1-2 is shown in SEQ ID NO: 6, and the heavy chain variable of HA1-2 is changed. The nucleotide sequence of the gene encoding the region is SEQ ID NO: 14, the amino acid sequence of the light chain variable region of HA1-2 is SEQ ID NO: 7, and the nucleotide sequence of the gene encoding the light chain variable region of HA1-2 is SEQ ID NO: 15. Shown in

[Example 5: Peptidase activity test with heavy and light chains of HA1-1 and HA1-2]
<Peptidase activity test using TP41-1 peptide>
In this experiment, all glassware, plastic articles and buffers were used after dry heat sterilization or autoclave sterilization. Those that could not be autoclaved were sterilized by filtration with a 0.2 μm filter. All experimental operations were performed in a clean bench.

  TP41-1 (TPRGPDRPEGIEEEGGERDRD) solution (final concentration: 120 μM in 15 mM PB) and HA1-1-H (or HA1-1-L or HA1-2-H or HA1-) prepared to a final concentration of 0.2 μM or 0.4 μM. 2-L). The reaction was performed at 25 ° C., and the disappearance of the TP41-1 peptide was followed by HPLC. Table 6 shows the composition of the reaction solution.

  FIG. 8 shows changes over time in the concentration of TP41-1 peptide when HA1-1-H and HA1-1-L are used, and FIG. 9 shows TP41- when HA1-2-H and HA1-2-L are used. The time course of 1 peptide concentration is shown. Further, FIG. 10 shows a chromatogram when the TP41-1 peptide is decomposed using HA1-1-H, and FIG. 11 shows a chromatogram when the TP41-1 peptide is decomposed using HA1-2-H. FIG. 12 shows a chromatogram when the TP41-1 peptide was decomposed using HA1-2-L. As shown in FIGS. 8 and 10, in HA1-1, the heavy chain passed through the induction period of about 25 hours, and the degradation was completed in about 95 hours. However, HA1-1-L was followed for more than 250 hours, but no decomposition was observed. Moreover, as shown in FIGS. 9 and 11, in HA1-2, the degradation of the peptide was completed in about 50 hours. Further, as shown in FIGS. 9 and 12, in the HA1-2 light chain, the degradation was completely completed in about 120 hours after going through the induction period of about 30 hours.

<Peptidase activity test using IAH peptide>
Mix IAH peptide solution (final concentration: 140 μM) and HA1-1-H (or HA1-1-L or HA1-2-H or HA1-2-L) prepared to a final concentration of 0.2 μM or 0.4 μM. did.

  The reaction was performed at 25 ° C. in the same manner as when the TP41-1 peptide was used, and the disappearance of the IAH peptide was followed by HPLC. Table 7 shows the composition of the reaction solution.

  FIG. 13 shows changes over time in IAH peptide concentrations when HA1-1-H and HA1-1-L are used, and FIG. 14 shows IAH peptide concentrations when HA1-2-H and HA1-2-L are used. The change with time is shown. Further, FIG. 15 shows a chromatogram when the IAH peptide is decomposed using HA1-1-H, FIG. 16 shows a chromatogram when the IAH peptide is decomposed using HA1-1-L, and FIG. A chromatogram when IAH peptide is decomposed using -2-H is shown, and FIG. 18 shows a chromatogram when IAH peptide is decomposed using HA1-2-L. As shown in FIGS. 13 and 15, in HA1-1, the heavy chain completely degraded the IAH peptide in about 55 hours. Further, as shown in FIGS. 13 and 16, even with the light chain that did not undergo a degradation reaction with the TP41-1 peptide, the degradation of the IAH peptide was completed in about 110 hours after going through the induction period of about 60 hours. Moreover, as shown in FIGS. 14 and 17, in HA1-2, the heavy chain has completed the degradation of the IAH peptide in about 65 hours. Further, as shown in FIGS. 14 and 18, in the light chain, the degradation of the IAH peptide is completed in about 75 hours after going through the induction period of about 30 hours. The degradation reaction of the IAH peptide is faster than that of the TP41-1 peptide for both the heavy chain and light chain of both the HA1-1 and HA1-2 antibodies. This is thought to be because the IAH peptide is the antigen itself. Moreover, the degradation fragment of IAH peptide by HA1-2-H was examined with a mass spectrometer. FIG. 19 shows the result. As shown in FIG. 19, the IAH peptide is between KV (between the 10th and 11th amino acids of the amino acid sequence shown in SEQ ID NO: 3) and between NS (12th of the amino acid sequence shown in SEQ ID NO: 3). And 13th amino acid), between E-K (between the 16th and 17th amino acids of the amino acid sequence shown in SEQ ID NO: 3), between KA (17th and 18th of the amino acid sequence shown in SEQ ID NO: 3). Between amino acids) and A-A (between the 18th and 19th amino acids of the amino acid sequence shown in SEQ ID NO: 3).

<Dynamic analysis>
Since antibody subunits exhibiting antigenolytic activity degrade peptides while drawing biphasic (induction and active) curves, it is difficult to determine the initial rate of degradation from this curve. Therefore, once the peptide was completely decomposed, the peptide was added again to eliminate the induction period and solve this problem. The initial rate was defined as less than 10% of the substrate change by the enzyme. HA1-2-L was adjusted to a constant concentration of 0.64 mM, and the substrate (TP41-1 peptide) concentration was changed. The kinetic parameters were calculated from the obtained Hanes-Woolf plot according to the Michaelis-Menten equation.

  FIG. 20 (a) shows the relationship between the substrate (TP41-1 peptide) concentration and the degradation rate, and FIG. 20 (b) shows the result of the Hanes-Woolf plot.

[Example 6: Influenza A hemagglutinin degradation test using heavy and light chains of HA1-1 and HA1-2]
<Influenza A hemagglutinin degradation test with heavy chain of HA1-1>
First, the TP41-1 peptide was completely degraded with the HA1-1 antibody heavy chain (HA1-1-H) (activation of HA1-1-H). Then, it was mixed with influenza A virus at a ratio of 1: 1 and reacted (influenza A virus 500 μL + activated HA1-1-H 500 μL). As a control, influenza A virus and 15 mM PB were mixed at a ratio of 1: 1 and reacted (influenza A virus 300 μL + 15 mM PB 300 μL). The time course of this was followed by SDS-PAGE.

  In addition, FIG. 21 shows the results of SDS-PAGE of influenza A virus, FIG. 22 shows the results of Western blot analysis for influenza A virus using HA1-1 antibody, and FIG. 23 shows influenza A by heavy chain of HA1-1 antibody. The degradation test result of hemagglutinin of type virus is shown.

  From FIGS. 21, 22 and 23, it can be seen that the disappearance of the HA2 band was specifically observed by the HA1-1 antibody heavy chain. In particular, from FIG. 23, the influenza A virus HA2 molecule is almost completely degraded by the HA1-1 antibody heavy chain at a reaction time of about 40% at 8 hours, 70% at 12 hours, 90% at 24 hours, and 48 hours. It can be seen that In the system in which the HA1-1 antibody heavy chain was not added, the HA2 molecule was not degraded at all. Further, it can be seen that the HA1-1 antibody heavy chain does not degrade the influenza A virus constituent protein other than the HA2 molecule, but has degraded the HA2 molecule with high specificity. From this, it can be said that the HA1-1 antibody heavy chain is an antibody enzyme that specifically recognizes and decomposes influenza A hemagglutinin.

<Influenza A hemagglutinin degradation test using HA1-1 light chain>
In the same manner as described above except that the HA1-1 antibody light chain (HA1-1-L) was used, degradation of the influenza virus constituent protein was attempted using the HA1-1 antibody light chain (HA1-1-L). FIG. 24 shows the results of the degradation test of hemagglutinin of influenza A virus using the HA1-1 antibody light chain. As shown in FIG. 24, only HA2 molecules were degraded by 20% in 4 hours and 40% in 8 hours.

<Influenza A hemagglutinin degradation test with heavy chain of HA1-2>
In the same manner as described above except that the HA1-2 antibody heavy chain (HA1-2-H) was used, degradation of the influenza virus constituent protein was attempted using the HA1-1 antibody heavy chain (HA1-2-H). FIG. 25 shows the results of the degradation test of hemagglutinin of influenza A virus by HA1-2 antibody heavy chain. As shown in FIG. 25, HA1-2H almost decomposed the HA2 molecule in about 4 hours from the start of the reaction. On the other hand, human serum albumin (HSA) used as a control was hardly degraded.

  If an antibody enzyme capable of recognizing a peptide constituting a highly conserved region of hemagglutinin HA1 and / or HA2 and cleaving and / or degrading HA can be obtained, it is not hindered by antigenic mutation that occurs annually. It is possible to recognize HA by the antibody enzyme and to cleave and / or decompose the recognized HA, which can be expected to contribute to prevention / treatment of influenza. Moreover, such an antibody enzyme is expected to be used for sensing and removing viruses in the atmosphere. For this reason, the antibody enzyme of the present invention can be used in the medical industry, pharmaceutical industry, reagent industry, medical equipment industry, food industry, etc., and is very useful.

In Example 1 which manufactures a monoclonal antibody, it is a figure which shows the result of the titer measurement after the first immunization. In Example 1 which manufactures a monoclonal antibody, it is a figure which shows the result of the titer measurement after the 2nd immunization. In Example 1 which manufactures a monoclonal antibody, it is a figure which shows the result of the titer measurement after the 3rd immunization. In Example 1 which manufactures a monoclonal antibody, it is a figure which shows the result of the titer measurement after the 4th immunization. In Example 1 which manufactures a monoclonal antibody, it is a figure which shows the result of the titer measurement after the last immunization. In Example 2, it is a figure which shows the result of the cross-reactivity test with TP41-1, ECL2A, HIV V3 loop, and RT-1. In Example 2, it is a figure which shows the result of the cross-reactivity test with H1N1, H2N2, and H3N2. It is a figure which shows the result of the peptidase activity test of HA1-1-H and HA1-1-L using TP41-1 peptide. It is a figure which shows the result of the peptidase activity test of HA1-2-H and HA1-2-L using TP41-1 peptide. It is a figure which shows a chromatogram when decomposing | disassembling TP41-1 peptide using HA1-1-H. It is a figure which shows a chromatogram when decomposing | disassembling TP41-1 peptide using HA1-2-H. It is a figure which shows a chromatogram when decomposing | disassembling TP41-1 peptide using HA1-2-L. It is a figure which shows the result of the peptidase activity test of HA1-1-H and HA1-1-L using IAH peptide. It is a figure which shows the result of the peptidase activity test of HA1-2-H and HA1-2-L using IAH peptide. It is a figure which shows a chromatogram when IAH peptide is decomposed | disassembled using HA1-1-H. It is a figure which shows a chromatogram when IAH peptide is decomposed | disassembled using HA1-1-L. It is a figure which shows a chromatogram when IAH peptide is decomposed | disassembled using HA1-2-H. It is a figure which shows a chromatogram when IAH peptide is decomposed | disassembled using HA1-2-L. It is a figure which shows the result of having investigated the degradation fragment of IAH peptide by HA1-2-H with the mass spectrometer. It is a figure which shows the result of the kinetic analysis in a peptidase activity test, (a) is a figure which shows the relationship between a substrate (TP41-1 peptide) density | concentration and degradation rate, (b) is a Hanes-Woolf plot. It is a figure which shows the result. It is a figure which shows the SDS-PAGE result of influenza A virus. It is a figure which shows the result of the Western blot analysis with respect to influenza A virus using HA1-1 antibody. It is a figure which shows the degradation test result of the hemagglutinin of influenza A virus by a HA1-1 antibody heavy chain. It is a figure which shows the degradation test result of the hemagglutinin of influenza A virus by a HA1-1 antibody light chain. It is a figure which shows the degradation test result of the hemagglutinin of influenza A virus by a HA1-2 antibody heavy chain. It is a figure which shows the amino acid sequence of the variable region of HA1-1, (a) is a figure which shows the amino acid sequence (sequence number 4) of the heavy chain variable region of HA1-1, (b) is a light figure of HA1-1. It is a figure which shows the amino acid sequence (sequence number 5) of a chain variable region. It is a figure which shows the amino acid sequence of the variable region of HA1-2, (a) is a figure which shows the amino acid sequence (sequence number 6) of the heavy chain variable region of HA1-2, (b) is a light figure of HA1-2. It is a figure which shows the amino acid sequence (sequence number 7) of a chain variable region.

Claims (6)

  An antibody against influenza virus having hemagglutinin, which recognizes hemagglutinin and has an activity of degrading hemagglutinin, or a fragment of an antibody enzyme containing a variable region thereof.   The amino acid sequence shown in SEQ ID NO: 1 present in the highly conserved region of hemagglutinin HA1 and / or the amino acid sequence shown in SEQ ID NO: 2 present in the highly conserved region of hemagglutinin HA2 are recognized. An antibody enzyme or a fragment of an antibody enzyme containing a variable region thereof.   The antigen peptide comprising the amino acid sequence shown in SEQ ID NO: 1 present in the highly conserved region of hemagglutinin HA1 and / or the amino acid sequence shown in SEQ ID NO: 2 present in the highly conserved region of hemagglutinin HA2, The antibody enzyme according to claim 1 or 2, or a fragment of an antibody enzyme comprising a variable region thereof.   4. The antibody enzyme according to claim 3, wherein the antigen peptide used as an antigen is obtained by covalently binding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to a protein. fragment. The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 in which one or several amino acids are substituted, deleted, inserted, and / or added. ,
The light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5 in which one or several amino acids are substituted, deleted, inserted and / or added. The antibody enzyme according to any one of claims 1 to 4, or a fragment of an antibody enzyme comprising a variable region thereof. The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 in which one or several amino acids are substituted, deleted, inserted, and / or added. ,
The light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 7. The antibody enzyme according to any one of claims 1 to 4, or a fragment of an antibody enzyme comprising a variable region thereof.