JP2020011916A - Cellular immunity inducers - Google Patents

Abstract

To provide novel cellular immunity inducers.SOLUTION: Disclosed herein is a composite comprising a first constituent and a second constituent, where the first constituent comprises dimer to pentamer of a cell attaching subunit of a bacterial toxin which comprises a toxic subunit monomer (toxin itself) and a pentamer of a host cell attaching subunit, and the second constituent comprises a MHC class I antigen peptide.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a conjugate having a cell-mediated immunity-inducing effect, and more particularly to a conjugate used as a drug such as an anticancer agent or an antiviral agent.

  Cellular immunity, especially cytotoxic T cells (sometimes called killer T cells or CTLs) and helper T cells, which are antigen-specific T cells, are central to the elimination of cancer cells and virus-infected cells by living organisms. Working. Antigen-specific T cells are composed of MHC molecules (also called HLA molecules in humans) on antigen-presenting cells such as dendritic cells and macrophages, and antigens that are fragment peptides of antigen proteins derived from cancers and viruses. Differentiates and proliferates by recognizing the binding complex with the peptide with the T cell receptor. It is known that the antigen peptide presented to the MHC molecule is usually about 8 to 20 amino acids in length. The differentiated and proliferated antigen-specific T cells specifically inhibit cancer cells and virus-infected cells presenting the antigen peptide-MHC molecule-binding complex, and produce various cytokines to produce anti- Exhibits tumor and antiviral effects.

  In order to efficiently induce specific immunity by a vaccine, it is effective to administer a non-specific immunostimulating substance in combination with a main antigen protein or antigen peptide. Substances and dosage forms that have the effect of enhancing the vaccine effect are called adjuvants. Currently, the most widely used adjuvant approved for human use is an aluminum preparation, which induces a so-called Th2-type immune response and enhances humoral immunity, but induces antigen-specific T cells. (Cytotoxic T cells and Th1-type immune response) are basically not induced. In addition, problems of side effects such as IgE production have been pointed out.

  Patent Document 1 discloses an antigenic peptide and two or more proteins selected from the B subunit of Shiga toxin 2e (Stx2eB), the B subunit of Escherichia coli heat-labile toxin (LTB), and the B subunit of cholera toxin (CTB). A fusion protein is disclosed, including that the fusion protein can be used as a vaccine. However, the pharmacological action of the fusion protein is to produce antibodies specifically reacting with the antigenic peptide and to enhance humoral immunity (Th2). Nor.

  On the other hand, Patent Literature 2 discloses a chimeric protein (B-X) obtained by fusing the B subunit of Shiga toxin with an antigenic peptide, thereby inducing cell-mediated immunity. However, the effect of fusing with the B subunit of Shiga toxin is not seen from the experimental data of FIGS. 3 and 5, and it is disclosed that two or more B subunits of Shiga toxin are linked and used. Absent.

WO2017 / 094793 US6613882B

  An object of the present invention is to provide an agent for inducing cell-mediated immunity.

As described above, Patent Document 1 discloses that a fusion protein containing an antigen peptide and two or more proteins selected from Stx2eB, LTB and CTB enhances humoral immunity and can be used as a vaccine. It is well known that adjuvants that enhance sexual immunity (Th2) do not always induce cellular immunity at the same time. For example, "Elucidation of mechanism and safety evaluation for next-generation adjuvant development" Takeshi Ishii, Chapter 1 of CMC Publishing (2017), page 1 mainly shows antibody production (B cell Activity), among CD4 + T cells, those that induce Th1 acquired immunity, those that induce Th2 type, those that induce Th17 type, or cytotoxic T cells (Cytotoxic T lymphocyte: CTL) It is disclosed that various acquired immunity directions, such as those that induce the activity of NKT cells, can be controlled. That is, it is known that the type and combination of adjuvants to be employed should be changed depending on the direction of acquired immunity. Therefore, two or more proteins selected from Stx2eB, LTB and CTB, which are known to have the effect of producing an antibody that specifically reacts with the antigen peptide, are used to induce cell-mediated immunity via MHC class I antigen peptides. There is no reason to use it.
The effect of the chimeric protein obtained by fusing one B subunit of Shiga toxin and an antigen peptide disclosed in Patent Document 2 is questionable for its effect as described above. Even if is correct, in the technical field of the present invention it is known that the type and combination of adjuvants to be adopted should be changed depending on the direction of acquired immunity, the technology of humoral immunity is different from the direction of acquired immunity There is no reason to combine techniques with cell-mediated immunity. Further, even if the combination is used, when the B subunit of Shiga toxin is changed to two or more, the effect of humoral immunity is considered to be exerted from the disclosure of Patent Document 1, There is no motivation for the two chimeric proteins to have more than one Shiga toxin B subunit.

  Under these circumstances, the present inventors have conducted intensive studies in order to solve the above problems, and as a result, a polypeptide in which 2 to 5 cell adhesion subunits of bacterial toxins such as Stx2eB and LTB are linked to an MHC class When administered in combination with an I antigen peptide, these polypeptides, which are involved in the enhancement of humoral immunity (Th2), surprisingly induce cellular immunity, especially the induction of cytotoxic T cells (killer T cells) Was also effective.

  That is, the present inventors considered that an antigen peptide containing an MHC class I antigen site, a B subunit of Shiga toxin 2e (Stx2eB), a B subunit of Escherichia coli heat-labile toxin (LTB) and a B subunit of cholera toxin ( By administering a polypeptide containing 2 to 5 cell adhesion subunits of a toxin protein selected from CTB) to an animal, the polypeptide containing 2 to 5 cell adhesion subunits can induce cellular immunity. Act as an adjuvant, and found that killer T cells, which are antigen-specific T cells for MHC class I antigen sites, are induced in the animal body.The complex of this MHC class I antigen peptide and the polypeptide was excellent. They have found that they can be used as inducers of cellular immunity, and have completed the present invention.

The present invention provides the following.
[1] Two to five cell adhesion subunits of a bacterial toxin consisting of a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer which is a toxin body were linked. A first component comprising a polypeptide;
A second component comprising an MHC class I antigen peptide.
[2] The complex of [1], wherein the MHC class I antigen peptide is at least one selected from the group consisting of an ovalbumin MHC class I antigen peptide, a cancer antigen peptide, and a virus antigen peptide.
[3] The cell adhesion subunit of the bacterial toxin is a cell adhesion subunit of one or more toxins selected from Shiga toxin, cholera toxin, Escherichia coli heat-labile toxin, pertussis toxin and subtilase cytotoxin. , [1] or [2].
[4] The complex according to any one of [1] to [3], wherein the cell adhesion subunit of the bacterial toxin contains at least the B subunit of Shiga toxin 2e.
[5] The complex according to any one of [1] to [4], wherein the cell adhesion subunit of the bacterial toxin contains only the B subunit of Shiga toxin 2e.
[6] In the first component, the 2 to 5 cell adhesion subunits are connected to each other via independent peptide linkers each consisting of 10 to 30 amino acid residues. ] The complex according to any one of [5].
[7] The composite according to any one of [1] to [6], wherein the first component and the second component are included as a composition.
[8] The complex according to any one of [1] to [6], wherein the first component and the second component are included as a fusion protein.
[9] The fusion protein is a fusion protein in which the first component and the second component are bound via a peptide linker consisting of 10 to 30 amino acid residues, [8] The complex according to any one of the above.
[10] Two to five cell adhesion subunits of a bacterial toxin consisting of a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer as a toxin body were linked. A DNA construct comprising a DNA encoding a fusion protein comprising the polypeptide and an MHC class I antigen peptide.
[11] The fusion protein according to [10], wherein the fusion protein is a fusion protein in which the polypeptide and the MHC class I antigen peptide are bound via a peptide linker consisting of 12 to 30 amino acid residues. The described DNA construct.
[12] A transformant into which the DNA construct according to [10] or [11] has been introduced.
[13] Two to five cell adhesion subunits of a bacterial toxin consisting of a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer as a toxin body were linked. A first DNA construct comprising DNA encoding a first component comprising a polypeptide,
A second DNA construct containing a DNA encoding a second component containing an MHC class I antigen peptide.
[14] A medicament comprising the complex according to any one of [1] to [9].
[15] Two to five cell adhesion subunits of a bacterial toxin consisting of a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer as a toxin body were linked. A first active ingredient containing a polypeptide;
And a second active ingredient comprising an MHC class I antigen peptide. [16] The medicament according to [14] or [15], which is a cell-mediated immunity inducer.
[17] The medicament according to any of [14] to [16], which is an anticancer agent or an antiviral agent. [18] The medicament according to any of [14] to [17], which is an injection.
[19] Two to five cell adhesion subunits of a bacterial toxin comprising a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer as a toxin body were linked. A first active ingredient containing a polypeptide;
A method for inducing cellular immunity in a non-human mammal, comprising administering a second active ingredient containing an MHC class I antigen peptide to the non-human mammal.

  According to the present invention, it is possible to provide an inducer of cellular immunity capable of inducing cytotoxic T cells (killer T cells) against MHC class I antigen, which has been considered difficult to induce. As a result, it is possible to prevent or treat diseases and cancers caused by viruses and intracellular parasitic bacteria, which are insufficiently effective only by inducing antibody production (B cell activity).

Graph (stimulated with OVAepi) showing the results of an ELISPOT test using ovalbumin MHC class I epitope (OVAepi) and spleen cells treated with LTB, LTB-Stx2eB, Stx2eB-Stx2eB or Freund's complete adjuvant (FCA). Graph showing the results of an ELISPOT test using spleen cells treated with a fusion protein of OVAepi and LTB-Stx2eB or Stx2eB-Stx2eB (stimulated with OVAepi). Graph showing the results of an ELISPOT test using spleen cells treated with a fusion protein of mouse survivin 57-64 (Msur57) and Stx2eB-Stx2eB (stimulated with various cancer cells). 4 is a graph showing cancer cytotoxicity of spleen cells treated with a fusion protein of Msur57 and Stx2eB-Stx2eB.

  Hereinafter, the present invention will be described.

<Composite>
The complex of the present invention comprises a bacterial toxin consisting of a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer which is a toxin main body. A first component comprising a polypeptide linked together,
And a second component comprising an MHC class I antigen peptide.

  Here, the complex may be any as long as it contains both the first component and the second component, and the manner in which the first component and the second component are contained is not particularly limited, and will be described later. In addition, the first component and the second component may be contained as a composition (mixture or the like) which exists independently, or at least one or more of the first component and the second component may be included. It may be contained as a fusion protein arranged and fused in tandem.

<First component>
The first component of the complex of the present invention is a cell adhesion subunit of a bacterial toxin comprising a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer which is a toxin body. Includes a polypeptide in which 2 to 5 units are linked. The bacterial toxin of the present invention is not particularly limited as long as it is a complex comprising the cell adhesion subunit pentamer and the toxic subunit monomer, and examples thereof include Escherichia coli, cholera bacteria, and B. pertussis. And toxin proteins produced by pathogenic bacteria.

  A bacterial toxin composed of a pentamer of a cell adhesion subunit involved in attachment to a host cell and a toxic monomer of a toxic subunit, which is a main body of the toxin, is a subunit involved in attachment to a host cell (also referred to as a B subunit). ) Binds to receptors on the cell surface, such as the intestinal mucosal cells of the host, thereby delivering a toxic subunit (also referred to as the A subunit) into the cell. Here, the term "attachment to a host cell" includes not only attachment to a cell membrane of a host cell and attachment to a protein on a cell surface, but also an embodiment in which the cell is attached to a host cell surface and then taken up into a cell. The mode of “attachment” is not particularly limited as long as the toxin subunit can be introduced into the host cell via the attachment of the attachment subunit to the host cell.

  Many bacterial holotoxins consisting of five cell adhesion subunits and one toxic subunit are known, and examples thereof include Shiga toxin, cholera toxin, Escherichia coli heat-labile toxin, pertussis toxin, and subtilase cell And toxins.

  By linking and using 2 to 5 cell adhesion subunits of such bacterial toxin, the function of a vector for transferring (transporting) the MHC class I antigen peptide as the second constituent substance into cells is expected. .

  Hereinafter, Shiga toxin, cholera toxin, Escherichia coli heat-labile toxin, pertussis toxin, subtilase cytotoxin, and their cell adhesion subunits will be described.

<Shiga toxin>
Shiga toxin (Stx) is a proteinaceous toxin produced by enterohemorrhagic Escherichia coli (EHEC, STEC) that causes edema disease, and is classified into type 1 (Stx1) and type 2 (Stx2). Stx1 is classified into subclasses a to d, and Stx2 is classified into subclasses ag. A holotoxin consisting of one molecule of the A subunit, which is a toxic substance, and five molecules of the B subunit, which acts to bind to the intestinal mucosa. It acts on eukaryotic ribosomes to inhibit protein synthesis.

In the present invention, the B subunit of Stx2 subclass e (Stx2eB) is preferably used, and examples of Stx2eB include a protein having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 shows the amino acid sequence of the mature region of Stx2e B subunit protein (GenBank Accession No. AAQ63639) (Ala19 to Asn87 excluding the secretory signal peptide to the periplasm). In addition, Stx2eB may have a sugar chain added thereto, or may be an unmodified sugar chain variant in which Asn73 (Asn residue at position 55 in the amino acid sequence of SEQ ID NO: 2) is substituted with a Ser residue.
In addition, as long as 2 to 5 Stx2eBs can be linked and exert a cell-mediated immunity inducing effect when combined with an MHC class I antigen peptide, one or several Stx2eBs in the amino acid sequence represented by SEQ ID NO: 2 May be substituted, deleted, inserted or added. The “several” is, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3.
In addition, Stx2eB has an identity of preferably 85% or more, more preferably 90% or more, more preferably 95% or more with the amino acid sequence represented by SEQ ID NO: 2, and 2 to 5 amino acids are linked. It may be one that can exert a cellular immunity inducing effect when combined with an MHC class I antigen peptide.

<Escherichia coli heat-labile toxin>
Escherichia coli diarrhea is caused by the proteinaceous toxin LT produced by enterotoxigenic Escherichia coli (ETEC), which is also called Escherichia coli heat-labile toxin. Escherichia coli heat-labile toxin (LT) is a holotoxin consisting of one molecule of A subunit and five molecules of B subunit, which are the toxic components. The A subunit of LT (LTA) penetrates into the cytoplasm, raises intracellular cAMP levels, and activates plasma membrane chloride channels, causing water leakage into the intestinal tract, a condition of diarrhea. The B subunit of LT (LTB) is non-toxic and is involved in the adhesion of LT toxin to intestinal cells.

LTB used in the present invention is represented, for example, by the amino acid sequence of SEQ ID NO: 4. Further, a sugar chain may be added to LTB. For example, an N-linked sugar chain is added to the Asn residue at position 90 of LTB (ie, position 90 of SEQ ID NO: 4).
Furthermore, as long as 2 to 5 LTBs can be linked and exert a cell-mediated immunity inducing effect when combined with an MHC class I antigen peptide, one or several LTBs in the amino acid sequence represented by SEQ ID NO: 4 May be substituted, deleted, inserted or added. The “several” is, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3. The amino acid sequence represented by SEQ ID NO: 4 has been registered as GenBank Accession No. AAL55672.
In addition, LTB has 85% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence represented by SEQ ID NO: 4, and has 2 to 5 ligated MHC class I Those which can exert a cellular immunity inducing effect when combined with an antigen peptide may be used.

<Cholera toxin>
Cholera toxin (CT) protein is a toxin produced by Vibrio cholerae that causes cholera illness characterized by symptoms such as diarrhea. One cholesterol A subunit (CTA), Consists of five B subunits (CTBs) involved in mucosal invasion.

The CTB used in the present invention is represented by, for example, the amino acid sequence of SEQ ID NO: 6. CTB is one or several amino acids in the amino acid sequence represented by SEQ ID NO: 6 as long as it can exert a cellular immunity-inducing effect when combined with 2 to 5 MHC class I antigen peptides. May be substituted, deleted, inserted or added. The “several” is, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3.
In addition, CTB has 85% or more, preferably 90% or more, more preferably 95% or more identity with the amino acid sequence represented by SEQ ID NO: 6, and has 2 to 5 ligated MHC class I Those which can exert a cellular immunity inducing effect when combined with an antigen peptide may be used.

<Pertussis toxin>
Pertussis toxin is a toxin produced by Bordetella pertussis, which causes pertussis, and is composed of a total of six subunits of S1, S2, S3, S4 (two molecules) and S5. The S1 subunit is the body of the toxin, and the remaining subunits are involved in binding to target cells. Therefore, in the present invention, any of the subunits S2, S3, S4, and S5 can be used. The amino acid sequences of these subunits can be obtained with reference to known sequence information.

<Saturase cytotoxin>
The subtilin cytotoxin is a toxin produced by Shiga toxin-producing Escherichia coli, and is composed of one A subunit, which is the main body of the toxin, and five B subunits involved in cell adhesion. Therefore, in the present invention, this B subunit can be used. The amino acid sequence of the B subunit of the subtilase cytotoxin can be obtained with reference to known sequence information.

The first component comprises two to five cell adhesion subunit proteins selected from the cell adhesion subunits of bacterial toxins as described above. The number is preferably two or three, and more preferably two. The 2 to 5 cell adhesion subunit proteins may be in a form in which 2 to 5 of any one type of cell adhesion subunit selected from Stx2eB, LTB, and the like as described above, or Stx2eB, An embodiment in which 2 to 5 cell adhesion subunits selected from LTB or the like are contained in total may be included.
More preferably, it is 2 to 5 selected from Stx2eB, LTB and CTB, even more preferably 2 to 5 selected from Stx2eB and LTB, and 2 to 3 selected from Stx2eB and LTB. .
When linking different types of cell attachment subunits, the order of linking the cell attachment subunits may be any order.
The first component preferably includes at least Stx2eB, and may include only Stx2eB.

  The 2 to 5 cell adhesion subunits constituting the first component of the complex of the present invention are preferably tandemly linked to each other via a peptide linker. That is, it is preferable that 2 to 5 cell adhesion subunits are polypeptides connected to each other via 1 to 4 peptide linkers.

Here, the peptide linker means a linker composed of a peptide in which amino acids are connected linearly. The number of amino acids in the peptide linker is, for example, 10 to 30, preferably 12 to 30, more preferably 12 to 25, and even more preferably 12 to 22. In the peptide linker used in the present invention, the content of proline is preferably 20 to 33.3%, more preferably 20 to 27%, and further preferably 20 to 25%.
In the peptide linker, the proline is preferably located flanking two or three other amino acids. The amino acids located between the prolines are preferably selected from glycine, serine, arginine. However, at one or both ends of the peptide, amino acids other than proline may be added in a range of 5 or less, preferably 4 or less. Such preferred peptide linkers are described, for example, in WO 2009/133882.

In the present invention, the peptide linker may preferably be a peptide (PG12) having the amino acid sequence represented by SEQ ID NO: 8, or PG12v2, PG17, or PG22 disclosed in WO2017 / 094793.
By using a peptide linker as described above, the stability of the polypeptide can be improved and the polypeptide can be highly accumulated in host cells.

<Second component>
The second component is an oligopeptide or polypeptide containing an MHC class I antigen peptide, but if the amino acid sequence of the oligopeptide or polypeptide contains at least one or more MHC class I antigen peptides, other amino acid sequences May be included. Further, two or more MHC class I antigen peptides may be contained. In this case, two or more MHC class I antigen peptides may be contained in the amino acid sequence of one oligopeptide or polypeptide, or two or more MHC class I antigen peptides are contained in another oligopeptide or polypeptide. You may.
MHC class I antigen peptide is presented on the cell surface by binding to MHC class I on the cell surface, which is recognized by the receptor of T cells, especially cytotoxic T cells (CTL), and Means a peptide capable of inducing
The antigenic peptide is not particularly limited as long as it is a peptide capable of eliciting a cellular immune response in the administered mammal, and its sequence and length are not particularly limited. Generally, the length is 8 to 20 amino acids. It is.
The second component can be chemically synthesized or can be produced by genetic engineering.

Examples of class I antigen peptides include MHC class I epitopes of ovalbumin (OVA) (eg, SEQ ID NO: 12), cancer antigen peptides, and viral antigen peptides.
Here, examples of the cancer antigen peptide (class I) include WT1, MUC1 (Mucin 1), HER-2 (Human epidermal growth factor receptor 2), Survivin (for example, SEQ ID NO: 15), Gp100, MAGE (Melanoma Many cancer antigen proteins such as -associated antigen) -A3, Tyrosinase and PSA (prostate-specific antigen) are known, and their class I antigen peptides can be used. However, the cancer antigen peptide can be appropriately selected according to the cancer to be treated, and is not limited thereto. In addition, a human antigen, a mouse antigen, or the like can be selected according to the treatment target.
Examples of the virus antigen peptide (class I) include Epstein-barr virus (EBV), Cytomegalovirus (CMV), Human Immunodeficiency virus (HIV), Human T-cell leukemia virus (HTLV), Influenza Virus, Respiratory syncytial virus A class I antigen peptide of a class I antigen peptide of a viral antigen protein such as (RSV) can be used. However, the virus antigen peptide can be appropriately selected according to the cancer to be treated, and is not limited thereto.

<Composite of the Present Invention to First Embodiment>
The first aspect of the composite of the present invention is an aspect containing the first component and the second component as separate components, that is, an aspect as a composition.
In this embodiment, the ratio of the first component to the second component is, for example, a molar ratio of 1: 5 to 5: 1, preferably 1: 2 to 2: 1, and particularly preferably 1: 1.

<Composite of the Present Invention to Second Embodiment>
A second embodiment of the complex of the present invention is an embodiment comprising the first component and the second component as a fusion protein. Either of the binding order of the first component and the second component in the fusion protein may be first (N-terminal). Here, the fusion protein means a multivalent protein containing two or more protein units in one polypeptide chain.
In the fusion protein, the first component and the second component may be directly linked, but are preferably linked via a peptide linker.
In this case, as the peptide linker, for example, a peptide linker as described in the above section <First component> can be used. Specifically, a peptide linker having 10 to 30 amino acids represented by PG12 can be used. It is preferred to use a peptide linker with a proline content of 20-33.3% and proline sandwiched between two or three other amino acids.

  The fusion protein used in the present invention has, for example, the amino acid sequence represented by SEQ ID NO: 23, 25 or 27. In the fusion protein having the amino acid sequence represented by SEQ ID NO: 23, LTB, Stx2eB, and OVAepi are tandemly linked via PG12. In the fusion protein having the amino acid sequence represented by SEQ ID NO: 25, Stx2eB, Stx2eB, and OVAepi are linked in tandem via PG12. In the fusion protein having the amino acid sequence represented by SEQ ID NO: 27, Stx2eB, Stx2eB, and Msur57 are tandemly linked via PG12.

  When the fusion protein used in the present invention is expressed in a plant or the like, a plant-derived secretory signal peptide or a chloroplast transit signal peptide may be added to the amino terminus thereof. The secretory signal peptide is preferably derived from tobacco β-D-glucan exohydrolase, tobacco 38 kDa peroxidase (GenBank Accession D42064). Preferred chloroplast transit signal peptides are described, for example, in WO 2009/004842 and WO 2009/133882.

Further, when the fusion protein used in the present invention is expressed in a plant or the like, a signal peptide such as an endoplasmic reticulum residual signal peptide and a vacuolar translocation signal peptide may be added to the carboxyl terminal.
Preferred ER residual signal peptides are described, for example, in WO 2009/004842 and WO 2009/133882, and the HDEL sequence (SEQ ID NO: 9) can be used.
Vacuolar translocation signal peptides are described, for example, in WO 2009/004842 and WO 2009/133882.

  The fusion protein used in the present invention can be chemically synthesized or can be produced by genetic engineering.

<DNA construct encoding fusion protein>
For production by genetic engineering, a DNA construct containing DNA encoding the fusion protein is used. The DNA construct used in the present invention is a DNA encoding an MHC class I antigen peptide such as OVAepi or Sur57, and a DNA encoding a polypeptide containing 2 to 5 cell adhesion subunits such as Stx2eB, LTB or CTB, It includes DNA linked in tandem via DNA encoding the linker peptide.
The DNA encoding the linker peptide is, for example, SEQ ID NO: 7 (PG12). As a DNA encoding Stx2eB, for example, a DNA encoding Stx2eB (SEQ ID NO: 1), as a DNA encoding LTB, for example, a DNA encoding LTB (SEQ ID NO: 3), and as a DNA encoding CTB, for example, the sequence And DNA having the nucleotide sequence of No. 5.
DNA encoding the MHC class I antigen peptide can be obtained by a technique such as PCR based on known information. For example, the DNA encoding OVAepi has the nucleotide sequence of nucleotide numbers 607 to 633 of SEQ ID NO: 22. Examples of the DNA encoding Msur57 include a DNA having a base sequence of base numbers 499 to 522 of SEQ ID NO: 26.

  The DNA encoding the MHC class I antigen peptide, the DNA encoding the linker peptide, the DNA encoding the cell adhesion subunit, and the like are linked such that the codon reading frames match, except for the termination codon. DNA ligation can be performed by a general genetic engineering technique such as a method using a restriction enzyme or DNA ligase, or a method using PCR.

The DNA encoding the fusion protein used in the present invention is represented by, for example, SEQ ID NO: 22, 24 or 26.
In addition, the DNA encoding the fusion protein is a DNA that hybridizes under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 22, 24 or 26 as long as it encodes a polypeptide having a cellular immunity inducing effect. You may. "Stringent conditions" refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, two DNAs having high identity, preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more hybridize with each other, but have a lower identity. Conditions under which the two DNAs do not hybridize are listed. For example, 2 × SSC (330 mM NaCl, 30 mM citric acid) at 42 ° C., preferably 0.1 × SSC (330 mM NaCl, 30 mM citric acid), 60 ° C.

It is also preferable that the DNA encoding the fusion protein be appropriately modified in codons indicating amino acids constituting the fusion protein so that the translation amount of the hybrid protein increases depending on the host cell that produces the protein.
As a method of codon modification, for example, the method of Kang et al. (Protein Expr Purif. 2004 Nov; 38 (1): 129-35.) Can be referred to. In addition, a method of selecting a codon frequently used in a host cell, selecting a codon having a high GC content, or selecting a codon frequently used in a housekeeping gene of a host cell can be mentioned.

In the DNA construct used in the present invention, preferably, the DNA encoding the fusion protein is operably linked to an enhancer. Here, “expressible” means that when the DNA construct used in the present invention is inserted into a vector containing an appropriate promoter and the vector is introduced into an appropriate host cell, the fusion protein is produced in the host cell. To be done. The term “ligation” is a concept including a case where two DNAs are directly linked and a case where two DNAs are linked via another base sequence.
Examples of the enhancer include a Kozak sequence and a 5′-untranslated region of a plant-derived alcohol dehydrogenase gene. When expressing in a plant or the like, preferably, the DNA encoding the hybrid protein is operably linked to the 5′-untranslated region of a plant-derived alcohol dehydrogenase gene. Methods for obtaining the 5'-untranslated region of a plant-derived alcohol dehydrogenase gene are described in, for example, JP-A-2012-19719 and WO2009 / 133882.

<Recombinant vector>
The recombinant vector used in the present invention is characterized by containing the DNA construct. The recombinant vector used in the present invention only needs to be inserted into the vector so that the DNA encoding the fusion protein can be expressed in a host cell into which the vector is introduced. The vector is not particularly limited as long as it can be replicated in the host cell, and includes, for example, a plasmid vector and a viral vector, and can be appropriately selected according to the host. Further, the vector preferably contains a selection marker such as a drug resistance gene. Commercially available plasmid vectors and virus vectors can be used.

  The promoter used in the vector can be appropriately selected according to the host cell into which the vector is introduced. When expressed in plant cells, for example, cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810), rice actin promoter (Zhang et al. 1991 Plant Cell 3: 1155), corn ubiquitin promoter (Cornejo et al.) al. 1993 Plant Mol. Biol. 23: 567) and the like are preferably used. The terminator used in the vector can also be appropriately selected according to the host cell into which the vector is to be introduced. When expressed in plant cells, for example, a nopaline synthase gene transcription terminator, a cauliflower mosaic virus 35S terminator, an Arabidopsis heat shock protein 18.2 gene terminator (HSP-T) and the like are preferably used.

  The recombinant vector used in the present invention can be prepared, for example, by cutting the DNA construct with an appropriate restriction enzyme or adding a restriction enzyme site by PCR and inserting the DNA construct into a restriction enzyme site or a multiple cloning site of the vector. .

<Transformant>
A first aspect of the transformant of the present invention is a transformant transformed with the recombinant vector.

On the other hand, a second embodiment of the transformant of the present invention is an embodiment in which a DNA encoding the first component and a DNA encoding the second component are separately introduced.
That is, a bacterial toxin consisting of a cell adhesion subunit pentamer involved in attachment to a host cell and a toxic subunit monomer which is a toxin main body, wherein a polysaccharide obtained by linking 2 to 5 cell adhesion subunits. A first DNA construct comprising DNA encoding a first component comprising a peptide,
And a second DNA construct containing a DNA encoding the second component containing the MHC class I antigen peptide.
The first DNA construct containing the DNA encoding the first component can be constructed based on the DNA sequence encoding each of the toxin cell attachment subunits described above. DNA constructs having sequences.
The second DNA construct containing the DNA encoding the second component can be constructed based on the DNA sequence encoding the amino acid sequence containing each MHC class I antigen peptide described above.
In order to obtain the transformant according to the second embodiment, usually, the first DNA construct and the second DNA construct are introduced into host cells using separate vectors, but the first DNA construct and the second DNA construct And may be introduced into host cells using a single vector capable of expressing them independently.

The host cell used for transformation may be either a eukaryotic cell or a prokaryotic cell.
Eukaryotic cells, mammalian cells, yeast cells, or in insect cells, plant cells are preferably used, among others Lactuca (Lactuca) Asteraceae such as (Asteraceae), Solanaceae, Brassicaceae, the Chenopodiaceae The cells of the plants to which they belong are preferably used. Furthermore, plant cells belonging to the genus Lactuca , particularly lettuce ( Lactuca sativa ) cells, are preferably used. When a lettuce cell is used as a host cell, a cauliflower mosaic virus 35S RNA promoter or the like can be used as a vector.
Prokaryotic cells are not particularly limited. For example, Escherichia coli
), Agrobacterium ( Agrobacterium tumefaciens ) and the like.
Although there is no particular limitation on the mammalian cells, for example, Chinese hamster oocyte (CHO) cells and the like are used.
There are no particular limitations on the yeast cell, e.g., Saccharomyces cerevisiae (Saccharomyces cerevisiae) or the like is used.

  The transformant used in the present invention can be prepared by introducing a vector used in the present invention into a host cell using a general genetic engineering technique. For example, an introduction method using Acrobacterium (Hood, et al., 1993, Transgenic, Res. 2: 218, Hiei, et al., 1994 Plant J. 6: 271), an electroporation method (Tada, et al.) Appl. Genet, 80: 475), polyethylene glycol method (Lazzeri, et al., 1991, Theor. Appl. Genet. 81: 437), particle gun method (Sanford, et al., 1987). , J. Part. Sci. Tech. 5:27), polycation method (Ohtsuki, et al., FEBS Lett. 1998 May 29; 428 (3): 235-40.). is there.

After introducing the recombinant vector into a host cell, the transformant can be selected according to the phenotype of the selectable marker. Further, by culturing the selected transformant, the first component, the second component, or a fusion protein thereof can be produced. The medium and conditions used for the culture can be appropriately selected depending on the type of the transformant.
Further, when the host cell is a plant cell, the plant can be regenerated by culturing the selected plant cell according to a conventional method, and the first constituent element inside the plant cell or outside the cell membrane of the plant cell, The second component, or their fusion protein, can accumulate. For example, depending on the type of plant cell, potatoes can be selected from Visser et al. (Theor. Appl. Genet.
78: 594 (1989)), and for tobacco, the method of Nagata and Takebe (Planta 99:12 (1971)).

The first component, the second component, and a fusion protein thereof can be purified from a transformant according to a conventional method.
However, purification is not essential, and the transformant can be used as it is or after processing such as drying and grinding.

<Medicine>
Since the complex of the present invention has an action of inducing cell-mediated immunity, it can be used as an active ingredient of a medicine. Note that the first component and the second component are not essential to be administered simultaneously, and may be administered sequentially, so that the medicament of the present invention includes the first component and the second component, An embodiment is also included in which either one is administered first to form a complex in vivo.

  The first component, the second component, or a fusion protein thereof may be used as a pharmaceutical as it is, or may be used as a pharmaceutical composition by being mixed with a carrier or an additive used for the pharmaceutical. Here, examples of the carrier include pharmacologically acceptable solvents, diluents, excipients, binders, and solvents. For preparing a liquid pharmaceutical composition, for example, water, saline, A buffer is used. Additives include stabilizers, pH adjusters, thickeners, antioxidants, isotonic agents, buffers, dissolution aids, suspending agents, preservatives, antifreezing agents, cryoprotectants, and lyophilization Protective agents, bacteriostatic agents and the like are exemplified.

The dosage form of the medicament of the present invention is exemplified by liquid preparations, tablets, ointments, granules and the like, but liquid preparations are preferable. Liquid pharmaceutical compositions obtained by suspending or dissolving in physiological saline, buffers, vegetable oils and the like, and adding additives such as stabilizers as necessary are preferred.
The concentration of the first component, the second component, or a fusion protein thereof in the medicament of the present invention is not particularly limited as long as it can exert a cell-mediated immunity-inducing effect, and for example, is 0.1 mg / mL to 10 mg / mL, respectively. It is.

INDUSTRIAL APPLICABILITY The pharmaceutical composition of the present invention can induce cell-mediated immunity, particularly CTL, in response to a peptide, and can be used as a pharmaceutical composition for treatment or prevention of cancer and viral diseases.
The type of cancer or viral disease to be treated or prevented is not particularly limited, and an antigen peptide can be selected according to the cancer or virus to be treated.

Subjects to be administered include humans and non-human mammals such as mice, rats, pigs, cows, chickens, sheep, goats, dogs and cats, and fish.
The administration method is not particularly limited, and includes oral administration, intravenous administration, local administration, and the like, and it is preferable to locally administer by injection or the like to a disease site. The dose is appropriately adjusted depending on the type, symptom, degree of the disease, age, sex, body weight, etc. of the patient. For example, a single dose is 0.1 mg to 100 mg per kg of body weight. The administration may be a single administration or a plurality of administrations, but a plurality of administrations is preferable, and for example, a method of administering a total of two to three times every 1 to 7 days can be mentioned.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the aspects of the examples.

<Gene design and preparation and purification of fusion protein>
(1) Gene Design LTB, LTB-Stx2eB in which LTB and Stx2eB were fused, or Stx2eB-Stx2eB in which Stx2eB was double-linked were used as a backbone for fusing the vaccine antigen. To the C-terminal of each skeleton, TCE (OVAepi or Msur57), a His tag for purification, and an endoplasmic reticulum residue signal (KDEL: SEQ ID NO: 9) for increasing the efficiency of transport to the endoplasmic reticulum in mammalian cells were added. As a recombinant protein expression vector, pET28a (Novagen) capable of inducing transcription by IPTG was used.

First, His tag and KDEL sequence were added based on the skeleton of LTB alone. Specifically, first, the L + expression plasmid of Patent Document (PCT / JP2014 / 081114) was cut with BglII and SacI. PG12-6His-F (5'-cctggttctggtcctggttctcct agatct ACC ACC TCC ACT AGT cat cac cat cac cat cac cat
After annealing aaa gat gaa-3 ′) (SEQ ID NO: 10) and pET-SacI-KDEL-R (5′-ggagagggttagggataggcttaccttcaagcttcgaatt gagctcta tta caa ttc atc ttt atg gtg atg gtg atg gtg atg-3 ′) (SEQ ID NO: 11), An extension reaction was performed using T4 DNA polymerase to prepare a His-KDEL fragment. The BglII-SacI-treated plasmid described above and the His-KDEL fragment were mixed, and homologous recombination was performed using GeneArt Seamless PLUS Cloning and Assembly Kit (Invitrogen) to construct LTB-His. On the other hand, a Stx2eB-His was constructed by introducing a BamHI-BglII fragment encoding Stx2eB into a gap obtained by treating LTB-His with BamHI-BglII.

Fusion of ovalbumin MHC class I epitope (OVAepi) (ESIINFEKL) (SEQ ID NO: 12) was performed as follows. OVAepi-F (5'-ggtcctggttctcctagatca GGT ACC ACC gag tct atc atc aac ttc gag aag ttg-3 ', KpnI underlined) (SEQ ID NO: 13) and OVAepi-R (5'-gtg atg gtg atg gtg atg act agt caa ctt) ctc gaa gtt gat gat-3 ′, SpeI underlined) (SEQ ID NO: 14) was annealed, and an extension reaction was performed using T4 DNA polymerase to prepare a DNA fragment corresponding to PG12-OVAepi-His. Next, the vector obtained by treating LTB-His or Stx2eB-His with BglII and SpeI and each PG12-OVAepi-His fragment were mixed, and GeneArt Seamless PLUS
The OVAepi-His-KDEL fragment was fused to LTB or Stx2eB by performing a homologous recombination reaction using a Cloning and Assembly Kit (Thermo Fisher Scientific).

  The skeleton was tandemized as follows. Stx2eB-OVAepi-His or Stx2eB-His was treated with XbaI-BamHI, and the XbaI-BglII fragment of LTB or Stx2eB was ligated to LTB-Stx2eB-OVAepi-His (SEQ ID NO: 23), Stx2eB-Stx2eB-OVAepi. -His (SEQ ID NO: 25) was prepared. Each of the resulting vectors was inserted into pET28a after adding NcoI site on the 5 'side and XhoI site on the 3' side.

The cancer antigen fusion protein was prepared as follows. Mouse Survivin 57-64 (Msur57) (CFFCFKEL) (SEQ ID NO: 15) was extended with T4 DNA polymerase after annealing the following F and R primers. The obtained fragment was treated with KpnI and SpeI, and replaced with OVAepi of Stx2eB-Stx2eB-OVAepi-His (Stx2eB-Stx2eB-Sur57) (SEQ ID NO: 27).
Msur57-F: 5'-tcctagatca GGT ACC ACC tgt ttt ttc tgt ttc aag gaa ctt-3 ', KpnI underlined (SEQ ID NO: 16)
Msur57 -R: 5'-gtg atg act agt aagttccttgaaacagaaaaaaca-3 ', SpeI underlined (SEQ ID NO: 17)

(2) Expression of recombinant protein using Escherichia coli The obtained expression plasmid was transformed into BL21 (DE3) strain, which is an Escherichia coli strain for protein expression. A colony of Escherichia coli having the recombinant plasmid was inoculated into four test tubes containing 5 ml of 2 × YT medium containing 100 mg / L ampicillin, and cultured overnight at 180 rpm at 37 ° C. 20 ml of this preculture was inoculated into 1 L of 2 × YT medium containing 100 mg / L ampicillin, and cultured at 180 rpm at 37 ° C. until the OD600 reached about 0.4. IPTG having a final concentration of 1 mM was added, and the cells were cultured at 22 ° C. and 120 rpm for 4 hours. The culture was centrifuged at 8,000 rpm for 5 minutes, and the obtained cells were stored at -80 ° C until use. LTB-Stx2eB-OVA was cultured at 10 ° C and 120 rpm for 12 hours after IPTG induction in order to suppress protein degradation during culture.

(3) Purification by affinity column Purification was performed using nickel-immobilized beads. For extraction of proteins from E. coli, PBS pH 7.4 containing 8M Urea was used.

<ELISPOT (Enzyme-Linked ImmunoSpot) test (1)-Stimulation by administering OVAepi and LTB-Stx2eB or Stx2eB-Stx2eB>
C57BL / 6 J female mice (Japanese SLC, 5 weeks old) were introduced into a laboratory maintained at 25 ° C., and then fed a basal diet for 7 days. After acclimation, the animals were randomly divided into groups, and each group was housed in a plastic cage.
At the start of the test, the antigen purified from E. coli was suspended in a predetermined volume of physiological saline, and a predetermined amount of the sample shown in Table 1 was intraperitoneally administered (first time). In the group to which the existing adjuvant, Freund's complete adjuvant (FCA) was added, a predetermined amount of purified antigen was injected into an equal volume of FCA at the time of the first immunization, and then a well-suspended emulsion was injected into the abdominal cavity. After normal breeding under free-feeding and free-drinking conditions for 2 weeks, a predetermined amount of the purified antigen was again administered subcutaneously (second time). After breeding normally for another week, a predetermined amount of the purified antigen was again administered subcutaneously (third time). After normal breeding for one week, an autopsy of the whole head was performed, and a spleen was collected.
Immediately after collection, the spleen was immersed in PRMI 1640 medium and cooled on ice. After preparing a cell suspension with a cell strainer, erythrocytes were removed using an ACK lysing buffer (Thermo Fisher Scientific). After washing twice with HBSS (Hank's balanced salt solution), the cells were suspended in PRMI 1640 medium, and the number of cells was counted. The obtained splenocytes were seeded on an IFN-γ ELISPOT kit (MABTECH). The method conformed to the instruction manual of MABTECH. The antigen was added / removed 3 times for each individual, and the OVAepi antigen was added at a concentration of 5 μg / mL in all cases.

Test results As a result, as shown in Table 2 and FIG. 1, compared with LTB, by immunizing a mixture of LTB-Stx2eB (SEQ ID NO: 19) or Stx2eB-Stx2eB (SEQ ID NO: 21) with the OVAepi antigen, It was found that cytotoxic T cells (killer T cells) specific to OVAepi antigen were strongly induced. It was also found that when Stx2eB-Stx2eB was mixed, the induction was even stronger.

<ELISPOT test (2)-LTB-Stx2eB-OVAepi or Stx2eB-Stx2eB-OVAepi administration and stimulation>
A predetermined amount of the sample shown in Table 3 was intraperitoneally administered in the same manner as in the above-mentioned Example, and an ELISPOT test was performed by stimulating OVAepi antigen using the removed spleen cells. Here, a fusion protein in which LTB-Stx2eB and Stx2eB-Stx2eB and the OVAepi antigen were linked by a peptide linker was used.

Test results As a result, as shown in Table 4 and FIG. 2, by immunizing LTB-Stx2eB or Stx2eB-Stx2eB with a fusion protein obtained by linking OVAepi antigen with a peptide linker, strongly OVAepi antigen-specific cytotoxic T cells (Killer T cells) were found to be induced.

<ELISPOT test (3)-stimulation by administration of Stx2eB-Stx2eB-Sur57>
The model antigen (OVAepi) used in the above examples was changed to the Sur57 peptide (57-64) of Survivin (survivin), which is a cancer-specific antigen, and a fusion protein (Stx2eB-) linked to Stx2eB-Stx2eB and a peptide linker. Stx2eB-Sur57) was prepared, and an ELISPOT test was performed in the same manner as in the above-mentioned example. The indicated cancer cells were added to the removed spleen cells as stimulants in an amount of 1/100 of the number of spleen cells.

Test Results As a result, as shown in Table 6 and FIG. 3, induction of Sur57 antigen-specific cytotoxic T cells (killer T cells) was confirmed for the stimulation of all the cancer cells used.

<Sur57 antigen-specific T cell cytotoxic activity>
A cell suspension of splenocytes obtained by immunizing a mouse with a fusion protein (Stx2eB-Stx2eB-Sur57) in which the Sur57 peptide (57-64) described in the above example was linked to Stx2eB-Stx2eB by a peptide linker was used to prepare Inoculated into a cm ² culture flask (cultured at 20 mL per flask). One repetition was performed for each individual, and the antigen (Sur57) was added to all flasks to a final concentration of 2 μg / mL. The medium was RPMI 1640 medium containing 10% FBS (containing penicillin / streptomycin), and culturing was performed at 37 ° C. in a 5% CO ₂ atmosphere. Two days after the start of the culture, 10 mL was collected from the flask, and 10 mL of the RPMI medium containing 100 U / mL Human IL-2 was added to continue the culture.
After the erythrocytes were removed from the spleen cells after 6 days of culture using ACK lysing buffer, the cells were washed twice with PBS, the cells were suspended in PBS, and the number of cells was counted (used as effector cells). On the other hand, 10 μL of 1 mM Dioc18 (staining the cell membrane) was added to 1 mL of the YAC-1 cell (lymphoma cell) suspension 2-3 days after the subculture, followed by staining at 37 ° C. for 15 minutes. After washing the YAC-1 cells three times with 1 mL of HBSS, the number of cells was counted. The above effector cells were mixed with YOC-1 cells fluorescently labeled with DiOC18 at a ratio of 5: 1 and 25: 1 (number of effector cells: number of YAC-1 cells) and incubated at 37 ° C. for 4 hours in a CO ₂ incubator. Cultured. After completion of the culture, 2 μL of Propidium iodide (PI: 2 mg / mL) was added to each well, and dead cells were stained. Immediately after staining, BD Biosciences
Analysis was performed on an Accuri C6 flow cytometer. The culture and analysis were performed in duplicate. The YAC-1 cell population (DiOC18 positive) was gated, the YAC-1 cell population was set to acquire 1000 events, and FACS analysis was performed. Cell death count of a sample in which YAC-1 cells and effector cells were co-cultured, cell death count of a sample in which YAC-1 cells were cultured alone (natural cell death count), and cells of a sample in which saponin was added to YAC-1 cells The number of dead cells (maximum cell death number) was calculated, and the CTL activity was calculated.

Test Results As a result, as shown in FIG. 4, by immunizing mice with the Sur57 peptide fusion protein (Stx2eB-Stx2eB-Sur57), the spleen cells have strong cytotoxic activity against lymphoma cells (YAC-1) It was shown that killer T cells were induced.

Claims (19)

A bacterial toxin consisting of a pentamer of a cell adhesion subunit involved in adhesion to a host cell and a monomer of a toxic subunit, which is a toxin body, is obtained by linking a polypeptide obtained by linking 2 to 5 cell adhesion subunits. A first component comprising:
A second component comprising an MHC class I antigen peptide.   The complex according to claim 1, wherein the MHC class I antigen peptide is at least one selected from the group consisting of an ovalbumin MHC class I antigen peptide, a cancer antigen peptide, and a virus antigen peptide.   The cell adhesion subunit of the bacterial toxin is a cell adhesion subunit of one or more toxins selected from Shiga toxin, cholera toxin, Escherichia coli heat-labile toxin, pertussis toxin and subtilase cytotoxin. 3. The complex according to 1 or 2.   The complex according to any one of claims 1 to 3, wherein the cell adhesion subunit of the bacterial toxin comprises at least the B subunit of Shiga toxin 2e.   The complex according to any one of claims 1 to 4, wherein the cell adhesion subunit of the bacterial toxin comprises only the B subunit of Shiga toxin 2e.   The said 1st component WHEREIN: The said 2-5 cell adhesion subunits were mutually connected via the respectively independent peptide linker which consists of 10-30 amino acid residues, The Claims 1-5 characterized by the above-mentioned. The conjugate according to any one of the above.   The composite according to any one of claims 1 to 6, wherein the first component and the second component are included as a composition.   The complex according to any one of claims 1 to 6, wherein the first component and the second component are included as a fusion protein.   9. The fusion protein according to claim 8, wherein the fusion protein is a fusion protein in which the first component and the second component are connected via a peptide linker consisting of 10 to 30 amino acid residues. Complex.   A bacterial toxin consisting of a cell adhesion subunit pentamer involved in adhesion to a host cell and a toxic subunit monomer which is a toxin main body, and a polypeptide in which 2 to 5 cell adhesion subunits are linked to each other; And a MHC class I antigen peptide.   The DNA according to claim 10, wherein the fusion protein is a fusion protein in which the polypeptide and the MHC class I antigen peptide are bound via a peptide linker consisting of 12 to 30 amino acid residues. Construct.   A transformant into which the DNA construct according to claim 10 or 11 has been introduced. A bacterial toxin consisting of a pentamer of a cell adhesion subunit involved in adhesion to a host cell and a monomer of a toxic subunit, which is a toxin body, is obtained by linking a polypeptide obtained by linking 2 to 5 cell adhesion subunits. A first DNA construct comprising a DNA encoding a first component comprising:
A second DNA construct containing a DNA encoding a second component containing an MHC class I antigen peptide.   A medicament comprising the complex according to any one of claims 1 to 9. A bacterial toxin consisting of a pentamer of a cell adhesion subunit involved in adhesion to a host cell and a monomer of a toxic subunit, which is a toxin body, is obtained by linking a polypeptide obtained by linking 2 to 5 cell adhesion subunits. Including the first active ingredient,
And a second active ingredient comprising an MHC class I antigen peptide.   The medicament according to claim 14 or 15, which is a cell-mediated immunity inducer.   The medicament according to any one of claims 14 to 16, which is an anticancer agent or an antiviral agent.   The medicament according to any one of claims 14 to 17, which is an injection. A bacterial toxin consisting of a pentamer of a cell adhesion subunit involved in adhesion to a host cell and a monomer of a toxic subunit, which is a toxin body, is obtained by linking a polypeptide obtained by linking 2 to 5 cell adhesion subunits. Including the first active ingredient,
A method for inducing cellular immunity in a non-human mammal, comprising administering a second active ingredient containing an MHC class I antigen peptide to the non-human mammal.