Improved peptide vaccines

Abstract

The present invention addresses the problem of providing a peptide vaccine in which a peptide vaccine has been complexed so as to enable specific delivery to the surface of a prescribed immune cell. The present invention also addresses the problem of providing a method for specifically delivering a peptide vaccine to the surface of a prescribed immune cell.　As a result of intensive investigations, the inventors of the present invention demonstrated that these problems can be solved by providing a peptide vaccine that has been combined with an IgG-binding peptide and can bind with an IgG that characteristically is an agonist for a molecule on the surface of a prescribed immune cell (for example, dendritic cells).

Description

[Technical Field]

[0001] The present invention relates to a new type of cancer peptide vaccine for treating or preventing cancer, more specifically, to a cancer peptide vaccine that can be delivered specifically to immune cells with which the cancer peptide vaccine is to be contacted. [Background technology]

[0002] Cancer peptide vaccines are being investigated as a new cancer treatment method. Cancer peptide vaccine therapy is a cancer treatment method that aims to prevent or treat cancer by activating and amplifying specific immune responses against cancer antigens expressed in cancer cells in the patient's body. The vaccine involves administering antigens made from peptides, which are part of cancer antigen proteins.

[0003] Among these studies, since the 1990s, research on many cancer antigens and the development of vaccines targeting these antigens have been progressing. Some cancer peptide vaccines have been reported to induce cancer-specific immunity at the laboratory level (Non-Patent Document 1: Fifis T. et al., Vaccine. 2004 Nov 25;23(2):258-66). Furthermore, some cancer vaccines have been reported to have exploratory efficacy in early clinical trials, such as immune induction and survival extension (Non-Patent Document 2: Cancer Sci. 2018 Sep;109(9):2660-2669; Non-Patent Document 3: Cancer Sci. 2017 Dec;108(12):2430-2437).

[0004] However, none of these cancer vaccines have been proven to be sufficiently effective (therapeutic effect) in confirmatory clinical trials, and none have yet been approved. One reason for this lack of effectiveness is thought to be the insufficient delivery of the vaccine to immune cells and the insufficient activation of immune cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2016 / 186206 [Patent Document 2] WO2018 / 230257 [Non-patent literature]

[0006] [Non-Patent Document 1] Fifis T. et al., Vaccine. 2004 Nov 25;23(2):258-66 [Non-patent document 2] Cancer Sci. 2018 Sep; 109(9): 2660-2669 [Non-patent document 3] Cancer Sci. 2017 Dec;108(12):2430-2437 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a peptide vaccine that is complexed so that the peptide vaccine can be delivered specifically to the surface of a specific immune cell. Another objective of the present invention is to provide a method for specifically delivering a peptide vaccine to the surface of a specific immune cell. [Means for solving the problem]

[0008] As a result of extensive research, the inventors of the present invention have demonstrated that the above-mentioned problems can be solved by providing a peptide vaccine that is combined with an IgG-binding peptide that can bind to IgG, which is characterized by being an antibody against a molecule on the surface of a specific immune cell (e.g., dendritic cell).

[0009] More specifically, the present application provides the following aspects to solve the above-mentioned problems: [1]: Peptide vaccines combined with IgG-binding peptides; [2]: The peptide vaccine according to [1], wherein the peptide vaccine is selected from the group consisting of cancer vaccines and infectious disease vaccines; [3]: The peptide vaccine according to [2], which is a cancer vaccine selected from the group consisting of cancer-associated antigens, cancer neoantigens, and personalized cancer neoantigens; [4]: The IgG-binding peptide is Xaa1 Xaa2 Xaa3 Cys Xaa4 Xaa5 His Xaa6 Gly Xaa7 Leu Val Trp Cys Xaa8 Xaa9 Xaa10(SEQ ID NO: 1) [In SEQ ID NO: 1, Xaa1, Xaa2, Xaa9, and Xaa10 are each any amino acid other than Cys or are absent, or Xaa1 and Xaa2 together are Arg Arg Gly Pro; each of Xaa3, Xaa4, Xaa5, and Xaa8 is any amino acid other than Cys; Xaa6 is Lys, Cys, Asp, Glu, Arg, Leu, 2-aminosuberic acid, or diaminopropionic acid; Xaa7 is Glu, Asn, or Gln; or Xaa11 Xaa12 Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Xaa13 Ile Xaa14 Ser Ile Arg Asp Asp Cys (SEQ ID NO: 2) [In SEQ ID NO: 2, Xaa11 is selected from the group consisting of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Lys, ornithine, Cys, Asp, Glu, β-Ala, 2-aminosuberic acid, diaminopropionic acid, and NH 2-(PEG)n-CO (n=1 to 50), or is absent; Xaa12 is selected from the group consisting of Lys, ornithine, Cys, Asp, Glu, Phe, 2-aminosuberic acid, and diaminopropionic acid; Xaa13 and Xaa14 are each independently selected from the group consisting of Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Tyr, and Cys. The peptide vaccine according to any one of [1] to [3], selected from the group consisting of: [5]: Xaa4 is selected from the group consisting of Ala, Ser, and Thr; Xaa5 is Tyr or Trp, The peptide vaccine according to any one of [1] to [4]. [6]: The IgG-binding peptide is Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr (SEQ ID NO: 3) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 4) Arg Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser (SEQ ID NO: 5) Gly Pro Arg Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser Phe His (SEQ ID NO: 6) Ser Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 7) Gly Asp Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 8) Gly Pro Ser Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 9) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser Phe His (SEQ ID NO: 10) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr His His (SEQ ID NO: 11) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 12) Ser Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 13) Ser Asp Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 14) Arg Gly Asn Cys Ala Tyr His Xaa6 Gly Gln Leu Val Trp Cys Thr Tyr His (SEQ ID NO: 15) Gly Xaa2 Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Xaa9 His (SEQ ID NO: 16) Arg Arg Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 17) Phe Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 18)、 Gly Phe Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 19), Lys Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 20), Gly Phe Asn Met Gln Cys Gln Lys Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 21), Lys Asn Met Gln Cys Gln Lys Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 22), Phe Asn Met Gln Gln Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp (SEQ ID NO: 23), or Gly Lys Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 24) The peptide vaccine according to [4], which is selected from the group consisting of: [7]: The IgG-binding peptide is Gly Pro Asp Cys Ala Tyr His Arg Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 25) Gly Pro Asp Cys Ala Tyr His Lys Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 26) Gly Pro Asp Cys Ala Trp His Arg Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 39) Gly Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 40) [6] The peptide vaccine according to [6], [8]: The peptide vaccine according to any one of [1] to [7], in which IgG is bound to an IgG-binding peptide; [9]: The peptide vaccine according to [8], wherein the IgG is an antibody against a target substance characteristic of immune cells that deliver the peptide vaccine;

[10] : The peptide vaccine according to [9], wherein the immune cells are selected from the group consisting of antigen-presenting cells, dendritic cells, and B cells;

[11] : The peptide vaccine according to any one of [8] to

[10] , wherein the IgG is selected from the group consisting of an anti-CD40 antibody, an anti-PD-L1 antibody, an anti-DEC205 antibody, an anti-DCIR antibody, an anti-Mannose receptor antibody, an anti-DC-SIGN antibody, an anti-CD11c antibody, and an anti-Dectin-1 antibody;

[12] : A method of delivering peptide vaccines to cells that have IgG-targeted molecules on their cell surface using IgG, IgG-binding peptides, or peptide vaccines combined with IgG-binding peptides;

[13] : The method according to

[12] , wherein the peptide vaccine is selected from the group consisting of cancer vaccines and infectious disease vaccines;

[14] : The method according to

[13] , wherein the peptide vaccine is a cancer vaccine selected from the group consisting of cancer-associated antigens, cancer neoantigens, and cancer-individualized neoantigens;

[15] : The IgG-binding peptide is Xaa1 Xaa2 Xaa3 Cys Xaa4 Xaa5 His Xaa6 Gly Xaa7 Leu Val Trp Cys Xaa8 Xaa9 Xaa10(SEQ ID NO: 1) [In SEQ ID NO: 1, Xaa1, Xaa2, Xaa9, and Xaa10 are each any amino acid other than Cys or are absent, or Xaa1 and Xaa2 together are Arg Arg Gly Pro; each of Xaa3, Xaa4, Xaa5, and Xaa8 is any amino acid other than Cys; Xaa6 is Lys, Cys, Asp, Glu, Arg, Leu, 2-aminosuberic acid, or diaminopropionic acid; Xaa7 is Glu, Asn, or Gln; or Xaa11 Xaa12 Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Xaa13 Ile Xaa14 Ser Ile Arg Asp Asp Cys (SEQ ID NO: 2) [In SEQ ID NO: 2, Xaa11 is selected from the group consisting of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Lys, ornithine, Cys, Asp, Glu, β-Ala, 2-aminosuberic acid, diaminopropionic acid, and NH 2-(PEG)n-CO (n=1 to 50), or is absent; Xaa12 is selected from the group consisting of Lys, ornithine, Cys, Asp, Glu, Phe, 2-aminosuberic acid, and diaminopropionic acid; Xaa13 and Xaa14 are each independently selected from the group consisting of Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Tyr, and Cys. The method according to any one of

[12] to

[14] , selected from the group consisting of:

[16] : Xaa4 is selected from the group consisting of Ala, Ser, and Thr; Xaa5 is Tyr or Trp, The peptide vaccine according to any one of

[12] to

[15] .

[17] : The IgG-binding peptide is Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr (SEQ ID NO: 3) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 4) Arg Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser (SEQ ID NO: 5) Gly Pro Arg Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser Phe His (SEQ ID NO: 6) Ser Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 7) Gly Asp Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 8) Gly Pro Ser Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 9) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser Phe His (SEQ ID NO: 10) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr His His (SEQ ID NO: 11) Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 12) Ser Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 13) Ser Asp Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 14) Arg Gly Asn Cys Ala Tyr His Xaa6 Gly Gln Leu Val Trp Cys Thr Tyr His (SEQ ID NO: 15) Gly Xaa2 Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Xaa9 His (SEQ ID NO: 16) Arg Arg Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 17) Phe Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 18)、 Gly Phe Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 19), Lys Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 20), Gly Phe Asn Met Gln Cys Gln Lys Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 21), Lys Asn Met Gln Cys Gln Lys Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 22), Phe Asn Met Gln Gln Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp (SEQ ID NO: 23), or Gly Lys Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 24) The method according to

[15] , selected from the group consisting of:

[18] : The IgG-binding peptide is Gly Pro Asp Cys Ala Tyr His Arg Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 25) or Gly Pro Asp Cys Ala Tyr His Lys Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 26) Gly Pro Asp Cys Ala Trp His Arg Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 39) Gly Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 40)

[17] , the method described in

[17] ;

[19] : The method according to any one of

[12] to

[18] , wherein the IgG is an antibody against a target substance characteristic of immune cells that deliver the peptide vaccine;

[20] : The method according to

[19] , wherein the immune cells are selected from the group consisting of antigen-presenting cells, dendritic cells, and B cells;

[21] : The method according to

[19] or

[20] , wherein the IgG is selected from the group consisting of an anti-CD40 antibody, an anti-PD-L1 antibody, an anti-DEC205 antibody, an anti-DCIR antibody, an anti-Mannose receptor antibody, an anti-DC-SIGN antibody, an anti-CD11c antibody, and an anti-Dectin-1 antibody. [Effects of the Invention]

[0010] The peptide vaccines disclosed in the present invention combined with IgG-binding peptides can efficiently deliver the peptide vaccine to the surface of specific immune cells (e.g., dendritic cells), enhancing their activation and enhancing the efficacy of the peptide vaccine. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing a peptide vaccine combined with an IgG-binding peptide. [Figure 2] FIG. 2 is a schematic diagram showing a complex (peptide vaccine-IgG complex) in which a peptide vaccine combined with an IgG-binding peptide is bound to IgG via the IgG-binding peptide moiety. [Figure 3] FIG. 3 shows the results of an in vivo analysis of the affinity of peptides for IgG, in which the affinity of H-2Kb-restricted OVA-derived peptides having various IgG-binding peptides prepared in Example 3 for IgG was examined. [Figure 4] FIG. 4 shows the results of an in vitro immune induction test (whole PBMC method) in which the antibody-peptide vaccine complexes (covalent and non-covalent types) prepared in Example 3 were examined in vitro to determine whether they have the ability to induce immunity. [Figure 5] FIG. 5 shows the results of an in vitro immune induction test (Monocyte derived dendritic cell (MDDC) method) in which the antibody-peptide vaccine complexes (covalent and non-covalent bond types) prepared in Example 3 were examined in vitro to determine whether they have the ability to induce immunity. [Figure 6] FIG. 6 shows the results of an in vivo evaluation of peptide uptake into dendritic cells in wild-type mice, investigating whether antibody-peptide complexes are taken up into dendritic cells when the antibody-peptide complexes are subcutaneously administered using the peptide vaccine prepared in Example 3. [Figure 7] FIG. 7 shows the schedule of an immunization induction test in wild-type mice. [Figure 8] FIG. 8 shows the results of an in vivo immune induction test in wild-type mice to examine whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has the ability to induce immunity. [Figure 9] FIG. 9 shows the results of an in vivo immune induction test in wild-type mice (a comparative test of antibody-vaccine mixture ratios) to examine whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has the ability to induce immunity. [Figure 10] FIG. 10 shows the results of an in vivo immune induction test (dose-dependent test) in wild-type mice, which investigated whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has the ability to induce immunity. [Figure 11] 11 shows the results of an in vivo test in tumor-bearing mice to examine whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has an antitumor effect. In this figure, the antibody:antigen peptide-IgG conjugate peptide group showed a high tumor growth inhibitory effect in vivo. [Figure 12] 12 shows the results of an in vivo test in tumor-bearing mice to examine whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has an antitumor effect. In this figure, the antibody:antigen peptide-IgG conjugated peptide group showed a significant effect of extending survival time. [Figure 13] FIG. 13 shows the schedule of an immunization induction test in wild-type mice. [Figure 14] 14 shows the results of an immune induction test in wild-type mice (neoantigen model), which was conducted using a test system with higher clinical applicability to examine whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has the ability to induce immunity. In this figure, the antibody:antigen-IgG bound peptide group showed the highest immune induction effect. [Figure 15] FIG. 15 shows the schedule of an immunization induction test in wild-type mice. [Figure 16]Figure 16 shows the results of an in vivo immune induction test in wild-type mice, in which the peptide vaccine prepared in Example 3 was used to examine whether antibody-peptide vaccine conjugates (non-covalently bound) combined with a CD40 antibody or a PD-L1 antibody have the ability to induce immunity. [Figure 17] Figure 17 shows the results of an in vivo immune induction test in wild-type mice, in which the peptide vaccine prepared in Example 3 was used to examine whether the antibody-peptide vaccine conjugate (non-covalently bound) combined with a CD40 antibody or a PD-L1 antibody has the ability to induce immunity. [Figure 18] Figure 18 shows the results of an in vivo study in tumor-bearing mice to examine whether antibody-peptide vaccine conjugates (non-covalent bond type) prepared by combining the peptide vaccine prepared in Example 3 with an anti-PD-L1 antibody have antitumor effects. In this figure, the antibody:antigen peptide-IgG conjugate group showed a high tumor growth inhibitory effect in vivo. [Figure 19] 19 shows the results of an in vivo study in tumor-bearing mice to examine whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has an antitumor effect. In this figure, the antibody:antigen peptide-IgG conjugated peptide group showed a significant effect of extending survival time. [Figure 20] FIG. 20 shows the schedule of an immunization induction test in wild-type mice. [Figure 21] Figure 21 shows the results of an immune induction test in wild-type mice in vivo to examine whether antibody-peptide vaccine conjugates (non-covalently bound) containing non-synonymous somatic mutant sequences of mouse tumor cell lines have antitumor effects. DETAILED DESCRIPTION OF THE INVENTION

[0012] The terms used in the present invention are defined as follows: (a) Cancer vaccine: A therapeutic drug that eliminates cancer by administering all or part of a protein (cancer antigen) that is expressed only in cancer cells or whose expression is enhanced in cancer cells; (b) Dendritic cells: immune cells that initially recognize foreign substances such as cancer antigens in the induction of the immune system; (c) T cells: immune cells that are endowed with the ability to specifically kill cancer cells based on information about cancer antigens recognized by dendritic cells; (d) CD40: A protein expressed on the surface of antigen-presenting cells, including dendritic cells. Binding of agonist molecules such as ligands can enhance the function of antigen-presenting cells. (e) Antibody vaccine: A complex of vaccine and antibody.

[0013] <Peptide vaccine> In one embodiment, the present invention discloses a peptide vaccine combined with an IgG-binding peptide (Figure 1). In this embodiment, the peptide vaccine can be used as a vaccine against pathogens, a vaccine against cancer (cancer vaccine), or the like, as long as it uses an antigenic peptide, regardless of the origin of the antigen. When using a vaccine against a pathogen as a peptide vaccine, antigenic peptides of pathogens such as HIV vaccine, HBV vaccine, and RSV vaccine can be used. When using a cancer vaccine as a peptide vaccine, antigenic peptides specific to cancers such as brain tumor, myeloma, gastric cancer, colorectal cancer, uterine cancer, head and neck cancer, gastric cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, splenomegaly, ovarian cancer, prostate cancer, and melanoma can be used. In general, antigenic peptides derived from cancer antigens that are considered suitable for immunotherapy can be used as antigens for cancer vaccines. For example, -Those expressed in certain tumor cells but only in the testis among normal tissues (NY-ESO-1, MAGEA1-4, PRAME, SSX2, CT83, etc.) -Those expressed at much higher levels in tumor cells than in normal cells (WT1, HER2, CEA, MUC1, cyclin B1, EGFR, mesothelin, telomerase, etc.) -expressed in tumor cells and only in a limited number of cell lineages in normal tissues (e.g., MART1, PSA, PSMA, CD19, GP100), - Those that are expressed in normal tissues but are specially modified in tumor cells (MUC1T, LSP1, etc.) - Novel proteins (BCR-ABL1, HRAS, KRAS, etc.) that are generated specifically in diseases due to abnormalities such as gene translocations and point mutations commonly observed in diseases. A group of peptides derived from mutant proteins (neoantigens) that are generated only in tumor cells as a result of gene mutations resulting from genomic instability, which is often observed in tumor cells. You can choose from, etc.

[0014] It is known that peptide vaccines, when administered alone, often fail to induce sufficient immunity. This is thought to be due to the inability to deliver the peptide vaccine to the target immune cells. The peptide vaccine of the present invention aims to overcome this common drawback of peptide vaccines by utilizing IgG to deliver the peptide vaccine to the target immune cells, and is provided in combination with an IgG-binding peptide.

[0015] The IgG-binding peptides used in the present invention can be any known peptide capable of binding IgG to a peptide vaccine peptide, including, but not limited to, those described in WO2016 / 186206 (Patent Document 1) (Type 1) and those described in WO2018 / 230257 (Patent Document 2) (Type 2). In the present invention, these IgG-binding peptides can be provided in combination with a peptide vaccine. IgG-binding peptides that can be used in one aspect of the present invention are: <Type 1> Xaa1 Xaa2 Xaa3 Cys Xaa4 Xaa5 His Xaa6 Gly Xaa7 Leu Val Trp Cys Xaa8 Xaa9 Xaa10(SEQ ID NO: 1) [In SEQ ID NO: 1, Xaa1, Xaa2, Xaa9, and Xaa10 are each any amino acid other than Cys or are absent, or Xaa1 and Xaa2 together are Arg Arg Gly Pro; Each of Xaa3, Xaa4, Xaa5, and Xaa8 is any amino acid other than Cys, and more preferably, Xaa4 may be selected from the group consisting of Ala, Ser, and Thr, and Xaa5 may be Tyr or Trp; Xaa6 is Lys, Cys, Asp, Glu, Arg, Leu, 2-aminosuberic acid, or diaminopropionic acid; Xaa7 is Glu, Asn, or Gln] <Type 2> Xaa11 Xaa12 Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Xaa13 Ile Xaa14 Ser Ile Arg Asp Asp Cys (SEQ ID NO: 2) [In SEQ ID NO: 2, Xaa11 is selected from the group consisting of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, Lys, ornithine, Cys, Asp, Glu, β-Ala, 2-aminosuberic acid, diaminopropionic acid, and NH 2-(PEG)n-CO (n=1 to 50), or is absent; Xaa12 is selected from the group consisting of Lys, ornithine, Cys, Asp, Glu, Phe, 2-aminosuberic acid, and diaminopropionic acid; Xaa13 and Xaa14 are each independently selected from the group consisting of Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Tyr, and Cys. These IgG-binding peptides can be used in combination in a peptide vaccine.

[0016] More specifically, IgG-binding peptides that can be used in one embodiment of the present invention include: <Type 1>: Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr (SEQ ID NO: 3); Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 4); Arg Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser (SEQ ID NO: 5); Gly Pro Arg Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser Phe His (SEQ ID NO: 6); Ser Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 7); Gly Asp Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 8); Gly Pro Ser Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 9); Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Ser Phe His (SEQ ID NO: 10); Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr His His (SEQ ID NO: 11); Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 12); Ser Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 13); Ser Asp Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe Tyr (SEQ ID NO: 14); Arg Gly Asn Cys Ala Tyr His Xaa6 Gly Gln Leu Val Trp Cys Thr Tyr His (SEQ ID NO: 15); Gly Xaa2 Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Xaa9 His (SEQ ID NO: 16); Arg Arg Gly Pro Asp Cys Ala Tyr His Xaa6 Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 17); <タイプ2>: Phe Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 18); Gly Phe Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 19); Lys Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 20); Gly Phe Asn Met Gln Cys Gln Lys Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 21); Lys Asn Met Gln Cys Gln Lys Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 22); Phe Asn Met Gln Gln Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp (SEQ ID NO: 23); Gly Lys Asn Met Gln Cys Gln Arg Arg Phe Tyr Glu Ala Leu His Asp Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Arg Ile Arg Ser Ile Arg Asp Asp Cys (SEQ ID NO: 24) is selected from the group consisting of:

[0017] Among these, particularly useful in the present invention are IgG-binding peptides in which Xaa6 is Arg, Lys, or Leu. For example, specific amino acid sequences of such useful IgG-binding peptides include: Gly Pro Asp Cys Ala Tyr His Arg Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 25) or Gly Pro Asp Cys Ala Tyr His Lys Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 26) Gly Pro Asp Cys Ala Trp His Arg Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 39) Gly Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Phe His(SEQ ID NO: 40) of Some examples include:

[0018] The IgG-binding peptides of the present invention are preferably those for binding to human IgG (which may be any IgG subclass, for example, human IgG1, IgG2, IgG3, or IgG4) or humanized IgG, but can also be used for IgG from animals other than humans, such as rats, mice, and rabbits.

[0019] The method of combining an IgG-binding peptide with a peptide vaccine can be any method, and peptide synthesis methods such as conventional liquid-phase synthesis methods and solid-phase synthesis methods, peptide synthesis using an automatic peptide synthesizer, etc. can be used to generate the IgG-binding peptide and the peptide of the peptide vaccine as one continuous peptide for combination, or it can be produced by a method of combining separately synthesized IgG-binding peptide and the peptide of the peptide vaccine via a linker. Alternatively, the peptide may be produced by a genetic recombination method using a nucleic acid encoding the peptide of the present invention. For example, by incorporating DNA encoding the amino acid sequence of one continuous peptide of the IgG-binding peptide and the peptide of the peptide vaccine of the present invention into an expression vector, introducing it into a host cell, and culturing it, the target peptide can also be produced.

[0020] When considering the ease of synthesis, etc., as a method of combination, it is preferable to synthesize the IgG-binding peptide and the peptide of the peptide vaccine as one continuous peptide. When adopting such a method, the target sequence of the peptide vaccine combined with the IgG-binding peptide can be designed and synthesized using a peptide synthesizer (for example, Symphony (registered trademark) X manufactured by Gyros Protein Technologies, etc.). Impurities contained when the peptide is synthesized can be removed by chromatography such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, etc., ammonium sulfate fractionation, ultrafiltration, and immunoabsorption method, etc., and the peptide can be purified. For purification by chromatography, equipment such as high performance liquid chromatography (HPLC) can be used.

[0021] <Complex with IgG> The present invention is characterized by providing a complex in which a peptide vaccine combined with an IgG-binding peptide is bound to IgG via the IgG-binding peptide moiety (Figure 2). The IgG used in the present invention can be selected depending on the immune cells to which the peptide vaccine is to be delivered. In the present invention, IgG intended to bind to target immune cells or IgG intended to bind to target immune cells and activate those immune cells can be used. For example, when dendritic cells are used as the immune cells, anti-CD40 antibody (IgG), anti-PD-L1 antibody (IgG), anti-DEC205 antibody (IgG), anti-DCIR antibody (IgG), anti-Mannose receptor antibody (IgG), anti-DC-SIGN antibody (IgG), anti-CD11c antibody (IgG), anti-Dectin-1 antibody (IgG), etc. can be used. However, other IgGs can also be used depending on the purpose.

[0022] When using a peptide vaccine against a pathogen or a cancer vaccine as a peptide vaccine, it is effective to deliver it to immune cells such as dendritic cells or B cells. Therefore, in the present invention, the IgG used for delivery to each cell can be an IgG known to target a substance characteristic of the immune cells to which the peptide vaccine is delivered. For delivery to dendritic cells, antibodies targeting CD40 (clinical development by J&J, Roche, Seattle Genetics, etc.), DEC205 (clinical development by Celldex), or DCIR (clinical development by Ascend Biopharmaceuticals) can be used.

[0023] In the present invention, binding of IgG to an IgG-binding peptide moiety that can be used to form a conjugate can be achieved by modifying specific amino acid residues in the IgG-binding peptide moiety of the present invention with a cross-linking agent and forming a cross-linked structure between the IgG-binding peptide moiety and specific amino acid residues in IgG Fc via the cross-linking agent. The IgG-binding peptide moiety of a peptide vaccine combined with an IgG-binding peptide of the present invention can be modified with a cross-linking agent by synthesizing the peptide vaccine combined with the IgG-binding peptide using amino acid residues modified with a cross-linking agent, or by synthesizing a peptide vaccine combined with the IgG-binding peptide and then specifically modifying the side chain of a specific amino acid residue in the IgG-binding peptide moiety.

[0024] Crosslinkers that can be used for conjugate formation in the present invention can be appropriately selected by those skilled in the art, and compounds having at least two sites capable of binding to specific amino acid residues in the IgG-binding peptide moiety can be used. Examples of crosslinkers include crosslinkers containing at least one, preferably two or more, succinimidyl groups, such as disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS); crosslinkers containing at least one, preferably two or more, imidic acid moieties, such as dimethyl adipimidate·2HCl (DMA), dimethyl pimelimidate·2HCl (DMP), and dimethyl suberimidate·2HCl (DMS); and dimethyl 3,3′-dithiobispropionimidate dihydrochloride (dimethyl Crosslinkers having an S-S bond in addition to the above-mentioned functional groups, such as 3,3'-dithiobispropionimidate·2HCl (DTBP) or dithiobis(succinimidyl propionate) (DSP) (WO2016 / 186206 (Patent Document 1), WO2018 / 230257 (Patent Document 2)), can be used, but are not limited to these.

[0025] Modification of specific amino acid residues with a crosslinker can be achieved by selecting a combination of the crosslinker and the type of specific amino acid residue (WO 2016 / 186206 (Patent Document 1), WO 2018 / 230257 (Patent Document 2)). For example, when a crosslinker containing a succinimidyl group, such as DSS or DSG, is used, it reacts with the amine (ε-amino group) in the side chain of a lysine residue and the primary amine (α-amino group) present at the N-terminus of a polypeptide. Therefore, by blocking the N-terminus of the IgG-binding peptide moiety and then reacting it with DSS or DSG, only the side chain of the lysine residue located as the specific amino acid residue in the IgG-binding peptide moiety can be specifically modified with DSS or DSG. Such combinations of amino acid residues and crosslinkers can be appropriately selected by those skilled in the art. Furthermore, when DSS or DSG is used, it is also possible to specifically modify the α-amino group in a peptide that does not contain a lysine residue by leaving the primary amine (α-amino group) present at the N-terminus of the polypeptide unmodified.

[0026] In the present invention, the peptide vaccine of the present invention, in which the IgG-binding peptide moiety has been modified with a cross-linking agent, can bind to IgG and form a complex by mixing with IgG. The conditions for this mixing step are not particularly limited, as long as they are conditions under which a cross-linking reaction occurs between the IgG-binding peptide moiety of the present invention modified with a cross-linking agent and the IgG. For example, the cross-linking reaction can be carried out by mixing the peptide vaccine of the present invention, which contains the IgG-binding peptide moiety modified with a cross-linking agent, with IgG in an appropriate buffer at room temperature (e.g., about 15°C to 30°C). If necessary, an appropriate amount of a catalyst that promotes the cross-linking reaction may be added to this mixing step.

[0027] In order to enhance the binding affinity between the IgG-binding peptide moiety and IgG, other reaction conditions in this mixing step may be adjusted as appropriate, taking into consideration the type of IgG and the peptide vaccine of the present invention comprising an IgG-binding peptide moiety modified with a crosslinker. For example, the pH during the reaction may be selected to be between pH 4.5 and 6.5 (e.g., pH 5.0 to 6.0, pH 5.2 to 5.8, pH 5.4 to 5.6, or about pH 5.5), or pH 6.5 to 8.5 (e.g., pH 6.9 to 7.9, pH 7.2 to 7.7, pH 7.3 to 7.5, or about pH 7.4). The mixing ratio of the peptide vaccine of the present invention comprising an IgG-binding peptide moiety modified with a crosslinker to IgG may be, for example, 1:1 to 20:1, 2:1 to 20:1, or 5:1 to 10:1, in molar ratio. The mixing time (reaction time) may be, for example, 1 minute to 5 hours, 10 minutes to 2 hours, or 15 minutes to 1 hour.

[0028] <Uses of the composite> In another aspect, the present invention provides a method for specifically delivering a peptide vaccine to cells that have, on their surface, a target molecule targeted by IgG, using a complex of the peptide vaccine of the present invention combined with the above-mentioned IgG-binding peptide moiety. By selecting immune cells as such desired cells and specifically delivering the peptide vaccine to these immune cells, it is possible to efficiently produce antibodies against the peptide vaccine and induce cellular immunity against the peptide vaccine, such as by inducing activation of T cells against the peptide vaccine.

[0029] In yet another aspect, the present invention provides a vaccine composition for treating or preventing a target disease, comprising a conjugate of the peptide vaccine of the present invention combined with the above-described IgG-binding peptide moiety and IgG. The target disease to be treated or prevented by the vaccine composition of the present invention refers to the target from which the antigen of the peptide vaccine of the present invention is derived, and examples include infectious diseases caused by pathogens and cancer. Furthermore, by selecting an IgG depending on the immune cells to which the peptide vaccine is to be delivered, an immune response appropriate to the immune cells can be induced. Therefore, the vaccine composition of the present invention can be used as a vaccine composition for efficiently inducing antibody production against the peptide vaccine, a vaccine composition for inducing T cell activation against the peptide vaccine, or a vaccine composition for inducing cellular immunity against the peptide vaccine.

[0030] When used for the purpose of treating a target disease, the vaccine composition of the present invention can contain a peptide vaccine against the target disease and IgG corresponding to immune cells that induce a desired immune response in a patient who has developed the target disease. By administering this vaccine composition of the present invention to a patient who has developed the target disease, the target disease can be treated in the patient's body.

[0031] When used for the purpose of preventing a target disease, the vaccine composition of the present invention can contain a peptide vaccine against the target disease and IgG corresponding to immune cells that induce a desired immune response against the target disease. By administering this vaccine composition of the present invention to a subject who has not developed the target disease, an immune response can be induced in the subject's body when the cause of the target disease invades or occurs in the body of the subject.

[0032] Since the vaccine composition of the present invention aims to deliver the peptide vaccine of the present invention to immune cells using IgG, it can be administered parenterally (for example, intravenous injection, intramuscular injection, subcutaneous administration, or intraperitoneal administration), and can be prepared in various formulations as dosage forms appropriate for the administration route. Those skilled in the art can appropriately select the administration method and dosage form depending on the patient's sex, age, weight, symptoms, etc.

[0033] The vaccine composition of the present invention can be formulated, including pharmaceutically acceptable carriers and additives, according to general knowledge in the art. For example, when used as an injectable formulation, the complex of the present invention can be dissolved in a solution containing, for example, physiological saline, a buffer solution, a glucose solution, etc., to which an agent for preventing container adsorption, such as Tween 80, Tween 20, gelatin, or human serum albumin, can be added. Alternatively, the complex can be lyophilized to form a dosage form that can be dissolved and reconstituted before use, and stabilizers for lyophilization can include sugar alcohols and / or saccharides, such as mannitol and glucose.

[0034] The present invention will be specifically illustrated by the following examples, which are not intended to limit the present invention in any way. [Example]

[0035] Example 1: Production of anti-CD40 antibodies In this example, dendritic cells that induce vaccine-specific T cells were selected as immune cells that deliver the peptide vaccine of the present invention, and agonistic antibodies against CD40, which is specific as the surface antigen of the selected cells, were produced with the aim of activating these cells.

[0036] (1) Production of anti-mouse CD40 antibody The anti-mouse CD40 antibody was constructed as a chimeric antibody by fusing the variable region of the FGK45 clone, a known anti-mouse CD40 agonist antibody, with the constant region of a human antibody. The variable region sequences of the FGK45 clone were published on NCBI (https: / / www.ncbi.nlm.nih.gov / ) (heavy chain: AEI27236.1, light chain: AEI27235.1). A plasmid was constructed for expression by linking a light chain expression element (EF-1α promoter, secretion signal, light chain variable region, and light chain constant region in tandem) and a heavy chain expression element (EF-1α promoter, secretion signal, heavy chain variable region, and heavy chain constant region in tandem) to pCI-neo.

[0037] The DNA sequences of the heavy and light chain variable regions were designed based on sequences obtained from the aforementioned databases and codons optimized for the hamster expression system. The amino acid sequences of the heavy and light chain constant regions can be determined from DNA sequences specifying the amino acid sequences of the heavy and light chain constant regions of known human antibodies, or variants thereof. In this example, the amino acid sequences used for the heavy and light chain constant regions were those of a mutant of human IgG1 (IgG1-lala) with L4A and L5A substitutions in the CH2 region and human IgG kappa, respectively.

[0038] ExpiCHO cells (Invitrogen A2910002) were used for antibody expression. The cells were cultured using ExpiCHO Expression Medium (Gibco, A2910002), and the antibody expression plasmid was transfected into ExpiCHO cells. TM After transfection using the Expression System (GIbco A29129), the cells were cultured for 14 days, and the culture supernatant was collected. The antibody was obtained by purifying the culture supernatant using a Protein A column (MonoSpin ProA, GL Science 7510-11314).

[0039] The purity and binding activity of the purified antibody were confirmed by SDS-PAGE and its binding to mouse spleen cells, and it was confirmed that the desired anti-mouse CD40 agonist antibody had been obtained.

[0040] (2) Production of anti-human CD40 antibody The anti-human CD40 antibody was produced as a chimeric antibody by fusing the variable region of the 21.4.1 clone, a known anti-human CD40 agonist antibody, with the constant region of a human antibody. The sequence of the 21.4.1 clone was that published in a patent document (Japanese Patent No. 4616555).

[0041] The vector for expressing the anti-human CD40 antibody and the antibody expression and purification were carried out in the same manner as used for the anti-mouse CD40 antibody described above in (1).

[0042] The purity and binding activity of the purified antibody were confirmed based on SDS-PAGE and its ability to activate targets in CD40-expressing cells, confirming that the desired anti-human CD40 agonist antibody had been obtained.

[0043] Example 2: Peptide production This example was carried out to prepare antigenic peptides against various target substances as examples of the peptide vaccine of the present invention.

[0044] As the vaccine peptide sequence, A 16-mer peptide (EQLESIINFEKLTEWT[peptide(2)], SEQ ID NO: 29) containing the antigen peptide sequence (SIINFEKL[peptide(1)], SEQ ID NO: 28] derived from H-2Kb-restricted OVA, which has been reported to induce antigen-specific T cells in mice (Vaccine. 2004 Nov 25;23(2):258-66). a 16-mer peptide (RQYDPVAALFFFDIDL [peptide (6)], SEQ ID NO: 33) containing the antigenic peptide sequence (PVAALFFF [peptide (5)], SEQ ID NO: 32) derived from human A24:02-restricted CMV (J Transl Med. 2005 May 26;3:23); or A 17-mer peptide (HLELASMTNMELMSSIV[peptide(9)], SEQ ID NO: 36) containing the H-2Db-restricted neoantigen Adpgk-derived peptide sequence (ASMTNMELM[peptide(8)], SEQ ID NO: 35) (Nature. 2014 Nov 27;515(7528):572-6). was used.

[0045] The IgG-binding peptide sequences used were the sequences reported in Patent Document PCT / JP2016 / 065061 (GPDCAYHRGELVWCTFH [IgG BP(1), (SEQ ID NO: 25)] or GPDCAYHKGELVWCTFH [IgG BP(2), (SEQ ID NO: 26)]) and newly created sequences (GPDCAWHRGELVWCTFH [IgG BP(3), (SEQ ID NO: 39)] or GPDCAWHLGELVWCTFH [IgG BP(4), (SEQ ID NO: 40)]). These sequences are the peptides shown in SEQ ID NO: 4, except that Xaa6 is Arg, Lys, or Leu. A negative control IgG-binding peptide sequence (GPDCAYHRGEAAACTFH [IgG BP(NC), SEQ ID NO: 27]) was prepared by partially mutating the IgG-binding peptide. These IgG-binding peptides are characterized by binding to IgG antibodies via non-covalent bonds to form complexes.

[0046] To form noncovalent peptide vaccine-antibody complexes (antibody-peptide vaccine complexes (noncovalent)), we synthesized peptides in which a vaccine peptide was linked in tandem to the N-terminus of an IgG-binding peptide, with two cysteines present in the IgG-binding peptide crosslinked by an SS bond [peptide (3-1) (SEQ ID NO: 30), peptides (7) (SEQ ID NO: 34), and peptide (10) (SEQ ID NO: 37)]. As control peptides, we synthesized peptides in which a vaccine peptide was linked in tandem to the N-terminus of a non-IgG-binding peptide (IgG BP(NC) (SEQ ID NO: 27)) with two cysteines present in the non-IgG-binding peptide sequence crosslinked by an SS bond [peptides (4) (SEQ ID NO: 31) and peptide (11) (SEQ ID NO: 38)].

[0047] These peptides were produced by chemical synthesis. Furthermore, as peptides for forming antibody-peptide vaccine conjugates (covalent bond type), we prepared a DBCO (dibenzylcyclooctyne) derivative peptide [peptide (6D)] in which a DBCO group was attached to the N-terminus of the antigen peptide, and an IgG-binding peptide derivative (IgG-binding peptide-PEG-N3 [peptide (2P)]) in which a PEG linker and an azide group were attached to the N-terminus of the IgG-binding peptide and two cysteines were crosslinked with an S-linkage bond.

[0048] [Table 1]

[0049] Example 3: Preparation of peptide vaccine-antibody conjugates This example was carried out with the aim of preparing a conjugate between a peptide vaccine and an antibody. To prepare the conjugate, a method for non-covalently conjugating an antibody with a peptide vaccine and a method for covalently conjugating the antibody with a peptide vaccine were used.

[0050] Non-covalent peptide vaccine / antibody conjugates (antibody-peptide vaccine conjugates (non-covalent)) were prepared by mixing the peptide vaccines combined with the IgG-binding peptides prepared in Example 2 (peptide (3-1) (SEQ ID NO: 30), peptide (3-2) (SEQ ID NO: 41), peptide (3-3) (SEQ ID NO: 42), peptide (3-4) (SEQ ID NO: 43), or peptide (7) (SEQ ID NO: 34), or peptide (10) (SEQ ID NO: 37)) with the anti-mouse CD40 antibody or anti-human CD40 antibody prepared in Example 1 or a commercially available human PD-L1 antibody (MedChemExpress CAS NO: 1380723-44-3) at room temperature.

[0051] Covalent peptide vaccine / antibody conjugates (antibody-peptide vaccine conjugates (covalent)) were prepared by first preparing DBCO derivative peptide [peptide (6D)] and IgG-binding peptide-PEG-N3 [IgG BP(2P)] at 10 mM and 10.8 mM concentrations, respectively, using DMSO. These were then mixed and incubated at room temperature for 2 hours to produce a fusion peptide of both peptides (CMV-IgG binding peptide). Next, disuccinimidyl glutarate (DSG) dissolved in acetonitrile was added at a molar ratio of 1.4 to the CMV-IgG binding peptide, and the mixture was incubated in the presence of pyridine at 50°C for 4 hours to produce a succinimidylated CMV-IgG binding peptide with the same sequence as peptide (7).

[0052] The peptide was purified using HPLC and then mixed with the anti-human CD40 antibody prepared in Example 1 or an isotype control antibody (human IgG1-lala antibody, an in-house preparation) at a molar ratio of 10:1 and incubated at room temperature for 3 hours. After confirming the formation of an antibody-peptide vaccine complex (covalent bond type) by electrophoresis, small molecules were removed by ultrafiltration.

[0053] Example 4: Analysis of peptide affinity to IgG The purpose of this example was to examine the affinity of H-2Kb-restricted OVA-derived peptides having various IgG-binding peptides, which were prepared in Example 3, for IgG.

[0054] The peptides evaluated were peptide (3-1) (SEQ ID NO: 30), peptide (3-2) (SEQ ID NO: 41), peptide (3-3) (SEQ ID NO: 42), peptide (3-4) (SEQ ID NO: 43), and peptide (4) (negative control, SEQ ID NO: 31).

[0055] The H-2Kb-restricted OVA-derived peptides having the four different IgG-binding peptide sequences were evaluated for their antibody binding activity. The IgG for which affinity was evaluated was the anti-CD40 antibody prepared in Example 1, which was immobilized on a CM5 sensor chip (GE Healthcare).

[0056] Specifically, a BIAcore 8K (GE Healthcare) was used to measure and analyze the peptides in single kinetics mode. Anti-CD40 antibodies were immobilized on a CM5 sensor chip. A three-fold dilution series of each peptide was prepared, ranging from 3 nM to 243 nM. The dissociation constants were calculated using Biacore Insight Evaluation software.

[0057] As a result, the dissociation rate constant (Kd) of peptide (3-3) was confirmed to be similar to that of peptide (3-1), and to have a higher affinity than peptide (3-2). In contrast, peptide (3-4) was confirmed to have an even higher affinity than peptides (3-2) and (3-3). Furthermore, the negative control peptide (4) had such low affinity for the anti-CD40 antibody that the dissociation rate constant and affinity could not be calculated (Figure 3, Table 2).

[0058] [Table 2]

[0059] Example 5: In vitro immune induction test 1 (whole PBMC method) The purpose of this example was to investigate in vitro whether antibody-peptide vaccine complexes (covalent and non-covalent) using the peptide vaccine prepared in Example 3 have the ability to induce immunity.

[0060] Peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers were cultured at 2 × 10 in RPMI 1640 medium (Thermo 61870-0362) containing 10% serum (BioWest, S4190). 6 The cells / mL were adjusted, and 500 μL of each was seeded into a 24-well plate (FALCON, 353047) and cultured at 37°C under 5% CO2 conditions.

[0061] Anti-CD40 antibody and peptide (7) (SEQ ID NO: 34) were adjusted to 20 μg / mL and 1 μg / mL, respectively, in RPMI 1640 medium containing 10% serum and 200 U / mL IL-2 (NIPRO, 87890), and 500 μL of each solution was added to wells seeded with PBMCs (final concentrations of 10 μg / mL and 0.5 μg / mL, respectively) (Group 2).

[0062] The antibody-peptide vaccine complex (covalently bound) consisting of anti-CD40 antibody and CMV peptide was adjusted to 21 μg / ml using RPMI 1640 medium containing the same additives as in Group 2 above, and 500 μL was added to wells seeded with PBMCs (final concentration 10.5 μg / ml) (Group 3).

[0063] As a control, Anti-CD40 antibody was adjusted to 20 μg / mL in RPMI1640 medium containing 10% serum and 200 U / mL IL-2 (NIPRO, 87890), and 500 μL of the antibody was added to each well containing PBMCs (final concentration: 10 μg / mL) (Group 1). An isotype control antibody (human IgG1-lala antibody, self-prepared) and peptide (7) were adjusted to 20 μg / mL and 1 μg / mL, respectively, in RPMI 1640 medium containing 10% serum and 200 U / mL IL-2 (NIPRO, 87890), and then added in 500 μL portions to wells seeded with PBMCs (final concentrations of 10 μg / mL and 0.5 μg / mL, respectively) (Group 4). An antibody-peptide vaccine conjugate (covalently bound) consisting of an isotype control antibody and the DSG-modified peptide prepared in Example 3 was adjusted to 21 μg / ml using RPMI 1640 medium containing the same additives as in Group 2 above, and 500 μl of this was added to wells seeded with PBMCs (final concentration 10.5 μg / ml) (Group 5). We have prepared the following.

[0064] LPS (SIGMA, L2762), R848 (Invivogen, vac-r848), and poly I:C (Invivogen, tlrl-pic) were adjusted to 800 ng / ml, 2 mM, and 4 μg / ml, respectively, in RPMI 1640 medium containing 10% serum and 200 U / mL IL-2, and 330 μl of each was added to wells seeded with PBMCs.

[0065] After 7 days of culture at 37°C and 5% CO2, the cells were harvested and stained with APC-CMV-tetramer (MBL, TS-0020-2C), BV421-anti-human CD3 antibody (Biolegend, 300434), and FITC-anti-human CD8 antibody (Biolegend, 300906), followed by FACS analysis.

[0066] The results are shown in Figure 4. Peptide-specific T cell induction was evaluated based on the percentage of tetramer-positive cells among CD3- and CD8-positive cells. The results showed that both the noncovalently conjugated (Group 2) and covalently conjugated (Group 3) anti-CD40 antibody-peptide vaccine conjugates induced vaccine-specific T cells more efficiently than the isotype control antibody-vaccine conjugates (Groups 4 and 5) (Figure 4).

[0067] Example 6: In vitro immune induction test 2 (Monocyte derived dendritic cell (MDDC) method) This example was conducted to investigate in vitro, using a method different from that used in Example 5, whether antibody-peptide vaccine complexes (covalent and non-covalent) using the peptide vaccine prepared in Example 3 have the ability to induce immunity.

[0068] CD14-positive monocytes were purified from PBMCs obtained from healthy volunteers using CD14 MACS beads (Miltenyl Biotec, 130-050-20110). CD14-negative cells were cryopreserved separately using cell cryopreservation medium (Takara, CB011).

[0069] Purified CD14-positive monocytes were cultured at 1 × 10 in RPMI 1640 medium (Thermo, 61870-036) containing 10% FBS (SIGMA, 172072-500ML), 10 mM HEPES (Nacalai, 17557-94), 50 μM 2-mercaptoethanol (2-ME; Nacalai, 21438-82), 6.5 ng / mL IL-4 (Peprotech, 200-04), and 100 ng / mL GM-CSF (R&D, 215-GM-500). 6 The cells were adjusted to a concentration of 1000 cells / mL, and 1 mL of each was seeded into a 24-well plate (FALCON, 353047) and cultured at 37°C under 5% CO2 conditions.

[0070] Six days after the start of culture, the cells were harvested and diluted to 6 × 10 in RPMI 1640 containing 10% FBS (SIGMA, 172072-500ML), non-essential amino acids (Nacalai, 06344-56), 1 mM sodium pyruvate (Nacalai, 06977-34), 10 mM HEPES (Nacalai, 17557-94), and 50 μM 2ME (Nacalai, 21438-82). 4 The concentration was adjusted to cells / mL, and 50 μL of each was seeded into a 96-well U-bottom plate (FALCON, 353077).

[0071] Anti-human CD40 antibody and peptide (7) (SEQ ID NO: 34) were adjusted to 40 μg / mL and 2 μg / mL, respectively, using the same medium as used to culture monocytes above, and 50 μL of each was added to a 96-well plate seeded with CD14-positive monocytes (final concentrations of 10 μg / mL and 0.5 μg / mL, respectively) (Group 2).

[0072] The antibody-peptide vaccine complex (covalently bound) consisting of anti-CD40 antibody and CMV peptide was adjusted to 42 μg / ml using RPMI 1640 medium containing the same additives as in Group 2 above, and 50 μl of the complex was added to a 96-well plate seeded with CD14-positive monocytes (final concentration 10.5 μg / ml) (Group 3).

[0073] As a control, Anti-human CD40 antibody was adjusted to 40 μg / mL in RPMI1640 medium containing 10% serum and 200 U / mL IL-2 (NIPRO, 87890), and 50 μL of this solution was added to a 96-well plate seeded with CD14-positive monocytes (final concentration: 10 μg / mL) (Group 1). An isotype control antibody (human IgG1-lala antibody, self-prepared) and peptide (7) were adjusted to 40 μg / mL and 2 μg / mL, respectively, in RPMI 1640 medium containing 10% serum and 200 U / mL IL-2 (NIPRO, 87890). 50 μL of each solution was added to a 96-well plate seeded with CD14-positive monocytes (final concentrations of 10 μg / mL and 0.5 μg / mL, respectively) (Group 4). The antibody-peptide vaccine complex (covalently bound) consisting of the isotype control antibody and the DSG-modified peptide prepared in Example 3 was adjusted to 42 μg / ml using RPMI 1640 medium containing the same additives as in Group 2 above, and 50 μl of each was added to a 96-well plate seeded with CD14-positive monocytes (final concentration 10.5 μg / ml) (Group 5). We have prepared the following.

[0074] Furthermore, as in Example 5, LPS, R848, poly I:C, and IL-2 were adjusted to 800 ng / ml, 2 mM, 4 μg / ml, and 80 U / ml, respectively, using the same medium, and then added in 50 μl portions.

[0075] On day 7 of culture at 37°C and 5% CO2, the frozen CD14-negative cells were awake and CD8-positive cells were purified using CD8 MACS beads (Miltenyl Biotec, 130-045-201). The cells were diluted to 1.8 × 10 in RPMI 1640 containing 10% FBS, non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, and 50 μM 2ME. 6 The concentration of the solution was adjusted to cells / mL, and 50 μL of each solution was added and cultured at 37°C. On day 14, the cells were harvested and stained with APC-CMV-tetramer (MBL, TS-0020-2C), BV421-anti-CD3 antibody (Biolegend, 300434), and FITC-anti-CD8 antibody (Biolegend, 300906), followed by FACS analysis.

[0076] The results are shown in Figure 5. Peptide-specific T cell induction was evaluated based on the percentage of tetramer-positive cells among CD3- and CD8-positive cells. The results showed that both the noncovalently conjugated (Group 2) and covalently conjugated (Group 3) anti-CD40 antibody-vaccine conjugates induced vaccine-specific T cells more efficiently than the isotype control antibody-vaccine conjugates (Groups 4 and 5) (Figure 5).

[0077] Example 7: Evaluation of peptide uptake into dendritic cells in wild-type mice The purpose of this example was to investigate in vivo whether antibody-peptide complexes using the peptide vaccine prepared in Example 3 are taken up by dendritic cells (classical dendritic cell 1 / 2, (cDC1 / cDC2)) present in lymph nodes near the administration site when the antibody-peptide complexes are subcutaneously administered.

[0078] In this example, the test peptide was an IgG BP(1) peptide (SEQ ID NO: 25) (total sequence: K*RRK*RRK*RRGPDCAYHRGELVWCTFH) to which a peptide (K*RRK*RRK*RR) labeled with the fluorescent dye FAM (6-Carboxyfluorescein) (K* represents a FAM-bound lysine) was added.

[0079] Seven-week-old female wild-type mice C57BL / 6 (CLEA Japan) were divided into three groups of three mice each, and the experiment was carried out according to the group composition shown in Table 3. Group 1: Adjuvant PolyIC:LC (Oncovir) only; Group 2: A mixture of fluorescently labeled IgG BP(1) peptide (test peptide) and adjuvant PolyIC:LC (Oncovir); Group 3: A mixture of the antibody-peptide vaccine complex (non-covalently bound) of the test peptide and anti-mouse CD40 antibody, and the adjuvant PolyIC:LC (Oncovir) added. The doses of each component were administered subcutaneously in the groin area as shown in Table 3.

[0080] [Table 3]

[0081] Each group of mice received a single dose of the drug. Five hours later, inguinal lymph nodes near the injection site were collected and the tissue was homogenized to prepare lymph node cells, which were then stained with anti-CD11c, anti-IA / IE, anti-XCR1, and anti-CD172a antibodies and subjected to flow cytometry analysis.

[0082] The uptake of fluorescently labeled IgG BP(1) peptide was evaluated based on the percentage of FAM-positive cells in cDC1 cells (CD11c+ / IA / I-E+ / XCR1+ / CD172-) and cDC2 cells (CD11c+ / IA / I-E+ / XCR1- / CD172+).

[0083] The results are shown in Figure 6. Comparisons between Groups 1 and 2 and between Groups 1 and 3 showed that peptide uptake into both cDC1 and cDC2 cells was enhanced in the group administered with the antibody-peptide vaccine complex (non-covalent bond type) (Group 3) compared to the group administered with the peptide vaccine alone without antibody (Group 2) (Figure 6).

[0084] Example 8: Immunity induction test 1 in wild-type mice (comparison test with conventional vaccine regimen) The purpose of this example was to investigate in vivo whether the antibody-peptide vaccine complex (non-covalent bond type) using the peptide vaccine prepared in Example 3 has the ability to induce immunity.

[0085] Seven-week-old female wild-type mice C57BL / 6 (CLEA Japan) were divided into five groups of six mice each, and the experiment was conducted according to the group composition shown in Table 4. Group 1: peptide vaccine (H-2Kb-restricted OVA-derived peptide (3-1) of Example 2, SEQ ID NO: 30) only; Group 2: a mixture of peptide (3-1) and adjuvant PolyIC:LC (Oncovir); Group 3: Antibody-peptide vaccine conjugate (non-covalently bound) consisting of peptide (3-1) and anti-mouse CD40 antibody; Group 4: A mixture of the antibody-peptide vaccine complex (non-covalently bound) of peptide (3-1) and anti-mouse CD40 antibody, and the adjuvant PolyIC:LC (Oncovir) added. Group 5: negative control containing no peptide (3-1), anti-mouse CD40 antibody, or adjuvant; The doses of each component are listed in Table 4.

[0086] The administration schedule is shown in Figure 7. Specifically, mice in each group received subcutaneous administration of the drug twice every seven days (days 0 and 7, with the first administration designated as day 0). On day 14, the spleens were harvested, and splenocytes were prepared by disruption. These cells were then stained with anti-CD8 antibody and PE-OVA Tetramer (MBL, TS-5001-1C) and subjected to flow cytometry analysis. The induction of OVA-specific T cells was evaluated based on the percentage of tetramer-positive cells among CD8-positive T cells.

[0087] [Table 4]

[0088] The results are shown in Figure 8. Comparisons between Groups 1 and 2 and between Groups 3 and 4 showed that the presence of the adjuvant PolyIC:LC demonstrated the ability to promote T cell induction (Groups 1 and 2, and Groups 3 and 4) (Figure 8). Furthermore, comparisons between Groups 2 and 4 in the presence of the adjuvant PolyIC:LC showed that the group administered the peptide vaccine alone without antibody (Group 2) also demonstrated the ability to promote T cell induction, while the group administered the antibody-peptide vaccine complex (non-covalently bound) (Group 4) demonstrated a significant ability to promote T cell induction compared to Group 2 (Groups 2 and 4) (Figure 8).

[0089] Example 9: Immunity induction test 2 in wild-type mice (comparison test of antibody-vaccine mixture ratios) The purpose of this example was to investigate in vivo the effect of the mixing ratio of antibody and peptide vaccine in the antibody-peptide vaccine complex (non-covalent type) using the peptide vaccine prepared in Example 3 on the immune induction ability.

[0090] Seven-week-old female wild-type mice C57BL / 6 (CLEA Japan) were divided into groups of four, and the experiment was carried out using the group composition shown in Table 5. Specifically, Group 1: A mixture of 2 equal amounts of peptide vaccine (peptide (3-1) derived from H-2Kb-restricted OVA in Example 2, SEQ ID NO: 30) and 1 equal amount of antibody-peptide vaccine complex (non-covalent bond type) made of anti-mouse CD40 antibody, further containing adjuvant PolyIC:LC (manufactured by Oncovir); Group 2: 2 equivalents of a control peptide, a non-IgG binding peptide (peptide (4), SEQ ID NO: 31), 1 equivalent of an anti-mouse CD40 antibody, and a mixture with the adjuvant PolyIC:LC (Oncovir); Group 3: A mixture of antibody-peptide vaccine complex (non-covalently bound) consisting of 4 equivalents of peptide (3-1) and 1 equivalent of anti-mouse CD40 antibody, and the adjuvant PolyIC:LC (Oncovir); Group 4: 4 equivalents of a control peptide (peptide (4)) containing a non-IgG binding peptide, 1 equivalent of an anti-mouse CD40 antibody, and a mixture of the adjuvant PolyIC:LC (Oncovir); The doses of each were as shown in Table 5.

[0091] The administration was performed according to the schedule shown in Figure 7. Specifically, the drug was administered subcutaneously to mice in each group twice every seven days (days 0 and 7, with the first administration day being designated as day 0). Spleens were collected on day 14, and induction of Ova-specific T cells was evaluated using the same method as in Example 8.

[0092] [Table 5]

[0093] The results are shown in Figure 9. When comparing the antibody-peptide vaccine conjugate (non-covalently bound) (anti-CD40 antibody: Ova-IgG-bound peptide) group with the mixture of antibody and Ova-IgG-unbound peptide group, the efficiency of immune induction in the conjugate was observed in both the 2-equivalent group (groups 1 and 2) and the 4-equivalent group (groups 3 and 4) (Figure 9). The results showed that when comparing the 2-equivalent group and the 4-equivalent group, stronger immune induction was observed in the 4-equivalent group in both the antibody and Ova-IgG-bound peptide group (groups 1 and 3) and the antibody and Ova-IgG-unbound peptide group (groups 2 and 4). It was revealed that the effect of the excess peptide added in the 4-equivalent group was additive.

[0094] Example 10: Immune induction test 3 in wild-type mice (dose-dependent test) This example was carried out with the aim of investigating the dose dependency of the antibody-peptide vaccine complex (non-covalent bond type) in vivo using the peptide vaccine prepared in Example 3.

[0095] Seven-week-old female wild-type mice C57BL / 6 (CLEA Japan) were divided into groups of four, and the experiment was conducted according to the group composition shown in Table 6. Specifically, groups 1 to 4 were administered with a high dose of peptide vaccine (10.5 μg of peptide vaccine), and groups 5 to 8 were administered with a medium dose of peptide vaccine (2.1 μg of peptide vaccine). Group 1: a mixture of a high dose (10.5 μg) of peptide vaccine (peptide (3-1) derived from H-2Kb-restricted OVA in Example 2, SEQ ID NO: 30) and adjuvant PolyIC:LC (Oncovir); Group 2: A mixture of a high dose (10.5 μg, equivalent to 2 equivalents) of peptide vaccine (peptide (3-1) in Example 2) and an anti-mouse CD40 antibody (200 μg, equivalent to 1 equivalent) as an antibody-peptide vaccine complex (non-covalently bound) and the adjuvant PolyIC:LC (manufactured by Oncovir); Group 3: a high dose (10.0 μg) of a non-IgG binding peptide (peptide (4), SEQ ID NO: 31) as a control peptide mixed with the adjuvant PolyIC:LC (Oncovir); · Group 4: a high dose (10.0 μg, equivalent to 2 equivalents) of IgG non-binding peptide (peptide (4)) as a control peptide, a mixture of anti-mouse CD40 antibody (200 μg, equivalent to 1 equivalent) and adjuvant PolyIC:LC (Oncovir); Group 5: a mixture of a medium dose (2.1 μg) of peptide vaccine (peptide (3-1) of Example 2) and adjuvant PolyIC:LC (Oncovir); Group 6: A mixture of a medium dose (2.1 μg, equivalent to 2 equivalents) of peptide vaccine (peptide (3-1) in Example 2) and an anti-mouse CD40 antibody (40 μg, equivalent to 1 equivalent) as an antibody-peptide vaccine complex (non-covalently bound) and the adjuvant PolyIC:LC (manufactured by Oncovir); Group 7: a medium dose (2.0 μg) of a non-IgG binding peptide (peptide (4)) as a control peptide mixed with the adjuvant PolyIC:LC (Oncovir); Group 8: a medium dose (2.0 μg, equivalent to 2 equivalents) of a non-IgG binding peptide (peptide (4)) as a control peptide, a mixture of anti-mouse CD40 antibody (40 μg, equivalent to 1 equivalent) and the adjuvant PolyIC:LC (Oncovir); The doses are shown in Table 6.

[0096] The administration was performed according to the schedule shown in Figure 7. Specifically, the drug was administered subcutaneously to mice in each group twice every seven days (the first administration was designated as day 0, and the drug was administered on days 0 and 7). The spleens were collected on day 14, and the induction of Ova-specific T cells was evaluated using the same method as in Example 8.

[0097] [Table 6]

[0098] The results are shown in Figure 10. The antibody:Ova-IgG bound peptide groups (Groups 2 and 6) showed a higher immune induction effect than the Ova-IgG bound peptide groups (Groups 1 and 5), the Ova-IgG non-bound peptide groups (Groups 3 and 7), and the antibody:Ova-IgG non-bound peptide groups (Groups 4 and 8). Furthermore, this effect was observed in a dose-dependent manner (Figure 10).

[0099] Example 11: Tumor-bearing mouse test 1 (Evaluation of antibody-peptide vaccine conjugate: conjugate with anti-CD40 antibody) This example was carried out with the aim of investigating in vivo whether an antibody-peptide vaccine complex (non-covalent bond type) prepared by combining the peptide vaccine prepared in Example 3 with an anti-CD40 antibody has an antitumor effect.

[0100] Seven-week-old female wild-type C57BL / 6 mice (CLEA Japan) were given 1 × 10 E.G7 (ATCC® CRL-2113), a mouse T-cell lymphoma cell line expressing the Ova antigen, per mouse. 6 The day of transplantation was designated Day 0, and on Day 3, the mice were divided into groups (6 mice per group) based on tumor volume. The experiment was conducted for each group with the group composition shown in Table 7. Specifically, for each group, Group 1: saline only (negative control); Group 2: Adjuvant PolyIC:LC (Oncovir) (negative control); Group 3: a mixture of peptide (3-1) (SEQ ID NO: 30) and adjuvant PolyIC:LC (Oncovir); Group 4: A mixture of an antibody-peptide vaccine complex (non-covalently bound) of peptide (3-1) and anti-mouse CD40 antibody and the adjuvant PolyIC:LC (Oncovir); Group 5: a mixture of a non-IgG binding peptide (peptide (4), SEQ ID NO: 31) as a control peptide and the adjuvant PolyIC:LC (Oncovir); Group 6: a mixture of IgG non-binding peptide (peptide (4)) as a control peptide, anti-mouse CD40 antibody, and adjuvant PolyIC:LC (Oncovir); The respective inoculation doses were as shown in Table 7.

[0101] The drugs were administered subcutaneously to the mice in each group twice in total, on day 3 and day 10, and tumor volume and body weight were observed twice a week from the start of drug administration.

[0102] [Table 7]

[0103] The results are shown in Figures 11 and 12. It was revealed that the antibody:Ova-IgG binding peptide group (Group 4) exhibited a higher tumor growth inhibitory effect in vivo compared to the Ova-IgG binding peptide group (Group 3), the Ova-IgG non-binding peptide group (Group 5), and the antibody:Ova-IgG non-binding peptide group (Group 6) (Figure 11).

[0104] Furthermore, when the survival period of each individual was confirmed, the antibody:Ova-IgG binding peptide group (Group 4) was found to have a significantly longer survival period compared to the Ova-IgG binding peptide group (Group 3), the Ova-IgG non-binding peptide group (Group 5), and the antibody:Ova-IgG non-binding peptide group (Group 6) (Figure 12).

[0105] Example 12: Immune induction test 4 in wild-type mice (neoantigen model) This example was conducted with the aim of evaluating whether an antibody-peptide vaccine complex (non-covalent type) using the peptide vaccine prepared in Example 3 has an antitumor effect in a test system with higher clinical applicability, specifically, a test system in which a peptide sequence selected based on cancer-specific mutations was used as the vaccine sequence.

[0106] Seven-week-old female wild-type mice C57BL / 6 (CLEA Japan) were divided into groups of four, and the experiment was carried out using the group composition shown in Table 8. Specifically, Group 1: a mixture of peptide vaccine (peptide (10) of Example 2, SEQ ID NO: 37) and adjuvant PolyIC:LC (Oncovir); Group 2: a mixture of an antibody-peptide vaccine complex (non-covalently bound) consisting of a peptide vaccine (peptide (10) in Example 2) and an anti-mouse CD40 antibody) and the adjuvant PolyIC:LC (Oncovir); Group 3: a mixture of a non-IgG binding peptide (peptide (11), SEQ ID NO: 38) as a control peptide and the adjuvant PolyIC:LC (Oncovir); · Group 4: a mixture of IgG non-binding peptide (peptide (11)) as a control peptide, anti-mouse CD40 antibody, and adjuvant PolyIC:LC (Oncovir); Group 5: Adjuvant PolyIC:LC only The doses are shown in Table 8.

[0107] [Table 8]

[0108] The administration was carried out according to the schedule shown in Figure 13. Specifically, the drug was administered subcutaneously to mice in each group three times every four days (the first administration day was designated as day 0, and the drugs were administered on days 0, 4, and 8).

[0109] On day 15, the spleen was harvested and 3 × 10 6The cells were suspended and seeded in 100 μl of splenocyte growth medium (RPMI-1640 (Thermo 61870-0362) containing 10% FBS (BioWest, S4190), 1 mM sodium pyruvate (Wako, 190-14881), 0.05 mM 2-ME (Wako, 131-14572), 20 mM HEPES (Wako, 345-06681), and 1% penicillin-streptomycin (Nakarai, 26253-84)). 100 μl of peptide (8) (SEQ ID NO: 35) was added to the cells at a final concentration of 10 μg / ml. The cells were then cultured at 37°C under 5% CO for 15 hours.

[0110] After adding 22 μl of 10× Brefeldin A (Biolegend 420601) and incubating for 3–4 hours, cells were collected by centrifugation at 2000 rpm and stained with Zombie Violet (BioLegend 423114), APC anti-mouse CD4 (BioLegend 100412), FITC anti-mouse CD8a (BioLegend 100706), and PE anti-mouse IFN-γ (BioLegend 505807) for flow cytometry analysis. The percentage of IFN-γ-positive cells among CD8+ cells was used to assess immune activation in response to the Adpgk peptide.

[0111] The results are shown in Figure 14. The antibody:Adpgk-IgG-bound peptide group (Group 2) showed a higher immune induction effect than the Adpgk-IgG-bound peptide group (Group 1), the non-Adpgk-IgG-bound peptide group (Group 3), and the antibody:Adpgk-IgG-unbound peptide group (Group 4). Thus, the antibody-bound neoantigen (Group 2) showed the highest immune induction, suggesting the usefulness of antibody vaccines in this model as well.

[0112] Example 13: Immune induction test 5 in wild-type mice (evaluation of multiple antibody-vaccine peptides) This example was carried out to investigate in vivo whether the antibody-peptide vaccine conjugate (non-covalent bond type) combined with a CD40 antibody or a PD-L1 antibody has the ability to induce immunity, using the peptide vaccine prepared in Example 3.

[0113] In this example, wild-type mice C57BL / 6 (CLEA Japan), 7 weeks old, female, were first divided into 4 groups of 5 mice each, and the experiment was carried out according to the group composition shown in Table 9. Specifically, Group 1: Adjuvant PolyIC:LC (Oncovir) only; Group 2: a mixture of peptide vaccine (peptide (3-1) derived from H-2Kb-restricted OVA in Example 2, SEQ ID NO: 30) and adjuvant PolyIC:LC (Oncovir); Group 3: A mixture of the peptide (3-1) and anti-mouse CD40 antibody-antibody peptide vaccine complex (non-covalently bound) and the adjuvant PolyIC:LC (Oncovir); Group 4: A mixture of the antibody-peptide vaccine complex (non-covalent bond) of peptide (3-1) and anti-PD-L1 antibody, and the adjuvant PolyIC:LC (Oncovir); were administered, respectively. In this example, the PD-L1 antibody used was a human PD-L1 antibody (MedChemExpress CAS NO: 1380723-44-3). The dosages of each component are listed in Table 9.

[0114] [Table 9]

[0115] The administration was performed according to the schedule shown in Figure 15. Specifically, the drug was administered subcutaneously to mice in each group twice every seven days (the first administration was designated as day 0, and the drug was administered on days 0 and 7). The spleens were collected on day 14, and the induction of Ova-specific T cells was evaluated using the same method as in Example 8.

[0116] The results are shown in Figure 16. Comparing Groups 2 and 3, and Groups 2 and 4, the group receiving the peptide vaccine together with an adjuvant without an antibody (Group 2) also showed the ability to promote T cell induction, but the group receiving the PD-L1 antibody-peptide vaccine noncovalent complex (Group 4), as well as the group receiving the CD40 antibody-peptide vaccine noncovalent complex (Group 3), showed a significant ability to promote T cell induction compared to Group 2 (Groups 3 and 4) (Figure 16).

[0117] Example 14: Immune induction test 6 in wild-type mice (evaluation of multiple antibody-vaccine peptides) Similar to Example 13, this Example was carried out using the peptide vaccine prepared in Example 3 to investigate in vivo whether antibody-peptide vaccine complexes (non-covalent bond type) combined with CD40 antibody or PD-L1 antibody have the ability to enhance immune induction compared to the combined administration of a peptide vaccine and antibody that do not form a complex.

[0118] Wild-type C57BL / 6 mice (Japan CLEA), 7 weeks old, female, were divided into 5 groups of 6 mice each, and the experiment was carried out with the group composition shown in Table 10. Specifically, Group 1: Adjuvant PolyIC:LC (Oncovir) only; Group 2: a mixture of a control peptide (peptide (4), SEQ ID NO: 31) and an anti-mouse CD40 antibody, and the adjuvant PolyIC:LC (Oncovir); Group 3: A mixture of the peptide (3-1) and anti-mouse CD40 antibody-antibody peptide vaccine complex (non-covalently bound) and the adjuvant PolyIC:LC (Oncovir); Group 4: A mixture of peptide (4) and anti-PD-L1 antibody, plus the adjuvant PolyIC:LC (Oncovir); Group 5: A mixture of the antibody-peptide vaccine complex (non-covalently bound) of peptide (3-1) and anti-PD-L1 antibody, and the adjuvant PolyIC:LC (Oncovir); were administered, respectively. In this example, the PD-L1 antibody used was a human PD-L1 antibody (MedChemExpress CAS NO: 1380723-44-3). The dosage of each component is as shown in Table 10.

[0119] [Table 10]

[0120] Administration was performed according to the schedule shown in Figure 15, as in Example 13. Specifically, the drug was subcutaneously administered to mice in each group twice every seven days (the first administration was designated as day 0, and administration was performed on days 0 and 7). Spleens were collected on day 14, and induction of Ova-specific T cells was evaluated using the same method as in Example 8.

[0121] The results are shown in Figure 17. Comparing Groups 2 and 3 and Groups 4 and 5, the groups that received a peptide vaccine that was not bound to the antibody together with the antibody (Groups 2 and 4) also showed T cell induction, but the groups that received the CD40 antibody-peptide vaccine conjugate (non-covalently bound) (Group 3) or the PD-L1 antibody-peptide vaccine conjugate (non-covalently bound) (Group 5) showed a significant enhancement in T cell induction (Figure 17).

[0122] Example 15: Tumor-bearing mouse study 2 (Evaluation of antibody-peptide vaccine conjugates: conjugates with anti-PD-L1 antibodies) This example was conducted to investigate in vivo whether the antibody-peptide vaccine conjugate (non-covalent bond type) prepared by combining the peptide vaccine prepared in Example 3 with an anti-PD-L1 antibody has an anti-tumor effect.

[0123] Seven-week-old female wild-type C57BL / 6 mice (CLEA Japan) were given 1 × 10 E.G7 (ATCC® CRL-2113), a mouse T-cell lymphoma cell line expressing the Ova antigen, per mouse. 6The day of transplantation was designated Day 0, and on Day 4, the mice were divided into groups (6 mice per group) based on tumor volume. The experiment was conducted for each group with the group composition shown in Table 11. Specifically, for each group, Group 1: Adjuvant PolyIC:LC (Oncovir) (negative control); Group 2: a mixture of peptide (3-1) (SEQ ID NO: 30) and adjuvant PolyICLC (Oncovir); Group 3: anti-PD-L1 antibody alone and the adjuvant PolyICLC (Oncovir) (control); Group 4: A mixture of an antibody-peptide vaccine conjugate (non-covalently bound) of peptide (3-1) and anti-PD-L1 antibody and the adjuvant PolyIC:LC (Oncovir); were subcutaneously administered. In this example, a human PD-L1 antibody (MedChemExpress CAS NO: 1380723-44-3) was used as the PD-L1 antibody. The respective inoculation doses are shown in Table 11.

[0124] The drugs were administered subcutaneously to the mice in each group twice in total, on day 4 and day 11, and tumor volume and body weight were observed twice a week from the start of drug administration.

[0125] [Table 11]

[0126] The results are shown in Figures 18 and 19. The antibody:Ova-IgG-binding peptide group (Group 4) was found to exhibit a higher tumor growth-inhibitory effect in vivo compared to the Ova-IgG-binding peptide group (Group 2) and the anti-PD-L1 antibody alone group (Group 3) (Figure 18).

[0127] Furthermore, when the survival time of each individual was confirmed, the antibody:Ova-IgG binding peptide group (Group 4) was found to have a significantly longer survival time than the Ova-IgG binding peptide group (Group 2) and the anti-PD-L1 antibody alone group (Group 3) (Figure 19).

[0128] Example 16: Immune induction test 7 in wild-type mice (neoantigen model) The purpose of this example was to investigate in vivo whether antibody-peptide vaccine conjugates (non-covalently bound) containing non-synonymous somatic mutant sequences of mouse tumor cell lines have antitumor effects.

[0129] First, based on the analysis results of the genomic sequence obtained from the mouse tumor cell line MC-38 as a non-synonymous somatic mutation sequence of the mouse tumor cell line, peptides containing the non-synonymous somatic mutation sequence (30 peptides, each 27mer, peptide (101) to peptide (130)) were created as shown in Table 12 below.

[0130] [Table 12]

[0131] Next, peptides (30 peptides, each 44mer, peptide (101BP) to peptide (130BP)) were prepared by adding IgG BP(1) (SEQ ID NO: 25) to the peptide vaccines (27mer, peptide (101) to peptide (130)) shown in the table above, as shown in Table 13 below.

[0132] [Table 13]

[0133] Seven-week-old female wild-type mice C57BL / 6 (CLEA Japan) were divided into two groups and the experiment was carried out according to the group composition shown in Table 14. Specifically, for each group: Group 1: A mixture of vaccine peptides prepared by pooling six peptides from the peptide vaccines (27mers, peptide (101) to peptide (130)) listed in Table 12 (e.g., Pool 01 (peptide (101) to peptide (106)) and Pool 06 (peptide (125) to peptide (130))) and the adjuvant PolyIC:LC (Oncovir); Group 2: Antibody-peptide vaccine conjugates (non-covalently bound) prepared by conjugating anti-mouse CD40 antibodies to the IgG-binding peptides (44mers each, peptide (101BP) to peptide (130BP)) listed in Table 13 above, antibody-peptide vaccine conjugates (non-covalently bound) prepared by pooling three peptides (e.g., Pool 11 (peptide (101BP) to peptide (103BP)), Pool 20 (peptide (128BP) to peptide (130BP)))) with the adjuvant PolyIC:LC (Oncovir); was subcutaneously administered to two mice per pool. The respective inoculation doses are shown in Table 14.

[0134] [Table 14]

[0135] The administration was carried out according to the schedule shown in Figure 20. Specifically, the drug was administered subcutaneously to mice in each group three times every seven days (the first administration was designated as day 0, and the drugs were administered on days 0, 7, and 14). On day 21, the spleens were collected, and then 2 × 10 6The cells were suspended in 100 μl of splenocyte growth medium (RPMI-1640 (Thermo 61870-0362) containing 10% FBS (BioWest, S4190), 1 mM sodium pyruvate (Wako, 190-14881), 0.05 mM 2-ME (Wako, 131-14572), 20 mM HEPES (Wako, 345-06681), and 1% penicillin-streptomycin (Nakarai, 26253-84)), and seeded onto a filter plate (Millipore) coated with mouse IFN-γ antibody (MabTech). The cells were then incubated with peptides (101e) to (130e), which are the epitope peptides of the respective peptides listed in Table 15 (SEQ ID NO: 74 to SEQ ID NO: 103) was dissolved individually and 100 μl of the solution was added to a final concentration of 10 μg / ml, and the mixture was cultured at 37°C under 5% CO2 for 15 hours.

[0136] [Table 15]

[0137] The spleen cells and each peptide were removed by aspiration. After washing the filter plate, 100 μL of biotin-labeled mouse IFN-γ antibody (MabTech) was added and incubated at 37°C for 2 hours. The filter plate was washed again, and 100 μL of streptavidin-ALP (MabTech) was added and incubated for an additional hour. The filter plate was washed again, and 100 μL of substrate solution (MabTech; BCIP / NBT-plus) was added to detect IFN-γ secreted by the cells.

[0138] The results are shown in Figure 21. Compared with the 27-mer peptide vaccine group (Group 1), the group administered with the antibody-peptide vaccine complex (non-covalently bound) made of anti-mouse CD40 antibody (Group 2) demonstrated a stronger immune response against the same epitope sequence. [Industrial Applicability]

[0139] The peptide vaccines disclosed in the present invention combined with IgG-binding peptides can efficiently deliver the peptide vaccine to the surface of specific immune cells (e.g., dendritic cells), enhancing their activation and enhancing the efficacy of the peptide vaccine.

Claims

1. An IgG-binding peptide-antigen peptide fusion peptide combining an IgG-binding peptide and an antigen peptide, wherein the IgG-binding peptide is Gly Pro Asp Cys Ala Tyr His Arg Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 25) or Gly Pro Asp Cys Ala Tyr His Lys Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 26) Gly Pro Asp Cys Ala Trp His Arg Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 39) Gly Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 40) IgG-binding peptide-antigen peptide fusion peptide selected from the group consisting of:

2. The IgG-binding peptide-antigenic peptide fusion peptide according to claim 1, wherein the antigenic peptide is selected from the group consisting of antigenic peptides that are cancer vaccines and antigenic peptides that are infectious disease vaccines.

3. The IgG-binding peptide-antigenic peptide fusion peptide of claim 2, which is a cancer vaccine, wherein the antigenic peptide is selected from the group consisting of a cancer-associated antigen, a cancer neoantigen, and a cancer-individualized neoantigen.

4. The IgG-binding peptide-antigen peptide fusion peptide according to any one of claims 1 to 3, wherein IgG is bound to the IgG-binding peptide.

5. The IgG-binding peptide-antigen peptide fusion peptide according to claim 4, wherein the IgG is an antibody against a target substance characteristic of immune cells that deliver the antigen peptide.

6. The IgG-binding peptide-antigen peptide fusion peptide of claim 5, wherein the immune cell is selected from the group consisting of an antigen-presenting cell, a dendritic cell, and a B cell.

7. The IgG-binding peptide-antigen peptide fusion peptide of any one of claims 4 to 6, wherein the IgG is selected from the group consisting of an anti-CD40 antibody, an anti-PD-L1 antibody, an anti-DEC205 antibody, an anti-DCIR antibody, an anti-Mannose receptor antibody, an anti-DC-SIGN antibody, an anti-CD11c antibody, and an anti-Dectin-1 antibody.

8. IgG, and an IgG-binding peptide-antigen peptide fusion peptide combining an IgG-binding peptide and an antigen peptide, wherein the IgG-binding peptide is Gly Pro Asp Cys Ala Tyr His Arg Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 25) or Gly Pro Asp Cys Ala Tyr His Lys Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 26) Gly Pro Asp Cys Ala Trp His Arg Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 39) Gly Pro Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Phe His (SEQ ID NO: 40) A method for delivering an antigen peptide to a cell having a molecule targeted by IgG on its cell surface in vitro, using an IgG-binding peptide-antigen peptide fusion peptide selected from the group consisting of:

9. The method according to claim 8, wherein the antigen peptide is selected from the group consisting of antigen peptides that are cancer vaccines and antigen peptides that are infectious disease vaccines.

10. The method of claim 9, wherein the antigen peptide is a cancer vaccine selected from the group consisting of a cancer-associated antigen, a cancer neoantigen, and a cancer-personalized neoantigen.

11. The method according to any one of claims 8 to 10, wherein the IgG is an antibody against a target substance characteristic of immune cells that deliver the antigen peptide.

12. The method of claim 11, wherein the immune cells are selected from the group consisting of antigen-presenting cells, dendritic cells, and B cells.

13. 13. The method of claim 11 or 12, wherein the IgG is selected from the group consisting of an anti-CD40 antibody, an anti-PD-L1 antibody, an anti-DEC205 antibody, an anti-DCIR antibody, an anti-Mannose receptor antibody, an anti-DC-SIGN antibody, an anti-CD11c antibody, and an anti-Dectin-1 antibody.

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