Cancer vaccine preparations

Abstract

The present invention provides a vaccine formulation for use in the prevention and / or treatment of cancer, which comprises a complex of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen.

Description

[Technical Field]

[0001] The present invention relates to a vaccine preparation for use in the prevention and / or treatment of cancer, which comprises a complex of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen. [Background technology]

[0002] Vaccine formulations for use in the prevention and / or treatment of cancer have been reported, which contain a hydrophobized polysaccharide such as cholesteryl pullulan (CHP) or cholesteryl mannan (CMP) and an antigen (Patent Document 1). Also reported are vaccine formulations for cancer treatment which contain a complex of a hydrophobized polysaccharide such as CHP with a synthetic long-chain peptide antigen having multiple T cell recognition epitopes, and an immunopotentiator (Patent Documents 2 and 3).

[0003] These vaccine preparations are formulated by phagocytosing antigens into peptides of various lengths by intracellular proteasomes, proteases, and peptidases. These peptides are then loaded onto major histocompatibility complex (MHC) class I or MHC class II molecules on the cell surface to form complexes, which then activate cytotoxic T cells (CTLs, CD8 + ) and helper T cells (CD4 + The principle is that these cells are specifically recognized by the IL-1 receptor agonist, IL-1, and activate these cells (Non-Patent Document 1).

[0004] On the other hand, it has been reported that the hyaluronic acid derivative that has introduced a group having a cholesteryl group as a hydrophobic group into hyaluronic acid forms fine particles by association in water, and forms a complex with a drug (Patent Document 4).In addition, it has been reported that the carboxyl group of the glucuronic acid part of hyaluronic acid is converted into an amide group by reacting with a specific amino acid, and then the steryl group, which is a hydrophobic group, is introduced into the remaining carboxyl group, and the hyaluronic acid derivative has both the properties of biodegradability and retention in blood, and the complex of this hyaluronic acid and drug has good properties as a pharmaceutical composition (Patent Document 5). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 1998 / 009650 [Patent Document 2] International Publication No. 2013 / 031882 [Patent Document 3] International Publication No. 2015 / 050158 [Patent Document 4] International Publication No. 2010 / 053140 [Patent Document 5] International Publication No. 2014 / 038641 [Non-patent literature]

[0006] [Non-Patent Document 1] ACS Nano, Vol. 8, pp. 9209-9218, 2014 Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need for vaccine formulations with even higher functionality than the previously reported CHP-based vaccine formulations, particularly cancer vaccine formulations with the ability to significantly induce antigen-specific cytotoxic T lymphocytes (CTLs) and have a strong antitumor effect. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve these problems and have discovered that a vaccine preparation containing a complex of a hyaluronic acid derivative with a hydrophobic group introduced therein and an antigen significantly induces antigen-specific CTLs and has high antitumor activity, thereby completing the present invention.

[0009] Specifically, the present invention relates to a complex formed by spontaneously associating in an aqueous solution to encapsulate an antigen, which exhibits improved antigen accumulation in lymph nodes and significantly induces antigen-specific CTLs, a complex with high antitumor activity, a vaccine preparation containing the complex, and methods for producing them. The present invention also relates to a complex formed by more dispersing in water to encapsulate an antigen, which exhibits improved antigen accumulation in lymph nodes and significantly induces antigen-specific CTLs, a vaccine preparation containing the complex, and methods for producing them.

[0010] In one aspect, the present invention provides the following vaccine preparations for use in the prevention and / or treatment of cancer [1] to

[13] and

[15] to

[27] , conjugates for use in the vaccine preparations

[14] and

[28] , and a method for producing the conjugate

[29] .

[0011] [1] A vaccine preparation for use in the prevention or treatment of cancer, comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen, The hyaluronic acid derivatives to which hydrophobic groups have been introduced are as follows: Formula (I)

[0012] [ka] [In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; R 5 is a hydrogen atom, formyl, or C 1-6 is alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any 2 to 30 amino acid residues; X 1 is the following formula: -NR b -R, -NR b -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -SR, -CO-Y a -SR, -O-CO-Y b -SR, -NR b -CO-Y b -SR, and -SSR, is a hydrophobic group selected from the group represented by R a , R b and R c are each independently a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, where the alkyl portion of the group is selected from -O- and -NR f -, and optionally 1 to 3 groups selected from -; R f is a hydrogen atom, C 1-12 Alkyl, Amino C 2-12 Alkyl and Hydroxy C 2-12 alkyl, the alkyl portion of which may be inserted with 1 to 2 groups selected from -O- and -NH-; R is a steryl group; Y is C 2-30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g - and -SS-; R g is a hydrogen atom, C 1-20 Alkyl, Amino C 2-20 Alkyl or hydroxy C 2-20alkyl, the alkyl portion of which may be inserted with 1 to 3 groups selected from -O- and -NH-; Y a is C 1-5 is alkylene; Y b is C 2-8 Alkylene or C 2-8 alkenylene; m is an integer selected from 1 to 100. A hyaluronic acid derivative containing one or more repeating units represented by the formula: Formula (II)

[0013] [ka] [In the formula, R 1a , R 2a , R 3a , and R 4a are independently hydrogen atoms, C 1-6 Alkyl, formyl, and C 1-6 alkylcarbonyl; R 5a is a hydrogen atom, formyl, or C 1-6 is alkylcarbonyl; X 1a is hydroxy, -O - Q + , C 1-6 Alkoxy, -NR 7 R 8 , or -NR 9 -Z 1 -Z 2 and; Q + represents the countercation; R 6a , R 7 , R 8 , and R 9 are independently hydrogen atoms, and C 1-6 alkyl; R aa is a hydrogen atom or C 1-6 alkyl, wherein the alkyl is independently hydroxy, carboxy, carbamoyl, C1-6 optionally substituted with one or more groups selected from alkylthio, aryl, and heteroaryl, wherein the aryl is optionally substituted with one or more hydroxy; Z 1 is C 2-30 Alkylene, or -(CH2CH2O) ma -CH2CH2-, wherein the alkylene is independently -O-, -NR ga - and -SS-, and 1 to 5 groups selected from -ma is an integer selected from 1 to 100; Z 2 is the following formula: -NR ba -Z 3 , -NR ba -COO-Z 3 , -NR ba -CO-Z 3 , -NR ba -CO-NR ca -Z 3 , -COO-Z 3 , -CO-NR ca -Z 3 , -O-CO-NR ca -Z 3 , -O-COO-Z 3 , -SZ 3 , -CO-Z a -SZ 3 , -O-CO-Z b -SZ 3 , -NR ba -CO-Z b -SZ 3 , and -SSZ 3 , is selected from the group represented by R ba and R ca are independently hydrogen atoms, C 1-20Alkyl, Amino C 2-20 Alkyl and Hydroxy C 2-20 alkyl, where the alkyl portion of the group is independently selected from -O- and -NR fa -, and optionally 1 to 3 groups selected from -; R fa are independently hydrogen atoms, C 1-12 Alkyl, Amino C 2-12 Alkyl and Hydroxy C 2-12 alkyl, the alkyl portion of which may be optionally interrupted by 1 to 2 groups independently selected from -O- and -NH-; R ga are independently hydrogen atoms, C 1-20 Alkyl, Amino C 2-20 Alkyl or hydroxy C 2-20 alkyl, the alkyl portion of which may be optionally interrupted by 1 to 3 groups independently selected from -O- and -NH-; Z 3 is a steryl group; Z a is C 1-5 is alkylene; Z b is C 2-8 Alkylene or C 2-8 alkenylene] A hyaluronic acid derivative containing a repeating unit represented by X 1a Ga-NR 9 -Z 1 -Z 2 When the repeating unit represented by formula (II) is not contained, the repeating unit represented by formula (III):

[0014] [ka] [In the formula, R 1b , R 2b , R 3b and R 4b are independently hydrogen atoms, C 1-6 Alkyl, formyl, and C 1-6 alkylcarbonyl; R5b is a hydrogen atom, formyl, or C 1-6 is alkylcarbonyl; X 2 is -NR 9 -Z 1 -Z 2 where R 9 , Z 1 , and Z 2 is as previously defined] A hyaluronic acid derivative containing a repeating unit represented by The vaccine preparation,

[0015] [2] The hyaluronic acid derivative having a hydrophobic group introduced therein is represented by the formula (IIIc)

[0016] [ka] [In the formula, R 1c , R 2c , R 3c and R 4c are each independently a hydrogen atom, C 1-6 Alkyl, formyl and C 1-6 alkylcarbonyl; R 5c is a hydrogen atom, formyl and C 1-6 alkylcarbonyl; X c is hydroxy and -O - Q + Selected from, where Q + represents the counter cation] The vaccine formulation according to [1], further comprising a repeating unit represented by:

[0017] [3] The vaccine preparation according to [1] or [2], which contains a hyaluronic acid derivative containing a repeating unit represented by formula (I), and the proportion of the repeating unit represented by formula (I) in the repeating units of the disaccharide present is 5 to 50%.

[0018] [4] A hyaluronic acid derivative containing a repeating unit represented by formula (II), wherein the repeating unit of the disaccharide is a group -NR 9 -Z 1 -Z 2 The vaccine formulation according to [1] or [2], wherein the proportion of disaccharide units containing

[0019] [5] A hyaluronic acid derivative containing a repeating unit represented by formula (I), wherein Z is a direct bond and Y is C 2-10 is alkylene, and X 1 The vaccine preparation according to any one of [1] to [3], wherein is —NH—COO—R, and R is a cholesteryl group.

[0020] [6] A hyaluronic acid derivative containing a repeating unit represented by formula (II), Z 1 But C 2-10 alkylene, and Z 2 But -NH-COO-Z 3 and Z 3 The vaccine formulation according to any one of [1], [2] and [4], wherein is a cholesteryl group.

[0021] [7] Hyaluronic acid derivatives are 1c , R 2c , R 3c , and R 4c are all hydrogen atoms, and R 5c is acetyl, and X c -O - Na + The vaccine preparation according to any one of [1] to [6], which is produced using hyaluronic acid composed only of disaccharide units represented by formula (IIIc) as defined in [2], and which has a weight-average molecular weight of 5 kilodaltons to 200 kilodaltons when calculated using the formula (IIIc).

[0022] [8] The vaccine preparation according to any one of [1] to [7], wherein the hyaluronic acid derivative and the antigen form a complex.

[0023] [9] The vaccine formulation according to any one of [1] to [8], which is to be administered in combination with one or more adjuvants.

[0024]

[10] The vaccine formulation according to any one of [1] to [9], wherein the antigen is an antigen peptide or an antigen protein.

[0025]

[11] The vaccine formulation according to

[10] , wherein the antigen peptide comprises two or more epitopes recognized by CD8-positive cytotoxic T cells or CD4-positive helper T cells.

[0026]

[12] The vaccine formulation according to

[11] , wherein the antigen peptide has an amino acid linker between epitopes.

[0027]

[13] The vaccine formulation according to any one of [1] to

[12] , which is to be administered in combination with one or more antibodies used in cancer treatment.

[0028]

[14] A complex formed from a hyaluronic acid derivative containing a repeating unit represented by formula (I) or formula (II) according to any one of [1] to [7] and an antigen used in a vaccine for the prevention or treatment of cancer.

[0029]

[15] Hyaluronic acid derivatives containing one or more repeating units represented by formula (I), wherein Y is -(CH2) n1 -or-(CH2CH2O) m1 The vaccine formulation according to any one of [1] to [3], [5] and [7] to

[13] , wherein n1 is an integer of 2 to 15, and m1 is an integer of 1 to 4.

[0030]

[16] Hyaluronic acid derivatives containing one or more repeating units represented by formula (I), wherein Y is -(CH2) n1 The vaccine formulation according to any one of [1] to [3], [5], [7] to

[13] and

[15] , wherein

[0031]

[17] Hyaluronic acid derivatives containing one or more repeating units represented by formula (I), wherein Y is -(CH2) n1 and n1 is 2, 6, 8, or 12.

[0032]

[18] A vaccine formulation according to any one of [1] to

[13] and

[15] to

[17] , for administration in combination with one or more adjuvants, wherein the adjuvant is i) a substance that activates an innate immune receptor (pattern recognition receptor), ii) an antigen-presenting cell stimulator, or iii) a substance that has the effect of inhibiting the acquisition of immunosuppressive activity by antigen-presenting cells.

[0033]

[19] A vaccine formulation according to any one of [1] to

[13] and

[15] to

[18] , for administration in combination with one or more adjuvants, wherein the adjuvant is CpG oligoDNA, PolyIC, QuilA, QS21, Sting, monophosphoryl lipid, R848, imiquimod, or MPL.

[0034]

[20] A vaccine formulation according to any one of [1] to

[13] and

[15] to

[19] , wherein the antigen is an antigen peptide, and the antigen peptide contains one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0035]

[21] The vaccine formulation according to any one of [1] to

[13] and

[15] to

[20] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 8 to 120.

[0036]

[22] The vaccine formulation according to any one of [1] to

[13] and

[15] to

[21] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 8 to 50.

[0037]

[23] The vaccine formulation according to any one of [1] to

[13] and

[15] to

[22] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 16 to 80.

[0038]

[24] The vaccine formulation according to any one of [1] to

[13] and

[15] to

[23] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 23 to 60.

[0039]

[25] A vaccine formulation according to any one of claims [1] to

[13] and

[15] to

[24] , for administration in combination with one or more antibodies used in cancer treatment, wherein the antibodies are antibodies that inhibit immunosuppressive signals from tumors or one or more antibodies that activate costimulatory signals in immune cells.

[0040]

[26] A vaccine formulation according to any one of claims [1] to

[13] and

[15] to

[25] , for administration in combination with one or more antibodies used in cancer treatment, wherein the antibody is an anti-CTLA4 antibody, an anti-PD1 antibody, an anti-PDL1 antibody, an anti-OX40 antibody, or an anti-4-1BB antibody.

[0041]

[27] A vaccine formulation according to any one of claims [1] to

[13] and

[15] to

[26] , for administration in combination with one or more antibodies used in cancer treatment, wherein the antibody is an anti-PDL1 antibody.

[0042]

[28] A complex formed from a hyaluronic acid derivative containing a repeating unit represented by formula (I) or formula (II) according to any one of

[15] to

[17] and an antigen used in a vaccine for the prevention or treatment of cancer.

[0043]

[29] A method for producing a complex of a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen, comprising the step of mixing in a solution a hyaluronic acid derivative containing a repeating unit represented by formula (I) or formula (II) described in any one of [1] to [7] and

[15] to

[17] with an antigen peptide used in a vaccine for use in the prevention or treatment of cancer.

[0044] In another aspect of the present invention, the following methods for preventing and / or treating cancer

[30] to

[46] and uses

[47] to

[63] are provided.

[0045]

[30] A method for preventing and / or treating cancer, comprising administering to a subject a vaccine preparation containing a hyaluronic acid derivative containing a repeating unit represented by formula (I) or formula (II) according to any one of [1] to [7] and

[15] to

[17] and an antigen.

[0046]

[31] The method according to

[30] , wherein a complex is formed between a hyaluronic acid derivative and an antigen.

[0047]

[32] The method of

[30] , wherein the vaccine formulation is administered in combination with one or more adjuvants.

[0048]

[33] The method according to

[30] , wherein the antigen is an antigen peptide or an antigen protein.

[0049]

[34] The method according to

[33] , wherein the antigen peptide comprises two or more epitopes recognized by CD8-positive cytotoxic T cells or CD4-positive helper T cells.

[0050]

[35] The method according to

[34] , wherein the antigen peptide has an amino acid linker between epitopes.

[0051]

[36] The method of

[30] , wherein the vaccine formulation is administered in combination with one or more antibodies used in cancer therapy.

[0052]

[37] The method described in

[30] , wherein the vaccine formulation is administered in combination with one or more adjuvants, and the adjuvants are i) substances that activate innate immune receptors (pattern recognition receptors), ii) antigen-presenting cell stimulators, or iii) substances that inhibit the acquisition of immunosuppressive activity of antigen-presenting cells.

[0053]

[38] The method according to

[30] , wherein the vaccine formulation is administered in combination with one or more adjuvants, and the adjuvant is CpG oligo DNA, PolyIC, QuilA, QS21, Sting, monophosphoryl lipid, R848, imiquimod, or MPL.

[0054]

[39] The method according to

[30] , wherein the antigen is an antigen peptide, and the antigen peptide contains one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0055]

[40] The method according to

[30] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 8 to 120.

[0056]

[41] The method according to any one of

[30] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 8 to 50.

[0057]

[42] The method according to

[30] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 16 to 80.

[0058]

[43] The method according to

[30] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 23 to 60.

[0059]

[44] The method described in

[30] , wherein the vaccine formulation is administered in combination with one or more antibodies used in cancer therapy, and the antibodies are antibodies that inhibit immunosuppressive signals from tumors or one or more antibodies that activate costimulatory signals in immune cells.

[0060]

[45] The method of

[30] , wherein the vaccine formulation is administered in combination with one or more antibodies used in cancer therapy, and the antibodies are anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-OX40, or anti-4-1BB antibody.

[0061]

[46] The method according to

[30] , wherein the vaccine preparation is administered in combination with one or more antibodies used in cancer therapy, and the antibodies are anti-PDL1 antibodies.

[0062]

[47] Use of a hyaluronic acid derivative and an antigen containing a repeating unit represented by formula (I) or formula (II) according to any one of [1] to [7] and

[15] to

[17] in the manufacture of a vaccine preparation for use in the prevention or treatment of cancer.

[0063]

[48] ​​The use according to

[47] , wherein the hyaluronic acid derivative and an antigen form a complex.

[0064]

[49] The use according to

[47] , wherein the vaccine formulation is administered in combination with one or more adjuvants.

[0065]

[50] The use according to

[47] , wherein the antigen is an antigen peptide or an antigen protein.

[0066]

[51] The use according to

[50] , wherein the antigen peptide comprises two or more epitopes recognized by CD8-positive cytotoxic T cells or CD4-positive helper T cells.

[0067]

[52] The use according to

[51] , wherein the antigen peptide has an amino acid linker between epitopes.

[0068]

[53] The use according to

[47] , wherein the vaccine formulation is administered in combination with one or more antibodies used in cancer therapy.

[0069]

[54] The use described in

[47] , wherein the vaccine formulation is administered in combination with one or more adjuvants, and the adjuvants are i) substances that activate innate immune receptors (pattern recognition receptors), ii) antigen-presenting cell stimulators, or iii) substances that inhibit the acquisition of immunosuppressive activity of antigen-presenting cells.

[0070]

[55] The use according to

[47] , wherein the vaccine formulation is administered in combination with one or more adjuvants, and the adjuvant is CpG oligo DNA, PolyIC, QuilA, QS21, Sting, monophosphoryl lipid, R848, imiquimod, or MPL.

[0071]

[56] The use according to

[47] , wherein the antigen is an antigen peptide, and the antigen peptide contains one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0072]

[57] The use according to

[47] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 8 to 120.

[0073]

[58] The use according to any one of

[47] , wherein the antigen is an antigen peptide and the number of amino acid residues of the antigen peptide is 8 to 50.

[0074]

[59] The use according to

[47] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 16 to 80.

[0075]

[60] The use according to

[47] , wherein the antigen is an antigen peptide, and the number of amino acid residues of the antigen peptide is 23 to 60.

[0076]

[61] The use described in

[47] , wherein the vaccine preparation is administered in combination with one or more antibodies used in cancer treatment, and the antibodies are antibodies that inhibit immunosuppressive signals from tumors or one or more antibodies that activate costimulatory signals in immune cells.

[0077]

[62] The use according to

[47] , wherein the vaccine formulation is administered in combination with one or more antibodies used in cancer treatment, and the antibodies are anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-OX40, or anti-4-1BB antibody.

[0078]

[63] The use according to

[47] , wherein the vaccine preparation is administered in combination with one or more antibodies used in cancer treatment, and the antibodies are anti-PDL1 antibodies. [Effects of the Invention]

[0079] By using the vaccine preparation of the present invention, which comprises the complex of the hyaluronic acid derivative with hydrophobic group introduced and antigen, the accumulation of antigen in lymph node is improved, and the remarkable induction of antigen-specific CTL can be achieved, and the anti-cancer effect of immunity can be enhanced.In addition, the complex and vaccine preparation of the present invention have excellent safety characteristics, especially the safety of long-term administration, because the hyaluronic acid derivative used is also excellent in safety. [Brief explanation of the drawings]

[0080] [Figure 1] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when mERK2 p121 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; CpG: spleen cells from mice administered with the antigenic peptide (mERK2 p121) and CpG oligo DNA; CHP+CpG: spleen cells from mice administered with a complex of CHP (CHP-80T; 80k) and the antigenic peptide and CpG oligo DNA; 99k41+CpG: spleen cells from mice administered with a complex of the antigenic peptide and an HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA. [Figure 2-1]This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when mERK2 p121 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; 99k41: spleen cells from mice administered a complex of an antigenic peptide (mERK2 p121) and an HA derivative (99k HA-C6-Chol-41%); 99k41+CpG: spleen cells from mice administered a complex of an antigenic peptide and the HA derivative and CpG oligo DNA; 99k41+polyIC: spleen cells from mice administered a complex of an antigenic peptide and the HA derivative and polyIC; 99k41+QuilA: spleen cells from mice administered a complex of an antigenic peptide and the HA derivative and QuilA; and 99k41+sting: spleen cells from mice administered a complex of an antigenic peptide and the HA derivative and Sting. [Figure 2-2] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD4+ cells to total CD4+ cells in spleen cells derived from BALB / c mice when mERK2 p121 was used as a CD4+ T cell-stimulating peptide. Each sample is the same as in Figure 2-1. [Figure 3-1] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when mERK2 p121 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells derived from untreated mice; 99k41+CpG: spleen cells derived from mice administered a complex of the antigenic peptide (mERK2 p121) and an HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA; 99k41+R848: spleen cells derived from mice administered a complex of the antigenic peptide and the HA derivative and R848; 99k41+MPL: spleen cells derived from mice administered a complex of the antigenic peptide and the HA derivative and MPL. [Figure 3-2]This graph shows the ratio (vertical axis) of interferon-gamma-producing CD4+ cells to total CD4+ cells in spleen cells derived from BALB / c mice when mERK2 p121 was used as a CD4+ T cell-stimulating peptide. The samples are the same as those in Figure 3-1. [Figure 4] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when MAGE-A4 p265 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; peptide+CpG: spleen cells from mice administered with the antigenic peptide (MAGE-A4 p264) and CpG oligo DNA; CHP / Wt+CpG: spleen cells from mice administered with a complex of CHP (80kJ) and the antigenic peptide and CpG oligo DNA; 99k41 / Wt+CpG: spleen cells from mice administered with a complex of the antigenic peptide and an HA derivative (99kJ HA-C6-Chol-41%) and CpG oligo DNA. [Figure 5] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when MAGE-A4 p265 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; CHP / Wt: spleen cells from mice administered a complex of CHP (80kJ) and antigen peptide (MAGE-A4 p264); 99k41 / Wt: spleen cells from mice administered a complex of antigen peptide and HA derivative (99kJ HA-C6-Chol-41%). [Figure 6]This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells from BALB / c mice when MAGE-A4 p265 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; 99k41 / Wt+CpG: spleen cells from mice administered a complex of the antigenic peptide (MAGE-A4 p264) and an HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA; 99k41 / LLLL+CpG: spleen cells from mice administered a complex of the antigenic peptide (MAGE-A4 p264-4L) and the HA derivative and CpG oligo DNA; and 99k41 / WWWW+CpG: spleen cells from mice administered a complex of the antigenic peptide (MAGE-A4 p264-4W) and the HA derivative and CpG oligo DNA. [Figure 7-1] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in splenocytes derived from C57BL / 6 mice when TRP1 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: splenocytes from untreated mice; CHP / 6Y+CpG: splenocytes from mice administered a complex of CHP and antigen peptide (TRP2TRP1gp100-6Y) and CpG oligo DNA; 99k43 / 6Y+CpG: splenocytes from mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA. [Figure 7-2] 7 is a graph showing the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in splenocytes derived from C57BL / 6 mice when TRP2 was used as a CD8+ T cell-stimulating peptide. Each sample is the same as in FIG. 7-1. [Figure 7-3] 7 is a graph showing the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in splenocytes derived from C57BL / 6 mice when gp100 was used as a CD8+ T cell-stimulating peptide. Each sample is the same as in FIG. 7-1. [Figure 8-1]This is a graph showing the ratio (vertical axis) of the number of interferon-gamma-producing CD8+ cells to the total number of CD8+ cells in splenocytes derived from C57BL / 6 mice when TRP1 was used as a CD8+ T cell-stimulating peptide. NT: spleen cells from untreated mice; 99k43 / 6G+CpG: spleen cells from mice administered a complex of antigenic peptide (TRP2TRP1gp100-6G) and HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA; 99k43 / 6Y+CpG: spleen cells from mice administered a complex of antigenic peptide (TRP2TRP1gp100-6Y) and HA derivative and CpG oligo DNA; 99k43 / 4L+CpG: spleen cells from mice administered a complex of antigenic peptide (TRP2TRP1gp100-4L) and HA derivative and CpG oligo DNA; 99k43 / 6L+CpG: spleen cells from mice administered a complex of antigenic peptide (TRP2TRP1gp100-6L) and HA derivative and CpG oligo DNA. [Figure 8-2] 8-1 is a graph showing the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in splenocytes derived from C57BL / 6 mice when TRP2 was used as a CD8+ T cell-stimulating peptide. Each sample is the same as in FIG. 8-1. [Figure 8-3] 8 is a graph showing the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in splenocytes derived from C57BL / 6 mice when gp100 was used as a CD8+ T cell-stimulating peptide. Each sample is the same as in FIG. 8-1. [Figure 9-1]This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when AH1 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; CHP / 6Y+CpG: spleen cells from mice administered a complex of CHP and antigen peptide (AH1gp70-6Y) and CpG oligo DNA; 99k43 / 6Y+CpG: spleen cells from mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA. [Figure 9-2] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD4+ cells to total CD4+ cells in spleen cells derived from BALB / c mice when gp70 was used as a CD4+ T cell-stimulating peptide. Each sample is the same as in Figure 9-1. [Figure 10-1] This graph shows the ratio (vertical axis) of interferon-gamma-producing CD8+ cells to total CD8+ cells in spleen cells derived from BALB / c mice when AH1 was used as a CD8+ T cell-stimulating peptide. The samples are as follows: NT: spleen cells from untreated mice; CHP / 6L+CpG: spleen cells from mice administered a complex of CHP and antigen peptide (AH1gp70-6L) and CpG oligo DNA; 99k43 / 6L+CpG: spleen cells from mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA. [Figure 10-2] 10-1 is a graph showing the ratio (vertical axis) of interferon-gamma-producing CD4+ cells to total CD4+ cells in spleen cells derived from BALB / c mice when gp70 was used as a CD4+ T cell-stimulating peptide. Each sample is the same as in FIG. 10-1. [Figure 11-1]This is a graph showing the ratio (vertical axis) of the number of interferon-gamma-producing CD8+ cells to the total number of CD8+ cells in spleen cells derived from BALB / c mice when AH1 was used as a peptide for stimulating CD8+ T cells. NT: spleen cells from untreated mice; 99k43 / 6G+CpG: spleen cells from mice administered a complex of the antigen peptide (AH1gp70-6G) and an HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA; 99k43 / 6Y+CpG: spleen cells from mice administered a complex of the antigen peptide (AH1gp70-6Y) and the HA derivative and CpG oligo DNA; 99k43 / 4L+CpG: spleen cells from mice administered a complex of the antigen peptide (AH1gp70-4L) and the HA derivative and CpG oligo DNA; 99k43 / 6L+CpG: spleen cells from mice administered a complex of the antigen peptide (AH1gp70-6L) and the HA derivative and CpG oligo DNA. [Figure 11-2] 11-1 shows the ratio (vertical axis) of interferon-gamma-producing CD4+ cells to total CD4+ cells in spleen cells derived from BALB / c mice when gp70 was used as a CD4+ T cell-stimulating peptide. Each sample is the same as in FIG. [Figure 12-1] This graph shows the change in mean tumor volume (vertical axis) over time in BALB / c mice bearing the murine fibrosarcoma CMS5a. The groups are as follows: Control: mice not administered any sample; Peptide: mice administered with antigen peptide (mERK2 p121) and CpG oligo DNA; Emulsion: mice administered with an emulsion formulation made with antigen peptide and CpG oligo DNA; CHP: mice administered with a complex of CHP and antigen peptide and CpG oligo DNA; 99k41: mice administered with a complex of antigen peptide and HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA. [Figure 12-2] 12 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of BALB / c mice bearing mouse fibrosarcoma CMS5a. Each group is the same as in FIG. 12-1. [Figure 13-1]This graph shows the change in mean tumor volume (vertical axis) over time in BALB / c mice (n=5) bearing the murine fibrosarcoma CMS5a. The groups are as follows: Control: Group of mice not administered sample, CpG: Group of mice administered CpG oligo DNA, CHP: Group of mice administered a complex of CHP and antigen peptide (mERK2 p121) and CpG oligo DNA, 99k41: Group of mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA. [Figure 13-2] 13 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of BALB / c mice (n=5) bearing the mouse fibrosarcoma CMS5a. Each group is the same as in FIG. 13-1. [Figure 14-1] This graph shows the change in mean tumor volume (vertical axis) over time in BALB / c mice (n=5) bearing the murine fibrosarcoma CMS5a. The groups are as follows: Control: A group of mice not administered any sample; aCTLA4 / aPD1 / aPDL1: A group of mice administered anti-CTLA4 antibody, anti-PD1 antibody, and anti-PDL1 antibody; 99k41: A group of mice administered a complex of antigen peptide (mERK2 p121) and HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA. [Figure 14-2] 14 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of BALB / c mice (n=5) bearing the mouse fibrosarcoma CMS5a. Each group is the same as in FIG. 14-1. [Figure 15-1]This graph shows the mean tumor volume (vertical axis) over time in BALB / c mice (n=5) bearing the murine fibrosarcoma CMS5a. The groups are as follows: Control: Mice not administered any sample; aCTLA4 / aPDL1 / aOX40 / a4-1BB: Mice administered with anti-CTLA4, anti-PDL1, anti-OX40, and anti-4-1BB antibodies; 99k41: Mice administered with a complex of antigen peptide (mERK2 p121) and HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA; 99k41+aCTLA4 / aPDL1 / aOX40 / a4-1BB: Mice administered with a complex of antigen peptide and HA derivative, CpG oligo DNA, and anti-CTLA4, anti-PDL1, anti-OX40, and anti-4-1BB antibodies. [Figure 15-2] 15-1 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of BALB / c mice (n=5) bearing the mouse fibrosarcoma CMS5a. Each group is the same as in FIG. 15-1. [Figure 16-1] This graph shows the change in the average tumor volume (vertical axis) over time in BALB / c mice bearing CT26 mouse colon cancer cells. The groups are as follows: Control: a group of mice not administered any sample; Emulsion: a group of mice administered an emulsion formulation made with antigen peptide (AH1gp70-6L) and CpG oligo DNA; CHP: a group of mice administered a complex of CHP and antigen peptide and CpG oligo DNA; 99k43: a group of mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA. [Figure 16-2] 16 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of BALB / c mice (n=5) bearing mouse colon cancer cell CT26 tumors. Each group is the same as in FIG. 16-1. [Figure 17-1]This graph shows the change in mean tumor volume (vertical axis) over time in C57BL / 6 mice bearing the mouse melanoma B16F10 tumor. The groups are as follows: Control: Group of mice not administered any sample; CHP: Group of mice administered a complex of CHP and antigen peptide (TRP2TRP1gp100-6Y) and CpG oligo DNA; 99k43: Group of mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-43%) and CpG oligo DNA. [Figure 17-2] 17 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of C57BL / 6 mice (n=5) bearing mouse melanoma B16F10 tumors. Each group is the same as in FIG. 17-1. [Figure 18-1] 1 is a graph showing the fluorescence intensity per cell in the lymph nodes of BALB / c mice 24 hours after sample administration in a lymph node migration test. The administered samples are as follows. NT: no sample administration, LPA: antigen peptide (fluorescently labeled mERK2 p121 (fluorescein-conjugated)), CHP: complex of CHP and antigen peptide, 10k17: complex of antigen peptide and HA derivative (10k HA-C6-Chol-17%), 10k25: complex of antigen peptide and HA derivative (10k HA-C6-Chol-25%), 10k42: complex of antigen peptide and HA derivative (10k HA-C6-Chol-42%), 50k23: complex of antigen peptide and HA derivative (50k HA-C6-Chol-23%), 50k43: complex of antigen peptide and HA derivative (50k HA-C6-Chol-43%), 99k24: complex of antigen peptide and HA derivative (99k HA-C6-Chol-24%), 99k43: complex of antigen peptide and HA derivative (99k HA-C6-Chol-43%) complex. [Figure 18-2] This is a graph showing the fluorescence intensity per cell in the lymph nodes of BALB / c mice 72 hours after sample administration in a lymph node migration test. Each administered sample is the same as in Figure 18-1. [Figure 19]1 is a graph showing the fluorescence intensity per cell in the lymph nodes of BALB / c mice 24 hours after sample administration in a lymph node migration test. The administered samples are as follows. NT: No sample administration, peptide: antigen peptide (fluorescently labeled mERK2 p121 (fluorescein-conjugated)), 99k41: complex of antigen peptide and HA derivative (99k HA-C6-Chol-41%), 10kHA-Ala-Chol30: complex of antigen peptide and HA derivative (10k HA-Ala-C6-Chol-30%), 10kHA-Ser-Chol28: complex of antigen peptide and HA derivative (10k HA-Ser-C6-Chol-28%), 10kHA-Gly-Chol32: complex of antigen peptide and HA derivative (10k HA-Gly-C6-Chol-32%), 10kHA-Thr-Chol32: complex of antigen peptide and HA derivative (10k HA-Thr-C6-Chol-32%), 10kHA-Asn-Chol20: complex of antigen peptide and HA derivative (10k HA-Asn-C6-Chol-20% complex, 10kHA-Asp-Chol32: complex of antigen peptide and HA derivative (10kHA-Asp-C6-Chol-32%), 10kHA-Phe-Chol31: complex of antigen peptide and HA derivative (10kHA-Phe-C6-Chol-31%), 10kHA-Tyr-Chol32: complex of antigen peptide and HA derivative (10kHA-Tyr-C6-Chol-32%), 10kHA-Ile-Chol28: complex of antigen peptide and HA derivative (10kHA-Ile-C6-Chol-28%), 10kHA-Leu-Chol31: complex of antigen peptide and HA derivative (10kHA-Leu-C6-Chol-31%), 10kHA-Val-Chol32: complex of antigen peptide and HA derivative (10k HA-Val-C6-Chol-32%) complex, 10kHA-Trp-Chol24: complex of antigen peptide and HA derivative (10kHA-Trp-C6-Chol-24%), 10kHA-Gln-Chol32: complex of antigen peptide and HA derivative (10kHA-Gln-C6-Chol-32%), 10kHA-Glu-Chol32: complex of antigen peptide and HA derivative (10kHA-Glu-C6-Chol-32%). [Figure 20-1] This graph shows the change in mean tumor volume (vertical axis) over time in C57BL / 6 mouse groups bearing the mouse melanoma B16F10. The groups are as follows: Control: Group of mice not administered any sample; 99k42: Group of mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-42%) and CpG oligo DNA; 50k42: Group of mice administered a complex of antigen peptide and HA derivative (50k HA-C6-Chol-42%) and CpG oligo DNA; 10k43: Group of mice administered a complex of antigen peptide and HA derivative (10k HA-C6-Chol-43%) and CpG oligo DNA. [Figure 20-2] This is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of C57BL / 6 mice (n=5) bearing mouse melanoma B16F10. Each group is the same as in Figure 20-1. [Figure 21-1] This graph shows the change in mean tumor volume (vertical axis) over time in C57BL / 6 mouse groups bearing the mouse melanoma B16F10. The groups are as follows: Control: Group of mice not administered any sample; 10kHA-Ala-Chol30: Group of mice administered a complex of antigen peptide and amino acid-modified HA derivative (10kHA-Ala-C6-Chol-30%) and CpG oligo DNA; 10kHA-Gln-Chol32: Group of mice administered a complex of antigen peptide and amino acid-modified HA derivative (10kHA-Gln-C6-Chol-32%) and CpG oligo DNA. [Figure 21-2] 21-1 is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of C57BL / 6 mice (n=5) bearing mouse melanoma B16F10. Each group is the same as in FIG. 21-1. [Figure 22-1] This graph shows the change in mean tumor volume (vertical axis) over time in C57BL / 6 mice bearing the mouse melanoma B16F10 tumor. The groups are as follows: Control: A group of mice that did not receive any sample; 99k42+PolyIC: A group of mice that received a complex of antigen peptide and HA derivative (99k HA-C6-Chol-42%) and PolyIC. [Figure 22-2] This is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of C57BL / 6 mice (n=5) bearing mouse melanoma B16F10. Each group is the same as in Figure 22-1. [Figure 23-1] This graph shows the change in mean tumor volume (vertical axis) over time in C57BL / 6 mouse groups bearing the mouse melanoma B16F10. The groups are as follows: Control: Group of mice not administered any sample; 99k42+Sting: Group of mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-42%) and a Sting agonist; 99k42+R848: Group of mice administered a complex of antigen peptide and HA derivative (99k HA-C6-Chol-42%) and R848. [Figure 23-2] This is a graph showing the time course of tumor volume (vertical axis) in each individual of a group of C57BL / 6 mice (n=5) bearing mouse melanoma B16F10. Each group is the same as in Figure 23-1. [Figure 24-1] This graph shows the ratio (vertical axis) of TRP2-specific CD8+ cells to total CD8+ cells in tumors or lymph nodes from mice bearing mouse melanoma B16F10, using H-2Kb TRP-2 Tetramer-SVYDFFVWL-APC as the tetramer. The samples are as follows: NT: cells from untreated tumor-bearing mice; 99k41: cells from tumor-bearing mice administered a complex of antigen peptide (TRP2TRP1gp100-6Y) and HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA. [Figure 24-2] This graph shows the ratio (vertical axis) of gp100-specific CD8+ cells to total CD8+ cells in tumors or lymph nodes derived from mice bearing mouse melanoma B16F10, when H-2Db gp100 Tetramer-EGSRNQDWL-PE was used as the tetramer. The samples are as follows: NT: cells derived from untreated tumor-bearing mice; 99k41: cells derived from tumor-bearing mice administered a complex of antigen peptide (TRP2TRP1gp100-6Y) and HA derivative (99k HA-C6-Chol-41%) and CpG oligo DNA. DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention will be described in more detail below.

[0082] The complex of the hyaluronic acid derivative that hydrophobic group is introduced into and antigen of the present invention is the hyaluronic acid derivative that comprises one or more disaccharide units (also repeating units) represented by formula (I), or the complex of the hyaluronic acid derivative that comprises one or more disaccharide units (also repeating units) represented by formula (II) and antigen, and can use this complex to prepare the vaccine preparation of the present invention.This specification also includes the disclosure of the method for preparing the complex of the hyaluronic acid derivative that hydrophobic group is introduced into and antigen of the present invention.

[0083] definition The term "steryl group" referred to herein is not particularly limited as long as it is a group having a steroid skeleton. Specific examples of steroids include cholesterol, dehydrocholesterol, coprostenol, coprostenol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, cimialenol, bile acids (cholanic acid, lithocholic acid, hyodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, apocholic acid, cholic acid, dehydrocholic acid, glycocholic acid, taurocholic acid), testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, and deoxycortisterone. Examples of the steryl group include a cholesteryl group, a stigmasteryl group, a lanosteryl group, an ergosteryl group, a cholanoyl group, and a choloyl group. Preferred examples include a cholesteryl group (particularly, a cholest-5-en-3β-yl group represented by the following formula) and a cholanoyl group (particularly, a 5β-cholan-24-oyl group represented by the following formula).

[0084] [ka] Here, the two asterisks represent the binding sites.

[0085] The term "C" referred to herein 1-20 The term "alkyl" refers to a straight or branched alkyl group having 1 to 20 carbon atoms, and examples of such alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, and t-butyl. 1-4 C includes "alkyl" and further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, and 2-ethylbutyl. 1-20 Alkyl has 1 to 12 carbon atoms, 1-12 alkyl, with 1 to 6 carbon atoms, 1-6 Also includes alkyl.

[0086] The term "C" referred to herein 1-6 The term "alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, and includes, for example, "C alkyl" such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, and t-butyl. 1-4 alkyl" is included.

[0087] The term "C" referred to herein 1-6 "Alkylcarbonyl" refers to a group in which the alkyl portion is one of the C groups already mentioned. 1-6 It means an alkylcarbonyl group, for example, acetyl, propionyl, n-propylcarbonyl, i-propylcarbonyl, n-butylcarbonyl, s-butylcarbonyl, i-butylcarbonyl, t-butylcarbonyl, etc. 1-4 alkylcarbonyl".

[0088] The term "C" referred to herein 1-6 "Alkoxy" refers to a group in which the alkyl portion is one of the C 1-6It means an alkyloxy group, for example, methoxy (H3C-O-), ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, i-butoxy, t-butoxy, etc. 1-4 Alkoxy" is included.

[0089] The term "C" referred to herein 1-6 "Alkylthio" means that the alkyl part is the C 1-6 It means an alkylthio group which is alkyl, and includes, for example, methylthio (H3C-S-), ethylthio, n-propylthio, i-propylthio, n-butylthio, s-butylthio, i-butylthio, t-butylthio, etc., and preferably methylthio.

[0090] The term "amino C" referred to herein 2-20 "Alkyl" means a linear or branched alkyl group having 2 to 20 carbon atoms and having an amino group as a substituent, and for example, the amino group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl has 2 to 12 carbon atoms, such as "amino C 2-12 Also includes alkyl.

[0091] The term "hydroxy C" referred to herein 2-20 "Alkyl" means a linear or branched alkyl group having 2 to 20 carbon atoms and having a hydroxy group as a substituent, and for example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. 2-20 Alkyl has 2 to 12 carbon atoms, including hydroxy C 2-12 Also includes alkyl.

[0092] The term "C" referred to herein 2-30 "Alkylene" means a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, including, for example, ethylene and propylene. 2-20 Alkylene, C 2-8 Alkylene, group -(CH2) n - (where n is 2 to 30, preferably 2 to 20, and more preferably 2 to 15).

[0093] The term "C" referred to herein 1-5 "Alkylene" means a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms, and includes, for example, methylene, ethylene (ethane-1,2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, butane-1,4-diyl, and pentane-1,5-diyl).

[0094] The term "C" referred to herein 2-10 "Alkylene" means a linear or branched divalent saturated hydrocarbon group having 2 to 10 carbon atoms, and includes, for example, ethylene (ethane-1,2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl), butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl, etc. 2-10 Alkylene is a group with 2 to 8 carbon atoms. 2-8 Alkylene" and "C 2-6 Contains "alkylene".

[0095] The term "C" referred to herein 2-8 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having 2 to 8 carbon atoms, and includes, for example, ethylene (ethane-1,2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl), butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl, and the like.

[0096] The term "C" referred to herein 2-8The term "alkenylene" refers to a divalent saturated hydrocarbon group having 2 to 8 carbon atoms, which is linear or branched and contains one or more double bonds, and includes, for example, -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, and octa-2,4,6-triene-1,8-diyl. When geometric isomers exist, each isomer and a mixture thereof are also included.

[0097] In the present invention, "aryl" means an aromatic carbocyclic group, for example, an aromatic carbocyclic group having 6 to 14 carbon atoms, and examples of aryl include phenyl, naphthyl (1-naphthyl and 2-naphthyl), etc. An example of aryl substituted with one or more hydroxy groups is 4-hydroxyphenyl.

[0098] In the present invention, "heteroaryl" refers to an aromatic ring group containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur atoms among the atoms constituting the ring, and may be partially saturated. The ring may be a monocyclic ring, or a bicyclic heteroaryl fused with a benzene ring or a monocyclic heteroaryl ring. The number of atoms constituting the ring is, for example, 4 to 15, preferably 5 to 14, and more preferably 6 to 10. Examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl, and preferably indol-2-yl.

[0099] The "divalent C" referred to in this specification 2-50The "hydrocarbon group" is not particularly limited, and examples thereof include linear, branched, cyclic and partially cyclic alkylene groups, alkenylene groups and alkynylene groups having 2 to 50 carbon atoms, and the groups may be divalent aromatic rings or may contain an aromatic ring as part of their structure.

[0100] The "divalent C" referred to in this specification 2-50 The "polyalkyleneoxy" is not particularly limited, and the alkylene group of the repeating unit may be a straight chain or a branched chain. 2-50 Examples of "polyalkyleneoxy" include divalent C 2-50 Polyethyleneoxy group, C 3-48 Polypropyleneoxy group, C 3-48 The group may be linked to other groups via an oxygen atom or a carbon atom, for example, C 2-50 The polyethyleneoxy group has -O(CH2CH2O) 1-25 -, -(CH2CH2O) 1-25 -, -(OCH2CH2) 1-25 -, -(CH2CH2O) 1-24 -(CH2CH2)- and so on.

[0101] The term "salt substance" referred to in this specification is not particularly limited as long as it is an inorganic substance soluble in water, and examples thereof include calcium salts such as calcium chloride and calcium phosphate, magnesium salts such as magnesium sulfate and magnesium chloride, aluminum salts such as aluminum sulfate and aluminum chloride, potassium salts such as potassium sulfate, potassium carbonate, potassium nitrate, potassium chloride, potassium bromide and potassium iodide, sodium salts such as sodium bicarbonate, sodium carbonate, sodium sulfate, sodium nitrate, sodium chloride, sodium bromide, sodium iodide, sodium silicate, trisodium phosphate, disodium phosphate, sodium borate, sodium acetate and sodium citrate, and lithium salts such as lithium chloride, lithium bromide, lithium iodide and lithium carbonate, and preferably includes sodium chloride, trisodium phosphate, disodium phosphate, potassium chloride, calcium chloride and magnesium chloride.

[0102] The term "antigen" referred to herein refers to a substance that induces immunity. For example, in lymph nodes or the spleen, it is a substance that can induce the activation of lymphocytes such as T cells and B cells by being presented by antigen-presenting cells. The term "antigen protein" refers to an antigen that is a protein. Furthermore, the term "antigen peptide" refers to an antigen that is a peptide. For example, an antigen peptide is a part of the amino acid sequence contained in an antigen protein, and includes a T cell-recognized epitope. It may also be a combination of multiple T cell-recognized epitopes.

[0103] The term "adjuvant" as referred to herein refers to a substance that enhances the immune response in a subject when administered to the subject in combination with a vaccine containing an antigen.

[0104] Hyaluronic acid derivatives containing repeating units represented by formula (I) In one embodiment, the hyaluronic acid derivative comprising a repeating unit represented by formula (I) is essentially composed of repeating units of (1) formula (I); and (2) formula (I) and formula (IIIc).

[0105] When the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) contains two or more repeating units of formula (I), the repeating units may be the same or different.The hyaluronic acid derivative may be modified at a position other than the repeating unit of formula (I), for example, the hydroxyl group may be -O(C 1-6 -O(alkyl), -O(formyl) and -O(C 1-6 The carboxyl group may be converted to an amide group or an ester group, or may form a salt.

[0106] According to one aspect, the group -ZN(R a )YX 1 is the following formula: -NH-(CH2) mz -NH-R; -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; -NH-(CH2) mz -COO-R; -NH-(CH2CH2O) m -CH2CH2-COO-R; -NH-(CH2) mz -O-COO-R; -NH-(CH2CH2O) m -CH2CH2-O-COO-R; -NH-(CH2) mz -SR; -NH-(CH2CH2O) m -CH2CH2-SR; -NH-(CH2) mz -O-CO-CH(R 10 )-CH2-SR; -NH-(CH2) mz -NHCO-CH(R 10 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-NHCO-CH(R 10 )-CH2-SR; -NH-(CH2CH2O) m -CH2CH2-O-CO-CH(R 10 )-CH2-SR; and -NH-(CH2) mz -SSR; -Z-NR a -Y-NR b -COO-R (where mz is an integer from 2 to 30, and R 10 is a hydrogen atom or a methyl group, and R and m are as previously defined herein. The group is preferably selected from groups represented by -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz-SSR (wherein mz, ​​R, and m are as previously defined herein). is a group selected from

[0107] In a preferred embodiment, Z in the above formula (I) is a direct bond. In addition, in one embodiment, when Z in the above formula (I) is a peptide linker, X 1 Ha-NR b -COO-R.

[0108] Specific examples of Y in the above formula (I) include -CH2CH2O-CH2CH2-SS-CH2CH2O-CH2CH2-, -(CH2CH2O)2-CH2CH2-SS-CH2CH2O-CH2CH2-, -CH2CH2O-CH2CH2-SS-(CH2CH2O)2-CH2CH2- and -(CH2CH2O)2-CH2CH2-SS-(CH2CH2O)2-CH2CH2-.

[0109] Y in the above formula (I) a As the alkyl group, -CH2- and -CH2-CH2- are preferred.

[0110] Y in the above formula (I) b Preferred examples of the alkyl group include -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, and octa-2,4,6-triene-1,8-diyl, and more preferred examples are -CH2-CH2- and -CH(CH3)CH2-.

[0111] In one embodiment, Z in the above formula (I) is —NH—[CH(—Z a )-CONH] n-1 -CH(-Z a )-CO-, where n is an integer of 2 to 30; Z a are each independently H2N-CH(-Z a The peptide linker is attached to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R)-COOH at the C-terminus.a )-YX 1 Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as natural (L-form) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as their D-forms. All α-amino acids, including synthetic amino acids, can be used. That is, Z a Examples of the linker include -CH3, H2NC(NH)NH(CH2)3-, and H2NCOCH2-. Furthermore, n Z's may be the same or different. n is an integer of 2 to 30, preferably 2 to 10, and more preferably 2 to 4. Preferred examples of the peptide linker include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, and -Phe-Gly-.

[0112] The group -ZN(R a )YX 1Specific examples of the cholesteryl include -NH-(CH2)2-NH-CO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C Examples include -NH-(CH)-NH-(CH)-COO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH-(CH)-NH-COO-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH-CO-NH-cholesteryl, -NH-(CH)-NH-(CH)-N(-(CH)-NH-CO-cholesteryl, and -NH-(CH)-NH-(CH)-N(-(CH)-NH-cholesteryl). a )YX 1 In a preferred embodiment, R a , R b and R c is a hydrogen atom, and Y is a linear C 2-30 Alkylene or -(CH2CH2O) m -CH2CH2-, and Y a However, linear C 1-5 alkylene, or Y b However, linear C 2-8 Alkylene or linear C 2-8 It is alkenylene.

[0113] In one embodiment, the group ZN(R a )YX 1 Z is a direct bond, R a is a hydrogen atom, Y is, for example, C 2-12 Alkylene, preferably C 2-6 Alkylene, more preferably C6 alkylene, X 1 Ga-NR b -COO-R, R b is a hydrogen atom and R is a cholesteryl group.

[0114] In one embodiment, the hyaluronic acid derivative comprising the repeating unit represented by formula (I) further comprises a repeating unit represented by formula (IIIc). When two or more repeating units represented by formula (IIIc) are contained, the repeating units may be the same or different.

[0115] Q of the above formula (IIIc) + is not particularly limited as long as it is a counter cation that forms a salt with a carboxyl group in water, and if it is divalent or higher, it forms a salt with multiple carboxyl groups depending on the valence. Examples of counter cations include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, and calcium ion; + R j R k R l R m (In the formula, R j , R k , R l and R m are each independently a hydrogen atom and C 1-6 Preferred examples include sodium ions, potassium ions, and tetraalkylammonium ions (e.g., tetra-n-butylammonium ions). j , R k , R l and R m is C 1-6 Preferably, they are identical groups selected from alkyl, and preferably n-butyl groups.

[0116] The group R of formula (I) 1 , R 2 , R 3 , and R 4 and R in the above formula (IIIc) 1a , R 2a , R 3a and R 4a are preferably all hydrogen atoms. a and R b are preferably all hydrogen atoms.

[0117] The group R of formula (I) 5 is preferably acetyl.

[0118] In one embodiment, the hyaluronic acid derivative comprising the repeating unit represented by the above formula (I) is substantially composed of the repeating units of formula (I) and (IIIc).In this hyaluronic acid derivative, for example, 80% or more, preferably 90% or more, more preferably 95% or more of the repeating units of the disaccharide consisting of D-glucuronic acid and N-acetylglucosamine contained in this derivative are the repeating units of formula (I) or (IIIc).In one embodiment, it is composed only of the repeating units represented by the above formula (I) and formula (IIIc).

[0119] Y defined in formula (I) can be, for example, -(CH2) na - (wherein na is selected from integers of 2 to 20, preferably 2 to 15, more preferably 2 to 12), and preferably -(CH2)2-, -(CH2)6-, -(CH2)8- and -(CH2) 12 -, and more preferably -(CH2)6-. These Y's are preferred from the viewpoint of precipitation and stable dispersion, which will be described later.

[0120] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by formula (I) has an introduction rate of hydrophobic groups relative to the repeating units of the disaccharide present in the derivative of, for example, 1 to 50%, preferably 7 to 50%, more preferably 17 to 50%, and even more preferably 20 to 45%.When the introduction rate is within the above range, the hyaluronic acid derivative containing the repeating unit represented by formula (I) can efficiently form a complex with an antigen in solution.

[0121] Here, the introduction rate of the hydrophobic group is calculated by the following formula:

[0122]

number

[0123] The hyaluronic acid derivative containing the repeating unit represented by the above formula (I) is preferably R 1c , R 2c , R 3c , and R 4c are all hydrogen atoms, and R 5c is acetyl, and X c -O - Na + The hyaluronic acid or its derivatives are synthesized from a raw material consisting solely of repeating units represented by formula (IIIc), which have a weight-average molecular weight of, for example, 1 kDa to 500 kDa, preferably 3 kDa to 500 kDa, and more preferably 5 kDa to 200 kDa. In one embodiment, the weight-average molecular weight of the raw material is preferably 1 kDa to 1000 kDa, and more preferably 3 kDa to 200 kDa, from the viewpoint of complex formation with an antigen. The weight-average molecular weight of the raw material is preferably 1 kDa to 1000 kDa, and more preferably 3 kDa to 500 kDa, and even more preferably 5 kDa to 200 kDa, from the viewpoint of lymph node migration of the complex.

[0124] In addition, since it is generally difficult to obtain hyaluronic acid and its derivatives as a single product, its molecular weight is calculated as number-average molecular weight or weight-average molecular weight.In the present invention, it is calculated as weight-average molecular weight.For the method of measuring weight-average molecular weight, various known methods can be used, such as light scattering method, osmotic pressure method, viscosity method, etc., as described in "Essential Polymer Science" by Nakahama Seiichi et al. (published by Kodansha, ISBN4-06-153310-X).The viscosity-average molecular weight shown in this specification can also be measured by the method commonly used in the technical field to which the present invention belongs, such as using Ubbelohde viscometer.When using commercially available hyaluronic acid and its derivatives that indicate molecular weight, the indicated numerical value can also be used as molecular weight.

[0125] The hyaluronic acid derivative that comprises the repeating unit of the above formula (I) is converted into amide group the carboxyl group of glucuronic acid, which is one of the disaccharides that constitute repeating unit, and introduces hydrophobic group.By adjusting the modification degree of hyaluronic acid derivative, it is also possible to control the pharmacokinetics of the preparation that uses this derivative to produce.

[0126] If the carboxyl group modification rate of the glucuronic acid moiety of the hyaluronic acid derivative containing the repeating unit of the above formula (I) is high, the binding to hyaluronic acid receptors such as CD44 is suppressed, and the hyaluronic acid derivative becomes a drug carrier (including vaccines) that remains in the body for a long time. Furthermore, by introducing targeting elements into the hyaluronic acid derivative, it can be targeted to various organs and cells, including lymph nodes. Targeting elements include, for example, target tissue-specific peptides, antibodies, fragmented antibodies, aptamers, RGD peptides for cancer cells, folic acid, anisamide, transferrin, galactose for the liver, tocopherol, etc.

[0127] The modification rate of the carboxyl group of the glucuronic acid moiety of the hyaluronic acid derivative containing the repeating unit of the above formula (I) with a hydrophobic group, i.e., the introduction rate of the hydrophobic group, is preferably 4 to 60%, more preferably 5 to 50%, even more preferably 7 to 50%, and even more preferably 7 to 45% from the viewpoint of complex formation with an antigen, and is preferably 6 to 60%, more preferably 17 to 50%, even more preferably 20 to 50%, and even more preferably 20 to 45% from the viewpoint of lymph node migration of the complex.

[0128] The molecular weight of the raw material of the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) and the introduction rate of hydrophobic groups are preferably 3kDa to 500kDa and 4 to 60%, more preferably 3kDa to 200kDa and 6 to 50%, more preferably 5kDa to 200kDa and 6 to 50%, and more preferably 5kDa to 150kDa and 6 to 45% from the viewpoint of complex formation with antigen. From the viewpoint of lymph node migration of the complex, preferably 3kDa to 500kDa and 4 to 60%, more preferably 3kDa to 200kDa and 6 to 50%, more preferably 5kDa to 200kDa and 6 to 50%, and more preferably 5kDa to 150kDa and 20 to 45%. From the viewpoint of stable dispersion, 3 kDa to 200 kDa and 4 to 60% are preferred, 3 kDa to 200 kDa and 6 to 50% are more preferred, 5 kDa to 200 kDa and 6 to 50% are even more preferred, and 5 kDa to 150 kDa and 17 to 45% are even more preferred. From the viewpoint of improving blood retention, 5 kDa to 27 kDa and 2 to 50% are preferred, 5 kDa to 27 kDa and 8 to 35% are even more preferred, 5 kDa to 18 kDa and 8 to 35% are even more preferred, and 5 kDa to 18 kDa and 15 to 22% are even more preferred. From the viewpoint of gelation, 5 kDa to 300 kDa and 2 to 30% are preferable, 5 kDa to 50 kDa and 2 to 22% are more preferable, 5 kDa to 27 kDa and 2 to 22% are even more preferable, and 5 kDa to 27 kDa and 7 to 22% are even more preferable.

[0129] Hyaluronic acid derivatives containing repeating units represented by formula (II) In one embodiment, the hyaluronic acid derivative containing the repeating unit represented by formula (II) is substantially composed of the repeating units of (1) formula (II); (2) formula (II) and formula (III); (3) formula (II) and formula (IIIc); or (4) formula (II), formula (III), and formula (IIIc). Of the repeating units of the disaccharide consisting of D-glucuronic acid and N-acetylglucosamine contained in the hyaluronic acid derivative, for example, 80% or more, preferably 90% or more, more preferably 95% or more are repeating units of formula (II), (III), or formula (IIIc). In one embodiment, the hyaluronic acid derivative is composed only of the repeating units of formula (1) formula (II); (2) formula (II) and formula (III); (3) formula (II) and formula (IIIc); or (4) formula (II), formula (III), and formula (IIIc).

[0130] The ratio of a specific disaccharide unit to the repeating disaccharide units present in a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) means the ratio of a specific disaccharide unit to all disaccharide units contained in a certain amount of a hyaluronic acid derivative containing a repeating unit represented by the above formula (II), which is a polysaccharide having disaccharide units as repeating units.

[0131] In the formula (II) representing the disaccharide unit contained in the hyaluronic acid derivative containing the repeating unit represented by the formula (II), R 1a , R 2a , R 3a , and R 4a Preferably, all of R are hydrogen atoms. 5a is a hydrogen atom or C 1-6 Preferably, R is alkylcarbonyl, more preferably hydrogen atom or acetyl, and even more preferably acetyl. In addition, in formulas (III) and (IIIc) representing the disaccharide unit contained in the hyaluronic acid derivative containing the repeating unit represented by formula (II), R 1b , R 2b , R 3b and R 4b , and R 1c , R 2c, R 3c and R 4c Preferably, all of R are hydrogen atoms. 5b and R 5c is a hydrogen atom or C 1-6 It is preferably an alkylcarbonyl, more preferably a hydrogen atom or acetyl, and most preferably both are acetyl.

[0132] R in formula (II) aa Specific examples include a hydrogen atom, methyl, hydroxymethyl, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, 1-methylpropyl, 2-methylpropyl, isopropyl, 2-carboxyethyl, 2-methylthioethyl, 2-carbamoylethyl, phenylmethyl, (4-hydroxyphenyl)methyl, and indol-3-ylmethyl.

[0133] group-CHR aa When - is an asymmetric center, each optically active substance and a mixture thereof are included, but H2N-CHR aa When written as -COOH (amino acid), it is preferably in the L form (natural form).

[0134] In formula (II), R 6a , R 7 , R 8 , and R 9 are, for example, independently a hydrogen atom or methyl, and preferably all are hydrogen atoms.

[0135] The group -CHR in formula (II) aa -CO-X 1a As an embodiment of the present invention, for example, a group -CHR aa Specific examples of the group include the following groups:

[0136] [ka] where the asterisk stands for -NR 6a - represents the bonding position (same below).

[0137] group-CHR aa Preferred examples of —COOH include the following groups:

[0138] [ka] group-CHR aa Preferred examples of —COOH include the following groups:

[0139] [ka] group-CHR aa Preferred examples of —COOH include the following groups:

[0140] [ka] group-CHR aa Preferred examples of —COOH include the following groups:

[0141] [ka] In terms of delivery of the conjugate to lymph nodes, the -CHR group aa Preferred examples of —COOH include the following groups:

[0142] [ka] More preferred examples include the following groups:

[0143] [ka] More preferred examples include the following groups:

[0144] [ka] The group -CHR as described above aa-COOH is a group in which part or all of it is a -CHR aa -CONH-Z 1 -Z 2 The group -Z may be converted to 1 -Z 2 Examples are given below.

[0145] The group -CHR in formula (II) aa -CO-X 1a In another embodiment, for example, the group -CHR aa Specific examples of the group include the following groups:

[0146] [ka] group-CHR aa Preferred examples of -CONH2 include the following groups:

[0147] [ka] group-CHR aa Preferred examples of -CONH2 include the following groups:

[0148] [ka] group-CHR aa Preferred examples of -CONH2 include the following groups:

[0149] [ka] group-CHR aa Preferred examples of -CONH2 include the following groups:

[0150] [ka] These groups are also preferred groups in that they have both biodegradability and blood retention.

[0151] In terms of having both biodegradability and blood retention, the -CHR group aa Preferred examples of -CONH2 also include the following groups:

[0152] [ka] In order to have both biodegradability and blood retention, the -CHR group aa More preferred examples of -CONH2 include the following groups:

[0153] [ka] From the viewpoint of better dispersibility in pure water, the group -CHR aa Preferred examples of -CONH2 include the following groups:

[0154] [ka] These two groups are also preferred examples from the viewpoint of being base materials for injection preparations for sustained subcutaneous administration.

[0155] From the viewpoint of a base material for a sustained subcutaneous injection formulation, the -CHR aa Preferred examples of -CONH2 include the following groups:

[0156] [ka] R 7 As the , a hydrogen atom and methyl are more preferred, and a hydrogen atom is even more preferred.

[0157] Carboxy as defined in formulas (II), (III) and (IIIc) is a group of formula -COO - Q + In this case, Q may form a salt represented by the formula: +is not particularly limited as long as it is a counter cation that forms a salt with carboxy in water, and when it is divalent or higher, it forms a salt with multiple carboxy depending on the valence. Examples of counter cations include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, and calcium ion; + R j R k R l R m (In the formula, R j , R k , R l and R m are each independently a hydrogen atom and C 1-6 Preferred examples include sodium ions, potassium ions, and tetraalkylammonium ions (e.g., tetra-n-butylammonium ions). j , R k , R l and R m is C 1-6 Preferably, they are identical groups selected from alkyl, and preferably n-butyl.

[0158] The group -CHR in formula (II) aa -CO-X 1a In another embodiment, for example, the group -CHR aa -CONH-Z 1 -Z 2 Specific examples of the group include the following groups.

[0159] [ka] Other specific examples of the group include the following groups.

[0160] [ka] group-CHR aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0161] [ka] group-CHR aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0162] [ka] group-CHR aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0163] [ka] In terms of delivery of the conjugate to lymph nodes, the -CHR group aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0164] [ka] More preferred groups include the following groups:

[0165] [ka] More preferred examples include the following groups:

[0166] [ka] In order to have both biodegradability and blood retention, the -CHR group aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0167] [ka] Group-Z 1 -Z 2 Examples of the group -(C 2-10 (Alkylene)-NH-COO-Z 3 In addition, the group -(C 2-12 (Alkylene)-NH-COO-Z 3 Here, C 2-12 Preferred examples of alkylene include -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2) 10 - and -(CH2) 12 -, more preferably -(CH2)2- and -(CH2)6-. Furthermore, the group -Z 1 -Z 2 An example is the group -(CH2CH2O) ma -CH2CH2-NH-Z 3 Here, ma is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. A specific example of a preferred ma is 2. The group -Z 1 -Z 2 As an example, the group -(hexane-1,6-diyl)-NH-COO-Z is preferably 3 , the group -(ethane-1,2-diyl)-NH-COO-Z 3 and the group -(CH2CH2O)2-CH2CH2-NH-Z 3 More preferred are the groups -(hexane-1,6-diyl)-NH-COO-cholesteryl, -(ethane-1,2-diyl)-NH-COO-cholesteryl, and -(CH2CH2O)2-CH2CH2-NH-cholanoyl, and even more preferred is the group -(hexane-1,6-diyl)-NH-COO-cholesteryl. 1 , Z 2 , group-Z 1 -Z 2 Examples of the hyaluronic acid derivatives containing the repeating unit represented by the above formula (I) include those in which Y and X 1 , group-YX 1 and the group -CO-NR ca -Z 3and the group -O-CO-NR ca -Z 3 An example of this is R ca and each of the groups represented by the formula (I) is a hydrogen atom.

[0168] In one embodiment, the hyaluronic acid derivative comprising a repeating unit of formula (II) is a group X 1a In X 1a Ga-NR 9 -Z 1 -Z 2 , R 9 is a hydrogen atom, Z 1 For example, C 2-12 Alkylene, preferably C 2-6 Alkylene, more preferably C6 alkylene, Z 2 Ga-NR ba -COO-Z 3 , R ba is a hydrogen atom, and Z 3 is a steryl group, and the group R aa is a hydrogen atom or C 1-6 alkyl, wherein the alkyl is independently hydroxy, carboxy, carbamoyl, C 1-6 It may be optionally substituted with one or more groups selected from alkylthio, aryl, and heteroaryl, wherein the aryl may be optionally substituted with one or more hydroxy.

[0169] Preferred group R in terms of lymph node delivery of the conjugate aa Specific examples of include methyl, hydroxymethyl, a hydrogen atom, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, benzyl, (4-hydroxyphenyl)methyl, 1-methylpropyl, 2-methylpropyl, isopropyl, indol-3-ylmethyl, 2-carbamoylethyl, and 2-carboxyethyl, and are preferably methyl, hydroxymethyl, 1-hydroxyethyl, 1-methylpropyl, and 2-carbamoylethyl, and more preferably methyl and 2-carbamoylethyl.

[0170] In one embodiment, a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) including a repeating unit represented by formula (III) is also preferred. In a more preferred embodiment, X in formula (II) 1a and X in formula (III) 2 In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is 1a Ga-NR 9 -Z 1 -Z 2 The compound may contain a repeating unit represented by formula (II), a repeating unit represented by formula (III) and a repeating unit represented by formula (IIIc).

[0171] In yet another aspect, the hyaluronic acid derivatives containing the repeating units of formula (II) above have a group -NR relative to the repeating units of the disaccharide present. 9 -Z 1 -Z 2 The proportion of repeating units of formula (II) and / or formula (III) having a hydrophobic group (hereinafter also referred to as a hydrophobic group) (introduction rate of hydrophobic group) may be 3 to 50%.

[0172] Here, the introduction rate of the hydrophobic group is calculated by the following formula:

[0173]

number

[0174] The introduction rate of hydrophobic groups in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is, for example, 3 to 50%, preferably 5 to 40%, more preferably 5 to 35%, even more preferably 5 to 25%, more preferably 5 to 20%, and even more preferably 5 to 10%. From the viewpoint of complex formation with antigen, it is preferably 3 to 60%, more preferably 7 to 50%, even more preferably 18 to 45%, and even more preferably 20 to 35%. From the viewpoint of lymph node migration of the complex, it is preferably 3 to 60%, more preferably 7 to 50%, even more preferably 18 to 45%, and even more preferably 20 to 35%.

[0175] In one embodiment, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is 1a Ga-NR 9 -Z 1 -Z 2In this case, the ratio of the disaccharide units of formula (II) to the disaccharide repeating units present in the hyaluronic acid derivative is, from the viewpoint of both biodegradability and blood retention, for example, 70% or more, preferably 75% or more, and more preferably 90% or more. The upper limit may be 100% or less. The range of this ratio is, for example, 70 to 100%, preferably 75 to 100%, and more preferably 90 to 100%. Furthermore, from the viewpoint of the ability to form a complex with an antigen, the ratio is, for example, 10% or more, preferably 20% or more, more preferably 50% or more, more preferably 70% or more, and even more preferably 90% or more. The upper limit may be 100% or less. The range of this ratio is, for example, 10 to 100%, preferably 20 to 100%, more preferably 50 to 100%, more preferably 70 to 100%, and even more preferably 90 to 100%. Furthermore, from the viewpoint of the complex having lymph node migration properties, the ratio is, for example, 10% or more, preferably 20% or more, more preferably 50% or more, even more preferably 70% or more, and even more preferably 90% or more. The upper limit may be 100% or less. The ratio range is, for example, 10 to 100%, preferably 20 to 100%, even more preferably 50 to 100%, even more preferably 70 to 100%, and even more preferably 90 to 100%. The hyaluronic acid derivative containing the repeating unit represented by the above formula (II) may further contain a repeating unit represented by formula (III).

[0176] In one embodiment, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is X 1 Ga-NR 9 -Z 1 -Z 2In this case, the sum of the proportion of the repeating units represented by formula (II) and the proportion of the repeating units represented by formula (III) in the repeating units of the disaccharide present is, for example, 70 to 100%, preferably 80 to 100%, and more preferably 90 to 100%. From the viewpoint of complex formation with an antigen, it is, for example, 7 to 100%, preferably 20 to 100%, and more preferably 30 to 100%. From the viewpoint of lymph node migration of the complex, it is, for example, 20 to 100%, preferably 30 to 100%, and more preferably 70 to 100%.

[0177] In the hyaluronic acid derivative containing the repeating unit represented by formula (II), the proportion of the repeating unit represented by formula (III) in the repeating units of the disaccharide present is preferably 3 to 50%, more preferably 5 to 40%, even more preferably 5 to 35%, even more preferably 5 to 25%, even more preferably 5 to 20%, and even more preferably 5 to 10%. From the viewpoint of complex formation with the antigen, it is preferably 3 to 60%, even more preferably 7 to 50%, even more preferably 18 to 45%, and even more preferably 20 to 35%. From the viewpoint of lymph node migration of the complex, it is preferably 3 to 60%, even more preferably 7 to 50%, even more preferably 18 to 45%, and even more preferably 20 to 35%.

[0178] In addition, in hyaluronic acid derivatives containing repeating units represented by formula (II), the proportion of repeating units represented by formula (II) in the repeating units of the disaccharides present is preferably 20 to 97%, more preferably 30 to 95%, even more preferably 35 to 95%, even more preferably 45 to 95%, even more preferably 50 to 95%, and even more preferably 60 to 95%. From the viewpoint of complex formation with antigen, it is, for example, 7 to 100%, preferably 20 to 100%, and more preferably 30 to 100%. From the viewpoint of lymph node migration of the complex, it is, for example, 20 to 100%, preferably 30 to 100%, and more preferably 70 to 100%.

[0179] Methods for producing hyaluronic acid derivatives containing repeating units represented by the above formula (I) and hyaluronic acid derivatives containing repeating units represented by the above formula (II) include the methods described in WO 2010 / 053140 and WO 2014 / 038641.

[0180] According to one aspect, hyaluronic acid derivatives containing repeating units represented by formula (I) or (II) are characterized by forming microparticles through association in water. It is believed that spontaneous association occurs in water due to the hydrophobic interaction of the introduced hydrophobic groups, forming microparticles. Taking advantage of this property, they can be used as vaccine carriers, lymph node delivery carriers, sustained blood release carriers, and targeting carriers. The particle size of the microparticles is not particularly limited, but is, for example, 1 μm or less, preferably 500 nm, more preferably 200 nm or less, even more preferably 100 nm or less, and even more preferably 50 nm or less. Methods for microparticulating hyaluronic acid derivatives containing repeating units represented by formula (I) and hyaluronic acid derivatives containing repeating units represented by formula (II) include the methods described in International Publication No. 2010 / 053140 and International Publication No. 2014 / 038641.

[0181] As the raw material for producing the hyaluronic acid derivative that comprises the repeating unit represented by the above formula (I) and the hyaluronic acid derivative that comprises the repeating unit represented by the above formula (II), hyaluronic acid or its salt or its derivative can be used.As hyaluronic acid salt, for example, alkali metal salt such as sodium salt, potassium salt, lithium salt and the like can be listed, and particularly preferred salt is the sodium salt that is commonly used as medicine.Hyaluronic acid or its pharmaceutically acceptable salt can be produced by various known methods such as extracting from the organism such as chicken comb or pig subcutaneous tissue or biological fermentation method, or can be obtained by purchasing commercially available products (for example, from Denka Co., Ltd., Shiseido Co., Ltd., Seikagaku Corporation, R&D system Co., Ltd., etc.).

[0182] Antigens complexed with hyaluronic acid derivatives containing repeating units represented by formula (I) or hyaluronic acid derivative microparticles containing repeating units represented by formula (II) may be released from the complex after administration due to degradation or disintegration of the hyaluronic acid derivative microparticles and the replacement of the antigen with biological components such as albumin. The release rate can be controlled by adjusting the hydrophobic group introduction rate, molecular weight, and amino acid introduction rate of the hyaluronic acid derivative. Alternatively, the antigen release rate can be controlled by chemically crosslinking and gelling the hyaluronic acid derivative using the method described in WO 2010 / 053140. Alternatively, the antigen release rate can be controlled by conjugating the hyaluronic acid derivative with the antigen using the method described in WO 2010 / 053140.

[0183] antigen Antigens used to produce vaccine preparations by forming complexes with hyaluronic acid derivatives containing repeating units represented by the above formula (I) or hyaluronic acid derivatives containing repeating units represented by the above formula (II) include, for example, antigen peptides and antigen proteins, as well as DNA and mRNA encoding these antigen sequences, preferably antigen peptides and antigen proteins, and more preferably antigen peptides.

[0184] The antigen may be a cancer antigen. A cancer antigen is an antigen that is highly expressed on cancer cells, and in some cases, is expressed only by cancer cells. A cancer antigen may be expressed within or on the surface of a cancer cell.

[0185] Antigen proteins that can be used in the present invention include, but are not limited to, MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-Xp4), MAGE-Xp5 (MAGE-Xp6), MAGE-Xp7 (MAGE-Xp8), MAGE-Xp9 (MAGE-Xp9), MAGE-Xp10 (MAGE-Xp11), MAGE-Xp22 (MAGE-Xp12), MAGE-Xp23 (MAGE-Xp14), MAGE-Xp24 (MAGE-Xp15), MAGE-Xp25 (MAGE-Xp16), MAGE-Xp26 (MAGE-Xp17), MAGE-Xp27 (MAGE-Xp18), MAGE-Xp28 (MAGE-Xp19), MAGE-Xp29 (MAGE-Xp2 ... -Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2, G AGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, Tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM 2 ganglioside, GD2 ganglioside, human papillomavirus proteins, the Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, and c-erbB-2.

[0186] The antigen protein may have its entire sequence or a sequence with a partial deletion.

[0187] The antigenic peptide is an antigenic peptide that contains, in the sequence of an antigenic protein, one or more epitopes recognizable by CD8-positive cytotoxic T cells and / or CD4-positive helper T cells. In one embodiment, the antigenic peptide is preferably an antigenic peptide that contains two or more epitopes, from the viewpoint of being loaded onto an MHC class I molecule or an MHC class II molecule after degradation in an antigen-presenting cell. Specifically, the antigenic peptide includes an antigenic peptide containing an epitope of an antigenic protein of a tumor cell.

[0188] In one aspect, the antigen peptide has, for example, 8 to 120 amino acids, preferably 8 to 80 amino acids, more preferably 15 to 80 amino acids, more preferably 16 to 80 amino acids, more preferably 23 to 80 amino acids, more preferably 23 to 60 amino acids, and more preferably 23 to 50 amino acids.

[0189] In one aspect, from the viewpoint of inducing activation of cytotoxic T cells (CTLs) by helper T cells, the antigen peptide is an antigen peptide that contains one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0190] In one aspect, when two or more epitopes are contained, an amino acid linker may be placed between the epitopes. The linker has, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, and more preferably 4 to 8 amino acids. Examples of amino acids used in the linker include glycine (G), tyrosine (Y), leucine (L), and tryptophan (W). Tyrosine (Y), leucine (L), and tryptophan (W) are preferred. Specific examples of amino acid linkers include -YYYY-(4Y), -LLLL-(4L), -WWWW-(4W), -GGGGGG-(6G), -YYYYYY-(6Y), -LLLLLL-(6L), -WWWWWW-(6W), -YYYYYYYY-(8Y), -LLLLLLLL-(8L) and -WWWWWWWW-(8W), with 6Y, 6L and 6W being preferred.

[0191] Complex The complex of the present invention between the hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen can be prepared by mixing the hyaluronic acid derivative containing the repeating unit represented by formula (I) or the hyaluronic acid derivative containing the repeating unit represented by formula (II) with an antigen in an appropriate solution. The solution used may be water, physiological saline, various buffer solutions, sugar solutions, DMSO, ethanol, DMF, or a combination thereof. Solvent-buffer exchange may be performed by dialysis or other methods.

[0192] For example, the complex of the present invention can be produced by mixing an antigen with microparticles of a hyaluronic acid derivative containing a repeating unit represented by the above formula (I) or a hyaluronic acid derivative containing a repeating unit represented by the above formula (II). Although not particularly limited, a complex of the hyaluronic acid derivative and the antigen can be produced by forming a complex between the microparticles of the hyaluronic acid derivative and the antigen. Furthermore, a complex of the hyaluronic acid derivative and the antigen can be produced by encapsulating the antigen in the microparticles of the hyaluronic acid derivative. The complex includes an antigen inclusion body in the microparticles of the hyaluronic acid derivative. In the inclusion body, the antigen is coated with the above hyaluronic acid derivative.

[0193] A method for complex formation includes adding an antigen solution to preformed microparticles of a hyaluronic acid derivative containing a repeating unit represented by formula (I) or a hyaluronic acid derivative containing a repeating unit represented by formula (II). In this method, the formed microparticles of the hyaluronic acid derivative and the antigen form complexes through interactions such as hydrophobic interactions, electrostatic interactions, and hydrogen bonds. The interactions can occur either on the surface of the microparticles or inside the microparticles. Conditions such as solvent, salt concentration, pH, temperature, time, and addition of denaturant can be appropriately selected to ensure stable and high-yield complex formation of the antigen. For example, the salt concentration and pH during complex formation affect the swelling degree and density of the hyaluronic acid derivative microparticles, as well as the ionization state of the antigen. Therefore, appropriate conditions can be selected depending on the combination of these factors. By performing complexation under low salt concentrations, electrostatic repulsion between the carboxyl groups of the hyaluronic acid derivative can be utilized to reduce the microparticle density, increase the amount of complexation, and allow complexation with antigens of higher molecular weight. After complex formation, increasing the salt concentration weakens electrostatic repulsion, increases the particle density, and makes the gel network smaller than the antigen size, thereby firmly retaining the antigen and delaying its release. In this case, the salt concentration can be set to physiological salt concentration. Ultrasonic irradiation can be performed using an ultrasonic homogenizer or a focused ultrasonic irradiation device to achieve micronization and uniform size. Ultrasonic irradiation can be performed after mixing the hyaluronic acid derivative and drug, or it can be performed on the hyaluronic acid derivative alone. Uncomplexed free antigen can be separated and removed using dialysis, size exclusion chromatography (SEC), or other methods.

[0194] In addition, as a complex formation method, a hyaluronic acid derivative containing a repeating unit represented by the above formula (I) or a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) can be dissolved in an aprotic polar organic solvent such as DMSO or DMF, mixed with an antigen, and then substituted with water, a salt solution, or various buffer solutions, thereby simultaneously forming microparticles and complexes. Substitution can be performed, for example, by dialysis. Ultrasonic irradiation can be performed using an ultrasonic homogenizer or a focused ultrasonic irradiation device to achieve micronization and uniform size. Ultrasonic irradiation can be performed after mixing the hyaluronic acid derivative and antigen, or on the hyaluronic acid derivative alone. It can also be performed before or after dialysis. This method makes the antigen complexed with the hyaluronic acid derivative less likely to be released from the complex. Conditions for mixing the hyaluronic acid derivative and antigen, such as the solvent type, salt concentration, pH, temperature, time, addition of a denaturant, hyaluronic acid derivative concentration, antigen concentration, and ratio of hyaluronic acid derivative to antigen, can be appropriately selected so as to ensure stable antigen and high-yield complex formation. Although not particularly limited, conditions such as the type of solvent, salt concentration, pH, temperature, time, number of times, whether or not a denaturant is added when replacing with water, a salt solution, or various buffer solutions, the hyaluronic acid derivative concentration, antigen concentration, and the ratio of hyaluronic acid derivative to antigen may be appropriately selected so that the antigen forms a stable complex with a high yield.The size of the microparticles can also be obtained by appropriately selecting these conditions.Free antigen that has not been complexed can be separated and removed by dialysis, size exclusion chromatography (SEC), or the like.

[0195] When the complex is in the form of particles, the particle size is not particularly limited, but from the viewpoint of lymph node transfer, it is, for example, 20 to 200 nm, preferably 20 to 100 nm, and more preferably 20 to 50 nm.

[0196] vaccine preparations The above complex can be used to produce the vaccine preparation of the present invention, specifically, a vaccine preparation for use in the prevention and / or treatment of cancer.

[0197] The vaccine formulation of the present invention may be administered via oral, parenteral, intranasal, intravaginal, intraocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intraarticular, intracerebral or intraoral routes, preferably via subcutaneous, intramuscular, intravenous or intradermal routes.

[0198] The vaccine preparation of the present invention can be administered in any suitable form depending on the intended route of administration as a pharmaceutical composition containing one or more pharmaceutically acceptable diluents, wetting agents, emulsifiers, dispersing agents, adjuvants, preservatives, buffers, binders, stabilizers, etc. The route of administration may be parenteral or oral.

[0199] The vaccine formulations of the present invention may be administered in combination with one or more adjuvants. The adjuvant used may be any substance that enhances the activity of antigen-presenting cells. More specifically, substances that activate innate immune receptors (pattern recognition receptors), other antigen-presenting cell stimulators, or substances that inhibit the acquisition of immunosuppressive activity by antigen-presenting cells may be selected. Innate immune receptors are classified into Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and cytoplasmic DNA sensors. Innate immune receptor agonist adjuvants can be selected from inactivated bacterial cells, bacterial cell extracts, nucleic acids, lipopolysaccharides, lipopeptides, and synthetic low-molecular-weight compounds. Preferred adjuvants include CpG oligonucleotides, poly(IC) RNA, imidazoquinones (e.g., R848 and imiquimod), saponins (e.g., QuilA and QS21), STING agonists (e.g., cyclic di-GMP), monophosphoryl lipids, and lipopeptides. Taxane drugs and anthracycline drugs can be used as adjuvants, which are antigen-presenting cell stimulators. Adjuvants, which are drugs that inhibit the acquisition of immunosuppressive activity by antigen-presenting cells, are selected from JAK / STAT inhibitors, indole deoxygenase (IDO) inhibitors, tryptophan deoxygenase (TDO) inhibitors, and the like. These inhibitors include compounds that antagonize the factor, as well as neutralizing antibodies, small interfering RNA (siRNA), and antisense DNA for the factor.

[0200] The adjuvant used in the vaccine formulation of the present invention is preferably an innate immune receptor agonist, and more preferably an agonist of Toll-like receptors (TLRs) or cytoplasmic DNA sensors. Preferred TLR agonists include CpG oligonucleotides, poly(IC) RNA, imidazoquinones such as R848 and imiquimod, saponins such as QuilA and QS21, STING agonists, and monophosphoryl lipids.

[0201] The adjuvant may be administered as a separate formulation from the vaccine formulation, or as a combined formulation. Alternatively, the adjuvant may be conjugated to a hyaluronic acid derivative containing a repeating unit represented by formula (I) using the method described in International Publication No. 2010 / 053140. When administered in combination, the vaccine formulation of the present invention may be administered at a dose of, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably, for example, 0.1 to 20 mg / dose, while the adjuvant may be administered at a dose of, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and more preferably 0.1 to 10 mg / kg body weight. The adjuvant may be administered at the same time as the vaccine formulation of the present invention (including when the vaccine formulation contains an adjuvant), or at different times. When administered at different times, it is preferable to administer one adjuvant followed by the other, for example, within 1 minute to 5 hours, preferably within 1 minute to 1 hour.

[0202] The vaccine formulation of the present invention may be administered in combination with one or more antibodies used in cancer treatment. The antibodies are, for example, antibodies that inhibit tumor-induced immunosuppressive signals or antibodies that activate immune cell costimulatory signals, preferably antibodies that inhibit tumor-induced immunosuppressive signals or antibodies that activate immune cell costimulatory signals. Specifically, the antibodies are one or more antibodies selected from anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-OX40, and anti-4-1BB antibody. When administered in combination, the dose of the vaccine formulation of the present invention is, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably 0.1 to 20 mg / dose, and the dose of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, and more preferably 1 to 40 mg / kg body weight. The antibody may be administered at the same time as the vaccine formulation of the present invention (including when the vaccine formulation contains an antibody) or at a different time. When they are administered at different times, it is preferable to administer one after the other within, for example, 1 minute to 24 hours, preferably 1 minute to 5 hours.

[0203] The vaccine formulation of the present invention may be administered in combination with both the adjuvant and the antibody. In this case, the dose of the vaccine formulation of the present invention is, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably, for example, 0.1 to 20 mg / dose. The dose of the adjuvant is, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and more preferably 0.1 to 10 mg / kg body weight. The dose of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, and more preferably 1 to 40 mg / kg body weight. The adjuvant and antibody may be administered at the same time as the vaccine formulation of the present invention (including cases where the vaccine formulation contains both the adjuvant and the antibody) or at different times. When administered at different times, it is preferable that the vaccine formulation, adjuvant, and antibody are all administered within, for example, 1 minute to 24 hours, preferably 1 minute to 5 hours. [Example]

[0204] Preferred specific embodiments of the present invention will be described below as examples.

[0205] [Example 1] Synthesis of hyaluronic acid (HA) derivatives It was synthesized by the method described in WO 2010 / 053140 or WO 2014 / 038641.

[0206] [Example 2] Obtaining antigen Antigen peptides were purchased from Sigma Genosys.

[0207] mERK2 p121 (amino acid sequence: NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 1)) The sequence comprises a 9-amino acid sequence from the 16th Q to the 24th L (SEQ ID NO: 2: QYIHSANVL) as an epitope recognized by CD8-positive cytotoxic T cells, and a 17-amino acid sequence from the 13th R to the 29th K (SEQ ID NO: 3: RGLQYIHSANVLHRDLK) as an epitope recognized by CD4-positive helper T cells.

[0208] MAGE-A4 p264 (amino acid sequence: GSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 4)) The sequence contains a 9-amino acid sequence from the 2nd S to the 10th L (SEQ ID NO: 5: SNPARYEFL) as an epitope recognized by CD8-positive cytotoxic T cells, and a 16-amino acid sequence from the 22nd V to the 37th I (SEQ ID NO: 6: VKVLEHVVRVNARVRI) as an epitope recognized by CD4-positive helper T cells.

[0209] MAGE-A4 p265 (amino acid sequence: SNPARYEFL (SEQ ID NO: 7)).

[0210] MAGE-A4 p285 (amino acid sequence: VKVLEHVVRVNARVRIAYP (SEQ ID NO: 8)).

[0211] TRP2TRP1gp100-6G (amino acid sequence: SVYDFFVWLGGGGGGTWHRYHLLGGGGGGEGSRNQDWL (SEQ ID NO: 9)).

[0212] The sequence contains the following epitope sequences recognized by CD8-positive cytotoxic T cells: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), with each epitope linked by six Gs.

[0213] TRP2TRP1gp100-6Y (amino acid sequence: SVYDFFVWLYYYYYYTWHRYHLLYYYYYYEGSRNQDWL (SEQ ID NO: 10)).

[0214] The sequence contains the following epitope sequences recognized by CD8-positive cytotoxic T cells: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), with each epitope linked by six Y residues.

[0215] TRP2TRP1gp100-4L (amino acid sequence: SVYDFFVWLLLLLTWHRYHLLLLLLEGSRNQDWL (SEQ ID NO: 11)).

[0216] The sequence contains the following epitope sequences recognized by CD8-positive cytotoxic T cells: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), with each epitope linked by four Ls.

[0217] TRP2TRP1gp100-6L (amino acid sequence: SVYDFFVWLLLLLLLTWHRYHLLLLLLLLEGSRNQDWL (SEQ ID NO: 12)).

[0218] The sequence contains the following epitope sequences recognized by CD8-positive cytotoxic T cells: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), with each epitope linked by six Ls.

[0219] TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)).

[0220] TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 14)).

[0221] gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)).

[0222] AH1gp70-6G (amino acid sequence: LVQFIKDRISVVQAGGGGGGSPSYVYHQF (SEQ ID NO: 16)).

[0223] The sequence includes the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as an epitope recognized by CD4-positive helper T cells, and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as an epitope recognized by CD8-positive cytotoxic T cells, with the epitopes linked by six Gs.

[0224] AH1gp70-6Y (amino acid sequence: LVQFIKDRISVVQAYYYYYYSPSYVYHQF (SEQ ID NO: 17)).

[0225] The sequence includes the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as an epitope recognized by CD4-positive helper T cells, and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as an epitope recognized by CD8-positive cytotoxic T cells, with the epitopes linked by six Ys.

[0226] AH1gp70-4L (amino acid sequence: LVQFIKDRISVVQALLLLSPSYVYHQF (SEQ ID NO: 18)).

[0227] The sequence includes the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as an epitope recognized by CD4-positive helper T cells, and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as an epitope recognized by CD8-positive cytotoxic T cells, with the epitopes linked by four Ls.

[0228] AH1gp70-6L (amino acid sequence: LVQFIKDRISVVQALLLLLLSPSYVYHQF (SEQ ID NO: 19)).

[0229] The sequence includes the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as an epitope recognized by CD4-positive helper T cells, and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as an epitope recognized by CD8-positive cytotoxic T cells, with the epitopes linked by six Ls.

[0230] AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)).

[0231] gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)).

[0232] MAGE-A4 p264-4L (amino acid sequence: GSNPARYEFLWGPRALLLLLYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 22)).

[0233] The sequence includes a 9-amino acid sequence from the second S to the tenth L (SEQ ID NO: 5: SNPARYEFL) as an epitope sequence recognized by CD8-positive cytotoxic T cells, and a 16-amino acid sequence from the 22nd V to the 37th I (SEQ ID NO: 6: VKVLEHVVRVNARVRI) as an epitope sequence recognized by CD4-positive helper T cells, with the epitopes linked by four Ls.

[0234] MAGE-A4 p264-4W (amino acid sequence: GSNPARYEFLWGPRALWWWWYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 23)).

[0235] The sequence includes a 9-amino acid sequence from the second S to the tenth L (SEQ ID NO: 5: SNPARYEFL) as an epitope sequence recognized by CD8-positive cytotoxic T cells, and a 16-amino acid sequence from the 22nd V to the 37th I (SEQ ID NO: 6: VKVLEHVVRVNARVRI) as an epitope sequence recognized by CD4-positive helper T cells, with the epitopes linked by four Ws.

[0236] [Example 3] Preparation of a complex of HA derivative and antigen (Example 3-1) Preparation of a complex of antigen and HA derivative HA derivatives (e.g., 99k HA-C6-Chol-42%) were dissolved in ultrapure water to a concentration of 12 mg / mL and sonicated (Covaris, E220X). Antigen peptides were dissolved in DMSO to a concentration of 50 mg / mL. The antigen peptide DMSO solution was added to the HA derivative solution and sonicated for 30 minutes using a bath sonicator. The solution was transferred to a dialysis kit (Slide-A-Lyzer, molecular weight cutoff 3.5K) and dialyzed sequentially against 5 mM carbonate buffer, pH 9, 10 mM phosphate buffer, pH 7, and 10 mM phosphate buffer, pH 7, containing 10% sucrose. The antigen peptide concentration was quantified by reverse-phase chromatography under the following conditions and adjusted to 250–500 μg / mL to prepare the dosing solution. If the desired concentration was not reached, the solution was concentrated using an ultraconcentrator (Vivaspin 6, 5000 MWCO, Sauturius). The HA derivatives were synthesized as described in Examples 1 and 2 of WO 2010 / 053140A1. For example, 99k HA-C6-Chol-42 shown in Table 1 of Example 4-1 is a compound represented by the formula (I) in which Z is a direct bond and R a is a hydrogen atom, Y is a C6 alkylene, and X 1 Ga-NR b -COO-R, R bThis indicates an HA derivative with a weight-average molecular weight of 99 kDa and a cholesteryl group introduction rate of 42%. Similarly, an HA derivative with a cholesteryl group introduction rate of 41% is designated as 99k HA-C6-Chol-41, and an HA derivative with a cholesteryl group introduction rate of 43% is designated as 99k HA-C6-Chol-43.

[0237] Reversed-phase chromatography analysis conditions Analytical column: PLRP-S 1000Å (Agilent) Column temperature: 40℃ Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: 0.1% TFA acetonitrile solution Flow rate: 2mL / min Detection: UV254nm Injection volume: 50μL [Comparative Example 1] Preparation of a complex of CHP and antigen Cholesterol-modified pullulan (abbreviated CHP) (CHP-80T; 1–2 cholesterols per 100 monosaccharides) was prepared according to International Publication No. 2015 / 050158. CHP was dissolved in phosphate-buffered saline (PBS) containing 6 M urea at a concentration of 10 mg / mL. The antigen peptide DMSO solution was added to the CHP solution and left overnight at room temperature in the dark. The mixture was transferred to a dialysis membrane (molecular weight cutoff: 3,500, Thermo Scientific) and dialyzed against PBS containing 0.6 M urea at a volumetric ratio of at least 100 times the volume of the external solution at 4°C for 2 hours to overnight. This was followed by dialysis against PBS containing 0.06 M urea at a volumetric ratio of at least 100 times the volume of the external solution at 4°C for 2 hours to overnight. This was followed by dialysis against PBS at a volumetric ratio of at least 100 times the volume of the external solution at 4°C for 2 hours to overnight. After collecting the dialyzed fluid, the antigen concentration was quantified and adjusted by the method described in Example 3-1 to prepare a dosing solution. If the target concentration was not reached, the solution was concentrated using an ultraconcentrator (Vivaspin6, 5000 MwCO, Sautorius).

[0238] [Example 4] Antigen-specific T cell induction test Experimental Method BALB / c mice (female, Japan SLC; body weight 15-25 g) or C57BL / 6 mice (female, Japan SLC; body weight 15-25 g) were subcutaneously administered in the right dorsal region with 0.05-0.1 mg of the antigen-equivalent complex of the antigen peptide and HA derivative prepared in Example 3-1 or the complex of CHP and antigen peptide prepared in Comparative Example 1. When an adjuvant (CpG oligo DNA (ODN1668, InvivoGen), 0.05 mg) was administered, it was subcutaneously administered in the vicinity of the dorsal region. Two doses were administered, with a one-week interval between doses. One week after the final dose, spleen cells were isolated from the administered mice as follows: Spleens were isolated from the mice, washed with RPMI 1640 medium containing 10% fetal bovine serum (FBS), and then bled. The spleens were ground, and the released cells were collected in FBS-containing RPMI 1640 medium. After centrifugation (300 × g, 5 minutes, 4°C), the supernatant was removed, and 1 to 2 mL of ACK solution (Sigma) was added. After 1 minute, 30 mL of FBS-containing RPMI 1640 medium was added, followed by centrifugation (300 × g, 5 minutes, 4°C). The supernatant was removed, and 10 mL of FBS-containing RPMI 1640 medium was added. The mixture was filtered through a 40 μm cell strainer and centrifuged (300 × g, 5 minutes, 4°C). The supernatant was removed, and 20 mL of FBS-containing RPMI 1640 medium was added, followed by centrifugation (300 × g, 5 minutes, 4°C). The supernatant was removed, and the cells were suspended in an appropriate amount of FBS-containing RPMI 1640 medium. The number of cells was counted, and a concentration of 2 × 10 7 The cells were suspended in FBS-containing RPMI1640 medium to a cell concentration of 10 cells / mL.

[0239] 5 × 10 mouse spleen cells were cultured in a 24-well culture plate (Nunc) at a rate of 10 × 10 per well. 6The cells were added at a concentration of 0.4 mL per well. 50 μL of 100 μg / mL T cell stimulatory peptide was added, and the cells were incubated at 37°C and 5% CO2 for 30 minutes. Subsequently, 50 μL of GoldiPlug (BD Biosciences) diluted 1:100 in FBS-containing RPMI 1640 medium was added per well, and the cells were incubated at 37°C and 5% CO2 for 5 hours. The cells were harvested and transferred to a 96-well V-bottom microplate (Nunc). After centrifugation (2000 rpm, 2 minutes, 4°C; the same conditions were used hereafter), the supernatant was removed, and the cells were suspended in 200 μL of staining buffer (PBS containing 0.5% bovine serum albumin (BSA)) per well. After centrifugation and removal of the supernatant, 50 μL of a solution containing fluorescently labeled anti-CD8 antibody (Invitrogen) and fluorescently labeled anti-CD4 antibody (BD Biosciences) was added, mixed, and then allowed to stand in the dark at 4°C for 15 minutes. After adding 150 μL of staining buffer and centrifuging to remove the supernatant, the cells were washed with an additional 200 μL of staining buffer. 100 μL of Cytofix / Cytoperm buffer (BD Biosciences) was added and gently mixed. After incubating in the dark at room temperature for 20 minutes, the cells were washed twice with staining buffer. 200 μL of staining buffer was added and the cells were stored overnight in a dark, refrigerated location. After centrifugation to remove the supernatant, 200 μL of Perm / Wash buffer (BD Biosciences) was added and the cells were incubated at room temperature for 3 minutes in the dark. After centrifugation and removal of the supernatant, the cells were gently suspended in 50 μL of Perm / Wash buffer containing various anti-cytokine antibodies and incubated in the dark at room temperature for 15 minutes. The cells were washed with 150 μL of Perm / Wash buffer, resuspended in 200 μL of staining buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a flow cytometer (FACS Canto II, BD Biosciences) and the accompanying analysis software (FACSDiva).

[0240] (Example 4-1) T cell induction test using mERK2 peptide BALB / c mice were used, and each sample in Table 1 was administered twice, and the test was carried out according to the method described in Example 4. CpG oligo DNA (ODN1668, manufactured by InvivoGen) was used as an adjuvant. + Total CD8 when mERK2 p121 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of cell numbers is shown in FIG.

[0241] [Table 1] It was confirmed that high levels of T cells can be induced by using HA derivatives as carriers.

[0242] (Example 4-2) T cell induction test using mERK2 peptide (adjuvant effect 1) BALB / c mice were used, and each sample in Table 2 was administered twice, and the test was carried out according to the method described in Example 4. CpG oligo DNA (ODN1668, InvivoGen), PolyIC (HMW VacciGrade, InvivoGen), QuilA (InvivoGen), and Sting agonist (2'3'-cGAM(PS)2(Rp / Sp), InvivoGen) were used as adjuvants. CD8 + Total CD8 when mERK2 p121 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of CD4 cells is shown in Figure 2-1. + Total CD4 when mERK2 p121 was used as a T cell stimulating peptide + Interferon-gamma-producing CD4 cells + The percentage of cell numbers is shown in Figure 2-2.

[0243] [Table 2] It was confirmed that T cells could be induced using either adjuvant.

[0244] (Example 4-3) T cell induction test using mERK2 peptide (adjuvant effect 2) BALB / c mice were used, and each sample in Table 3 was administered twice, and the test was carried out according to the method described in Example 4. CpG oligo DNA (ODN1668, manufactured by InvivoGen), R848 (VacciGrade, manufactured by InvivoGen), and MPL (MPLAs VacciGrade, manufactured by InvivoGen) were used as adjuvants. + Total CD8 when mERK2 p121 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of CD4 cells is shown in Figure 3-1. + Total CD4 when mERK2 p121 was used as a T cell stimulating peptide + Interferon-gamma-producing CD4 cells + The percentage of cell numbers is shown in Figure 3-2.

[0245] [Table 3] It was confirmed that T cells could be induced using either adjuvant.

[0246] (Example 4-4) T cell induction test using MAGE-A4 p264 peptide BALB / c mice were administered with each of the samples in Table 4 twice, and the test was carried out according to the method described in Example 4. + Total CD8 when MAGE-A4 p265 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of cell numbers is shown in FIG.

[0247] [Table 4] It was confirmed that high levels of T cells can be induced by using HA derivatives.

[0248] (Example 4-5) T cell induction test using MAGE-A4 p264 peptide (without adjuvant) BALB / c mice were administered with each of the samples in Table 5 twice, and the test was carried out according to the method described in Example 4. + Total CD8 when MAGE-A4 p265 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of cell numbers is shown in FIG.

[0249] [Table 5] It was confirmed that by using HA derivatives as carriers, it is possible to induce high levels of T cells even without the use of adjuvants.

[0250] (Example 4-6) T cell induction test using MAGE-A4 p264 peptide BALB / c mice were administered with each of the samples in Table 6 twice, and the test was carried out according to the method described in Example 4. + Total CD8 when MAGE-A4 p265 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of cell numbers is shown in FIG.

[0251] [Table 6] It was revealed that a high level of T cells could be induced using peptides containing any of the amino acid linkers.

[0252] (Examples 4-7) T cell induction test using TRP2TRP1gp100 peptide C57BL / 6 mice were administered with each of the samples in Table 7 twice, and the test was carried out in the same manner as described in Example 4. + Total CD8 T cells stimulated by TRP1, TRP2, and gp100 peptides + Interferon-gamma-producing CD8 cells + The percentages of cell numbers are shown in Figures 7-1, 7-2, and 7-3, respectively.

[0253] [Table 7] It was confirmed that the use of HA derivatives as carriers enabled the induction of high levels of T cells against TRP1, TRP2, and gp100. (Examples 4-8) T cell induction test using TRP2TRP1gp100 peptide C57BL / 6 mice were administered with each of the samples in Table 8 twice, and the test was carried out in the same manner as described in Example 4. + Total CD8 T cells stimulated by TRP1, TRP2, and gp100 peptides + Interferon-gamma-producing CD8 cells + The percentages of cell numbers are shown in Figures 8-1, 8-2, and 8-3, respectively.

[0254] [Table 8] It was revealed that high levels of T cells can be induced regardless of the peptide linker used.

[0255] (Example 4-9) T cell induction test using AH1gp70 peptide BALB / c mice were administered with each of the samples in Table 9 twice, and the test was carried out according to the method described in Example 4. + Total CD8 when AH1 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of total CD4 T cells when gp70 was used as a peptide for stimulating CD4+ T cells is shown in Figure 9-1. + Interferon-gamma-producing CD4 cells + The percentage of cell numbers is shown in Figure 9-2.

[0256] [Table 9] It was confirmed that the use of HA derivatives makes it possible to induce high levels of T cells against both AH1 and gp70.

[0257] (Example 4-10) T cell induction test using AH1gp70 peptide BALB / c mice were administered with each of the samples in Table 10 twice, and the test was carried out according to the method described in Example 4. + Total CD8 when AH1 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of CD4 cells is shown in Figure 10-1. + Total CD4 when gp70 was used as a T cell stimulating peptide + Interferon-gamma-producing CD4 cells + The percentage of cell numbers is shown in Figure 10-2.

[0258] [Table 10] It was confirmed that the use of HA derivatives makes it possible to induce high levels of T cells against both AH1 and gp70.

[0259] (Example 4-11) T cell induction test using AH1gp70 peptide BALB / c mice were administered with each of the samples in Table 11 twice, and the test was carried out in the same manner as in Example 4. + Total CD8 when AH1 was used as a T cell stimulating peptide + Interferon-gamma-producing CD8 cells + The percentage of CD4 cells is shown in Figure 11-1. + Total CD4 when gp70 was used as a T cell stimulating peptide + Interferon-gamma-producing CD4 cells + The percentage of cell numbers is shown in Figure 11-2.

[0260] [Table 11] It was revealed that a high level of T cells could be induced using any peptide species containing any amino acid linker.

[0261] Example 5: Mouse tumor growth test Experimental Method The mouse fibrosarcoma CMS5a cell line expressing mERK2, the mouse colon cancer CT26 cell line expressing AH1 and gp70, and the mouse melanoma B16F10 cell line expressing TRP2, TRP1, and gp100 were subcutaneously implanted into BALB / c mice (female; body weight 15-25 g) at a concentration of 1 × 10 6 The cells were subcutaneously transplanted into C57BL / 6 mice (female; body weight 15-25 g) at a volume of 2 × 10 cells / 100 μL per mouse. 5 The tumors were subcutaneously transplanted at a volume of 100 μL per individual, and the tumor volume was then measured over time.

[0262] (Comparative Example 5-1) Preparation of emulsion formulation An emulsion was prepared using two hard glass syringes with locking mechanisms, each containing 0.5 mL of antigen peptide, Freund's incomplete adjuvant (Invivogen), and 0.5 mL of saline, connected by a syringe connector. The antigen peptide concentration was adjusted to 0.5 mg / mL.

[0263] (Example 5-1) Mouse fibrosarcoma CMS5a A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 12 to mice bearing mouse fibrosarcoma CMS5a tumor prepared by the method described in Example 5 at the frequency shown in Table 12. The time course of the average tumor volume is shown in Figure 12-1, and the time course for each individual is shown in Figure 12-2.

[0264] [Table 12] The HA derivative (99k41) group was shown to have a higher tumor growth inhibitory effect than the peptide group, emulsion group, and CHP group.

[0265] (Example 5-2) Mouse fibrosarcoma CMS5a A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 13 to mice bearing mouse fibrosarcoma CMS5a tumor prepared by the method described in Example 5 at the frequency shown in Table 13. The time course of the average tumor volume is shown in Figure 13-1, and the time course for each individual is shown in Figure 13-2.

[0266] [Table 13] The HA derivative (99k41) group was shown to have a higher tumor growth inhibitory effect than the CpG alone group and the CHP group.

[0267] (Example 5-3) Mouse fibrosarcoma CMS5a A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 14 at the frequency shown in Table 14 to mice bearing murine fibrosarcoma CMS5a tumors prepared by the method described in Example 5. The antibodies were administered intraperitoneally. The time course of the mean tumor volume is shown in Figure 14-1, and the time course for each individual is shown in Figure 14-2. The anti-CTLA4 antibody, anti-PD1 antibody, and anti-PDL1 antibody were obtained from BioX Cell, Inc.

[0268] [Table 14] The HA derivative (99k41) group showed a high tumor growth inhibitory effect even when administered from the 7th day onwards, and its effect was shown to be higher than that of the antibody.

[0269] (Example 5-4) Mouse fibrosarcoma CMS5a A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 15 at the frequency shown in Table 15 to mice bearing murine fibrosarcoma CMS5a tumors prepared by the method described in Example 5. The antibodies were administered intraperitoneally. The time course of the mean tumor volume is shown in Figure 15-1, and the time course for each individual is shown in Figure 15-2. The anti-CTLA4 antibody, anti-PDL1 antibody, anti-OX40 antibody, and anti-4-1BB antibody were obtained from BioX Cell.

[0270] [Table 15] It was shown that the HA derivative (99k41) had a higher tumor growth inhibitory effect than the mERK2 peptide when combined with antibodies (aCTLA4 / aPDL1 / aOX40 / a4-1BB).

[0271] (Example 5-5) Mouse colon cancer cell line CT26 A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 16 to mice bearing CT26 colon cancer cells prepared by the method described in Example 5 at the frequency shown in Table 16. The time course of the average tumor volume is shown in Figure 16-1, and the time course for each individual is shown in Figure 16-2.

[0272] [Table 16] The HA derivative (99k43) group was shown to have a higher tumor growth inhibitory effect than the emulsion group and CHP group.

[0273] (Examples 5-6) Mouse melanoma B16F10 A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 17 at the frequency shown in Table 17 to mice bearing mouse melanoma B16F10 tumors prepared by the method described in Example 5. The time course of the average tumor volume is shown in Figure 17-1, and the time course for each individual is shown in Figure 17-2.

[0274] [Table 17] The HA derivative (99k43) group was shown to have a higher tumor growth inhibitory effect than the CHP group.

[0275] Example 6: Lymph node delivery Experimental Method Fluorescently labeled mERK2 p121 (fluorescein-conjugated) was used to prepare a sample as described in Example 3. BALB / c mice (15-25 g body weight) were subcutaneously administered to the right dorsal region. After 24 and 72 hours, the right inguinal lymph nodes were excised and stained with anti-CD11b and anti-F4 / 80 antibodies. The uptake of fluorescently labeled peptides into F4 / 80+CD11b+ cells was analyzed by flow cytometry. Cells were analyzed using a flow cytometer (FACS Canto II, BD Biosciences) with the accompanying analysis software (FACSDiva).

[0276] (Example 6-1) Lymph node delivery of various HA derivatives The samples listed in Table 18 were administered and analyzed by the method described in Example 6, and a lymph node migration test was performed. The fluorescence intensity per cell after 24 hours is shown in Figure 18-1, and the fluorescence intensity per cell after 72 hours is shown in Figure 18-2.

[0277] [Table 18] The HA derivative group showed greater translocation of mERK2 to lymph nodes than the peptide and CHP groups.

[0278] (Example 6-2) Lymph node delivery of various amino acid-modified HA derivatives The samples listed in Table 19 were administered and analyzed by the method described in Example 6, and a lymph node migration test was conducted. The fluorescence intensity per cell after 24 hours is shown in Figure 19. The amino acid-modified HA derivative was synthesized as described in Example 1 of WO 2014 / 038641. For example, 10kHA-Ala-C6-Chol-30% in Table 19 below has the formula (II) R aa is methyl (C1 alkyl), R 6a is a hydrogen atom, X 1a Ga-NR 9 -Z 1 -Z 2 , R 9 is a hydrogen atom, Z 1 is C6 alkylene, Z 2 Ga-NR ba -COO-Z 3 , Rba is a hydrogen atom, and Z 3 The amino acid-modified HA derivative is described in Examples 3-8 of WO 2014 / 038641.

[0279] [Table 19] The amino acid-modified HA derivatives were shown to promote the translocation of mERK2 to lymph nodes.

[0280] Example 7: Mouse tumor growth test (Example 7-1) Mouse melanoma B16F10 A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 20 at the frequency shown in Table 20 to mice bearing mouse melanoma B16F10 tumors prepared by the method described in Example 5. The time course of the average tumor volume is shown in Figure 20-1, and the time course for each individual is shown in Figure 20-2.

[0281] [Table 20] The HA derivative (50k42) group and the HA derivative (10k43) group, which have different HA molecular weights from the HA derivative (99k42) group, were also shown to have a high tumor growth inhibitory effect similar to that of the HA derivative (99k42) group.

[0282] (Example 7-2) Mouse melanoma B16F10 A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 21 at the frequency shown in Table 21 to mice bearing mouse melanoma B16F10 tumors prepared by the method described in Example 5. The time course of the average tumor volume is shown in Figure 21-1, and the time course for each individual is shown in Figure 21-2.

[0283] [Table 21] Amino acid-modified HA derivatives (10kHA-Ala-Chol30, 10kHA-Gln-Chol32) were also shown to have a high tumor growth inhibitory effect.

[0284] (Example 7-3) Mouse melanoma B16F10 A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 22 at the frequency shown in Table 22 to mice bearing mouse melanoma B16F10 tumors prepared by the method described in Example 5. The time course of the average tumor volume is shown in Figure 22-1, and the time course for each individual is shown in Figure 22-2.

[0285] [Table 22] It was shown that PolyIC, when used as an adjuvant, also had a high tumor growth inhibitory effect.

[0286] (Example 7-4) Mouse melanoma B16F10 A mouse tumor growth test was conducted by subcutaneously administering the samples in Table 23 at the frequency shown in Table 23 to mice bearing mouse melanoma B16F10 tumors prepared by the method described in Example 5. The time course of the average tumor volume is shown in Figure 23-1, and the time course for each individual is shown in Figure 23-2.

[0287] [Table 23] It was shown that the use of Sting and R848 as adjuvants also had a high tumor growth inhibitory effect.

[0288] Example 8: Tumor and lymph node analysis The samples in Table 24 were subcutaneously administered to mice bearing mouse melanoma B16F10 tumors prepared by the method described in Example 5 at the frequency shown in Table 24. Tumors and lymph nodes (four locations in total, the left and right axillae and the left and right inguinal regions) were collected on days 12, 14, and 18 after transplantation, and a tetramer assay (described below) was performed. The collected tumors and lymph nodes from two mice were pooled, and two samples (four mice) from each group were analyzed. CD8 + TRP-2 antigen-specific CD8 cells +The percentage of cells is shown in Figure 24-1. + gp100 antigen-specific CD8 cells + The percentage of cell numbers is shown in Figure 24-2.

[0289] [Table 24] TRP-2 antigen-specific CD8 in tumors and lymph nodes + Cell- and gp100 antigen-specific CD8 + Cell infiltration was observed, indicating that the antitumor effect of the HA derivatives was mediated by antigen-specific CD8 cells.

[0290] Tetramer assay The collected tumor tissue samples were placed in a 6-well plate containing 1 mL of RPMI-1640, minced with scissors into pieces 2 mm or smaller, and then collected in a gentleMACS C-tube (Miltenyi Biotec). After adding Enzyme Mix (Miltenyi Biotec), the cells were placed in a gentleMACS Dissociator (Miltenyi Biotec) and homogenized. The cells were then incubated at 37°C for 40 minutes and homogenized again in the gentleMACS Dissociator. The resulting cell suspension was passed through a strainer and centrifuged (300 × g, 5 minutes, 4°C). The pellet was suspended in MACS Buffer (Miltenyi Biotec) to prepare a cell suspension.

[0291] The collected lymph nodes were crushed using the tail of the syringe plunger to prepare a cell suspension.

[0292] 5×10 7 A tumor cell suspension or lymph node cell suspension (cells / mL) was added to a 96-well V-bottom microplate at 20 μL / well. A 50-fold diluted mouse FcR blocking reagent was added at 10 μL / well and incubated at 4°C for 5 minutes. b TRP-2 Tetramer-SVYDFFVWL-APC (MBL), H-2D bgp100 Tetramer-EGSRNQDWL-PE (MBL) was added at 10 μL / well and incubated at 4°C for 30 minutes in the dark. MACS Buffer (200 μL / well) was added, and the plate was centrifuged (310–400 × g, 5 minutes, 4°C) and the supernatant was removed. This procedure was repeated twice. Next, an antibody solution containing fluorescently labeled anti-CD8 antibody (MBL) was added at 20 μL / well and incubated at 4°C for 30 minutes in the dark. MACS Buffer (200 μL / well) was added, and the plate was centrifuged (310–400 × g, 5 minutes, 4°C) and the supernatant was removed. This procedure was repeated twice. MACS Buffer (200 μL / well) was added, and the plate was suspended in MACS Buffer. The plate was analyzed using a flow cytometer (BD LSRFortessa X-20, BD Biosciences) with the accompanying analysis software (FACSDiva).

Claims

1. A vaccine preparation for use in the prevention or treatment of cancer, comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen, The hyaluronic acid derivative having a hydrophobic group introduced therein is as follows: Formula (I) 【Chemistry 1】 [In the formula, R １ , R ２ , R ３ , and R ４ are each independently a hydrogen atom, C １－６ Alkyl, formyl and C １－６ alkylcarbonyl; R ５ is a hydrogen atom, formyl, or C １－６ alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any of 2 to 30 amino acid residues; X １ is the following formula: -NR ｂ -R、 -NR ｂ -COO-R、 -NR ｂ -CO-R、 -NR ｂ -A--Rc-R、 -COO-R, -O-COO-R, -S-R, -CO-Y ａ -S-R、 -O-C-Y ｂ -S-R、 -NR ｂ -CO-Y ｂ -S-R, and -S-S-R, is a hydrophobic group selected from the group represented by R ａ , R ｂ and R ｃ are each independently a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, where the alkyl portion of the group is selected from —O— and —NR ｆ - may be inserted with 1 to 3 groups selected from; R ｆ is a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ Alkyl and hydroxy C ２－１２ alkyl, the alkyl portion of which may be interrupted by 1 to 2 groups selected from -O- and -NH-; R is a cholesteryl group; Y is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍ -CH ２ CH ２ -, where the alkylene is -O-, -NR ｇ optionally containing 1 to 5 groups selected from - and -S-S-; Rg is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl or hydroxy C ２－２０ alkyl, the alkyl portion of which may be interrupted by 1 to 3 groups selected from -O- and -NH-; Y ａ is C １－５ alkylene; Y ｂ is C ２－８ Alkylene or C ２－８ alkenylene; m is an integer selected from 1 to 100. and one or more repeating units represented by The following formula (IIIc): 【Chemistry 2】 wherein R 1c , R 2c , R 3c and R 4c are each independently selected from a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; R 5c is selected from a hydrogen atom, formyl and C 1-6 alkylcarbonyl; X c is selected from hydroxy and —O − Q + , where Q + represents a counter cation. and a repeating unit represented by a hyaluronic acid derivative produced using hyaluronic acid composed only of disaccharide units represented by formula (IIIc), in which R 1c , R 2c , R 3c , and R 4c are all hydrogen atoms, R 5c is acetyl, and X c is —O − Na + , and the weight-average molecular weight is 3 kDa to 200 kDa; the antigen is an antigen peptide or an antigen protein, 80% or more of the number of disaccharide repeating units present in the hyaluronic acid derivative are repeating units represented by the formula (I) or repeating units represented by the formula (IIIc), The vaccine preparation, wherein the ratio of the number of repeating units having a cholesteryl group to the number of repeating disaccharide units present in the hyaluronic acid derivative is 20 to 50%.

2. The hyaluronic acid derivative contains a repeating unit represented by the formula (I), wherein Z is a direct bond and Y is C ２－１０ alkylene, and X １ The vaccine formulation of claim 1, wherein is -NH-COO-R.

3. A vaccine preparation for use in the prevention or treatment of cancer, comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen, The hyaluronic acid derivative having a hydrophobic group introduced therein is as follows: Formula (II) 【Transformation 3】 [In the formula, R １ａ , R ２ａ , R ３ａ , and R ４ａ are independently a hydrogen atom, C １－６ Alkyl, formyl, and C １－６ alkylcarbonyl; R ５ａ is a hydrogen atom, formyl, or C １－６ alkylcarbonyl; X １ａ is hydroxy, -O － Q ＋ , -NR ７ R ８ , or -NR ９ -Z １ -Z ２ and Q ＋ represents a counter cation; R ６ａ , R ７ , R ８ , and R ９ are independently a hydrogen atom, and C １－６ alkyl; R ａａ is a hydrogen atom, methyl, hydroxymethyl, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, 1-methylpropyl, 2-methylpropyl, isopropyl, 2-carboxyethyl, 2-methylthioethyl, 2-carbamoylethyl, phenylmethyl, (4-hydroxyphenyl)methyl, or indol-3-ylmethyl; Z １ is C ２－３０ Alkylene, or -(CH ２ CH ２ O) ｍａ -CH ２ CH ２ -, wherein the alkylene is independently -O-, -NR ｇａ - and -S-S-, and 1 to 5 groups may be inserted, and ma is an integer selected from 1 to 100; Z ２ is the following formula: -NR ｂａ -Z ３ 、 -NR ｂａ -COO-Z ３ 、 -NR ｂａ -CO-Z ３ 、 -NR ｂａ -CO-NR ｃａ -Z ３ 、 -COO-Z ３ 、 -CO-NR ｃａ -Z ３ 、 -O-CO-NR ｃａ -Z ３ 、 -O-COO-Z ３ 、 -S-Z ３ 、 -CO-Z ａ -S-Z ３ 、 -O-CO-Z ｂ -S-Z ３ 、 -NR ｂａ -CO-Z ｂ -S-Z ３ , and -S-S-Z ３ 、 is selected from the group represented by R ｂａ and R ｃａ are independently a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl and hydroxy C ２－２０ alkyl, where the alkyl portion of the group is independently selected from —O— and —NR ｆａ - may be inserted with 1 to 3 groups selected from; R ｆａ are independently a hydrogen atom, C １－１２ Alkyl, Amino C ２－１２ is selected from alkyl and hydroxyC2-12 alkyl, the alkyl portion of which may be optionally interrupted by 1 to 2 groups independently selected from -O- and -NH-; R ｇａ are independently a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl or hydroxy C ２－２０ alkyl, the alkyl portion of which is optionally interrupted by 1 to 3 groups independently selected from —O— and —NH—; Z ３ is a cholesteryl group; Z ａ is C １－５ alkylene; Z ｂ is C ２－８ Alkylene or C ２－８ alkenylene] and a repeating unit represented by The following formula (IIIc): 【Chemistry 4】 wherein R 1c , R 2c , R 3c and R 4c are each independently selected from a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; R 5c is selected from a hydrogen atom, formyl and C 1-6 alkylcarbonyl; X c is selected from hydroxy and —O − Q + , where Q + represents a counter cation. and a repeating unit represented by X １ａ Ga-NR ９ -Z １ -Z ２ When the repeating unit represented by formula (II) is not contained, the repeating unit represented by formula (III): 【Transformation 5】 [In the formula, R １ｂ , R ２ｂ , R ３ｂ and R ４ｂ are independently a hydrogen atom, C １－６ Alkyl, formyl, and C １－６ alkylcarbonyl; R ５ｂ is a hydrogen atom, formyl, or C １－６ alkylcarbonyl; X ２ is -NR ９ -Z １ -Z ２ where R ９ , Z １ , and Z ２ is as previously defined. The repeating unit is represented by the ratio of the repeating units of formula (II) and / or formula (III) having the group -NR 9 -Z 1 -Z 2 to the number of repeating disaccharide units present in the hyaluronic acid derivative is 20 to 50%, 80% or more of the number of disaccharide repeating units present in the hyaluronic acid derivative are repeating units represented by the formula (II), repeating units represented by the formula (III), or repeating units represented by the formula (IIIc), the antigen is an antigen peptide or an antigen protein, The hyaluronic acid derivative is R １ｃ , R ２ｃ , R ３ｃ , and R ４ｃ are all hydrogen atoms, and R ５ｃ is acetyl, and X ｃ W-O － Na ＋ A vaccine preparation produced using hyaluronic acid composed only of disaccharide units represented by formula (IIIc), which has a weight-average molecular weight of 5 kDa to 200 kDa when the formula is expressed as follows:

4. The vaccine formulation of claim 3, wherein R aa is methyl, hydroxymethyl, 1-hydroxyethyl, 1-methylpropyl, or 2-carbamoylethyl.

5. The hyaluronic acid derivative contains a repeating unit represented by formula (II), and Z １ But C ２－１０ alkylene, and Z ２ But -NH-COO-Z ３ 5. The vaccine formulation according to claim 3 or 4, wherein

6. The vaccine preparation according to any one of claims 1 to 5, wherein the hyaluronic acid derivative and the antigen form a complex.

7. A vaccine formulation according to any one of claims 1 to 6 for administration in combination with one or more adjuvants.

8. A vaccine formulation described in any one of claims 1 to 7, wherein the antigen is an antigen peptide containing two or more epitopes recognized by CD8-positive cytotoxic T cells or CD4-positive helper T cells.

9. The vaccine formulation of claim 8 , wherein the antigenic peptide has an amino acid linker between epitopes.

10. The vaccine formulation of claim 9, wherein the amino acid linker consists of 2 to 10 amino acids selected from the group consisting of glycine, tyrosine, leucine, and tryptophan.

11. The vaccine formulation according to any one of claims 1 to 9, for administration in combination with one or more antibodies used in cancer therapy.

12. A complex formed from a hyaluronic acid derivative containing a repeating unit represented by formula (I) according to claim 1 or 2, or a hyaluronic acid derivative containing a repeating unit represented by formula (II) according to any one of claims 3 to 5, and an antigen used in a vaccine for use in the prevention or treatment of cancer.

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