Cancer vaccine formulation

Abstract

To provide a vaccine formulation for use in the prevention and / or treatment of a cancer, comprising a complex of a hyaluronic acid derivative having an introduced hydrophobic group and an antigen.SOLUTION: The present invention provides a vaccine formulation for use in the prevention or treatment of a cancer, comprising a hyaluronic acid derivative having an introduced hydrophobic group including a specific repeating unit, and an antigen; and a complex formed from a hyaluronic acid derivative including a specific repeating unit and an antigen for use in a vaccine for preventing or treating a cancer.SELECTED DRAWING: Figure 12-1

Description

Technical Field

[0001] The present invention relates to a vaccine preparation for preventing and / or treating cancer, which contains a complex of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen.

Background Art

[0002] Vaccine preparations for preventing and / or treating cancer, which contain a hydrophobic polysaccharide such as cholesterylated pullulan (CHP) or cholesterylated mannan (CMP) and an antigen, have been reported (Patent Document 1). Further, vaccine preparations for cancer treatment, which contain a complex of a hydrophobic polysaccharide such as CHP and a synthetic long-chain peptide antigen having a plurality of T cell recognition epitopes and an immunopotentiator, have been reported (Patent Documents 2 and 3).

[0003] In these vaccine preparations, the antigen phagocytosed by antigen-presenting cells is cleaved into peptides of various lengths by intracellular proteasomes, proteases, and peptidases, and is loaded as an antigen epitope peptide onto major histocompatibility complex (MHC) class I molecules or MHC class II molecules on the cell surface to form a complex, whereby cytotoxic T cells (CTL, CD8 + ) and helper T cells (CD4 + ) are specifically recognized to activate these cells (Non-Patent Document 1).

[0004] On the other hand, it has been reported that a hyaluronic acid derivative in which a group having a cholesteryl group as a hydrophobic group is introduced into hyaluronic acid forms fine particles by association in water and forms a complex with a drug (Patent Document 4). Further, a hyaluronic acid derivative in which the carboxyl group of the glucuronic acid moiety of hyaluronic acid is reacted with a specific amino acid to be converted into an amide group, and a sterol group, which is a hydrophobic group, is introduced into the remaining carboxyl group has both biodegradability and blood retention properties, and a complex of the hyaluronic acid and a drug has good properties as a pharmaceutical composition (Patent Document 5).

Prior Art Documents

Patent Document

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Document

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] There is a need for a vaccine formulation with even higher functionality than the previously reported vaccine formulations using CHP. In particular, the development of a cancer vaccine formulation that significantly induces antigen-specific cytotoxic T lymphocytes (CTL) and has a high antitumor effect is desired.

Means for Solving the Problems

[0008] The present inventors diligently conducted research to solve such problems, and as a result, found that a vaccine formulation containing a complex of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen significantly induces antigen-specific CTL and has a high antitumor activity, thereby completing the present invention.

[0009] That is, the present invention relates to a complex formed by spontaneously associating in an aqueous solution to encapsulate an antigen, which improves the accumulation of the antigen in lymph nodes and significantly induces antigen-specific CTLs, a complex having high antitumor activity, a vaccine preparation containing the complex, and methods for producing them. Further, the present invention relates to a complex formed by being more dispersed in water to encapsulate an antigen, which improves the accumulation of the antigen in lymph nodes and significantly induces antigen-specific CTLs, a vaccine preparation containing the complex, and methods for producing them.

[0010] In one aspect of the present invention, there are provided a vaccine preparation for use in the prevention and / or treatment of cancers of the following [1] to

[13] and

[15] to

[27] , a complex for use in the vaccine preparation of

[14] and

[28] , and a method for producing the complex of

[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, wherein the hyaluronic acid derivative having a hydrophobic group introduced therein is the following: formula (I)

[0012]

Chemical formula

[0013] [In the formula, R 1 , R 2 , R 3 , and R 4 are each independently selected from 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 2 to 30 arbitrary amino acid residues; X 1 is the following formula: -NR b -R, -NRb -COO-R, -NR b -CO-R, -NR b -CO-NR c -R, -COO-R, -O-COO-R, -S-R, -CO-Y a -S-R, -O-CO-Y b -S-R, -NR b -CO-Y b -S-R, and -S-S-R, is a hydrophobic group selected from the groups 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 may have 1 to 3 groups selected from -O- and -NR f - inserted; R f is a hydrogen atom, C 1-12 alkyl, amino C 2-12 alkyl and hydroxy C 2-12 alkyl, and the alkyl portion of the group may have 1 to 2 groups selected from -O- and -NH- inserted; R is a sterol group; Y is C 2-30 alkylene, or -(CH 2 CH 2 O)m-CH 2 CH 2 -, where the alkylene may have 1 to 5 groups selected from -O-, -NR g - and -S-S- inserted; R g is a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl or hydroxy C2-20 selected from alkyl, and 1 to 3 groups selected from -O- and -NH- may be inserted into the alkyl moiety of the group; 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] a hyaluronic acid derivative containing at least one repeating unit represented by; or formula (II)

[0014]

Chemical formula

[0015] [wherein, R 1a , R 2a , R 3a , and R 4a are independently selected from 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 + , C 1-6 alkoxy, -NR 7 R 8 , or -NR 9 - Z 1 -Z 2 ; Q + represents a counter cation; R 6a , R 7 , R 8 , and R 9 are independently selected from a hydrogen atom and C 1-6 alkyl; R aa is a hydrogen atom or C 1-6is alkyl, where the alkyl is independently optionally substituted with one or more groups selected from hydroxy, carboxy, carbamoyl, C 1-6 alkylthio, aryl, and heteroaryl, where the aryl is optionally substituted with one or more hydroxy; Z 1 is C 2-30 alkylene, or -(CH 2 CH 2 O) ma -CH 2 CH 2 -, where the alkylene is independently optionally inserted with 1 to 5 groups selected from -O-, -NR ga -, and -S-S-, 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 ,Selected from the groups 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 said group may independently have 1 to 3 groups selected from -O- and -NR fa - inserted therein; R fa is, independently, a hydrogen atom, C 1-12 alkyl, amino C 2-12 alkyl and hydroxy C 2-12 alkyl, where the alkyl portion of said group may independently have 1 to 2 groups selected from -O- and -NH- inserted therein; R ga is, independently, a hydrogen atom, C 1-20 alkyl, amino C 2-20 alkyl or hydroxy C 2-20 alkyl, where the alkyl portion of said group may independently have 1 to 3 groups selected from -O- and -NH- inserted therein; Z 3 is a sterol group; Z a is C 1-5 alkylene; Z b is C 2-8 alkylene or C 2-8 alkenylene] A hyaluronic acid derivative containing a repeating unit represented by, wherein X 1a is -NR 9 -Z 1 - Z 2 When the repeating unit represented by formula (II) is not included, further formula (III):

[0016]

Chemical formula

[0017] [wherein, R 1b, R 2b , R 3b and R 4b are each independently selected from 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 wherein R 9 , Z 1 , and Z 2 are as defined above] The vaccine preparation is a hyaluronic acid derivative containing a repeating unit represented by [2] A hyaluronic acid derivative into which a hydrophobic group is introduced has the formula (IIIc)

[0018] [Chemical formula]

[0019] [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] The vaccine preparation according to [1], further comprising a repeating unit represented by [3] A vaccine preparation according to [1] or [2], comprising a hyaluronic acid derivative containing a repeating unit represented by formula (I), wherein the proportion of the repeating unit represented by formula (I) in the repeating unit of the disaccharide present is 5 to 50%. [4] A vaccine preparation according to [1] or [2], comprising a hyaluronic acid derivative containing a repeating unit represented by formula (II), wherein the proportion of the disaccharide unit containing 9 -NR 1 -Z 2 is 5 to 50%. [5] A vaccine preparation according to any one of [1] to [3], comprising a hyaluronic acid derivative containing a repeating unit represented by formula (I), wherein Z is a direct bond, Y is C 2-10 alkylene, X 1 is -NH-COO-R, and R is a cholesteryl group. [6] A vaccine preparation according to any one of [1], [2] and [4], comprising a hyaluronic acid derivative containing a repeating unit represented by formula (II), wherein Z 1 is C 2-10 alkylene, Z 2 is -NH-COO-Z 3 and Z 3 is a cholesteryl group. [7] When all of R 1c , R 2c , R 3c , and R 4c are hydrogen atoms, R 5c is acetyl, and X c is -O-Na + , a vaccine preparation according to any one of [1] to [6], produced using hyaluronic acid composed only of the disaccharide unit represented by formula (IIIc) defined in [2], having a weight average molecular weight of 5 kilodaltons to 200 kilodaltons. [8] A vaccine preparation according to any one of [1] to [7], wherein the hyaluronic acid derivative forms a complex with the antigen. [9] A vaccine preparation according to any one of [1] to [8], for administration in combination with one or more adjuvants.

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

[11] The vaccine preparation according to

[10] , wherein the antigen peptide contains two or more CD8-positive cytotoxic T cell recognition epitopes or CD4-positive helper T cell recognition epitopes.

[12] The vaccine preparation according to

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

[13] For administration in combination with one or more antibodies used for cancer treatment, [1 to

[12] The vaccine preparation according to any one of.

[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 preventing or treating cancer.

[15] Containing a hyaluronic acid derivative containing one or more repeating units represented by formula (I), where Y is -(CH 2 ) n1 - or -(CH 2 CH 2 O) m1 -CH 2 CH 2 -, n1 is an integer from 2 to 15, and m1 is an integer from 1 to 4, the vaccine preparation according to any one of [1] to [3], [5], and [7] to

[13] .

[16] Containing a hyaluronic acid derivative containing one or more repeating units represented by formula (I), where Y is -(CH 2 ) n1 -, the vaccine preparation according to any one of [1] to [3], [5], [7] to

[13] , and

[15] .

[17] Containing a hyaluronic acid derivative containing one or more repeating units represented by formula (I), where Y is -(CH 2 ) n1 , and n1 is 2, 6, 8, or 12, the vaccine preparation according to any one of [1] to [3], [5], [7] to

[13] ,

[15] , and

[16] .

[18] A vaccine preparation 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 natural immune receptors (pattern recognition receptors), ii) an antigen-presenting cell stimulant, or iii) a substance that has an action of inhibiting the acquisition of immunosuppressive activity of antigen-presenting cells, said vaccine preparation.

[19] A vaccine preparation 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 oligodeoxynucleotide, PolyIC, QuilA, QS21, Sting, monophosphoryl lipid, R848, imiquimod, or MPL, said vaccine preparation.

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

[13] and

[15] to

[19] , wherein the antigen is an antigenic peptide, and the antigenic peptide contains one or more CD8-positive cytotoxic T cell recognition epitopes and one or more CD4-positive helper T cell recognition epitopes respectively.

[21] A vaccine preparation according to any one of [1] to

[13] and

[15] to

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

[22] A vaccine preparation according to any one of [1] to

[13] and

[15] to

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

[23] A vaccine preparation according to any one of [1] to

[13] and

[15] to

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

[24] A vaccine preparation according to any one of [1] to

[13] and

[15] to

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

[25] A vaccine preparation according to any one of [1] to

[13] and

[15] to

[24] , for administration in combination with one or more antibodies used for cancer treatment, wherein the antibody is an antibody that inhibits an immunosuppressive signal by a tumor, or one or more antibodies that activate a co-stimulatory signal of immune cells, said vaccine preparation. A vaccine preparation according to any one of [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, anti-OX40, or an anti-4-1BB antibody, said vaccine preparation.

[27] A vaccine preparation according to any one of [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, said vaccine preparation.

[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 preventing or treating cancer.

[29] A method for producing a complex of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen, comprising the step of mixing in solution 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 peptide used in a vaccine for preventing or treating cancer.

[0020] In another aspect of the present invention, there are provided methods for preventing and / or treating cancers of

[30] to

[46] below, and uses of

[47] to

[63] .

[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.

[31] The method according to

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

[32] The method according to

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

[33] The method according to

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

[34] The method according to

[33] , wherein the antigen peptide contains two or more CD8-positive cytotoxic T cell recognition epitopes or CD4-positive helper T cell recognition epitopes.

[35] The method according to

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

[36] The method according to

[30] , wherein the vaccine preparation is administered in combination with one or more antibodies used for cancer treatment.

[37] The method according to

[30] , wherein the vaccine preparation is administered in combination with one or more adjuvants, and the adjuvant is a substance that activates i) innate immune receptors (pattern recognition receptors), ii) antigen-presenting cell stimulants, or iii) a substance having an action of inhibiting the acquisition of immunosuppressive activity of antigen-presenting cells.

[38] The method according to

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

[39] The antigen is an antigen peptide, and the antigen peptide contains at least one CD8-positive cytotoxic T cell recognition epitope and at least one CD4-positive helper T cell recognition epitope.

[30] The method according to

[30] .

[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.

[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.

[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.

[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.

[44] The method according to

[30] , wherein the vaccine preparation is administered in combination with one or more antibodies used for cancer treatment, and the antibody is an antibody that inhibits an immunosuppressive signal by a tumor or one or more antibodies that activate a costimulatory signal of immune cells.

[45] The method according to

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

[46] The method according to

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

[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.

[48] The use according to

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

[49] The use according to

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

[50] The use according to

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

[51] The use according to

[50] , wherein the antigen peptide contains two or more CD8-positive cytotoxic T cell recognition epitopes or CD4-positive helper T cell recognition epitopes.

[52] The use according to

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

[53] The use according to

[47] , wherein the vaccine preparation is administered in combination with one or more antibodies used for cancer treatment.

[54] The use according to

[47] , wherein the vaccine preparation is administered in combination with one or more adjuvants, and the adjuvant is i) a substance that activates innate immune receptors (pattern recognition receptors), ii) an antigen-presenting cell stimulant, or iii) a substance that has an effect of inhibiting the acquisition of immunosuppressive activity of antigen-presenting cells.

[55] The use according to

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

[56] The use according to

[47] , wherein the antigen is an antigenic peptide, and the antigenic peptide contains at least one CD8-positive cytotoxic T cell recognition epitope and at least one CD4-positive helper T cell recognition epitope.

[57] The use according to

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

[58] The use according to any one of

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

[59] The use according to

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

[60] The use according to

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

[61] The use according to

[47] , wherein the vaccine preparation is administered in combination with one or more antibodies used for cancer treatment, and the antibody is an antibody that inhibits an immunosuppressive signal by a tumor or one or more antibodies that activate a costimulatory signal of immune cells.

[62] The use according to

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

[63] The use according to

[47] , wherein the vaccine preparation is administered in combination with one or more antibodies used for cancer treatment, and the antibody is an anti-PDL1 antibody.

Advantages of the Invention

[0021] By using the vaccine preparation containing the complex of the hyaluronic acid derivative introduced with the hydrophobic group of the present invention and the antigen, the accumulation of the antigen in the lymph nodes is improved, and significant induction of antigen-specific CTL becomes possible, and it becomes possible to enhance the anti-cancer effect by immunity. In addition, since the complex and the vaccine preparation of the present invention are excellent in terms of safety of the hyaluronic acid derivative used, they have excellent characteristics in terms of safety, particularly in long-term administration.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2-1

Figure 2-2

Figure 3-1

Figure 3-2

Figure 4

Figure 5

Figure 6

Figure 7-1

Figure 7-2

Figure 7-3

Figure 8-1

Figure 8-2

Figure 8-3

Figure 9-1

Figure 9-2

Figure 10-1

Figure 10-2

Figure 11-1

Figure 11-2

Figure 12-1

Figure 12-2

Figure 13-1

Figure 13-2

Figure 14-1

Figure 14-2

Figure 15-1

Figure 15-2

Figure 16-1

Figure 16-2

Figure 17-1

Figure 17-2

Figure 18-1

Figure 18-2

Figure 19

Figure 20-1

Figure 20-2

Figure 21-1

Figure 21-2

Figure 22-1

Figure 22-2

Figure 23-1

Figure 23-2

Figure 24-1

Figure 24-2

Mode for Carrying Out the Invention

[0023] Hereinafter, the present invention will be described more specifically.

[0024] The complex of a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen of the present invention is a hyaluronic acid derivative containing one or more disaccharide units (which are also repeating units) represented by the formula (I), or a complex of a hyaluronic acid derivative containing one or more disaccharide units (which are also repeating units) represented by the formula (II) and an antigen, and a vaccine preparation of the present invention can be produced using the complex. In the present specification, disclosure of a method for producing the complex of a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen of the present invention is also included.

[0025] Definition The term "steryl group" referred to in this specification is not particularly limited as long as it is a group having a steroid skeleton. Here, steroids specifically include cholesterol, dehydrocholesterol, coprostanol, coprosterol, cholestanol, campestanol, ergosterol, stigmasterol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, simiarenol, bile acids (cholic 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, deoxycorticosterone, and the like. Examples of steryl groups include cholesteryl group, stigmasteryl group, lanosteryl group, ergosteryl group, cholanoyl group, coloyl group, etc. Preferably, a cholesteryl group (particularly, a cholesta-5-en-3β-yl group represented by the following formula) and a cholanoyl group (particularly, a 5β-cholan-24-oil group represented by the following formula) are mentioned.

[0026]

Chem.

[0027] Here, the two asterisks represent the bonding positions.

[0028] The term "C 1-20 alkyl" referred to in this specification means a linear or branched alkyl group having 1 to 20 carbon atoms. For example, "C1-4 alkyl" such as methyl, ethyl, n-propyl, i-propyl, n -butyl, s-butyl, i-butyl, t-butyl, etc. are included. Further, 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, etc. are included. C 1-20 alkyl includes "C" having 1 to 12 carbon atoms.1-12 "alkyl", "C" with 1 to 6 carbon atoms 1-6 also includes "alkyl".

[0029] The term "C 1-6 alkyl" as referred to in this specification means a linear or branched alkyl group having 1 to 6 carbon atoms, for example, "C 1-4 alkyl" such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, etc. is included.

[0030] The term "C 1-6 alkylcarbonyl" as referred to in this specification means an alkylcarbonyl group in which the alkyl part is the C 1-6 alkyl already mentioned, for example, "C 1-4 alkylcarbonyl" such as acetyl, propionyl, n-propylcarbonyl, i-propylcarbonyl, n-butylcarbonyl, s-butylcarbonyl, i-butylcarbonyl, t-butylcarbonyl, etc. is included.

[0031] The term "C 1-6 alkoxy" as referred to in this specification means an alkyloxy group in which the alkyl part is the C 1-6 alkyl already mentioned, for example, methoxy (H 3 C-O-), ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, i-butoxy, t-butoxy, etc. of "C 1-4 alkoxy" is included.

[0032] The term "C 1-6 alkylthio" as referred to in this specification means an alkylthio group in which the alkyl part is the C 1-6 alkyl already mentioned, for example, methylthio (H 3 C-S-), ethylthio, n-propylthio, i-propylthio, n-butylthio, s-butylthio, i-butylthio, t-butylthio, etc. are included, and preferably methylthio is mentioned.

[0033] The term "amino C 2-20"Alkyl" means a linear or branched alkyl group having 2 to 20 carbon atoms and having an amino group as a substituent. For example, the amino group may be located on the terminal carbon atom of the alkyl group. Amino C 2-20 "Alkyl" includes "amino C 2-12 alkyl" having 2 to 12 carbon atoms.

[0034] The term "hydroxy C 2-20 alkyl" as referred to in this specification means a linear or branched alkyl group having 2 to 20 carbon atoms and having a hydroxy group as a substituent. For example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. Hydroxy C 2-20 "Alkyl" includes "hydroxy C 2-12 alkyl" having 2 to 12 carbon atoms.

[0035] The term "C 2-30 alkylene" as referred to in this specification means a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, and includes, for example, ethylene, propylene, etc. C 2-20 alkylene, C 2-8 alkylene, the group -(CH 2 ) n -(where n is 2 to 30, preferably 2 to 20, more preferably 2 to 15).

[0036] The term "C 1-5 alkylene" as referred to in this specification 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, etc.).

[0037] The term "C 2-10"Alkylene" means a linear or branched divalent saturated hydrocarbon group having 2 to 10 carbon atoms, 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. are included. "C 2-10 "Alkylene" is "C" having 2 to 8 carbon atoms 2-8 "Alkylene" and "C" having 2 to 6 carbon atoms 2-6 "Alkylene" are included.

[0038] The term "C 2-8 "Alkylene" mentioned in this specification means a linear or branched divalent saturated hydrocarbon group having 2 to 8 carbon atoms, 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. are included.

[0039] The term "C 2-8 "Alkenylene" mentioned in this specification means a linear or branched divalent saturated hydrocarbon group having 2 to 8 carbon atoms and containing one or more double bonds, for example, -CH=CH-, -C(CH 3 )=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, and octa-2,4,6-triene-1,8-diyl, etc. are included. When geometric isomers exist, each isomer and their mixtures are also included.

[0040] In the present invention, "aryl" means an aromatic carbocyclic group, for example, an aromatic carbocyclic group having 6 to 14 carbon atoms. Examples of aryl include phenyl, naphthyl (1-naphthyl, and 2-naphthyl), etc. Examples of aryl substituted with one or more hydroxy include 4-hydroxyphenyl.

[0041] In the present invention, "heteroaryl" means an aromatic ring group containing one or more heteroatoms selected from nitrogen atoms, oxygen atoms, 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 condensed 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, for example, 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, imidazopyridyl, etc., and preferably indol-2-yl.

[0042] The "divalent C 2-50 hydrocarbon group" mentioned in this specification 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 group may be a divalent aromatic ring or may contain an aromatic ring as part of the structure.

[0043] The "divalent C 2-50 polyalkyleneoxy" mentioned in this specification is not particularly limited, and the alkylene group of the repeating unit may be linear or branched. Examples of the "divalent C 2-50 polyalkyleneoxy" include divalent C 2-50 polyethyleneoxy group, C 3-48 polypropyleneoxy group, C 3-48 polybutyleneoxy group, etc. The group may be linked to another group via an oxygen atom or a carbon atom. For example, C2-50 The polyethyleneoxy group includes -O(CH 2 CH 2 O) 1-25 -, -(CH 2 CH 2 O) 1-25 -, -(OCH 2 CH 2 ) 1-25 -, -(CH 2 CH 2 O) 1-24 -(CH 2 CH 2 )- and the like.

[0044] The term "salt substance" referred to in this specification is not particularly limited as long as it is an inorganic substance soluble in water. For example, 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 hydrogen carbonate, 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 can be mentioned. Preferably, sodium chloride, trisodium phosphate, disodium phosphate, potassium chloride, calcium chloride, magnesium chloride, etc. can be mentioned.

[0045] The term "antigen" referred to in this specification refers to a substance that elicits an immune response. For example, in lymph nodes and the spleen, it is a substance that can induce the activation of lymphocytes such as T cells and B cells by being presented to antigen-presenting cells. The term "antigen protein" refers to an antigen that is a protein. Also, "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 contains a T cell recognition epitope. It may be a combination of multiple T cell recognition epitopes.

[0046] As used herein, the term "adjuvant" 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.

[0047] Hyaluronic acid derivative containing a repeating unit represented by formula (I) In one aspect, the hyaluronic acid derivative containing the repeating unit represented by formula (I) is substantially composed of (1) formula (I); and (2) the repeating units of formula (I) and formula (IIIc).

[0048] 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 positions other than the repeating unit of formula (I). For example, a hydroxy group may be converted to -O(C 1-6 alkyl), -O(formyl), and -O(C 1-6 alkylcarbonyl), etc., and a carboxyl group may be converted to an amide group, an ester group, or form a salt.

[0049] According to one aspect, the group -Z-N(R a )Y-X 1 of the above formula (I) is the following formula: -NH-(CH 2 ) mz -NH-R; -NH-(CH 2 ) mz -NH-COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -NH-COO-R; -NH-(CH 2 ) mz -COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -COO-R; -NH-(CH 2 ) mz -O-COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -O-COO-R; -NH-(CH 2 ) mz -S-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -S-R; -NH-(CH 2 ) mz -O-CO-CH(R 10 )-CH 2 -S-R; -NH-(CH 2 ) mz -NHCO-CH(R 10 )-CH 2 -S-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -NHCO-CH(R 10 )-CH 2 -S-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -O-CO-CH(R 10 )-CH 2 -S-R; and -NH-(CH 2 ) mz -S-S-R; -Z-NR a -Y-NR b -COO-R (where mz is an integer from 2 to 30, R 10 is a hydrogen atom or a methyl group, and R and m are as defined hereinbefore) selected from the groups represented by. The group is preferably -NH-(CH 2 ) mz -NH-COO-R; -NH-(CH 2 CH 2 O) m -CH 2 CH 2 -NH-COO-R; and -NH-(CH 2 ) mz -S-S-R (wherein mz, R, and m are as defined hereinbefore).

[0050] In a preferred embodiment, Z in the above formula (I) is a direct bond. Also, in one embodiment, when Z in the above formula (I) is a peptide linker, X 1 is -NR b -COO-R.

[0051] Specific examples of Y in the above formula (I) include -CH 2 CH 2 O-CH 2 CH 2 -S-S-CH 2 CH 2 O-CH 2 CH 2 -, -(CH 2 CH 2 O) 2 -CH 2 CH 2 -S-S-CH 2 CH 2 O-CH 2 CH 2 -, -CH 2 CH 2 O-CH 2 CH 2 -S-S-(CH 2 CH 2 O) 2 -CH 2 CH 2 - and -(CH 2 CH 2 O) 2 -CH 2 CH 2-S-S-(CH 2 CH 2 O) 2 -CH 2 CH 2 - may be mentioned.

[0052] Y in the above formula (I) a Examples include -CH 2 - and -CH 2 CH 2 - are preferred.

[0053] Y in the above formula (I) b Examples include -CH 2 CH 2 -, -CH(CH 3 )CH 2 -, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl and octa-2,4,6-triene-1,8-diyl are preferred, and -CH 2 CH 2 - and -CH(CH 3 )CH 2 - are more preferred.

[0054] In one aspect, Z in the above formula (I) is a peptide linker represented by -NH-[CH(-Z a )-CONH] n-1 -CH(-Z a )-CO-, where n is an integer from 2 to 30, and Z a each independently represents a substituent in an α-amino acid represented as H 2 N-CH(-Z a )-COOH. The peptide linker is bonded to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R a )-Y-X 1is coupled to. Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as natural-type (L-type) amino acids like 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, valine, and their D-forms, etc. All α-amino acids including synthetic amino acids can be used. That is, Z a include, for example, -CH 3 , H 2 NC(NH)NH(CH 2 ) 3 , H 2 NCOCH 2 -, etc. Also, n Zs may be the same or different. n is an integer from 2 to 30, preferably from 2 to 10, and more preferably from 2 to 4. Preferred examples of the peptide linker include, for example, -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, -Phe-Gly-, etc.

[0055] Specific examples of the group -Z-N(R a )Y-X 1 in the above formula (I) include -NH-(CH 2 ) 2 -NH-CO-cholesteryl, -NH-(CH 2 ) 4 -NH-(CH 2 ) 3 -NH-(CH 2 ) 3 -NH-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -NH-(CH 2 ) 3 -NH-COO-cholesteryl, -NH-(CH 2 ) 4 -NH-(CH 2 ) 3 -NH-COO-cholesteryl, -NH-(CH2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH-(CH 2 ) 3 -NH 2 )-COO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-CO-NH-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-CO-cholesteryl, -NH-(CH 2 ) 3 -NH-(CH 2 ) 4 -N(-(CH 2 ) 3 -NH 2 )-cholesteryl may be mentioned. In a preferred embodiment of the group -Z-N(R a )Y-X 1 , R a , R b and Rc are hydrogen atoms, Y is a linear C 2-30 alkylene or -(CH 2 CH 2 O) m -CH 2 CH 2 -, and Y ais a linear C 1-5 alkylene, or Y b is a linear C 2-8 alkylene or a linear C 2-8 alkenylene.

[0056] In one aspect, the group Z-N(R a )Y-X 1 is such that 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 C 6 alkylene, X 1 is -NR b -COO-R, R b is a hydrogen atom, and R is a cholesteryl group.

[0057] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) further contains a repeating unit represented by the formula (IIIc). When two or more repeating units represented by the formula (IIIc) are included, the repeating units may be the same or different.

[0058] Q in 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 in the case of a divalent or higher valence, it forms a salt with a plurality of carboxyl groups according to the valence. Examples of the counter cation include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, calcium ion; the formula: N + R j R k R l R m (wherein R j , R k , R l and R m are each independently a hydrogen atom and C 1-6Examples include ammonium ions represented by (selected from alkyl), preferably sodium ions, potassium ions, tetraalkylammonium ions (e.g., tetra n-butylammonium ions, etc.). R j 、R k 、R l and R m are preferably the same group selected from C 1-6 alkyl, and preferably an n-butyl group.

[0059] The group R 1 、R 2 、R 3 、and R 4 in the above formula (I), and R 1a 、R 2a 、R 3a and R 4a in the above formula (IIIc) are all preferably hydrogen atoms. Also, R a and R b are both preferably hydrogen atoms.

[0060] The group R5 in the above formula (I) is preferably acetyl.

[0061] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) is substantially composed of the repeating units of formulas (I) and (IIIc). Among the repeating units of the disaccharide composed 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 the repeating units of formula (I) or (IIIc). In one aspect, it is composed only of the repeating units represented by the above formula (I) and formula (IIIc).

[0062] Y defined by formula (I) may be, for example, -(CH 2 ) na -(where na is selected from integers of 2 to 20, preferably 2 to 15, more preferably 2 to 12), and preferably -(CH 2 ) 2-, -(CH 2 ) 6 -, -(CH 2 ) 8 - and -(CH 2 ) 12 - and more preferably -(CH 2 ) 6 -. These Ys are preferable from the viewpoints of precipitate formation and stable dispersion, which will be described later.

[0063] According to one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) has an introduction rate of a hydrophobic group with respect to the repeating unit of the disaccharide present in the derivative of, for example, 1 to 50%, preferably 7 to 50%, more preferably 17 to 50%, and still more preferably 20 to 45%. When the introduction rate is within the above range, the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) can efficiently form a complex with an antigen in a solution.

[0064] Here, the introduction rate of the hydrophobic group is calculated by the following formula:

[0065]

Number

[0066] Herein, the "repeating unit of the disaccharide present in the derivative" includes the repeating unit of formula (I) in which the carboxyl group is converted to an amide group and a hydrophobic group is introduced, and the repeating unit of formula (IIIc) in which no hydrophobic group is introduced. The introduction rate can be controlled by reaction conditions, for example, the ratio of reagents, and can be determined by, for example, NMR measurement.

[0067] The hyaluronic acid derivative containing the repeating unit represented by the above formula (I) preferably has all of R 1c , R 2c , R 3c , and R 4c as hydrogen atoms, R 5c as acetyl, and X c as -O-Na +When used, the weight-average molecular weight is, for example, 1 kDa to 500 kDa, preferably 3 kDa to 500 kDa, more preferably 5 kDa to 200 kDa, and it is synthesized using hyaluronic acid or a derivative thereof composed only of the repeating unit represented by formula (IIIc). As one aspect, the weight-average molecular weight of the raw material preferably from the viewpoint of complex formation with an antigen is 1 kDa to 1000 kDa, more preferably 3 kDa to 200 kDa. The weight-average molecular weight of the above raw material preferably from the viewpoint of lymph node metastasis of the complex is 1 kDa to 1000 kDa, more preferably 3 kDa to 500 kDa, still more preferably 5 kDa to 200 kDa, still more preferably 5 kDa to 150 kDa.

[0068] In general, since it is difficult to obtain hyaluronic acid and its derivatives as a single product, their molecular weights are calculated as the number-average molecular weight or the weight-average molecular weight. In the present invention, it is calculated as the weight-average molecular weight. Regarding the method for measuring the weight-average molecular weight, for example, various known methods such as the light scattering method, osmotic pressure method, viscosity method, etc. described in "Essential Polymer Science" written by Seiichi Nakahama et al. (published by Kodansha, ISBN 4-06-153310-X) can be used, and the viscosity-average molecular weight shown in this specification can also be measured by a method commonly used in the technical field to which the present invention belongs, such as using an Ubbelohde viscometer. When using hyaluronic acid and its derivatives commercially available with the molecular weight specified, the specified numerical value can also be used as the molecular weight.

[0069] In the hyaluronic acid derivative containing the repeating unit of the above formula (I), the carboxyl group of glucuronic acid, which is one of the disaccharides constituting the repeating unit, is converted to an amide group to introduce a hydrophobic group. By adjusting the degree of modification of the hyaluronic acid derivative, it is also possible to control the in vivo behavior of the preparation produced using the derivative.

[0070] 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, 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 a targeting element into the hyaluronic acid derivative, it is possible to target various organs and cells including lymph nodes. Examples of the targeting element include a target tissue-specific peptide, an antibody, a fragmented antibody, an aptamer, an RGD peptide for cancer cells, folic acid, anisamide, transferrin, galactose for the liver, tocopherol, and the like.

[0071] From the viewpoint of complex formation with an antigen, 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) by a hydrophobic group, that is, the introduction rate of the hydrophobic group, is preferably 4 to 60%, more preferably 5 to 50%, still more preferably 7 to 50%, and still more preferably 7 to 45%. From the viewpoint of lymph node migration of the complex, it is preferably 6 to 60%, more preferably 17 to 50%, still more preferably 20 to 50%, and still more preferably 20 to 45%.

[0072] From the perspective of complex formation with an antigen, the combination of 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 the hydrophobic group is preferably 3 kDa to 500 kDa and 4 to 60%, more preferably 3 kDa to 200 kDa and 6 to 50%, still more preferably 5 kDa to 200 kDa and 6 to 50%, and still more preferably 5 kDa to 150 kDa and 6 to 45%. From the perspective of lymph node metastasis of the complex, it is preferably 3 kDa to 500 kDa and 4 to 60%, more preferably 3 kDa to 200 kDa and 6 to 50%, still more preferably 5 kDa to 200 kDa and 6 to 50%, and still more preferably 5 kDa to 150 kDa and 20 to 45%. From the perspective of stable dispersion, it is preferably 3 kDa to 200 kDa and 4 to 60%, more preferably 3 kDa to 200 kDa and 6 to 50%, still more preferably 5 kDa to 200 kDa and 6 to 50%, and still more preferably 5 kDa to 150 kDa and 17 to 45%. From the perspective of improving blood retention, it is preferably 5 kDa to 27 kDa and 2 to 50%, more preferably 5 kDa to 27 kDa and 8 to 35%, still more preferably 5 kDa to 18 kDa and 8 to 35%, still more preferably 5 kDa to 18 kDa and 8 to 35%, and still more preferably 5 kDa to 18 kDa and 15 to 22%. From the perspective of gelation, it is preferably 5 kDa to 300 kDa and 2 to 30%, more preferably 5 kDa to 50 kDa and 2 to 22%, still more preferably 5 kDa to 27 kDa and 2 to 22%, and still more preferably 5 kDa to 27 kDa and 7 to 22%.

[0073] Hyaluronic acid derivative containing a repeating unit represented by formula (II) In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is substantially composed of the repeating units of (1) the above formula (II); (2) the above formula (II) and formula (III); (3) the above formula (II) and formula (IIIc); or (4) the above formula (II), formula (III), and formula (IIIc). In the hyaluronic acid derivative, for example, 80% or more, preferably 90% or more, more preferably 95% or more of the repeating units of the disaccharide composed of D-glucuronic acid and N-acetylglucosamine contained in the derivative are the repeating units of formula (II), (III), or formula (IIIc). In one aspect, the hyaluronic acid derivative is composed only 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).

[0074] The ratio of a specific disaccharide unit to the repeating unit of the disaccharide present in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) means the ratio of the specific disaccharide unit to all the disaccharide units contained in a certain amount of the hyaluronic acid derivative containing the repeating unit represented by the above formula (II), which is a polysaccharide having the disaccharide unit as the repeating unit.

[0075] In formula (II) representing the disaccharide unit contained in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II), R 1a , R 2a , R 3a , and R 4a are all preferably hydrogen atoms. R 5a is preferably a hydrogen atom or C 1-6 alkylcarbonyl, more preferably a hydrogen atom or acetyl, and even more preferably acetyl. Also, in formulas (III) and (IIIc) representing the disaccharide unit contained in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II), R 1b , R 2b , R 3b and R 4b , and R 1c , R 2c, R 3c and R 4c are preferably all hydrogen atoms. R 5b and R 5c are preferably a hydrogen atom or C 1-6 alkylcarbonyl, more preferably a hydrogen atom or acetyl, and even more preferably both are acetyl.

[0076] Specific examples of R aa in formula (II) 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 indole-3-ylmethyl.

[0077] When the group -CHR aa - becomes an asymmetric center, each optical isomer and mixtures thereof are included, but when described as H 2 N-CHR aa -COOH (amino acid), it is preferably the L-form (natural form).

[0078] 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.

[0079] As an embodiment of the group -CHR aa -CO-X 1a in formula (II), for example, the group -CHR aa -COOH is mentioned. Specific examples of this group include the following groups.

[0080]

Chemical formula

[0081] Here, the asterisk represents the bonding position with -NR 6a - (the same applies hereinafter).

[0082] The group -CHR aa Preferred examples of the group -COOH include the following groups.

[0083] [Chemical formula]

[0084] The group -CHR aa Preferred examples of the group -COOH include the following groups.

[0085] [Chemical formula]

[0086] The group -CHR aa Preferred examples of the group -COOH include the following groups.

[0087] [Chemical formula]

[0088] The group -CHR aa Preferred examples of the group -COOH include the following groups.

[0089] [Chemical formula]

[0090] From the perspective of delivering the complex to the lymph nodes, the group -CHR aa Preferred examples of the group -COOH include the following groups.

[0091] [Chemical formula] Even more preferred examples include the following groups.

[0092] [Chemical formula]

[0093] More preferable examples include the following groups.

[0094] [Chemical formula]

[0095] The above-mentioned group -CHR aa -COOH, in each case, part or all of it may be converted to the group -CHR aa -CONH-Z 1 -Z 2 Examples of the group -Z 1 -Z 2 are as described below.

[0096] In formula (II), the group -CHR aa -CO-X 1a In another embodiment, for example, the group -CHR aa -CONH 2 is mentioned. Specific examples of this group include the following groups.

[0097] [Chemical formula]

[0098] The group -CHR aa -CONH 2 Preferable examples of this group include the following groups.

[0099] [Chemical formula]

[0100] The group -CHR aa -CONH 2 Preferable examples of this group include the following groups.

[0101] [Chemical formula]

[0102] Group - CHR aa -CONH 2 Preferred examples thereof include the following groups.

[0103] [Chemical formula]

[0104] Group - CHR aa -CONH 2 Preferred examples thereof include the following groups.

[0105] [Chemical formula]

[0106] From the viewpoint of having both biodegradability and blood retention properties, these groups are also preferred groups.

[0107] From the viewpoint of having both biodegradability and blood retention properties, preferred examples of the group - CHR aa -CONH 2 also include the following groups.

[0108] [Chemical formula]

[0109] From the viewpoint of having both biodegradability and blood retention properties, aa -CONH 2 even more preferred examples of the group include the following groups.

[0110] [Chemical formula]

[0111] From the perspective of more suitable dispersibility in pure water, the group -CHR aa -CONH 2 Preferred examples thereof include the following groups.

[0112]

Chemical formula

[0113] These two groups are also preferred examples from the perspective of being a base material for sustained - release subcutaneous injection preparations.

[0114] From the perspective of being a base material for sustained - release subcutaneous injection preparations, for the group -CHR aa -CONH 2 Preferred examples thereof include the following groups.

[0115]

Chemical formula

[0116] R 7 is more preferably a hydrogen atom and methyl, and even more preferably a hydrogen atom.

[0117] The carboxy defined in formulas (II), (III) and (IIIc) may form a salt represented by the formula -COO-Q + Here, Q + is not particularly limited as long as it is a counter - cation that forms a salt with carboxy in water. In the case of being divalent or higher, it forms a salt with a plurality of carboxy according to the valence. Examples of the counter - cation include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, calcium ion; the formula: N+R j R k R l R m (wherein R j 、R k 、R l and R m are each independently a hydrogen atom and C 1-6Examples include ammonium ions represented by (selected from alkyl), and preferably, sodium ions, potassium ions, tetraalkylammonium ions (e.g., tetra-n-butylammonium ions, etc.). R j , R k , R l and R m are preferably the same group selected from C 1-6 alkyl, and preferably n-butyl.

[0118] In formula (II), the group -CHR aa -CO-X 1a In another aspect, for example, the group -CHR aa -CONH-Z 1 -Z 2 is exemplified. Specific examples of the group include the following groups.

[0119]

Chemical formula

[0120] Another specific example of the group includes the following groups.

[0121]

Chemical formula

[0122] The group -CHR aa -CONH-Z 1 -Z 2 Preferred examples include the following groups.

[0123]

Chemical formula

[0124] The group -CHR aa -CONH-Z 1 -Z 2 Preferred examples include the following groups.

[0125] [Chemical formula]

[0126] Group - CHR aa -CONH-Z 1 -Z 2 Preferred examples of these groups are as follows.

[0127] [Chemical formula]

[0128] From the perspective of delivering the complex to the lymph nodes, for the groups - CHR aa -CONH-Z 1 -Z 2 Preferred examples of these groups are as follows.

[0129] [Chemical formula]

[0130] Even more preferred groups are as follows.

[0131] [Chemical formula]

[0132] Even more preferred examples are as follows.

[0133] [Chemical formula]

[0134] From the perspective of having both biodegradability and blood retention properties, for the groups - CHR aa -CONH-Z 1 -Z 2 Preferred examples of these groups are as follows.

[0135] [Chemical]

[0136] Group -Z 1 -Z 2 Examples of this include the group -(C 2-10 alkylene)-NH-COO-Z 3 Also included is the group -(C 2-12 alkylene)-NH-COO-Z 3 Here, examples of C 2-12 alkylene preferably include -(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 8 -, -(CH 2 ) 10 - and -(CH 2 ) 12 -; more preferably -(CH 2 ) 2 - and -(CH 2 ) 6 - are also included. Further, examples of the group -Z 1 -Z 2 include the group -(CH 2 CH 2 O) ma -CH 2 CH 2 -NH-Z 3 Here, ma is preferably from 1 to 20, more preferably from 1 to 10, and even more preferably from 1 to 3. A specific example of a preferred ma is 2. Examples of the group -Z 1 -Z 2 preferably include the group -(hexane-1,6-diyl)-NH-COO-Z 3 the group -(ethane-1,2-diyl)-NH-COO-Z 3 and the group -(CH 2 CH 2 O) 2 -CH 2 CH 2 -NH-Z 3include, more preferably, a group -(hexane-1,6-diyl)-NH-COO-cholesteryl, a group -(ethane-1,2-diyl)-NH-COO-cholesteryl, and a group -(CH 2 CH 2 O) 2 -CH 2 CH 2 -NH-colanoyl, more preferably, a group -(hexane-1,6-diyl)-NH-COO-cholesteryl. Z 1 、Z 2 、Examples of the group -Z 1 -Z 2 include those corresponding to Y and X of the hyaluronic acid derivative containing the repeating unit represented by the above formula (I), respectively. Examples of the group -CO-NR 1 、the group -Y-X 1 can also be listed respectively. Examples of the group -CO-NR ca -Z 3 and the group -O-CO-NR ca -Z 3 include groups in which R ca is a hydrogen atom, respectively.

[0137] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the formula (II) has, in the group X 1a where X 1a is -NR 9 -Z 1 -Z 2 、R 9 is a hydrogen atom, Z 1 is, for example, C 2-12 alkylene, preferably C 2-6 alkylene, more preferably C 6 alkylene, Z 2 is -NR ba -COO-Z 3 、R ba is a hydrogen atom, and Z 3 is a sterol group, and the group R aa is a hydrogen atom or C 1-6 alkyl, where the alkyl is independently hydroxy, carboxy, carbamoyl, C 1-6It may be substituted with one or more groups selected from alkylthio, aryl, and heteroaryl, wherein the aryl may be substituted with one or more hydroxy groups.

[0138] Group R preferred from the perspective of lymph node delivery of the complex aa Specific examples of the group include, for example, methyl, hydroxymethyl, hydrogen atom, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, benzyl, (4-hydroxyphenyl)methyl, 1-methylpropyl, 2-methylpropyl, isopropyl, indole-3-ylmethyl, 2-carbamoylethyl, and 2-carboxyethyl. Preferably, they are methyl, hydroxymethyl, 1-hydroxyethyl, 1-methylpropyl, and 2-carbamoylethyl, and more preferably methyl and 2-carbamoylethyl.

[0139] In one aspect, a hyaluronic acid derivative containing the repeating unit represented by formula (III) and containing the repeating unit represented by the above formula (II) is also preferred. In a more preferred aspect, X in formula (II) 1a and X in formula (III) 2 are the same. In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) has X 1a being -NR 9 -Z 1 -Z 2 and may contain the repeating unit represented by formula (II), the repeating unit represented by formula (III), and the repeating unit represented by formula (IIIc).

[0140] According to still another aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) has a ratio (hydrophobic group introduction rate) of the repeating unit of formula (II) and / or formula (III) having the group -NR 9 -Z 1 -Z 2 (hereinafter also referred to as a hydrophobic group) of 3 to 50% with respect to the repeating unit of the disaccharide present.

[0141] Here, the hydrophobic group introduction rate is calculated by the following formula:

[0142] [Number]

[0143] It is calculated by. Here, the "repeating unit of the disaccharide present in the derivative" includes the repeating units of formula (II) and formula (III), and the repeating unit of formula (IIIc). The introduction rate can be controlled by reaction conditions, for example, the ratio of reagents, and can be determined by, for example, NMR measurement.

[0144] In the hyaluronic acid derivative containing the repeating unit represented by the above formula (II), the introduction rate of the hydrophobic group is, for example, 3 to 50%, preferably 5 to 40%, more preferably 5 to 35%, still more preferably 5 to 25%, even more preferably 5 to 20%, and still more preferably 5 to 10%. From the viewpoint of complex formation with an antigen, 3 to 60% is preferable, 7 to 50% is more preferable, 18 to 45% is more preferable, and 20 to 35% is more preferable. From the viewpoint of lymph node metastasis of the complex, 3 to 60% is preferable, 7 to 50% is more preferable, 18 to 45% is more preferable, and 20 to 35% is more preferable.

[0145] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) has X in formula (II) 1a being -NR 9 -Z 1 -Z 2is the case. In this case, the ratio of the disaccharide unit of formula (II) to the repeating unit of the disaccharide present in the hyaluronic acid derivative is, from the viewpoint of having both biodegradability and blood retention properties, for example, 70% or more, preferably 75% or more, more preferably 90% or more. The upper limit may be 100% or less. The range of the ratio is, for example, 70 to 100%, preferably 75 to 100%, more preferably 90 to 100%. Further, from the viewpoint of having the property of forming a complex with an antigen, for example, 10% or more, preferably 20% or more, more preferably 50% or more, still more preferably 70% or more, even more preferably 90% or more. The upper limit may be 100% or less. The range of the ratio is, for example, 10 to 100%, preferably 20 to 100%, more preferably 50 to 100%, still more preferably 70 to 100%, even more preferably 90 to 100%. Furthermore, from the viewpoint of the complex having the property of lymph node migration, for example, 10% or more, preferably 20% or more, more preferably 50% or more, still more preferably 70% or more, even more preferably 90% or more. The upper limit may be 100% or less. The range of the ratio is, for example, 10 to 100%, preferably 20 to 100%, more preferably 50 to 100%, still more preferably 70 to 100%, even more preferably 90 to 100%. Incidentally, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) may further contain the repeating unit represented by the formula (III).

[0146] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is X 1 is -NR 9 -Z 1 -Z 2It does not contain the repeating unit represented by formula (II). In this case, the sum of the proportion of the repeating unit represented by formula (II) and the proportion of the repeating unit represented by formula (III) in the repeating unit of the existing disaccharide is, for example, 70 to 100%, preferably 80 to 100%, 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%, more preferably 30 to 100%. From the viewpoint of lymph node metastasis of the complex, it is, for example, 20 to 100%, preferably 30 to 100%, more preferably 70 to 100%.

[0147] Here, in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II), the proportion of the repeating unit represented by formula (III) in the repeating unit of the existing disaccharide is preferably 3 to 50%, more preferably 5 to 40%, still more preferably 5 to 35%, still more preferably 5 to 25%, still more preferably 5 to 20%, still more preferably 5 to 10%. From the viewpoint of complex formation with an antigen, 3 to 60% is preferable, 7 to 50% is more preferable, 18 to 45% is still more preferable, and 20 to 35% is still more preferable. From the viewpoint of lymph node metastasis of the complex, 3 to 60% is preferable, 7 to 50% is more preferable, 18 to 45% is still more preferable, and 20 to 35% is still more preferable.

[0148] Also, in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II), the proportion of the repeating unit represented by formula (II) in the repeating unit of the existing disaccharide is preferably 20 to 97%, more preferably 30 to 95%, still more preferably 35 to 95%, still more preferably 45 to 95%, still more preferably 50 to 95%, still more preferably 60 to 95%. From the viewpoint of complex formation with an antigen, it is, for example, 7 to 100%, preferably 20 to 100%, more preferably 30 to 100%. From the viewpoint of lymph node metastasis of the complex, it is, for example, 20 to 100%, preferably 30 to 100%, more preferably 70 to 100%.

[0149] Examples of methods for producing a hyaluronic acid derivative containing a repeating unit represented by the above formula (I) and a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) include the methods described in International Publication No. 2010 / 053140 and International Publication No. 2014 / 038641.

[0150] According to one aspect, 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) is characterized by forming fine particles by association in water. Although not particularly limited, it is considered that spontaneous association occurs in water due to the hydrophobic interaction of the introduced hydrophobic group, forming fine particles. Taking advantage of this property, it can be used as a vaccine carrier, a lymph node delivery carrier, a sustained release carrier in blood, and a targeting carrier. The particle size of the fine particles is not particularly limited, but for example, it is 1 μm or less, preferably 500 nm, more preferably 200 nm or less, still more preferably 100 nm or less, and even more preferably 50 nm or less. Examples of methods for micronizing a hyaluronic acid derivative containing a repeating unit represented by the above formula (I) and a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) include the methods described in International Publication No. 2010 / 053140 and International Publication No. 2014 / 038641.

[0151] As a raw material for producing a hyaluronic acid derivative containing a repeating unit represented by the above formula (I) and a hyaluronic acid derivative containing a repeating unit represented by the above formula (II), hyaluronic acid or its salt or its derivative can be used. Examples of hyaluronic acid salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt, and a particularly preferred salt is the sodium salt that is frequently used as a pharmaceutical. Hyaluronic acid or its pharmaceutically acceptable salt can be produced using various known methods such as a method of extracting from biological sources such as chicken combs and porcine subcutaneous tissues and a bioconversion method, or it can be purchased commercially (for example, from Denka Co., Ltd., Shiseido Co., Ltd., Seikagaku Corporation, R&D system, etc.).

[0152] An antigen that forms a complex with hyaluronic acid derivative microparticles containing the repeating unit represented by the above formula (I) or hyaluronic acid derivative microparticles containing the repeating unit represented by the above formula (II) may be released from the complex after administration by decomposition and disintegration of the hyaluronic acid derivative microparticles and substitution of the antigen with a biological component such as albumin. The rate of this release can be controlled by the introduction rate of the hydrophobic group of the hyaluronic acid derivative, the molecular weight, and the amino acid introduction rate. Also, it is possible to control the antigen release rate by chemically cross-linking and gelling the hyaluronic acid derivative by the method described in International Publication No. 2010 / 053140. Further, it is possible to control the antigen release rate by conjugating the hyaluronic acid derivative and the antigen by the method described in International Publication No. 2010 / 053140.

[0153] Antigen As an antigen used for producing a vaccine preparation as a complex with a hyaluronic acid derivative containing the repeating unit represented by the above formula (I) or a hyaluronic acid derivative containing the repeating unit represented by the above formula (II), for example, antigen peptides and antigen proteins, as well as DNA and mRNA encoding these antigen sequences can be mentioned, preferably antigen peptides and antigen proteins, and more preferably antigen peptides.

[0154] The antigen may be a cancer antigen. A cancer antigen is an antigen that is highly expressed in cancer cells and in some cases is expressed only by cancer cells. The cancer antigen can be expressed inside or on the surface of cancer cells.

[0155] 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-related 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-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2, GAGE-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, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad family of tumor antigens, lmp-1, P1A, Epstein-Barr virus-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.

[0156] As the above antigen protein, all of its sequences may be used, or sequences with some deletions may be used.

[0157] The above antigenic peptide is an antigenic peptide containing one or more CD8-positive cytotoxic T cell recognition epitopes and / or CD4-positive helper T cell recognition epitopes among the sequences of the antigen protein. In one aspect, from the perspective of being loaded onto MHC class I molecules or MHC class II molecules via degradation in antigen-presenting cells, the above antigenic peptide is preferably an antigenic peptide containing two or more epitopes. Specifically, examples of the above antigenic peptide include antigenic peptides containing epitopes of tumor cell antigen proteins.

[0158] In one aspect, the above antigenic 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, more preferably 23 to 50 amino acids.

[0159] In one aspect, from the perspective of inducing the activation of cytotoxic T cells (CTL) by helper T cells, the above antigenic peptide is an antigenic peptide containing one or more CD8-positive cytotoxic T cell recognition epitopes and one or more CD4-positive helper T cell recognition epitopes respectively.

[0160] In one aspect, when two or more epitopes are included, an amino acid linker may be arranged between the epitopes. The linker has, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, more preferably 4 to 8 amino acids. Examples of the amino acids used for the linker include glycine (G), tyrosine (Y), leucine (L), tryptophan (W), etc. Preferably, they are tyrosine (Y), leucine (L), tryptophan (W). Specific examples of the amino acid linker include, for example, -YYYY- (4Y), -LLLL- (4L), -WWWW- (4W), -GGGGGG- (6G), -YYYYYY- (6Y), -LLLLLL- (6L), -WWWWWW- (6W), -YYYYYYYY- (8Y), -LLLLLLLL- (8L) and -WWWWWWWW- (8W), and preferably, 6Y, 6L and 6W.

[0161] Complex The complex of the hyaluronic acid derivative introduced with the hydrophobic group of the present invention and the antigen can be produced by mixing the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) or the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) and the antigen in an appropriate solution. Examples of the solution to be used include water, physiological saline, various buffer solutions, sugar solutions, DMSO, ethanol, DMF, and combinations thereof, and the solvent and buffer solution may be exchanged by methods such as dialysis.

[0162] For example, the complex of the present invention can be produced by mixing fine particles of the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) or the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) and the antigen. Although not particularly limited, when the fine particles of the hyaluronic acid derivative and the antigen form a complex, a complex of the hyaluronic acid derivative and the antigen can be obtained. Further, by encapsulating the antigen in the fine particles of the hyaluronic acid derivative, a complex of the hyaluronic acid derivative and the antigen can be obtained. The complex includes an antigen encapsulate in the fine particles of the hyaluronic acid derivative. In the encapsulate, the antigen is coated with the hyaluronic acid derivative.

[0163] As a complex formation method, there is a method of adding an antigen solution to fine particles 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) that have been formed in advance. In this method, the formed fine particles of the hyaluronic acid derivative and the antigen form a complex by interactions such as hydrophobic interaction, electrostatic interaction, and hydrogen bond. The interaction occurs either on the surface of the fine particles or inside the fine particles. Although not particularly limited, conditions such as the solvent, salt concentration, pH, temperature, time, and addition of a denaturing agent may be appropriately selected so that the antigen forms a complex stably and in a high yield. For example, since the swelling degree and density of the hyaluronic acid derivative fine particles change depending on the salt concentration and pH during complex formation, and the ionization state of the antigen also changes, appropriate conditions may be used according to the combination. By performing complexation under a low salt concentration, the electrostatic repulsion between the carboxyl groups of the hyaluronic acid derivative is utilized to reduce the fine particle density, increase the amount of complexation, or complex with a higher molecular weight antigen. After complex formation, increasing the salt concentration weakens the electrostatic repulsion, increases the fine particle density, and making the gel network smaller than the antigen size enables the antigen to be firmly retained and the release to be delayed. At this time, the salt concentration can also be set to the physiological salt concentration. Ultrasonic irradiation may be performed using an ultrasonic homogenizer or a point-focus type ultrasonic irradiation device for miniaturization and size uniformity. Ultrasonic irradiation may be performed after mixing the hyaluronic acid derivative and the drug, or may be performed only on the hyaluronic acid derivative. Free antigen that has not been complexed can be separated and removed by a dialysis method, a size exclusion chromatography (SEC) method, or the like.

[0164] Also, as a method for forming a complex, 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) is dissolved in an aprotic polar organic solvent such as DMSO or DMF, mixed with an antigen, and then replaced with water, an aqueous salt solution, or an aqueous solution of various buffers, whereby fine particle formation and complex formation can be carried out simultaneously. The replacement can be carried out, for example, by dialysis. Ultrasonic irradiation may be performed using an ultrasonic homogenizer or a point-focused ultrasonic irradiation device for miniaturization and size uniformity. The ultrasonic irradiation may be performed after mixing the hyaluronic acid derivative and the antigen, or may be performed only on the hyaluronic acid derivative. Also, it may be before or after dialysis. By this method, the antigen complexed with the hyaluronic acid derivative becomes more difficult to be released from the complex. Although not particularly limited, conditions such as the solvent species, salt concentration, pH, temperature, time, addition of a denaturing agent, hyaluronic acid derivative concentration, antigen concentration, and ratio of hyaluronic acid derivative to antigen at the time of mixing the hyaluronic acid derivative and the antigen may be appropriately selected so that the antigen forms a complex stably and in a high yield. Although not particularly limited, conditions such as the solvent species, salt concentration, pH, temperature, time, number of times, presence or absence of addition of a denaturing agent, hyaluronic acid derivative concentration, antigen concentration, and ratio of hyaluronic acid derivative to antigen when replacing with water, an aqueous salt solution, or an aqueous solution of various buffers may be appropriately selected so that the antigen forms a complex stably and in a high yield. The size of the fine particles can also be obtained as desired by appropriately selecting these conditions. The free antigen that has not been complexed may be separated and removed by a dialysis method, a size exclusion chromatography (SEC) method, or the like.

[0165] When the above complex is in the form of particles, its particle diameter is not particularly limited, but from the viewpoint of lymph node migration, for example, it is 20 to 200 nm, preferably 20 to 100 nm, more preferably 20 to 50 nm.

[0166] Vaccine preparation By using the above complex, a vaccine preparation of the present invention, specifically, a vaccine preparation for use in the prevention and / or treatment of cancer can be produced.

[0167] The vaccine preparation of the present invention may be administered via oral, parenteral, intranasal, intravaginal, intraocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intra-articular, intracerebral or intraoral routes. Preferably, it may be administered via subcutaneous, intramuscular, intravenous or intradermal routes.

[0168] The vaccine preparation of the present invention can be administered in any appropriate form according to the intended route of administration as a pharmaceutical composition containing one or more pharmaceutically acceptable diluents, wetting agents, emulsifying agents, dispersing agents, adjuvants, preservatives, buffering agents, binding agents, stabilizers and the like. The route of administration may be either a parenteral route or an oral route.

[0169] The vaccine preparation of the present invention may be administered in combination with one or more adjuvants. The adjuvant to be used may be any substance having an action of enhancing the activity of antigen-presenting cells. More specifically, substances that activate innate immune receptors (pattern recognition receptors), other antigen-presenting cell stimulants, and substances having an action of inhibiting the acquisition of immunosuppressive activity of antigen-presenting cells are 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. As adjuvants that are agonists of innate immune receptors, inactivated bacteria, bacterial extracts, nucleic acids, lipopolysaccharides, lipopeptides, synthetic low-molecular compounds, etc. can be selected. Preferably, CpG oligodeoxynucleotides, polyIC RNA, imidazoquinones (e.g., R848 and imiquimod), saponins (e.g., QuilA and QS21), STING agonists (e.g., cyclic di-GMP), monophosphoryl lipid, lipopeptides, etc. are used. As adjuvants that are antigen-presenting cell stimulants, taxane-based drugs and anthracycline-based drugs can be used. As adjuvants that are drugs having an action of inhibiting the acquisition of immunosuppressive activity of antigen-presenting cells, inhibitors of JAK / STAT, inhibitors of indole dioxygenase (IDO), inhibitors of tryptophan dioxygenase (TDO), etc. are selected. These inhibitors include, in addition to compounds having an antagonistic action against the factor, neutralizing antibodies, small interfering RNAs (siRNAs), and antisense DNAs of the factor.

[0170] The adjuvant used in the vaccine preparation of the present invention is preferably an adjuvant that is an agonist of an innate immune receptor, and more preferably an agonist of Toll-like receptor (TLR) and cytoplasmic DNA sensor. As TLR agonists, CpG oligodeoxynucleotides, polyIC RNA, imidazoquinones such as R848 and imiquimod, saponins such as QuilA and QS21, STING agonists, and monophosphoryl lipid are preferred.

[0171] The adjuvant may be administered as a formulation separate from the vaccine formulation or as a combined formulation. Further, according to the method described in International Publication No. 2010 / 053140, the adjuvant may be conjugated to a hyaluronic acid derivative containing a repeating unit represented by formula (I) and used. When administered in combination, the dosage of the vaccine formulation of the present invention is, for example, 0.01 to 100 mg per administration, preferably 0.1 to 50 mg per administration, more preferably, for example, 0.1 to 20 mg per administration, and the dosage of the adjuvant is, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight. The adjuvant may be administered at the same timing as the vaccine formulation of the present invention (including the case where the adjuvant is contained in the vaccine formulation) or at different timings. When administered at different timings, it is preferable to administer the other one within, for example, 1 minute to 5 hours, preferably within 1 minute to 1 hour after administering one of them.

[0172] The vaccine formulation of the present invention may be administered in combination with one or more antibodies used for cancer treatment. The antibody is, for example, an antibody that inhibits an immunosuppressive signal caused by a tumor or one or more antibodies that activate a co-stimulatory signal of immune cells, preferably an antibody that inhibits an immunosuppressive signal caused by a tumor or an antibody that activates a co-stimulatory signal of immune cells. Specifically, it is 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 dosage of the vaccine formulation of the present invention is, for example, 0.01 to 100 mg per administration, preferably 0.1 to 50 mg per administration, more preferably 0.1 to 20 mg per administration, and the dosage of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, more preferably 1 to 40 mg / kg body weight. The antibody may be administered at the same timing as the vaccine formulation of the present invention (including the case where the antibody is contained in the vaccine formulation) or at different timings. When administered at different timings, it is preferable to administer the other one within, for example, 1 minute to 24 hours, preferably within 1 minute to 5 hours after administering one of them.

[0173] The vaccine preparation of the present invention may be administered in combination with both the above adjuvant and antibody. In that case, the dosage of the vaccine preparation of the present invention is, for example, 0.01 to 100 mg / time, preferably 0.1 to 50 mg / time, more preferably, for example, 0.1 to 20 mg / time. The dosage of the adjuvant is, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight. The dosage of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, more preferably 1 to 40 mg / kg body weight. The adjuvant and antibody may be administered at the same timing as the vaccine preparation of the present invention (including the case where the adjuvant and antibody are included in the vaccine preparation), or at different timings. When administered at different timings, it is preferable to administer all of the vaccine preparation, adjuvant, and antibody, for example, within 1 minute to 24 hours, preferably within 1 minute to 5 hours.

Examples

[0174] Hereinafter, preferred specific embodiments of the present invention will be described as examples.

[0175] [Example 1] Synthesis of hyaluronic acid (HA) derivative It was synthesized by the method described in International Publication No. 2010 / 053140 or International Publication No. 2014 / 038641.

[0176] [Example 2] Obtaining of antigen The antigen peptide was purchased from Sigma Genosys.

[0177] mERK2 p121 (amino acid sequence: NDHIAYFLYQILRGLQYIHSA NVLHRDLKPSNLLLNT (SEQ ID NO: 1)) When the said sequence contains, as the CD8-positive cytotoxic T cell recognition epitope sequence, a 9-amino acid sequence from the 16th Q to the 24th L (SEQ ID NO: 2: QYIHSANVL), and, as the CD4-positive helper T cell recognition epitope sequence, a 17-amino acid sequence from the 13th R to the 29th K (SEQ ID NO: 3: RGLQYIHSANVLHRDLK).

[0178] MAGE-A4 p264 (amino acid sequence: GSNPARYEFLWGPRALAET SYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 4)) When the said sequence contains, as the CD8-positive cytotoxic T cell recognition epitope sequence, a 9-amino acid sequence from the 2nd S to the 10th L (SEQ ID NO: 5: SNPARYEFL), and, as the CD4-positive helper T cell recognition epitope sequence, a 16-amino acid sequence from the 22nd V to the 37th I (SEQ ID NO: 6: VKVLEHVVRVNARVRI).

[0179] MAGE-A4 p265 (amino acid sequence: SNPARYEFL (SEQ ID NO: 7)).

[0180] MAGE-A4 p285 (amino acid sequence: VKVLEHVVRVNARVRIAYP (SEQ ID NO: 8)).

[0181] TRP2TRP1gp100-6G (amino acid sequence: SVYDFFVWLGGGGGG TWHRYHLLGGGGGGEGSRNQDWL (SEQ ID NO: 9)).

[0182] When the said sequence contains, as the CD8-positive cytotoxic T cell recognition epitope sequences, the following: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWH RYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), with six Gs connecting between each epitope.

[0183] TRP2TRP1gp100-6Y (amino acid sequence: SVYDFFVWLYYYYYY TWHRYHLLYYYYYYEGSRNQDWL (SEQ ID NO: 10)).

[0184] This sequence contains the following as CD8-positive cytotoxic T cell recognition epitope sequences: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWH RYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), and the epitopes are linked by six Ys.

[0185] TRP2TRP1gp100-4L (amino acid sequence: SVYDFFVWLLLLLTW HRYHLLLLLLEGSRNQDWL (SEQ ID NO: 11)).

[0186] This sequence contains the following as CD8-positive cytotoxic T cell recognition epitope sequences: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWH RYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), and the epitopes are linked by four Ls.

[0187] TRP2TRP1gp100-6L (amino acid sequence: SVYDFFVWLLLLLLL TWHRYHLLLLLLLLEGSRNQDWL (SEQ ID NO: 12)).

[0188] This sequence contains the following as CD8-positive cytotoxic T cell recognition epitope sequences: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)), TRP1 (amino acid sequence: TWH RYHLL (SEQ ID NO: 14)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)), and the epitopes are linked by six Ls.

[0189] TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 13)).

[0190] TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 14)).

[0191] gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 15)).

[0192] AH1gp70-6G (amino acid sequence: LVQFIKDRISVVQAGGGGGGS PSYVYHQF (SEQ ID NO: 16)).

[0193] The said sequence contains the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as a CD4-positive helper T cell recognition epitope and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as a CD8-positive cytotoxic T cell recognition epitope sequence, and the epitopes are linked by six Gs.

[0194] AH1gp70-6Y (amino acid sequence: LVQFIKDRISVVQAYYYYYYS PSYVYHQF (SEQ ID NO: 17)).

[0195] The said sequence contains the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as a CD4-positive helper T cell recognition epitope and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as a CD8-positive cytotoxic T cell recognition epitope sequence, and the epitopes are linked by six Ys.

[0196] AH1gp70-4L (amino acid sequence: LVQFIKDRISVVQALLLLSPS YVYHQF (SEQ ID NO: 18)).

[0197] The said sequence contains the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as a CD4-positive helper T cell recognition epitope and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as a CD8-positive cytotoxic T cell recognition epitope sequence, and the epitopes are linked by four Ls.

[0198] AH1gp70-6L (amino acid sequence: LVQFIKDRISVVQALLLLLLSPSYVYHQF (SEQ ID NO: 19)).

[0199] This sequence contains the following gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)) as a CD4-positive helper T cell recognition epitope, and the following AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)) as a CD8-positive cytotoxic T cell recognition epitope sequence, with the epitopes linked by six Ls.

[0200] AH1 (amino acid sequence: SPSYVYHQF (SEQ ID NO: 20)). gp70 (amino acid sequence: LVQFIKDRISVVQA (SEQ ID NO: 21)).

[0201] MAGE-A4 p264-4L (amino acid sequence: GSNPARYEFLWGPRALLLLYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 22)).

[0202] This sequence contains a 9-amino acid sequence from the second S to the tenth L (SEQ ID NO: 5: SNPARYEFL) as a CD8-positive cytotoxic T cell recognition epitope sequence, and a 16-amino acid sequence from the 22nd V to the 37th I (SEQ ID NO: 6: VKVLEHVVRVNARVRI) as a CD4-positive helper T cell recognition epitope sequence, with the epitopes linked by four Ls.

[0203] MAGE-A4 p264-4W (amino acid sequence: GSNPARYEFLWGPRALWWWWYVKVLEHVVRVNARVRIAYP (SEQ ID NO: 23)).

[0204] When the array contains a 9 - amino acid sequence from the 2nd S to the 10th L (SEQ ID NO: 5: SNPARYEFL) as a CD8 - positive cytotoxic T - cell recognition epitope sequence and a 16 - amino acid sequence from the 22nd V to the 37th I (SEQ ID NO: 6: VKVLEHVVRVNARVRI) as a CD4 - positive helper T - cell recognition epitope sequence, and the epitopes are linked by 4 Ws.

[0205] [Example 3] Preparation of complex of HA derivative and antigen (Example 3-1) Preparation of complex of antigen and HA derivative HA derivative (99k HA - C 6 -Chol - 42% etc.) was dissolved in ultrapure water to a concentration of 12 mg / mL, and ultrasonic treatment (manufactured by Covaris, E220X) was performed. The antigen peptide was dissolved in DMSO to a concentration of 50 mg / mL. The antigen peptide DMSO solution was added to the HA derivative aqueous solution, and ultrasonic treatment was performed for 30 minutes using a bath - type sonicator. It was transferred to a dialysis kit (Slide - A - Lyzer, molecular weight cut - off 3.5K) and sequentially dialyzed 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 analysis under the following conditions, and the concentration was adjusted to 250 - 500 μg / mL to obtain an administration solution. When the target concentration was not reached, it was concentrated using an ultrafiltration concentrator (Vivaspin6, 5000 MwCO, Sartorius). The HA derivative was synthesized as described in Examples 1 and 2 of International Publication No. WO2010 / 053140A1. For example, 99k HA - C 6 -Chol - 42, in formula (I), Z is a direct bond, R a is a hydrogen atom, Y is C 6 alkylene, X 1 is - NR b -COO - R, R b is a hydrogen atom, R is a cholesteryl group (that is, cholesteryl 6 - aminohexylcarbamate is introduced), the weight - average molecular weight is 99 kDa, and the introduction rate of the cholesteryl group is 42% of the HA derivative. Similarly, the HA derivative with a cholesteryl group introduction rate of 41% is 99k HA - C6 -Chol-41, which represents an HA derivative with a cholesterol group introduction rate of 43%, is 99k HA-C 6 -Chol-43 is represented as such.

[0206] Reverse-phase chromatography analysis conditions Analysis column: PLRP-S 1000Å (Agilent) Column temperature: 40 °C Mobile phase A: 0.1% TFA aqueous solution, Mobile phase B: 0.1% TFA acetonitrile solution Flow rate: 2 mL / min Detection: UV254 nm Injection volume: 50 μL

[0207] [Comparative Example 1] Preparation of complex of CHP and antigen Prepared according to International Publication No. WO2015 / 050158. Cholesterol-modified pullulan (abbreviation CHP) (CHP-80T; 1 to 2 cholesterols are introduced per 100 monosaccharides) was obtained from NOF Corporation. CHP was dissolved in phosphate-buffered saline (PBS) containing 6 M urea at a concentration of 10 mg / mL. An antigen peptide DMSO solution was added to the CHP solution, and the mixture was left at room temperature in the dark overnight. The mixture was transferred to a dialysis membrane (molecular weight cut-off: 3,500, Thermo Scientific), and dialyzed against PBS containing 0.6 M urea at 4 °C for 2 hours to overnight with an external dialysis solution volume ratio of 100 times or more. Subsequently, it was dialyzed against PBS containing 0.06 M urea at 4 °C for 2 hours to overnight with an external dialysis solution volume ratio of 100 times or more. Again, it was dialyzed against PBS at 4 °C for 2 hours to overnight with an external dialysis solution volume ratio of 100 times or more. After collecting the internal dialysis solution, the antigen concentration was quantified by the method described in Example 3-1, adjusted in concentration, and used as an administration solution. If the target concentration was not reached, it was concentrated using an ultrafiltration concentrator (Vivaspin6, 5000 MwCO, Sartorius).

[0208] [Example 4] Antigen-specific T cell induction test Experimental method Into the subcutaneous tissue of the right posterior back of BALB / c mice (female, SLC Japan; body weight 15 - 25 g) or C57BL / 6 mice (female, SLC Japan; body weight 15 - 25 g), a complex of the antigen peptide prepared in Example 3-1 and the HA derivative or a complex of CHP prepared in Comparative Example 1 and the antigen peptide was administered in an amount equivalent to 0.05 - 0.1 mg of antigen. When administering the adjuvant (CpG oligodeoxynucleotide (ODN1668, manufactured by InvivoGen), 0.05 mg), it was subsequently administered subcutaneously nearby. The number of administrations was 2 times, and the administration interval was 1 week. One week after the final administration, spleen cells were isolated from the administered mice in the following manner. The spleen was isolated from the mice, washed with RPMI1640 medium containing 10% fetal bovine serum (FBS) to remove blood. After grinding the spleen, the released cells were collected in RPMI1640 medium containing FBS. After centrifugation (300×g, 5 minutes, 4°C), the supernatant was removed, and 1 - 2 mL of ACK solution (manufactured by Sigma) was added. After 1 minute, 30 mL of RPMI1640 medium containing FBS was added, and centrifugation (300×g, 5 minutes, 4°C) was performed. The supernatant was removed, 10 mL of RPMI1640 medium containing FBS was added, filtered through a 40 μm cell strainer, and centrifuged (300×g, 5 minutes, 4°C). The supernatant was removed, 20 mL of RPMI1640 medium containing FBS was added, and centrifugation (300×g, 5 minutes, 4°C) was performed. The supernatant was removed, and the cells were suspended in an appropriate amount of RPMI1640 medium containing FBS. After counting the number of cells, they were suspended in RPMI1640 medium containing FBS to a cell concentration of 2×10 7 cells / mL.

[0209] Mouse spleen cells were added to a 24-well culture plate (Nunc) at 5×10 6 cells / 0.4 mL per well. 50 μL of a peptide for T cell stimulation at 100 μg / mL was added, and the cells were cultured at 37°C, 5% CO 2 for 30 minutes. Then, 50 μL of GoldiPlug (BD Biosciences) diluted 100-fold with RPMI1640 medium containing FBS was added per well, and the cells were cultured at 37°C, 5% CO 2They were cultured for 5 hours. The cells were collected and transferred to a 96-well V-bottom microplate (Nunc). After centrifugation (2000 rpm, 2 minutes, 4°C; same conditions hereinafter) to remove the supernatant, the cells were suspended in 200 μL of staining buffer (PBS containing 0.5% bovine serum albumin (BSA)) per well. After centrifugation to remove the supernatant, 50 μL of a solution containing a fluorescently labeled anti-CD8 antibody (manufactured by Invitrogen) and a fluorescently labeled anti-CD4 antibody (BD Biosciences) was added, mixed, and then left standing in the dark at 4°C for 15 minutes. 150 μL of staining buffer was added, and after centrifugation to remove the supernatant, the cells were further washed with 200 μL of staining buffer. 100 μL of Cytofix / Cyto perm buffer (BD Biosciences) was added and gently mixed. After leaving standing in the dark at room temperature for 20 minutes, they were washed twice with staining buffer. 200 μL of staining buffer was added and stored refrigerated in the dark overnight. After centrifugation to remove the supernatant, 200 μL of Perm / Wash buffer (BD Biosciences) was added and left standing at room temperature in the dark for 3 minutes. After centrifugation and removal of the supernatant, 50 μL of Perm / Wash buffer containing various anti-cytokine antibodies was added to the cells and gently suspended, and then left standing in the dark at room temperature for 15 minutes. After washing the cells with 150 μL of Perm / Wash buffer, they were 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) with the attached analysis software (FACSDiva).

[0210] (Example 4-1) T cell induction test using mERK2 peptide Using BALB / c mice, each sample in Table 1 was administered twice, and the test was conducted by the method described in Example 4. CpG oligodeoxynucleotide (ODN1668, manufactured by InvivoGen) was used as an adjuvant. CD8 + When mERK2 p12 1 was used as a peptide for stimulating CD8 + T cells, the ratio of the number of interferon-gamma-producing CD8 + cells to all CD8

[0211]

Table 1

[0212] It was confirmed that high-level induction of T cells is possible by using an HA derivative as a carrier.

[0213] (Example 4-2) T cell induction test using mERK2 peptide (adjuvant effect 1) Using BALB / c mice, each sample in Table 2 was administered twice, and the test was conducted by the method described in Example 4. CpG oligodeoxynucleotide (ODN1668, manufactured by InvivoGen), PolyIC (HMW VacciGrade, manufactured by InvivoGen), QuilA (manufactured by InvivoGen), and Sting agonist (2’3’-cGAM(PS)2(Rp / Sp), manufactured by InvivoGen) were used as adjuvants. CD8 + The ratio of the number of interferon-gamma-producing CD8 + cells to all CD8 + cells when mERK2 p121 was used as a peptide for stimulating CD8 + T cells is shown in Fig. 2-1. CD4 + The ratio of the number of interferon-gamma-producing CD4 + cells to all CD4

[0214]

Table 2

[0215] It was confirmed that T cell induction is possible regardless of which adjuvant is used.

[0216] (Example 4-3) T cell induction test using mERK2 peptide (adjuvant effect 2) Using BALB / c mice, each sample in Table 3 was administered twice, and the test was conducted by the method described in Example 4. As adjuvants, CpG oligodeoxynucleotide (ODN1668, manufactured by InvivoGen), R848 (VacciGrade, manufactured by VacciGrade, InvivoGen), and MPL (MPLAs VacciGrade, manufactured by InvivoGen) were used respectively. When mERK2 p121 was used as the CD8 + peptide for stimulating T cells, the proportion of interferon-gamma-producing CD8 + cells among all CD8 + cells is shown in Fig. 3-1. When mERK2 p121 was used as the CD4 + peptide for stimulating T cells, the proportion of interferon-gamma-producing CD4 + cells among all CD4 + cells is shown in Fig. 3-2.

[0217]

Table 3

[0218] It was confirmed that T cell induction was possible regardless of which adjuvant was used.

[0219] (Example 4-4) T cell induction test using MAGE-A4 p264 peptide Using BALB / c mice, each sample in Table 4 was administered twice, and the test was conducted by the method described in Example 4. When MAGE-A4 p265 was used as the CD8 + peptide for stimulating T cells, the proportion of interferon-gamma-producing CD8 + cells among all CD8 + cells is shown in Fig. 4.

[0220]

Table 4

[0221] It was confirmed that high-level T cell induction was possible by using the HA derivative.

[0222] (Example 4-5) T cell induction test using MAGE-A4 p264 peptide (without adjuvant) Using BALB / c mice, each sample in Table 5 was administered twice, and the test was conducted by the method described in Example 4. CD8 + When MAGE-A4 p265 was used as the peptide for CD8 + T cell stimulation, the ratio of the number of interferon-gamma-producing CD8 + cells to all CD8

[0223]

Table 5

[0224] It was confirmed that high-level induction of T cells was possible even without using an adjuvant by using an HA derivative as a carrier.

[0225] (Example 4-6) T cell induction test using MAGE-A4 p264 peptide Using BALB / c mice, each sample in Table 6 was administered twice, and the test was conducted by the method described in Example 4. CD8 + When MAGE-A4 p265 was used as the peptide for CD8 + T cell stimulation, the ratio of the number of interferon-gamma-producing CD8 + cells to all CD8

[0226]

Table 6

[0227] It was revealed that high-level induction of T cells was possible regardless of the peptide containing any amino acid linker.

[0228] (Example 4-7) T cell induction test using TRP2TRP1gp100 peptide Using C57BL / 6 mice, each sample in Table 7 was administered twice, and the test was conducted by the method described in Example 4. CD8 +When using TRP1, TRP2, and gp100 as peptides for T cell stimulation, the proportion of interferon-gamma-producing CD8 + cells among all CD8 + cells is shown in Figures 7-1, 7-2, and 7-3 respectively.

[0229]

Table 7

[0230] By using an HA derivative as a carrier, it was confirmed that high-level induction of T cells against each of TRP1, TRP2, and gp100 is possible.

[0231] (Example 4-8) T cell induction test using TRP2TRP1gp100 peptide Using C57BL / 6 mice, each sample in Table 8 was administered twice, and the test was conducted by the method described in Example 4. CD8 + When using TRP1, TRP2, and gp100 as peptides for T cell stimulation, the proportion of interferon-gamma-producing CD8 + cells among all CD8 + cells is shown in Figures 8-1, 8-2, and 8-3 respectively.

[0232]

Table 8

[0233] It was revealed that high-level induction of T cells is possible regardless of the peptide linker used.

[0234] (Example 4-9) T cell induction test using AH1gp70 peptide Using BALB / c mice, each sample in Table 9 was administered twice, and the test was conducted by the method described in Example 4. CD8 + When using AH1 as a peptide for T cell stimulation, the proportion of interferon-gamma-producing CD8 + cells among all CD8 + cells is shown in Figure 9-1. CD4 +The ratio of the number of interferon gamma-producing CD4 + cells to all CD4 + cells when gp70 is used as a T cell-stimulating peptide is shown in Fig. 9-2.

[0235] [Table 9]

[0236] It was confirmed that high levels of T cell induction against AH1 and gp70, respectively, were possible by using HA derivatives.

[0237] (Example 4-10) T cell induction test using AH1gp70 peptide Using BALB / c mice, each sample in Table 10 was administered twice, and the test was conducted by the method described in Example 4. CD8 + The ratio of the number of interferon gamma-producing CD8 + cells to all CD8 + cells when AH1 is used as a T cell-stimulating peptide is shown in Fig. 10-1. CD4 + The ratio of the number of interferon gamma-producing CD4 + cells to all CD4 + cells when gp70 is used as a T cell-stimulating peptide is shown in Fig. 10-2.

[0238] [Table 10]

[0239] It was confirmed that high levels of T cell induction against AH1 and gp70, respectively, were possible by using HA derivatives.

[0240] (Example 4-11) T cell induction test using AH1gp70 peptide Using BALB / c mice, each sample in Table 11 was administered twice, and the test was conducted by the method described in Example 4. CD8 + The ratio of the number of interferon gamma-producing CD8 +Percentage of interferon-gamma-producing CD8 cells relative to the total number of cells is shown in Fig. 11-1. CD4 + Percentage of interferon-gamma-producing CD4 cells relative to the total number of cells when gp70 is used as a peptide for CD4 T cell stimulation is shown in Fig. 11-2. + + +

[0241]

Table 11

[0242] It was revealed that high-level induction of T cells is possible regardless of the peptide species containing any amino acid linker.

[0243] [Example 5] Mouse tumor growth test Experimental method Subcutaneous transplantation was performed on mouse fibrosarcoma CMS5a cell line expressing mERK2, AH1, mouse colon cancer CT26 cell line expressing gp70, and mouse melanoma B16F10 cell line expressing TRP2, TRP1, gp100. The CMS5a cell line and CT26 cell line were subcutaneously transplanted into BALB / c mice (female; body weight 15-25 g) at a dose of 1×10 6 cells / 100 μL / individual. B16F10 was subcutaneously transplanted into C57BL / 6 mice (female; body weight 15-25 g) at a dose of 2×10 5 cells / 100 μL / individual. Thereafter, the tumor volume was measured over time.

[0244] (Comparative Example 5-1) Preparation of emulsion preparation An emulsion was prepared using two hard glass syringes with locking fittings connected by a syringe connector, containing 0.5 mL of antigen peptide, 0.5 mL of Freund's incomplete adjuvant (manufactured by InvivoGen), and 0.5 mL of physiological saline. The antigen peptide concentration was adjusted to 0.5 mg / mL.

[0245] (Example 5-1) Mouse fibrosarcoma CMS5a ​​​The mouse fibrosarcoma CMS5a-bearing mice prepared by the method described in Example 5 were subcutaneously administered with the samples in Table 12 at the frequencies described in Table 12, and a mouse tumor growth test was conducted. The transition of the average value of the tumor volume is shown in Fig. 12-1, and the transition of each individual is shown in Fig. 12-2.

[0246] [Table 12]

[0247] The HA derivative (99k41) group was shown to have a high tumor growth inhibitory effect compared with the peptide group, the emulsion group, and the CHP group.

[0248] (Example 5-2) Mouse fibrosarcoma CMS5a The mouse fibrosarcoma CMS5a-bearing mice prepared by the method described in Example 5 were subcutaneously administered with the samples in Table 13 at the frequencies described in Table 13, and a mouse tumor growth test was conducted. The transition of the average value of the tumor volume is shown in Fig. 13-1, and the transition of each individual is shown in Fig. 13-2.

[0249] [Table 13]

[0250] The HA derivative (99k41) group was shown to have a high tumor growth inhibitory effect compared with the CpG alone group and the CHP group.

[0251] (Example 5-3) Mouse fibrosarcoma CMS5a The mouse fibrosarcoma CMS5a-bearing mice prepared by the method described in Example 5 were subcutaneously administered with the samples in Table 14 at the frequencies described in Table 14, and a mouse tumor growth test was conducted. The antibodies were administered intraperitoneally. The transition of the average value of the tumor volume is shown in Fig. 14-1, and the transition of each individual is shown in Fig. 14-2. The anti-CTLA4 antibody, anti-PD1 antibody, and anti-PDL1 antibody were each obtained from BioX Cell.

[0252] [Table 14]

[0253] The HA derivative (99k41) group showed a high tumor growth inhibitory effect even when administered from the 7th day, and the effect was shown to be higher than that of the antibody.

[0254] (Example 5-4) Mouse fibrosarcoma CMS5a The samples in Table 15 were subcutaneously administered to mouse fibrosarcoma CMS5a-bearing mice prepared by the method described in Example 5 at the frequencies described in Table 15, and a mouse tumor growth test was conducted. The antibody was administered intraperitoneally. The transition of the average value of the tumor volume is shown in Fig. 15-1, and the transition of each individual is shown in Fig. 15-2. The anti-CTLA4 antibody, anti-PDL1 antibody, anti-OX40 antibody, and anti-4-1BB antibody were obtained from BioX Cell, respectively.

[0255]

Table 15

[0256] The HA derivative (99k41) was shown to have a higher tumor growth inhibitory effect by higher mERK2 peptide when combined with the antibody (aCTLA4 / aPDL1 / aOX40 / a4-1BB).

[0257] (Example 5-5) Mouse colon cancer cell CT26 The samples in Table 16 were subcutaneously administered to mouse colon cancer cell CT26-bearing mice prepared by the method described in Example 5 at the frequencies described in Table 16, and a mouse tumor growth test was conducted. The transition of the average value of the tumor volume is shown in Fig. 16-1, and the transition of each individual is shown in Fig. 16-2.

[0258]

Table 16

[0259] The HA derivative (99k43) group was shown to have a high tumor growth inhibitory effect compared with the emulsion group and the CHP group.

[0260] (Example 5-6) Mouse melanoma B16F10 The samples in Table 17 were subcutaneously administered to mouse melanoma B16F10-bearing mice prepared by the method described in Example 5 at the frequencies described in Table 17, and a mouse tumor growth test was conducted. The transition of the average value of the tumor volume is shown in Figure 17-1, and the transition of each individual is shown in Figure 17-2.

[0261]

Table 17

[0262] The HA derivative (99k43) group was shown to have a high tumor growth inhibitory effect compared with the CHP group.

[0263] [Example 6] Lymph node delivery Experimental method After 24 hours and 72 hours, the right inguinal lymph nodes were excised, stained with anti-CD11b antibody and anti-F4 / 80 antibody, and the uptake of the fluorescently labeled peptide into F4 / 80+CD11b+ cells was analyzed by flow cytometry. The cells were analyzed using the attached analysis software (FACSDiva) with a flow cytometer (FACS Canto II, BD Biosciences).

[0264] (Example 6-1) Lymph node delivery of various HA derivatives The samples described in Table 18 were administered and analyzed by the method described in Example 6, and a lymph node metastasis test was conducted. 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.

[0265]

Table 18

[0266] The HA derivative group was shown to have more mERK2 migrating to the lymph nodes compared with the peptide group and the CHP group.

[0267] (Example 6-2) Lymph node delivery of various HA derivatives with amino acid modifications The samples described in Table 19 were administered and analyzed by the method described in Example 6, and a lymph node metastasis 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 International Publication No. WO2014 / 038641. For example, 10kHA-Ala-C in Table 19 below 6 -Chol-30% means that in formula (II), R aa is methyl (C 1 alkyl), R 6a is a hydrogen atom, X 1a is -NR 9 -Z 1 -Z 2 , R 9 is a hydrogen atom, Z 1 is C 6 alkylene, Z 2 is -NR ba -COO-Z 3 , R ba is a hydrogen atom, and Z 3 is a cholesteryl group (that is, alanine and cholesteryl 6-aminohexylcarbamate are introduced), indicating that it is an HA derivative with a weight average molecular weight of 10 kDa and a cholesteryl group introduction rate of 30%. The synthesized amino acid-modified HA derivatives are described in Examples 3-8 of International Publication No. WO2014 / 038641.

[0268]

Table 19

[0269] The group of amino acid-modified HA derivatives was shown to cause mERK2 to migrate to the lymph nodes.

[0270] [Example 7] Mouse tumor growth test (Example 7-1) Mouse melanoma B16F10 The samples in Table 20 were subcutaneously administered to mouse melanoma B16F10-bearing mice prepared by the method described in Example 5 at the frequencies described in Table 20, and a mouse tumor growth test was conducted. The transition of the average value of the tumor volume is shown in Figure 20-1, and the transition of each individual is shown in Figure 20-2.

[0271]

Table 20

[0272] The group of HA derivatives (99k42), the group of HA derivatives (50k42) with different molecular weights of HA, and the group of HA derivatives (10k43) were shown to have a high tumor growth inhibitory effect similar to that of the group of HA derivatives (99k42).

[0273] (Example 7-2) Mouse melanoma B16F10 Samples in Table 21 were subcutaneously administered to mouse melanoma B16F10-bearing mice prepared by the method described in Example 5 at the frequency described in Table 21, and a mouse tumor growth test was performed. The transition of the average value of the tumor volume is shown in Fig. 21-1, and the transition of each individual is shown in Fig. 21-2.

[0274]

Table 21

[0275] The group of amino acid-modified HA derivatives (10kHA-Ala-Chol30, 10kHA-Gln-Chol32) was also shown to have a high tumor growth inhibitory effect.

[0276] (Example 7-3) Mouse melanoma B16F10 Samples in Table 22 were subcutaneously administered to mouse melanoma B16F10-bearing mice prepared by the method described in Example 5 at the frequency described in Table 22, and a mouse tumor growth test was performed. The transition of the average value of the tumor volume is shown in Fig. 22-1, and the transition of each individual is shown in Fig. 22-2.

[0277]

Table 22

[0278] It was shown that even when PolyIC was used as an adjuvant, it had a high tumor growth inhibitory effect.

[0279] (Example 7-4) Mouse melanoma B16F10 The samples in Table 23 were subcutaneously administered to mouse melanoma B16F10-bearing mice prepared by the method described in Example 5 at the frequencies described in Table 23, and a mouse tumor growth test was conducted. The transition of the average value of the tumor volume is shown in Fig. 23-1, and the transition of each individual is shown in Fig. 23-2.

[0280]

Table 23

[0281] It was shown that Sting and R848 used as adjuvants also have a high tumor growth inhibitory effect.

[0282] [Example 8] Tumor and lymph node analysis The samples in Table 24 were subcutaneously administered to mouse melanoma B16F10-bearing mice prepared by the method described in Example 5 at the frequencies described in Table 24. On the 12th, 14th, and 18th days after transplantation, tumors and lymph nodes (a total of 4 sites in the left and right axillary regions and the left and right inguinal regions) were collected, and a tetramer assay (described below) was conducted. The collected tumors and lymph nodes were pooled in two portions, and two samples (4 portions) in each group were analyzed. CD8 + The ratio of the number of TRP-2 antigen-specific CD8 + cells to CD8 + cells is shown in Fig. 24-1, and the ratio of the number of gp100 antigen-specific CD8 + cells to CD8

[0283]

Table 24

[0284] It was shown that TRP-2 antigen-specific CD8 + cells and gp100 antigen-specific CD8 + cells infiltrated into the tumors and lymph nodes. This indicates that the antitumor effect by the HA derivative is caused by antigen-specific CD8 cells.

[0285] Tetramer assay The collected tumor tissue samples were placed in a 6-well plate containing 1 mL of RPMI-1640, minced into pieces smaller than 2 mm square with scissors, and then collected into a gentleMACS C-tube (Miltenyi Biotec). After adding Enzyme Mix (Miltenyi Biotec), it was set in a gentleMACS Dissociator (Miltenyi Biotec) and disrupted. Then, it was incubated at 37°C for 40 minutes, set in the gentleMACS Dissociator again and disrupted. The obtained cell suspension was passed through a strainer and then centrifuged (300×g, 5 minutes, 4°C), and the pellet was suspended in MACS Buffer (Miltenyi Biotec) to obtain a cell suspension.

[0286] The collected lymph nodes were crushed using the tail of the syringe barrel to obtain a cell suspension.

[0287] 5×10 7 The tumor cell suspension or lymph node cell suspension at 5×10 cells / mL was added to a 96-well V-bottom microplate at 20 μL / well. 10 μL / well of a 50-fold diluted mouse FcR blocking reagent was added and reacted at 4°C for 5 minutes. 10 μL / well of each of H-2Kb TRP-2 Tetramer-SVYDFFVWL-APC (MBL) and H-2Dbgp100 Tetramer-EGSRNQDWL-PE (MBL) was added and reacted at 4°C in the dark for 30 minutes. 200 μL / well of MACS Buffer was added, and the operation of centrifuging (310 - 400×g, 5 minutes, 4°C) to remove the supernatant was repeated twice. Next, 20 μL / well of an antibody solution containing a fluorescently labeled anti-CD8 antibody (MBL) was added and reacted at 4°C in the dark for 30 minutes. 200 μL / well of MACS Buffer was added, and the operation of centrifuging (310 - 400×g, 5 minutes, 4°C) to remove the supernatant was repeated twice. 200 μL / well of MACS Buffer was added and suspended, and analysis was performed using the attached analysis software (FACSDiva) on a flow cytometer (BD LSRFortessa X-20, BD Biosciences).

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 Each independently represents 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 of any length from 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、 -NRb-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 Each independently represents 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 contain 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 steryl 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 1 to 5 groups selected from - and -S-S- may be inserted; R ｇ is a hydrogen atom, C １－２０ Alkyl, Amino C ２－２０ Alkyl or Hydroxy C ２－２０ alkyl, the alkyl portion of which is selected from -O- and -NH-. 1 to 3 groups selected from the group may be inserted; 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) 【Chemistry 2】 [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 ＋ , C １－６ Alkoxy, -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 From alkyl Selected; R ａａ is a hydrogen atom or C １－６ alkyl, wherein the alkyl is independently hydroxy, carboxy, carbamoyl, C １－６ 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 -(CH ２ CH ２ O) ｍａ -CH ２ CH ２ -, wherein the alkylene is independently -O-, -NR ｇａ 1 to 5 groups selected from - and -S-S- 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-NRca-Z ３ 、 -COO-Z ３ 、 -CO-NR ｃａ -Z ３ 、 -O-CO-NR ｃａ -Z ３ 、 -O-COO-Z ３ 、 -S-Z ３ 、 -CO-Za-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 contain 1 to 3 groups selected from; R ｆａ are independently 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 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 may be independently interrupted by 1 to 3 groups 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 the formula: １ａ -NR ９ -Z １ -Z ２ When the repeating unit represented by formula (II) is not included, the repeating unit represented by formula (III): 【Chemistry 3】 [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, Z １ , and Z ２ is as already defined. A hyaluronic acid derivative comprising a repeating unit represented by the formula: The vaccine preparation.

2. The hyaluronic acid derivative having a hydrophobic group introduced therein is represented by the formula (IIIc): 【Chemistry 4】 [In the formula, R １ｃ , R ２ｃ , R ３ｃ and R ４ｃ Each independently represents 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 ＋ where Q is selected from ＋ represents a counter cation. The vaccine formulation of claim 1, further comprising a repeating unit represented by:

3. 3. The vaccine preparation according to claim 1 or 2, comprising a hyaluronic acid derivative containing a repeating unit represented by formula (I), wherein the proportion of the repeating unit represented by formula (I) in the repeating units of the disaccharide present is 5 to 50%.

4. The hyaluronic acid derivatives contain a repeating unit of formula (II), and the repeating unit of the disaccharide present is preferably a group -NR ９ -Z １ -Z ２ The vaccine formulation according to claim 1 or 2, wherein the proportion of disaccharide units comprising is between 5 and 50%.

5. The compound according to the present invention comprises a hyaluronic acid derivative comprising 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 formulation of any one of claims 1 to 3, wherein is -NH-COO-R, and R is a cholesteryl group.

6. The hyaluronic acid derivative contains a repeating unit represented by formula (II), Z １ But, C ２－１０ alkylene; Z ２ But -NH-COO-Z ３ And Z ３ The vaccine formulation of any one of claims 1, 2 and 4, wherein is a cholesteryl group.

7. 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 -O-Na ＋ The vaccine preparation according to any one of claims 1 to 6, which is produced using hyaluronic acid composed only of disaccharide units represented by formula (IIIc) as defined in claim 2, and which has a weight-average molecular weight of 5 kilodaltons to 200 kilodaltons when calculated using the formula (IIIc).

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

9. A vaccine formulation according to any one of claims 1 to 8 for administration in combination with one or more adjuvants.

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

11. The vaccine formulation according to claim 10, wherein the antigen peptide comprises two or more epitopes recognized by CD8-positive cytotoxic T cells or CD4-positive helper T cells.

12. The vaccine formulation of claim 11 , wherein the antigenic peptide has an amino acid linker between epitopes.

13. The vaccine formulation according to any one of claims 1 to 12, for administration in combination with one or more antibodies used in cancer therapy.

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

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