Pharmaceutical composition

Abstract

Provided is a pharmaceutical composition which can exhibit an anti-tumor effect more potently, more efficiently, more sustainably, and / or in a wider range. A composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen is used in combination with lymphocytes each expressing an immunoreceptor for the antigen.

Description

Pharmaceutical Composition

[0001] The present invention relates to a pharmaceutical composition and the like.

[0002] Cancer vaccines are generally administered subcutaneously, intradermally, or intramuscularly. However, to induce tumor-specific immune responses in the immunosuppressive environment of cancer patients, it is important to efficiently deliver cancer vaccines to nearby lymph nodes and create an environment in which antigen-presenting cells in the lymph nodes can present antigens to T cells, which is thought to lead to improved therapeutic efficacy. Many of the proteins, peptides, and nucleic acids used in cancer vaccines generally have low stability and are easily degraded and inactivated when administered in the body. Therefore, there is currently a need for the development of drug delivery systems (DDS) that can stably deliver and sustainably release vaccines to the target site.

[0003] Hyaluronic acid derivatives with introduced hydrophobic groups self-assemble to form complexes, typically nanoparticle-shaped nanogels. These complexes, due to their crosslinked three-dimensional network structure and hydrophobic interactions, can easily encapsulate proteins, peptides, and poorly soluble low-molecular-weight compounds, stably delivering and sustained-release the encapsulated substances to the target site, and are expected to be applied to novel DDS. Patent Document 1 reports that a cancer vaccine made from the above complex was prepared and administered subcutaneously to mice, where it rapidly and efficiently migrated to nearby lymph nodes and induced an antitumor effect.

[0004] Meanwhile, although research and development into cancer vaccines is currently underway worldwide, the effectiveness of these vaccines is not yet fully satisfactory.

[0005] International Publication No. 2020 / 158771

[0006] An objective of the present invention is to provide a pharmaceutical composition that can exert a stronger, more efficient, more sustained, and / or more extensive antitumor effect.

[0007] The present inventor has carried out intensive research in view of the above-mentioned problems, and as a result, has found that by combining the composition that contains the hyaluronic acid derivative that has been introduced with hydrophobic group and an antigen, and the lymphocyte that expresses the immune receptor for the antigen, the above-mentioned problems can be solved.The present inventor has carried out intensive research based on this finding, and has completed the present invention.That is, the present invention includes the following aspects:

[0008] Item 1. A pharmaceutical composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen, for use in combined administration with lymphocytes expressing an immune receptor for the antigen.

[0009] Item 2. The pharmaceutical composition according to Item 1, wherein the hydrophobic group is a steryl group.

[0010] Item 3. The pharmaceutical composition according to Item 1 or 2, wherein the hyaluronic acid derivative and the antigen form a complex.

[0011] Item 4. The pharmaceutical composition according to any one of Items 1 to 3, wherein the antigen is a cancer antigen.

[0012] Item 5. The pharmaceutical composition according to any one of Items 1 to 4, wherein the antigen is an antigenic peptide or antigenic protein.

[0013] Item 6. The pharmaceutical composition according to Item 5, wherein the antigen comprises an epitope recognized by CD8-positive cytotoxic T cells and / or an epitope recognized by CD4-positive helper T cells.

[0014] Item 7. The pharmaceutical composition according to any one of Items 1 to 6, wherein the immunoreceptor is a T cell receptor or a chimeric antigen receptor.

[0015] Item 8. The pharmaceutical composition according to any one of Items 1 to 7, which further contains an adjuvant or is for use in combination with an adjuvant.

[0016] Item 9. The pharmaceutical composition according to any one of Items 1 to 8, which is used for the prevention or treatment of cancer.

[0017] Item 10. A pharmaceutical composition for use in combined administration of a hyaluronic acid derivative having a hydrophobic group introduced therein and a composition containing the antigen, the composition comprising the antigen, the hyaluronic acid derivative containing a lymphocyte expressing an immunoreceptor for the antigen.

[0018] Item 11. A cancer treatment kit comprising a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, and lymphocytes expressing an immune receptor for the cancer antigen.

[0019] Item 12. A T cell activating vaccine comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen.

[0020] Item 13. A cancer treatment kit comprising T cells expressing an immune receptor for a cancer antigen and a pharmaceutical composition, wherein the pharmaceutical composition contains a hyaluronic acid derivative having a hydrophobic group introduced therein, an antigen peptide or antigen protein derived from the cancer antigen, and an adjuvant, wherein the hyaluronic acid derivative is a compound represented by formula (I): [In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1－6 Alkyl, formyl and C 1－6 alkylcarbonyl; R 5 is a hydrogen atom, formyl, or C 1－6 Z represents a direct bond or a peptide linker consisting of any of 2 to 30 amino acid residues; X 1 is represented by the following formula: b -R, -NR b -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, a hydrophobic group selected from the group represented by a , R b and R c are each independently a hydrogen atom, C 1－20 Alkyl, Amino C 2－20 Alkyl and Hydroxy C 2－20alkyl, where the alkyl portion of the group is selected from —O— and —NR f - may be inserted with 1 to 3 groups selected from; R f is a hydrogen atom, C 1－12 Alkyl, Amino C 2－12 Alkyl and Hydroxy C 2－12 alkyl, the alkyl portion of which may be interrupted by 1 to 2 groups selected from -O- and -NH-; R is a steryl group; Y is C 2－30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g - and -S-S-; 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 may be inserted with 1 to 3 groups selected from —O— and —NH—; Y a is C 1－5 alkylene; Y b is C 2－8 Alkylene or C 2－8 and m is an integer selected from 1 to 100.

[0021] Item 14. The cancer treatment kit according to Item 13, wherein the modification rate of carboxyl groups in glucuronic acid moieties of the hyaluronic acid derivative with hydrophobic groups is 5 to 50%.

[0022] Item 15. Z is a direct bond and Y is C 2－12 is alkylene, and X 1 Item 15. The cancer treatment kit according to Item 14, wherein is —NH—COO—R and R is a cholesteryl group.

[0023] Item 16. The cancer treatment kit according to Item 13, wherein the weight-average molecular weight of the hyaluronic acid derivative is 5,000 to 2,000,000.

[0024] Item 17. The hyaluronic acid derivative is represented by formula (IIIc): [In the formula, R 1c , R 2c , R 3c and R 4c are each independently a hydrogen atom, C 1－6 Alkyl, formyl and C 1－6 alkylcarbonyl; R 5c is a hydrogen atom, formyl and C 1－6 alkylcarbonyl; c is hydroxy and -O-Q + Selected from, where Q + Item 14. The cancer treatment kit according to Item 13, comprising a repeating unit represented by the formula:

[0025] Item 18. The cancer treatment kit according to Item 13, wherein the pharmaceutical composition contains a complex of the cancer antigen and the hyaluronic acid derivative.

[0026] Item 19. The cancer treatment kit according to Item 13, wherein the antigen peptide comprises two or more epitopes recognized by CD8-positive cytotoxic T cells and / or CD4-positive helper T cells.

[0027] Item 20. The cancer treatment kit according to Item 19, wherein the antigen peptide is a long-chain peptide antigen comprising two or more epitopes recognized by CD8-positive cytotoxic T cells, or one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0028] Item 21. The cancer treatment kit according to Item 13, which is used in T cell infusion therapy for a cancer with low immunogenicity, a metastatic cancer, or a cancer with a low number of intratumoral T cells.

[0029] Item 22. The cancer treatment kit according to Item 13, wherein the pharmaceutical composition is administered before administering the T cells.

[0030] Item 23. The cancer treatment kit according to Item 22, wherein the pharmaceutical composition is administered at least once after one unit or two units have been administered, where one administration of the pharmaceutical composition followed by one infusion of the T cells constitutes a unit of administration.

[0031] Item 24. The cancer treatment kit according to Item 22, wherein the pharmaceutical composition is administered subcutaneously or intravenously at least twice, and the T cells are infused after the first administration of the pharmaceutical composition and before the second administration of the pharmaceutical composition.

[0032] Item 25. A method for treating cancer, comprising administering to a subject having cancer a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, in combination with lymphocytes expressing an immune receptor for the cancer antigen.

[0033] Item 26. A cancer treatment kit for use in cancer treatment, comprising a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, and lymphocytes expressing an immune receptor for the cancer antigen.

[0034] Item 27. A pharmaceutical composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, for use in cancer therapy by administering lymphocytes expressing an immune receptor for a cancer antigen.

[0035] Item 28. A pharmaceutical composition comprising lymphocytes expressing an immune receptor for a cancer antigen, for use in cancer treatment by administering a pharmaceutical composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen.

[0036] Item 29. Use of a combination of a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, and lymphocytes expressing an immune receptor for the cancer antigen, for the manufacture of a cancer treatment kit.

[0037] Item 30. Use of a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen for the manufacture of a cancer therapeutic agent to be administered in combination with lymphocytes expressing an immune receptor for the cancer antigen.

[0038] Item 31. Use of lymphocytes expressing an immune receptor for a cancer antigen in the manufacture of a cancer therapeutic agent to be administered in combination with a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen.

[0039] According to the present invention, it is possible to provide a pharmaceutical composition that can exert a stronger, more efficient, more sustained, and / or more extensive antitumor effect.

[0040] 1 shows a graph showing the therapeutic effect (tumor growth inhibition) of CMS5a tumors subcutaneously implanted in BALB / c mice when left untreated, or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 1). The plots represent the average tumor area for each group. 1 shows a graph showing the therapeutic effect (tumor growth inhibition) of CMS5a tumors subcutaneously implanted in BALB / c mice when left untreated, or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 1). The plots represent the measured tumor area for each group. This photograph shows the tumor recurrence suppression effect (tumor growth suppression) when a CMS5a tumor was subcutaneously transplanted into a BALB / c mouse and then administered with a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells, and then re-transplanted (CR-Re) the CMS5a tumor into an individual that had completely regressed and shown no recurrence. WT is a photograph showing the size of the tumor when the same tumor was transplanted into a wild-type BALB / c mouse (WT) at the same time (Example 1). This graph shows the cell counts in the regional lymph nodes (inguinal lymph nodes) at the vaccine administration site in BALB / c mice that were untreated, or administered with a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) for CMS5a tumors subcutaneously transplanted into the mouse. These photographs show the regional lymph nodes (inguinal lymph nodes) at the vaccine administration site and the size of the tumor when CMS5a tumors were subcutaneously implanted in BALB / c mice and the mice were either untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 2).Graphs showing the percentage of dendritic cells or macrophages in lymph nodes (inguinal lymph nodes) at the site of vaccine administration in BALB / c mice subcutaneously implanted with CMS5a tumors, either untreated or administered with a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both therapies (HANG-V and TCR-T group) (Example 2). Also shown are graphs showing the percentage of CD207-positive Langerhans cells relative to total CD11c-positive cells. Graphs showing the number of T cells, B cells, NK cells, dendritic cells, and macrophages in lymph nodes (inguinal lymph nodes) at the site of vaccine administration in BALB / c mice subcutaneously implanted with CMS5a tumors, either untreated or administered with a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group) (Example 2). 1 is a graph showing the numbers of T cells, B cells, and NK cells in lymph nodes (inguinal lymph nodes) at the site of vaccine administration when CMS5a tumors were subcutaneously implanted in BALB / c mice that were either untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group) (Example 2). 1 is a graph showing the numbers of dendritic cells and macrophages in lymph nodes (inguinal lymph nodes) at the site of vaccine administration when CMS5a tumors were subcutaneously implanted in BALB / c mice that were either untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group) (Example 2). 1 is a graph showing the ratio of CD90.1-positive infused cells to all CD8-positive T cells in lymph nodes (inguinal lymph nodes) draining the vaccine administration site when CMS5a tumors were subcutaneously implanted in BALB / c mice and the mice were either left untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 2).1 is a graph showing the percentage of interferon gamma (IFN-γ)-producing cells relative to total CD90.1-positive CD8-positive T cells or CD90.1-negative CD8-positive T cells in lymph nodes (inguinal lymph nodes) at the site of vaccine administration in BALB / c mice treated with a CMS5a tumor implanted subcutaneously, either untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 2). BALB / c mice treated with a CMS5a tumor implanted subcutaneously were either untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), or the HANG-V group received antigen-specific T cell infusion therapy, each at a different cell number (2 x 10). 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 5 1 is a graph showing the therapeutic effect (tumor growth inhibition) of a combination of 2 × 10 cells (HANG-V + TCR-T group) and 2 × 10 cells (HANG-V + TCR-T group) (Example 3). The plots represent the average tumor area of ​​individuals in each group. BALB / c mice were subcutaneously implanted with CMS5a tumors, and the mice were either left untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group). The HANG-V group received antigen-specific T cell infusion therapy, with different cell numbers (2 × 10 cells). 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 5 1 is a graph showing the therapeutic effect (tumor growth inhibition) when a combination of 2 × 10 cells (HANG-V + TCR-T group) and 2 × 10 cells (HANG-V + TCR-T group) was administered (Example 3). The plots represent the measured tumor area of ​​individuals in each group. BALB / c mice were subcutaneously implanted with CMS5a tumors, and the mice were either left untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group). The HANG-V group was also administered antigen-specific T cell infusion therapy (TCR-T) at different cell numbers (2 × 10 cells). 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 51 is a graph showing the cell counts in the lymph nodes (inguinal lymph nodes) at the site of vaccine administration when a combination of a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA was administered to BALB / c mice subcutaneously implanted with CMS5a tumors (HANG-V group), either untreated or administered with a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), or antigen-specific T cell infusion therapy (TCR-T) in the HANG-V group (Example 3). 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 5 10 ) in combination (HANG-V + TCR-T group) (Example 3). It is also a graph showing the percentage of CD207-positive Langerhans cells among all CD11c-positive cells. CMS5a tumors were subcutaneously implanted in BALB / c mice. The HANG-V group was left untreated, or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA. The HANG-V group received antigen-specific T cell infusion therapy (TCR-T) with different cell numbers (2 × 10 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 5 1 is a graph showing the ratio of CD90.1-positive cells or CD90.2-negative cells to all CD8-positive T cells in lymph nodes (inguinal lymph nodes) at the vaccine administration site when a combination of 2 × 10 cells (2 × 10 cells) and 1 × 10 cells (HANG-V + TCR-T group) was administered (Example 3). BALB / c mice were treated with a CMS5a tumor subcutaneously implanted in them with either no treatment or administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), or the HANG-V group received antigen-specific T cell infusion therapy (TCR-T). 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 51 is a graph showing the ratio of interferon gamma (IFN-γ)-producing cells to all CD90.1-positive CD8-positive T cells or CD90.2-negative CD8-positive T cells in lymph nodes (inguinal lymph nodes) at the site of vaccine administration when a combination of 2×10 cells (2×10 cells) and 1×10 cells (HANG-V + TCR-T group) was administered (Example 3). BALB / c mice were treated with a CMS5a tumor subcutaneously implanted in them with either no treatment or administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), or the HANG-V group received antigen-specific T cell infusion therapy (TCR-T). 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 5 1 is a graph showing the ratio of interferon gamma (IFN-γ)-producing cells to all CD90.2-positive CD8-positive T cells or CD90.1-negative CD8-positive T cells in lymph nodes (inguinal lymph nodes) draining the vaccine administration site when a combination of a long-chain peptide antigen-loaded HA nanogel cancer vaccine and a CpG oligoDNA (HANG-V group), an antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) was administered (Example 3). Figure 4 shows micrographs of CD8-positive T cells in tumors of CMS5a tumors subcutaneously implanted in BALB / c mice, where the tumors were left untreated, or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and a CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 4). CMS5a tumors were subcutaneously implanted in BALB / c mice. The HANG-V group received either no treatment or a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA. The HANG-V group received antigen-specific T cell infusion therapy (TCR-T) at different cell numbers (2 × 10 6 pieces, 1×10 6 pieces, 5×10 5 pieces, 2.5×10 51 is a graph showing the ratio of CD90.1-positive cells or CD90.2-negative cells to total CD8-positive T cells in peripheral blood cells when a combination of a long-chain peptide antigen-loaded HA nanogel cancer vaccine and a CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), and a combination of both treatments (HANG-V + TCR-T group) were administered to B16F10 tumors subcutaneously implanted in C57BL / 6 mice (Example 6). The plots show the average tumor area for each group. Graphs showing the therapeutic effects (tumor growth inhibition) of B16F10 tumors subcutaneously implanted in C57BL / 6 mice after administration of either untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 6). Plots represent measured tumor area for individuals in each group. Photographs showing tumor size and shape after B16F10 tumors subcutaneously implanted in C57BL / 6 mice after administration of long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group) or a combination of both treatments with antigen-specific T cell infusion therapy (HANG-V + TCR-T group) (Example 6). Graph showing the therapeutic effect (prolonged survival time) of B16F10 tumors subcutaneously implanted in C57BL / 6 mice when untreated, administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), administered antigen-specific T cell infusion therapy (TCR-T group), or administered both treatments in combination (HANG-V + TCR-T group) (Example 6). The plots represent the survival rate of individuals in each group. Photographs showing C57BL / 6 mice in which tumors completely regressed after administration of a long-chain peptide antigen-loaded cancer vaccine, CpG oligoDNA, and administered antigen-specific T cell infusion therapy (Example 6).1 is a graph showing the therapeutic effect (tumor growth inhibition) of B16F10 tumors subcutaneously implanted in C57BL / 6 mice when either untreated or administered a long-chain peptide antigen-carrying cancer vaccine (HANG-V or CHP-V) and CpG oligoDNA in combination with antigen-specific T cell infusion therapy (HANG-V + TCR-T group or CHP-V + TCR-T group) (Example 7). The plots represent the average tumor area for each group.

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[0078] [ Photographs showing the size and shape of tumors in C57BL / 6 mice subcutaneously implanted with B16F10 tumors were administered with a long-chain peptide antigen-carrying cancer vaccine (HANG-V or CHP-V) and CpG oligoDNA, followed by antigen-specific T cell infusion therapy (HANG-V + TCR-T group or CHP-V + TCR-T group) (Example 7). Graphs showing the therapeutic effect (prolonged survival) of C57BL / 6 mice subcutaneously implanted with B16F10 tumors were either untreated or administered with a long-chain peptide antigen-carrying cancer vaccine (HANG-V or CHP-V) and CpG oligoDNA, followed by antigen-specific T cell infusion therapy (HANG-V + TCR-T group or CHP-V + TCR-T group) (Example 7). The plots represent the survival rates of individuals in each group. 10 is a graph showing the percentage of lymph node (inguinal lymph node) cells at the site of vaccine administration in C57BL / 6 mice that had subcutaneously implanted B16F10 tumors and were either left untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 8).10 is a graph showing the percentage of gp100 tetramer-positive (tet+) CD8+ T cells relative to the total CD8+ T cells in lymph nodes (inguinal lymph nodes) draining the vaccine administration site in C57BL / 6 mice treated with either untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 8). 10 is a graph showing the expression intensity of CD44 or KLRG1 in gp100 tetramer-positive CD8-positive T cells in lymph nodes (inguinal lymph nodes) draining the vaccine administration site when B16F10 tumors subcutaneously implanted in C57BL / 6 mice were left untreated, or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 8). 10 is a graph showing the ratio of interferon gamma (IFN-γ)-producing cells to CD90.1-positive CD8-positive T cells in lymph nodes (inguinal lymph nodes) draining the vaccine administration site when B16F10 tumors were subcutaneously implanted in C57BL / 6 mice and the mice were either left untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligoDNA (HANG-V group), antigen-specific T cell infusion therapy (TCR-T group), or a combination of both treatments (HANG-V + TCR-T group) (Example 8). FIG. 10 is a graph showing the ratio of CD90.1-positive CD8-positive infused T cells to all CD8-positive T cells in each tissue (draining lymph node (dLN) at the vaccine administration site, non-draining lymph node (NdLN), spleen, tumor (TIL), and peripheral blood (PBMC)) when a long-chain peptide antigen-loaded cancer vaccine, CpG oligoDNA, and antigen-specific T cell infusion therapy were administered in combination to a B16F10 tumor subcutaneously implanted in a C57BL / 6 mouse (Example 9).Graphs showing the percentage of gp100 tetramer-positive (tet+) CD8+ T cells relative to total CD8+ T cells in tissues (draining lymph nodes (dLN) at the vaccine administration site, non-draining lymph nodes (NdLN), spleen, tumor (TIL), and peripheral blood (PBMC)) when a long-chain peptide antigen-loaded cancer vaccine, CpG oligoDNA, and antigen-specific T cell infusion therapy were administered in combination to a B16F10 tumor subcutaneously implanted in a C57BL / 6 mouse (Example 9). Graphs showing the percentage of CD90.1-positive and / or gp100 tetramer (tet)-positive CD8+ infused T cells relative to total CD8+ T cells in peripheral blood from individuals who achieved complete tumor regression and were relapse-free (CR) after administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells to a B16F10 tumor subcutaneously implanted in a C57BL / 6 mouse (Example 10). 10 is a graph showing the percentage of CD90.1-positive and / or gp100 tetramer (tet)-positive CD8-positive infused T cells relative to total CD8-positive T cells in the peripheral blood of C57BL / 6 mice that underwent complete tumor regression and no recurrence after administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells to subcutaneously transplanted B16F10 tumors, and then were either re-transplanted with B16F10 tumors (CR-Re) or re-vaccinated with the vaccine (CR-Vac) (Example 10). 10 is a graph showing the percentage of CD44+CD62L-negative (effector memory T cells) and CD44+CD62L-positive (central memory T cells) among gp100 tetramer-positive, CD8-positive, CD90.1-positive infused T cells in the peripheral blood of C57BL / 6 mice that were administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells to subcutaneously transplanted B16F10 tumors, and then showed complete tumor regression and no recurrence. The percentages were then compared to mice that were re-transplanted with B16F10 tumors (CR-Re) or re-vaccinated with the vaccine (CR-Vac) (Example 10).10 is a graph showing the expression intensity (High / Mid / Low) of CD44+ cells relative to CD8+ T cells in the peripheral blood of C57BL / 6 mice that had undergone subcutaneous administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells to a B16F10 tumor, and then underwent re-implantation of the B16F10 tumor (CR-Re) or revaccination of the vaccine (CR-Vac) in mice that had completely regressed and were subsequently free of recurrence (Example 10). 11 is a photograph showing the recurrence-suppressing effect (tumor growth inhibition) of C57BL / 6 mice that had undergone subcutaneous administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells to a B16F10 tumor, and then underwent re-implantation of the C57BL / 6 tumor (CR-Re) or revaccination of the vaccine (CR-Vac) in mice that had completely regressed and were subsequently free of recurrence (Example 10). WT is a photograph showing the tumor size when the same tumor was transplanted into wild-type B57BL / 6 mice (WT) at the same time (Example 10). This graph shows the therapeutic effect (tumor growth inhibition) of untreated or administered a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo DNA, and antigen-specific T cells to CMS5a tumors subcutaneously transplanted into BALB / c mice (Example 11). The plot represents the average tumor area for each group. This graph (left) compares the therapeutic effect (tumor growth inhibition) of vaccine readministration, antigen-specific T cells, and immune checkpoint inhibitors (ICIs) in mice that showed tumor regrowth after a response following administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo DNA, and antigen-specific T cells to CMS5a tumors subcutaneously transplanted into BALB / c mice, compared with untreated or responding mice (Example 11). In addition, a separate test similar to that of Example 1 or Example 11 was conducted, and the graph (right) shows tumor growth in individuals in which tumor regrowth was observed when no additional treatment such as vaccine re-administration was administered. The plots represent the measured tumor area of ​​individuals in each group.1 shows a graph showing the recurrence-inhibitory effect (tumor growth inhibition) of CMS5a tumors re-implanted in BALB / c mice after administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo DNA, and antigen-specific T cells to CMS5a tumors subcutaneously, in individuals showing complete tumor regression (CR). WT shows a graph showing tumor growth when the same tumors were implanted in wild-type BALB / c mice (WT) at the same time (Example 12). The plots represent the average tumor area for each group. 1 shows a graph showing the recurrence-inhibitory effect (tumor growth inhibition) of CMS5a tumors re-implanted in BALB / c mice after administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo DNA, and antigen-specific T cells to CMS5a tumors subcutaneously, in individuals showing complete tumor regression (CR). WT shows a graph showing tumor growth when the same tumors were implanted in wild-type BALB / c mice (WT) at the same time (Example 12). The plots represent the measured tumor area of ​​individuals in each group. This graph shows the therapeutic effect (tumor growth inhibition) of untreated or single-administered long-peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo DNA, and antigen-specific T cells on CMS5a tumors subcutaneously implanted in BALB / c mice (Example 13). The plots represent the average tumor area of ​​individuals in each group. This graph shows the therapeutic effect (tumor growth inhibition) of untreated or single-administered long-peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo DNA, and antigen-specific T cells on CMS5a tumors subcutaneously implanted in BALB / c mice (Example 13). The plots represent the measured tumor area of ​​individuals in each group. 14 shows a graph comparing the therapeutic effect (tumor growth inhibition) of BALB / c mice in which tumor regrowth was observed after administration of a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cells to CMS5a tumors subcutaneously implanted in the mice. Vaccine re-administration and additional treatment with an immune checkpoint inhibitor (ICI) were then performed on the mice, comparing the therapeutic effect (tumor growth inhibition) with that of untreated or responding mice (Example 14). The plots show the measured tumor area of ​​each group of mice.15 is a graph showing the percentage of CD44+CD62L-negative (effector memory T cells) and CD44+CD62L-positive (central memory T cells) among all CD8+ T cells in peripheral blood cells in BALB / c mice that showed complete tumor regression (CR) after a combination of a long-peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cell infusion therapy against a CMS5a tumor subcutaneously implanted in the mouse, when the HA vaccine was re-administered (Vac) or not (UT) (Example 15). 16 is a graph showing the percentage of CD44+CD62L-negative cells (effector memory T cells) and CD44+CD62L-positive cells (central memory T cells) relative to total CD8+ T cells in peripheral blood cells when the HA vaccine was re-administered (Vac) or not (UT) in individuals (PD) who underwent additional treatment due to tumor regrowth after successful treatment with a long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligoDNA, and antigen-specific T cell infusion therapy against CMS5a tumors subcutaneously implanted in BALB / c mice (Example 16). The sequence of mMAGE#17zG CAR is shown. A schematic diagram of the structure of mMAGE#17zG CAR is shown. The sequence of mMAGE 28z CAR is shown. A schematic diagram of the structure of mMAGE 28z CAR is shown. The sequence of the MAGE-A4 expression vector is shown. A schematic diagram of the structure of the MAGE-A4 expression vector is shown. The sequence of the HHD-A2 expression vector is shown. A schematic diagram of the structure of the HHD-A2 expression vector is shown. Graphs showing the percentage of CD4-positive or CD8-positive T cells in cultured cells and the percentage of CAR expression in each T cell when the MAGE#17 zG CAR gene was introduced into spleen cells derived from HLA-A2 Tg mice cultured using anti-mouse CD3 antibody and CD28 antibody (Example 17). Graphs showing the percentage of expression of both the MAGE-A4 gene and the HHD-A2 gene when introduced into MC38 and CT26 cells (Example 17). Graphs showing the amount of IFN-γ produced in the culture supernatant when mMAGE#17 zG CAR-introduced T cells or cells not introduced with the CAR gene were added to MC38 and CT26 cells and co-cultured (Example 17).

[0041] In one aspect, the present invention relates to a combination of a composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen (sometimes referred to herein as an "HA-antigen composition") and lymphocytes expressing an immune receptor for the antigen (sometimes referred to herein as "antigen-specific lymphocytes"). This is described below.

[0042] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0043] The term "C" referred to herein 1－20 "Alkyl" means a straight or branched chain alkyl group having 1 to 20 carbon atoms, and examples thereof include "C alkyl" such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, and t-butyl. 1－4 C includes "alkyl" and further includes n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, and 2-ethylbutyl. 1－20 Alkyl has 1 to 12 carbon atoms, "C 1－12 alkyl, with 1 to 6 carbon atoms, 1－6 "Alkyl" is also included.

[0044] The term "C" referred to herein 1－6 "Alkyl" means a straight or branched chain alkyl group having 1 to 6 carbon atoms, and examples thereof include "C alkyl" such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, and t-butyl. 1－4 alkyl" is included.

[0045] The term "C" referred to herein 1－6 "Alkylcarbonyl" refers to a group in which the alkyl portion is one of the C groups already mentioned. 1－6It means an alkylcarbonyl group, for example, acetyl, propionyl, n-propylcarbonyl, i-propylcarbonyl, n-butylcarbonyl, s-butylcarbonyl, i-butylcarbonyl, t-butylcarbonyl, etc. 1－4 alkylcarbonyl".

[0046] The term "C" referred to herein 1－6 "Alkoxy" refers to a group in which the alkyl portion is one of the C 1－6 It means an alkyloxy group, for example, methoxy (H3C-O-), ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, i-butoxy, t-butoxy, etc. 1－4 Alkoxy" is included.

[0047] The term "C" referred to herein 1－6 "Alkylthio" means that the alkyl part is the C 1－6 It means an alkylthio group, which is alkyl, and includes, for example, methylthio (H3C-S-), ethylthio, n-propylthio, i-propylthio, n-butylthio, s-butylthio, i-butylthio, t-butylthio, etc., and preferably methylthio.

[0048] The term "amino C" referred to herein 2－20 "Alkyl" means a linear or branched alkyl group having 2 to 20 carbon atoms and having an amino group as a substituent, and for example, the amino group may be located on the terminal carbon atom of the alkyl group. 2－20 Alkyl has 2 to 12 carbon atoms, such as "amino C 2－12 "Alkyl" is also included.

[0049] The term "hydroxy C" referred to herein 2－20 "Alkyl" means a linear or branched alkyl group having 2 to 20 carbon atoms and having a hydroxy group as a substituent, and for example, the hydroxy group may be located on the terminal carbon atom of the alkyl group. 2－20 Alkyl has 2 to 12 carbon atoms, such as "hydroxy C 2－12 "Alkyl" is also included.

[0050] The term "C" referred to herein 2－30 "Alkylene" means a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, including, for example, ethylene, propylene, etc. 2－20 Alkylene, C 2－8 Alkylene includes the group -(CH2)n- where n is 2 to 30, preferably 2 to 20, more preferably 2 to 15.

[0051] The term "C" referred to herein 1－5 The term "alkylene" refers to a linear or branched divalent saturated hydrocarbon group having from 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).

[0052] The term "C" referred to herein 2－10 "Alkylene" means a linear or branched divalent saturated hydrocarbon group having 2 to 10 carbon atoms, and includes, for example, ethylene (ethane-1,2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl), butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl, and the like. 2－10 Alkylene is a group of 2 to 8 carbon atoms. 2－8 Alkylene" and "C" 2－6 Contains "alkylene".

[0053] The term "C" referred to herein 2－8 "Alkylene" means a linear or branched divalent saturated hydrocarbon group having from 2 to 8 carbon atoms, and includes, for example, ethylene (ethane-1,2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl), butane-1,4-diyl, pentane-1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl, and the like.

[0054] The term "C" referred to herein2－8 The term "alkenylene" means a divalent saturated hydrocarbon group having 2 to 8 carbon atoms, which is linear or branched and contains one or more double bonds, and includes, for example, -CH=CH-, -C(CH3)=CH-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, and octa-2,4,6-triene-1,8-diyl. When geometric isomers exist, each isomer and a mixture thereof are also included.

[0055] In the present invention, "aryl" means an aromatic carbocyclic group, for example, an aromatic carbocyclic group having 6 to 14 carbon atoms, and examples of aryl include phenyl, naphthyl (1-naphthyl and 2-naphthyl), etc. An example of aryl substituted with one or more hydroxy groups is 4-hydroxyphenyl.

[0056] In the present invention, "heteroaryl" refers to an aromatic ring group containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur atoms among the atoms constituting the ring, and may be partially saturated. The ring may be a monocyclic ring or a bicyclic heteroaryl fused with a benzene ring or a monocyclic heteroaryl ring. The number of atoms constituting the ring is, for example, 4 to 15, preferably 5 to 14, and more preferably 6 to 10. Examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, and imidazopyridyl, and preferably indol-2-yl.

[0057] The "divalent C" referred to in this specification 2－50The "hydrocarbon group" is not particularly limited, and examples thereof include linear, branched, cyclic, and partially cyclic alkylene, alkenylene, and alkynylene groups having 2 to 50 carbon atoms, and such groups may be divalent aromatic rings or may contain an aromatic ring as part of their structure.

[0058] The "divalent C" referred to in this specification 2－50 The "polyalkyleneoxy" is not particularly limited, and the alkylene group of the repeating unit may be a straight chain or a branched chain. 2－50 Examples of "polyalkyleneoxy" include divalent C 2－50 Polyethyleneoxy group, C 3－48 Polypropyleneoxy group, C 3－48 The group may be linked to other groups via an oxygen atom or a carbon atom, for example, C 2－50 The polyethyleneoxy group has -O(CH2CH2O) 1－25 -, -(CH2CH2O) 1－25 -, -(OCH2CH2) 1－25 -, -(CH2CH2O) 1－24 -(CH2CH2)- and the like.

[0059] The term "salt substance" referred to in this specification is not particularly limited as long as it is an inorganic substance soluble in water, and examples thereof include calcium salts such as calcium chloride and calcium phosphate, magnesium salts such as magnesium sulfate and magnesium chloride, aluminum salts such as aluminum sulfate and aluminum chloride, potassium salts such as potassium sulfate, potassium carbonate, potassium nitrate, potassium chloride, potassium bromide and potassium iodide, sodium salts such as sodium bicarbonate, sodium carbonate, sodium sulfate, sodium nitrate, sodium chloride, sodium bromide, sodium iodide, sodium silicate, trisodium phosphate, disodium phosphate, sodium borate, sodium acetate and sodium citrate, and lithium salts such as lithium chloride, lithium bromide, lithium iodide and lithium carbonate, and preferably includes sodium chloride, trisodium phosphate, disodium phosphate, potassium chloride, calcium chloride and magnesium chloride.

[0060] The term "antigen" referred to herein refers to a substance that induces immunity. For example, in lymph nodes or the spleen, it is a substance that can induce the activation of lymphocytes such as T cells and B cells by being presented by antigen-presenting cells. The term "antigen protein" refers to an antigen that is a protein. Furthermore, the term "antigen peptide" refers to an antigen that is a peptide. For example, an antigen peptide is a part of the amino acid sequence contained in an antigen protein and includes a T cell-recognized epitope. It may also be a combination of multiple T cell-recognized epitopes. What is recognized as an antigen can be, for example, the protein or peptide itself, a complex of the protein or peptide with another molecule (e.g., major histocompatibility complex (MHC)), or the major histocompatibility complex (MHC) itself.

[0061] The term "adjuvant" as referred to herein refers to a substance that enhances the immune response in a subject when administered to the subject in combination with a vaccine containing an antigen.

[0062] 2. HA-antigen composition <2-1. Hyaluronic acid derivative having a hydrophobic group introduced therein> The hyaluronic acid derivative having a hydrophobic group introduced therein is not particularly limited, as long as it is hyaluronic acid or a derivative thereof, has a hydrophobic group, and is capable of forming a complex with an antigen (particularly, an antigen peptide or antigen protein).

[0063] The hydrophobic group is a group having hydrophobicity, and is not particularly limited as long as the hyaluronic acid derivative can form a complex with an antigen (especially, antigen peptide, antigen protein).Preferably, the hydrophobic group includes a hydrophobic group having a sterol skeleton (steryl group), a hydrocarbon group, etc., and particularly preferably, the steryl group.

[0064] The sterol skeleton is an alcohol with a hydroxy group bonded to a cyclopentahydrophenanthrene ring as shown in formula I. The symbols A to D in formula I represent the individual rings that make up the cyclopentahydrophenanthrene ring.

[0065]

[0066] The sterol skeleton may have a double bond in the cyclopentahydrophenanthrene ring, and the position of the hydroxyl group is not particularly limited. Sterols having a hydroxyl group bonded to the C-3 position and a double bond in the B ring, or stanols having a hydroxyl group bonded to the C-3 position and a saturated ring, are preferred. Examples of hydrophobic groups include groups derived from compounds (steroids) in which the sterol skeleton is modified, for example, by substituting a hydrocarbon group (e.g., a linear or branched alkyl group having 1 to 20 carbon atoms) at a ring carbon atom. Here, the term "derived group" refers to a group obtained by removing a functional group such as a hydrogen atom or a hydroxyl group from a compound.

[0067] Specific examples of steroids include cholesterol, dehydrocholesterol, coprostenol, coprostanol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, cimialenol, bile acids (cholanic acid, lithocholic acid, hyodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, apocholic acid, cholic acid, dehydrocholic acid, glycocholic acid, taurocholic acid), testosterone, estradiol, progesterone, cortisol, cortisone, aldosterone, corticosterone, and deoxycortisterone. Examples of the steryl group include a cholesteryl group, a stigmasteryl group, a lanosteryl group, an ergosteryl group, a cholanoyl group, and a choloyl group, and preferred examples include a cholesteryl group (particularly, a cholest-5-en-3β-yl group represented by the following formula) and a cholanoyl group (particularly, a 5β-cholan-24-oyl group represented by the following formula), in which the two asterisks indicate the bonding position.

[0068]

[0069] The hydrocarbon group is not particularly limited, and examples thereof include chain (preferably linear) hydrocarbon groups (preferably alkyl groups) having 8 to 50 carbon atoms (preferably 10 to 30, more preferably 12 to 20).

[0070] The weight-average molecular weight of the hyaluronic acid derivative into which a hydrophobic group has been introduced is not particularly limited as long as it can form a complex with an antigen, but is, for example, 5,000 to 2,000,000, preferably 10,000 to 1,000,000, more preferably 20,000 to 500,000, even more preferably 40,000 to 250,000, and even more preferably 80,000 to 125,000.

[0071] The introduction rate of hydrophobic groups is, for example, 1 to 50%, preferably 7 to 50%, more preferably 17 to 50%, and even more preferably 20 to 45%. When the introduction rate is within the above range, the hyaluronic acid derivative can efficiently form a complex with an antigen in solution. The introduction rate of hydrophobic groups can be measured and calculated according to or in accordance with the method described below.

[0072] Specific examples of the hyaluronic acid derivatives into which hydrophobic groups have been introduced include hyaluronic acid derivatives that contain one or more disaccharide units (also repeating units) represented by formula (I) or hyaluronic acid derivatives that contain one or more disaccharide units (also repeating units) represented by formula (II).Hyaluronic acid derivatives include, for example, the following: formula (I)

[0073]

[0074] [In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1－6 Alkyl, formyl and C 1－6 alkylcarbonyl; R 5 is a hydrogen atom, formyl, or C 1－6 Z represents a direct bond or a peptide linker consisting of any of 2 to 30 amino acid residues; X 1 is represented by the following formula: b -R, -NR b -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, a hydrophobic group selected from the group represented by a , R b and R c are each independently a hydrogen atom, C 1－20 Alkyl, Amino C 2－20 Alkyl and Hydroxy C 2－20 alkyl, where the alkyl portion of the group is selected from —O— and —NR f - may be inserted with 1 to 3 groups selected from; R f is a hydrogen atom, C 1－12 Alkyl, Amino C 2－12 Alkyl and Hydroxy C 2－12 alkyl, the alkyl portion of which may be interrupted by 1 to 2 groups selected from -O- and -NH-; R is a steryl group; Y is C 2－30 Alkylene, or -(CH2CH2O) m -CH2CH2-, where the alkylene is -O-, -NR g - and -S-S-; 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 may be inserted with 1 to 3 groups selected from —O— and —NH—; Y a is C 1－5 alkylene; Y b is C 2－8 Alkylene or C 2－8 and m is an integer selected from 1 to 100; or a hyaluronic acid derivative comprising one or more repeating units represented by formula (II):

[0075]

[0076] [In the formula, R 1a , R 2a , R 3a , and R 4a are independently hydrogen atoms, C 1－6 Alkyl, formyl, and C 1－6 alkylcarbonyl; R 5a is a hydrogen atom, formyl, or C 1－6 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 hydrogen atoms, and C 1－6 alkyl; R aa is a hydrogen atom or C 1－6 alkyl, wherein the alkyl is independently hydroxy, carboxy, carbamoyl, 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 -(CH2CH2O) ma -CH2CH2-, wherein the alkylene is independently -O-, -NR ga - and -S-S-, and ma is an integer selected from 1 to 100; Z 2 is represented by the following formula: ba -Z 3 , -NR ba -COO-Z 3 , -NR ba -CO-Z 3 , -NR ba -CO-NR ca -Z 3 , -COO-Z3 , -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 R ba and R ca are independently hydrogen atoms, C 1－20 Alkyl, Amino C 2－20 Alkyl and Hydroxy C 2－20 alkyl, where the alkyl portion of the group is independently selected from —O— and —NR fa - may be inserted with 1 to 3 groups selected from; R fa are independently hydrogen atoms, C 1－12 Alkyl, Amino C 2－12 Alkyl and Hydroxy C 2－12 alkyl, the alkyl portion of which may be optionally interrupted by 1 to 2 groups independently selected from —O— and —NH—; R ga are independently hydrogen atoms, C 1－20 Alkyl, Amino C 2－20 Alkyl or hydroxy C 2－20 alkyl, the alkyl portion of which may be optionally interrupted by 1 to 3 groups independently selected from —O— and —NH—; Z 3 is a steryl group; Z a is C 1－5 alkylene; Z b is C 2－8 Alkylene or C 2－8 A hyaluronic acid derivative containing a repeating unit represented by the formula: X is an alkenylene. 1a Ga-NR 9 -Z 1 -Z 2When the repeating unit represented by formula (II) is not contained, the repeating unit represented by formula (III):

[0077]

[0078] [In the formula, R 1b , R 2b , R 3b and R 4b are independently hydrogen atoms, C 1－6 Alkyl, formyl, and C 1－6 alkylcarbonyl; R 5b is a hydrogen atom, formyl, or C 1－6 alkylcarbonyl; X 2 is -NR 9 -Z 1 -Z 2 where R 9 , Z 1 , and Z 2 is as defined above].

[0079] The hyaluronic acid derivative having a hydrophobic group introduced therein is represented by the formula (IIIc):

[0080]

[0081] [In the formula, R 1c , R 2c , R 3c and R 4c are each independently a hydrogen atom, C 1－6 Alkyl, formyl and C 1－6 alkylcarbonyl; R 5c is a hydrogen atom, formyl and C 1－6 alkylcarbonyl; c is hydroxy and -O-Q + Selected from, where Q + represents a counter cation].

[0082] In one embodiment, the hyaluronic acid derivative comprising a repeating unit represented by formula (I) is essentially composed of repeating units of (1) formula (I); and (2) formula (I) and formula (IIIc).

[0083] When the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) contains two or more repeating units of formula (I), the repeating units may be the same or different. The hyaluronic acid derivative may be modified at a position other than the repeating unit of formula (I), for example, the hydroxy group may be modified by -O(C 1－6 alkyl), -O(formyl) and -O(C 1－6 The carboxyl group may be converted to an amide group or an ester group, or may form a salt.

[0084] According to one aspect, the group -Z-N(R a ) Y-X 1 is represented by the formula: -NH-(CH2) mz -NH-R; -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; -NH-(CH2) mz -COO-R; -NH-(CH2CH2O) m -CH2CH2-COO-R; -NH-(CH2) mz -O-COO-R; -NH-(CH2CH2O) m -CH2CH2-O-COO-R; -NH-(CH2) mz -S-R; -NH-(CH2CH2O) m -CH2CH2-S-R; -NH-(CH2) mz -O-CO-CH(R 10 )-CH2-S-R; -NH-(CH2) mz -NHCO-CH(R 10 )-CH2-S-R; -NH-(CH2CH2O) m -CH2CH2-NHCO-CH(R 10 )-CH2-S-R;-NH-(CH2CH2O) m -CH2CH2-O-CO-CH(R10 )-CH2-S-R; and -NH-(CH2) mz -S-S-R; -Z-NR a -Y-NR b -COO-R (where mz is an integer from 2 to 30, and R 10 is a hydrogen atom or a methyl group, and R and m are as defined above in this specification. The group is preferably selected from the group represented by: -NH-(CH2) mz -NH-COO-R; -NH-(CH2CH2O) m -CH2CH2-NH-COO-R; and -NH-(CH2) mz -S-S-R, where mz, R, and m are as previously defined herein.

[0085] In a preferred embodiment, Z in the above formula (I) is a direct bond. In addition, in one embodiment, when Z in the above formula (I) is a peptide linker, X 1 Ha-NR b Specific examples of Y in the above formula (I) include -CH2CHO-CH2CH2-S-S-CH2CHO-CH2CH2-, -(CH2CHO)2-CH2CH2-S-S-CH2CHO-CH2CH2-, -CH2CHO-CH2CH2-S-S-(CH2CHO)2-CH2CH2-, -CH2CHO-CH2CH2-S-S-(CH2CHO)2-CH2CH2-, and -(CH2CHO)2-CH2CH2-S-S-(CH2CHO)2-CH2CH2-.

[0086] Y in the above formula (I) a As the alkyl group, -CH2- and -CH2-CH2- are preferred.

[0087] Y in the above formula (I) b Preferred examples of the alkyl group include -CH2-CH2-, -CH(CH3)CH2-, 2-butene-1,4-diyl, hepta-2,4-diene-1,6-diyl, and octa-2,4,6-triene-1,8-diyl, and more preferred examples are -CH2-CH2- and -CH(CH3)CH2-.

[0088] In one embodiment, Z in the above formula (I) is —NH—[CH(—Z a )-CONH]n -1-CH(-Z a )-CO-, where n is an integer from 2 to 30; Z a are each independently H2N-CH(-Z a The peptide linker is attached to the carboxyl group of the glucuronic acid moiety at the N-terminus and to the group -N(-R)-COOH at the C-terminus. a )-Y-X 1 Examples of amino acids that can be used as amino acid residues of the peptide linker include α-amino acids, such as natural (L-form) amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, as well as D-forms thereof, and all α-amino acids, including synthetic amino acids, can be used. That is, Z a Examples of the linker include -CH3, H2NC(NH)NH(CH2)3-, and H2NCOCH2-. The n Zs may be the same or different. n is an integer of 2 to 30, preferably 2 to 10, and more preferably 2 to 4. Preferred examples of the peptide linker include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, and -Phe-Gly-.

[0089] The group -Z-N(R a ) Y-X 1Specific examples of the alkyl group include -NH-(CH2)2-NH-CO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-NH-(CH2)3-NH-COO-cholesteryl, -NH-(CH2)4-N(-(CH2)3-NH2)-COO-cholesteryl, -NH-(CH2)3-NH-(CH2)4-N(-(C Examples include -Z-N(R a ) Y-X 1 In a preferred embodiment, R a , R b and R c is a hydrogen atom, and Y is a linear C 2－30 Alkylene or -(CH2CH2O) m -CH2CH2- and Y a However, linear C 1－5 alkylene, or Y b However, linear C 2－8 Alkylene or linear C 2－8 It is alkenylene.

[0090] In one embodiment, the group Z—N(R a ) Y-X 1 Z is a direct bond, R a is a hydrogen atom, Y is, for example, C 2－12 Alkylene, preferably C 2－6 Alkylene, more preferably C6 alkylene, X 1 Ga-NR b -COO-R, R b is a hydrogen atom and R is a cholesteryl group.

[0091] In one embodiment, the hyaluronic acid derivative that comprises the repeating unit represented by formula (I) further comprises a repeating unit represented by formula (IIIc).When the repeating unit represented by formula (IIIc) comprises two or more, the repeating units may be the same or different.

[0092] Q of the above formula (IIIc) ＋ is not particularly limited as long as it is a counter cation that forms a salt with a carboxyl group in water, and if it is divalent or higher, it forms a salt with multiple carboxyl groups depending on the valence. Examples of counter cations include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, and calcium ion; j R k R l R m (In the formula, R j , R k , R l and R m are each independently a hydrogen atom and C 1－6 Preferred examples include sodium ions, potassium ions, and tetraalkylammonium ions (e.g., tetra-n-butylammonium ions). j , R k , R l and R m is C 1－6 Preferably, they are identical groups selected from alkyl, and preferably n-butyl groups.

[0093] The group R of formula (I) 1 , R 2 , R 3 , and R 4 and R in the above formula (IIIc) 1a , R 2a , R 3a and R 4a are preferably all hydrogen atoms. a and R b are preferably all hydrogen atoms.

[0094] The group R of formula (I) 5is preferably acetyl.

[0095] In one embodiment, a hyaluronic acid derivative containing repeating units represented by formula (I) is substantially composed of repeating units of formulas (I) and (IIIc). Of the repeating units of a disaccharide consisting of D-glucuronic acid and N-acetylglucosamine contained in the hyaluronic acid derivative, for example, 80% or more, preferably 90% or more, and more preferably 95% or more are repeating units of formula (I) or (IIIc). In one embodiment, the hyaluronic acid derivative is composed solely of repeating units represented by formula (I) and formula (IIIc).

[0096] Y defined in formula (I) may be, for example, —(CH2) na - (where na is selected from integers of 2 to 20, preferably 2 to 15, more preferably 2 to 12, and even more preferably 2 to 10), and preferably -(CH2)2-, -(CH2)6-, -(CH2)8- and -(CH2) 12 -, and more preferably -(CH2)6-. These Y's are preferred from the viewpoint of precipitation and stable dispersion, which will be described later.

[0097] In one aspect, the hyaluronic acid derivative containing the repeating unit represented by formula (I) has a hydrophobic group introduction rate relative to the disaccharide repeating unit present in the derivative of, for example, 1 to 50%, preferably 7 to 50%, more preferably 17 to 50%, and even more preferably 20 to 45%. When the introduction rate is within this range, the hyaluronic acid derivative containing the repeating unit represented by formula (I) can efficiently form a complex with an antigen in solution.

[0098] Here, the introduction rate of the hydrophobic group is calculated by the following formula:

[0099]

[0100] Here, the "disaccharide repeating unit present in the derivative" includes the repeating unit of formula (I) in which the carboxyl group has been converted to an amide group and a hydrophobic group has been introduced, and the repeating unit of formula (IIIc) in which no hydrophobic group has been introduced. The introduction rate can be controlled by the reaction conditions, for example, the ratio of reagents, and can be determined, for example, by NMR measurement.

[0101] The hyaluronic acid derivative containing the repeating unit represented by the above formula (I) is preferably R 1c , R 2c , R 3c , and R 4c are all hydrogen atoms, and R 5c is acetyl, and X c wo-O-N a＋ The hyaluronic acid or its derivatives are synthesized from a starting material consisting solely of repeating units represented by formula (IIIc), which has a weight-average molecular weight of, for example, 1 kDa to 500 kDa, preferably 3 kDa to 500 kDa, and more preferably 5 kDa to 200 kDa. In one embodiment, the weight-average molecular weight of the starting material is preferably 1 kDa to 1000 kDa, more preferably 3 kDa to 200 kDa, from the viewpoint of complex formation with an antigen. The weight-average molecular weight of the starting material is preferably 1 kDa to 1000 kDa, more preferably 3 kDa to 500 kDa, even more preferably 5 kDa to 200 kDa, from the viewpoint of lymph node migration of the complex.

[0102] Generally, since it is difficult to obtain hyaluronic acid and its derivatives as a single product, their molecular weights are calculated as number-average molecular weights or weight-average molecular weights. In the present invention, they are calculated as weight-average molecular weights. As for the method for measuring weight-average molecular weights, various known methods can be used, such as light scattering, osmotic pressure, and viscosity methods, as described in "Essential Polymer Science" by Nakahama Seiichi et al. (published by Kodansha, ISBN4-06-153310-X). The viscosity-average molecular weights shown in this specification can also be measured by methods commonly used in the technical field to which the present invention pertains, such as using an Ubbelohde viscometer. When using commercially available hyaluronic acid and its derivatives with their molecular weights clearly indicated, the indicated numerical value can also be used as the molecular weight.

[0103] The hyaluronic acid derivative that comprises the repeating unit of the above formula (I) is converted into amide group the carboxyl group of glucuronic acid, which is one of the disaccharides that constitute repeating unit, and introduces hydrophobic group.By adjusting the modification degree of hyaluronic acid derivative, it is also possible to control the pharmacokinetics of the preparation that uses this derivative to produce.

[0104] If the carboxyl group modification rate of the glucuronic acid portion 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 targeting elements into the hyaluronic acid derivative, it is possible to target various organs and cells, including lymph nodes. Examples of targeting elements include target tissue-specific peptides, antibodies, fragmented antibodies, aptamers, and R-antibodies against cancer cells. g These include D-peptide, folic acid, anisamide, transferrin, galactose for the liver, and tocopherol.

[0105] The modification rate of the carboxyl group of the glucuronic acid moiety of the hyaluronic acid derivative containing the repeating unit of the above formula (I) with a hydrophobic group, i.e., the introduction rate of the hydrophobic group, is preferably 4-60%, more preferably 5-50%, more preferably 7-50%, and even more preferably 7-45% from the viewpoint of complex formation with an antigen. From the viewpoint of lymph node migration of the complex, it is preferably 6-60%, more preferably 17-50%, more preferably 20-50%, and even more preferably 20-45%.

[0106] The molecular weight of the raw material of the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) and the introduction rate of hydrophobic groups are preferably 3kDa to 500kDa and 4 to 60%, more preferably 3kDa to 200kDa and 6 to 50%, more preferably 5kDa to 200kDa and 6 to 50%, and more preferably 5kDa to 150kDa and 6 to 45%.In terms of the lymph node migration of the complex, preferably 3kDa to 500kDa and 4 to 60%, more preferably 3kDa to 200kDa and 6 to 50%, more preferably 5kDa to 200kDa and 6 to 50%, and more preferably 5kDa to 150kDa and 20 to 45%. From the viewpoint of stable dispersion, 3 kDa to 200 kDa and 4 to 60% are preferred, 3 kDa to 200 kDa and 6 to 50% are more preferred, 5 kDa to 200 kDa and 6 to 50% are even more preferred, and 5 kDa to 150 kDa and 17 to 45% are even more preferred. From the viewpoint of improving blood retention, 5 kDa to 27 kDa and 2 to 50% are preferred, 5 kDa to 27 kDa and 8 to 35% are even more preferred, 5 kDa to 18 kDa and 8 to 35% are even more preferred, and 5 kDa to 18 kDa and 15 to 22% are even more preferred. From the viewpoint of gelation, 5 kDa to 300 kDa and 2 to 30% are preferred, 5 kDa to 50 kDa and 2 to 22% are more preferred, 5 kDa to 27 kDa and 2 to 22% are even more preferred, and 5 kDa to 27 kDa and 7 to 22% are even more preferred.

[0107] In one embodiment, the hyaluronic acid derivative containing the repeating unit represented by formula (II) is substantially composed of repeating units of formula (1) (II); (2) (II) and (III); (3) (II) and (IIIc); or (4) (II), (III), and (IIIc). Of the repeating units of the disaccharide consisting of D-glucuronic acid and N-acetylglucosamine contained in the hyaluronic acid derivative, for example, 80% or more, preferably 90% or more, and more preferably 95% or more are repeating units of formula (II), (III), or (IIIc). In one embodiment, the hyaluronic acid derivative is composed exclusively of repeating units of formula (1) (II); (2) (II) and (III); (3) (II) and (IIIc); or (4) (II), (III), and (IIIc).

[0108] The ratio of a specific disaccharide unit to the repeating disaccharide units present in a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) means the ratio of a specific disaccharide unit to all disaccharide units contained in a certain amount of a hyaluronic acid derivative containing a repeating unit represented by the above formula (II), which is a polysaccharide having disaccharide units as repeating units.

[0109] In the formula (II) representing the disaccharide unit contained in the hyaluronic acid derivative containing the repeating unit represented by the formula (II), R 1a , R 2a , R 3a , and R 4a Preferably, all of R are hydrogen atoms. 5a is a hydrogen atom or C 1－6 Preferably, R is alkylcarbonyl, more preferably hydrogen atom or acetyl, and even more preferably acetyl. In addition, in the 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 R4c Preferably, all of R are hydrogen atoms. 5b and R 5c is a hydrogen atom or C 1－6 It is preferably an alkylcarbonyl, more preferably a hydrogen atom or acetyl, and most preferably both are acetyl.

[0110] R in formula (II) aa Specific examples include a hydrogen atom, methyl, hydroxymethyl, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, 1-methylpropyl, 2-methylpropyl, isopropyl, 2-carboxyethyl, 2-methylthioethyl, 2-carbamoylethyl, phenylmethyl, (4-hydroxyphenyl)methyl, and indol-3-ylmethyl.

[0111] base-CHR aa When - is an asymmetric center, each optically active substance and a mixture thereof are included, but H2N-CHR aa When written as --COOH (amino acid), it is preferably in the L form (natural form).

[0112] 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.

[0113] The group —CHR in formula (II) aa -CO-X 1a As an embodiment of the present invention, for example, the group -CHR aa Specific examples of the group include the following:

[0114]

[0115] where the asterisk stands for -NR 6a - represents the bonding position (same below).

[0116] base-CHR aa Preferred examples of —COOH include the following groups:

[0117]

[0118] base-CHR aa Preferred examples of —COOH include the following groups:

[0119]

[0120] base-CHR aa Preferred examples of —COOH include the following groups:

[0121]

[0122] base-CHR aa Preferred examples of —COOH include the following groups:

[0123]

[0124] In terms of delivery of the conjugate to lymph nodes, the group -CHR aa Preferred examples of —COOH include the following groups:

[0125]

[0126] More preferred examples include the following groups:

[0127]

[0128] More preferred examples include the following groups:

[0129]

[0130] The group -CHR as described above aa Any of the -COOH groups may be partly or entirely -CHR groups. aa -CONH-Z 1 -Z 2 The group -Z may be converted to 1 -Z 2 Examples of the group -CHR in formula (II) are as follows: aa -CO-X 1a In another embodiment, for example, the group -CHR aa Specific examples of the group include the following:

[0131]

[0132] base-CHR aa Preferred examples of -CONH2 include the following groups:

[0133]

[0134] base-CHR aa Preferred examples of -CONH2 include the following groups:

[0135]

[0136] base-CHR aa Preferred examples of -CONH2 include the following groups:

[0137]

[0138] base-CHR aa Preferred examples of -CONH2 include the following groups:

[0139]

[0140] These groups are also preferred groups in that they have both biodegradability and blood retention.

[0141] In terms of having both biodegradability and blood retention, the -CHR group aa Preferred examples of -CONH2 also include the following groups:

[0142]

[0143] In order to have both biodegradability and blood retention, the -CHR group aa More preferred examples of -CONH2 include the following groups:

[0144]

[0145] From the viewpoint of better dispersibility in pure water, the group -CHR aa Preferred examples of -CONH2 include the following groups:

[0146]

[0147] These two groups are also preferred examples from the viewpoint of being base materials for injection preparations for sustained subcutaneous administration.

[0148] From the viewpoint of a base material for a sustained subcutaneous injection formulation, the group -CHR aa Preferred examples of -CONH2 include the following groups:

[0149]

[0150] R 7 As the , a hydrogen atom and methyl are more preferred, and a hydrogen atom is even more preferred.

[0151] Carboxy as defined in formulas (II), (III) and (IIIc) is a group of formula -COO-Q ＋ In this case, Q may form a salt represented by the formula: ＋ is not particularly limited as long as it is a counter cation that forms a salt with carboxy in water, and if it is divalent or higher, it forms a salt with multiple carboxy depending on the valence. Examples of counter cations include metal ions such as lithium ion, sodium ion, rubidium ion, cesium ion, magnesium ion, and calcium ion; j R k R l R m (In the formula, R j , R k , R l and R m are each independently a hydrogen atom and C 1－6 Preferred examples include sodium ions, potassium ions, and tetraalkylammonium ions (e.g., tetra-n-butylammonium ions). j , R k , R l and R m is C 1－6 Preferably, they are identical groups selected from alkyl, and preferably n-butyl.

[0152] The group —CHR in formula (II) aa -CO-X 1a In another embodiment, for example, the group -CHR aa -CONH-Z 1 -Z 2Specific examples of the group include the following groups.

[0153]

[0154] Other specific examples of the group include the following groups.

[0155]

[0156] base-CHR aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0157]

[0158] base-CHR aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0159]

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

[0161]

[0162] In terms of delivery of the conjugate to lymph nodes, the group -CHR aa -CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0163]

[0164] More preferred groups include the following groups:

[0165]

[0166] More preferred examples include the following groups:

[0167]

[0168] In order to have both biodegradability and blood retention, the -CHR group aa-CONH-Z 1 -Z 2 Preferred examples of include the following groups:

[0169]

[0170] Group-Z 1 -Z 2 Examples of the group -(C 2－10 (Alkylene)-NH-COO-Z 3 In addition, the group -(C 2－12 (Alkylene)-NH-COO-Z 3 Here, C 2－12 Preferred examples of alkylene include -(CH2)2-, -(CH2)6-, -(CH2)8-, -(CH2)10- and -(CH2)12-, and more preferred examples include -(CH2)2- and -(CH2)6-. Furthermore, the group -Z 1 -Z 2 An example of this is the group -(CH2CH2O) ma -CH2CH2-NH-Z 3 Here, ma is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. A specific example of a preferred ma is 2. Group -Z 1 -Z 2 As an example, the group -(hexane-1,6-diyl)-NH-COO-Z is preferred. 3 , the group -(ethane-1,2-diyl)-NH-COO-Z 3 and the group -(CH2CH2O)2-CH2CH2-NH-Z 3 More preferred are the groups -(hexane-1,6-diyl)-NH-COO-cholesteryl, -(ethane-1,2-diyl)-NH-COO-cholesteryl and -(CH2CH2O)2-CH2CH2-NH-cholanoyl, and even more preferred is the group -(hexane-1,6-diyl)-NH-COO-cholesteryl. Z 1 , Z 2 , group-Z 1 -Z 2 Examples of the hyaluronic acid derivatives containing the repeating unit represented by the above formula (I) include those in which Y and X 1 , group-Y-X 1Also included are groups corresponding to the following: ca -Z 3 and the group -O-CO-NR ca -Z 3 An example of this is R ca and each of the groups represented by the formula (I) is a hydrogen atom.

[0171] In one embodiment, the hyaluronic acid derivative comprising a repeating unit represented by formula (II) is a group X 1a In X 1a Ga-NR 9 -Z 1 -Z 2 , R 9 is a hydrogen atom, Z 1 For example, C 2－12 Alkylene, preferably C 2－6 Alkylene, more preferably C6 alkylene, Z 2 Ga-NR ba -COO-Z 3 , R ba is a hydrogen atom, and Z 3 is a steryl group, and the group R aa is a hydrogen atom or C 1－6 alkyl, wherein the alkyl is independently hydroxy, carboxy, carbamoyl, C 1－6 It may be optionally substituted with one or more groups selected from alkylthio, aryl, and heteroaryl, wherein the aryl may be optionally substituted with one or more hydroxy.

[0172] Preferred group R in terms of lymph node delivery of the conjugate aa Specific examples of include methyl, hydroxymethyl, a hydrogen atom, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, benzyl, (4-hydroxyphenyl)methyl, 1-methylpropyl, 2-methylpropyl, isopropyl, indol-3-ylmethyl, 2-carbamoylethyl, and 2-carboxyethyl, and are preferably methyl, hydroxymethyl, 1-hydroxyethyl, 1-methylpropyl, and 2-carbamoylethyl, and more preferably methyl and 2-carbamoylethyl.

[0173] In one embodiment, a hyaluronic acid derivative containing a repeating unit represented by the above formula (II) including a repeating unit represented by formula (III) is also preferred. In a more preferred embodiment, X in formula (II) 1a and X in formula (III) 2 In one aspect, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is 1a Ga-NR 9 -Z 1 -Z 2 The repeating unit may contain a repeating unit represented by formula (II), a repeating unit represented by formula (III) and a repeating unit represented by formula (IIIc).

[0174] In yet another aspect, the hyaluronic acid derivatives containing the repeating units of formula (II) above have a group -NR relative to the repeating units of the disaccharide present. 9 -Z 1 -Z 2 The proportion of repeating units of formula (II) and / or formula (III) having a hydrophobic group (hereinafter also referred to as a hydrophobic group) (introduction rate of hydrophobic groups) may be 3 to 50%.

[0175] Here, the introduction rate of the hydrophobic group is calculated by the following formula:

[0176]

[0177] Here, the "repeating units of the disaccharide present in the derivative" include the repeating units of formula (II) and formula (III) as well as the repeating unit of formula (IIIc). The introduction rate can be controlled by reaction conditions, such as the ratio of reagents, and can be determined, for example, by NMR measurement.

[0178] The introduction rate of hydrophobic groups in hyaluronic acid derivatives containing repeating units represented by the above formula (II) is, for example, 3 to 50%, preferably 5 to 40%, more preferably 5 to 35%, even more preferably 5 to 25%, more preferably 5 to 20%, and even more preferably 5 to 10%. From the viewpoint of complex formation with antigen, 3 to 60% is preferred, 7 to 50% is more preferred, 18 to 45% is more preferred, and 20 to 35% is even more preferred. From the viewpoint of lymph node migration of the complex, 3 to 60% is preferred, 7 to 50% is more preferred, 18 to 45% is more preferred, and 20 to 35% is even more preferred.

[0179] In one embodiment, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is 1a Ga-NR 9 -Z 1 -Z 2In this case, the ratio of the disaccharide units of formula (II) to the disaccharide repeating units present in the hyaluronic acid derivative is, from the viewpoint of both biodegradability and blood retention, for example, 70% or more, preferably 75% or more, and more preferably 90% or more. The upper limit may be 100% or less. The range of this ratio is, for example, 70 to 100%, preferably 75 to 100%, and more preferably 90 to 100%. Furthermore, from the viewpoint of the ability to form a complex with an antigen, the ratio is, for example, 10% or more, preferably 20% or more, more preferably 50% or more, even more preferably 70% or more, and even more preferably 90% or more. The upper limit may be 100% or less. The range of this ratio is, for example, 10 to 100%, preferably 20 to 100%, more preferably 50 to 100%, even more preferably 70 to 100%, and even more preferably 90 to 100%. Furthermore, from the viewpoint of the complex having lymph node migration properties, the ratio is, for example, 10% or more, preferably 20% or more, more preferably 50% or more, even more preferably 70% or more, and even more preferably 90% or more. The upper limit may be 100% or less. The ratio range is, for example, 10 to 100%, preferably 20 to 100%, even more preferably 50 to 100%, even more preferably 70 to 100%, and even more preferably 90 to 100%. The hyaluronic acid derivative containing the repeating unit represented by formula (II) above may further contain a repeating unit represented by formula (III).

[0180] In one embodiment, the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is X 1 Ga-NR 9 -Z 1 -Z 2In this case, the sum of the proportion of repeating units represented by formula (II) and the proportion of repeating units represented by formula (III) in the repeating units of the disaccharide present is, for example, 70 to 100%, preferably 80 to 100%, and more preferably 90 to 100%. From the viewpoint of complex formation with an antigen, it is, for example, 7 to 100%, preferably 20 to 100%, and more preferably 30 to 100%. From the viewpoint of lymph node migration of the complex, it is, for example, 20 to 100%, preferably 30 to 100%, and more preferably 70 to 100%.

[0181] In the hyaluronic acid derivative containing the repeating unit represented by formula (II), the proportion of the repeating unit represented by formula (III) in the repeating units of the disaccharide present is preferably 3-50%, more preferably 5-40%, even more preferably 5-35%, even more preferably 5-25%, even more preferably 5-20%, even more preferably 5-10%. From the viewpoint of complex formation with antigen, 3-60% is preferred, 7-50% is more preferred, 18-45% is more preferred, and 20-35% is even more preferred. From the viewpoint of lymph node migration of the complex, 3-60% is preferred, 7-50% is more preferred, 18-45% is more preferred, and 20-35% is even more preferred.

[0182] In addition, in hyaluronic acid derivatives containing repeating units represented by formula (II) above, the proportion of repeating units represented by formula (II) in the repeating units of the disaccharides present is preferably 20-97%, more preferably 30-95%, even more preferably 35-95%, even more preferably 45-95%, even more preferably 50-95%, and even more preferably 60-95%. From the viewpoint of complex formation with antigen, it is, for example, 7-100%, preferably 20-100%, and more preferably 30-100%. From the viewpoint of lymph node migration of the complex, it is, for example, 20-100%, preferably 30-100%, and more preferably 70-100%.

[0183] Methods for producing hyaluronic acid derivatives into which hydrophobic groups have been introduced include those described in WO 2010 / 053140 and WO 2014 / 038641.

[0184] According to one aspect, hyaluronic acid derivatives having hydrophobic groups introduced therein are characterized by forming microparticles through association in water. Although not particularly limited, it is believed that spontaneous association occurs in water due to the hydrophobic interaction of the introduced hydrophobic groups, forming microparticles. Utilizing this property, they can be used as vaccine carriers, lymph node delivery carriers, sustained blood release carriers, and targeting carriers. The particle diameter of the microparticles is not particularly limited, but is, for example, 1 μm or less, preferably 500 nm, more preferably 200 nm or less, even more preferably 100 nm or less, and even more preferably 50 nm or less. Methods for microparticulating hyaluronic acid derivatives having hydrophobic groups introduced therein include the methods described in International Publication No. 2010 / 053140 and International Publication No. 2014 / 038641.

[0185] As the raw material for producing the hyaluronic acid derivative that hydrophobic group is introduced, hyaluronic acid or its salt or its derivative can be used.As hyaluronic acid salt, for example, alkali metal salt such as sodium salt, potassium salt, lithium salt can be listed, and particularly preferred salt is the sodium salt that is commonly used as medicine.Hyaluronic acid or its pharmaceutically acceptable salt can be produced by various known methods such as the method of extracting from the organism such as chicken comb or pig subcutaneous tissue or biological fermentation method, or can also be obtained by purchasing commercially available products (for example, from Denka Co., Ltd., Shiseido Co., Ltd., Seikagaku Kogyo Co., Ltd., R&D system Co., Ltd., etc.).

[0186] The antigen that forms a complex with the hyaluronic acid derivative microparticles that have been introduced with hydrophobic groups can be released from the complex after administration due to the decomposition and disintegration of the hyaluronic acid derivative microparticles, and the substitution of the antigen with biological components such as albumin.This release rate can be controlled by the introduction rate of the hydrophobic groups of the hyaluronic acid derivative, molecular weight, and the introduction rate of amino acids.In addition, according to the method described in WO 2010 / 053140, the hyaluronic acid derivative can be chemically crosslinked and gelled to control the antigen release rate.In addition, according to the method described in WO 2010 / 053140, the antigen release rate can be controlled by conjugating the hyaluronic acid derivative with the antigen.

[0187] <2-2. Antigens> Antigens to be combined with the hyaluronic acid derivatives having hydrophobic groups introduced therein include, for example, antigen peptides and antigen proteins, as well as DNA and mRNA encoding these antigen sequences. Antigen peptides and antigen proteins are preferred, and antigen peptides are even more preferred.

[0188] The antigen may be a cancer antigen. A cancer antigen is an antigen that is highly expressed on cancer cells, and in some cases, is expressed only by cancer cells. A cancer antigen may be expressed within or on the surface of a cancer cell.

[0189] Antigen proteins that can be used in the present invention include, but are not limited to, ERK1, ERK2, MART-1 / Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-Xp4), MAGE-Xp5 (MAGE-Xp6), MAGE-Xp7 (MAGE-Xp8), MAGE-Xp9 (MAGE-Xp9), MAGE-Xp10 (MAGE-Xp11), MAGE-Xp22 (MAGE-Xp13), MAGE-Xp14 (MAGE-Xp15), MAGE-Xp16 (MAGE-Xp16), MAGE-Xp17 (MAGE-Xp18), MAGE-Xp19 (MAGE-Xp19), MAGE-Xp23 (MAGE-Xp19), MAGE-Xp24 (MAGE-Xp19), MAGE-Xp25 (MAGE-Xp26), MAGE-Xp27 (MAGE-Xp19), MAGE-Xp28 (MAGE-Xp29), MAGE-Xp29 (MAGE-Xp29), MAGE- -Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2, G AGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, Tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM 2 ganglioside, GD2 ganglioside, human papillomavirus proteins, the Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, and c-erbB-2.

[0190] The antigen protein may have its entire sequence or a sequence with a partial deletion.

[0191] The antigenic peptide is an antigenic peptide that contains one or more epitopes recognized by CD8-positive cytotoxic T cells and / or CD4-positive helper T cells in the sequence of the antigenic protein. In one embodiment, the antigenic peptide preferably contains two or more epitopes (e.g., 2, 3, 4, 5, 6, 7, or 8) from the viewpoint of being loaded onto an MHC class I molecule or an MHC class II molecule after degradation in an antigen-presenting cell. Specifically, the antigenic peptide includes an antigenic peptide containing an epitope of an antigenic protein of a tumor cell.

[0192] In one embodiment, the 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.

[0193] In one aspect, from the viewpoint of inducing activation of cytotoxic T cells (CTLs) by helper T cells, the antigenic peptide is an antigenic peptide that contains one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

[0194] In one aspect, when two or more epitopes are contained, an amino acid linker may be placed between the epitopes. The linker may contain, for example, 2 to 10 amino acids, preferably 4 to 10 amino acids, and more preferably 4 to 8 amino acids. Amino acids used in the linker include glycine (G), tyrosine (Y), leucine (L), tryptophan (W), etc. Preferred are tyrosine (Y), leucine (L), and tryptophan (W). Specific examples of amino acid linkers include -YYYY-(4Y), -LLLL-(4L), -WWWW-(4W), -GGGGGG-(6G), -YYYYYY-(6Y), -LLLLLL-(6L), -WWWWWW-(6W), -YYYYYYYY-(8Y), -LLLLLLLL-(8L), and -WWWWWWWW-(8W), with 6Y, 6L, and 6W being preferred.

[0195] <2-3. Complex> The complex of hyaluronic acid derivatives that have been introduced with hydrophobic groups and antigen can be produced by mixing the hyaluronic acid derivatives that have been introduced with hydrophobic groups and antigen in suitable solution.The solution that can be used can include water, physiological saline, various buffer solutions, sugar solution, DMSO, ethanol, DMF, and their combinations, and can also be carried out solvent / buffer exchange by methods such as dialysis.

[0196] For example, the complex of the present invention can be produced by mixing the microparticles of hyaluronic acid derivatives into which hydrophobic groups have been introduced with an antigen.Although not particularly limited, the microparticles of the hyaluronic acid derivatives and the antigen can form a complex to form a complex of the hyaluronic acid derivative and the antigen.In addition, the antigen can be encapsulated in the microparticles of the hyaluronic acid derivatives to form a complex of the hyaluronic acid derivative and the antigen.The complex includes an antigen inclusion body in the microparticles of the hyaluronic acid derivatives.In this inclusion body, the antigen is coated with the hyaluronic acid derivative.

[0197] A method for forming a complex includes adding an antigen solution to microparticles of hyaluronic acid derivatives into which hydrophobic groups have been previously introduced. In this method, the formed microparticles of the hyaluronic acid derivative and the antigen form complexes through interactions such as hydrophobic interaction, electrostatic interaction, and hydrogen bonding. The interaction occurs either on the surface of the microparticles or inside the microparticles. Conditions such as solvent, salt concentration, pH, temperature, time, and addition of denaturant can be appropriately selected to ensure stable and high-yield complex formation of the antigen. For example, the salt concentration and pH during complex formation affect the swelling degree and density of the hyaluronic acid derivative microparticles, and also affect the ionization state of the antigen, so appropriate conditions can be used depending on the combination. By performing complexation under a low salt concentration, the electrostatic repulsion between the carboxyl groups of the hyaluronic acid derivative can be utilized to reduce the microparticle density, increase the amount of complexation, and complex with a higher molecular weight antigen. After complex formation, increasing the salt concentration weakens electrostatic repulsion, increases the particle density, and makes the gel network smaller than the antigen size, thereby firmly retaining the antigen and delaying its release. In this case, the salt concentration can be set to physiological salt concentration. Ultrasonic irradiation can be performed using an ultrasonic homogenizer or a focused ultrasonic irradiation device to achieve micronization and uniform size. Ultrasonic irradiation can be performed after mixing the hyaluronic acid derivative and drug, or it can be performed on the hyaluronic acid derivative alone. Uncomplexed free antigen can be separated and removed using dialysis, size exclusion chromatography (SEC), or other methods.

[0198] In addition, as a complex formation method, a hyaluronic acid derivative having a hydrophobic group introduced therein is dissolved in an aprotic polar organic solvent such as DMSO or DMF, mixed with an antigen, and then the solution is replaced with water, a salt solution, or various buffer solutions, thereby simultaneously forming microparticles and complexes. The replacement can be performed, for example, by dialysis. Ultrasonic irradiation can be performed using an ultrasonic homogenizer or a focused ultrasonic irradiation device to achieve miniaturization and uniform size. Ultrasonic irradiation can be performed after mixing the hyaluronic acid derivative and antigen, or on the hyaluronic acid derivative alone. It can also be performed before or after dialysis. This method makes the antigen complexed with the hyaluronic acid derivative less likely to be released from the complex. Conditions for mixing the hyaluronic acid derivative and antigen, such as the solvent type, salt concentration, pH, temperature, time, addition of a denaturant, hyaluronic acid derivative concentration, antigen concentration, and ratio of hyaluronic acid derivative to antigen, can be appropriately selected so as to ensure stable antigen and high-yield complex formation. Although not particularly limited, the conditions such as solvent type, salt concentration, pH, temperature, time, number of times, whether or not denaturant is added when replacing with water, salt solution, or various buffer solution aqueous solution, hyaluronic acid derivative concentration, antigen concentration, and the ratio of hyaluronic acid derivative to antigen can be appropriately selected so that the antigen forms a stable complex with high yield.The size of the microparticles can also be obtained by appropriately selecting these conditions.The free antigen that is not complexed can be separated and removed by dialysis, size exclusion chromatography (SEC), etc.

[0199] When the complex is in the form of particles, the particle size is not particularly limited, but from the viewpoint of lymph node transfer, it is, for example, 20 to 200 nm, preferably 20 to 100 nm, more preferably 20 to 50 nm.

[0200] 3. Antigen-Specific Lymphocytes <3-1. Immune Receptors> The immune receptor is not particularly limited as long as it is capable of transmitting a signal required for immune cell effector function, i.e., a receptor capable of transmitting a signal required for immune cell activation when the antigen-binding domain binds to an antigen. Preferred immune receptors include T cell receptors and chimeric antigen receptors (CARs). The antigen recognized by a chimeric antigen receptor may be a cell surface antigen or a complex of an intracellular antigen and a major histocompatibility complex.

[0201] An immunoreceptor typically comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain.

[0202] The antigen-binding domain is a domain that is located extracellularly when an immune receptor is located on the cell membrane, and is not particularly limited as long as it is a domain that can recognize and bind to an antigen. An antigen-binding domain typically comprises one or more, preferably two, three, four, five or more, and more preferably all six, of the CDRs of an antibody or T cell receptor against the antigen (e.g., heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 in the case of an antibody). More preferably, the antigen-binding region comprises the variable region of an antibody or T cell receptor against the antigen (e.g., in the case of an antibody, the heavy chain variable region and / or the light chain variable region).

[0203] The antigen-binding domain can have a single-chain antibody structure, such as an scFv structure. When the domain contains a heavy chain variable region and a light chain variable region, as in the scFv structure, the heavy chain variable region and the light chain variable region are typically linked via a linker. The linker is not particularly limited and can be any linker as long as it does not significantly impair antigen binding. A preferred linker is a linker composed of glycine or glycine and serine (e.g., a GGS linker, a GS linker, a GGG linker, etc.). The length of the linker is not particularly limited. The linker may have, for example, 5 to 30 amino acid residues, preferably 10 to 25. The positional relationship between the heavy chain variable region and the light chain variable region is not particularly limited, and either the light chain variable region at the N-terminus or the heavy chain variable region at the N-terminus may be employed.

[0204] The transmembrane domain is a domain that is located within the cell membrane when an immune receptor is positioned on the cell membrane, and is not particularly limited as long as it is capable of constituting an immune receptor. Examples of transmembrane domains that can be used include transmembrane regions of TCR α / β chains, CD28, CD3ε, CD8α, CD3, CD4, 4-1BB, and the like. The transmembrane domains of each factor are publicly known or can be easily determined from publicly known sequence information (e.g., using a transmembrane domain prediction program, etc.). Alternatively, a transmembrane domain consisting of an artificially constructed polypeptide may be used. Appropriate mutations may be introduced into these transmembrane domains as long as they do not significantly inhibit the function of the chimeric antigen receptor.

[0205] The antigen-binding domain and the transmembrane domain are linked directly or via a spacer. When a spacer is used, its sequence is not particularly limited, and examples of the spacer include the hinge region or a portion thereof of IgG, preferably human IgG (e.g., subtypes IgG1 and IgG4), the hinge region and a portion of CH2, or a portion of a factor used in the transmembrane domain (e.g., CD28). Using the sequence of the hinge region or a portion thereof is expected to enable the construction of a highly flexible spacer.

[0206] The intracellular signaling domain is a domain that is located intracellularly when an immune receptor is located on the cell membrane, and is capable of transmitting a signal required for immune cells to exhibit their effector functions; that is, a domain that is capable of transmitting a signal required for immune cell activation when the antigen-binding domain binds to an antigen.

[0207] The intracellular signal domain preferably includes an intracellular activation domain. Examples of the intracellular activation domain that can be used include intracellular domains such as CD3ζ and FcεRIγ. CD3ζ is preferably used. CD3 may be appropriately mutated as long as it does not inhibit the function of the immunoreceptor. When mutating CD3, it is preferable to introduce a mutation so that it contains an ITAM (immunoreceptor tyrosine-based activation motif).

[0208] The immunoreceptor may contain regions other than those described above. Examples of such regions include a costimulatory factor intracellular domain, a leader sequence (signal peptide) (e.g., the leader sequence of the GM-CSF receptor) that promotes transport of the immunoreceptor onto the cell membrane, and a spacer / linker (e.g., between the transmembrane region and the intracellular signal domain, or between each domain within the intracellular signal domain). The intracellular domain of the costimulatory factor is not particularly limited, as long as it is derived from a costimulatory factor possessed by T cells or the like. For example, one or more species selected from the group consisting of OX40, 4-1BB, GITR, CD27, CD278, and CD28 can be appropriately selected and used.

[0209] An immunoreceptor may be a molecule consisting of a single polypeptide or a complex of two or more polypeptides. Furthermore, an immunoreceptor may be a molecule consisting of a polypeptide or a complex thereof, or a polypeptide or a complex thereof linked to another substance (e.g., a fluorescent substance, a radioactive substance, an inorganic particle, etc.).

[0210] 3-2. Lymphocytes Antigen-specific lymphocytes are not particularly limited, as long as they express an immunoreceptor for the antigen in the HA-antigen composition (i.e., capable of binding (preferably specifically binding) to the antigen). In a more specific embodiment, antigen-specific lymphocytes express an immunoreceptor on the cell membrane, with the antigen-binding domain exposed outside the cell membrane.

[0211] Examples of lymphocytes include T cells (e.g., CD8-positive cells, CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, αβ-T cells, γδ-T cells, etc.), NK cells, NKT cells, etc.

[0212] Antigen-specific lymphocytes can be obtained by introducing into cells a polynucleotide containing a coding sequence for an immune receptor.

[0213] It is preferable to activate the immunoreceptor-expressing cells before introducing the polynucleotide. For example, the immunoreceptor-expressing cells can be activated by stimulation with anti-CD3 and anti-CD28 antibodies. For example, stimulation with anti-CD3 and anti-CD28 antibodies can be added by culturing the cells in a culture vessel (e.g., a culture dish) whose culture surface has been coated with anti-CD3 and anti-CD28 antibodies. This stimulation can also be performed using magnetic beads coated with anti-CD3 and anti-CD28 antibodies (e.g., Dynabeads T-Activator CD3 / CD28, available from VERITAS).

[0214] To increase the cell survival rate / proliferation rate, it is preferable to use a culture medium supplemented with a T cell growth factor during activation treatment. IL-2, IL-15, IL-7, etc. can be used as T cell growth factors. IL-2, IL-15, IL-7, etc. can be prepared according to standard methods. Commercially available products can also be used. While this does not exclude the use of T cell growth factors from animal species other than humans, T cell growth factors derived from humans (which may be recombinant) are usually used.

[0215] Typically, for application (administration to patients), the cells (polynucleotide-introduced lymphocytes) after polynucleotide introduction are expanded. For example, the polynucleotide-introduced lymphocytes are cultured (subcultured as necessary) in a culture medium supplemented with a T cell growth factor. In addition to this culture, a treatment (reactivation) similar to that used for activating immune receptor-expressing cells may be performed.

[0216] Although serum (e.g., human serum, fetal bovine serum) may be added to culture the immunoreceptor-expressing cells and the polynucleotide-transfected lymphocytes, serum-free medium or medium with a low serum concentration allows for the preparation of cells with the advantages of high safety for clinical application and reduced variation in culture efficiency due to differences between serum lots. When serum is used, autologous serum, i.e., serum collected from a patient receiving immunoreceptor gene-transfected lymphocytes, is preferably used. Furthermore, feeder cells inactivated by irradiation (e.g., 25-35 Gy) of peripheral blood mononuclear cells (e.g., pooled peripheral blood mononuclear cells from several healthy individuals) can be used for culture.

[0217] More specifically, the method for obtaining antigen-specific lymphocytes is as follows.

[0218] Nucleic acids encoding immunoreceptors can be linked to other nucleic acids so that they are expressed under the control of an appropriate promoter. Promoters that can be used include those that constitutively promote expression and those that are inducible by drugs (e.g., tetracycline or doxorubicin). For example, mammalian promoters such as the phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, and RNA polymerase II promoter, as well as viral promoters such as the SV40 early promoter, cytomegalovirus promoter, herpes simplex virus thymidine kinase promoter, and various retroviral LTR promoters, can be used. To achieve efficient transcription of the nucleic acid, a nucleic acid containing other regulatory elements (e.g., enhancer sequences or terminator sequences) that cooperate with the promoter or transcription initiation site may be linked. Furthermore, genes that can serve as markers for confirming nucleic acid expression (e.g., drug resistance genes, genes encoding reporter enzymes, or genes encoding fluorescent proteins) may also be incorporated.

[0219] Nucleic acids encoding immune receptors can be introduced into cells using vectors.

[0220] The vector is operably linked to an appropriate control sequence to express the nucleic acid in a suitable host. Such control sequences include a promoter that transcribes the nucleic acid, an operator sequence that controls transcription, a sequence encoding a ribosome binding site, an enhancer, a polyadenylation sequence, and a sequence that controls the termination of transcription and translation. Various known sequences (e.g., restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences, etc.) can be used in the vector. Various sequences can be appropriately selected and used depending on the type of polypeptide to be expressed, the host cell, the culture medium, and other conditions. Vectors that can be used include those that integrate into the host genome, those that do not, and episomal vectors that exist in the cytoplasm and replicate autonomously. Examples of such vectors include viral vectors such as retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Viral vectors are preferably replication-deficient so that the virus cannot replicate in infected cells. Non-viral vectors can also be used in combination with condensing agents such as liposomes and cationic lipids. Nucleic acids can also be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation, and particle bombardment.

[0221] Antigen-specific lymphocytes can be prepared by introducing nucleic acids encoding immunoreceptors into cells. Cells expressing immunoreceptors are activated by intracellular signal transduction upon binding to antigens via the immunoreceptor. Cell activation varies depending on the type of host cell and intracellular domain, but can be confirmed using indicators such as cytokine release, increased cell proliferation, and changes in cell surface molecules. For example, the release of cytotoxic cytokines (e.g., tumor necrosis factor, lymphotoxin) from activated cells leads to the destruction of tumor cells expressing the complex. Furthermore, cytokine release and changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages. Therefore, immunoreceptor-expressing cells are useful in adoptive immunotherapy.

[0222] The process of introducing a nucleic acid encoding a CAR into cells can be carried out ex vivo or in vivo. Cells to be introduced can be derived from mammals, such as humans, or non-human mammals, such as monkeys, mice, rats, pigs, cows, and dogs. Cell types that can be used include, for example, cells collected, isolated, purified, or induced from body fluids such as blood (peripheral blood, umbilical cord blood, etc.), bone marrow, or other body fluids, tissues, or organs. PBMCs, immune cells [dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes or NK cells, blood cells (neutrophils, basophils, monocytes)], hematopoietic stem cells, umbilical cord blood mononuclear cells, fibroblasts, preadipocytes, hepatocytes, blood cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, pluripotent stem cells, various cancer cell lines, and neural stem cells. In the present invention, T cells, T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), pluripotent stem cells, or cell populations containing these are particularly preferred. T cells include CD8-positive T cells, CD4-positive T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell precursors include PBMCs. The cells used in the present invention may be collected from a living organism, expanded, or established as a cell line. When it is desired to transplant nucleic acid-introduced cells or cells differentiated from such cells into a living organism, it is preferable to introduce the nucleic acid into cells collected from the living organism itself or from the same organism.

[0223] The antigen-specific lymphocytes may be cultured and / or stimulated using an appropriate medium and / or stimulatory molecules before administration to a subject. Stimulatory molecules include cytokines, appropriate proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN-γ, with IL-2 being preferred. The concentration of IL-2 in the medium is not particularly limited, but may be, for example, 0.01 to 1 x 105 U / mL, more preferably 1 to 1 x 10 4U / mL. Examples of suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody. Other lymphocyte-stimulating factors, such as lectins, can also be added. Furthermore, serum or plasma may be added to the medium. While the amount of these to be added to the medium is not particularly limited, examples include 0 to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. The serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous (meaning the same origin as the cells to be cultured) or non-autologous (meaning a different origin from the cells to be cultured), although autologous origin is preferred from the standpoint of safety.

[0224] The cell culture equipment used for cell culture is not particularly limited, and examples thereof include petri dishes, flasks, bags, large culture tanks, and bioreactors. CO2 gas-permeable bags for cell culture can be used as bags. Large culture tanks can be used for industrial production of large amounts of cell populations. Culture can be performed in either an open or closed system, but it is preferable to culture in a closed system from the viewpoint of the safety of the resulting cell population.

[0225] 4. Uses The combined use of an HA-antigen composition and antigen-specific lymphocytes can exert a stronger, more efficient, more sustained, and / or broader anti-tumor effect. Therefore, in some aspects, the present invention relates to a pharmaceutical composition containing an HA-antigen composition for use in combination with antigen-specific lymphocytes, a pharmaceutical composition containing antigen-specific lymphocytes for use in combination with an HA-antigen composition, a pharmaceutical composition characterized by comprising a combination of an HA-antigen composition and antigen-specific lymphocytes, etc. Furthermore, the HA-antigen composition can also be used as a T cell activating vaccine, etc.

[0226] The subject to which the present invention is administered (for example, an animal (particularly a mammal) such as a human, mouse, monkey, dog, or cat) can be, for example, a subject with cancer.

[0227] The pharmaceutical composition can be used to prevent or treat (or improve) cancer (target disease). Target diseases include solid cancers and hematological cancers. Examples of target diseases include various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative disorders, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumors, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma. "Treatment" includes alleviating (alleviating) symptoms characteristic of or associated symptoms of the target disease, preventing or delaying the progression of symptoms, etc. "Prevention" refers to preventing or delaying the onset / manifestation of a disease (disorder) or its symptoms, or reducing the risk of onset / manifestation. On the other hand, "amelioration" refers to the alleviation (reducing severity), improvement, remission, or cure (including partial cure) of a disease (disorder) or its symptoms.

[0228] Although immune checkpoint inhibitors have shown excellent efficacy against melanoma and lung cancer, the therapeutic effect remains at around 20%. Currently, there is a need for a treatment for cold tumors, known as immunodeserts or immunoexcluded tumors, which have a low number of T cells in the tumor.

[0229] The mouse melanoma cell line B16F10 has very low MHC expression (Oncoimmunology. 2017; 6(6):e1259049), low immunogenicity (Cancer Res. 2000; 60: 5514-5521, Curr Opin Genet Dev. 2014; 24: 46-51), and is a typical cold tumor with low intratumoral T cell counts (Genomics 21, 2 (2020)). At the same time, it is highly metastatic (J Immunogenet 1989; 16(4-5):291-303). Preclinical models using B16F10 have been widely used as preclinical models for cancers with low immunogenicity, high metastatic potential, and low intratumoral T cell counts, making it difficult to demonstrate antitumor effects.

[0230] The pharmaceutical composition of the present invention showed high efficacy in this model using B16F10, and is therefore also effective against cancers with low immunogenicity.

[0231] Here, a low-immunogenic cancer is a cancer in which MHC class I expression in cancer cells is reduced or absent, tumor antigen presentation is reduced, or the cancer is not presented, resulting in low immunogenicity. Specifically, the proportion of cancer cells expressing MHC class I among all cancer cells is, for example, 50% or less. As the expression level of MHC class I decreases, cancer treatment becomes more difficult, and treatment is difficult when the expression level is 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, and particularly 1% or less. The expression level of MHC class I can be measured by immunohistochemical staining or FCS analysis.

[0232] The pharmaceutical composition of the present invention showed high efficacy in this model using B16F10, and is therefore also effective against metastatic cancer.

[0233] Here, primary tumor sites of metastatic cancer include, but are not limited to, the bladder, bone, bone marrow, brain, breast, cervix, colon, endometrium, esophagus, intestine, kidney, liver, lung, mouth, muscle, ovary, skin, pancreas, prostate, skin, stomach, testes, thyroid, uterus, and any hyperproliferative tissue including, for example, vascular structures (e.g., endothelial cells, smooth muscle cells, pericytes, scar, fibrotic tissue, surgical adhesion tissue, or hyperproliferative bone lesions). Here, metastatic sites of metastatic cancer include, but are not limited to, the adrenal gland, bone, brain, kidney, liver, lung, lymph node, ovary, peritoneum, skin, spleen, etc.

[0234] The pharmaceutical composition of the present invention showed high efficacy in this model using B16F10, and therefore can exert a favorable effect even against cancers with a low number of intratumoral T cells.

[0235] Here, a cancer with a low number of intratumoral T cells refers to a cancer with very few or no T cells present in the tumor. Specifically, the ratio of T cells to cancer cells is, for example, 10% or less. The lower the ratio of T cells, the less effective anticancer drugs such as immune checkpoint inhibitors become. Treatment is difficult when the ratio is 5% or less, 3% or less, 1% or less, 0.5% or less, 0.3% or less, or 0.1% or less. The ratio of T cells can be measured by immunohistochemical staining or FCS analysis.

[0236] The combined administration of the HA-antigen composition and antigen-specific lymphocytes may be simultaneous, on the same day, or at an interval. In a preferred embodiment, the antigen-specific lymphocytes are administered after the administration of the HA-antigen composition (more preferably after the administration of the HA-antigen composition and an adjuvant). In this case, the interval between administrations of the HA-antigen composition and the antigen-specific lymphocytes (the interval between administrations in the case of multiple administrations) can be, for example, 1 day to 1 week, 1 to 3 days, or 1 to 2 days.

[0237] A pharmaceutical composition comprising a combination of an HA-antigen composition and antigen-specific lymphocytes can also be in the form of a kit, for example, a cancer treatment kit containing the HA-antigen composition and antigen-specific lymphocytes.

[0238] 4-1. Use of HA-antigen composition The HA-antigen composition may be administered orally, parenterally, intranasally, intravaginally, intraocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intra-articularly, intracerebrally, intraorally, intraadipose tissue, intramammary tissue, or by inhalation, etc. Preferably, it may be administered subcutaneously, intramuscularly, intravenously, or intradermally.

[0239] The HA-antigen composition can be administered in any suitable form depending on the intended administration route as a pharmaceutical composition containing one or more pharmaceutically acceptable diluents, wetting agents, emulsifying agents, dispersing agents, adjuvants, preservatives, buffers, binders, stabilizers, etc. The administration route may be parenteral or oral.

[0240] The dosage of the HA-antigen composition can be determined appropriately. For example, a typical dosage is approximately 0.1 mg to 100 mg of antigen per dose. The appropriate number of doses is 2 to 20. The administration interval can be selected from the range of 1 day to 2 weeks.

[0241] The HA-antigen composition may be administered in combination with one or more adjuvants. Adjuvants used may be any substance that enhances the activity of antigen-presenting cells. More specifically, substances that activate innate immune receptors (pattern recognition receptors), other antigen-presenting cell stimulators, or substances that inhibit the acquisition of immunosuppressive activity by antigen-presenting cells may be selected. Innate immune receptors are classified into Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and cytoplasmic DNA sensors. Innate immune receptor agonist adjuvants can be selected from inactivated bacterial cells, bacterial cell extracts, nucleic acids, lipopolysaccharides, lipopeptides, and synthetic low-molecular-weight compounds. Preferred adjuvants include CpG oligonucleotides, poly(IC) RNA, imidazoquinones (e.g., R848 and imiquimod), saponins (e.g., QuilA and QS21), STING agonists (e.g., cyclic di-GMP), monophosphoryl lipids, and lipopeptides. Taxanes and anthracyclines can be used as adjuvants, which stimulate antigen-presenting cells. Adjuvants, which inhibit the acquisition of immunosuppressive activity by antigen-presenting cells, are selected from JAK / STAT inhibitors, indole deoxygenase (IDO) inhibitors, and tryptophan deoxygenase (TDO) inhibitors. These inhibitors include compounds that antagonize the factor, as well as neutralizing antibodies, small interfering RNA (siRNA), and antisense DNA.

[0242] The adjuvant used in the HA-antigen composition is preferably an innate immune receptor agonist, more preferably an agonist of Toll-like receptors (TLRs) or cytoplasmic DNA sensors. Preferred TLR agonists include CpG oligoDNA, polyIC RNA, imidazoquinones such as R848 and imiquimod, saponins such as QuilA and QS21, STING agonists, and monophosphoryl lipids.

[0243] The adjuvant may be administered as a separate formulation from the HA-antigen composition, or as a combined formulation. Alternatively, the adjuvant may be conjugated to a hyaluronic acid derivative having a hydrophobic group introduced therein, as described in International Publication No. WO 2010 / 053140. When administered in combination, the dose of the HA-antigen composition is, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably, 0.1 to 20 mg / dose. The dose of the adjuvant is, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and more preferably 0.1 to 10 mg / kg body weight. The adjuvant may be administered at the same time as the HA-antigen composition (including when the adjuvant is included in the formulation), or at different times. When administered at different times, it is preferable to administer one after the other, for example, within 1 minute to 5 hours, preferably within 1 minute to 1 hour.

[0244] The HA-antigen composition may be administered in combination with one or more antibodies used in cancer treatment. The antibodies may be, for example, antibodies that inhibit tumor-induced immunosuppressive signals or antibodies that activate immune cell costimulatory signals, preferably antibodies that inhibit tumor-induced immunosuppressive signals or antibodies that activate immune cell costimulatory signals. Specifically, the antibodies may be one or more antibodies selected from anti-CTLA4, anti-PD1, anti-PDL1, anti-OX40, and anti-4-1BB. When administered in combination, the dosage of the HA-antigen composition is, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably 0.1 to 20 mg / dose, and the dosage of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, and more preferably 1 to 40 mg / kg body weight. The antibody may be administered at the same time as the HA-antigen composition (including when the antibody is included in the formulation) or at different times. When the two are administered at different times, it is preferable to administer one after the other, for example, within 1 minute to 24 hours, preferably within 1 minute to 5 hours.

[0245] The HA-antigen composition may be administered in combination with both the adjuvant and the antibody. In this case, the dose of the HA-antigen composition is, for example, 0.01 to 100 mg / dose, preferably 0.1 to 50 mg / dose, and more preferably, 0.1 to 20 mg / dose. The dose of the adjuvant is, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and more preferably 0.1 to 10 mg / kg body weight. The dose of the antibody is, for example, 0.01 to 200 mg / kg body weight, preferably 0.1 to 100 mg / kg body weight, and more preferably 1 to 40 mg / kg body weight. The adjuvant and antibody may be administered at the same time as the HA-antigen composition (including cases where the adjuvant and antibody are contained in the formulation), or at different times. When administered at different times, it is preferable that the vaccine formulation, adjuvant, and antibody are all administered within, for example, 1 minute to 24 hours, preferably 1 minute to 5 hours.

[0246] In a preferred embodiment of the present invention, the HA-antigen composition is administered before the administration of antigen-specific lymphocytes. In a preferred embodiment of the present invention, when one administration of the HA-antigen composition followed by one infusion of antigen-specific lymphocytes is defined as a unit of administration, the HA-antigen composition is preferably administered at least once after one or two units have been administered.

[0247] In a preferred embodiment of the present invention, the interval between the first and second administrations of the HA-antigen composition can be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or 14 days or more. Furthermore, the interval can be, for example, 28 days or less, 24 days or less, 21 days or less, 17 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, or 2 days or less. 4-2. Use of Antigen-Specific Lymphocytes Antigen-specific lymphocytes can be widely used as a cell preparation for the treatment, prevention, or amelioration of tumors / cancers (target diseases) that express the target antigen of the antigen-binding domain.

[0248] The content of antigen-specific lymphocytes in the cell preparation is not particularly limited, but is preferably 1×10 3 ~1×10 11 1 × 10 4 ~1×10 10 More preferably, 1×10 5 ~2×10 9 The dosage of the therapeutic agent containing the cell population as an active ingredient is not particularly limited, but is preferably 1×10 cells / mL for an adult. 6 ~1×10 12 1×10 7 ~5×10 11 More preferably, 1 x 10 8 ~2×10 11 The cell preparation may contain components such as dimethyl sulfoxide (DMSO) or serum albumin for the purpose of protecting the cells, antibiotics for the purpose of preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of activating, proliferating, or inducing differentiation of the cells.

[0249] The route of administration of antigen-specific lymphocytes is not particularly limited. For example, they may be administered by intravenous injection, intraarterial injection, intraportal vein injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Local administration may be used instead of systemic administration. An example of local administration is direct injection into the target tissue, organ, or tissue. The administration schedule may be determined taking into account the gender, age, weight, and pathological condition of the subject (patient). In addition to a single administration, multiple administrations may be administered continuously or periodically. The appropriate number of administrations is 2 to 20. The administration interval can be selected between 1 day and 2 weeks.

[0250] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0251] Materials and Methods (Examples 1-5) (1) Cells. RPMI1640 medium (supplemented with 2-mercaptoethanol) was purchased from the Cell Science Institute. Fetal bovine serum (FBS) was purchased from Gibco. The mouse fibrosarcoma CMS5a cell line was provided by Memorial Sloan-Kettering Cancer Institute and passaged at Mie University. The mouse fibrosarcoma CMS5a cell line expresses a mutant ERK2 protein. A peptide containing the mutated portion of the mutant ERK2 protein (QYIHSANVL (SEQ ID NO: 1)) is recognized by CD8+ cytotoxic T cells in BALB / c mice. A T cell receptor (TCR) that recognizes this peptide has been isolated, and transgenic mice (DUC18 mice) transfected with the TCR have been generated. The long-chain peptide antigen used in this example contains the epitope sequence (QYIHSANVL) of the mutant ERK2 that recognizes CD8+ cytotoxic T cells.

[0252] (2) Test animals: Female BALB / c mice (CD90.2 positive) aged 6 to 8 weeks were purchased from Japan SLC. DUC18 transgenic mice were provided by the University of Washington and bred at Mie University. Both mice were housed in the Animal Experiment Facility at the Advanced Science Research Support Center, Mie University. The animal experiment protocol was approved by the Ethics Committee of the Mie University School of Medicine.

[0253] (3) Hyaluronic acid nanogel (HANG) Hyaluronic acid nanogel with cholesteryl groups introduced (weight-average molecular weight: 99 kDa, cholesteryl group introduction rate: 42%) was synthesized as described in Examples 1 and 2 of WO 2010 / 053140.

[0254] (4) Long-chain peptide antigen. A synthetic long-chain peptide was purchased from Sigma Genosys. The sequence was as follows: NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 2).

[0255] The sequence includes a 9-amino acid sequence (QYIHSANVL (SEQ ID NO: 1)) from the 16th Q to the 24th L as an epitope sequence recognized by CD8-positive cytotoxic T cells, and a 17-amino acid sequence (RGLQYIHSANVLHRDLK (SEQ ID NO: 3)) from the 13th R to the 29th K as an epitope sequence recognized by CD4-positive helper T cells.

[0256] (5) Preparation of Hyaluronic Acid Nanogel and Antigen Complex Hyaluronic acid nanogel was dissolved in ultrapure water to a concentration of 12 mg / mL and sonicated (Covaris, E220X). 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 sonicated for 30 minutes using a bath sonicator. The solution was transferred to a dialysis kit (Slide-A-Lyzer, molecular weight cutoff 3.5K) and dialyzed sequentially against 5 mM carbonate buffer, pH 9, 10 mM phosphate buffer, pH 7, and 10 mM phosphate buffer, pH 7, containing 10% sucrose. The antigen peptide concentration was quantified by reversed-phase chromatography and adjusted to 250–500 μg / mL to prepare the dosing solution. If the desired concentration was not reached, it was concentrated using an ultraconcentrator (Vivaspin 6, 5000 MwCO, Sauturius). The resulting complex is sometimes referred to as a "long-chain peptide antigen-loaded HA nanogel cancer vaccine" or "HA nanogel cancer vaccine."

[0257] (6) Other Reagents - Adjuvant CpG oligo DNA1668 was purchased from Ajinomoto Bio-Pharma Services Gene Design.

[0258] Fluorescently labeled antibodies: eFluor450-labeled anti-mouse CD8a antibody (clone 53-6.7), eFluor450-labeled anti-mouse KLRG1 antibody (clone 2F1), and APC-labeled anti-mouse CD45R (B220) antibody (clone RA3-6B2) were purchased from eBioscience. APC-labeled anti-mouse CD8a antibody (clone 53-6.7), APC-labeled anti-mouse CD4 antibody (clone RM4-5), PerCP-Cy5.5-labeled anti-mouse CD90.1 antibody (clone OX-7), PerCP-Cy5.5-labeled anti-mouse F4 / 80 antibody (clone BM8), PE-labeled anti-mouse CD207 antibody (clone 4C7), Pacific blue-labeled anti-mouse CD90.2 antibody (clone 30-H12), and purified CD16 / 32 antibody (clone 93) were purchased from BioLegend. FITC-conjugated anti-mouse CD11c antibody (clone HL3) and PerCP-Cy5.5-conjugated anti-mouse CD3 antibody (clone 145-2C11) were purchased from BD Biosciences. APC-conjugated anti-mouse IFN-γ antibody (clone XMG1.2) was purchased from eBioscience or TONBO. Anti-mouse CD8a antibody (clone 4SM15) for tissue staining was purchased from Invitrogen, and Alexa488-conjugated anti-rat IgG (H+L) antibody was purchased from Cell Signaling.

[0259] Short peptide antigen: A synthetic short peptide for stimulating CD8-positive T cells was purchased from Sigma Genosys. The sequence was as follows: QYIHSANVL (SEQ ID NO: 1).

[0260] Example 1. Tumor Growth Test Using a T75 culture flask (Corning), mouse fibrosarcoma CMS5a cell line was cultured in RPMI1640 medium containing 10% FBS. The cultured cells were detached using PBS containing 0.5% trypsin and suspended in RPMI1640 medium containing 10% FBS. The suspension was centrifuged (400 × g, 5 minutes, 4 °C), and the supernatant was removed. Then, the cells were washed twice with RPMI1640 medium and 1 × 10 6 The cells were suspended in RPMI1640 medium at a concentration of 100 μL / cell and subcutaneously implanted into the right anterior dorsum of BALB / c mice at a dose of 100 μL / cell (5 mice per group).

[0261] When administering the long-chain peptide antigen-loaded HA nanogel cancer vaccine, 50 μg of the long-chain peptide antigen-loaded HA nanogel cancer vaccine was administered subcutaneously to the right dorsal region of the mice along with 50 μg of CpG oligo DNA dissolved in PBS 7 and 11 days after tumor inoculation (HANG-V group).

[0262] For the administration of antigen-specific T cells, CD8+ T cells were isolated from the spleens of DUC18 mice (CD90.1-positive or CD90.2-negative) transfected with mutant ERK2-specific TCR genes using CD8a microbeads (Miltenyi) 8 and 12 days after tumor inoculation, and then cultured in RPMI1640 medium at 2 × 10 6 After suspending the cells at a concentration of 200 μL per 200 μL, 200 μL was infused into the tail vein of each mouse (TCR-T group).Tumor area was then measured over time.

[0263] Example 1: Results To demonstrate the antitumor effect of a HA nanogel cancer vaccine in combination with antigen-specific T cell infusion therapy, we used mice bearing the immune checkpoint inhibitor (ICI)-resistant fibrosarcoma cell line CMS5a tumor. We constructed a long-chain peptide antigen-loaded HA nanogel cancer vaccine by conjugating HA nanogel with a long-chain peptide antigen containing the CD8 epitope of the mutant ERK2 antigen (mERK2) expressed in CMS5a. We then investigated the therapeutic efficacy of this vaccine in combination with mERK2-recognizing TCR gene-transduced T cell infusion therapy. As shown in Figures 1-1 and 1-2, the HANG-V and TCR-T combined treatment group significantly suppressed CMS5a tumor growth and caused tumor regression compared with the HANG-V or TCR-T treatment groups alone. Tumor disappearance was observed in all mice 7 days after treatment (Figures 1-1 and 1-2). This effect persisted for a long time; even when CMS5a was re-implanted into the same mice, no tumor growth was observed, and the mice survived for a long time without recurrence (Figures 1-3). These results demonstrate that the combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy has a strong antitumor effect and can cure ICI-resistant tumors. Furthermore, this combination was shown to suppress tumor recurrence even after tumor cure, suggesting that the HA nanogel cancer vaccine may enhance the antitumor effect of antigen-specific T cells.

[0264] Example 2. Analysis of antitumor immune response in lymph nodes related to the vaccine administration site Fourteen days after tumor inoculation in the test of Example 1, lymph nodes related to the vaccine administration site (right inguinal lymph nodes) were collected, and the lymph nodes were ground using a glass slide, and the cells were suspended in RPMI1640 medium. At this time, cells from two animals per group were pooled. After centrifugation (400 × g, 5 minutes, 4°C), the supernatant was removed, washed twice with RPMI1640 medium, and then suspended in RPMI1640 medium containing 10% FBS. The cell count was measured using a hemocytometer (Figure 2-1), and the cell concentration was 2 × 10 6 The concentration was adjusted to 1 / mL.

[0265] Staining of myeloid cells (Figures 2-3 and 2-4): Add 4 x 10 cells per well of the cell suspension to a 96-well V-bottom microplate (Nunc). 5 The cells were added at a concentration of 200 μL per cell. The cell suspension was centrifuged (2000 rpm, 2 minutes, 4°C), the supernatant removed, and the cells were washed twice with 200 μL of staining buffer (PBS containing 0.5% FBS). After centrifugation and removal of the supernatant, 50 μL of a solution containing 50-fold diluted anti-mouse CD16 / CD32 antibodies was added and the mixture was left to stand in the dark at 4°C for 10 minutes. 150 μL of staining buffer was added, the mixture was centrifuged, the supernatant removed, and the cells were washed twice with 200 μL of staining buffer. 50 μL of a solution containing FITC-labeled anti-mouse CD11c antibody, PerCP-Cy5.5-labeled anti-mouse F4 / 80 antibody, or PE-labeled anti-mouse CD207 antibody was added at the manufacturer's recommended concentrations, mixed, and left to stand in the dark at 4°C for 15 minutes. After adding 150 μL of staining buffer and centrifuging to remove the supernatant, the cells were washed twice with 200 μL of staining buffer. After centrifuging to remove the supernatant, the cells were resuspended in 200 μL of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences) and the accompanying analysis software (FACSDiva). Dendritic cells were detected as CD11c-positive, macrophages as F4 / 80-positive, and Langerhans cells as CD11c- and CD207-positive populations.

[0266] Lymphocyte staining (Figure 2-4): Add 4 x 10 cells per well of the cell suspension to a 96-well V-bottom microplate (Nunc). 5 The cells were added at a concentration of 200 μL per cell. The cell suspension was centrifuged (2000 rpm, 2 minutes, 4°C) and the supernatant removed. The cells were then washed twice with staining buffer (PBS containing 0.5% FBS) and resuspended. Following the manufacturer's recommended concentrations for each antibody, 50 μL of a solution containing PerCP-Cy5.5-labeled anti-mouse CD3 antibody, APC-labeled anti-mouse CD45R (B220) antibody, and eFluor450-labeled anti-mouse KLRG1 antibody was added to the cell suspension and mixed. The mixture was then incubated in the dark at 4°C for 15 minutes. The cells were washed twice with 200 μL of staining buffer, resuspended in 200 μL of staining buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences) and the accompanying analysis software (FACSDiva). T cells were detected as CD3-positive, B cells as CD45R(B220)-positive, and NK cells as KLRG1-positive populations.

[0267] Staining of CD8-positive infused T cells (Figures 2-5): Add 4 x 10 cells per well of the cell suspension to a 96-well V-bottom microplate (Nunc). 5 The cells were added at a concentration of 200 μL per cell. The cell suspension was centrifuged (400 × g, 5 minutes, 4°C) and the supernatant removed. The cells were then washed twice with staining buffer (PBS containing 0.5% FBS) and resuspended. 50 μL of a solution containing eFluor450-labeled anti-mouse CD8 antibody, PerCP-Cy5.5-labeled anti-mouse CD90.1 antibody, and APC-labeled anti-mouse CD4 antibody, at the manufacturer's recommended concentrations, was added to the cell suspension, mixed, and incubated in the dark at 4°C for 15 minutes. The cells were washed twice with 200 μL of staining buffer, resuspended in 200 μL of staining buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences) and the accompanying analysis software (FACSDiva). The CD90.1- and CD8-positive populations were detected for the infused CD8+ T cells.

[0268] Antigen-specific T cell induction test (intracellular cytokine staining) (Figure 2-6): In a 48-well culture plate (Nunc), 4 × 10 cells were added to each well of the cell suspension. 5The cells were added at a concentration of 200 μL per well. 50 μL of mERK2 9m peptide (10 μg / mL) was added as a short-chain peptide for stimulating CD8+ T cells, and the cells were cultured at 37°C and 5% CO2 for 1 hour. Then, 50 μL of Goldi Plug (BD Biosciences) diluted 167-fold with RPMI 1640 medium containing 10% FBS was added per well, and the cells were cultured at 37°C and 5% CO2 for 5 hours. The cells were harvested and transferred to a 96-well V-bottom microplate (Nunc). After centrifugation (2000 rpm, 2 minutes, 4°C) to remove the supernatant, the cells were suspended in 200 μL of staining buffer per well. After washing the cells twice with 200 μL of staining buffer, eFluor450-labeled anti-CD8 antibody and PerCP-Cy5.5-labeled anti-CD90.1 antibody were added to the cell suspension at the manufacturer's recommended concentrations, mixed, and left to stand for 15 minutes in the dark at 4°C. 150 μL of staining buffer was added, centrifuged, and the supernatant was removed. The cells were then washed twice with 200 μL of staining buffer, followed by the addition of 100 μL of Cytofix / Cytoperm buffer (BD Biosciences) and gentle mixing. After leaving the cells to stand for 20 minutes in the dark at room temperature, they were washed twice with staining buffer, suspended in 200 μL of staining buffer, and stored overnight in a dark, refrigerated container. After centrifugation and removal of the supernatant, the cells were washed with 200 μL of Perm / Wash buffer (BD Biosciences). A 50-fold diluted APC-conjugated anti-mouse IFN-γ antibody was added to the cells, mixed, and incubated at room temperature in the dark for 15 minutes. The cells were washed twice with 200 μL of Perm / Wash buffer, resuspended in 200 μL of staining buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences) and the accompanying analysis software (FACSDiva). IFN-γ-producing CD8+ infused T cells were detected as IFN-γ-positive, CD90.1-positive, CD8-positive, or IFN-γ-positive, CD90.2-negative, CD8-positive populations. IFN-γ-producing, CD8+ host T cells were detected as IFN-γ-positive, CD90.1-negative, CD8-positive, or IFN-γ-positive, CD90.2-positive, CD8-positive populations.

[0269] Example 2: Results To clarify the effect of HA nanogel cancer vaccines in combination with antigen-specific T cell infusion therapy on antitumor immune responses, lymph nodes were collected from the HA vaccine administration site 14 days after tumor inoculation, and lymph node cell analysis was performed. Results showed significant lymph node swelling in the HANG-V monotherapy group and the HANG-V and TCR-T combination group, with both groups showing approximately a 4- to 8-fold increase in lymph node cell counts compared to the untreated group (Figure 2-1). Lymph node swelling continued even 22 days after tumor inoculation (Figure 2-2). Next, the proportion of myeloid antigen-presenting cells that had inflowed and accumulated in the regional lymph nodes was examined. An increase in myeloid antigen-presenting cells was observed in both the HANG-V monotherapy group and the HANG-V and TCR-T combination group. A particularly significant increase in Langerhans cells was observed in the combination group (Figure 2-3). Furthermore, to clarify the effect of the HA nanogel cancer vaccine on regional lymph node cell numbers, we analyzed lymphocytes and myeloid cells in the regional lymph nodes. As shown in Figure 2-4, the HANG-V group showed a several- to several-tens-fold increase in lymphocytes (T cells, B cells, and NK cells) and myeloid cells (Figure 2-4). Furthermore, we examined the influx of CD8+ infused T cells into regional lymph nodes. The HANG-V and TCR-T combination group showed a significantly higher proportion of CD8+ infused T cells in the regional lymph nodes than the TCR-T alone group (Figure 2-5), and approximately 80% of these cells were found to produce IFN-γ in specific response to tumor antigens (Figure 2-6). Furthermore, the HANG-V and TCR-T combination group also showed an increase in the proportion of host IFN-γ-producing CD8+ T cells (Figure 2-6). These results suggest that HA nanogel cancer vaccines may be able to induce large numbers of antigen-presenting cells and lymphocytes, such as dendritic cells, macrophages, and Langerhans cells, to flow into and accumulate in the regional lymph nodes in the immunosuppressive environment of tumor-bearing hosts, thereby creating an efficient tumor antigen presentation environment.Furthermore, by supplementing this environment of improved tumor antigen presentation with T cells through antigen-specific T cell infusion therapy, the CD8-positive infused T cells that flowed into the regional lymph nodes were able to efficiently receive antigen presentation, and the CD8-positive infused T cells were induced to proliferate as qualitatively and quantitatively potent tumor antigen-specific cytotoxic T cells (CTLs), suggesting the possibility that a sustainable synergistic anti-tumor immune environment was constructed in the regional lymph nodes.

[0270] Example 3. Therapeutic effect and anti-tumor immune response analysis by gradually decreasing the number of infused cells For the mouse tumor growth test (Example 1) and the anti-tumor immune response analysis (Example 2), the number of antigen-specific T cells was 2 × 10 6 From 1 x 10 cells / 200 μL 6 pieces / 200μL, 5×10 5 pieces / 200μL, 2.5×10 5 A similar test was performed with the dose gradually decreasing to a concentration of 1000 cells / 200 μL.

[0271] <Results of Example 3> To clarify the influence of the number of CD8-positive T cells infused on the therapeutic effect of the combined HA nanogel cancer vaccine and antigen-specific infusion T cell therapy, the number of CD8-positive T cells infused was gradually decreased, and the therapeutic effect and anti-tumor immune response were examined. As a result, the number of CD8-positive T cells infused was gradually decreased from 1 × 10 6 pieces, 5×10 5 pieces, 2.5×10 5 Even when the dose is gradually reduced to 2 x 10 6 The therapeutic effect was equivalent to that of administration of 2 × 10 (Figures 3-1 and 3-2). Furthermore, 14 days after tumor inoculation, the effects on the total cell count and myeloid antigen-presenting cells in the regional lymph nodes were examined, and these cells were significantly observed even when the gradually decreasing dose was administered (Figures 3-3 and 3-4). Furthermore, the proportion of CD8-positive infused cells and tumor antigen-specific IFN-γ-producing cells in the regional lymph nodes was examined, and the proportion of CD8-positive infused cells was 2 × 10 (Figures 3-3 and 3-4). 6Although the highest activity was observed when 1000 CD8+ T cells were administered, it was also revealed that tumor antigen-specific IFN-γ-producing cells, both infused and host T cells, had equivalent activity even when gradually decreasing amounts of CD8+ infused T cells were administered (Figures 3-5 and 3-6). These results demonstrate that the HA nanogel vaccine can induce tumor antigen-specific immune responses and potently cure ICI-resistant tumors even when administered in combination with CD8+ infused T cells, which is far less than the number of conventional CD8+ infused T cells, nearly 1 / 10.

[0272] Example 4. Observation of Tumor-Infiltrating CD8-Positive T Cells Tumors were excised 14 days after tumor implantation in the study described in Example 1 and subjected to immunohistofluorescence staining as follows. Tumors embedded in OCT compound (Sakura Fine Tech) and frozen were sliced ​​at 5 μm thicknesses, and the resulting tumor sections were air-dried. The dried tumor sections were fixed in ice-cold acetone for 10 minutes and used for immunostaining. After washing the tumor sections three times with PBS, they were immersed in Blocking One Histo (Nacalai Tesque) and blocked for 30 minutes in an incubation chamber. Next, the tumor sections were stained for 1 hour at room temperature in an incubation chamber using anti-mouse CD8 antibody diluted 1:100 with Antibody Diluent with Background Reducing Component (Dako). After washing three times with PBS containing 0.1% Tween 20, the tumor sections were stained with Alexa488-conjugated anti-rat antibody diluted 1:1000 in PBS in an incubation chamber for 1 hour at room temperature. Finally, after washing three times with PBS containing 0.1% Tween 20, the tumor sections were immersed in Prolong Gold antifade reagent with DAPI (Life Technologies) and CD8+ T cells infiltrating the tumor were observed using a fluorescence microscope BZ-710 (Keyence).

[0273] Example 4 Results To clarify the effect of the combined use of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy on the intratumoral environment in tumor-bearing hosts, tumors were excised from each mouse 14 days after tumor inoculation, and CD8+ T cells infiltrating the tumor were observed under a fluorescent microscope. The results showed that CD8+ T cells were significantly more abundant in the tumors of the HANG-V and TCR-T combination group than in the untreated or monotherapy groups (Figure 4). These results suggest that the combined use of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy may induce tumor antigen-specific cytotoxic T cells (CTLs) within tumors and eliminate tumors.

[0274] Example 5. Analysis of CD8+ Infused T Cells in Peripheral Blood. Peripheral blood was collected from mouse orbits 21 days after tumor inoculation in the study described in Example 3 and suspended in RPMI containing 10% heparin. The suspension was then slowly added to 500 μL of Ficoll (GE Healthcare) in an Eppendorf tube and centrifuged (4000 rpm × 10 min, 25°C, AC:1, DE:1). The mononuclear cell layer was then recovered in staining buffer (PBS containing 0.5% BSA). The cell suspension was centrifuged (5000 rpm × 1 min, 25°C), the supernatant was removed using an aspirator, and the cells were washed twice with PBS containing 0.5% FBS. APC-labeled anti-mouse CD8 antibody, PerCP-Cy5.5-labeled anti-mouse CD90.1 antibody, and Pacific Blue-labeled anti-mouse CD90.2 antibody were added at the manufacturer-recommended concentrations, mixed, and then allowed to stand in the dark at 4°C for 15 minutes. The cells were washed twice with 200 μL of staining buffer, resuspended in 200 μL of staining buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and the accompanying analysis software (FACSDiva).

[0275] <Results of Example 5> To clarify the persistence of CD8-positive infused T cells throughout the body after infusion of HA nanogel cancer vaccine and antigen-specific T cells, peripheral blood cells were collected 21 days after tumor inoculation, and the proportion of CD8-positive infused T cells present in the peripheral blood was examined. As a result, it was revealed that CD8-positive infused T cells were circulating throughout the body and present in the blood even 21 days after tumor inoculation. In addition, the number of CD8-positive infused T cells was 2.5 x 10 5 Even when administered at a low dose, the CD8+ T cells infused were 2 × 10 6 These results demonstrate that CD8+ T cells continue to circulate in the peripheral blood and persist throughout the body, regardless of the number of cells transfused.

[0276] Summary of Examples 1-5: These results demonstrate that the combined use of HA nanogel cancer vaccines and antigen-specific T cell infusion therapy promotes the influx and accumulation of antigen-presenting cells and infused T cells in regional lymph nodes through the HA nanogel cancer vaccine, which then induces the infused T cells to transform into tumor antigen-specific cytotoxic T cells (CTLs) through antigen presentation, resulting in the elimination and cure of ICI-resistant tumors. This therapy not only has the advantage of qualitatively and quantitatively enhancing the infused T cells, but also acts on host T cells, shifting the host's immunosuppressive environment to an antitumor environment. Furthermore, this effect was comparable with a much smaller number of infused cells than conventional infusions, and persisted throughout the body for a long period after cure, suppressing tumor recurrence and ensuring long-term host survival.

[0277] Cancer vaccines are generally a treatment that aims to enhance the antigen-presenting ability of antigen-presenting cells and stimulate the anti-tumor effects of T cells. However, because the immune environment of many cancer patients is suppressed, even if a vaccine can present tumor antigens to T cells, the therapeutic effect is limited if there are few T cells in the body, making it necessary to replenish T cells at the same time as the vaccine. On the other hand, antigen-specific T cell infusion therapy is a treatment in which cancer patient's T cells are modified ex vivo into antigen-specific T cells and then returned to the body. However, the infused T cells gradually become exhausted and weakened, and in order to sustain the therapeutic effect, it is important to continue the infusion of T cells and maintain high-quality T cells in the body.

[0278] In this invention, by complementing the weaknesses of each treatment method, an environment for efficient tumor antigen presentation was created in the regional lymph nodes by the HA nanogel cancer vaccine, and T cells were further added to this environment by T cell infusion therapy. As a result, an anti-tumor immune environment was created in which tumor antigen-specific CTLs were induced qualitatively, quantitatively, and sustainably, and it is believed that a synergistic therapeutic effect was successfully achieved.

[0279] Materials and Methods (Examples 6-16) (1) Cells. RPMI1640 medium (supplemented with 2-mercaptoethanol) was purchased from the Cell Science Institute. D-MEM medium was purchased from Wako Pure Chemical Industries, Ltd., and fetal bovine serum (FBS) was purchased from Gibco. The mouse fibrosarcoma cell line CMS5a was provided by Memorial Sloan-Kettering Cancer Institute, and the mouse metastatic melanoma cell line B16F10 was purchased from ATCC and passaged at Mie University. The mouse fibrosarcoma cell line CMS5a expresses a mutant ERK2 protein. A peptide containing the mutated portion of the mutant ERK2 protein (QYIHSANVL (SEQ ID NO: 1)) is recognized by CD8+ cytotoxic T cells in BALB / c mice. A T cell receptor (TCR) that recognizes this peptide has been isolated, and transgenic mice (DUC18 mice) have been generated by introducing the TCR gene. The long peptide antigen used in this example contains the mutant ERK2 CD8-positive cytotoxic T cell-recognized epitope sequence (QYIHSANVL). The mouse metastatic melanoma cell line B16F10 expresses the tumor antigen gp100 protein, and the p25-33 peptide (EGSRNQDWL (SEQ ID NO: 4)) contained in this protein is recognized by CD8-positive cytotoxic T cells of C57BL / 6 mice. Transgenic mice (Pmel-1 mice) have been generated by introducing a TCR gene that recognizes this CD8-positive cytotoxic T cell-recognized epitope sequence. The long peptide antigen used in this example contains this sequence.

[0280] (2) Test animals Female BALB / c mice and female C57BL / 6 mice aged 6 to 8 weeks were purchased from Japan SLC. DUC18 transgenic mice were provided by the University of Washington and bred at the Mie University Animal Experiment Facility. Pmel-1 transgenic mice were purchased from the Jackson Laboratory and bred at the Mie University Animal Experiment Facility. All mice were housed at the Mie University Animal Experiment Facility. The animal experiment protocol was approved by the Ethics Committee of the Mie University School of Medicine.

[0281] (3) Preparation of a complex using HA nanogel with a molecular weight of 10 kDa and peptide mERK2 (used in Examples 11 to 16) (3-1) Hyaluronic acid nanogel (HANG) Hyaluronic acid nanogels with introduced cholesteryl groups (weight-average molecular weight of 10 kDa, introduction rates of cholesteryl groups of 44 and 41%) were synthesized as described in Examples 1 and 2 of WO 2010 / 053140.

[0282] (3-2) Long-chain peptide antigen. A synthetic long-chain peptide was purchased from Biologica. The sequence was as follows: NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT (SEQ ID NO: 2). This sequence contains a 9-amino acid sequence from Q at position 16 to L at position 24 (QYIHSANVL (SEQ ID NO: 1)) as an epitope recognized by CD8-positive cytotoxic T cells, and a 17-amino acid sequence from R at position 13 to K at position 29 (RGLQYIHSANVLHRDLK (SEQ ID NO: 3)) as an epitope recognized by CD4-positive helper T cells.

[0283] (3-3) Preparation of Hyaluronic Acid Nanogel and Antigen Complexes. The lyophilized hyaluronic acid nanogel was weighed, and water for injection was added to a concentration of 5 mg / mL. The mixture was stirred overnight to allow for complete dissolution. Meanwhile, in a separate container, the peptide was dissolved in dimethyl sulfoxide to a concentration of 50 mg / mL. The hyaluronic acid nanogel aqueous solution and the peptide dimethyl sulfoxide solution were then mixed at a volume ratio of 100:1 and stirred at room temperature for 24 hours to obtain a composition containing a peptide-hyaluronic acid nanogel complex. Solid purified sucrose (Fujifilm Wako Co., Ltd., product number 198-18385) was then added to the composition containing the peptide-hyaluronic acid nanogel complex to a concentration of 10% sucrose by mass, and the mixture was stirred for at least 1 hour. The solution was then diluted with 10% by mass sucrose-containing 25 mM phosphate buffer (pH 7.4) to a theoretical peptide concentration of 0.3 mg / mL, and sterilized through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a composition containing a peptide-hyaluronic acid nanogel complex. The peptide concentration in the composition containing the peptide-hyaluronic acid nanogel complex was quantified by reverse phase chromatography, and the concentration was then adjusted to 250 μg / mL with 10% sucrose, 10 mM phosphate buffer (pH 7.4), to obtain the administration solution.

[0284] (4) Preparation of a complex using HA nanogel with a molecular weight of 10 kDa and peptide gp100 (used in Examples 6 to 10) (4-1) Hyaluronic acid nanogel (HANG) Hyaluronic acid nanogel with introduced cholesteryl groups (weight-average molecular weight of 10 kDa, introduction rate of cholesteryl groups: 44%) was synthesized as described in Examples 1 and 2 of WO 2010 / 053140.

[0285] (4-2) Long-Chain Peptide Antigens Using the hyaluronic acid nanogel obtained above, a peptide-hyaluronic acid nanogel complex was prepared as follows. The gp100 TRP2 TRP1_6Y peptide was purchased from Biologica. Its sequence is as follows: SVYDFFVWLYYYYYYTWHRYHLLYYYYYY EGSRNQDWL (SEQ ID NO: 5). This sequence contains the following epitope sequences recognized by CD8-positive cytotoxic T cells: TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 6)), TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 7)), and gp100 (amino acid sequence: EGSRNQDWL (SEQ ID NO: 4)), with each epitope linked by six Y residues.

[0286] (4-3) Preparation of Hyaluronic Acid Nanogel and Antigen Complex. The lyophilized hyaluronic acid nanogel was weighed, and water for injection was added to a concentration of 5 mg / mL. The mixture was stirred overnight to allow for complete dissolution. Meanwhile, in a separate container, the peptide was dissolved in dimethyl sulfoxide to a concentration of 50 mg / mL. After confirming dissolution, the hyaluronic acid nanogel aqueous solution was mixed with 1 mol / L sodium hydroxide aqueous solution at a volume ratio of 100:2 at room temperature. The hyaluronic acid nanogel aqueous solution was then mixed with a peptide dimethyl sulfoxide solution at a volume ratio of 102:1 at room temperature and stirred for 24 hours to obtain a composition containing a peptide-hyaluronic acid nanogel complex. Solid purified sucrose (Fujifilm Wako Co., Ltd., product number 198-18385) was then added to the composition containing the peptide-hyaluronic acid nanogel complex to a concentration of 10% sucrose by mass, and the mixture was stirred for at least 1 hour. The solution was then diluted with 10% by mass sucrose-containing 25 mM phosphate buffer (pH 7.4) to a theoretical peptide concentration of 0.3 mg / mL, and sterilized through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a composition containing a peptide-hyaluronic acid nanogel complex. The peptide concentration in the composition containing the peptide-hyaluronic acid nanogel complex was quantified by reverse phase chromatography, and the concentration was then adjusted to 250 μg / mL with 10% sucrose, 10 mM phosphate buffer (pH 7.4), to obtain the administration solution.

[0287] (5) Preparation of Complex Using CHP and Peptide gp100 (Used in Example 7) (5-1) Cholesteryl-Modified Pullulan (CHP) CHP (cholesteryl-modified pullulan, product number CHP-80T) manufactured by NOF Corporation was used.

[0288] (5-2) Long-Chain Peptide Antigen The gp100 peptide (SEQ ID NO: 5) was used.

[0289] (5-3) Preparation of Hyaluronic Acid Nanogel and Antigen Complex: CHP powder was dissolved in 6 M urea-containing phosphate-buffered saline (pH 7.4) to a concentration of 10 mg / mL. Separately, in a separate container, peptide was dissolved in dimethyl sulfoxide to a concentration of 50 mg / mL. The 6 M urea-containing CHP aqueous solution and the peptide dimethyl sulfoxide solution were mixed at a volume ratio of 100:1 and incubated at room temperature for at least 12 hours to obtain a composition containing peptide-CHP complexes. The resulting solution was then transferred to a dialysis cassette (Thermo, Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL) and dialyzed against 0.6 M urea-containing phosphate-buffered saline (pH 7.4). This was followed by dialysis against 0.06 M urea-containing phosphate-buffered saline (pH 7.4) and then against phosphate-buffered saline (pH 7.4). The mixture was further concentrated to the desired concentration using an ultraconcentrator (Vivaspin 20, MWCO: 10,000, Sartorius). Finally, it was sterile filtered through a 0.2 μm PES (polyethersulfone) filter (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a composition for administration containing a complex of CHP and antigen. The peptide concentration was quantified by reversed-phase chromatography.

[0290] (6) Other Reagents - Adjuvant CpG oligo DNA1668 was purchased from Ajinomoto Bio-Pharma Services Gene Design.

[0291] Fluorescently labeled antibodies: eFluor 450-labeled anti-mouse CD8a antibody (clone 53-6.7), eFluor 450-labeled anti-mouse KLRG1 antibody (clone 2F1), APC-labeled anti-mouse CD44 antibody (clone IM7), and APC-labeled anti-mouse IFN-γ antibody (clone XMG1.2) were purchased from eBioscience. APC-labeled anti-mouse CD8a antibody (clone 53-6.7), APC-labeled anti-mouse CD4 antibody (clone RM4-5), PerCP-Cy5.5-labeled anti-mouse CD90.1 antibody (clone OX-7), Pacific Blue-labeled anti-mouse CD90.2 antibody (clone 30-H12), FITC-labeled anti-mouse CD8 antibody (clone 53-6.7), Brilliant Violet 510-labeled anti-mouse CD90.1 antibody (clone OX-7), Pacific Blue-labeled anti-mouse CD44 (clone IM7), and purified CD16 / 32 antibody (clone 93) were purchased from BioLegend. PE-labeled anti-mouse CD62L antibody (clone MEL-14) was purchased from BD Biosciences. APC-labeled H-2Db gp100 Tetramer and Clear Back for tetramer staining were purchased from MBL.

[0292] Immune checkpoint inhibitors (ICIs): Anti-mouse PD-1 antibody (clone RMPI-14) was purchased from BioCell. Anti-mouse CTLA-4 antibody (clone 9D9) was produced at Mie University using a hybridoma provided by MD Anderson Cancer Center.

[0293] Short peptide antigens: Synthetic short peptides (QYIHSANVL (SEQ ID NO: 1)) for stimulating CD8-positive T cells were purchased from Sigma Genosys. EGSRNQDWL (SEQ ID NO: 4) and VYDGREHTV (SEQ ID NO: 9) were purchased from Eurofins.

[0294] Example 6. Tumor Growth Test (Metastatic Malignant Melanoma Cell Line B16F10) The metastatic malignant melanoma cell line B16F10 was cultured in 10% FBS-containing D-MEM medium using a T75 culture flask (Corning). The cultured cells were detached using 0.5% trypsin-containing PBS and suspended in 10% FBS-containing D-MEM medium. The suspension was centrifuged (400 × g, 5 minutes, 4°C), and the supernatant was removed. The cells were then washed twice with D-MEM medium and 2 × 10 5 The cells were suspended in D-MEM medium at a concentration of 100 μL / 100 cells / 100 μL and subcutaneously implanted into the right anterior dorsum of C57BL / 6 mice at a dose of 100 μL / mouse (5 mice per group).

[0295] When administering the long-chain peptide antigen-loaded HA nanogel cancer vaccine, 50 μg of the long-chain peptide antigen-loaded HA nanogel cancer vaccine (75 μg on the 15th day only) was administered subcutaneously to the right dorsal region of mice on days 7, 11, and 15 after tumor inoculation, together with 50 μg of CpG oligo DNA dissolved in PBS (HANG-V group).

[0296] For administration of antigen-specific T cells, CD8+ T cells were isolated from the spleen of Pmel-1 transgenic mice (CD90.1-positive) carrying a gp100-recognizing TCR gene on days 8 and 12 after tumor inoculation using CD8a microbeads (Miltenyi). 2 × 10 cells were then added to RPMI1640 medium. 6 After suspending the cells at a concentration of 200 μL per 200 μL, 200 μL was infused into the tail vein of each mouse (TCR-T group).Tumor area was then measured over time.

[0297] Example 6 Results Similar to Example 1 (ICI-resistant fibrosarcoma cell line CMS5a), to clarify the antitumor effect of a combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy against metastatic melanoma, we used C57BL / 6 mice bearing metastatic melanoma cell line B16F10 tumors to create a long-chain peptide antigen-loaded HA nanogel cancer vaccine by conjugating HA nanogel with a long-chain peptide antigen containing the CD8 epitope of the gp100 tumor antigen overexpressed in B16F10. We then investigated the therapeutic effect of this vaccine in combination with gp100-recognizing TCR gene-transduced T cell infusion therapy. The results showed that the HANG-V treatment group exhibited suppression of B16F10 tumor growth compared with the untreated group and the TCR-T alone group (Figures 6-1 and 6-2). In particular, the HANG-V and TCR-T combination group showed a significant improvement in tumor growth, even at a tumor size of 100 mm. 2 The antitumor effect was also observed against B16F10 tumors, which had reached a tumor size of approximately 100 μg / mL (Figure 6-2). All mice showed significant tumor regression (Figure 6-2), and two mice showed complete tumor disappearance. In both the HANG-V monotherapy and TCR-T combination therapy groups, all mice developed crater-like cavities (tumor necrosis) in the tumor center, likely due to the tumor-killing effect of cytotoxic T cells (CTLs) (Figure 6-3). Furthermore, while all mice in the untreated and TCR-T monotherapy groups died within 25 days of tumor inoculation, 100% of mice in the HANG-V and TCR-T combination therapy group and 80% of mice in the HANG-V group survived (Figure 6-4). This effect persisted for a long time, and the two mice that showed complete tumor disappearance survived without recurrence for more than five months after tumor inoculation.

[0298] These results suggest that the combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy exerts a strong antitumor effect not only on immune checkpoint inhibitor (ICI)-resistant tumors, but also on malignant melanomas with poor prognosis and metastasis, such as B16F10, and may be capable of curing tumors, making it a groundbreaking treatment for "cold tumors" and "immune desert tumors."

[0299] Example 7. Tumor growth test (comparison between HA nanogel vaccine and CHP nanogel vaccine) For the mouse tumor growth test (Example 6), the long-chain peptide antigen-loaded nanogel cancer vaccine (HANG-V or CHP-V) was used in combination with antigen-specific T cell infusion therapy. For both HANG-V and CHP-V, 50 μg of the long-chain peptide antigen-loaded nanogel cancer vaccine (75 μg from the third time onwards) was subcutaneously administered to the right dorsal region of mice on days 7, 11, 15, 29 and 36 after tumor inoculation, together with 50 μg of CpG oligo DNA dissolved in PBS, to induce the induction of antigen-specific T cells (4 × 10 6 ) were infused.

[0300] Example 7 Results To determine whether the combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy is superior to the CHP nanogel cancer vaccine and antigen-specific T cell infusion therapy (prior art) in terms of antitumor efficacy, a tumor growth test was conducted to compare the therapeutic effects of both. As in Example 6, the HANG-V and TCR-T combination group significantly suppressed B16F10 tumor growth and achieved strong tumor regression in all animals, whereas the CHP-V and TCR-T combination group did not demonstrate a similar therapeutic effect (Figures 7-1 and 7-2). Furthermore, the HANG-V and TCR-T combination group significantly suppressed tumor growth, not only in terms of tumor size (area) but also in terms of tumor growth and growth (height), compared with the CHP-V and TCR-T combination group. Crater-like cavities (tumor necrosis), thought to be due to the tumor-damaging effect of CTLs, were observed in the central part of the tumor in all animals (Figure 7-3). Subsequently, of all animals showing regression, two showed complete disappearance of the tumor, with the tumor area becoming scab-like, and the remaining three showed regression to the point where it was barely visible to the naked eye without recurrence of tumors. On the other hand, in the CHP-V and TCR-T combination group, tumor growth continued unsuppressed and accelerated, and all animals died within 50 days, whereas the HANG-V and TCR-T combination group maintained a 100% survival rate (Figure 7-4).

[0301] These results demonstrate that the combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy has superior therapeutic effects in all aspects, including tumor growth inhibition, tumor damage, and survival extension, compared to the prior art combination therapy of CHP nanogel cancer vaccine and T cell infusion therapy.

[0302] Example 8. Analysis of gp100 antigen-specific CD8 T cells in lymph nodes regionally related to the vaccine administration site. A similar test to that in Example 6 was performed. Lymph nodes regionally related to the vaccine administration site (right inguinal lymph nodes) were collected 14 days after tumor inoculation (2-3 mice per group). The lymph nodes were ground using a glass slide, and the cells were suspended in RPMI1640 medium. After centrifugation (400 × g, 5 minutes, 4°C), the supernatant was removed, and the cells were washed twice with RPMI1640 medium and then suspended in RPMI1640 medium containing 10% FBS. The cell count was measured (Figure 8-1), and the cell concentration was 2 × 10 6 The concentration was adjusted to 1 / mL.

[0303] gp100 tetramer cell staining (Figures 8-2 and 8-3): Add 4 x 10 cells per well of the cell suspension to a 96-well V-bottom microplate (Nunc). 5 The cell suspension was centrifuged (2000 rpm, 2 minutes, 4°C), the supernatant was removed, and the cells were washed twice with 200 μL of staining buffer (PBS containing 0.5% FBS) and then suspended. 50 μL of the cell suspension was added per well.

[0304] Clear Back (MBL) was added to the cell suspension at 10 μL / well and incubated for 10 minutes. Then, H-2Db gp100 Tetramer (MBL) was added at 10 μL / well and incubated for 30 minutes in the dark. Next, FITC-labeled anti-mouse CD8 antibody, Brilliant Violet 510-labeled anti-mouse CD90.1 antibody, Pacific Blue-labeled anti-mouse CD44 antibody, or eFlow450-labeled anti-mouse KLRG1 antibody were added to the final concentrations recommended by the antibody manufacturer, mixed, and incubated at 4°C in the dark for 15 minutes. After adding 150 μL of staining buffer, the cells were centrifuged, the supernatant removed, and then washed twice with 200 μL of staining buffer. After centrifuging and removing the supernatant, the cells 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) and the accompanying analysis software (FACSDiva). gp100 antigen-specific CD8+ infused T cells were detected as a gp100 tetramer-positive, CD90.1-positive, CD8-positive population.

[0305] Antigen-specific T cell induction test (intracellular cytokine staining) (Figure 8-4): In a 48-well culture plate (Nunc), 4 × 10 cells were added to each well of the cell suspension. 5The cells were added at a concentration of 200 μL per well. 50 μL of gp100 peptide, a short-chain peptide for stimulating CD8+ T cells, was added to the wells to a final concentration of 10 μM, and the cells were cultured for 1 hour in a 37°C, 5% CO2 incubator. Then, 50 μL of Goldi Plug (BD Biosciences) diluted 167-fold with 10% FBS-containing RPMI 1640 medium was added per well, and the cells were cultured for 5 hours in a 37°C, 5% CO2 incubator. The cells were harvested, transferred to a 96-well V-bottom microplate (Nunc), centrifuged (2000 rpm, 2 minutes, 4°C), and the supernatant was removed. The cells were then suspended in 200 μL of staining buffer per well. After washing the cells twice with 200 μL of staining buffer, FITC-labeled anti-CD8 antibody and Brilliant Violet 510-labeled anti-mouse CD90.1 antibody were added to the cell suspension at the manufacturer's recommended concentrations, mixed, and then left to stand for 15 minutes in the dark at 4°C. 150 μL of staining buffer was added, centrifuged, and the supernatant was removed. The cells were then washed twice with 200 μL of staining buffer, followed by the addition of 100 μL of Cytofix / Cytoperm buffer (BD Biosciences) and gentle mixing. After leaving the cells to stand for 20 minutes in the dark at room temperature, they were washed twice with staining buffer, suspended in 200 μL of staining buffer, and stored overnight in a dark, refrigerated container. After centrifugation and removal of the supernatant, the cells were washed with 200 μL of Perm / Wash buffer (BD Biosciences). A 50-fold diluted APC-labeled anti-mouse IFN-γ antibody was added to the cells, mixed, and incubated at room temperature in the dark for 15 minutes. The cells were washed twice with 200 μL of Perm / Wash buffer, resuspended in 200 μL of staining buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences) and the accompanying analysis software (FACSDiva). IFN-γ-producing CD8+ infused T cells were detected as an IFN-γ-positive, CD90.1-positive, CD8-positive population.

[0306] Example 8: Results To investigate the effect of the HA nanogel cancer vaccine on gp100-specific CD8+ T cells in combination with antigen-specific T cell infusion therapy, regional lymph nodes were collected 14 days after tumor inoculation and analyzed for CD8 T cells. Results showed significant lymph node swelling in the HANG-V monotherapy and HANG-V / TCR-T combination groups, with both groups showing approximately 9- to 16-fold increases in lymph node cells compared with the untreated group (Figure 8-1). Furthermore, analysis of the proportion of gp100-specific tetramer-positive CD8 T cells that had inflowed and accumulated in the regional lymph nodes revealed a significant increase in these CD8 T cells in both groups compared with the untreated HANG-V group (Figure 8-2), demonstrating a highly activated effector T cell phenotype (KLRG1, CD44) (Figure 8-3). Furthermore, in the HANG-V and TCR-T combination group, it was revealed that IFN-γ was significantly produced by the CD8-positive infused T cells in a gp100 antigen-specific manner (Figure 8-4).

[0307] These results suggest that the HA nanogel cancer vaccine may potently induce the infused CD8-positive T cells in the regional lymph nodes to become gp100 antigen-specific CTLs in the immunosuppressive environment of the tumor-bearing host, thereby suppressing B16F10 tumors.

[0308] Example 9. Examination of localization of antigen-specific CD8-positive infused T cells in tumors and throughout the body After carrying out a test similar to that in Example 6, lymph nodes (right inguinal lymph nodes), non-regional lymph nodes (left inguinal lymph nodes), spleen, tumor, and peripheral blood were collected from the vaccine administration site on day 19 after tumor inoculation. Each type of cell was separated by the method described below, and the proportion of CD8-positive infused T cells and gp100 tetramer-positive CD8-positive T cells present in each tissue was examined.

[0309] Isolation of lymph node cells and spleen cells: Lymph nodes (draining and non-draining) and spleens were crushed using a glass slide, and the released cells were collected in RPMI 1640 medium. After centrifugation (400 × g, 5 minutes, 4°C), the supernatant was removed. For spleen cells, 2 mL of red blood cell lysis solution was added and the cells were treated for 1 minute. 18 mL of RPMI 1640 medium was then added, followed by centrifugation (400 × g, 5 minutes, 4°C). The supernatant was removed, and the cells were suspended in RPMI 1640 medium (primary cell suspension).

[0310] Isolation of tumor-infiltrating immune cells (TIL): Tumor tissue was homogenized and then suspended in RPMI 1640 medium. Collagenase D (2 mg / mL, Roche) was added to the suspended cells and incubated at 37°C for 30 minutes. Then, the cells were filtered through a 0.22 μm pore size filter while adding RPMI 1640 medium. After centrifugation (400 × g, 5 minutes, 4°C), the supernatant was removed and the cells were suspended in RPMI 1640 medium (primary cell suspension).

[0311] Isolation of peripheral blood mononuclear cells (PBMCs): Peripheral blood mononuclear cells were collected by the same method as in Example 5. The collected primary cell suspensions from each tissue were stained and measured by the same method as in Example 7. CD8-positive infused T cells were detected as a CD90.1-positive CD8-positive population, and gp100 antigen-specific CD8-positive T cells were detected as a gp100 tetramer-positive CD8-positive population.

[0312] Example 9 Results To determine the persistence of antigen-specific CD8+ T cells in the tumor and systemic areas after HA nanogel cancer vaccine and antigen-specific T cell infusion, secondary lymphoid tissues, tumor tissues, and peripheral blood were collected 19 days after tumor inoculation, and the proportion of infused T cells was examined. The results revealed that infused T cells were significantly present not only in regional lymph nodes, but also in other secondary lymphoid tissues (spleen and non-regional lymph nodes), tumor tissue, and systemic peripheral blood (Figure 9-1). Furthermore, gp100 antigen-specific CD8 T cells were also widely and significantly present in each tissue (Figure 9-2), suggesting that these systemically localized antigen-specific CD8 T cells may be responsible for suppressing not only local tumor growth but also systemic metastasis.

[0313] Example 10. Recurrence suppression effect after treatment After the treatment in Example 6, peripheral blood was collected from two animals in which complete tumor regression was observed (five months after tumor transplantation), and the proportion of gp100 antigen-specific CD8-positive infused T cells was examined. Subsequently, one of the two animals was inoculated with B16F10 tumor (2 × 10 5 One mouse was re-implanted with a B16F10 tumor (2 x 10 cells), and the other mouse was subcutaneously injected with 75 μg of a long-chain peptide antigen-loaded HA nanogel cancer vaccine together with 50 μg of CpG oligo DNA dissolved in PBS into the right dorsal region three times (every 3 to 4 days). Peripheral blood was then collected again, and the percentage, activation, and phenotype of each gp100 antigen-specific CD8-positive T cell infusion were examined. Furthermore, the same mice were inoculated with B16F10 tumor (2 x 10 cells). 5 Peripheral blood mononuclear cells (PBMCs) were collected as in Example 5, and tetramer staining was performed as in Example 8. The cells were then stained and measured by the following method.

[0314] 50 μL of a solution containing FITC-labeled anti-mouse CD8 antibody, Brilliant Violet 510-labeled anti-mouse CD90.1 antibody, Pacific Blue-labeled anti-mouse CD44 antibody, and PE-labeled anti-mouse CD62L antibody was added at the manufacturer's recommended concentrations and mixed. The mixture was then incubated at 4°C for 15 minutes in the dark. 150 μL of staining buffer was added, and the mixture was centrifuged to remove the supernatant. The cells were then washed twice with 200 μL of staining buffer. After centrifugation and removal of the supernatant, the cells were resuspended in 200 μL of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences) and the accompanying analysis software (FACSDiva). gp100 antigen-specific CD8-positive infused T cells were detected as gp100 tetramer-positive, CD90.1-positive, CD8-positive populations, effector memory cells as CD44-positive, CD62L-negative populations, and central memory cells as CD44-positive, CD62L-positive populations.

[0315] Example 10 Results: Measurement of gp100 antigen-specific CD8+ infused T cells in the peripheral blood of an individual who underwent HA nanogel cancer vaccine and antigen-specific T cell infusion therapy and experienced complete tumor regression and no recurrence revealed that these CD8+ infused T cells were still present in the peripheral blood 5 months after recovery (Figure 10-1). Furthermore, after the same individual was reimplanted with B16F10 tumor or revaccinated, the proportion of gp100 antigen-specific CD8+ infused T cells in the peripheral blood was measured again, and a significant increase in these cells was observed in the peripheral blood (Figure 10-2). These cells exhibited the phenotypes of effector memory T cells (CD44+CD62L-negative) and central memory T cells (CD44+CD62L-positive) (Figure 10-3). In particular, explosive proliferation of these cells was evident in individuals after revaccination, revealing the presence of strongly CD44+ effector cells (Figure 10-4). Furthermore, when B16F10 was re-implanted into the same mouse, no tumor engraftment or growth was observed, demonstrating that the mouse would not relapse (Figure 10-5).

[0316] These results demonstrate that in individuals who successfully received HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, a certain number of antigen-specific CD8+ infused T cells remain in the peripheral blood for a long period of time even after tumor cure, and that in the event of tumor recurrence, these memory cells rapidly proliferate, recognize tumor antigens, exert antitumor effects, and suppress recurrence. Furthermore, it was demonstrated that regular administration of this vaccine after successful treatment can induce highly activated antigen-specific effector / memory T cells and prevent tumor recurrence.

[0317] When used in combination with antigen-specific T cell infusion therapy, this vaccine exerts a powerful antitumor effect against malignant melanomas with poor prognosis and metastasis, such as B16F10, and is also effective in preventing tumor recurrence. It is expected that this vaccine will become an innovative cancer vaccine that combines both therapeutic and preventive aspects.

[0318] Example 11. Therapeutic Effect of Vaccine, etc. on Individuals Whose Response Changed to Regrowth After Treatment After conducting a test similar to the mouse tumor growth test (Example 1) (9 mice per treatment group), an individual (1 out of 9 mice) in which tumor regrowth was observed was administered 75 μg of a long-chain peptide antigen-loaded HA nanogel cancer vaccine together with 50 μg of CpG oligo DNA dissolved in PBS subcutaneously to the right dorsal region of the mouse on days 27, 32, 36, 40, and 43 after tumor implantation. Furthermore, on day 41 after tumor implantation, antigen-specific CD8+ T cells (2 × 10 ) isolated from the spleen of a mutant ERK2-recognizing TCR transgenic mouse (DUC18) using CD8a microbeads (Miltenyi) were administered. 6 On day 49 after tumor inoculation, ICI (200 μg of anti-mouse PD-1 antibody and 100 μg of anti-mouse CTLA-4 antibody) was administered intraperitoneally, and tumor area was measured over time.

[0319] <Results of Example 11> After receiving HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, individuals who initially responded to treatment but subsequently experienced tumor regrowth were given vaccine re-administration, re-infusion, and ICI. As a result, although relapsed tumors generally grow rapidly and have a poor prognosis (Figure 11-2, right), it was revealed that repeated administration of the vaccine significantly suppressed tumor growth, and that combined use with other immunotherapies (ICI) could suppress tumor progression (Figure 11-1 and Figure 11-2, left).

[0320] These results suggest that even if treatment is initially successful after administration of an HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, but tumor regrowth is subsequently observed, re-administration of the vaccine, re-infusion, and additional ICI therapy can significantly suppress tumor growth and may be an effective treatment for tumor relapse.In addition, this study used hyaluronic acid nanogels with cholesteryl group introduction rates of 44 and 41%, and both showed high antitumor effects.

[0321] Example 12. Recurrence-inhibitory effect on individuals who achieved complete response (complete regression) after treatment. After the treatment in Example 11, individuals (n=8) who showed complete regression of the tumor were injected with CMS5a tumor (1×10 6The tumors were then re-implanted and the tumor area was measured.

[0322] <Results of Example 12> After administering the HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, CMS5a tumors were re-implanted into mice that had shown complete tumor regression. As a result, tumor growth was suppressed in all mice, and 6 out of 8 mice showed complete tumor regression (75%) (Figures 12-1 and 12-2).

[0323] These results suggest that this treatment can cure tumors resistant to ICI therapy, and that, as with the results of Example 10, memory immunity against the tumor is acquired after successful treatment (memory formation). When the tumor recurs, antigen-specific memory T cells can rapidly recognize and destroy these T cells, suppressing tumor recurrence. Furthermore, as with Example 10, revaccination with the vaccine can induce activated antigen-specific CD8 memory T cells with strong effector function, further preventing recurrence.

[0324] Example 13. Effect of combined use of vaccine and antigen-specific T cell infusion therapy (single administration) A similar experiment to the mouse tumor growth test (Example 1) was performed, except that the antigen-specific T cell infusion therapy was reduced from two times to one time (9 mice per treatment group).

[0325] <Results of Example 13> To clarify the effect of the number of CD8+ T cell infusions on the antitumor effect in the combined use of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, the number of CD8+ T cell infusions was gradually reduced from two as in the conventional method to one, and the therapeutic effect was examined. As a result, tumor regression was observed in all individuals, even when CD8+ T cells were infused once, similar to the two-infusion method used in the conventional method and the prior art, and tumors completely disappeared in 8 of 9 individuals (response rate: 100%, complete response: 89%, partial response: 11%) (Figures 13-1 and 13-2).

[0326] These results demonstrate that a single infusion of HA nanogel cancer vaccines is powerful enough to cure ICI-resistant tumors, eliminating the need for two infusions of antigen-specific T cells. Currently, the physical and economic burden on patients undergoing T cell infusion therapy (TCR-T or CAR-T) is a problem, and improving this is a challenge. However, a regimen of single cell infusion and multiple doses of this vaccine could potentially cure ICI-resistant tumors and be a treatment with less physical and economic burden on patients.

[0327] Example 14. Therapeutic Effect of Vaccine, etc. on Individuals Showing a Change from Response to Regrowth After Treatment

[0111] For one of nine mice in which tumor regrowth was observed after the treatment in Example 13, 75 μg of a long-chain peptide antigen-loaded HA nanogel cancer vaccine was administered subcutaneously to the right dorsal region of the mouse along with 50 μg of CpG oligoDNA dissolved in PBS on days 26, 30, 33, and 36 after tumor inoculation. Furthermore, ICI (200 μg of anti-mouse PD-1 antibody and 100 μg of anti-mouse CTLA-4 antibody) was administered intraperitoneally on days 33, 36, and 39 after tumor inoculation, and tumor area was measured over time.

[0328] Example 14 Results: Vaccine and ICI were administered to individuals who had initially responded to a single HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, but subsequently experienced tumor regrowth. The results demonstrated that while recurrent tumors generally grow rapidly and have a poor prognosis, repeated administration of the vaccine and combined administration of an ICI significantly suppressed tumor growth (Figure 14).

[0329] Based on the above results, even if treatment was initially successful after administration of the HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, but tumor regrowth was subsequently observed, repeated administration of this vaccine, reinfusion, and additional ICI therapy can significantly suppress tumor growth and may be an effective treatment for tumor relapse.

[0330] Example 15. Memory T cell induction effect by vaccine re-administration in individuals who achieved a response (complete regression) after treatment. Peripheral blood from individuals (n = 8) who showed complete tumor regression after treatment in Example 13 was collected using the same method as in Example 5 (day 36 after tumor inoculation), and the memory phenotype of CD8+ T cells was analyzed. Furthermore, 75 μg of a long-chain peptide antigen-loaded HA nanogel cancer vaccine was subcutaneously administered to the right dorsal region of the same mice (n = 4) together with 50 μg of CpG oligo DNA dissolved in PBS (days 36 and 39 after tumor inoculation), and the memory phenotype of the cells was reanalyzed (day 41 after tumor inoculation). Cell staining and measurement were performed using the same method as in Example 10.

[0331] <Results of Example 15> Analysis of CD8+ T cells present in the peripheral blood of an individual that showed complete tumor regression after administration of an HA nanogel cancer vaccine and antigen-specific T cell infusion therapy (single dose) revealed that the individual contained CD8+ T cells that displayed effector memory and central memory phenotypes, and that the proportion of both types of cells significantly increased after revaccination (Figure 15).

[0332] These results suggest that re-administration of this vaccine after successful treatment with a single HA nanogel cancer vaccine and antigen-specific T cell infusion therapy can significantly increase effector memory and central memory CD8+ T cells, potentially enabling the sustainable induction of memory T cells.

[0333] Example 16. Memory T cell induction effect by vaccine re-administration in individuals whose response changed to regrowth after treatment (additional treatment) Peripheral blood from individuals undergoing additional treatment as described in Example 14 was collected using the same method as in Example 5 (36 days after tumor inoculation), and the memory phenotype of CD8+ T cells was analyzed. Furthermore, 75 μg of a long-chain peptide antigen-loaded HA nanogel cancer vaccine was administered subcutaneously to the right dorsal region of the same mice (on the same day after blood collection) together with 50 μg of CpG oligo DNA dissolved in PBS, and the memory phenotype of the cells was reanalyzed (41 days after tumor inoculation). Cell staining and measurement were performed using the same method as in Example 10.

[0334] <Results of Example 16> Analysis of CD8+ T cells present in the peripheral blood of individuals who had changed from a response to regrowth after receiving a single dose of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy (currently undergoing additional treatment) revealed that, like individuals who had shown complete regression, individuals undergoing additional treatment also contained CD8+ T cells displaying effector memory and central memory phenotypes, and that the proportion of both types of cells significantly increased after revaccination (Figure 16).

[0335] These results suggest that even when tumor regrowth occurs after successful treatment with an HA nanogel cancer vaccine and antigen-specific T cell infusion therapy (single dose), administration of this vaccine can significantly increase and induce effector memory and central memory CD8+ T cells, potentially enabling the sustainable induction of memory T cells and therapeutic effects.

[0336] Discussion of Examples 6 to 16 The results of Examples 6 to 16 revealed or suggested the following eight points in particular: 1. This treatment exerts a strong therapeutic effect on difficult-to-treat malignant melanoma, which is characterized by high metastatic potential, high lethality and rapid lethality, low immunogenicity, and low T cell infiltration into the tumor (cold tumor, immune desert tumor) (Example 6). 2. While the prior art CHP vaccine fails to suppress tumor growth or death in these difficult-to-treat tumors, this treatment exerts a strong antitumor effect, suppressing tumors and maintaining survival (Example 7). 3. This treatment significantly increases antigen-specific CD8 T cells in lymph nodes draining the vaccine administration site and can induce highly activated antigen-specific cytotoxic CD8 T cells (CTL) (Example 8). 4. The infused antigen-specific CD8 T cells infiltrate tumors well and are prominently localized throughout the body, potentially suppressing tumor metastasis (Example 9). 5. In addition to its potent therapeutic effect, this treatment can also prevent recurrence after successful treatment, combining the aspects of both a cancer treatment and a recurrence prevention vaccine (Examples 10 and 12). 6. Furthermore, this treatment can rapidly induce strong and sustainable memory T cells by re-administering the vaccine after successful treatment or during additional treatment (Examples 10 and 15-16). 7. Furthermore, even if tumor regrowth is observed after successful treatment, tumor growth can be suppressed by combining it with repeated vaccine administration or other immunotherapies (Examples 11 and 14). 8. Compared to conventional methods and prior art (two infusion therapies), this treatment exhibits significant therapeutic effects even with a single administration, potentially reducing the physical and economic burden on patients (Example 13).

[0337] Example 17. Antitumor Test Using Lymphocytes Expressing Chimeric Antigen Receptors and an Administration Composition Comprising a Hyaluronic Acid Derivative and an Antigen. The test results of Examples 1 to 16 for administration compositions containing TCR-T, a hyaluronic acid derivative, and an antigen suggest that one of the mechanisms of the high antitumor effect is that antigen-presenting cells incorporating a hyaluronic acid derivative and an antigen efficiently present the antigen on the cell surface, and that these antigen-presenting cells activate and proliferate the infused T cells. A previous report (Blood (2018) 132 (11): 1134-1145, "Antitumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination") reported that the antitumor effect was enhanced by infusing lymphocytes expressing chimeric antigen receptors and antigen-incorporated antigen-presenting cells together. A previous report (Science. 2020 Jan 24;367(6476):446-453, An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors) reported that the antitumor effect was enhanced by injecting lymphocytes expressing chimeric antigen receptors and expressing antigens recognized by the chimeric antigen receptors in antigen-presenting cells. Therefore, when injecting not only TCR-T but also chimeric antigen receptor-expressing lymphocytes, a similarly high antitumor effect can be expected by administering a composition containing a hyaluronic acid derivative that produces antigen-uptaken antigen-presenting cells in the body and the antigen.

[0338] By using the chimeric antigen receptor-expressing lymphocytes, tumors expressing antigens that can be recognized by the chimeric antigen receptor, and HLA transgenic mice shown below, it is possible to demonstrate that lymphocytes expressing the chimeric antigen receptor, and an administered composition containing a hyaluronic acid derivative and an antigen have a high antitumor effect.

[0339] <Materials and Methods> (1) Cells The mouse colon cancer cell lines MC38 and CT26 were purchased from ATCC and passaged at Mie University. The retrovirus packaging cell line Plat-E was purchased from CELL BIOLABS. RPMI1640 medium was purchased from the Cell Science Institute. High-glucose D-MEM medium was purchased from Wako Pure Chemical Industries. Opti-MEM and fetal bovine serum (FBS) were purchased from Gibco. Albumin was purchased from CSL Behling, and human IL-2 was purchased from Novartis.

[0340] (2) Test animals: HLA-A2 transgenic (Tg) mice were generated by injecting HHD-A2 plasmid DNA provided by the Pasteur Institute into fertilized eggs of C57BL / 6 mice at the Mie University Animal Experimental Facility. These mice were bred at the Mie University Animal Experimental Facility and further backcrossed with BALB / c mice to generate HLA-A2 Tg mice (BALB / c background). These were kept at the Mie University Animal Experimental Facility and used in the experiments.

[0341] (3) Antibodies: Anti-mouse CD3e antibody (clone 145-2C11), anti-mouse CD28 antibody (clone 37.51), and Alexa Fluor 647-labeled anti-rabbit IgG antibody (clone Poly4064) were purchased from Invitrogen. APC-labeled anti-mouse CD4 antibody (clone RM4-5) and APC / Cyanine7-labeled anti-mouse CD8 antibody (clone 53-6.7) were purchased from BioLegend. MAGE-A4 (E7O1U) XP Rabbit mAb was purchased from Cell Signaling Technology. PE-labeled anti-human HLA-A2 antibody (clone BB7.2) was purchased from MBL. Purified Rat Anti-Mouse IFN-γ antibody and Biotin Rat Anti-Mouse IFN-γ antibody were purchased from BD Biosciences.

[0342] (4) Reagents: Lipofectamine 3000 Reagent, P300 reagent, and human IFN-γ uncoated ELISA kit were purchased from Invitrogen. pCL-ECO Retrovirus Packaging Vector was purchased from NOVUS. A2 MAGE-A4 tetramer was prepared using Streptavidin-R-Phycoerithrin (Agilent). RetroNectin was purchased from Takara Bio.

[0343] 1. Construction of Retroviral Vector Plasmids 1-1 For Chimeric Antigen Receptors (1) mMAGE#17 zG CAR The MAGE#17scFv used in CAR construction was isolated by reacting a human antibody library with an artificially constructed HLA-A*02:01 MAGE-A4 p230-239 peptide-MHC complex (Patent Application No. 2019-523909 and others / Antigen-binding protein that recognizes MAGE-A4-derived peptides). A human VH leader sequence was linked to the 5' end of the scFv, and mCD8 hinge, mCD28 TM, mCD28 ICD, mCD3z, and mGITR ICD were linked to the 3' end, creating an artificial gene (ordered by Genscript). This gene was inserted into a retroviral expression plasmid to create the mMAGE zG viral vector plasmid (Figures 17-1a and 17-1b).

[0344] (2) An artificial gene was constructed by linking a human VH leader sequence to the 5' end of mMAGE 28z CAR scFv and mCD8 hinge, mCD28 TM, mCD28 ICD, and mCD3z to the 3' end (ordered by Genscript). This gene was then inserted into a retroviral expression plasmid to generate the mMAGE zG viral vector plasmid (Figures 17-2a and 17-2b).

[0345] 1-2 For MAGE-A4 and HHD-A2 expressing MC38 cells and CT26 cells (1) mNGFR co-expressing MAGE-A4 An artificial gene was created by linking the human MAGE-A4 gene with the P2A peptide sequence and a C-terminal truncated mouse NGFR gene (ordered from Genscript). This gene was excised by digestion with the restriction enzymes NotI and XhoI and inserted into a retroviral expression plasmid (Figure 17-3a and Figure 17-3b).

[0346] (2) HHD-A2: The intron portion was deleted from the MHC sequence of the HHD-A2 plasmid, and an mRNA-type HHD-A2 expression gene was designed to create an artificial gene (ordered by Genscript). This gene was excised with NotI-XhoI and inserted into a retroviral expression plasmid (Figure 17-4a and Figure 17-4b).

[0347] 2. Generation of chimeric antigen receptor-expressing lymphocytes 2-1. Generation of retroviruses Retrovirus packaging cell line Plat-E (1.2 × 10 6 (100 cells / ml) were cultured in high-glucose D-MEM medium in a 6-well plate for 24 hours and mixed with Opti-MEM (250 μL) and Lipofectamine 3000 Reagent (7 μL) (Solution A). Opti-MEM (250 μL), the mMAGE#17 zG CAR viral vector plasmid (prepared in 1-1(1)) (3 μL), pCL-ECO Retrovirus Packaging Vector (1 μL), and P300 reagent (6 μL) were then mixed with Solution A and allowed to stand at room temperature for 15 minutes. Half of the medium was removed from the 6-well plate, and the mixture was added. The plate was then cultured for 6 hours in a 37°C, 5% CO2 incubator. The medium was then completely replaced, and the medium was collected after 24, 48, and 72 hours, sterilized by filtration through a 0.45 μm filter, and cryopreserved.

[0348] 2-2 Retroviral gene transfer into mouse spleen cells. Spleens were collected from BALB / c mice and crushed using a slide glass. The cells were then collected in RPMI1640 medium. After centrifugation (400 × g, 5 min, 4°C), the supernatant was removed, and 2 mL of red blood cell lysis solution was added and the cells were treated for 30 seconds. 18 mL of RPMI1640 medium was then added, followed by centrifugation (400 × g, 5 min, 4°C), the supernatant was removed, and 3 × 10 cells were collected. 6 The cells were suspended in RPMI1640 medium containing 10% FBS at a concentration of 1.5 × 10 cells / mL. Anti-mouse CD3e antibody (1 μg / mL) and anti-mouse CD28 antibody (2.5 μg / mL) were added to a 6-well plate the day before and left to stand overnight at 4°C. The plate was washed three times with PBS and then added with 5 mL of the cell suspension (1.5 × 10 cells). 7 The cells were seeded and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0349] The day before gene transfection, RetroNectin was adjusted to 20 μg / mL with PBS, and 500 μL was added to each well of a 24-well plate (Non-Treated Multidishes, Nunc) and left to stand overnight at 4°C. The next day, after washing three times with PBS, the virus solution prepared in 2-1 was diluted two-fold, and 1 mL was added to the plate and centrifuged (2000 × g, 2 hours, 32°C). The plate was washed twice with PBS containing 1.5% albumin, and mouse spleen cells were collected from the CO2 incubator and 3.3 × 10 5 The cells were suspended in RPMI1640 containing 10% FBS and human IL-2 (600 IU / μL) at a concentration of 1 / mL, and 1.5 mL of the cells were seeded onto a plate. After centrifugation (1000 × g, 10 minutes, 32°C), the cells were cultured in a 37°C, 5% CO2 incubator for 5 days.

[0350] 2-3 Confirmation of chimeric antigen receptor-expressing lymphocytes. Cells were harvested and transferred to a 96-well V-bottom microplate (Nunc). After centrifugation (2000 rpm, 2 minutes, 4°C), the supernatant was removed and the cells were suspended in 200 μL of staining buffer (PBS containing 0.5% BSA) per well. After washing the cells twice with 200 μL of staining buffer, 30 μL of 100-fold diluted A2 MAGE-A4 tetramer was added and incubated in the dark for 30 minutes. After washing the cells twice with staining buffer, APC-labeled anti-mouse CD4 antibody and APC / Cyanine7-labeled anti-mouse CD8 antibody were added to the cell suspension at the concentrations recommended by the antibody manufacturers, mixed, and incubated in the dark at 4°C for 15 minutes. After adding 150 μL of staining buffer and centrifuging to remove the supernatant, the cells were washed twice with 200 μL of staining buffer, resuspended in 200 μL of buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a flow cytometer LSRFortessa Cell Analyzer (BD Biosciences) and the accompanying analysis software (FACSDiva) (Figure 17-5).

[0351] 3. Preparation of target cells (MAGE-A4- and HHD-A2-expressing MC38 cells and CT26 cells) expressing antigens that can be recognized by chimeric antigen receptors, and confirmation of recognition and reactivity 3-1 Preparation of retroviruses Retroviruses were prepared in the same manner as described in 2-1, except that the mNGFR-coexpressing MAGE-A4 viral vector plasmid prepared in 1-2(1) or the HHD-A2 viral vector plasmid prepared in 1-2(2) was used instead of the mMAGE#17 zG CAR viral vector plasmid prepared in 1-1(1).

[0352] 3-2 Preparation of MAGEA4- and HHD-A2-Expressing MC-38 and CT26 Cells. Mouse colon cancer cell lines, MC38 and CT26, were cultured in RPMI1640 medium containing 10% FBS using T75 culture flasks (Corning). The cultured cells were detached using PBS containing 0.5% trypsin and suspended in RPMI1640 medium containing 10% FBS. The suspension was centrifuged (400 × g, 5 minutes, 4°C), and the supernatant was removed. Subsequently, the cells were washed twice with RPMI1640 medium containing 10% FBS, and 3.3 × 10 5 The cells were suspended in RPMI1640 medium containing 10% FBS at a concentration of 10 cells / mL.

[0353] The day before transfection, RetroNectin was adjusted to 20 μg / mL with PBS, and 500 μL was added to each well of a 24-well plate (Non-Treated Multidishes, Nunc) and left to stand overnight at 4°C. The next day, after washing three times with PBS, each virus solution prepared in 3-1 was diluted two-fold, and 1 mL was added to the plate and centrifuged (2000 × g, 2 hours, 32°C). The plate was washed twice with PBS containing 1.5% albumin, and MC38 cells (3.3 × 10 5 After centrifugation (1000 × g, 10 minutes, 32°C), the cells were cultured at 37°C in a 5% CO2 incubator for 3 days (the same procedure was used for the CT26 cell line).

[0354] Both cell lines were harvested and transferred to a 96-well V-bottom microplate (Nunc). After centrifugation (2000 × rpm, 2 minutes, 4°C), the supernatant was removed, and the cells were suspended in 200 μL of staining buffer per well. After washing the cells twice with 200 μL of staining buffer, staining and expression of HHD-A2 and MAGE-A4 were confirmed as follows. HHD-A2: PE-labeled anti-HLA-A2 antibody was added to the cell suspension at the manufacturer's recommended concentration and incubated in the dark at 4°C for 15 minutes. After adding 150 μL of staining buffer and centrifuging to remove the supernatant, the cells were washed twice with 200 μL of staining buffer, resuspended in 200 μL of buffer, and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a flow cytometer (FACS LSR Fortessa Cell Analyzer) (BD Biosciences) and the accompanying analysis software (FACSDiva) (Figure 17-6).

[0355] MAGE A4 intracellular staining: 1 x 10 5 The cells were adjusted to individual cells and mixed thoroughly with 100 μL of cell fixative (Cytofix Cytoperm: BD Biosciences) and left to stand for 20 minutes in the dark at room temperature. After washing with Perm / Wash buffer (BD Biosciences) diluted 1:10 with PBS, 0.25 μL of MAGE-A4 (E7O1U) XP Rabbit mAb was added to 50 μL of Perm / Wash buffer and left to stand for 20 minutes in the dark at room temperature. After washing twice with Perm / Wash buffer, 0.25 μL of Alexa Fluor 647-labeled anti-rabbit IgG antibody was added to 50 μL of Perm / Wash buffer and mixed, and left to stand for 20 minutes in the dark at room temperature. After washing twice with Perm / Wash buffer, the cells were suspended in staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). The cells were analyzed using a flow cytometer LSRFortessa Cell Analyzer (BD Biosciences) and the accompanying analysis software (FACSDiva) (Figure 17-6).

[0356] 3-3 Confirmation of recognition and reactivity of mMAGE zG CAR T cells to target cells The target cells prepared in 3-2 (1. MC38 cells, 2. HHD-A2-transfected MC38 cells, 3. HHD-A2-transfected MC38 cells (pulsed with 10 μM of MAGE-A4 CD8 epitope peptide (SEQ ID NO: 9)), 4. HHD-A2- and MAGE-A4-transfected MC38 cells) were diluted to 5 × 10 4 The cells were suspended in RPMI1640 medium containing 10% FBS at a concentration of 1 × 10 cells / 100 μl, and mMAGE zG CAR T cells (1 × 10 cells) also suspended in RPMI1640 medium containing 10% FBS were added. 5 CAR T cells (mMAGE zG cells / 100 μl) or non-CAR gene-transfected cells were added to the plate and co-cultured for 24 hours, after which the culture supernatant was collected. CT26 cells were also co-cultured with mMAGE zG CAR T cells, and the culture supernatant was collected. The collected culture supernatant was evaluated for IFN-γ production by the ELISA method described below.

[0357] IFN-γ ELISA: 50 μL of purified rat anti-mouse IFN-γ antibody, adjusted to 10 μg / mL in 0.05% NaN3 PBS, was added to a 96-well flat-bottom plate and incubated overnight at 4°C for immobilization. The next day, the plate was washed three times with PBS, and 50 μL of the recovered cell culture supernatant and standard solution were added to each well. The plate was incubated at room temperature for 2 hours. IFN-γ standard solution was prepared by adjusting the stock solution to 1000 pg / mL with standard solution and then diluting it five-fold in four steps. After washing three times with PBS, 100 μL of biotin rat anti-mouse IFN-γ antibody, adjusted to 0.5 μL / mL in ELISA buffer prepared by diluting 5x ELISA / Elispot Diluent (ELISA kit) five-fold with sterile water, was added and incubated at room temperature for 1 hour. After washing three times with PBS, 100 μL of streptoavidin-HRP adjusted to 0.5 μL / mL in ELISA buffer was added and incubated at room temperature for 30 minutes. After washing three times with 0.05% PBS-T and twice with PBS, 100 μL of TMB substrate solution was added and incubated for 2 minutes at room temperature in the dark. The reaction was stopped by adding 100 μL of 0.18 M H2SO4. The absorbance was immediately measured at 450 nm using a microplate reader Model 680 (Bio-Rad) (Figure 17-7).

[0358] 4. Preparation of HA nanogel cancer vaccine loaded with long-chain peptide antigen 4-1 Preparation of a complex using HA nanogel with a molecular weight of 10 kDa and the peptide MAGE-A4 29mer Hyaluronic acid nanogel (HANG) Hyaluronic acid nanogel with introduced cholesteryl groups (weight-average molecular weight of 10 kDa, introduction rate of cholesteryl groups: 44%) was synthesized as described in Examples 1 and 2 of WO 2010 / 053140.

[0359] Long-chain peptide antigen: Using the hyaluronic acid nanogel obtained above, a peptide-hyaluronic acid nanogel complex was prepared as follows. MAGE-A4 peptide was purchased from Eurofins. Its sequence is as follows: AMEGDSASEEEIWEELGVMGVYDGREHTV (SEQ ID NO: 8). This sequence contains GVYDGREHTV (SEQ ID NO: 9) as an epitope recognized by CD8-positive cytotoxic T cells.

[0360] Preparation of Hyaluronic Acid Nanogel and Antigen Complex: The lyophilized hyaluronic acid nanogel was weighed, and water for injection was added to a concentration of 5 mg / mL. The mixture was stirred overnight to allow complete dissolution. Meanwhile, in a separate container, the peptide was dissolved in dimethyl sulfoxide to a concentration of 50 mg / mL. After confirming dissolution, the hyaluronic acid nanogel aqueous solution was mixed with 1 mol / L sodium hydroxide aqueous solution at a volume ratio of 100:2 at room temperature. The hyaluronic acid nanogel aqueous solution was then mixed with a peptide dimethyl sulfoxide solution at a volume ratio of 102:1 and stirred at room temperature for 24 hours to obtain a composition containing a peptide-hyaluronic acid nanogel complex. Solid purified sucrose (Fujifilm Wako Co., Ltd., product number 198-18385) was then added to the composition containing the peptide-hyaluronic acid nanogel complex to a concentration of 10% sucrose by mass, and the mixture was stirred for at least 1 hour. Subsequently, the solution was diluted with 25 mM phosphate buffer (pH 7.4) containing 10% by mass sucrose to a theoretical peptide concentration of 0.3 mg / mL, and sterilized through 0.2 μm PES (polyethersulfone) (Pall, Acrodisc syringe filter, 25 mm diameter) to obtain a composition containing a peptide-hyaluronic acid nanogel complex. The peptide concentration in the composition containing the peptide-hyaluronic acid nanogel complex was quantified by reverse phase chromatography analysis, and the peptide concentration was adjusted to 250 μg / mL to prepare an administration solution.

[0361] 4-2 Preparation of a complex using 10kJ HA nanogel and MAGE-A4 20mer peptide The method described in 4-1 above was used to prepare the complex, except that the long-chain peptide antigen shown below was used. The peptide concentration was adjusted to 250 μg / mL to prepare the administration solution. Long-chain peptide antigen MAGE-A4 20mer MAGE-A4 20mer was purchased from Eurofins. Its sequence is as follows: GVYDGREHTVGVYDGREHTV (SEQ ID NO: 10). This sequence contains the following MAGEA4 (amino acid sequence: GVYDGREHTV (SEQ ID NO: 9)) as the CD8-positive cytotoxic T cell-recognized epitope sequence.

[0362] 5. Antitumor test 5 × 10 MC38 cells expressing MAGE-A4 / HHD-A2 prepared in 3-2 were injected into the right anterior dorsal side of HLA-A2 Tg mice. 6 On days 5 and 10 after tumor inoculation, the chimeric antigen receptor-expressing lymphocytes prepared in 2-2 were subcutaneously inoculated into the mice at a concentration of 3 × 10 cells / mouse. 6 After suspending the vaccine at a concentration of 50 μg / 200 μL, 200 μL is infused into the tail vein of the mouse. On days 4 and 9 (and 14) after tumor implantation, 50 μg of the long-chain peptide antigen-loaded HA nanogel cancer vaccine prepared in 4-1 and 4-2 is subcutaneously administered to the right dorsal region of the mouse along with 50 μg of CpG oligo DNA dissolved in PBS. The tumor area is then measured over time.

Claims

1. A pharmaceutical composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen, for use in combination administration with lymphocytes expressing an immune receptor for the antigen.

2. The pharmaceutical composition of claim 1 , wherein the hydrophobic group is a steryl group.

3. The pharmaceutical composition according to claim 1 or 2, wherein the hyaluronic acid derivative and the antigen form a complex.

4. The pharmaceutical composition according to claim 1 or 2, wherein the antigen is a cancer antigen.

5. The pharmaceutical composition according to claim 1 or 2, wherein the antigen is an antigenic peptide or an antigenic protein.

6. The pharmaceutical composition according to claim 5 , wherein the antigen comprises an epitope recognized by CD8-positive cytotoxic T cells and / or an epitope recognized by CD4-positive helper T cells.

7. The pharmaceutical composition according to claim 1 or 2, wherein the immune receptor is a T cell receptor or a chimeric antigen receptor.

8. 3. The pharmaceutical composition according to claim 1 or 2, further comprising an adjuvant or for use in combination with an adjuvant.

9. The pharmaceutical composition according to claim 1 or 2, which is for the prevention or treatment of cancer.

10. A pharmaceutical composition for use in combination administration of a hyaluronic acid derivative having a hydrophobic group introduced therein, the hyaluronic acid derivative containing lymphocytes expressing an immune receptor for an antigen, and a composition containing the antigen.

11. A cancer treatment kit comprising a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, and lymphocytes expressing an immune receptor for the cancer antigen.

12. A T cell activating vaccine comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and an antigen.

13. A cancer treatment kit comprising T cells expressing an immune receptor for a cancer antigen and a pharmaceutical composition, The pharmaceutical composition comprises a hyaluronic acid derivative having a hydrophobic group introduced therein, an antigen peptide or an antigen protein derived from the cancer antigen, and an adjuvant; The hyaluronic acid derivative has the formula (I): [In the formula, R 1 , R 2 , R 3 , and R 4 are each independently a hydrogen atom, C 1－6 Alkyl, formyl and C 1－6 alkylcarbonyl; R 5 is a hydrogen atom, formyl, or C 1－6 is alkylcarbonyl; Z represents a direct bond or a peptide linker of any length from 2 to 30 amino acid residues; 1 is the following formula: -NO b -R、 -NR b -COO-R、 -NR b -CO-R、 -NO b -CO-NR c -R、 -COO-R, -O-COO-R, -S-R, -WHAT-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 group represented by R a , R b and R c are each independently a hydrogen atom, C 1－20 Alkyl, Amino C 2－20 Alkyl and Hydroxy C 2－20 alkyl, where the alkyl portion of the group is selected from -O- and -NR f - may optionally 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 Optionally, 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 may be interrupted by 1 to 3 groups selected from -O- and -NH-; Y a is C 1－5 is alkylene; Y b is C 2－8 Alkylene or C 2－8 alkenylene; m is an integer selected from 1 to 100. A cancer treatment kit comprising one or more repeating units represented by the formula:

14. The cancer treatment kit according to claim 13, wherein the modification rate of the carboxyl groups of the glucuronic acid portion of the hyaluronic acid derivative with hydrophobic groups is 5 to 50%.

15. Z is a direct bond and Y is C 2－12 is alkylene, and X 1 The cancer treatment kit of claim 14, wherein is -NH-COO-R and R is a cholesteryl group.

16. The cancer treatment kit according to claim 13, wherein the weight average molecular weight of the hyaluronic acid derivative is 5,000 to 2,000,000.

17. The hyaluronic acid derivative has the formula (IIIc): [In the formula, R 1c , R 2c , R 3c and R 4c are each independently a hydrogen atom, C 1－6 Alkyl, formyl and C 1－6 alkylcarbonyl; R 5c is a hydrogen atom, formyl and C 1－6 alkylcarbonyl; c is hydroxy and -O-Q + are selected from, where Q + represents a counter cation. The cancer treatment kit of claim 13, comprising a repeating unit represented by:

18. The cancer treatment kit according to claim 13 , wherein the pharmaceutical composition contains a complex of the cancer antigen and the hyaluronic acid derivative.

19. The cancer treatment kit according to claim 13, wherein the antigen peptide comprises two or more epitopes recognizing CD8-positive cytotoxic T cells and / or CD4-positive helper T cells.

20. The cancer treatment kit according to claim 19, wherein the antigen peptide is a long-chain peptide antigen comprising two or more epitopes recognized by CD8-positive cytotoxic T cells, or one or more epitopes recognized by CD8-positive cytotoxic T cells and one or more epitopes recognized by CD4-positive helper T cells.

21. The cancer treatment kit according to claim 13, which is used in T cell infusion therapy for a cancer with low immunogenicity, a metastatic cancer, or a cancer with a low number of intratumoral T cells.

22. The cancer treatment kit of claim 13 , wherein the pharmaceutical composition is administered prior to administration of the T cells.

23. The cancer treatment kit according to claim 22, wherein the pharmaceutical composition is administered at least once after administration of 1 unit or 2 units, where the unit of administration is one administration of the pharmaceutical composition followed by one infusion of the T cells.

24. 23. The cancer treatment kit of claim 22, wherein the pharmaceutical composition is administered subcutaneously or intravenously at least twice, and the T cells are infused after a first administration of the pharmaceutical composition and before a second administration of the pharmaceutical composition.

25. A method for treating cancer, comprising administering to a subject having cancer a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, in combination with lymphocytes expressing an immune receptor for the cancer antigen.

26. A cancer treatment kit for use in cancer treatment, comprising a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, and lymphocytes expressing an immune receptor for the cancer antigen.

27. A pharmaceutical composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, for use in cancer treatment involving administration of lymphocytes expressing an immune receptor for a cancer antigen.

28. A pharmaceutical composition comprising lymphocytes expressing an immune receptor for a cancer antigen, for use in cancer treatment comprising administering a pharmaceutical composition comprising a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen.

29. Use of a combination of a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen, and lymphocytes expressing an immune receptor for the cancer antigen, for the manufacture of a cancer treatment kit.

30. Use of a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen for the manufacture of a cancer therapeutic agent to be administered in combination with lymphocytes expressing an immune receptor for a cancer antigen.

31. Use of lymphocytes expressing an immune receptor for a cancer antigen for the manufacture of a cancer therapeutic agent to be administered in combination with a pharmaceutical composition containing a hyaluronic acid derivative having a hydrophobic group introduced therein and a cancer antigen.

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