WO2022255384A1 - 医薬組成物 - Google Patents
医薬組成物 Download PDFInfo
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- WO2022255384A1 WO2022255384A1 PCT/JP2022/022211 JP2022022211W WO2022255384A1 WO 2022255384 A1 WO2022255384 A1 WO 2022255384A1 JP 2022022211 W JP2022022211 W JP 2022022211W WO 2022255384 A1 WO2022255384 A1 WO 2022255384A1
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Definitions
- the present invention relates to pharmaceutical compositions and the like.
- cancer vaccines are often administered subcutaneously, intradermally, or intramuscularly. It is important to deliver well and create an environment in which antigen-presenting cells can present antigens to T cells in lymph nodes, and it is thought that this will lead to improved therapeutic effects.
- Many of the proteins, peptides, and nucleic acids used in cancer vaccines generally have low stability, and have the property of being easily decomposed and deactivated when administered into the body. Therefore, the development of a drug delivery system (DDS) that can stably deliver the vaccine to the target site and release it in a controlled manner is now required.
- DDS drug delivery system
- a hyaluronic acid derivative with a hydrophobic group introduced self-associates to form a complex, typically a nanoparticle-like nanogel.
- This complex enables easy embedding and encapsulation of proteins, peptides, and sparingly soluble low-molecular-weight compounds due to the cross-linked three-dimensional network structure and hydrophobic interaction, and is stably embedded in the target site. It is expected to be applied to new DDS by delivering and sustaining substances.
- Patent Document 1 reports that when a cancer vaccine of the above complex was prepared and subcutaneously administered to mice, it rapidly and highly efficiently migrated to nearby lymph nodes and induced an antitumor effect.
- An object of the present invention is to provide a pharmaceutical composition capable of exhibiting a stronger, more efficient, more sustained, and/or broader antitumor effect.
- the present inventors combined a composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen with lymphocytes expressing immunoreceptors for the antigen. It has been found that the above problems can be solved by using The present inventor has completed the present invention as a result of intensive research based on this knowledge. That is, the present invention includes the following aspects.
- Item 1 A pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen, for use in combination administration with lymphocytes expressing immunoreceptors for the antigen.
- Item 5 Items 1 to 4, wherein the antigen is an antigen peptide or antigen protein.
- Item 6 wherein the antigen comprises a CD8-positive cytotoxic T-cell recognition epitope and/or a CD4-positive helper T-cell recognition epitope.
- Item 7 Items 1 to 6, wherein the immune receptor is a T cell receptor or a chimeric antigen receptor.
- Item 8 The pharmaceutical composition according to any one of Items 1 to 7, further containing an adjuvant or for use in combination administration with an adjuvant.
- Item 9 The pharmaceutical composition according to any one of Items 1 to 8, which is for prevention or treatment of cancer.
- Item 10 A pharmaceutical composition for combined administration with a composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced, which contains lymphocytes expressing immunoreceptors for an antigen, and the antigen.
- a cancer treatment kit comprising a pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen, and lymphocytes expressing immunoreceptors for the cancer antigen.
- T-cell activating vaccine containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen.
- a cancer treatment kit comprising T cells expressing immunoreceptors for cancer antigens and a pharmaceutical composition, wherein the pharmaceutical composition comprises a hyaluronic acid derivative having a hydrophobic group introduced therein, and the cancer antigen and an adjuvant, wherein the hyaluronic acid derivative has the formula (I): [wherein R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; R 5 is a hydrogen atom, formyl, or C 1-6 alkylcarbonyl; Z represents a direct bond or a peptide linker consisting of any 2-30 amino acid residues; X 1 has the following formula: -NR b -R, -NRb -COO-R, -NRb -CO-R, -NRb -CO- NRc -R, -COO-R, -O-COO-R, -SR,
- Item 14 wherein the modification rate of the carboxyl group of the glucuronic acid portion of the hyaluronic acid derivative with a hydrophobic group is 5 to 50%.
- Item 15 The cancer treatment kit according to Item 14, wherein Z is a direct bond, Y is C 2-12 alkylene, X 1 is -NH-COO-R, and R is a cholesteryl group.
- Item 16 Item 13, wherein the hyaluronic acid derivative has a weight average molecular weight of 5,000 to 2,000,000.
- the hyaluronic acid derivative has the formula (IIIc): [wherein R 1c , R 2c , R 3c and R 4c are each independently selected from a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; R 5c is selected from hydrogen atom, formyl and C 1-6 alkylcarbonyl; X c is selected from hydroxy and —O—Q + where Q + represents a counter cation] Item 14.
- Item 18 wherein the pharmaceutical composition contains a complex of the cancer antigen and the hyaluronic acid derivative.
- Item 19 wherein the antigen peptide comprises two or more CD8-positive cytotoxic T-cell recognition epitopes and/or CD4-positive helper T-cell recognition epitopes.
- the antigenic peptide is a long-chain peptide comprising two or more CD8-positive cytotoxic T-cell recognition epitopes, or one or more CD8-positive cytotoxic T-cell recognition epitopes and one or more CD4-positive helper T-cell recognition epitopes.
- Item 20. The cancer treatment kit according to Item 19, which is an antigen.
- Item 21 The cancer treatment kit according to Item 13, which is used for T cell infusion therapy for low immunogenic cancer, metastatic cancer, or cancer with a small number of intratumoral T cells.
- Item 22 Item 13, wherein the pharmaceutical composition is administered before administering the T cells.
- Item 23 When one administration of the pharmaceutical composition followed by one infusion of the T cells is taken as the unit of administration, after administration of one unit or two units, the pharmaceutical composition is administered at least once. 23.
- Item 25 A pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen, and a lymphocyte expressing an immune receptor for a cancer antigen are administered in combination to a subject with cancer.
- methods of treating cancer including;
- the cancer treatment kit for use in cancer treatment comprises a pharmaceutical composition containing a hyaluronic acid derivative to which a hydrophobic group has been introduced and a cancer antigen, and an immunoreceptor for the cancer antigen.
- a cancer treatment kit comprising lymphocytes.
- Item 27 A pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen for use in cancer therapy in which lymphocytes expressing immunoreceptors for cancer antigens are administered.
- Item 28 A pharmaceutical composition containing lymphocytes expressing immunoreceptors to cancer antigens for use in cancer treatment by administering a pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen thing.
- Item 29 A combination of a pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen, and a lymphocyte expressing an immunoreceptor for the cancer antigen, for the production of a cancer treatment kit Use of.
- Item 30 A pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen for the production of a therapeutic agent for cancer to be administered in combination with lymphocytes expressing immunoreceptors for cancer antigens. use.
- Lymphocytes expressing immunoreceptors to cancer antigens for the production of cancer therapeutic agents to be administered in combination with a pharmaceutical composition containing a hyaluronic acid derivative into which a hydrophobic group has been introduced and a cancer antigen. use.
- HANG-V group For CMS5a tumors subcutaneously implanted in BALB/c mice, administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), antigen-specific T cell infusion therapy (TCR- T group) and combined treatment (HANG-V+TCR-T group) are graphs showing the therapeutic effect (tumor growth inhibition) (Example 1). The plot represents the mean tumor area of individuals in each group.
- HANG-V group For CMS5a tumors subcutaneously implanted in BALB/c mice, administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), antigen-specific T cell infusion therapy (TCR- T group) and combined treatment (HANG-V+TCR-T group) are graphs showing the therapeutic effect (tumor growth inhibition) (Example 1). Plots represent measurements of tumor area for individuals in each group. CMS5a tumors subcutaneously implanted in BALB/c mice were treated with long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells. FIG.
- WT is a photographic diagram showing the effect of suppressing tumor recurrence (suppression of tumor growth) when reimplanting (CR-Re).
- WT is a photographic diagram showing the size of tumors when the same tumors were transplanted into wild-type BALB/c mice (WT) at the same time (Example 1).
- HANG-V group untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA
- TCR- T group antigen-specific T cell infusion therapy
- HANG-V+TCR-T group both treatments
- HANG-V group untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA
- TCR- T group antigen-specific T cell infusion therapy
- HANG-V + TCR-T group combined treatment
- FIG. 2 a photographic diagram showing regional lymph nodes (inguinal lymph nodes) of the vaccine administration site and tumor size
- HANG-V group For CMS5a tumors subcutaneously implanted in BALB/c mice, administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), antigen-specific T cell infusion therapy (TCR- T group), combined treatment (HANG-V + TCR-T group), a graph showing the ratio of dendritic cells or macrophages in the regional lymph node (inguinal lymph node) cells of the vaccine administration site (Example 2) ). Also, it is a graph showing the ratio of CD207-positive Langerhans cells to all CD11c-positive cells.
- Regional lymph nodes at the site of vaccination in BALB/c mice subcutaneously implanted with CMS5a tumors after administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group) (Inguinal lymph node) is a graph showing the number of T cells or B cells, NK cells, dendritic cells, and macrophages in cells (Example 2).
- HANG-V group untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA
- TCR- T group antigen-specific T cell infusion therapy
- HANG-V + TCR-T group combined treatment showing the ratio of CD90.1-positive transfused cells to all CD8-positive T cells in regional lymph node (inguinal lymph node) cells at the vaccine administration site It is a graph (Example 2).
- FIG. 2 is a graph showing the ratio of interferon gamma (IFN- ⁇ )-producing cells to T cells (Example 2).
- CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group Therapeutic effect ( tumor (Example 3).
- the plot represents the mean tumor area of individuals in each group.
- CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group Therapeutic effect ( tumor (Example 3).
- Plots represent measurements of tumor area for individuals in each group.
- HANG-V group CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group
- infusion therapy TCR-T
- different cell numbers 2 ⁇ 10 6 , 1 ⁇ 10 6 , 5 ⁇ 10 5 , 2.5 ⁇ 10 5
- HANG-V + TCR-T group 3 is a graph showing the number of cells in regional lymph nodes (inguinal lymph nodes) at vaccine administration sites (Example 3).
- HANG-V group CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group
- infusion therapy TCR-T
- different cell numbers 2 ⁇ 10 6 , 1 ⁇ 10 6 , 5 ⁇ 10 5 , 2.5 ⁇ 10 5
- HANG-V + TCR-T group 3 is a graph showing the percentage of dendritic cells or macrophages in the cells of the regional lymph nodes (inguinal lymph nodes) at the vaccine administration site (Example 3).
- FIG. 1 is a graph showing the ratio of CD207-positive Langerhans cells to all CD11c-positive cells.
- CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group
- infusion therapy TCR-T
- different cell numbers 2 ⁇ 10 6 , 1 ⁇ 10 6 , 5 ⁇ 10 5 , 2.5 ⁇ 10 5
- HANG-V + TCR-T group 3 is a graph showing the ratio of CD90.1-positive cells or CD90.2-negative cells to all CD8-positive T cells in regional lymph node (inguinal lymph node) cells at the vaccine administration site (Example 3).
- HANG-V group CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group
- infusion therapy TCR-T
- different cell numbers 2 ⁇ 10 6 , 1 ⁇ 10 6 , 5 ⁇ 10 5 , 2.5 ⁇ 10 5
- HANG-V + TCR-T group A graph showing the ratio of interferon-gamma (IFN- ⁇ )-producing cells to all CD90.1-positive CD8-positive T cells or CD90.2-negative CD8-positive T cells in regional lymph node (inguinal lymph node) cells at the vaccine administration site.
- Example 3 CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group
- infusion therapy TCR-T
- different cell numbers 2 ⁇ 10 6 , 1 ⁇ 10 6 , 5 ⁇ 10 5 , 2.5 ⁇ 10 5
- HANG-V + TCR-T group A graph showing the ratio of interferon gamma (IFN- ⁇ )-producing cells to all CD90.2-positive CD8-positive T cells or CD90.1-negative CD8-positive T cells in regional lymph node (inguinal lymph node) cells at the vaccine administration site.
- IFN- ⁇ interferon gamma
- Example 3 For CMS5a tumors subcutaneously implanted in BALB/c mice, administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), antigen-specific T cell infusion therapy (TCR- T group) and combined treatment (HANG-V+TCR-T group), showing micrographs of intratumoral CD8-positive T cells (Example 4).
- HANG-V group CMS5a tumors subcutaneously implanted in BALB/c mice were treated with untreated or long peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group), antigen-specific T cells in HANG-V group
- infusion therapy TCR-T
- different cell numbers 2 ⁇ 10 6 , 1 ⁇ 10 6 , 5 ⁇ 10 5 , 2.5 ⁇ 10 5
- HANG-V + TCR-T group 3 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 (Example 5).
- HANG-V group For B16F10 tumors subcutaneously implanted in C57BL/6 mice, administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), antigen-specific T cell infusion therapy (TCR- T group) and combined treatment (HANG-V+TCR-T group) are graphs showing the therapeutic effect (tumor growth inhibition) (Example 6). The plot represents the mean tumor area of individuals in each group.
- HANG-V group untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA
- TCR- T group antigen-specific T cell infusion therapy
- HANG-V+TCR-T group combined treatment
- HANG-V group Long-chain peptide antigen loaded HA nanogel cancer vaccine and CpG oligo DNA administration (HANG-V group) or antigen-specific T cell infusion therapy (HANG-V + TCR) for B16F10 tumors subcutaneously implanted in C57BL/6 mice -T group) is a photograph showing the size and shape of the tumor (Example 6).
- HANG-V group untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA
- TCR- T group antigen-specific T cell infusion therapy
- HANG-V+TCR-T group combined treatment
- FIG. 10 is a photographic diagram showing a C57BL/6 mouse subcutaneously implanted with a B16F10 tumor that underwent complete regression of the tumor by combined use of a long-chain peptide antigen-loaded cancer vaccine, CpG oligo-DNA, and antigen-specific T cell infusion therapy.
- Antigen-specific T cell infusion therapy by administering untreated or long peptide antigen-loaded cancer vaccine (HANG-V or CHP-V) and CpG oligo DNA to B16F10 tumor subcutaneously implanted in C57BL/6 mice (HANG-V+TCR-T group or CHP-V+TCR-T group), showing the therapeutic effect (tumor growth inhibition) (Example 7).
- the plot represents the mean tumor area of individuals in each group.
- Antigen-specific T cell infusion therapy by administering untreated or long peptide antigen-loaded cancer vaccine (HANG-V or CHP-V) and CpG oligo DNA to B16F10 tumor subcutaneously implanted in C57BL/6 mice (HANG-V+TCR-T group or CHP-V+TCR-T group), showing the therapeutic effect (tumor growth inhibition) (Example 7).
- Plots represent measurements of tumor area for individuals in each group.
- FIG. 10 is an example of a photographic diagram showing the size and shape of tumors in (HANG-V+TCR-T group or CHP-V+TCR-T group) (Example 7).
- Antigen-specific T cell infusion therapy by administering untreated or long peptide antigen-loaded cancer vaccine (HANG-V or CHP-V) and CpG oligo DNA to B16F10 tumor subcutaneously implanted in C57BL/6 mice (HANG-V+TCR-T group or CHP-V+TCR-T group), showing the therapeutic effect (prolongation of survival period) (Example 7).
- Plots represent survival rates of individuals in each group.
- HANG-V group For B16F10 tumors subcutaneously implanted in C57BL/6 mice, administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA (HANG-V group), antigen-specific T cell infusion therapy (TCR- T group) and combined treatment (HANG-V+TCR-T group) are graphs showing the percentage of regional lymph node (inguinal lymph node) cells at the vaccine administration site (Example 8).
- HANG-V group antigen-specific T cell infusion therapy
- HANG-V + TCR-T group antigen-specific T cell infusion therapy
- HANG-V group untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine and CpG oligo DNA
- TCR- T group antigen-specific T cell infusion therapy
- HANG-V + TCR-T group combined treatment
- Fig. 10 is a graph showing the percentage of producing cells (Example 8).
- B16F10 tumors subcutaneously implanted in C57BL/6 mice were treated with long peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells.
- ) is a graph showing the ratio of CD90.1-positive and/or gp100 tetramer (tet)-positive CD8-positive transfused T cells to total CD8-positive T cells in peripheral blood (Example 10).
- Fig. 10 is a graph showing the percentage of positive transfused T cells (Example 10).
- B16F10 tumors implanted subcutaneously in C57BL/6 mice, after administration of long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells, no recurrence after complete tumor regression shows the expression intensity (High/Mid/Low) of CD44-positive cells relative to CD8-positive T cells in the peripheral blood of individuals re-implanted with B16F10 tumors (CR-Re) or vaccinated (CR-Vac).
- FIG. 2 is a photographic diagram showing the recurrence inhibitory effect (tumor growth inhibition) when a C57BL/6 tumor was reimplanted (CR-Re) or re-inoculated with a vaccine (CR-Vac) and then reimplanted with a C57BL/6 tumor.
- WT is a photographic diagram showing the size of tumors when the same tumors were transplanted to wild-type B57BL/6 mice (WT) at the same time (Example 10).
- Therapeutic effect of administration of untreated or long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells to CMS5a tumor subcutaneously implanted in BALB/c mice (tumor growth suppression) (Example 11).
- the plot represents the mean tumor area of individuals in each group.
- BALB/c mice subcutaneously implanted with CMS5a tumors were treated with HA nanogel cancer vaccine loaded with long-chain peptide antigens, CpG oligo-DNA, and antigen-specific T cells.
- a graph (left) compares the therapeutic effect (tumor growth suppression) when re-administration of vaccine, antigen-specific T cells and immune checkpoint inhibitor (ICI) is used in combination with untreated or responding individuals ( Example 11).
- ICI immune checkpoint inhibitor
- a graph ( right) Plots represent measurements of tumor area for individuals in each group.
- BALB/c mice subcutaneously implanted with CMS5a tumor showed complete tumor regression after administration of long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells (CR ), is a graph showing the recurrence inhibitory effect (tumor growth inhibition) when the CMS5a tumor was reimplanted.
- WT is a graph showing tumor growth when the same tumors were transplanted into wild-type BALB/c mice (WT) at the same time (Example 12).
- the plot represents the mean tumor area of individuals in each group.
- BALB/c mice subcutaneously implanted with CMS5a tumor showed complete tumor regression after administration of long-chain peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells (CR ), is a graph showing the recurrence inhibitory effect (tumor growth inhibition) when the CMS5a tumor was reimplanted.
- WT is a graph showing tumor growth when the same tumors were transplanted into wild-type BALB/c mice (WT) at the same time (Example 12).
- Plots represent measurements of tumor area for individuals in each group.
- Therapeutic effect of single administration of untreated or long peptide antigen-loaded HA nanogel cancer vaccine, CpG oligo-DNA, and antigen-specific T cells to CMS5a tumors subcutaneously implanted in BALB/c mice (tumor growth (Example 13).
- the plot represents the mean tumor area of individuals in each group.
- Plots represent measurements of tumor area for individuals in each group.
- tumor re-growth was observed 14 is a graph comparing the therapeutic effect (tumor growth suppression) when , re-administration of vaccine and additional treatment with an immune checkpoint inhibitor (ICI) were performed, with those of untreated or responding individuals (Example 14).
- Plots represent measurements of tumor area for individuals in each group.
- Fig. 15 is a graph showing the percentage of CD62L-positive (central memory T cells) (Example 15).
- CD44-positive CD62L-negative cells effector memory Fig. 10 is a graph showing the ratio of CD44-positive CD62L-positive cells (central memory T cells) (Example 16).
- the sequence of mMAGE#17 zG CAR is shown.
- the sequence 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.
- FIG. 10 is a graph showing the expression ratio of MAGE-A4 gene and HHD-A2 gene when introduced into MC38 and CT26 cells (Example 17).
- Fig. 4 is a graph showing the amount of IFN- ⁇ produced in the culture supernatant when mMAGE#17 zG CAR-introduced T cells or CAR gene-unintroduced cells were added to MC38 and CT26 cells and co-cultured (Example 17). .
- a composition comprising a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen (also referred to herein as an "HA-antigen composition”); combination with lymphocytes expressing immunoreceptors for antigens (herein also referred to as “antigen-specific lymphocytes”).
- an antigen also referred to herein as an "HA-antigen composition”
- lymphocytes expressing immunoreceptors for antigens herein also referred to as “antigen-specific lymphocytes”
- C 1-20 alkyl referred to herein means straight or branched chain alkyl groups having 1 to 20 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl , n-butyl, s-butyl, i-butyl, t-butyl, etc., and furthermore, n - pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethyl propyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl, 2-ethylbutyl and the like.
- C 1-20 alkyl also includes “C 1-12 alkyl” having 1 to 12 carbon atoms and “C 1-6 alkyl” having 1 to 6 carbon atoms.
- C 1-6 alkyl referred to herein means a linear or branched alkyl group having 1 to 6 carbon atoms, for example methyl, ethyl, n-propyl, i-propyl , n-butyl, s-butyl, i-butyl, t-butyl, and other “C 1-4 alkyl”.
- C 1-6 alkylcarbonyl referred to herein means an alkylcarbonyl group wherein the alkyl portion is C 1-6 alkyl as already mentioned, for example acetyl, propionyl, n-propylcarbonyl, i -propylcarbonyl, n-butylcarbonyl, s-butylcarbonyl, i-butylcarbonyl, t-butylcarbonyl and the like “C 1-4 alkylcarbonyl”.
- C 1-6 alkoxy means an alkyloxy group wherein the alkyl portion is C 1-6 alkyl as already mentioned, for example methoxy (H 3 C—O—), ethoxy , n-propoxy, i-propoxy, n-butoxy, s-butoxy, i-butoxy, t - butoxy and the like.
- C 1-6 alkylthio referred to herein means an alkylthio group wherein the alkyl portion is C 1-6 alkyl as already mentioned, for example methylthio (H 3 C—S—), ethylthio, n-propylthio, i-propylthio, n-butylthio, s-butylthio, i-butylthio, t-butylthio and the like, preferably methylthio.
- amino C 2-20 alkyl referred to herein means a linear or branched C 2-20 alkyl group having an amino group as a substituent, for example, the amino group is an alkyl It may be located on the terminal carbon atom of the group. Amino C 2-20 alkyl also includes "amino C 2-12 alkyl" having 2 to 12 carbon atoms.
- hydroxy C 2-20 alkyl referred to herein means a straight or branched C 2-20 alkyl group having a hydroxy group as a substituent, for example, the hydroxy group is an alkyl It may be located on the terminal carbon atom of the group. Hydroxy C 2-20 alkyl also includes "hydroxy C 2-12 alkyl" having 2 to 12 carbon atoms.
- C 2-30 alkylene referred to herein means a linear or branched divalent saturated hydrocarbon group having 2 to 30 carbon atoms, and includes, for example, ethylene, propylene and the like. , C 2-20 alkylene, C 2-8 alkylene, the group —(CH 2 )n—, where n is 2-30, preferably 2-20, more preferably 2-15.
- C 1-5 alkylene means a linear or branched divalent saturated hydrocarbon group having 1 to 5 carbon atoms, such as methylene, ethylene (ethane- 1,2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, butane-1,4-diyl, and pentane-1,5-diyl, etc.) including.
- C 2-10 alkylene means a linear or branched divalent saturated hydrocarbon group having 2 to 10 carbon atoms, such as 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- Including 1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl and the like.
- “C 2-10 alkylene” includes “C 2-8 alkylene” having 2 to 8 carbon atoms and “C 2-6 alkylene” having 2 to 6 carbon atoms.
- C 2-8 alkylene means a linear or branched divalent saturated hydrocarbon group having 2 to 8 carbon atoms, for example, ethylene (ethane-1, 2-diyl, ethane-1,1-diyl), propylene (propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl), butane-1,4-diyl, pentane- Including 1,5-diyl, hexane-1,6-diyl, heptane-1,7-diyl, octane-1,8-diyl and the like.
- aryl means an aromatic carbocyclic group, for example, an aromatic carbocyclic group having 6 to 14 carbon atoms, examples of aryl are phenyl, naphthyl (1-naphthyl and 2-naphthyl) etc. Examples of aryl substituted with one or more hydroxy include 4-hydroxyphenyl.
- heteroaryl means an aromatic ring group containing one or more heteroatoms selected from nitrogen, oxygen and sulfur atoms in the ring-constituting atoms. and may be partially saturated.
- the ring may be monocyclic or a bicyclic heteroaryl fused to a benzene ring or a monocyclic heteroaryl ring.
- the number of atoms constituting the ring is, for example, 4-15, preferably 5-14, more preferably 6-10.
- heteroaryl examples include, for example, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazinyl, benzofuranyl, benzothienyl, Benzothiadiazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzodioxolyl, indolizinyl, imidazopyr
- the "divalent C 2-50 hydrocarbon group" referred to in this specification is not particularly limited, and examples thereof include linear, branched, cyclic and partially cyclic alkylene having 2 to 50 carbon atoms. groups, alkenylene groups and alkynylene groups, which groups may be divalent aromatic rings or may include aromatic rings as part of their structure.
- the “divalent C 2-50 polyalkyleneoxy” referred to in this specification is not particularly limited, and the alkylene group of the repeating unit may be linear or branched.
- Examples of "divalent C 2-50 polyalkyleneoxy” include divalent C 2-50 polyethyleneoxy group, C 3-48 polypropyleneoxy group, C 3-48 polybutyleneoxy group and the like.
- Such groups may be linked to other groups via an oxygen or carbon atom, for example a C 2-50 polyethyleneoxy group includes --O(CH 2 CH 2 O) 1-25 --, --(CH 2CH 2 O) 1-25 -, -(OCH 2 CH 2 ) 1-25 -, -(CH 2 CH 2 O) 1-24 -(CH 2 CH 2 )- and the like are included.
- salt substance referred to in this specification is not particularly limited as long as it is a water-soluble inorganic substance.
- Aluminum, aluminum salts such as aluminum chloride, potassium salts such as potassium sulfate, potassium carbonate, potassium nitrate, potassium chloride, potassium bromide, potassium iodide, sodium hydrogen carbonate, sodium carbonate, sodium sulfate, sodium nitrate, sodium chloride, bromide
- Sodium salts such as sodium, sodium iodide, sodium silicate, trisodium phosphate, disodium phosphate, sodium borate, sodium acetate, sodium citrate, lithium chloride, lithium bromide, lithium iodide, lithium carbonate, etc.
- Examples include lithium salts, preferably sodium chloride, trisodium phosphate, disodium phosphate, potassium chloride, calcium chloride, magnesium chloride, and the like.
- antigen refers to a substance that induces immunity. For example, in lymph nodes and spleen, it is a substance that can induce the activation of lymphocytes such as T cells and B cells by being presented to antigen-presenting cells.
- antigenic protein refers to antigens that are proteins.
- antigen peptide refers to an antigen that is a peptide.
- an antigenic peptide is a portion of the amino acid sequence contained in an antigenic protein and includes T cell-recognized epitopes. It may be a combination of multiple T cell recognition epitopes.
- an antigen may be, for example, a protein or peptide itself, a complex of the protein or peptide with another molecule (e.g., major histocompatibility antigen (MHC)), or the major histocompatibility antigen (MHC) itself.
- MHC major histocompatibility antigen
- MHC major histocompatibility antigen
- adjuvant refers to a substance that enhances the immune response in a subject by administering it to the subject in combination with a vaccine containing an antigen.
- HA-antigen composition ⁇ 2-1. Hyaluronic Acid Derivative Introduced with a Hydrophobic Group>
- a hyaluronic acid derivative into which a hydrophobic group has been introduced is hyaluronic acid or a derivative thereof, as long as it has a hydrophobic group and can form a complex with an antigen (especially antigenic peptide, antigenic protein). , is not particularly limited.
- a hydrophobic group is a group having hydrophobicity, and is not particularly limited as long as a hyaluronic acid derivative can form a complex with an antigen (especially antigen peptide, antigen protein).
- the hydrophobic group preferably includes a hydrophobic group having a sterol skeleton (steryl group), a hydrocarbon group, and the like, and particularly preferably a steryl group.
- the sterol skeleton is an alcohol in which a hydroxy group is attached to the cyclopentahydrophenanthrene ring shown in Formula I.
- the symbols A to D in formula I represent each ring constituting the cyclopentahydrophenanthrene ring.
- the sterol skeleton may have a double bond in the cyclopentahydrophenanthrene ring, and the bonding position of the hydroxyl group is not particularly limited.
- sterols having a hydroxy group at the C-3 position and a double bond at the B ring, or stanols having a saturated ring with a hydroxy group at the C-3 position is.
- the hydrophobic group is a compound in which the sterol skeleton is modified, for example, a compound in which the ring-constituting carbon is substituted with a hydrocarbon group (e.g., a linear or branched alkyl group having 1 to 20 carbon atoms, etc.) ( steroid).
- the term “derived group” refers to a group obtained by removing a hydrogen atom or a functional group such as a hydroxyl group from a certain compound.
- steroids include cholesterol, dehydrocholesterol, coprostenol, coprosterol, cholestanol, campestanol, ergostanol, stigmastanol, coprostanol, stigmasterol, sitosterol, lanosterol, ergosterol, and cimilenol.
- 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, Gestrone, cortisol, cortisone, aldosterone, corticosterone, deoxycortiserone and the like.
- the steryl group includes a cholesteryl group, a stigmasteryl group, a lanosteryl group, an ergosteryl group, a cholanoyl group, a coloyl group, etc., and preferably a cholesteryl group (particularly, a cholest-5-en-3 ⁇ -yl group represented by the following formula). , a cholanoyl group (in particular, a 5 ⁇ -cholan-24-oil group represented by the following formula). where the two asterisks represent the point of attachment.
- the hydrocarbon group is not particularly limited, and is, for example, a chain (preferably linear) hydrocarbon group (preferably an alkyl group) having 8 to 50 carbon atoms (preferably 10 to 30, more preferably 12 to 20 carbon atoms). is mentioned.
- 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, and is, for example, 5,000 to 2,000,000.
- the weight average molecular weight is preferably 10,000 to 1,000,000, more preferably 20,000 to 500,000, still more preferably 40,000 to 250,000, and even more preferably 80,000 to 125,000.
- the introduction rate of the hydrophobic group is, for example, 1-50%, preferably 7-50%, more preferably 17-50%, more preferably 20-45%. When the introduction rate is within the above range, the hyaluronic acid derivative can efficiently form a complex with the antigen in solution.
- the introduction rate of the hydrophobic group can be measured and calculated according to or according to the method described below.
- hyaluronic acid derivative into which a hydrophobic group is introduced include a hyaluronic acid derivative containing one or more disaccharide units (also repeating units) represented by the formula (I), or a hyaluronic acid derivative represented by the formula (II) and hyaluronic acid derivatives containing one or more disaccharide units (also repeating units).
- Hyaluronic acid derivatives are, for example: Formula (I)
- R 1 , R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl;
- R 5 is a hydrogen atom, formyl, or C 1-6 alkylcarbonyl;
- Z represents a direct bond or a peptide linker consisting of any 2-30 amino acid residues;
- X 1 has the following formula: -NR b -R, -NRb -COO-R, -NRb -CO-R, -NRb -CO- NRc -R, -COO-R, -O-COO-R, -SR, -CO-Y a -S-R, -O-CO-Y b -S-R, -NRb -CO-Yb -S -R and -S-S-R, is a hydrophobic group selected from the groups represented by; R a , R b and R c are each
- R 1a , R 2a , R 3a , and R 4a are independently selected from a hydrogen atom, C 1-6 alkyl, formyl, and C 1-6 alkylcarbonyl;
- R 5a is a hydrogen atom, formyl, or C 1-6 alkylcarbonyl;
- X 1a is hydroxy, —O—Q + , C 1-6 alkoxy, —NR 7 R 8 , or —NR 9 —Z 1 —Z 2 ;
- Q + represents the counter cation;
- R 6a , R 7 , R 8 and R 9 are independently selected from a hydrogen atom and C 1-6 alkyl;
- R aa is a hydrogen atom or C 1-6 alkyl, wherein said alkyl is independently substituted with one or more groups selected from hydroxy, carboxy, carbamoyl, C 1-6 alkylthio, aryl, and heteroaryl, wherein said aryl is substituted with one or more hydroxy may be
- R 1b , R 2b , R 3b and R 4b are independently selected from a hydrogen atom, C 1-6 alkyl, formyl, and C 1-6 alkylcarbonyl;
- R 5b is a hydrogen atom, formyl, or C 1-6 alkylcarbonyl;
- X 2 is -NR 9 -Z 1 -Z 2 , where R 9 , Z 1 and Z 2 are as defined above], a hyaluronic acid derivative comprising a repeating unit represented by be.
- a hyaluronic acid derivative introduced with a hydrophobic group has the formula (IIIc)
- R 1c , R 2c , R 3c and R 4c are each independently selected from a hydrogen atom, C 1-6 alkyl, formyl and C 1-6 alkylcarbonyl; R 5c is selected from hydrogen atom, formyl and C 1-6 alkylcarbonyl; X c is selected from hydroxy and —O—Q + where Q + represents a counter cation] It is preferable to further include a repeating unit represented by
- the hyaluronic acid derivative comprising repeating units represented by formula (I) consists essentially of (1) repeating units of formula (I); and (2) repeating units of formula (I) and formula (IIIc). be done.
- the repeating units may be the same or different.
- the hyaluronic acid derivative may be modified at positions other than the repeating unit of formula (I), for example, the hydroxy group is —O(C 1-6 alkyl), —O (formyl) and —O(C 1 -6 alkylcarbonyl), etc., and the carboxyl group may be converted to an amide group or an ester group, or may form a salt.
- the group —Z—N(R a )Y—X 1 of formula (I) above is represented by the following formula: -NH-( CH2 ) mz -NH-R; -NH-( CH2 ) mz -NH-COO-R; -NH-( CH2CH2O ) m - CH2CH2 - NH-COO-R ; -NH-( CH2 ) mz -COO-R; -NH-( CH2CH2O ) m - CH2CH2 - COO-R ; -NH-( CH2 ) mz -O-COO-R; -NH-( CH2CH2O ) m - CH2CH2 - O-COO-R ; -NH-( CH2 ) mz -S-R; -NH-( CH2CH2O ) m - CH2CH2 -S-R ; -NH-( CH2CH2O
- Z in formula (I) above is a direct bond.
- X 1 is -NR b -COO-R.
- Y in the above formula (I) include -CH2CH2O - CH2CH2 -S- S - CH2CH2O - CH2CH2- , - ( CH2CH2O ) 2 -CH2CH2 - S - S - CH2CH2O - CH2CH2- , -CH2CH2O - CH2CH2-S - S- ( CH2CH2O ) 2 - CH2CH 2- and -( CH2CH2O ) 2 - CH2CH2 -S - S-( CH2CH2O ) 2 - CH2CH2- .
- Y a in formula (I) above is preferably —CH 2 — and —CH 2 —CH 2 —.
- Y b in formula (I) above includes —CH 2 —CH 2 —, —CH(CH 3 )CH 2 —, 2-butene-1,4-diyl, hepta-2,4-diene-1,6 -diyl and octa-2,4,6-triene-1,8-diyl are preferred, and -CH 2 -CH 2 - and -CH(CH 3 )CH 2 - are more preferred.
- Z in formula (I) above is a peptide linker represented by -NH-[CH(-Z a )-CONH] n -1-CH(-Z a )-CO-, wherein , n is an integer from 2 to 30, and each Z a independently represents a substituent in an ⁇ -amino acid represented as H 2 N—CH(—Z a )—COOH.
- the peptide linker is attached at the N-terminus to the carboxyl group of the glucuronic acid moiety and at the C-terminus to the group -N(-R a )-YX 1 .
- amino acids that can be used as amino acid residues of the peptide linker include ⁇ -amino acids such as alanine, arginine, asparagine (Asn), aspartic acid, cysteine, glutamine, glutamic acid, glycine (Gly), histidine, isoleucine, leucine (Leu ), natural (L-type) amino acids such as lysine, methionine, phenylalanine (Phe), proline, serine, threonine, tryptophan, tyrosine, and valine, their D forms, etc.
- - Amino acids can be used.
- Z a includes, for example, —CH 3 , H 2 NC(NH)NH(CH 2 ) 3 —, H 2 NCOCH 2 —, and the like.
- n Zs may be the same or different.
- n is an integer of 2-30, preferably 2-10, more preferably 2-4.
- Preferred examples of peptide linkers include -Gly-Phe-Leu-Gly-, -Asn-Phe-Phe-, -Phe-Phe-, -Phe-Gly- and the like.
- R a , R b and R c are hydrogen atoms and Y is linear C 2-30 alkylene or -(CH 2 CH 2 O) m —CH 2 CH 2 — and Y a is linear C 1-5 alkylene, or Y b is linear C 2-8 alkylene or linear C 2-8 alkenylene.
- the group Z—N(R a )Y—X 1 is such that Z is a direct bond, R a is a hydrogen atom and Y is for example C 2-12 alkylene, preferably C 2-6 alkylene, more preferably C 6 alkylene, X 1 is --NR b --COO--R, R b is a hydrogen atom, and R is a cholesteryl group.
- the hyaluronic acid derivative containing the repeating unit represented by formula (I) above further contains a repeating unit represented by formula (IIIc).
- the repeating units may be the same or different.
- Q + in the above formula (IIIc) is not particularly limited as long as it is a counter cation that forms a salt with a carboxyl group in water.
- counter cations include metal ions such as lithium ions, sodium ions, rubidium ions , cesium ions, magnesium ions, calcium ions ;
- l and R m are each independently selected from a hydrogen atom and C 1-6 alkyl) and include ammonium ions and the like, preferably sodium ion, potassium ion, tetraalkylammonium ion (e.g., tetra-n-butylammonium ion, etc.).
- R j , R k , R l and R m are preferably the same group selected from C 1-6 alkyl, preferably n-butyl group.
- R 1 , R 2 , R 3 and R 4 of formula (I) above and R 1a , R 2a , R 3a and R 4a of formula (IIIc) above are preferably all hydrogen atoms. Both R a and R b are preferably hydrogen atoms.
- the group R 5 of formula (I) above is preferably acetyl.
- the hyaluronic acid derivative containing the repeating unit represented by formula (I) is substantially composed of repeating units represented by formulas (I) and (IIIc).
- the hyaluronic acid derivative of the disaccharide repeating units composed of D-glucuronic acid and N-acetylglucosamine contained in the derivative, for example, 80% or more, preferably 90% or more, more preferably 95%
- the above is the repeating unit of the formula (I) or (IIIc). In one embodiment, it is composed only of repeating units represented by formulas (I) and (IIIc) above.
- Y defined in formula (I) is, for example, —(CH 2 ) na — (wherein na is 2 to 20, preferably 2 to 15, more preferably 2 to 12, even more preferably 2 to 10 integers), preferably -(CH 2 ) 2 -, -(CH 2 ) 6 -, -(CH 2 ) 8 - and -(CH 2 ) 12 -, and further -(CH 2 ) 6 - is preferred.
- Y are preferable from the viewpoint of precipitate formation and stable dispersion, which will be described later.
- the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) has a hydrophobic group introduction rate with respect to the disaccharide repeating unit present in the derivative, for example, 1 to 50%, It is preferably 7-50%, more preferably 17-50%, more preferably 20-45%.
- the introduction rate is within the above range, the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) can efficiently form a complex with an antigen in solution.
- the introduction rate of the hydrophobic group is the following formula:
- the "disaccharide repeating unit present in the derivative” includes a repeating unit of formula (I) in which a hydrophobic group is introduced by converting a carboxyl group into an amide group, and a hydrophobic group is introduced. It includes repeating units of formula (IIIc) that are not The rate of introduction can be controlled by reaction conditions, such as the ratio of reagents, and can be determined, for example, by NMR measurement.
- the hyaluronic acid derivative containing a repeating unit represented by the above formula (I) preferably has R 1c , R 2c , R 3c , and R 4c all hydrogen atoms, R 5c acetyl, and X c
- the weight average molecular weight when -O-N a + is, for example, 1 kDa to 500 kDa, preferably 3 kDa to 500 kDa, more preferably 5 kDa to 200 kDa, composed only of repeating units represented by formula (IIIc) Synthesized from hyaluronic acid or its derivatives.
- the weight-average molecular weight of the raw 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 preferred weight average molecular weight of the raw material is 1 kDa to 1000 kDa, more preferably 3 kDa to 500 kDa, still more preferably 5 kDa to 200 kDa, still more preferably 5 kDa to 150 kDa. .
- weight-average molecular weights are calculated as number-average molecular weights or weight-average molecular weights. In the present invention, it is calculated as a weight average molecular weight.
- Methods for measuring the weight-average molecular weight include, for example, the light scattering method, osmotic pressure method, viscosity method, etc. described in "Essential Polymer Science" by Seiichi Nakahama et al.
- the viscosity average molecular weight shown in this specification can also be measured by a method commonly used in the technical field to which the present invention belongs, such as using an Ubbelohde viscometer.
- the specified numerical value can be used as the molecular weight.
- a hydrophobic group is introduced by converting the carboxyl group of glucuronic acid, which is one of the disaccharides constituting the repeating unit, into an amide group.
- the carboxyl group modification rate of the glucuronic acid moiety of the hyaluronic acid derivative containing the repeating unit of the above formula (I) is high, the binding to hyaluronic acid receptors including CD44 is suppressed, and the hyaluronic acid derivative stays in the body for a long time.
- drug carrier including vaccines.
- a targeting element into a hyaluronic acid derivative, it is possible to target each organ and cell including lymph nodes.
- target elements include target tissue-specific peptides, antibodies, fragmented antibodies, aptamers, RgD peptides for cancer cells, folic acid, anisamide, transferrin, and galactose and tocopherol for liver.
- 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 is 4 to 60 from the viewpoint of complex formation with an antigen. %, more preferably 5-50%, more preferably 7-50%, even more preferably 7-45%. From the viewpoint of lymph node migration of the complex, 6 to 60% is preferable, 17 to 50% is more preferable, 20 to 50% is more preferable, and 20 to 45% is more preferable.
- the combination of the molecular weight of the raw material of the hyaluronic acid derivative containing the repeating unit represented by the above formula (I) and the introduction rate of the hydrophobic group is 3 kDa to 500 kDa and 4 to 60% from the viewpoint of complex formation with the antigen. is preferred, 3 kDa to 200 kDa and 6 to 50% is more preferred, 5 kDa to 200 kDa and 6 to 50% is more preferred, 5 kDa to 150 kDa and 6 to 45% is more preferred.
- 3 kDa to 500 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 more preferred, 5 kDa to 150 kDa and 20 ⁇ 45% is more preferred.
- 3 kDa to 200 kDa and 4 to 60% are preferable, 3 kDa to 200 kDa and 6 to 50% are more preferable, 5 kDa to 200 kDa and 6 to 50% are more preferable, 5 kDa to 150 kDa and 17 to 45%. is more preferred.
- 5 kDa to 27 kDa and 2 to 50% are preferable, 5 kDa to 27 kDa and 8 to 35% are more preferable, 5 kDa to 18 kDa and 8 to 35% are more preferable, 5 kDa to 18 kDa and 8 to 35% is more preferred, 5kDa-18kDa and 15-22% are even more preferred.
- 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 more preferred, and 5 kDa to 27 kDa and 7 to 22% are preferred. More preferred.
- the hyaluronic acid derivative comprising a repeating unit represented by formula (II) above is composed of (1) formula (II) above; (2) formula (II) and formula (III) above; (II) and formula (IIIc); or (4) consisting essentially of repeating units of formula (II), formula (III), and formula (IIIc) above.
- the disaccharide repeating units composed of D-glucuronic acid and N-acetylglucosamine contained in the derivative for example, 80% or more, preferably 90% or more, more preferably 95 % or more are repeating units of formula (II), (III), or formula (IIIc).
- the ratio of the specific disaccharide unit to the disaccharide repeating unit present in the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is the above formula ( It means the ratio of a specific disaccharide unit to all disaccharide units contained in a given amount of the hyaluronic acid derivative containing the repeating unit represented by II).
- R 1a , R 2a , R 3a and R 4a are all hydrogen atoms. is preferred.
- R 5a is preferably a hydrogen atom or C 1-6 alkylcarbonyl, more preferably a hydrogen atom or acetyl, even more preferably acetyl.
- R 1b , R 2b , R 3b and R 4b , and R 1c , R 2c , R 3c and R 4c are preferably all hydrogen atoms.
- R 5b and R 5c are preferably a hydrogen atom or C 1-6 alkylcarbonyl, more preferably a hydrogen atom or acetyl, and more preferably both acetyl.
- R aa in formula (II) include a hydrogen atom, methyl, hydroxymethyl, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, 1-methylpropyl, 2-methylpropyl, isopropyl, 2-carboxyethyl, 2 -methylthioethyl, 2-carbamoylethyl, phenylmethyl, (4-hydroxyphenyl)methyl, and indol-3-ylmethyl.
- each optically active form and a mixture thereof are also included, but when described as H 2 N-CHR aa -COOH (amino acid), L-type (natural type) It is preferable that
- R 6a , R 7 , R 8 and R 9 are, for example, independently hydrogen atoms or methyl, preferably all hydrogen atoms.
- Embodiments of the group —CHR aa —CO—X 1a in formula (II) include, for example, the group —CHR aa —COOH. Specific examples of the group include the following groups.
- the asterisk represents the binding position with -NR 6a - (same below).
- Preferred examples of the group —CHR aa —COOH include the following groups.
- Preferred examples of the group —CHR aa —COOH include the following groups.
- Preferred examples of the group —CHR aa —COOH include the following groups.
- Preferred examples of the group —CHR aa —COOH include the following groups.
- Preferred examples of the group —CHR aa —COOH from the viewpoint of delivery of the complex to lymph nodes include the following groups.
- any of the above groups -CHR aa -COOH may be partially or wholly converted to the group -CHR aa -CONH-Z 1 -Z 2 .
- Examples of groups -Z 1 -Z 2 are given below.
- Another embodiment of the group —CHR aa —CO—X 1a in formula (II) includes, for example, the group —CHR aa —CONH 2 . Specific examples of the group include the following groups.
- Preferred examples of the group —CHR aa —CONH 2 include the following groups.
- Preferred examples of the group —CHR aa —CONH 2 include the following groups.
- Preferred examples of the group —CHR aa —CONH 2 include the following groups.
- Preferred examples of the group —CHR aa —CONH 2 include the following groups.
- These groups are also preferable groups from the viewpoint of having both biodegradability and blood retention properties.
- Preferred examples of the group —CHR aa —CONH 2 also include the following groups from the viewpoint of having both biodegradability and blood retention properties.
- Preferred examples of the group —CHR aa —CONH 2 from the standpoint of better dispersibility in pure water include the following groups.
- Preferred examples of the group —CHR aa —CONH 2 include the following groups from the standpoint of the base material for injection preparations for sustained subcutaneous administration.
- R 7 is more preferably a hydrogen atom and methyl, more preferably a hydrogen atom.
- Carboxy defined in formulas (II), (III) and (IIIc) may form a salt represented by the formula -COO-Q + .
- Q + is not particularly limited as long as it is a counter cation that forms a salt with carboxy in water, and if it has a valence of two or more, it forms a salt with multiple carboxys depending on the valence.
- counter cations include metal ions such as lithium ions, sodium ions, rubidium ions , cesium ions, magnesium ions, calcium ions ; l and R m are each independently selected from a hydrogen atom and C 1-6 alkyl) and include ammonium ions and the like, preferably sodium ion, potassium ion, tetraalkylammonium ion (e.g., tetra-n-butylammonium ion, etc.).
- R j , R k , R l and R m are preferably the same group selected from C 1-6 alkyl, preferably n-butyl.
- Another embodiment of the group —CHR aa —CO—X 1a in formula (II) includes, for example, the group —CHR aa —CONH—Z 1 —Z 2 .
- Specific examples of the group include the following groups.
- Another specific example of the group is the following group.
- Preferred examples of the group —CHR aa —CONH—Z 1 —Z 2 include the following groups.
- Preferred examples of the group —CHR aa —CONH—Z 1 —Z 2 include the following groups.
- Preferred examples of the group —CHR aa —CONH—Z 1 —Z 2 include the following groups.
- Preferred examples of the group —CHR aa —CONH—Z 1 —Z 2 from the viewpoint of delivery of the complex to lymph nodes include the following groups.
- Preferred examples of the group —CHR aa —CONH—Z 1 —Z 2 from the viewpoint of having both biodegradability and blood retention properties include the following groups.
- Examples of groups -Z 1 -Z 2 include the group -(C 2-10 alkylene)-NH-COO-Z 3 . Also included is the group —(C 2-12 alkylene)—NH—COO—Z 3 . Examples of C 2-12 alkylene here are preferably —(CH 2 ) 2 —, —(CH 2 ) 6 —, —(CH 2 ) 8 —, —(CH 2 ) 10 — and —(CH 2 ) )12-, more preferably -(CH 2 ) 2 - and -(CH 2 )6-. Further examples of the group -Z1 - Z2 include the group -( CH2CH2O ) ma - CH2CH2 -NH- Z3 .
- ma is preferably from 1 to 20, more preferably from 1 to 10, even more preferably from 1 to 3.
- a specific example of preferable ma is 2.
- Examples of the group -Z 1 -Z 2 are preferably the group -(hexane-1,6-diyl)-NH-COO-Z 3 and the group -(ethane-1,2-diyl)-NH-COO-Z 3 and the group -(CH 2 CH 2 O) 2 -CH 2 CH 2 -NH-Z 3 , more preferably the group -(hexane-1,6-diyl)-NH-COO-cholesteryl, the group -(ethane -1,2-diyl)-NH-COO-cholesteryl and the group -(CH 2 CH 2 O) 2 -CH 2 CH 2 -NH-cholanoyl, more preferably the group -(hexane-1,6-diyl )-NH-COO-cholesteryl.
- Examples of Z 1 , Z 2 and the group -Z 1 -Z 2 correspond to Y, X 1 and the group -Y-X 1 of the hyaluronic acid derivative containing the repeating unit represented by formula (I) above. You can also list things. Examples of the group —CO—NR ca —Z 3 and the group —O—CO—NR ca —Z 3 include groups in which R ca is a hydrogen atom, respectively.
- the hyaluronic acid derivative comprising a repeating unit represented by formula (II) has a group X 1a in which X 1a is —NR 9 —Z 1 —Z 2 , R 9 is a hydrogen atom, and Z 1 is, for example, C 2-12 alkylene, preferably C 2-6 alkylene, more preferably C6 alkylene, Z 2 is —NR ba —COO-Z 3 , R ba is a hydrogen atom and Z 3 is a steryl group, the group R aa is , a hydrogen atom, or C 1-6 alkyl, wherein said alkyl is independently substituted with one or more groups selected from hydroxy, carboxy, carbamoyl, C 1-6 alkylthio, aryl, and heteroaryl , where said aryl is optionally substituted with one or more hydroxy.
- preferred groups R aa from the viewpoint of lymph node delivery of the conjugate include, for example, methyl, hydroxymethyl, hydrogen atom, 1-hydroxyethyl, carbamoylmethyl, carboxymethyl, benzyl, (4-hydroxyphenyl)methyl, 1 -methylpropyl, 2-methylpropyl, isopropyl, indol-3-ylmethyl, 2-carbamoylethyl and 2-carboxyethyl, preferably methyl, hydroxymethyl, 1-hydroxyethyl, 1-methylpropyl and 2-carbamoyl Ethyl, more preferably methyl and 2-carbamoylethyl.
- a hyaluronic acid derivative containing a repeating unit represented by formula (II) above, which contains a repeating unit represented by formula (III), is also preferred.
- X 1a of formula (II) and X 2 of formula (III) are the same.
- the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) is a repeating unit represented by the formula (II), wherein X 1a is -NR 9 -Z 1 -Z 2 , the formula ( It may contain repeating units represented by III) and repeating units represented by formula (IIIc).
- the hyaluronic acid derivative containing the repeating unit represented by the above formula (II) has a group —NR 9 —Z 1 —Z 2 (hereinafter also referred to as a hydrophobic group) for the existing disaccharide repeating unit.
- the ratio of the repeating units of formula (II) and/or formula (III) having the formula (hydrophobic group introduction ratio) may be 3 to 50%.
- the introduction rate of the hydrophobic group is the following formula:
- a repeating unit of a disaccharide present in the derivative includes repeating units of formulas (II) and (III), and repeating units of formula (IIIc).
- the rate of introduction can be controlled by reaction conditions, such as the ratio of reagents, and can be determined, for example, by NMR measurement.
- the introduction rate of the hydrophobic group in the hyaluronic acid derivative containing the repeating unit represented by the formula (II) is, for example, 3 to 50%, preferably 5 to 40%, more preferably 5 to 35%. Yes, more preferably 5 to 25%, more preferably 5 to 20%, even more preferably 5 to 10%. From the viewpoint of complex formation with an 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 to 60% is preferable, 7 to 50% is more preferable, 18 to 45% is more preferable, and 20 to 35% is more preferable.
- X 1a is —NR 9 —Z 1 —Z 2 in formula (II).
- the ratio of the disaccharide unit of formula (II) to the disaccharide repeating unit present in the hyaluronic acid derivative is, for example, 70% or more, from the viewpoint of having both biodegradability and blood retention properties. It is preferably 75% or more, more preferably 90% or more. The upper limit may be 100% or less. The range of the ratio is, for example, 70-100%, preferably 75-100%, more preferably 90-100%.
- the complex has the property of lymph node migration, it is, for example, 10% or more, preferably 20% or more, more preferably 50% or more, still more preferably 70% or more, and still more preferably 90% or more.
- the upper limit may be 100% or less.
- the range of the ratio is, for example, 10 to 100%, preferably 20 to 100%, more preferably 50 to 100%, more preferably 70 to 100%, still more preferably 90 to 100%.
- the hyaluronic acid derivative containing the repeating unit represented by formula (II) may further contain a repeating unit represented by formula (III).
- the hyaluronic acid derivative containing a repeating unit represented by formula (II) above does not contain a repeating unit represented by formula (II) in which X 1 is —NR 9 —Z 1 —Z 2 .
- the sum of the proportion of the repeating unit represented by formula (II) and the proportion of the repeating unit represented by formula (III) in the disaccharide repeating units present is, for example, 70 to 100%, preferably 80 to 100%, more preferably 90-100%.
- it is, for example, 7-100%, preferably 20-100%, more preferably 30-100%.
- lymph node migration of the complex it is, for example, 20-100%, preferably 30-100%, more preferably 70-100%.
- the ratio of the repeating unit represented by the formula (III) to the existing disaccharide repeating units is preferably 3 to 50%, and It is preferably 5 to 40%, more preferably 5 to 35%, still more preferably 5 to 25%, still more preferably 5 to 20%, still more preferably 5 to 10%. From the viewpoint of complex formation with an antigen, 3-60% 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 to 60% is preferable, 7 to 50% is more preferable, 18 to 45% is more preferable, and 20 to 35% is more preferable.
- the ratio of the repeating unit represented by the formula (II) to the existing disaccharide repeating units is preferably 20 to 97%, more preferably is 30 to 95%, more preferably 35 to 95%, more preferably 45 to 95%, still more preferably 50 to 95%, still more preferably 60 to 95%.
- the ratio of the repeating unit represented by the formula (II) to the existing disaccharide repeating units is preferably 20 to 97%, more preferably is 30 to 95%, more preferably 35 to 95%, more preferably 45 to 95%, still more preferably 50 to 95%, still more preferably 60 to 95%.
- it is, for example, 7-100%, preferably 20-100%, more preferably 30-100%.
- lymph node migration of the complex it is, for example, 20-100%, preferably 30-100%, more preferably 70-100%.
- Methods for producing a hyaluronic acid derivative into which a hydrophobic group has been introduced include the methods described in International Publication Nos. 2010/053140 and 2014/038641.
- the hyaluronic acid derivative into which a hydrophobic group is introduced is characterized by forming fine particles by association in water.
- the hydrophobic interactions of the introduced hydrophobic groups cause spontaneous association in water to form fine particles.
- the particle size of the fine particles is not particularly limited, it is, for example, 1 ⁇ m or less, preferably 500 nm or less, more preferably 200 nm or less, even more preferably 100 nm or less, and even more preferably 50 nm or less.
- Methods for microparticulating a hyaluronic acid derivative into which a hydrophobic group has been introduced include the method described in International Publication No. 2010/053140 and the method described in International Publication No. 2014/038641.
- hyaluronic acid As a raw material for producing a hyaluronic acid derivative into which a hydrophobic group has been introduced, hyaluronic acid, a salt thereof, or a derivative thereof can be used.
- hyaluronic acid salts include alkali metal salts such as sodium salts, potassium salts and lithium salts, and particularly preferred salts are sodium salts which are frequently used as pharmaceuticals.
- Hyaluronic acid or a pharmaceutically acceptable salt thereof can be produced using various known methods such as a method of extracting biological substances such as chicken combs and pig subskin, biological fermentation methods, etc., or commercially available products. (for example, from Denka Co., Ltd., Shiseido Co., Ltd., Seikagaku Corporation, R&D System, etc.).
- the antigen complexed with the hyaluronic acid derivative microparticles into which a hydrophobic group has been introduced is released from the complex through decomposition/disintegration of the hyaluronic acid derivative microparticles and substitution of the antigen with biological components such as albumin.
- the rate of this release can be controlled by the introduction rate of the hydrophobic group, the molecular weight, and the amino acid introduction rate of the hyaluronic acid derivative.
- it is also possible to control the antigen release rate by chemically cross-linking and gelling a hyaluronic acid derivative by the method described in International Publication No. 2010/053140. It is also possible to control the antigen release rate by conjugating a hyaluronic acid derivative and an antigen by the method described in WO2010/053140.
- Antigens to be combined with hyaluronic acid derivatives having a hydrophobic group introduced include, for example, antigenic peptides and proteins, and DNAs and mRNAs encoding these antigenic sequences, preferably antigenic peptides and proteins, and more preferably antigenic peptides and proteins. is the antigenic peptide.
- the antigen may be a cancer antigen.
- Cancer antigens are antigens that are commonly expressed on cancer cells and, in some cases, only by cancer cells. Cancer antigens can be expressed within cancer cells or on the surface of cancer cells.
- 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 (M
- the entire sequence may be used, or a partially deleted sequence may be used.
- the antigen peptide is an antigen peptide containing one or more CD8-positive cytotoxic T-cell recognition epitopes and/or CD4-positive helper T-cell recognition epitopes in the sequence of the antigen protein.
- the antigen peptide includes an antigen peptide containing an epitope of a tumor cell antigen protein.
- the antigenic peptide is for example 8-120 amino acids, preferably 8-80 amino acids, more preferably 15-80 amino acids, more preferably 16-80 amino acids, more preferably 23-80 amino acids, more preferably 23 It has ⁇ 60 amino acids, more preferably 23-50 amino acids.
- the antigen peptide has one CD8-positive cytotoxic T-cell recognition epitope and one CD4-positive helper T cell recognition epitope. It is an antigenic peptide containing the above.
- an amino acid linker may be placed between the epitopes.
- the linker has for example 2-10 amino acids, preferably 4-10 amino acids, more preferably 4-8 amino acids.
- Amino acids used for the linker include glycine (G), tyrosine (Y), leucine (L), tryptophan (W) and the like. Preferred are tyrosine (Y), leucine (L) and tryptophan (W).
- amino acid linkers include -YYYY-(4Y), -LLLL-(4L), -WWWW-(4W), -GGGGGG-(6G), -YYYYYY-(6Y), -LLLLLL-(6L ), -WWWWW-(6W), -YYYYYYY-(8Y), -LLLLLLLL-(8L) and -WWWWWWWW-(8W), preferably 6Y, 6L and 6W.
- a complex of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen can be produced by mixing a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen in a suitable solution.
- Solutions to be used include water, physiological saline, various buffer solutions, sugar solutions, DMSO, ethanol, DMF, combinations thereof, and the like, and solvent/buffer solutions may be exchanged by methods such as dialysis.
- the complex of the present invention can be produced by mixing fine particles of a hyaluronic acid derivative into which a hydrophobic group has been introduced and an antigen.
- a complex of the hyaluronic acid derivative and the antigen can be obtained by forming a complex between the fine particles of the hyaluronic acid derivative and the antigen.
- a complex of the hyaluronic acid derivative and the antigen can be obtained.
- the complex includes an antigen inclusion body in the hyaluronic acid derivative microparticles. In the inclusion body, the antigen is coated with the hyaluronic acid derivative.
- a method of forming a complex includes a method of adding an antigen solution to fine particles of a hyaluronic acid derivative into which preformed hydrophobic groups have been introduced.
- the formed fine particles of the hyaluronic acid derivative and the antigen form complexes through interactions such as hydrophobic interactions, electrostatic interactions, and hydrogen bonds. Interactions occur both on the microparticle surface and inside the microparticle.
- conditions such as solvent, salt concentration, pH, temperature, time, and addition of denaturant may be appropriately selected so that the antigen forms a stable complex with high yield.
- the degree of swelling and density of the hyaluronic acid derivative fine particles and the ionization state of the antigen also change depending on the salt concentration and pH during complex formation, so appropriate conditions may be used depending on the combination.
- electrostatic repulsion between carboxyl groups of hyaluronic acid derivatives can be used to reduce the density of fine particles, increase the amount of complexation, and increase the amount of complexation.
- the electrostatic repulsion is weakened by increasing the salt concentration, the fine particle density is increased, and the gel network is made smaller than the antigen size, thereby firmly retaining the antigen and delaying the release.
- the salt concentration can also be the physiological salt concentration.
- Ultrasonic irradiation may be performed using an ultrasonic homogenizer or a single-point concentrated ultrasonic irradiation apparatus for miniaturization and size uniformity. Ultrasonic irradiation may be performed after mixing the hyaluronic acid derivative and the drug, or may be performed only on the hyaluronic acid derivative. Uncomplexed free antigens can be separated and removed by dialysis, size exclusion chromatography (SEC), or the like.
- SEC size exclusion chromatography
- 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 mixed with water, an aqueous salt solution, or various buffer solutions.
- an aqueous solution fine particle formation and complex formation can be carried out at the same time. Replacement can be performed, for example, by dialysis.
- Ultrasonic irradiation may be performed using an ultrasonic homogenizer or a single-point concentrated ultrasonic irradiation apparatus for miniaturization and size uniformity.
- Ultrasonic irradiation may be performed after mixing the hyaluronic acid derivative and the antigen, or may be performed only on the hyaluronic acid derivative. Moreover, it may be before dialysis or after dialysis. An antigen complexed with a hyaluronic acid derivative by this method is more difficult to release from the complex.
- Conditions for mixing the hyaluronic acid derivative and antigen include, but are not limited to, solvent species, salt concentration, pH, temperature, time, addition of denaturant, hyaluronic acid derivative concentration, antigen concentration, ratio of hyaluronic acid derivative and antigen, and the like. may be appropriately selected such that the antigen is stable and complexes in high yield.
- solvent type such as solvent type, salt concentration, pH, temperature, time, number of times, addition of denaturant, hyaluronic acid derivative concentration, antigen concentration, hyaluron when replacing with water, salt solution, or various buffer solution aqueous solutions
- Conditions such as the ratio of the acid derivative to the antigen may be appropriately selected so that the antigen is stable and forms a complex with high yield.
- the desired particle size can also be obtained by appropriately selecting these conditions.
- Free antigens that have not been complexed can be separated and removed by dialysis, size exclusion chromatography (SEC), or the like.
- the particle size is not particularly limited, but from the viewpoint of lymph node migration, it is, for example, 20 to 200 nm, preferably 20 to 100 nm, more preferably 20 to 50 nm.
- Immunoreceptors are capable of transmitting signals necessary for the exertion of effector functions of immune cells, that is, signals necessary for activating immune cells when antigen-binding domains bind to antigens. There is no particular limitation as long as it is a receptor.
- Preferred immunoreceptors include T cell receptors, chimeric antigen receptors (CAR), and the like.
- the antigen recognized by the chimeric antigen receptor may be a cell surface antigen or a complex of intracellular antigen and major histocompatibility complex.
- An immunoreceptor typically contains an antigen-binding domain, a transmembrane domain, and an intracellular domain.
- the antigen-binding domain is a domain that is located extracellularly when the immunoreceptor is placed on the cell membrane, and is not particularly limited as long as it is a domain capable of recognizing and binding to the antigen.
- Antigen-binding domains are usually CDRs of antibodies or T-cell receptors against antigens (for example, in the case of antibody CDRs, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR1). 1 or more, preferably 2, 3, 4, 5 or more, more preferably all 6, of chain CDR3).
- the antigen-binding region more preferably comprises a variable region of an antibody against an antigen or a T-cell receptor (in the case of an antibody, for example, a heavy chain variable region and/or a light chain variable region).
- the antigen-binding domain can adopt a single-chain antibody structure, such as a scFv structure.
- a heavy chain variable region and a light chain variable region are included like the scFv structure, the heavy chain variable region and the light chain variable region are usually linked via a linker.
- the linker is not particularly limited as long as it does not significantly impair antigen-binding properties, and is arbitrary.
- the linker is preferably glycine or a linker composed of glycine and serine (GGS linker, GS linker, GGG linker, etc.).
- the length of the linker is not particularly limited.
- the number of amino acid residues of the linker is, for example, 5-30, preferably 10-25.
- the positional relationship between the heavy chain variable region and the light chain variable region is not particularly limited, and it is possible to adopt either an embodiment in which the N-terminal side is the light chain variable region or a case in which the N-terminal side is the heavy chain variable region.
- the transmembrane domain is a domain that is located within the cell membrane when the immunoreceptor is located on the cell membrane, and is not particularly limited as long as it can constitute the immunoreceptor.
- transmembrane domain for example, transmembrane regions such as TCR ⁇ chain/ ⁇ chain, CD28, CD3 ⁇ , CD8 ⁇ , CD3, CD4, 4-1BB can be used.
- the transmembrane region of each factor is known or can be easily determined from known sequence information (for example, using a transmembrane region prediction program or the like).
- a transmembrane domain composed of an artificially constructed polypeptide may be used. These transmembrane domains may be appropriately mutated as long as they do not significantly inhibit the function of the chimeric antigen receptor.
- the antigen-binding domain and transmembrane domain are linked directly or via a spacer.
- a spacer When a spacer is used, its sequence is not particularly limited. A sequence such as a part of the factor to be used (for example, CD28) can be used as a spacer.
- a highly flexible spacer can be constructed by utilizing the arrangement of the hinge portion or a part thereof.
- Intracellular signal domain is a domain that is placed in the cell when the immunoreceptor is placed on the cell membrane, and transmits the signal necessary for the effector function of the immune cell, that is, the antigen-binding domain is a domain capable of transducing the signals necessary for the activation of immune cells when it binds to an antigen.
- the intracellular signal domain preferably contains an intracellular activation domain.
- an intracellular domain such as Fc ⁇ RI ⁇ can be used.
- CD3 ⁇ is used.
- CD3 may be appropriately mutated as long as it does not inhibit the function of immune receptors.
- ITAM immunomunoreceptor tyrosine-based activation motif
- the immune receptor can contain regions other than those mentioned above. Other regions include, for example, co-stimulatory factor intracellular domains, leader sequences (signal peptides) to facilitate transport of immunoreceptors onto the cell membrane (e.g., GM-CSF receptor leader sequences), spacers/linkers (e.g., between a transmembrane region and an intracellular signaling domain, between domains within an intracellular signaling domain), and the like.
- the intracellular domain of the co-stimulatory factor is not particularly limited as long as it is an intracellular domain derived from a co-stimulatory factor possessed by T cells or the like. For example, one or more selected from the group consisting of OX40, 4-1BB, GITR, CD27, CD278, CD28 and the like can be appropriately selected and used.
- An immunoreceptor may be a molecule consisting of a single polypeptide, or a molecule consisting of a complex of two or more polypeptides.
- the immunoreceptor may be a molecule composed of a polypeptide or a complex thereof, or a polypeptide or a complex thereof may be linked to another substance (for example, a fluorescent substance, a radioactive substance, an inorganic particle, etc.). It can be anything.
- Antigen-specific lymphocytes are not particularly limited as long as they express immunoreceptors for the antigen in the HA-antigen composition (that is, capable of binding (preferably specifically binding) to the antigen).
- the antigen-specific lymphocyte expresses an immunoreceptor on the cell membrane, and the immunoreceptor is expressed with an antigen-binding domain exposed outside the cell membrane.
- Lymphocytes include, for example, T cells (e.g., CD8-positive cells, CD4-positive cells, CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, ⁇ -T cells, ⁇ -T cells, etc.), NK cells, NKT cells, and the like.
- T cells e.g., CD8-positive cells, CD4-positive cells, CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, T cells prepared from iPS cells, ⁇ -T cells, ⁇ -T cells, etc.
- NK cells e.g., NK cells, NKT cells, and the like.
- Antigen-specific lymphocytes can be obtained by introducing into cells a polynucleotide containing an immune receptor coding sequence.
- stimulation with anti-CD3 and anti-CD28 antibodies can activate immunoreceptor-expressing cells.
- stimulation with anti-CD3 antibody and anti-CD28 antibody can be applied by culturing in a culture vessel (eg, culture dish) whose culture surface is coated with anti-CD3 antibody and anti-CD28 antibody.
- the stimulation can also be performed using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody (for example, Dynabeads T-Activator CD3/CD28 provided by VERITAS).
- T cell growth factors In order to increase the survival rate/proliferation rate of cells, it is preferable to use a culture medium to which T cell growth factors have been added during the activation treatment.
- IL-2, IL-15, IL-7 and the like can be used as T cell growth factors.
- T cell growth factors such as IL-2, IL-15 and IL-7 can be prepared according to conventional methods. Moreover, a commercial item can also be utilized. Although the use of T-cell growth factors derived from animals other than humans is not excluded, T-cell growth factors of human origin (which may be recombinant) are usually used.
- the cells (the polynucleotide-introduced lymphocytes) after introduction of the polynucleotide are proliferated.
- the polynucleotide-introduced lymphocytes are cultured (if necessary, subcultured) using a culture medium supplemented with a T cell growth factor.
- the same treatment (reactivation) as in the activation of immunoreceptor-expressing cells may be performed.
- a medium supplemented with serum human serum, fetal bovine serum, etc.
- serum human serum, fetal bovine serum, etc.
- autologous serum that is, serum collected from a patient receiving immunoreceptor gene-introduced lymphocytes.
- feeder cells that are inactivated by irradiation (e.g., 25-35 Gy) from peripheral blood mononuclear cells (e.g., pooled peripheral blood mononuclear cells from several healthy individuals). can do.
- irradiation e.g., 25-35 Gy
- peripheral blood mononuclear cells e.g., pooled peripheral blood mononuclear cells from several healthy individuals.
- the method for obtaining antigen-specific lymphocytes is as follows.
- a nucleic acid encoding an immune receptor can be linked to another nucleic acid so that it is expressed under the control of an appropriate promoter.
- a promoter either one that promotes expression constitutively, or one that is induced by drugs (eg, tetracycline or doxorubicin) can be used.
- mammalian promoters such as phosphoglycerate kinase (PGK) promoter, Xist promoter, ⁇ -actin promoter, RNA polymerase II promoter, SV40 early promoter, cytomegalovirus promoter, herpes simplex virus thymidine kinase promoter, various retroviruses Virus-derived promoters such as the LTR promoter can be used.
- PGK phosphoglycerate kinase
- Xist promoter Xist promoter
- ⁇ -actin promoter RNA polymerase II promoter
- SV40 early promoter SV40 early promoter
- Nucleic acids containing promoters or other regulatory elements (eg, enhancer or terminator sequences) that cooperate with the transcription initiation site may be ligated to achieve efficient transcription of the nucleic acids.
- a gene that can serve as a marker for confirming the expression of the nucleic acid for example, a drug resistance gene, a gene encoding a reporter enzyme, a gene encoding a fluorescent protein, etc. may be incorporated.
- Nucleic acids encoding immune receptors can be introduced into cells using vectors.
- a vector is operably linked to suitable control sequences so as to express the nucleic acid in a suitable host.
- control sequences include promoters that transcribe nucleic acids, operator sequences that control transcription, sequences that encode ribosome binding sites, enhancers, polyadenylation sequences, sequences that control the termination of transcription/translation, and the like.
- Various known sequences eg, restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences, etc.
- sequences can be appropriately selected and used according to conditions such as the type of polypeptide to be expressed, host cells, and media.
- a vector that integrates into the host genome a vector that does not integrate, an episomal vector that exists in the cytoplasm and replicates autonomously, and the like can be used.
- Such vectors include, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, pseudotype vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Vector, Epstein-Barr virus (EBV) vector, virus vector such as HSV vector can be exemplified.
- retroviral vectors including oncoretroviral vectors, lentiviral vectors, pseudotype vectors
- adenoviral vectors adeno-associated virus (AAV) vectors
- AAV adeno-associated virus vectors
- simian virus vectors simian virus vectors
- vaccinia virus vectors Sendai virus vectors
- Vector Epstein-Bar
- Non-viral vectors can also be used in combination with condensing agents such as liposomes and cationic lipids.
- nucleic acids can be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation, particle bombardment.
- Antigen-specific lymphocytes can be prepared by introducing a nucleic acid encoding an immune receptor into cells.
- a cell expressing an immunoreceptor is activated by intracellular signal transmission by binding to an antigen via the immunoreceptor.
- Cell activation varies depending on the type of host cell and intracellular domain, but can be confirmed using the release of cytokines, an increase in cell proliferation rate, changes in cell surface molecules, and the like as indicators. For example, release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells results in the destruction of tumor cells expressing the complex. It also stimulates other immune cells such as B cells, dendritic cells, NK cells, macrophages, etc. by cytokine release and changes in cell surface molecules. Immunoreceptor-expressing cells are therefore useful in adoptive immunotherapy.
- the step of introducing a nucleic acid encoding a CAR into a cell is performed ex vivo or in vivo.
- Cells into which nucleic acids are introduced can be cells derived from mammals, such as humans, or cells derived from non-human mammals such as monkeys, mice, rats, pigs, cows, and dogs.
- Cell types that can be used include, for example, blood (peripheral blood, umbilical cord blood, etc.), body fluids such as bone marrow, and cells collected, isolated, purified, and induced from tissues or organs.
- PBMC immune cells
- B cells hematopoietic stem cells, macrophages, monocytes or NK cells
- blood cells neurotrophils, basophils, monocytes
- hematopoietic stem cells cord blood mononuclear cells, fibers Blast cells, pre-adipocytes, hepatocytes, blood cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, pluripotent stem cells, various cancer cell lines or neural stem cells can be used.
- T cells T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), pluripotent stem cells, or cell populations containing these.
- T-cells include CD8-positive T-cells or CD4-positive T-cells, regulatory T-cells, cytotoxic T-cells, and tumor-infiltrating lymphocytes.
- Cell populations containing T cells and T cell progenitor cells include PBMCs.
- the cells used in the present invention may be those collected from living organisms, those obtained by expanding the cells, or those established as cell lines. When it is desired to transplant into a living body cells into which nucleic acids have been introduced or cells differentiated from the cells, it is preferable to introduce the nucleic acids into cells harvested from the living body itself or from the same kind of living body.
- Antigen-specific lymphocytes may be cultured and/or stimulated using appropriate media and/or stimulatory molecules prior to administration to a subject.
- Stimulatory molecules include cytokines, suitable proteins, and other components.
- cytokines include IL-2, IL-7, IL-12, IL-15, IFN- ⁇ and the like, preferably IL-2.
- the concentration of IL-2 in the medium is not particularly limited, but is, for example, 0.01 to 1 ⁇ 10 5 U/mL, more preferably 1 to 1 ⁇ 10 4 U/mL.
- Suitable proteins also include, for example, CD3 ligand, CD28 ligand, and anti-IL-4 antibody.
- a lymphocyte stimulating factor such as lectin can also be added.
- serum or plasma may be added to the medium.
- the amount of these agents added to the medium is not particularly limited, but 0 to 20% by volume is exemplified, and the amount of serum or plasma used can be changed according to the culture stage. Decreasing serum or plasma concentrations can be used.
- the serum or plasma may be derived from either autologous (meaning that the origin is the same as the cultured cells) or non-autologous (meaning that the origin is different from the cultured cells), but is preferably safe. From the point of view of sex, autologous ones are used.
- the cell culture equipment used for cell culture is not particularly limited, and for example, petri dishes, flasks, bags, large culture tanks, bioreactors and the like can be used.
- As the bag a CO 2 gas permeable bag for cell culture can be used.
- a large culture vessel can be used. Cultivation can be performed in either an open system or a closed system, but it is preferable to culture in a closed system from the viewpoint of the safety of the resulting cell population.
- the present invention provides a pharmaceutical composition for use in combination administration with antigen-specific lymphocytes, containing an HA-antigen composition, an HA-antigen composition containing antigen-specific lymphocytes, - a pharmaceutical composition for combined administration with an antigen composition, a pharmaceutical composition characterized by comprising a combination of an HA-antigen composition and antigen-specific lymphocytes, etc.
- the HA-antigen composition can also be used as a T cell activation vaccine and the like.
- An administration subject eg, animals such as humans, mice, monkeys, dogs, and cats (especially mammals)
- animals such as humans, mice, monkeys, dogs, and cats (especially mammals)
- mammals can be, for example, a subject with cancer.
- Target diseases include solid tumors and hematological cancers.
- Target diseases include, for example, various B-cell malignant lymphomas (B-cell acute lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), Myeloproliferative disease, myelodysplastic/myeloproliferative neoplasm (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndrome, acute myelogenous leukemia, multiple myeloma, lung cancer, colorectal cancer, ovarian cancer cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma,
- B-cell malignant lymphomas B-cell acute lymphocy
- Treatment includes mitigation (mitigation) of symptoms characteristic of the target disease or associated symptoms, prevention or delay of exacerbation of symptoms, and the like.
- Prevention means preventing or delaying the onset/development of a disease (disorder) or its symptoms, or reducing the risk of onset/development.
- improved means mitigation (lightening), amelioration, remission, or cure (including partial cure) of a disease (disorder) or its symptoms.
- Immuno-checkpoint inhibitors have shown excellent efficacy against melanoma and lung cancer, but their therapeutic efficacy remains at around 20%.
- therapeutic methods for cold tumors with a small number of intratumoral T cells, called immunodesert and immunoexcluded, are desired.
- the pharmaceutical composition of the present invention showed high efficacy in this model using B16F10, it can exhibit good efficacy even against low-immunogenic cancers.
- a low immunogenic cancer has reduced or lost expression of MHC class I in cancer cells, reduced or no tumor antigen presentation, immunogenicity is low cancer.
- the percentage of cancer cells expressing MHC class I in all cancer cells is, for example, 50% or less.
- the expression level of MHC class I can be measured by immunohistochemical staining or FCS analysis.
- the pharmaceutical composition of the present invention showed high efficacy in this model using B16F10, it can exhibit good efficacy even against metastatic cancer.
- the primary tumor site of metastatic cancer includes, but is not limited to, bladder, bone, bone marrow, brain, breast, neck, colon, endometrium, esophagus, intestine, kidney, liver, lung, mouth, muscle. , ovary, skin, pancreas, prostate, skin, stomach, testis, thyroid, uterus, and any hyperproliferative tissue, including, for example, vascular structures (e.g., endothelial cells, smooth muscle cells, pericytes, scars, fibrous cells). diseased tissue, surgically adherent tissue, or hyperproliferative bone lesions).
- the metastatic site of metastatic cancer is not limited, but includes adrenal gland, bone, brain, kidney, liver, lung, lymph node, ovary, peritoneum, skin, spleen, and the like.
- the pharmaceutical composition of the present invention showed high efficacy in this model using B16F10, it can exhibit good efficacy even against cancers with a small number of intratumoral T cells.
- cancers with a low number of T cells in the tumor are cancers with very few or no T cells in the tumor.
- the ratio of T cells to cancer cells is, for example, 10% or less.
- the percentage of T cells can be measured by immunohistochemical staining or FCS analysis.
- the combined administration of the HA-antigen composition and antigen-specific lymphocytes may be simultaneous administration, administration on the same day, or administration at intervals.
- antigen-specific lymphocytes are preferably administered after administration of the HA-antigen composition (more preferably after administration of the HA-antigen composition and adjuvant).
- the administration interval between the HA-antigen composition and the antigen-specific lymphocytes is, for example, 1 day to 1 week, 1 to 3 days, 1 to 2 can be days.
- a pharmaceutical composition comprising a combination of an HA-antigen composition and antigen-specific lymphocytes can also be in the form of a kit.
- the pharmaceutical composition can be, for example, a cancer treatment kit comprising the HA-antigen composition and antigen-specific lymphocytes.
- the HA-antigen composition may be administered orally, parenterally, intranasally, intravaginally, intraocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intraarticularly, intracerebral, intraorally, intrafat, mammary. It may be administered via routes such as intra-tissue, inhalation, and the like. Preferably, it may be administered subcutaneously, intramuscularly, intravenously or intradermally.
- HA-antigen compositions are pharmaceutical compositions containing one or more pharmaceutically acceptable diluents, wetting agents, emulsifying agents, dispersing agents, adjuvants, preservatives, buffers, binders, stabilizers, etc. As such, it can be administered in any appropriate form depending on the intended administration route.
- the route of administration may be parenteral or oral.
- the dosage of the HA-antigen composition can be determined appropriately. For example, as a usual dosage, an amount of about 0.1 mg/time to 100 mg/time can be used as the antigen. Appropriate administration frequency is 2 to 20 times. Dosing intervals can be selected between 1 day and 2 weeks.
- the HA-antigen composition may be administered in combination with one or more adjuvants.
- the adjuvant to be used may be any substance that has the effect of enhancing the activity of antigen-presenting cells, and more specifically, substances that activate innate immune receptors (pattern recognition receptors) and other antigen-presenting cell stimulating substances. , a substance that inhibits the acquisition of immunosuppressive activity by antigen-presenting cells is selected. Innate immune receptors are classified as Toll-like receptors (TLR), C-type lectin receptors (CLR), NOD-like receptors (NLR), RIG-I-like receptors (RLR) and cytoplasmic DNA sensors.
- TLR Toll-like receptors
- CLR C-type lectin receptors
- NLR NOD-like receptors
- RIG-I-like receptors RLR
- Adjuvants that are innate immune receptor agonists can be selected from inactivated bacterial cells, bacterial cell extracts, nucleic acids, lipopolysaccharides, lipopeptides, synthetic low-molecular-weight compounds, etc., preferably CpG oligo DNA, poly IC RNA, imidazoquinone (eg, R848, imiquimod, etc.), saponin (eg, QuilA, QS21, etc.), STING agonist (eg, cyclic di-GMP, etc.), monophosphoryl lipid, lipopeptide and the like are used.
- Taxane drugs and anthracycline drugs can be used as adjuvants, which are antigen-presenting cell stimulators.
- Adjuvants which are drugs that inhibit the acquisition of immunosuppressive activity by antigen-presenting cells, include JAK/STAT inhibitors, indole deoxygenase (IDO) inhibitors, and tryptophan deoxygenase (TDO) inhibitors. selected. These inhibitory substances include not only compounds having an antagonistic effect on the factor, but also neutralizing antibodies, small interfering RNA (siRNA), and antisense DNA of the factor.
- the adjuvant used in the HA-antigen composition is preferably an innate immune receptor agonist, more preferably a Toll-like receptor (TLR) or cytoplasmic DNA sensor agonist.
- TLR agonists include CpG oligo DNA and poly IC RNA, imidazoquinones such as R848 and imiquimod, saponins such as QuilA and QS21, STING agonists, and monophosphoryl lipids.
- the adjuvant may be administered as a formulation separate from or together with the HA-antigen composition.
- the adjuvant may be used by conjugating it to a hyaluronic acid derivative having a hydrophobic group introduced by the method described in International Publication No. 2010/053140.
- the dosage of the HA-antigen composition when administered in combination is, for example, 0.01 to 100 mg/dose, preferably 0.1 to 50 mg/dose, more preferably, for example, 0.1 to 20 mg/dose. is, for example, 0.01 to 100 mg/kg body weight, preferably 0.1 to 50 mg/kg body weight, more preferably 0.1 to 10 mg/kg body weight.
- the adjuvant may be administered at the same time as the HA-antigen composition (including when the adjuvant is included in the formulation) or at different times. When they are administered at different timings, it is preferable to administer one after administration of the other, for example, within 1 minute to 5 hours, preferably within 1 minute to 1 hour.
- the HA-antigen composition may be administered in combination with one or more antibodies used for cancer therapy.
- the antibody is, for example, an antibody that inhibits an immunosuppressive signal by a tumor or one or more antibodies that activate a co-stimulatory signal of immune cells, preferably an antibody that inhibits an immunosuppressive signal by a tumor or co-stimulatory immune cells.
- An antibody that activates a signal is one or more antibodies selected from anti-CTLA4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-OX40, and anti-4-1BB antibody.
- the dosage of the HA-antigen composition when administered in combination is, for example, 0.01-100 mg/dose, preferably 0.1-50 mg/dose, more preferably 0.1-20 mg/dose.
- the dosage is, for example, 0.01-200 mg/kg body weight, preferably 0.1-100 mg/kg body weight, more preferably 1-40 mg/kg body weight.
- the antibody may be administered at the same time as the HA-antigen composition (including when the antibody is contained in the formulation) or at different times. When they are administered at different timings, it is preferable to administer one after administration of the other, for example, within 1 minute to 24 hours, preferably within 1 minute to 5 hours.
- the HA-antigen composition may be administered in combination with both the above-mentioned adjuvant and antibody.
- 50 mg/dose, more preferably 0.1 to 20 mg/dose, and 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, more preferably 0.1 to 100 mg/kg body weight. It is 1 to 10 mg/kg body weight, and the dosage of the antibody is, for example, 0.01 to 200 mg/kg body weight, preferably 0.1 to 100 mg/kg body weight, more preferably 1 to 40 mg/kg body weight.
- the adjuvant and antibody may be administered at the same time as the HA-antigen composition (including when the adjuvant and antibody are included in the formulation) or at different times. When administered at different times, it is preferred that all of the vaccine formulation, adjuvant, and antibody be administered within, for example, 1 minute to 24 hours, preferably 1 minute to 5 hours.
- the HA-antigen composition is preferably administered prior to administration of antigen-specific lymphocytes.
- administration of 1 unit or 2 units is performed when one administration of the HA-antigen composition followed by one infusion of antigen-specific lymphocytes is taken as the unit of administration. It is preferred that the HA-antigen composition is administered at least once after having been made.
- the interval between the first administration and the second administration of the HA-antigen composition is, 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, It can be 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.
- the interval is, 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.
- Antigen-specific lymphocytes can be widely used as cell preparations for the treatment, prevention, or amelioration of tumors/cancers (target diseases) that express the target antigen of the antigen-binding domain.
- the content of antigen-specific lymphocytes in the cell preparation is not particularly limited, but is preferably 1 ⁇ 10 3 to 1 ⁇ 10 11 cells/mL, more preferably 1 ⁇ 10 4 to 1 ⁇ 10 10 cells/mL. Preferably, 1 ⁇ 10 5 to 2 ⁇ 10 9 cells/mL are more preferred.
- the dosage of the therapeutic agent containing the cell population as an active ingredient is not particularly limited, but is preferably 1 ⁇ 10 6 to 1 ⁇ 10 12 cells/day, and 1 ⁇ 10 7 to 5 ⁇ 10 11 per adult. more preferably 1 ⁇ 10 8 to 2 ⁇ 10 11 pieces/day.
- DMSO Dimethyl sulfoxide
- serum albumin for the purpose of protecting cells
- antibiotics for the purpose of preventing bacterial contamination
- various ingredients for the purpose of activating, proliferating or inducing differentiation of cells
- cytokines for the purpose of activating, proliferating or inducing differentiation of cells
- steroids for the purpose of preventing bacterial contamination
- vitamins for the purpose of activating, proliferating or inducing differentiation of cells
- the route of administration of antigen-specific lymphocytes is not particularly limited.
- administration is by intravenous, intraarterial, intraportal, intradermal, subcutaneous, intramuscular, or intraperitoneal injection.
- Local administration may be used instead of systemic administration. Examples of local administration include direct injection into a target tissue/organ/organ.
- the administration schedule may be prepared in consideration of the sex, age, body weight, disease state, etc. of the subject (patient).
- multiple doses may be administered continuously or periodically.
- Appropriate administration frequency is 2 to 20 times. Dosing intervals can be selected between 1 day and 2 weeks.
- Example 5 ⁇ Materials and methods (Examples 1 to 5)>
- Cellular RPMI1640 medium (2-mercaptoethanol added) was purchased from Cell Science Institute.
- Fetal bovine serum (FBS) was purchased from Gibco.
- Mouse fibrosarcoma CMS5a cell line was obtained from Memorial Sloan Kettering Cancer Institute and subcultured at Mie University.
- the mouse fibrosarcoma CMS5a cell line expresses a mutant ERK2 protein.
- a peptide (QYIHSANVL (SEQ ID NO: 1)) containing a mutant portion of the mutant ERK2 protein is recognized by CD8-positive cytotoxic T cells of BALB/c mice.
- TCR T-cell receptor
- DUC18 mice transgenic mice
- the long-chain peptide antigen used in this example contains the CD8-positive cytotoxic T cell recognition epitope sequence (QYIHSANVL) of the mutant ERK2.
- mice Female BALB/c mice (CD90.2-positive) were purchased from Japan SLC at the age of 6-8 weeks. DUC18 transgenic mice were obtained from the University of Washington and bred at Mie University. Both mice were bred at Mie University Advanced Science Research Support Center Animal Experiment Facility. The animal experiment protocol was approved by the Ethics Committee of Mie University School of Medicine.
- Hyaluronic acid nanogel As described in Examples 1 and 2 of WO 2010/053140, hyaluronic acid nanogels with cholesteryl groups introduced therein (weight average molecular weight of 99 kDa, cholesteryl group introduction rate of 42%) were synthesized.
- the sequence consists of a 9 amino acid sequence from 16th Q to 24th L (QYIHSANVL (SEQ ID NO: 1)) as a CD8 positive cytotoxic T cell recognition epitope sequence and a CD4 positive helper T cell recognition epitope sequence. It contains a 17 amino acid sequence from the 13th R to the 29th K (RGLQYIHSANVLHRDLK (SEQ ID NO: 3)).
- Hyaluronic acid nanogel was dissolved in ultrapure water to a concentration of 12 mg/mL, and subjected to ultrasonic treatment (E220X, manufactured by Covaris).
- the antigen peptide was dissolved in DMSO to 50 mg/mL.
- the antigen peptide DMSO solution was added to the HA derivative aqueous solution, and the mixture was sonicated with a bath sonicator for 30 minutes.
- CpG oligo DNA1668 was purchased from Ajinomoto Bio-Pharma Service Gene Design.
- Fluorescent-labeled 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 BioLegend 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) purchased from FITC-labeled anti-mouse CD11c antibody (clone HL3) and PerCP-Cy5.5-labeled anti-mouse CD3 antibody (clone 145-2C11) were purchased from BD Biosciences.
- APC-labeled anti-mouse IFN- ⁇ 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-labeled anti-rat IgG (H+L) antibody was purchased from Cell Signaling.
- ⁇ Short-chain peptide antigen A synthetic short-chain peptide for stimulating CD8-positive T cells was purchased from Sigma Genesis. The sequence was: QYIHSANVL (SEQ ID NO: 1).
- Example 1 Using a tumor growth test T75 culture flask (Corning), the 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. After centrifuging the suspension (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed. After that, they were washed twice with RPMI1640 medium and suspended in RPMI1640 medium at a concentration of 1 ⁇ 10 6 cells/100 ⁇ L. A dose of 100 ⁇ L/individual was subcutaneously implanted into the right anterior dorsum of BALB/c mice (5 per group).
- CD8-positive T cells from the spleens of mutant ERK2-specific TCR transgenic DUC18 mice (CD90.1-positive or CD90.2-negative) were infected with CD8a. It was isolated using microbeads (Miltenyi), suspended in RPMI1640 medium at a concentration of 2 ⁇ 10 6 cells/200 ⁇ L, and 200 ⁇ L was injected through the tail vein of mice (TCR-T group). Thereafter, the tumor area was measured over time.
- Example 1 To clarify the antitumor effect of HA nanogel cancer vaccine combined with antigen-specific T cell infusion therapy, CMS5a expressed in immune checkpoint inhibitor (ICI)-resistant fibrosarcoma cell line CMS5a tumor-bearing mice.
- ICI immune checkpoint inhibitor
- a long-chain peptide antigen-loaded HA nanogel cancer vaccine was prepared by combining a long-chain peptide antigen containing the CD8 epitope of mutant ERK2 antigen (mERK2) with HA nanogel, and it was used in combination with mERK2-recognizing TCR gene-transferred T cell infusion therapy. The therapeutic effect was examined.
- the HANG-V and TCR-T combined treatment group reduced CMS5a tumor growth compared to the HANG-V and TCR-T single treatment groups. It markedly suppressed the tumors and caused tumor regression, and tumor disappearance was observed in all mice 7 days after the treatment (Figs. 1-1 and 1-2). This effect persisted for a long period of time, and even when CMS5a was reimplanted into the same mice, no tumor growth was observed, and the mice survived for a long period of time without recurrence (Fig. 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. In addition, it was revealed that this combination can suppress tumor recurrence even after tumor healing, suggesting the possibility that HA nanogel cancer vaccine enhances the antitumor effect of antigen-specific T cells.
- Example 2 Anti-tumor immune response analysis of lymph nodes draining vaccine administration site 14 days after tumor transplantation in the test of Example 1, the draining lymph nodes (right inguinal lymph nodes) of the vaccine administration site were collected, and the lymph nodes were crushed using a slide glass. After that, the cells were suspended in RPMI1640 medium. At this time, cells for 2 mice per group were pooled. After centrifugation (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed, washed twice with RPMI1640 medium, and suspended in RPMI1640 medium containing 10% FBS. The number of cells was measured using a cell hemocytometer (Fig. 2-1), and the cell concentration was adjusted to 2 x 106 cells/mL.
- Staining of myeloid cells (Fig. 2-3, Fig. 2-4): A cell suspension was added to a 96-well V-bottom microplate (Nunc) at 4 x 105 cells/200 ⁇ L per well. The cell suspension was centrifuged (2000 rpm, 2 minutes, 4° C.), the supernatant was removed, and the cells were washed twice with 200 ⁇ L of staining buffer (0.5% FBS-containing PBS) and suspended. After centrifugation and removal of the supernatant, 50 ⁇ L of a solution containing a 50-fold diluted anti-mouse CD16/CD32 antibody was added and allowed to stand in the dark at 4° C. for 10 minutes.
- staining buffer (0.5% FBS-containing PBS
- 150 ⁇ L of staining buffer was added, the cells were centrifuged, the supernatant was removed, and the cells were washed twice with 200 ⁇ L of staining buffer.
- 150 ⁇ L of staining buffer was added, the cells were centrifuged, the supernatant was removed, and the cells were washed twice with 200 ⁇ L of staining buffer.
- the cells were resuspended in 200 ⁇ L of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and accompanying analysis software (FACSDiva). Dendritic cells were detected as CD11c-positive, macrophages as F4/80-positive, and Langerhans cells as CD11c-positive and CD207-positive populations.
- FIGS. 2-4 Staining of lymphocytes (FIGS. 2-4): Cell suspensions were added to 96-well V-bottom microplates (Nunc) at 4 ⁇ 10 5 cells/200 ⁇ L per well. The cell suspension was centrifuged (2000 rpm, 2 minutes, 4°C), the supernatant was removed, and the cells were washed twice with a staining buffer (0.5% FBS-containing PBS) and suspended.
- a staining buffer (0.5% FBS-containing PBS
- T cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and accompanying analysis software (FACSDiva). T cells were detected as CD3-positive, B cells as CD45 R (B220)-positive, and NK cells as KLRG1-positive populations.
- Antigen-specific T cell induction test (intracellular cytokine staining) (Fig. 2-6): A cell suspension was added to a 48-well culture plate (Nunc) at 4 x 105 cells/200 ⁇ L per well. 50 ⁇ L of mERK2 9m peptide was added at a concentration of 10 ⁇ g/mL as a short-chain peptide for stimulating CD8-positive T cells, and cultured at 37° C., 5% CO 2 for 1 hour. After that, 50 ⁇ L of Goldi Plug (BD Biosciences) diluted 167-fold with RPMI1640 medium containing 10% FBS was added per well and cultured at 37° C. and 5% CO 2 for 5 hours.
- Cells were harvested and transferred to 96-well V-bottom microplates (Nunc). After centrifugation (2000 ⁇ rpm, 2 minutes, 4° C.) to remove the supernatant, the cells were suspended in 200 ⁇ L of staining buffer per well. After washing the cells twice with 200 ⁇ L of staining buffer, add eFluor450-labeled anti-CD8 antibody and PerCP-Cy5.5-labeled anti-CD90.1 antibody to the cell suspension according to the concentration recommended by each antibody manufacturer. After mixing, the mixture was allowed to stand in the dark at 4°C for 15 minutes.
- the cells are washed with 200 ⁇ L of Perm/Wash buffer (BD Biosciences), and APC-labeled anti-mouse IFN- ⁇ antibody diluted 50-fold with Perm/Wash buffer is added to the cells and mixed. , and allowed to stand in the dark at room temperature for 15 minutes. After washing the cells twice with 200 ⁇ L of Perm/Wash buffer, they were resuspended in 200 ⁇ L of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and accompanying analysis software (FACSDiva).
- IFN- ⁇ -producing CD8-positive infused T cells are IFN- ⁇ -positive, CD90.1-positive, CD8-positive or IFN- ⁇ -positive, CD90.2-negative, CD8-positive, IFN- ⁇ -producing CD8-positive host T cells are IFN- ⁇ -positive, CD90.1-negative CD8-positive or IFN- ⁇ -positive CD90.2-positive CD8-positive populations were detected.
- Example 2 In order to clarify the effects of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy on antitumor immune responses, regional lymph nodes were collected from the HA vaccine administration site 14 days after tumor transplantation. , analyzed lymph node cells. As a result, the lymph nodes of the HANG-V alone group and the HANG-V + TCR-T combination group were significantly swollen, and in both groups, an increase of about 4 to 8 times the lymph node cells was observed compared to the untreated group. ( Figure 2-1). In addition, swollen lymph nodes were still observed 22 days after tumor implantation (Fig. 2-2).
- the HA nanogel cancer vaccine induces a large amount of influx and accumulation of antigen-presenting cells such as dendritic cells, macrophages, Langerhans cells, and lymphocytes of the host in the immunosuppressive environment of the tumor-bearing host. , suggesting the possibility of creating an efficient tumor antigen presentation environment. Furthermore, in this environment with improved tumor antigen presentation, as a result of supplementing T cells with antigen-specific T cell infusion therapy, CD8-positive infused T cells that flowed into the regional lymph nodes were able to efficiently receive antigen presentation.
- antigen-presenting cells such as dendritic cells, macrophages, Langerhans cells, and lymphocytes of the host in the immunosuppressive environment of the tumor-bearing host.
- Example 3 Therapeutic effect and anti-tumor immune response analysis by gradually decreasing the number of infused cells Regarding mouse tumor growth test (Example 1) and anti-tumor immune response analysis (Example 2), the number of antigen-specific T cells was reduced from 2 ⁇ 10 6 /200 ⁇ L. A similar test was performed by gradually decreasing the concentration to 1 ⁇ 10 6 cells/200 ⁇ L, 5 ⁇ 10 5 cells/200 ⁇ L, and 2.5 ⁇ 10 5 cells/200 ⁇ L.
- Example 4 Observation of Intratumor-Infiltrating CD8-Positive T Cells 14 days after the tumor transplantation in the test of Example 1, the tumor was excised and immunohistofluorescent staining was carried out by the following method.
- a tumor embedded in an OCT compound (Sakura Fine Tech) and frozen was sliced at a thickness of 5 ⁇ m, and the obtained tumor slice was air-dried.
- the dried tumor sections were fixed with ice-cold acetone for 10 minutes and used for immunostaining. After washing the tumor section with PBS three times, it was immersed in Blocking One Histo (Nacalai Tesque) and blocked in an incubation chamber for 30 minutes.
- Blocking One Histo Nacalai Tesque
- Anti-mouse CD8 antibody diluted 1:100 with Antibody Diluent with Background reducing Component (Dako) was then used to stain the tumor sections for 1 hour at room temperature in an incubation chamber. Furthermore, after washing three times with 0.1% Tween20-containing PBS, the tumor section was stained with an Alexa488-labeled anti-rat antibody diluted 1000-fold with PBS at room temperature for 1 hour in an incubation chamber. Finally, after washing three times with 0.1% Tween20-containing PBS, the tumor sections were immersed in Prolong Gold antifade reagent with DAPI (Life Technologies), and CD8 infiltrating into the tumor was examined using a fluorescence microscope BZ-710 (Keyence). Positive T cells were observed.
- Prolong Gold antifade reagent with DAPI Life Technologies
- Example 4 In order to clarify the effects of combined use of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy on the intratumoral environment of tumor-bearing hosts, the tumors of each mouse were excised 14 days after tumor implantation and infiltrated into the tumor. CD8-positive T cells were observed under a fluorescence microscope. As a result, in the HANG-V and TCR-T combination group, CD8-positive T cells were significantly observed in the tumor compared to the untreated or monotherapy group (Fig. 4). These results suggest that the combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy may induce tumor antigen-specific cytotoxic T cells (CTL) in the tumor and eliminate the tumor. .
- CTL tumor antigen-specific cytotoxic T cells
- Example 5 Analysis of CD8-positive transfused T cells in peripheral blood Twenty-one days after tumor implantation in the test of Example 3, peripheral blood was collected from the mouse orbit, suspended in 10% heparin-containing RPMI, and mixed. After that, the suspension was slowly added to 500uL of Ficoll (GE Healthcare) in an Eppendorf tube, and after centrifugation (4000rpm x 10min, 25°C, AC:1, DE:1), the mononuclear cell layer was stained. buffer (0.5% BSA-containing PBS). After centrifuging the cell suspension (5000 rpm ⁇ 1 min, 25° C.), the supernatant was removed with an aspirator and washed twice with 0.5% FBS-containing PBS.
- Ficoll GE Healthcare
- HA nanogel cancer vaccine and antigen-specific T cell infusion therapy promotes the influx and accumulation of antigen-presenting cells and infused T cells into regional lymph nodes, resulting in antigen presentation. It was found that transfused T cells can be induced into tumor antigen-specific cytotoxic T cells (CTL) through the transfusion, and ICI-resistant tumors can be eliminated and cured. In addition to the advantage of being able to qualitatively and quantitatively increase the number of infused T cells, this therapy also works on the host's T cells, making it possible to shift the host's immunosuppressive environment to an anti-tumor environment.
- CTL tumor antigen-specific cytotoxic T cells
- cancer vaccines enhance the antigen-presenting ability of antigen-presenting cells and are expected to have an antitumor effect on T cells. Even if it can be presented to cells, the therapeutic effect is limited if there are few T cells in the body, and it is necessary to replenish T cells at the same time as the vaccine.
- antigen-specific T cell infusion therapy is a treatment method in which the T cells of cancer patients are transformed into antigen-specific T cells outside the body and returned to the body. To sustain this, continuous infusion of T cells and maintenance of good quality T cells in the body are important issues.
- the HA nanogel cancer vaccine creates an efficient tumor antigen presentation environment in the regional lymph nodes, and T cell infusion therapy supplements this environment with T cells.
- T cell infusion therapy supplements this environment with T cells.
- Example 6 to 16 ⁇ Materials and methods (Examples 6 to 16)>
- Cellular RPMI1640 medium (2-mercaptoethanol added) was purchased from Cell Science Institute.
- D-MEM medium was purchased from Wako Pure Chemicals, and fetal bovine serum (FBS) was purchased from Gibco.
- Mouse fibrosarcoma cell line CMS5a was obtained from Memorial Sloan Kettering Cancer Institute, and mouse metastatic melanoma cell line B16F10 was purchased from ATCC and subcultured at Mie University.
- the mouse fibrosarcoma cell line CMS5a expresses a mutant ERK2 protein.
- a peptide (QYIHSANVL (SEQ ID NO: 1)) containing a mutant portion of the mutant ERK2 protein is recognized by CD8-positive cytotoxic T cells of 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 CD8-positive cytotoxic T cell recognition epitope sequence (QYIHSANVL) of the mutant ERK2.
- 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 inhibits CD8-positive cytotoxicity in C57BL/6 mice.
- Transgenic mice (Pmel-1 mice) transgenic with a TCR that is recognized by T cells and recognizes this CD8-positive cytotoxic T cell recognition epitope sequence have been generated.
- the long peptide antigen used in this example contains this sequence.
- mice Female BALB/c mice and female C57BL/6 mice aged 6 to 8 weeks were purchased from Japan SLC. DUC18 transgenic mice were obtained from the University of Washington and bred at the Mie University Animal Research Facility. Pmel-1 transgenic mice were purchased from The Jackson Laboratory and bred at the Mie University Animal Research Facility. All mice were bred at the Mie University Animal Research Facility. The animal experiment protocol was approved by the Ethics Committee of Mie University School of Medicine.
- solid purified sucrose manufactured by FUJIFILM Wako Co., Ltd., product number 198-18385, exclusive for manufacturing
- the composition containing the peptide-hyaluronic acid nanogel complex was 10 mass% sucrose, and allowed to stand for 1 hour or longer. Stirred.
- the peptide was diluted with 25 mM phosphate buffer (pH 7.4) containing 10% by mass of sucrose so that the theoretical concentration of the peptide was 0.3 mg/mL, and 0.2 ⁇ m PES (polyethersulfone) (manufactured by Pall, Acrodisc) was added.
- a composition containing the peptide-hyaluronic acid nanogel complex was obtained by sterile filtration with a syringe filter (25 mm ⁇ ). After quantifying the peptide concentration in the composition containing the peptide-hyaluronic acid nanogel complex at this time by reverse phase chromatography analysis, 10% sucrose 10 mM phosphate buffer (pH 7.4) was added so that the peptide concentration was 250 ⁇ g / mL. ) to adjust the concentration and prepare a dosing solution.
- a peptide-hyaluronic acid nanogel complex was prepared as follows.
- the gp100 TRP2 TRP1_6Y peptide was purchased from Biologica. The sequence is: SVYDFFVWLYYYYYYTWHRYHLLYYYYYY EGSRNQDWL (SEQ ID NO:5).
- the sequence includes the following TRP2 (amino acid sequence: SVYDFFVWL (SEQ ID NO: 6)), TRP1 (amino acid sequence: TWHRYHLL (SEQ ID NO: 7)), gp100 (amino acid sequence: EGSRNQDWL (sequence number 4)), with six Ys connecting each epitope.
- the hyaluronic acid nanogel aqueous solution and the peptide dimethylsulfoxide solution were mixed at a volume ratio of 102:1 and stirred at room temperature for 24 hours to obtain a composition containing the peptide-hyaluronic acid nanogel complex.
- solid purified sucrose manufactured by FUJIFILM Wako Co., Ltd., product number 198-18385, exclusive for manufacturing
- the composition containing the peptide-hyaluronic acid nanogel complex was 10 mass% sucrose, and allowed to stand for 1 hour or longer. Stirred.
- the peptide was diluted with 25 mM phosphate buffer (pH 7.4) containing 10% by mass of sucrose so that the theoretical concentration of the peptide was 0.3 mg/mL, and 0.2 ⁇ m PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe) was added. Filter, 25 mm ⁇ ) to obtain a composition containing a peptide-hyaluronic acid nanogel complex.
- PES polyethersulfone
- CpG oligo DNA1668 was purchased from Ajinomoto Bio-Pharma Service Gene Design.
- ⁇ Fluorescent-labeled eFluor450-labeled anti-mouse CD8a antibody (clone 53-6.7), eFluor450-labeled anti-mouse KLRG1 antibody (clone 2F1), APC-labeled anti-mouse CD44 (clone IM7) and APC-labeled anti-mouse IFN- ⁇ 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.
- Anti-mouse PD-1 antibody (clone RMPI-14) was purchased from BioCell.
- Short peptide antigen A synthetic short peptide (QYIHSANVL (SEQ ID NO: 1) for stimulating CD8-positive T cells was purchased from Sigma Genosys.
- EGSRNQDWL (SEQ ID NO: 4) and VYDGREHTV (SEQ ID NO: 9) were purchased from Eurofins. did.
- Example 6 Tumor growth test (metastatic melanoma cell line B16F10) Metastatic melanoma cell line B16F10 was cultured in D-MEM medium containing 10% FBS using a T75 culture flask (Corning). The cultured cells were detached using PBS containing 0.5% trypsin and suspended in D-MEM medium containing 10% FBS. After centrifuging the suspension (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed. After that, they were washed twice with D-MEM medium and suspended in D-MEM medium at a concentration of 2 ⁇ 10 5 cells/100 ⁇ L. A dose of 100 ⁇ L/individual was subcutaneously implanted into the right anterior dorsum of C57BL/6 mice (5 per group).
- CD8-positive T cells were extracted from the spleen of gp100-recognizing TCR transgenic mouse Pmel-1 (CD90.1-positive) on days 8 and 12 after tumor transplantation using CD8a microbeads. (Miltenyi), suspended in RPMI1640 medium at a concentration of 2 ⁇ 10 6 cells/200 ⁇ L, and then injected at 200 ⁇ L from the mouse tail vein (TCR-T group). Thereafter, the tumor area was measured over time.
- Example 6 As in Example 1 (ICI-resistant fibrosarcoma cell line CMS5a), metastatic Long-chain peptide antigen-loaded HA nanogel cancer vaccine using malignant melanoma cell line B16F10 tumor-bearing C57BL/6 mice. was prepared and combined with gp100-recognizing TCR gene transfected T cell infusion therapy to examine the therapeutic effect. As a result, in the HANG-V treatment group, suppression of B16F10 tumor growth was observed compared to the untreated group and the TCR-T alone group (Figs. 6-1 and 6-2).
- the combined HANG-V and TCR-T group exhibited an antitumor effect even against B16F10 tumors that once reached about 100 mm 2 , and the tumors regressed significantly in all individuals (Fig. 6-2). Two of them showed complete tumor disappearance.
- a crater-shaped cavity tumor necrosis was observed in the center of the tumor, which is thought to be caused by the tumor-damaging action of cytotoxic T cells (CTL). ( Figure 6-3).
- HA nanogel cancer vaccine and antigen-specific T cell infusion therapy is effective not only for immune checkpoint inhibitor (ICI) treatment-resistant tumors, but also for malignant melanoma with poor prognosis with metastasis such as B16F10. It also exerts a strong anti-tumor effect against and can cure tumors, suggesting the possibility of becoming an epoch-making treatment for “Cold tumor” and “Immune desert tumor”.
- ICI immune checkpoint inhibitor
- Example 7 Tumor growth test (comparison of HA nanogel vaccine and CHP nanogel vaccine) Regarding 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.
- HANG-V or CHP-V 50 ⁇ g (75 ⁇ g after the 3rd time) was dissolved in PBS on days 7, 11, 15, 29 and 36 after tumor transplantation, and 50 ⁇ g of CpG was administered.
- the oligo DNA was administered subcutaneously to the right hind dorsal region of mice and transfused with antigen-specific T cells (4 ⁇ 10 6 ).
- Example 7 To clarify whether the combination of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy is superior to CHP nanogel cancer vaccine and antigen-specific T cell infusion therapy (prior art) in terms of antitumor effect A study was conducted to compare the therapeutic effects of both. As a result, as in Example 6, in the HANG-V and TCR-T combination group, the growth of B16F10 tumors was significantly suppressed in all individuals, and the tumors could be strongly regressed, whereas CHP-V and TCR-T Similar therapeutic effects were not observed in the T combination group ( Figures 7-1 and 7-2).
- the HANG-V and TCR-T combination group showed not only tumor size (area) but also upward progression and growth (height). Tumors were remarkably suppressed, and crater-like cavities (tumor necrosis), which were considered to be caused by the tumor-damaging action of CTL, were observed in the central part of the tumor in all individuals (Fig. 7-3). After that, of all the animals that showed regression, the tumor part became scab-like in 2 animals, and the tumor disappeared completely. Regressed. On the other hand, in the CHP-V and TCR-T combination group, tumor growth was promoted without being suppressed, and all individuals died within 50 days, whereas in the HANG-V and TCR-T combination group 100% viability was maintained (Figure 7-4).
- the combined use of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy has a tumor growth inhibitory effect, tumor toxic effect, and survival time compared to the prior art CHP nanogel cancer vaccine and T cell infusion combination therapy. It was found to have a superior therapeutic effect in all prolongation effects.
- Example 8 Analysis of gp100 antigen-specific CD8 T cells in the regional lymph nodes of the vaccine administration site The same test as in Example 6 was performed, and the regional lymph nodes of the vaccine administration site (right inguinal lymph node) were collected 14 days after tumor transplantation. (2-3 animals per group), the lymph nodes were ground using a slide glass, and the cells were suspended in RPMI1640 medium. After centrifugation (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed, washed twice with RPMI1640 medium, and suspended in RPMI1640 medium containing 10% FBS. The number of cells was measured (Fig. 8-1), and the cell concentration was adjusted to 2 x 106 cells/mL.
- FIGS. 8-2 and 8-3 Staining of gp100 tetramer cells (FIGS. 8-2 and 8-3): A cell suspension was added to a 96-well V-bottom microplate (Nunc) at 4 ⁇ 10 5 cells/200 ⁇ L per well. Centrifuge the cell suspension (2000 rpm, 2 minutes, 4 ° C), remove the supernatant, wash twice with 200 ⁇ L of staining buffer (PBS containing 0.5% FBS), suspend, and obtain a cell suspension. was added at 50 ⁇ L per well.
- PBS containing 0.5% FBS staining buffer
- Antigen-specific T cell induction test (intracellular cytokine staining) (Fig. 8-4): A cell suspension was added to a 48-well culture plate (Nunc) at 4 x 105 cells/200 ⁇ L per well. 50 ⁇ L of gp100 peptide as a short-chain peptide for stimulating CD8-positive T cells was added to a final concentration of 10 ⁇ M, and cultured at 37° C. in a 5% CO 2 incubator for 1 hour. After that, 50 ⁇ L of Goldi Plug (BD Biosciences) diluted 167-fold with RPMI1640 medium containing 10% FBS was added per well and cultured at 37° C. in a 5% CO 2 incubator for 5 hours.
- Goldi Plug diluted 167-fold with RPMI1640 medium containing 10% FBS was added per well and cultured at 37° C. in a 5% CO 2 incubator for 5 hours.
- Cells were collected, transferred to a 96-well V-bottom microplate (Nunc), centrifuged (2000 ⁇ rpm, 2 minutes, 4°C) to remove the supernatant, and then suspended in 200 ⁇ L of staining buffer per well. did. After washing the cells twice with 200 ⁇ L of staining buffer, FITC-labeled anti-CD8 antibody and Brilliant Violet 510-labeled anti-mouse CD90.1 antibody were added to the cell suspension and mixed according to the concentration recommended by the manufacturer of each antibody. After that, it was allowed to stand in a dark place at 4°C for 15 minutes.
- the cells After centrifuging and removing the supernatant, the cells are washed with 200 ⁇ L of Perm/Wash buffer (BD Biosciences), and APC-labeled anti-mouse IFN- ⁇ antibody diluted 50-fold with Perm/Wash buffer is added to the cells and mixed. It was allowed to stand in a dark place at room temperature for 15 minutes. After washing the cells twice with 200 ⁇ L of Perm/Wash buffer, they were resuspended in 200 ⁇ L of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and accompanying analysis software (FACSDiva). IFN- ⁇ -producing CD8-positive infused T cells were detected as an IFN- ⁇ -positive CD90.1-positive CD8-positive population.
- Perm/Wash buffer BD Biosciences
- Example 8 To clarify the effects of HA nanogel cancer vaccine and antigen-specific T cell infusion therapy on gp100 antigen-specific CD8-positive T cells, regional lymph nodes were collected 14 days after tumor transplantation. Then, the same CD8 T cells were analyzed. As a result, the lymph nodes in the HANG-V alone group and the HANG-V + TCR-T combination group showed significant swelling, and in both groups, an increase in lymph node cells of approximately 9 to 16 times was observed compared to the untreated group. ( Figure 8-1).
- HA nanogel cancer vaccine strongly induces gp100 antigen-specific CTL in the regional lymph nodes of CD8-positive infused T cells in the immunosuppressive environment of tumor-bearing hosts, and suppresses B16F10 tumors. gender was suggested.
- Example 9 Examination of localization of antigen-specific CD8-positive infused T cells in the tumor and in the whole body .
- Non-draining lymph nodes left inguinal lymph nodes
- spleens spleens
- tumors and peripheral blood were collected, each cell was separated by the method shown below, and CD8-positive transfused T cells and gp100 tetramer-positive CD8-positive T cells present in each tissue were analyzed. The percentage of cells was examined.
- lymph node cells and spleen cells Separation of lymph node cells and spleen cells: Lymph nodes (affiliated and non-affiliated) and spleens were ground using a slide glass, and the liberated cells were collected in RPMI1640 medium. After centrifugation (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed and, for splenocytes, 2 mL of erythrocyte lysing solution was added to treat the cells for 1 minute. After that, 18 mL of RPMI1640 medium was added, centrifuged (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed, and the cells were suspended in RPMI1640 medium (primary cell suspension).
- RPMI1640 medium primary cell suspension
- TIL tumor-infiltrating immune cells
- Peripheral blood mononuclear cell (PBMC) separation Peripheral blood mononuclear cells were collected in the same manner as in Example 5. The collected primary cell suspension of each tissue was stained and measured in the same manner 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.
- PBMC Peripheral blood mononuclear cell
- Example 10 Effect of suppressing recurrence after treatment
- peripheral blood was collected from 2 individuals in whom complete tumor regression was observed (5 months after tumor transplantation), and gp100 antigen-specific CD8-positive infused T cells were collected.
- one of the two individuals was reimplanted with B16F10 tumors (2 ⁇ 10 5 ), and the other individual was given 50 ⁇ g of 75 ⁇ g of long-chain peptide antigen-loaded HA nanogel cancer vaccine dissolved in PBS.
- peripheral blood was recollected, and changes in the percentage, activation, and transfusion of each gp100 antigen-specific CD8-positive T cell The phenotype was examined. Furthermore, B16F10 tumors (2 ⁇ 10 5 ) were reimplanted in the same mice, and the presence or absence of recurrence was examined.
- Peripheral blood mononuclear cells (PBMC) were collected in the same manner as in Example 5, and tetramer staining was performed in the same manner as in Example 8. Cells were stained and measured by the following method.
- the cells were resuspended in 200 ⁇ L of staining buffer and transferred to a round-bottom polystyrene tube (BD Biosciences). Cells were analyzed using a flow cytometer FACS Canto II (BD Biosciences) and accompanying analysis software (FACSDiva). gp100 antigen-specific CD8-positive infused T cells were detected as gp100 tetramer-positive, CD90.1-positive, CD8-positive, effector memory cells as CD44-positive, CD62L-negative, and central memory cells as CD44-positive and CD62L-positive.
- this vaccine Combined with antigen-specific T cell infusion therapy, this vaccine exerts a strong anti-tumor effect against malignant melanoma with poor prognosis with metastasis such as B16F10, and also has a preventive effect on tumor recurrence. It is expected that it can become an innovative cancer vaccine that combines both type and preventive type.
- Example 11 Effect of vaccine treatment on individuals who changed from response to re-growth after treatment. 27, 32, 36, 40, and 43 days after tumor transplantation, 75 ⁇ g of long-chain peptide antigen-loaded HA nanogel cancer vaccine was injected with 50 ⁇ g of CpG oligo DNA dissolved in PBS into the right side of the mouse. It was administered subcutaneously on the back.
- Antigen-specific CD8-positive T cells (2 ⁇ 10 6 ) were isolated from the spleen of mutant ERK2-recognizing TCR transgenic mice (DUC18) using CD8a microbeads (Miltenyi) 41 days after tumor transplantation. was injected via the tail vein of the mouse. On the 49th day after tumor implantation, ICI (anti-mouse PD-1 antibody 200 ⁇ g and anti-mouse CTLA-4 antibody 100 ⁇ g) was intraperitoneally administered, and the tumor area was measured over time.
- Example 11 After HA nanogel cancer vaccine and antigen-specific T-cell infusion therapy were performed, the vaccine was re-administered, re-infusion, and ICI were performed for individuals who were once successfully treated but whose tumor re-growth was later observed. As a result, relapsed tumors generally grow rapidly and have a poor prognosis (Fig. 11-2, right). It was found that tumor aggravation could be suppressed (FIGS. 11-1 and 11-2 left).
- Example 12 Recurrence inhibitory effect on individuals who achieved a complete response (complete regression) after treatment (1 ⁇ 10 6 cells) were reimplanted and the area of the tumor was measured.
- Example 12 After HA nanogel cancer vaccine and antigen-specific T cell infusion therapy, the CMS5a tumor was re-implanted in mice that showed complete tumor regression. As a result, tumor growth was suppressed in all mice, and 8 mice. Six of them achieved complete tumor regression (75%) (Figs. 12-1 and 12-2).
- this treatment can cure ICI treatment-resistant tumors, and at the same time, similar to the results of Example 10, memory immunity is acquired (memory formation) against the tumor after the treatment is effective, and the same tumor recurs. Antigen-specific memory T cells rapidly recognize and injure them, and suppress tumor recurrence. Further, as in Example 10, re-inoculation of the vaccine is thought to induce activated antigen-specific CD8 memory T cells with strong effector functions and further prevent recurrence.
- Example 13 Regarding the combined effect of vaccine and antigen-specific T cell infusion therapy (single dose) mouse tumor growth test (Example 1), the antigen-specific T cell infusion therapy was reduced from 2 times to 1 time, and a similar test was conducted (treatment 9 per group).
- the HA nanogel cancer vaccine can sufficiently cure ICI-resistant tumors with a single infusion without having to perform two antigen-specific T cell infusions.
- TCR-T and CAR-T T-cell infusion therapy
- improvement is an issue. can cure ICI-resistant tumors, and can be a treatment with less physical and economic burden on patients.
- Example 14 Therapeutic effect of vaccines, etc. on individuals who changed from response to re-growth after treatment Example 13.
- 75 ⁇ g of the long-chain peptide antigen-loaded HA nanogel cancer vaccine was subcutaneously administered to the right hind dorsal region of the mouse together with 50 ⁇ g of CpG oligo DNA dissolved in PBS.
- ICI anti-mouse PD-1 antibody 200 ⁇ g and anti-mouse CTLA-4 antibody 100 ⁇ g
- Example 14 After the HA nanogel cancer vaccine and antigen-specific T cell infusion therapy (single dose), the vaccine and ICI were administered to individuals whose tumors were found to re-grow even though the treatment was successful once. As a result, relapsed tumors generally grow rapidly and have a poor prognosis, whereas continuous administration of vaccines and combined use of ICI can remarkably suppress tumor growth (Fig. 14).
- Example 15 After the HA nanogel cancer vaccine and antigen-specific T cell infusion therapy (single dose), CD8-positive T cells present in the peripheral blood of individuals in whom complete tumor regression was observed were analyzed. CD8-positive T cells exhibiting memory and central memory phenotypes were included, and revaccination revealed a marked increase in the proportion of both cells ( Figure 15).
- Example 16 Memory T cell induction effect by re-administration of vaccine to individuals who changed from response to repopulation after treatment (additional treatment) After 36 days), the memory phenotype of CD8-positive T cells was analyzed. In addition, 75 ⁇ g of long-chain peptide antigen-loaded HA nanogel cancer vaccine was administered subcutaneously to the right dorsal region of the mouse together with 50 ⁇ g of CpG oligo DNA dissolved in PBS (on the same day after blood collection), and the memory phenotype of the same cells was re-examined. analyzed (41 days after tumor implantation). Cell staining and measurement were performed in the same manner as in Example 10.
- Example 16 After the HA nanogel cancer vaccine and antigen-specific T cell infusion therapy (single dose), the CD8 positive T cells present in the peripheral blood of individuals (under additional treatment) who changed from response to repopulation were analyzed. Individuals undergoing additional treatment, as well as individuals with regressions, contain CD8-positive T cells exhibiting effector memory and central memory phenotypes, and revaccination markedly increases the proportion of both cells. (Fig. 16).
- the vaccination of this vaccine is effective for effector memory and central memory CD8 positive T cells. It was suggested that the cells could be remarkably increased and induced, and that memory T cells and therapeutic effects could be induced sustainably.
- This treatment can significantly increase antigen-specific CD8 T cells in the regional lymph nodes at the site of vaccination and induce highly activated antigen-specific cytotoxic CD8 T cells (CTL) (Example 8 ).
- CTL cytotoxic CD8 T cells
- the transfused antigen-specific CD8 T cells well infiltrate into the tumor and are remarkably localized throughout the body, and may suppress tumor metastasis (Example 9).
- this treatment can prevent recurrence after treatment success, and has both aspects of a cancer treatment type vaccine and a recurrence prevention type vaccine (Examples 10 and 12). 6.
- memory T cells can be strongly and sustainably induced rapidly by re-administration of the vaccine after the therapeutic response by this treatment or during additional treatment (Example 10 and Examples 15 to 16). 7.
- tumor growth can be suppressed by continuous vaccination or combined use with other immunotherapies (Examples 11 and 14).
- this treatment exhibits a remarkable therapeutic effect even with a single treatment, and can reduce the physical and economic burden on the patient (Example 13).
- Example 17 Antitumor Test Using Lymphocytes Expressing Chimeric Antigen Receptors and Administration Compositions Containing Hyaluronic Acid Derivatives and Antigens Test Results of Administration Compositions Containing TCR-T, Hyaluronic Acid Derivatives, and Antigens in Examples 1 to 16 Therefore, antigen-presenting cells that have incorporated hyaluronic acid derivatives and antigens efficiently present antigens on the cell surface, and the activation and proliferation of transfused T cells by these antigen-presenting cells is the mechanism of high antitumor effects.
- RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors, infused lymphocytes expressing chimeric antigen receptors
- an antitumor effect is enhanced by expressing an antigen recognized by a chimeric antigen receptor in an antigen-presenting cell. Therefore, not only TCR-T but also chimeric antigen receptor-expressing lymphocytes can be transfused by administering an administration composition containing the antigen and a hyaluronic acid derivative that produces antigen-incorporated antigen-presenting cells in the body. can be expected to have a high antitumor effect.
- lymphocytes By using the following chimeric antigen receptor-expressing lymphocytes, tumors expressing antigens that can be recognized by chimeric antigen receptors, and HLA transgenic mice, lymphocytes expressing chimeric antigen receptors, hyaluronic acid derivatives and antigens It is possible to demonstrate that an administration composition comprising has a high anti-tumor effect.
- ⁇ Materials and methods> Cells Mouse colon cancer cell lines MC38 and CT26 were purchased from ATCC and subcultured at Mie University.
- the retrovirus packaging cell line Plat-E was purchased from CELL BIOLABS.
- RPMI1640 medium was purchased from 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.
- Albuminar was purchased from CSL Behling and human IL-2 from Novartis.
- HLA-A2 transgenic mice were produced by injecting HHD-A2 plasmid DNA provided by the Pasteur Institute into fertilized eggs of C57BL/6 mice at the Mie University Animal Research Facility. The same mice were bred at the Mie University Animal Research Facility, and HLA-A2 Tg mice (BALB/c background) that were back-crossed with BALB/c mice were generated and bred. Used for experiments.
- 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- ⁇ antibody and Biotin Rat Anti-Mouse IFN- ⁇ antibody were purchased from BD Biosciences.
- Lipofectamine 3000 Reagent, P300 reagent and human IFN- ⁇ uncoated ELISA kit were purchased from Invitrogen.
- the pCL-ECO Retrovirus Packaging Vector was purchased from NOVUS.
- A2 MAGE-A4 tetramer was made using Streptavidin-R-Phycoerithrin (Agilent).
- RetroNectin was purchased from Takara Bio.
- mMAGE#17 zG CAR MAGE#17scFv used for CAR production was isolated by reacting a human antibody library with an artificially produced HLA-A*02:01 MAGE-A4 p230-239 peptide MHC complex (particularly Application 2019-523909, etc./Antigen-binding protein that recognizes MAGE-A4-derived peptides).
- a human VH leader sequence was ligated to the 5' side of scFv, and mCD8 hinge, mCD28 TM, mCD28 ICD, mCD3z and mGITR ICD were designed to ligate to the 3' side, and an artificial gene was constructed (accepted by Genscript). This gene was incorporated into a retroviral expression plasmid to prepare an mMAGE zG viral vector plasmid (Figs. 17-1a and 17-1b).
- MAGE-A4 HHD-A2-expressing MC38 cells and CT26 cells
- mNGFR co-expressing MAGE-A4 The human MAGE-A4 gene, the P2A peptide sequence, and the mouse NGFR gene of the C-terminal truncate were ligated to design and create an artificial gene (accepted by Genscript). This gene was digested with restriction enzymes NotI and XhoI, excised, and integrated into a retrovirus expression plasmid to prepare a plasmid (Figs. 17-3a and 17-3b).
- HHD-A2 We deleted the intron part from the MHC sequence of the HHD-A2 plasmid, designed an mRNA-type HHD-A2 expression gene, and produced an artificial gene (accepted by Genscript). This gene was excised with NotI-XhoI and incorporated into a retroviral expression plasmid (Figs. 17-4a and 17-4b).
- Retrovirus packaging cell line Plat-E (1.2 ⁇ 10 6 cells) was cultured in a 6-well plate in high-glucose D-MEM medium for 24 hours.
- Opti-MEM 250 ⁇ L
- Lipofectamine 3000 Reagent 7 ⁇ L
- Opti-MEM 250 ⁇ L
- mMAGE#17 zG CAR viral vector plasmid 3 ⁇ L
- pCL-ECO Retrovirus Packaging Vector (1 ⁇ L)
- P300 reagent (6 ⁇ L) were mixed and mixed. and allowed to stand at room temperature for 15 minutes.
- the mixed solution was added, and the cells were cultured at 37°C in a 5% CO 2 incubator for 6 hours. After that, the medium in the 6-well plate was completely replaced, and after 24 hours, 48 hours and 72 hours, the medium was collected, filtered and sterilized with a 0.45 ⁇ m filter, and cryopreserved.
- Anti-mouse CD3e antibody (1 ⁇ g/mL) and anti-mouse CD28 antibody (2.5 ⁇ g/mL) were added to a 6-well plate the day before, left overnight at 4°C, and the immobilized plate was washed 3 times with PBS. After that, 5 mL of the cell suspension (1.5 ⁇ 10 7 cells) was seeded and cultured in a 37° C., 5% CO 2 incubator for 24 hours.
- RetroNectin was adjusted to 20 ⁇ g/mL with PBS, 500 ⁇ L was added to each well of a 24-well plate (Non-Treated Multidishes, Nunc), and allowed 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, added to the plate by 1 mL, and centrifuged (2000 ⁇ g, 2 hours, 32° C.). After washing the plate twice with PBS supplemented with 1.5% albumin, mouse spleen cells were collected from the CO 2 incubator and treated with 10% FBS supplemented with human IL-2 (600IU/ ⁇ L) at a concentration of 3.3 ⁇ 10 5 cells/mL.
- Retrovirus 1-1(1 ) instead of the mMAGE#17 zG CAR viral vector plasmid prepared in 1-2(1), mNGFR co-expressing MAGE-A4 viral vector plasmid prepared in 1-2(2), or the HHD-A2 viral vector plasmid prepared in 1-2(2)
- a retrovirus was prepared in the same manner as described in 2-1, except that .
- mice colon cancer cell lines MC38 and CT26 were 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. After centrifuging the suspension (400 ⁇ g, 5 minutes, 4° C.), the supernatant was removed. Thereafter, the cells were washed twice with RPMI1640 medium containing 10% FBS and suspended in RPMI1640 medium containing 10% FBS at a concentration of 3.3 ⁇ 10 5 cells/mL.
- RetroNectin was adjusted to 20 ⁇ g/mL with PBS, 500 ⁇ L was added to each well of a 24-well plate (Non-Treated Multidishes, Nunc), and allowed to stand overnight at 4°C.
- each virus solution prepared in 3-1 was diluted two-fold, added to the plate by 1 mL, and centrifuged (2000 ⁇ g, 2 hours, 32° C.).
- 1.5 mL of MC38 cells (3.3 ⁇ 10 5 cells/mL) suspended in RPMI1640 medium containing 10% FBS were seeded on the plate. After centrifugation (1000 ⁇ g, 10 minutes, 32° C.), the cells were cultured at 37° C. in a 5% CO 2 incubator for 3 days (the same applies to the CT26 cell line).
- HHD-A2 PE-labeled anti-HLA-A2 antibody was added to the cell suspension according to the concentration recommended by the manufacturer, and allowed to stand in the dark at 4°C for 15 minutes.
- Intracellular staining of MAGE A4 Cells adjusted to 1 ⁇ 10 5 cells were well mixed with 100 ⁇ L of cell fixative (Cytofix Cytoperm: BD Biosciences) and allowed to stand in the dark at room temperature for 20 minutes. After washing with Perm/Wash buffer (BD Biosciences) diluted 10-fold with PBS, add 0.25 ⁇ L of MAGE-A4 (E7O1U) XP Rabbit mAb to 50 ⁇ L of Perm/Wash buffer and let stand at room temperature in the dark for 20 minutes. did.
- the plate was washed twice with Perm/Wash buffer, and 0.25 ⁇ L of Alexa Fluor 647-labeled anti-rabbit IgG antibody was added to 50 ⁇ L of Perm/Wash buffer and mixed, and allowed to stand in the dark at room temperature for 20 minutes. After washing twice with Perm/Wash buffer, the cells were suspended in staining buffer and transferred to a round-bottomed polystyrene tube (BD Biosciences). Cells were analyzed using a flow cytometer LSRFortessa Cell Analyzer (BD Biosciences) and attached analysis software (FACSDiva) (Fig. 17-6).
- HHD-A2 and MAGE-A4-introduced MC38 cells were suspended in RPMI1640 medium containing 10% FBS at a concentration of 5 ⁇ 10 4 cells/100 ⁇ l.
- mMAGE zG CAR T cells (1 ⁇ 10 5 cells/100 ⁇ l) suspended in RPMI1640 medium containing % FBS or CAR gene-unintroduced cells were added to the plate, co-cultured for 24 hours, and the culture supernatant was collected.
- CT26 cells were similarly co-cultured with mMAGE zG CAR T cells, and the culture supernatant was collected. The collected culture supernatant was evaluated for IFN- ⁇ production by the ELISA method shown below.
- IFN- ⁇ ELISA 50 ⁇ L of Purified Rat Anti-Mouse IFN- ⁇ antibody adjusted to 10 ⁇ g/mL with 0.05% NaN 3 PBS was added to a 96-well flat-bottom plate and allowed to stand overnight at 4° C. for immobilization. On the following day, after washing the plate three times with PBS, 50 ⁇ L of the collected cell culture supernatant and standard solution were added to each well and allowed to stand at room temperature for 2 hours. The IFN- ⁇ standard solution was prepared by adjusting the stock solution to 1000 pg/mL with the standard solution and diluting it in four 5-fold steps.
- a peptide-hyaluronic acid nanogel complex was prepared as follows.
- MAGE-A4 peptide was purchased from Eurofins.
- the sequence is: AMEGDSASEEEIWEELGVMGVYDGREHTV (SEQ ID NO:8).
- the sequence contains GVYDGREHTV (SEQ ID NO: 9) as a CD8 positive cytotoxic T cell recognition epitope sequence.
- Hyaluronic Acid Nanogel-Antigen Complex Lyophilized hyaluronic acid nanogel was weighed, added with water for injection so as to give a concentration of 5 mg/mL, and stirred overnight to fully dissolve. On the other hand, in another container, the peptide was dissolved in dimethylsulfoxide to a concentration of 50 mg/mL. After confirming the dissolution, the hyaluronic acid nanogel aqueous solution and 1 mol/L sodium hydroxide aqueous solution were mixed at room temperature at a volume ratio of 100:2.
- the hyaluronic acid nanogel aqueous solution and the peptide dimethylsulfoxide solution were mixed at a volume ratio of 102:1 and stirred at room temperature for 24 hours to obtain a composition containing the peptide-hyaluronic acid nanogel complex.
- solid purified sucrose manufactured by FUJIFILM Wako Co., Ltd., product number 198-18385, exclusive for manufacturing
- the composition containing the peptide-hyaluronic acid nanogel complex was 10 mass% sucrose, and allowed to stand for 1 hour or longer. Stirred.
- the peptide was diluted with 25 mM phosphate buffer (pH 7.4) containing 10% by mass of sucrose so that the theoretical concentration of the peptide was 0.3 mg/mL, and 0.2 ⁇ m PES (polyethersulfone) (manufactured by Pall, Acrodisc syringe) was added. Filter, 25 mm ⁇ ) 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 reversed-phase chromatography analysis, and the peptide concentration was adjusted to 250 ⁇ g/mL to prepare an administration solution.
- the MAGE-A4/HHD-A2-expressing MC38 cells prepared in 3-2 are subcutaneously transplanted to the right front dorsal region of HLA-A2 Tg mice for antitumor test at 5 ⁇ 10 6 cells/mouse.
- the chimeric antigen receptor-expressing lymphocytes prepared in 2-2 are suspended in PBS at a concentration of 3 ⁇ 10 6 cells/200 ⁇ L, and 200 ⁇ L is injected into the tail vein of the mouse.
- the long-chain peptide antigen-loaded HA nanogel cancer vaccine prepared in 4-1 and 4-2 50 ⁇ g of the long-chain peptide antigen-loaded HA nanogel cancer vaccine was dissolved in PBS on days 4, 9 (and 14) after tumor transplantation. Mice are administered subcutaneously on the right hind dorsum with 50 ⁇ g of CpG oligo DNA. Tumor area is then measured over time.
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Abstract
Description
[式中、R1、R2、R3、およびR4は、それぞれ独立に、水素原子、C1-6アルキル、ホルミルおよびC1-6アルキルカルボニルから選択され;
R5は、水素原子、ホルミル、またはC1-6アルキルカルボニルであり;
Zは、直接結合、または2~30個の任意のアミノ酸残基からなるペプチドリンカーを表し;X1は、以下の式:
-NRb-R、
-NRb-COO-R、
-NRb-CO-R、
-NRb-CO-NRc-R、
-COO-R、
-O-COO-R、
-S-R、
-CO-Ya-S-R、
-O-CO-Yb-S-R、
-NRb-CO-Yb-S-R、および
-S-S-R、
により表される基から選択される疎水性基であり;
Ra、RbおよびRcは、それぞれ独立に、水素原子、C1-20アルキル、アミノC2-20アルキルおよびヒドロキシC2-20アルキルから選択され、ここで当該基のアルキル部分は、-O-および-NRf-から選択される1~3個の基が挿入されていてもよく;
Rfは、水素原子、C1-12アルキル、アミノC2-12アルキルおよびヒドロキシC2-12アルキルから選択され、当該基のアルキル部分は-O-および-NH-から選択される1~2個の基が挿入されていてもよく;
Rは、ステリル基であり;
Yは、C2-30アルキレン、または-(CH2CH2O)m-CH2CH2-であり、ここで、当該アルキレンは、-O-、-NRg-および-S-S-から選択される1~5の基が挿入されていてもよく;
Rgは、水素原子、C1-20アルキル、アミノC2-20アルキルまたはヒドロキシC2-20アルキルから選択され、当該基のアルキル部分は-O-および-NH-から選択される1~3個の基が挿入されていてもよく;
Yaは、C1-5アルキレンであり;
Ybは、C2-8アルキレンまたはC2-8アルケニレンであり;
mは、1~100から選択される整数である]
で表される繰り返し単位を1以上含む、がん治療キット。
[式中、R1c、R2c、R3c及びR4cは、それぞれ独立に、水素原子、C1-6アルキル、ホルミルおよびC1-6アルキルカルボニルから選択され;
R5cは、水素原子、ホルミルおよびC1-6アルキルカルボニルから選択され; Xcは、ヒドロキシおよび-O-Q+から選択され、ここでQ+は、カウンターカチオンを表す]
で表わされる繰り返し単位を含む、項13に記載のがん治療キット。
本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。
<2-1.疎水性基が導入されてなるヒアルロン酸誘導体>
疎水性基が導入されてなるヒアルロン酸誘導体は、ヒアルロン酸又はその誘導体であって、疎水性基を有し、抗原(特に、抗原ペプチド、抗原タンパク質)と複合体を形成可能なものである限り、特に制限されない。
式(I)
R5は、水素原子、ホルミル、またはC1-6アルキルカルボニルであり;
Zは、直接結合、または2~30個の任意のアミノ酸残基からなるペプチドリンカーを表し;X1は、以下の式:
-NRb-R、
-NRb-COO-R、
-NRb-CO-R、
-NRb-CO-NRc-R、
-COO-R、
-O-COO-R、
-S-R、
-CO-Ya-S-R、
-O-CO-Yb-S-R、
-NRb-CO-Yb-S-R、および
-S-S-R、
により表される基から選択される疎水性基であり;
Ra、RbおよびRcは、それぞれ独立に、水素原子、C1-20アルキル、アミノC2-20アルキルおよびヒドロキシC2-20アルキルから選択され、ここで当該基のアルキル部分は、-O-および-NRf-から選択される1~3個の基が挿入されていてもよく;
Rfは、水素原子、C1-12アルキル、アミノC2-12アルキルおよびヒドロキシC2-12アルキルから選択され、当該基のアルキル部分は-O-および-NH-から選択される1~2個の基が挿入されていてもよく;
Rは、ステリル基であり;
Yは、C2-30アルキレン、または-(CH2CH2O)m-CH2CH2-であり、ここで、当該アルキレンは、-O-、-NRg-および-S-S-から選択される1~5の基が挿入されていてもよく;
Rgは、水素原子、C1-20アルキル、アミノC2-20アルキルまたはヒドロキシC2-20アルキルから選択され、当該基のアルキル部分は-O-および-NH-から選択される1~3個の基が挿入されていてもよく;
Yaは、C1-5アルキレンであり;
Ybは、C2-8アルキレンまたはC2-8アルケニレンであり;
mは、1~100から選択される整数である]
で表される繰り返し単位を1以上含む、ヒアルロン酸誘導体;または
式(II)
R5aは、水素原子、ホルミル、またはC1-6アルキルカルボニルであり;
X1aは、ヒドロキシ、-O-Q+、C1-6アルコキシ、-NR7R8、または-NR9-Z1-Z2であり;
Q+は、カウンターカチオンを表し;
R6a、R7、R8、およびR9は、独立に、水素原子、およびC1-6アルキルから選択され;Raaは、水素原子、またはC1-6アルキルであり、ここで該アルキルは、独立に、ヒドロキシ、カルボキシ、カルバモイル、C1-6アルキルチオ、アリール、およびヘテロアリールから選択される1以上の基で置換されていてもよく、ここで該アリールは1以上のヒドロキシで置換されていてもよく;
Z1は、C2-30アルキレン、または-(CH2CH2O)ma-CH2CH2-であり、ここで、該アルキレンは、独立に、-O-、-NRga-および-S-S-から選択される1~5の基が挿入されていてもよく、maは、1~100から選択される整数であり;
Z2は、以下の式:
-NRba-Z3、
-NRba-COO-Z3、
-NRba-CO-Z3、
-NRba-CO-NRca-Z3、
-COO-Z3、
-CO-NRca-Z3、
-O-CO-NRca-Z3、
-O-COO-Z3、
-S-Z3、
-CO-Za-S-Z3、
-O-CO-Zb-S-Z3、
-NRba-CO-Zb-S-Z3、および
-S-S-Z3、
により表される基から選択され;
RbaおよびRcaは、独立に、水素原子、C1-20アルキル、アミノC2-20アルキルおよびヒドロキシC2-20アルキルから選択され、ここで当該基のアルキル部分は、独立に、-O-および-NRfa-から選択される1~3個の基が挿入されていてもよく;
Rfaは、独立に、水素原子、C1-12アルキル、アミノC2-12アルキルおよびヒドロキシC2-12アルキルから選択され、当該基のアルキル部分は、独立に、-O-および-NH-から選択される1~2個の基が挿入されていてもよく;
Rgaは、独立に、水素原子、C1-20アルキル、アミノC2-20アルキルまたはヒドロキシC2-20アルキルから選択され、当該基のアルキル部分は、独立に、-O-および-NH-から選択される1~3個の基が挿入されていてもよく;
Z3は、ステリル基であり;
Zaは、C1-5アルキレンであり;
Zbは、C2-8アルキレンまたはC2-8アルケニレンである]
で表される繰り返し単位を含むヒアルロン酸誘導体であって、X1aが-NR9-Z1-Z2である式(II)で表される繰り返し単位が含まれない場合、さらに式(III):
R5bは、水素原子、ホルミル、またはC1-6アルキルカルボニルであり;
X2は、-NR9-Z1-Z2であり、ここで、R9、Z1、およびZ2は既に定義したとおりである]で表される繰り返し単位を含む、ヒアルロン酸誘導体、である。
R5cは、水素原子、ホルミルおよびC1-6アルキルカルボニルから選択され; Xcは、ヒドロキシおよび-O-Q+から選択され、ここでQ+は、カウンターカチオンを表す]
で表わされる繰り返し単位をさらに含むことが好ましい。
-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(R10)-CH2-S-R;
-NH-(CH2)mz-NHCO-CH(R10)-CH2-S-R;
-NH-(CH2CH2O)m-CH2CH2-NHCO-CH(R10)-CH2-S-R;-NH-(CH2CH2O)m-CH2CH2-O-CO-CH(R10)-CH2-S-R;および-NH-(CH2)mz-S-S-R;
-Z-NRa-Y-NRb-COO-R
(ここで、mzは、2~30の整数であり、R10は、水素原子またはメチル基であり、R、およびmは、本明細書で既に定義したとおりである)
で表される基から選択される。当該基は、好ましくは、
-NH-(CH2)mz-NH-COO-R;
-NH-(CH2CH2O)m-CH2CH2-NH-COO-R;および
-NH-(CH2)mz-S-S-R
(ここで、mz、R、およびmは、本明細書で既に定義したとおりである)
から選択される基である。
上記式(I)のYの具体例としては、-CH2CH2O-CH2CH2-S-S-CH2CH2O-CH2CH2-、-(CH2CH2O)2-CH2CH2-S-S-CH2CH2O-CH2CH2-、-CH2CH2O-CH2CH2-S-S-(CH2CH2O)2-CH2CH2-および-(CH2CH2O)2-CH2CH2-S-S-(CH2CH2O)2-CH2CH2-が挙げられる。
式(II)における基-CHRaa-CO-X1aの別の態様として、例えば、基-CHRaa-CONH2が挙げられる。該基の具体例として以下の基が挙げられる。
疎水性基が導入されてなるヒアルロン酸誘導体と組み合わせる抗原としては、例えば、抗原ペプチドおよび抗原タンパク、ならびにこれらの抗原配列をコードしたDNAおよびmRNAが挙げられ、好ましくは抗原ペプチドおよび抗原タンパク、さらに好ましくは抗原ペプチドである。
疎水性基が導入されてなるヒアルロン酸誘導体と抗原の複合体は、疎水性基が導入されてなるヒアルロン酸誘導体と抗原を適当な溶液中で混合することで製造することができる。用いる溶液としては、水、生理食塩水、各種緩衝液、糖溶液、DMSO、エタノール、DMF、それらの組み合わせなどが挙げられ、透析などの方法により溶媒・緩衝液交換を行ってもよい。
<3-1.免疫受容体>
免疫受容体は、免疫細胞のエフェクター機能の発揮に必要なシグナルを伝達すること、即ち、抗原結合性ドメインが抗原と結合した際、免疫細胞の活性化に必要なシグナルを伝達することが可能な受容体である限り、特に制限されない。免疫受容体としては、好ましくはT細胞受容体、キメラ抗原受容体(CAR)等が挙げられる。キメラ抗原受容体が認識する抗原としては、細胞表面抗原であっても、細胞内抗原と主要組織適合遺伝子複合体の複合体であってもよい。
抗原特異的リンパ球は、HA-抗原組成物における抗原に対する(すなわち、当該抗原に結合(好ましくは特異的に結合)可能な)免疫受容体を発現するものである限り、特に制限されない。より具体的な態様においては、抗原特異的リンパ球は、免疫受容体が細胞膜上に発現しており、また免疫受容体が抗原結合性ドメインを細胞膜外に露出した状態で発現している。
ベクターとしては、宿主ゲノムに組み込まれるベクター・組み込まれないベクター、細胞質に存在して自律的に複製するエピソーマルベクターなどが使用できる。そのようなベクターとしては、例えばレトロウイルスベクター(オンコレトロウイルスベクター、レンチウイルスベクター、シュードタイプベクターを含む)、アデノウイルスベクター、アデノ随伴ウイルス(AAV)ベクター、シミアンウイルスベクター、ワクシニアウイルスベクター、センダイウイルスベクター、エプスタイン・バーウイルス(EBV)ベクター、HSVベクターなどのウイルスベクターが例示できる。ウイルスベクターとしては、感染した細胞中でウイルスが自己複製できないように複製能を欠損させたものが好ましく使用できる。また、リポソーム及び陽イオン脂質などの縮合剤との併用により、非ウイルスベクターも使用できる。更に、リン酸カルシウム形質移入、DEAE-デキストラン、エレクトロポレーション、パーティクルボンバードメントにより細胞に核酸を導入できる。
HA-抗原組成物と抗原特異的リンパ球を組み合わせて用いることにより、より強く、より効率的に、より持続的に、及び/又はより広範囲において抗腫瘍効果を発揮することができる。このため、本発明は、その一部の態様において、HA-抗原組成物を含有する、抗原特異的リンパ球との併用投与に用いるための医薬組成物、抗原特異的リンパ球を含有する、HA-抗原組成物との併用投与に用いるための医薬組成物、HA-抗原組成物と、抗原特異的リンパ球との組合せからなることを特徴とする、医薬組成物、等に関する。さらには、HA-抗原組成物は、T細胞活性化ワクチン等として利用することもできる。
HA-抗原組成物は、経口、腸管外、鼻腔内、膣内、眼内、皮下、静脈内、筋肉内、皮内、腹腔内、関節腔内、脳内、口腔内、脂肪組織内、乳腺組織内、吸入等の経路を経て投与されてもよい。好ましくは、皮下、筋肉内、静脈内、皮内を経て投与されてもよい。
<4-2.抗原特異的リンパ球の使用>
抗原特異的リンパ球は、細胞製剤として、抗原結合性ドメインの標的抗原を発現する腫瘍/がん(標的疾患)の治療、予防、又は改善に広く利用することができる。
(1)細胞
RPMI1640培地(2-メルカプトエタノール添加)は細胞科学研究所から購入した。ウシ胎児血清(FBS)はGibcoから購入した。マウス線維肉腫CMS5a 細胞株はメモリアルスローンケタリングがん研究所より分譲され、三重大学で継代したものを用いた。マウス線維肉腫CMS5a細胞株は、変異型ERK2蛋白質を発現している。変異型ERK2蛋白質の変異部分を含むペプチド(QYIHSANVL(配列番号1))は、BALB/cマウスのCD8陽性細胞傷害性T細胞に認識される。このペプチドを認識するT細胞受容体(TCR)が単離され、TCRを遺伝子導入したトランスジェニックマウス(DUC18マウス)が作出されている。本実施例で用いた長鎖ペプチド抗原は、前記変異型ERK2のCD8陽性細胞傷害性T細胞認識エピトープ配列(QYIHSANVL)が含まれている。
雌性BALB/cマウス(CD90.2陽性)は6週齢~8週齢のものを日本SLCから購入した。DUC18トランスジェニックマウスはワシントン大学より分譲され、三重大学で繁殖させたものを用いた。両マウスは三重大学先端科学研究支援センター動物実験施設にて飼育した。動物実験のプロトコールは、三重大学医学部の倫理委員会の承認を得た。
国際公開第2010/053140号の実施例1及び2に記載のとおりに、コレステリル基が導入されてなるヒアルロン酸ナノゲル(重量平均分子量が99kDa、コレステリル基の導入率が42%)を合成した。
合成長鎖ペプチドはシグマジェノシスから購入した。配列は次の通りであった:NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(配列番号2)。
ヒアルロン酸ナノゲルを12mg/mLとなるように超純水に溶解させ、超音波処理(コバリス社製、E220X)を実施した。抗原ペプチドは50mg/mLとなるようにDMSOに溶解させた。HA誘導体水溶液に対し、抗原ペプチドDMSO溶液を加え、バス型ソニケーターにて30分間超音波処理した。透析キット(Slide-A-Lyzer、分画分子量3.5K)に移し、5mM炭酸緩衝液pH9、10mMリン酸緩衝液pH7、10%スクロース入り10mMリン酸緩衝液pH7に対して順次透析した。逆相クロマトグラフィー分析により抗原ペプチド濃度を定量し、250~500ug/mLとなるように濃度調整し、投与溶液とした。目的の濃度に達しない場合は、限外濃縮器(Vivaspin6、5000MwCO、ザウトリウス)にて濃縮した。得られた複合体を、「長鎖ペプチド抗原搭載HAナノゲルがんワクチン」又は「HAナノゲルがんワクチン」と示すこともある。
・アジュバント
CpGオリゴDNA1668は味の素バイオファーマサービスジーンデザインから購入した。
eFluor450標識抗マウスCD8a抗体(クローン53-6.7)、eFluor450標識抗マウスKLRG1抗体(クローン2F1)、APC標識抗マウスCD45R(B220)抗体(クローンRA3-6B2)は、eBioscienceから購入した。APC標識抗マウスCD8a抗体(クローン53-6.7)、APC標識抗マウスCD4抗体(クローンRM4-5)、PerCP-Cy5.5標識抗マウスCD90.1抗体(クローンOX-7)、PerCP-Cy5.5標識抗マウスF4/80抗体(クローンBM8)、PE標識抗マウスCD207抗体(クローン4C7)、Pacific blue標識抗マウスCD90.2抗体(クローン30-H12)、Purified CD16/32抗体(クローン93)はBioLegendから購入した。FITC標識抗マウスCD11c抗体(クローンHL3)、PerCP-Cy5.5標識抗マウスCD3抗体(クローン145-2C11)はBDバイオサイエンスから購入した。APC標識抗マウスIFN-γ抗体(クローンXMG1.2)はeBioscienceあるいはTONBOから購入した。組織染色用の抗マウスCD8a抗体(クローン4SM15)はインビトロジェンから、Alexa488標識抗ラットIgG(H+L)抗体はCell Signalingから購入した。
CD8陽性T細胞刺激用の合成短鎖ペプチドはシグマジェノシスから購入した。配列は次の通りであった:QYIHSANVL(配列番号1)。
T75培養フラスコ(コーニング)を用い、マウス線維肉腫CMS5a細胞株を10%FBS含有RPMI1640培地中で培養した。培養した細胞は0.5%トリプシン含有PBSを用いて剥離し、10%FBS含有RPMI1640培地に懸濁した。懸濁液を遠心分離(400×g、5分、4℃)した後に上清を除いた。その後、RPMI1640培地を用いて2回洗浄し、1×106個/100μLの濃度でRPMI1640培地に懸濁した。100μL/個体の用量でBALB/cマウスの右側前背部に皮下移植した(1群あたり5匹)。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用による抗腫瘍効果について明らかにするため、免疫チェックポイント阻害剤(ICI)抵抗性繊維肉腫細胞株CMS5a担癌マウスを用いて、CMS5aに発現する変異型ERK2抗原(mERK2)のCD8エピトープを含む長鎖ペプチド抗原とHAナノゲルを複合化した長鎖ペプチド抗原搭載HAナノゲルがんワクチンを作製し、mERK2認識TCR遺伝子導入T細胞輸注療法と併用して治療効果を検討した。その結果、図1-1及び図1-2に示すように、HANG-V及びTCR-T併用治療群は、HANG-V単独治療群あるいはTCR-T単独治療群と比べ、CMS5a腫瘍の増殖を著しく抑制し、腫瘍を退縮させ、治療7日後には全てのマウスにおいて腫瘍の消失が認められた(図1-1及び図1-2)。本効果は長期間持続し、同マウスにCMS5aを再移植しても腫瘍増殖は認められず、マウスは再発することなく長期間生存した(図1-3)。これらの結果より、HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用は強力な抗腫瘍作用を有し、ICI抵抗性腫瘍を治癒できることが明らかとなった。また、本併用は腫瘍治癒後も腫瘍の再発を抑制し得ることが明らかとなり、HAナノゲルがんワクチンは抗原特異的T細胞の抗腫瘍作用を増強させる可能性が示唆された。
実施例1の試験の腫瘍移植14日後にワクチン投与部位の所属リンパ節(右鼠径リンパ節)を採取し、スライドガラスを用いてリンパ節を磨砕後、細胞をRPMI1640培地に懸濁した。このとき、1群2匹分の細胞をプールした。遠心分離(400×g、5分、4℃)後に上清を除去し、RPMI1640培地を用いて2回洗浄後、10%FBS含有RPMI1640培地に懸濁した。細胞血球計を用いて細胞数を測定し(図2-1)、細胞濃度が2×106個/mL になるよう調製した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用におけるHAナノゲルがんワクチンの抗腫瘍免疫応答に及ぼす影響について明らかにするため、腫瘍移植14日後にHAワクチン投与部位の所属リンパ節を採取し、リンパ節細胞の解析を行なった。その結果、HANG-V単独群及びHANG-V、TCR-T併用群のリンパ節は著しく腫脹し、両群では未治療群と比較して約4~8倍のリンパ節細胞の増加が認められた(図2-1)。また、リンパ節の腫脹は、腫瘍移植22日後においても継続して認められた(図2-2)。次に、所属リンパ節に流入・集積したミエロイド系抗原提示細胞の割合について検討した結果、HANG-V単独群及びHANG-V、TCR-T併用群の両群において、ミエロイド系抗原提示細胞の増加が認められた。特に併用群においてはランゲルハンス細胞の増加が顕著に認められた(図2-3)。さらに、HAナノゲルがんワクチンが所属リンパ節細胞数に及ぼす影響について明らかにするため、所属リンパ節のリンパ球やミエロイド系細胞の解析を行なった。その結果、図2-4に示すように、HANG-V群ではリンパ球(T細胞、B細胞、NK細胞)とミエロイド系細胞の数倍から数十倍の増加が認められた(図2-4)。また、CD8陽性輸注T細胞の所属リンパ節への流入について検討した結果、HANG-V、TCR-T併用群は、TCR-T単独群と比較して所属リンパ節のCD8陽性輸注T細胞の割合が著しく上昇しており(図2-5)、その内約80%が腫瘍抗原に特異的に反応してIFN-γを産生していることが明らかとなった(図2-6)。また、HANG-V、TCR-T併用群は、宿主のIFN-γ産生CD8陽性T細胞割合の上昇も認められた(図2-6)。これらの結果より、HAナノゲルがんワクチンは、担癌宿主の免疫抑制環境において、宿主の樹状細胞やマクロファージ、ランゲルハンス細胞等の抗原提示細胞及びリンパ球を所属リンパ節に大量に流入・蓄積させ、効率的な腫瘍抗原提示環境を創出する可能性が示唆された。さらにこの腫瘍抗原提示が向上した環境に、抗原特異的T細胞輸注療法によりT細胞を補った結果、所属リンパ節に流入したCD8陽性輸注T細胞が効率よく抗原提示を受けることが可能となり、CD8陽性輸注T細胞を質的・量的に強力な腫瘍抗原特異的細胞傷害性T細胞(CTL)に誘導、増殖させ、所属リンパ節で相乗的な抗腫瘍免疫環境がサステナブルに構築された可能性が示唆された。
マウス腫瘍増殖試験(実施例1)及び抗腫瘍免疫応答解析(実施例2)について、抗原特異的T細胞数を2×106個/200μLから1×106個/200μL、5×105個/200μL、2.5×105個/200μLの濃度に漸減投与して、同様の試験を実施した。
HAナノゲルがんワクチン及び抗原特異的輸注T細胞療法併用治療効果におけるCD8陽性輸注T細胞数の影響を明らかにするため、CD8陽性輸注T細胞数を漸減投与し、治療効果及び抗腫瘍免疫応答について検討した。その結果、CD8陽性輸注T細胞数を1×106個、5×105個、2.5×105個に漸減し少量投与した場合も、従来の2×106個投与した場合と同等に治療効果が認められた(図3-1、図3-2)。また、腫瘍移植14日後に所属リンパ節の総細胞数やミエロイド系抗原提示細胞に及ぼす影響について検討した結果、漸減少量投与した場合においてもこれらの細胞が顕著に認められた(図3-3、図3-4)。さらに、所属リンパ節におけるCD8陽性輸注細胞と腫瘍抗原特異的IFN-γ産生細胞の割合について検討した結果、CD8陽性輸注細胞の割合は従来の2×106個投与した場合が最も多かったが、腫瘍抗原特異的IFN-γ産生細胞は輸注及び宿主T細胞共にCD8陽性輸注T細胞を漸減少量投与した場合も、同等の活性を有することが明らかとなった(図3-5、図3-6)。これらの結果より、HAナノゲルワクチンは従来のCD8陽性輸注T細胞数より遥かに少ない1/10近いCD8陽性輸注T細胞との併用においても、腫瘍抗原特異的免疫応答を誘導し、強力にICI抵抗性腫瘍を治癒できることが明らかとなった。
実施例1の試験の腫瘍移植14日後に腫瘍を摘出し、次に示す方法で免疫組織蛍光染色を実施した。O.C.T.コンパウンド(サクラファインテック)に包埋し凍結した腫瘍を5 μm厚で薄切し、得られた腫瘍切片について風乾を行った。乾燥した腫瘍切片に対して氷冷したアセトンで10分間の固定を行い、免疫染色に用いた。腫瘍切片をPBSで3回洗浄した後、ブロッキングワン Histo(ナカライテスク)に浸漬し、インキュベーションチャンバー内で30分間ブロッキングを行った。次にAntibody Diluent with Background reducing Component (Dako)で100倍希釈した抗マウスCD8抗体を用いて、インキュベーションチャンバーで腫瘍切片に対し室温で1時間染色を行なった。さらに、0.1%Tween20含有PBSで3回洗浄を行なった後、PBSで1000倍希釈したAlexa488標識抗ラット抗体を用いて、インキュベーションチャンバーで腫瘍切片に対し室温で1時間染色を行なった。最後に0.1%Tween20含有PBSで3回洗浄を行ない、腫瘍切片をProlong Gold antifade reagent with DAPI(ライフテクノロジーズ) に浸漬し、蛍光顕微鏡BZ-710(キーエンス)を用いて腫瘍内に浸潤しているCD8陽性T細胞を観察した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用が担癌宿主の腫瘍内環境に及ぼす影響について明らかにするため、腫瘍移植14日後に各々のマウスの腫瘍を摘出し、腫瘍内に浸潤しているCD8陽性T細胞を蛍光顕微鏡で観察した。その結果、HANG-V及びTCR-T併用群は、未治療あるいは単独治療群と比較し、腫瘍内においてCD8陽性T細胞が顕著に認められた(図4)。本結果より、HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法の併用は、腫瘍内に腫瘍抗原特異的細胞傷害性T細胞(CTL)を誘導し、腫瘍を消失させる可能性が示唆された。
実施例3の試験の腫瘍移植21日後にマウス眼窩より末梢血を採取し、10%ヘパリン含有RPMIに懸濁し、混和した。その後、懸濁液をエッペンチューブに添加したFicoll(GEヘルスケア)500uLにゆっくり重曹し、遠心分離(4000rpm×10 min、25 ℃、AC:1、DE:1)後、単核球層を染色用バッファー(0.5% BSA含有PBS)に回収した。細胞懸濁液を遠心分離(5000rpm×1 min、25 ℃)後、上清をアスピレーターで除去し、0.5% FBS含有PBSを用いて2回洗浄した。各抗体のメーカーが推奨する使用濃度に従い、APC標識抗マウスCD8抗体、PerCP-Cy5.5標識抗マウスCD90.1抗体、Pacific blue標識抗マウスCD90.2抗体を添加し混合した後、4℃の暗所にて15分間静置した。200μLの染色用バッファーで細胞を2回洗浄した後、200μLの染色用バッファーに再懸濁を行い、丸底ポリスチレンチューブ(BDバイオサイエンス)へ移した。細胞はフローサイトメーターFACS Canto II(BDバイオサイエンス)及び付属の解析ソフトウェア(FACSDiva)を用いて解析した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注後の全身におけるCD8陽性輸注T細胞の生存持続性(persistence)を明らかにするため、腫瘍移植21日後に末梢血球を採取し、末梢血中に存在しているCD8陽性輸注T細胞の割合について検討した。その結果、CD8陽性輸注T細胞は腫瘍移植21日後も全身を循環し血中に存在していることが明らかとなった。また、CD8陽性輸注T細胞数を2.5×105個に漸減し、少量投与した場合もCD8陽性輸注T細胞は2×106個輸注の際と同等に血中に認められた(図5)。これらの結果より、CD8陽性輸注T細胞は輸注細胞数に関わらず、輸注後も継続して末梢血中を循環し、全身に持続して存在することが明らかとなった。
以上の結果より、HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用は、HAナノゲルがんワクチンにより所属リンパ節への抗原提示細胞や輸注T細胞の流入・集積が促進され、抗原提示を介して輸注T細胞を腫瘍抗原特異的細胞傷害性T細胞(CTL)に誘導し、ICI抵抗性腫瘍を消失させて、治癒できることが明らかとなった。本療法は、輸注T細胞を質的・量的に高めることができる利点に加え、宿主のT 細胞にも働き、宿主の免疫抑制環境を抗腫瘍環境へとシフトできることが明らかとなった。
さらに、本効果は従来の輸注細胞数よりも遥かに少ない輸注細胞数で同等の効果を示し、治癒後も全身において長期間持続し、腫瘍の再発を抑え、宿主は長期間生存できることが明らかとなった。
(1)細胞
RPMI1640培地(2-メルカプトエタノール添加)は細胞科学研究所から購入した。D-MEM培地は和光純薬から購入し、ウシ胎児血清(FBS)はGibcoから購入した。マウス線維肉腫細胞株CMS5aはメモリアルスローンケタリングがん研究所より分譲され、マウス転移性悪性黒色腫細胞株B16F10はATCCより購入し、三重大学で継代したものを用いた。マウス線維肉腫細胞株CMS5aは、変異型ERK2蛋白質を発現している。変異型ERK2蛋白質の変異部分を含むペプチド(QYIHSANVL(配列番号1))は、BALB/cマウスのCD8陽性細胞傷害性T細胞に認識される。このペプチドを認識するT細胞受容体(TCR)が単離され、TCRを遺伝子導入したトランスジェニックマウス(DUC18マウス)が作出されている。本実施例で用いた長鎖ペプチド抗原は、前記変異型ERK2のCD8陽性細胞傷害性T細胞認識エピトープ配列(QYIHSANVL)が含まれている。マウス転移性悪性黒色腫細胞株B16F10は、腫瘍抗原gp100蛋白質を発現しており、本蛋白質に含まれるp25-33ペプチド(EGSRNQDWL(配列番号4))は、C57BL/6マウスのCD8陽性細胞傷害性T細胞に認識され、本CD8陽性細胞傷害性T細胞認識エピトープ配列を認識するTCRを遺伝子導入したトランスジェニックマウス(Pmel-1マウス)が作出されている。本実施例で用いた長鎖ペプチド抗原には、本配列が含まれている。
雌性BALB/cマウス及び雌性C57BL/6マウスは6週齢~8週齢のものを日本SLCから購入した。DUC18トランスジェニックマウスはワシントン大学より分譲され、三重大学動物実験施設で繁殖させたものを用いた。Pmel-1トランスジェニックマウスはJackson研究所から購入し、三重大学動物実験施設で繁殖させたものを用いた。全てのマウスは三重大学動物実験施設にて飼育した。動物実験のプロトコールは、三重大学医学部の倫理委員会の承認を得た。
(3-1)ヒアルロン酸ナノゲル(HANG)
国際公開第2010/053140号の実施例1及び2に記載のとおりに、コレステリル基が導入されてなるヒアルロン酸ナノゲル(重量平均分子量が10kDa、コレステリル基の導入率が44および41%)を合成した。
合成長鎖ペプチドはバイオロジカから購入した。配列は次の通りであった:NDHIAYFLYQILRGLQYIHSANVLHRDLKPSNLLLNT(配列番号2)。当該配列は、CD8陽性細胞傷害性T細胞認識エピトープ配列として16番目のQから24番目のLまでの9個のアミノ酸配列(QYIHSANVL(配列番号1))を、CD4陽性ヘルパーT細胞認識エピトープ配列として13番目のRから29番目のKまでの17個のアミノ酸配列(RGLQYIHSANVLHRDLK(配列番号3))を含む。
ヒアルロン酸ナノゲルの凍結乾燥体を秤量し、5mg/mLの濃度となるように注射用水を添加し、一晩攪拌して十分に溶解させた。一方、別の容器にて、ペプチドの濃度が50mg/mLとなるようにジメチルスルホキシドへ溶解させた。続いて上記ヒアルロン酸ナノゲル水溶液とペプチドのジメチルスルホキシド溶液を容量比100:1の割合で混合し、室温で24時間攪拌することで、ペプチド-ヒアルロン酸ナノゲル複合体を含む組成物を得た。その後、固体の精製スクロース(富士フイルム和光社製、製造専用、製品番号198-18385)をペプチド-ヒアルロン酸ナノゲル複合体を含む組成物が10質量%スクロースとなるように添加して、1時間以上攪拌した。続いて、10質量%スクロース含有25 mMリン酸緩衝液(pH 7.4)にてペプチドの理論濃度が0.3 mg/mLとなるように希釈し、0.2μmPES(ポリエーテルスルフォン)(Pall社製、アクロディスクシリンジフィルター、25mmΦ)で滅菌ろ過し、ペプチド-ヒアルロン酸ナノゲル複合体を含む組成物を得た。この時のペプチド-ヒアルロン酸ナノゲル複合体を含む組成物中のペプチド濃度を逆相クロマトグラフィー分析により定量した後に、ペプチド濃度が250ug/mLとなるように10%スクロース10mMリン酸緩衝液(pH 7.4)で濃度調整し、投与溶液とした。
(4-1)ヒアルロン酸ナノゲル(HANG)
国際公開第2010/053140号の実施例1及び2に記載のとおりに、コレステリル基が導入されてなるヒアルロン酸ナノゲル(重量平均分子量が10kDa、コレステリル基の導入率が44%)を合成した。
上記で得たヒアルロン酸ナノゲルを用いて、ペプチド-ヒアルロン酸ナノゲル複合体の調製を次の通り行った。gp100 TRP2 TRP1_6Yペプチドはバイオロジカから購入した。配列は次の通りである:SVYDFFVWLYYYYYYTWHRYHLLYYYYYY EGSRNQDWL(配列番号5)。当該配列は、CD8陽性細胞傷害性T細胞認識エピトープ配列として下記TRP2(アミノ酸配列:SVYDFFVWL(配列番号6))、TRP1(アミノ酸配列:TWHRYHLL(配列番号7))、gp100(アミノ酸配列:EGSRNQDWL(配列番号4))を含み、各エピトープ間を6つのYで結合している。
ヒアルロン酸ナノゲルの凍結乾燥体を秤量し、5mg/mLの濃度となるように注射用水を添加し、一晩攪拌して十分に溶解させた。一方、別の容器にて、ペプチドの濃度が50mg/mLとなるようにジメチルスルホキシドへ溶解させた。溶解を確認した後に、室温で、上記ヒアルロン酸ナノゲル水溶液と1mol/Lの水酸化ナトリウム水溶液を容量比100:2の割合で混合した。続いて上記ヒアルロン酸ナノゲル水溶液とペプチドのジメチルスルホキシド溶液を容量比102:1の割合で混合し、室温で24時間攪拌することで、ペプチド-ヒアルロン酸ナノゲル複合体を含む組成物を得た。その後、固体の精製スクロース(富士フイルム和光社製、製造専用、製品番号198-18385)をペプチド-ヒアルロン酸ナノゲル複合体を含む組成物が10質量%スクロースとなるように添加して、1時間以上攪拌した。続いて、10質量%スクロース含有25mMリン酸緩衝液(pH 7.4)にてペプチドの理論濃度が0.3 mg/mLとなるように希釈し、0.2μmPES(ポリエーテルスルフォン)(Pall社製、アクロディスクシリンジフィルター、25mmΦ)で滅菌ろ過し、ペプチド-ヒアルロン酸ナノゲル複合体を含む組成物を得た。この時のペプチド-ヒアルロン酸ナノゲル複合体を含む組成物中のペプチド濃度を逆相クロマトグラフィー分析により定量した後に、ペプチド濃度が250ug/mLとなるように10%スクロース10mMリン酸緩衝液(pH 7.4)で濃度調整し、投与溶液とした。
(5-1)コレステリル修飾プルラン(CHP)
日油社製のCHP(コレステリル修飾プルラン、製品番号CHP-80T)を用いた。
上記gp100ペプチド(配列番号5)を用いた。
CHPの粉体を10mg/mLの濃度になるように6M尿素含有リン酸緩衝生理食塩水pH 7.4に溶解させた。一方、別の容器にて、ペプチドの濃度が50mg/mLとなるようにジメチルスルホキシドへ溶解させた。上記6M尿素含有CHP水溶液とペプチドのジメチルスルホキシド溶液を容量比で100:1の割合で混合し、室温で12時間以上インキュベートし、ペプチド-CHP複合体を含む組成物を得た。その後、透析カセット(Thermo社製、Slide-A-Lyzer G2 Dialysis Cassettes、3.5K MWCO、15mL)に移し、0.6M尿素含有リン酸緩衝生理食塩水pH 7.4で透析した。続いて、0.06M尿素含有リン酸緩衝生理食塩水pH 7.4、リン酸緩衝生理食塩水pH 7.4の順に透析を行った。さらに所望の濃度まで限外濃縮器(Vivaspin20、MWCO:10,000、ザルトリウス)にて濃縮した。 最後に、0.2μmPES(ポリエーテルスルフォン)(Pall社製、アクロディスクシリンジフィルター、25mmΦ)で滅菌ろ過し、CHPと抗原の複合体を含む投与組成物を得た。ペプチドの濃度定量は逆相クロマトグラフィー分析により行った。
・アジュバント
CpGオリゴDNA1668は味の素バイオファーマサービスジーンデザインから購入した。
eFluor450標識抗マウスCD8a抗体(クローン53-6.7)、eFluor450標識抗マウスKLRG1抗体(クローン2F1)、APC標識抗マウスCD44(クローンIM7)及びAPC標識抗マウスIFN-γ抗体(クローンXMG1.2)は、eBioscienceから購入した。APC標識抗マウスCD8a抗体(クローン53-6.7)、APC標識抗マウスCD4抗体(クローンRM4-5)、PerCP-Cy5.5標識抗マウスCD90.1抗体(クローンOX-7)、Pacific blue標識抗マウスCD90.2抗体(クローン30-H12)、FITC標識抗マウスCD8抗体(クローン53-6.7)、Brilliant Violet 510標識抗マウスCD90.1抗体(クローンOX-7)、Pacific Blue標識抗マウスCD44(クローンIM7)及びPurified CD16/32抗体(クローン93)は、BioLegendから購入した。PE標識抗マウスCD62L抗体(クローンMEL-14)はBDバイオサイエンスから購入した。テトラマー染色用のAPC標識H-2Db gp100 Tetramer及びClear BackはMBLより購入した。
抗マウスPD-1抗体(クローンRMPI-14)は、Bio Cellから購入した。抗マウスCTLA-4抗体(クローン9D9)は、MDアンダーソンがんセンターより分譲されたハイブリドーマを用いて三重大学で作製した。
CD8陽性T細胞刺激用の合成短鎖ペプチド(QYIHSANVL(配列番号1)は、シグマジェノシスから購入した。EGSRNQDWL(配列番号4)及びVYDGREHTV(配列番号9)はユーロフィンから購入した。
T75培養フラスコ(コーニング)を用い、転移性悪性黒色腫細胞株B16F10を10%FBS含有D-MEM培地中で培養した。培養した細胞は0.5%トリプシン含有PBSを用いて剥離し、10%FBS含有D-MEM培地に懸濁した。懸濁液を遠心分離(400×g、5分、4℃)した後に上清を除いた。その後、D-MEM培地を用いて2回洗浄し、2×105個/100μLの濃度でD-MEM培地に懸濁した。100μL/個体の用量でC57BL/6マウスの右側前背部に皮下移植した(1群当たり5匹)。
実施例1(ICI抵抗性繊維肉腫細胞株CMS5a)同様、転移性悪性黒色腫(メラノーマ)に対するHAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用による抗腫瘍効果について明らかにするため、転移性悪性黒色腫細胞株B16F10担癌C57BL/6マウスを用いて、B16F10に過剰発現するgp100腫瘍抗原のCD8エピトープを含む長鎖ペプチド抗原とHAナノゲルを複合化した長鎖ペプチド抗原搭載HAナノゲルがんワクチンを作製し、gp100認識TCR遺伝子導入T細胞輸注療法と併用し治療効果を検討した。その結果、HANG-V治療群は、未治療群やTCR-T単独群と比べ、B16F10腫瘍の増殖抑制が認められた(図6-1及び図6-2)。とりわけ、HANG-V及びTCR-T併用群は、一旦100mm2程度まで到達したB16F10腫瘍に対しても、抗腫瘍効果を発揮し、全ての個体で腫瘍が著しく退縮し(図6-2)、その内の2匹は完全に腫瘍の消失が認められた。HANG-V単独群及びTCR-T併用の両群では、全ての個体で腫瘍中央に細胞傷害性T細胞(CTL)の腫瘍傷害作用に起因すると考えられるクレーター状の空洞孔(腫瘍壊死)が認められた(図6-3)。また、未治療群やTCR-T単独群では腫瘍移植25日以内に全ての個体が死亡したのに対し、HANG-V、TCR-T併用群では100%、HANG-V群では80%の個体の生存が認められた(図6-4)。本効果は長期間持続し、腫瘍の完全消失が認められた個体2匹は、腫瘍移植5ヶ月以上経過後も再発することなく生存した。
マウス腫瘍増殖試験(実施例6)について、長鎖ペプチド抗原搭載ナノゲルがんワクチン(HANG-V又はCHP-V)に抗原特異的T細胞輸注療法を併用し同試験を実施した。長鎖ペプチド抗原搭載ナノゲルがんワクチンは、HANG-V、CHP-V共に腫瘍移植後7、11、15、29及び36日目に50μg(3回目以降は75μg)をPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に皮下投与し、抗原特異的T細胞(4×106個)を輸注した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用がCHPナノゲルがんワクチンと抗原特異的T細胞輸注療法(先行技術)に対し、抗腫瘍効果において優れているかどうか明らかにするため、腫瘍増殖試験を実施し、両者の治療効果を比較した。その結果、実施例6同様、HANG-V及びTCR-T併用群では、全ての個体においてB16F10腫瘍の増殖が著しく抑制され、腫瘍を強力に退縮し得たのに対し、CHP-V及びTCR-T併用群においては、同様の治療効果は認められなかった(図7-1及び図7-2)。また、HANG-V、TCR-T併用群は、CHP-V、TCR-T併用群と比べ、腫瘍の大きさ(面積)のみならず、上方向への進展・増殖(高さ)においても、腫瘍を著しく抑制し、全ての個体で腫瘍の中央部分にCTLの腫瘍傷害作用に起因すると考えられるクレーター状の空洞孔(腫瘍壊死)が認められた(図7-3)。その後、退縮が認められた全個体の内、2匹は腫瘍部分が瘡蓋状となり腫瘍が完全に消失し、残りの3匹についても腫瘍が再増悪することなく肉眼で確認できるか分からない程度まで退縮した。一方でCHP-V及びTCR-T併用群は、その後も腫瘍の増殖が抑制されることなく促進し、50日以内に全個体が死亡したのに対し、HANG-V及びTCR-T併用群は100%の生存率が維持された(図7-4)。
実施例6と同様の試験を実施し、腫瘍移植後14日目にワクチン投与部位の所属リンパ節(右鼠径リンパ節)を採取し(一群当たり2~3匹)、スライドガラスを用いてリンパ節を磨砕後、細胞をRPMI1640培地に懸濁した。遠心分離(400×g、5分、4℃)後に上清を除去し、RPMI1640培地を用いて2回洗浄後、10%FBS含有RPMI1640培地に懸濁した。細胞数を測定し(図8-1)、細胞濃度が2×106個/mL になるよう調製した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用におけるHAナノゲルがんワクチンのgp100抗原特異的CD8陽性T細胞に及ぼす影響について明らかにするため、腫瘍移植後14日目に所属リンパ節を採取し、同CD8 T細胞の解析を行なった。その結果、HANG-V単独群及びHANG-V、TCR-T併用群のリンパ節は著しく腫脹し、両群では未治療群と比較して約9~16倍のリンパ節細胞の増加が認められた(図8-1)。また、所属リンパ節に流入・集積したgp100抗原特異的テトラマー陽性CD8 T細胞の割合について解析した結果、両群はHANG-V未治療群と比較して、同CD8 T細胞の著しい増加が認められ(図8-2)、同細胞は非常に活性化したエフェクターT細胞フェノタイプ(KLRG1、CD44)を示すことが明らかとなった(図8-3)。さらにHANG-V、TCR-T併用群では、CD8陽性輸注T細胞よりgp100抗原特異的にIFN-γが顕著に産生されていることが明らかとなった(図8-4)。
実施例6と同様の試験を実施後、腫瘍移植後19日目にワクチン投与部位の所属リンパ節(右鼠径リンパ節)、非所属リンパ節(左鼠径リンパ節)、脾臓、腫瘍及び末梢血を採取し、次に示す方法にて各細胞を分離し、各組織に存在するCD8陽性輸注T細胞及びgp100テトラマー陽性CD8陽性T細胞の割合について検討した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注後の腫瘍局所及び全身における抗原特異的CD8陽性輸注T細胞の生存持続性(persistence)を明らかにするため、腫瘍移植後19日目に二次リンパ組織、腫瘍組織及び末梢血を採取し、同T細胞の割合について検討した。その結果、輸注T細胞は所属リンパ節に加え、他の二次リンパ組織(脾臓及び非所属リンパ節)や腫瘍組織内、全身の末梢血にも著しく存在していることが明らかとなった(図9-1)。また、gp100抗原特異的CD8 T細胞についても、各組織に広く顕著に存在しており(図9-2)、これら全身に局在する抗原特異的CD8 T細胞により、本治療が腫瘍局所の増殖抑制のみならず、全身への転移を抑制している可能性が示唆された。
実施例6施行後、腫瘍の完全退縮が認められた2匹の個体の末梢血を採取し(腫瘍移植後5ヶ月経過)、gp100抗原特異的CD8陽性輸注T細胞の割合について検討した。その後、2匹の個体の内1匹にB16F10腫瘍(2×105個)を再移植し、もう1匹の個体には長鎖ペプチド抗原搭載HAナノゲルがんワクチン75μgをPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に計3回(3日~4日間隔)皮下投与後、末梢血を再採取し、各々のgp100抗原特異的CD8陽性輸注T細胞の割合変化、活性化及びフェノタイプについて検討した。さらに、同マウスにB16F10腫瘍(2×105個)を再移植し、再発の有無について検討した。末梢血単核球(PBMC)の回収は、実施例5同様に、テトラマー染色は実施例8同様に施行後、下記の方法により細胞の染色と測定を行なった。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法を実施し、腫瘍完全退縮後無再発の個体の末梢血中のgp100抗原特異的CD8陽性輸注T細胞を測定した結果、治癒5ヶ月後も末梢血中には同CD8陽性輸注T細胞が存在することが明らかとなった(図10-1)。さらに、同個体にB16F10腫瘍を再移植又はワクチンを再接種後、再び末梢血中のgp100抗原特異的CD8陽性輸注T細胞の割合について測定した結果、末梢血中には同細胞の著しい増加が認められた(図10-2)。本細胞はエフェクターメモリーT細胞(CD44陽性CD62L陰性)及びセントラルメモリーT細胞(CD44陽性CD62L陽性)フェノタイプを示し(図10-3)、特にワクチン再接種後の個体には、本細胞が爆発的に増殖しており、CD44強陽性エフェクター細胞が存在することが明らかとなった(図10-4)。また、同個体にB16F10を再移植しても腫瘍の生着・増殖は認められず、同マウスは再発しないことが明らかとなった。(図10-5)。
マウス腫瘍増殖試験(実施例1)と同様の試験を実施後(治療群当たり9匹)、腫瘍の再増殖が認められた個体(9匹中1匹)に対し、腫瘍移植後27、32、36、40、43日目に長鎖ペプチド抗原搭載HAナノゲルがんワクチン75μgをPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に皮下投与した。また、腫瘍移植後41日目に、変異型ERK2認識TCR遺伝子導入マウス(DUC18)の脾臓からCD8a マイクロビーズ(ミルテニー)を用いて単離した抗原特異的CD8陽性T細胞(2×106個)をマウス尾静脈内より輸注した。また、腫瘍移植後49日目に、ICI(抗マウスPD-1抗体200μg及び抗マウスCTLA-4抗体100μg)を腹腔内投与し、経時的に腫瘍面積を測定した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法を実施後、一度治療が奏効したものの、その後腫瘍の再増殖が認められた個体に対し、ワクチン再投与、再輸注及びICIを実施した。その結果、一般的に再燃した腫瘍は、増殖速度が速く予後不良だが(図11-2右図)、ワクチン連投により著しく腫瘍の増殖を抑制し、他の免疫療法(ICI)との併用により、腫瘍の増悪を抑え得ることが明らかとなった(図11-1及び図11-2左図)。
実施例11施行後、腫瘍の完全退縮が認められた個体(n=8)に対し、腫瘍移植2ヶ月後に同個体にCMS5a腫瘍(1×106個)を再移植し、腫瘍の面積を測定した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法を実施後、腫瘍の完全退縮が認められた個体に対しCMS5a腫瘍を再移植した結果、全てのマウスで腫瘍の増殖抑制が認められ、8匹中6匹は腫瘍の完全退縮(75%)に至った(図12-1及び図12-2)。
マウス腫瘍増殖試験(実施例1)について、抗原特異的T細胞輸注療法を2回から1回に減らし、同様の試験を実施した(治療群当たり9匹)。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法併用におけるCD8陽性輸注T細胞の輸注回数が抗腫瘍効果に及ぼす影響について明らかにするため、CD8陽性輸注T細胞の輸注回数を従来法の2回から1回に漸減し、治療効果について検討した。その結果、CD8陽性輸注T細胞を単回輸注した場合においても、従来法や先行技術の2回輸注の場合と同等に、全ての個体で腫瘍の退縮が認められ、9匹の個体の内、8匹で完全に腫瘍が消失した(奏効率:100%、完全奏効:89%、部分奏効:11%)(図13-1及び図13-2)。
実施例13施行後、腫瘍の再増殖が認められた個体(9匹中1匹)に対し、腫瘍移植後26、30、33及び36日目に長鎖ペプチド抗原搭載HAナノゲルがんワクチン75μgをPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に皮下投与した。また、腫瘍移植後33、36及び39日目にICI(抗マウスPD-1抗体200μg及び抗マウスCTLA-4抗体100μg)を腹腔内投与し、経時的に腫瘍面積を測定した。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法(単回)実施後、一度治療が奏効したものの、その後腫瘍の再増殖が認められた個体に対し、ワクチン及びICIを投与した。その結果、一般的に再燃した腫瘍は、増殖速度が速く予後不良なのに対し、ワクチン連投やICI併用により著しく腫瘍の増殖を抑制できることが明らかとなった(図14)。
実施例13施行後、腫瘍の完全退縮が認められた個体(n=8)の末梢血を実施例5と同様の方法により採取(腫瘍移植後36日目)し、CD8 陽性T細胞のメモリーフェノタイプについて解析した。さらに、同マウス(n=4)に長鎖ペプチド抗原搭載HAナノゲルがんワクチン75μgをPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に皮下投与(腫瘍移植後36及び39日目)し、同細胞のメモリーフェノタイプについて再解析した(腫瘍移植後41日目)。細胞染色及び測定は実施例10に従い同様の方法で行なった。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法(単回)施行後、腫瘍の完全退縮が認められた個体の末梢血中に存在するCD8陽性T細胞について解析した結果、同個体にはエフェクターメモリー及びセントラルメモリーフェノタイプを示すCD8陽性T細胞が含まれ、ワクチンの再接種により、両者の細胞の割合が著しく増加することが明らかとなった(図15)。
実施例14により追加治療中の個体の末梢血を実施例5と同様の方法により採取(腫瘍移植後36日目)し、CD8 陽性T細胞のメモリーフェノタイプについて解析した。さらに、同マウスに長鎖ペプチド抗原搭載HAナノゲルがんワクチン75μgをPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に皮下投与(採血後同日)し、同細胞のメモリーフェノタイプについて再解析した(腫瘍移植後41日目)。細胞染色及び測定は実施例10に従い、同様の方法で行なった。
HAナノゲルがんワクチン及び抗原特異的T細胞輸注療法(単回)施行後、奏効から再増殖に転じた個体(追加治療中)の末梢血中に存在するCD8陽性T細胞について解析した結果、完全退縮が認められた個体同様に、追加治療中の個体においてもエフェクターメモリー及びセントラルメモリーフェノタイプを示すCD8陽性T細胞が含まれ、ワクチンの再接種により、両者の細胞の割合が著しく増加することが明らかとなった(図16)。
実施例6~16の結果から、特に、以下の8点が判明し又は示唆された。
1.本治療は、高転移性で高致死性且つ速致死性、低免疫原性、腫瘍にT細胞が浸潤しにくい(Cold tumor, Immune desert tumor)などの特徴を有する治療困難な悪性黒色腫(メラノーマ)に対し、強力に治療効果を発揮する(実施例6)。
2.上記の治療困難な腫瘍に対し、先行技術であるCHPワクチンでは腫瘍増殖や致死を抑えられないのに対し、本治療は強力に抗腫瘍効果を発揮し腫瘍を抑え、生存を維持し得る(実施例7)。
3.本治療は、ワクチン投与部位の所属リンパ節で抗原特異的CD8 T細胞を顕著に増加させ、非常に活性化された抗原特異的細胞傷害性CD8 T細胞(CTL)を誘導し得る(実施例8)。
4.輸注した抗原特異的CD8 T細胞は、腫瘍内にもよく浸潤し、且つ全身にも顕著に局在しており、腫瘍の転移を抑えている可能性がある(実施例9)。
5.本治療は、強力な治療効果に加え、治療奏効後の再発も予防可能で、がん治療型及び再発予防型ワクチンの両側面を併せ持つ(実施例10及び実施例12)。
6.さらに本治療により治療奏効後あるいは追加治療中のワクチン再投与により、迅速にメモリーT細胞を強くサステナブルに誘導し得る(実施例10及び実施例15~16)。
7.又、治療奏効後、腫瘍の再増殖が認められた場合においても、ワクチン連投や他の免疫療法との併用により腫瘍の増殖を抑えられる(実施例11及び実施例14)。
8.従来法や先行技術(輸注療法2回)に対し、本治療は単回でも顕著に治療効果を発揮し、患者の身体面及び経済面での負担を軽減し得る(実施例13)。
実施例1~16におけるTCR-Tとヒアルロン酸誘導体及び抗原を含む投与組成物の試験結果より、ヒアルロン酸誘導体と抗原を取り込んだ抗原提示細胞が、抗原を細胞表面に効率的に提示し、この抗原提示細胞が輸注されたT細胞を活性化ならびに増殖させることが高い抗腫瘍効果のメカニズムの一つであると考えられる。既報1(Blood (2018) 132 (11): 1134-1145、Antitumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination)では、キメラ抗原受容体を発現するリンパ球を輸注すると共に、抗原を取り込んだ抗原提示細胞を輸注することで抗腫瘍効果が高まることが報告されている。既報2(Science. 2020 Jan 24;367(6476):446-453、An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors)では、キメラ抗原受容体を発現するリンパ球を輸注すると共に、キメラ抗原受容体に認識される抗原を抗原提示細胞に発現させることで抗腫瘍効果が高まることが報告されている。従って、TCR-Tのみならずキメラ抗原受容体発現リンパ球を輸注する際においても、抗原を取り込んだ抗原提示細胞を体内で作るヒアルロン酸誘導体及び抗原を含む投与組成物を投与することにより、同様に高い抗腫瘍効果が期待できる。
(1)細胞
マウス大腸がん細胞株MC38及びCT26 は、ATCCから購入し、三重大学で継代したものを用いた。レトロウイルスパッケージング細胞株Plat-Eは、CELL BIOLABSから購入したRPMI1640培地は、細胞科学研究所から購入した。高グルコースD-MEM培地は、和光純薬から購入した。Opti-MEM及びウシ胎児血清(FBS)は、Gibcoから購入した。アルブミナーはCSL Behlingから、ヒトIL-2はノバルティスから購入した。
HLA-A2トランスジェニック(Tg)マウスは、パスツール研究所より分譲されたHHD-A2プラスミドDNAを三重大学動物実験施設にてC57BL/6マウスの受精卵に注入し作製した。同マウスを三重大学動物実験施設で繁殖させ、さらにBALB/cマウスとバッククロスしたHLA-A2 Tgマウス(BALB/cバックグラウンド)を作製、繁殖させ、これらを三重大学動物実験施設で飼育し、実験に使用した。
抗マウスCD3e抗体(クローン145-2C11)、抗マウスCD28抗体(クローン37.51)及びAlexa Fluor 647標識抗ウサギIgG抗体(クローンPoly4064)は、invitrogenから購入した。APC標識抗マウスCD4 抗体(クローンRM4-5)及びAPC/Cyanine7標識抗マウスCD8抗体(クローン53-6.7)は、BioLegendから購入した。MAGE-A4 (E7O1U) XP Rabbit mAbは、Cell Signaling technologyから購入した。PE標識抗ヒトHLA-A2抗体(クローンBB7.2)は、MBLから購入した。Purified Rat Anti-Mouse IFN-γ抗体及びBiotin Rat Anti-Mouse IFN-γ抗体は、BDバイオサイエンスから購入した。
Lipofectamine 3000 Reagent、P300 reagent及びhuman IFN-γ uncoated ELISA kitは、invitrogenから購入した。pCL-ECO Retrovirus Packaging Vectorは、NOVUSから購入した。A2 MAGE-A4 tetramerは、Streptavidin-R-Phycoerithrin(Agilent)を使用し作製した。RetroNectinはタカラバイオから購入した。
1-1 キメラ抗原受容体用
(1)mMAGE#17 zG CAR
CAR作製に使用したMAGE#17scFvは、人工的に作製したHLA-A*02:01 MAGE-A4 p230-239ペプチドMHC複合体に対してヒト抗体ライブラリを反応させて単離したものである(特願2019-523909他/MAGE-A4由来ペプチドを認識する抗原結合性タンパク質)。scFvの5’側にヒトVH leader配列を連結し、3’側にmCD8 hinge、mCD28 TM、mCD28 ICD、mCD3z及びmGITR ICDを連結するよう設計し、人工遺伝子を作製した(Genscriptに受注)。本遺伝子をレトロウイルス発現プラスミドに組み込み、mMAGE zGウイルスベクタープラスミドを作製した(図17-1a及び図17-1b)。
scFvの5’側にヒトVH leader配列を連結し、3’側にmCD8 hinge、mCD28 TM、mCD28 ICD及びmCD3zを連結するよう設計し、人工遺伝子を作製した(Genscriptに受注)。本遺伝子をレトロウイルス発現プラスミドに組み込み、mMAGE zGウイルスベクタープラスミドを作製した(図17-2a及び図17-2b)。
(1)mNGFR共発現MAGE-A4
ヒトMAGE-A4遺伝子とP2Aペプチド配列、C末truncateのmouse NGFR遺伝子を連結したものを設計し、人工遺伝子を作製した(Genscriptに受注)。本遺伝子を制限酵素NotIとXhoIにて消化して切り出し、レトロウイルス発現プラスミドに組み込んだものを作製した(図17-3a及び図17-3b)。
HHD-A2プラスミドのMHC配列よりintron部分を削除し、mRNA型HHD-A2発現遺伝子を設計し、人工遺伝子を作製した(Genscriptに受注)。本遺伝子をNotI-XhoIで切り出しレトロウイルス発現プラスミドに組み込んだものを作製した(図17-4a及び図17-4b)。
2-1 レトロウイルスの作製
レトロウイルスパッケージング細胞株Plat-E(1.2×106個)を高グルコースD-MEM培地を用いて6 well plateで24時間培養し、Opti-MEM(250μL)及びLipofectamine 3000 Reagent(7μL)と混合した(A液)。さらにOpti-MEM(250μL)、1-1(1)で調製したmMAGE#17 zG CARウイルスベクタープラスミド(3μL)、pCL-ECO Retrovirus Packaging Vector (1μL)及びP300 reagent(6μL)を混合し、A液と混合後15分室温で静置した。6 well plate から培地を半量抜き取り、混合液を添加し、37℃、5% CO2インキュベーターにて6時間培養した。その後、6 well plateの培地を全て交換し、24時間、48時間及び72時間後に培地を回収し、0.45μmフィルターで濾過滅菌を行い、凍結保存した。
BALB/cマウスより脾臓を採取し、スライドガラスを用いて磨砕後、細胞をRPMI1640培地中に回収した。遠心分離(400×g、5分、4℃)後、上清を除去し、2mLの赤血球溶血溶液を加えて細胞を30秒間処理した。その後18mLのRPMI1640培地を加え、遠心分離(400×g、5分、4℃)し、上清を除き、3×106個/mLの濃度で細胞を10%FBS含有RPMI1640培地に懸濁した。予め前日に抗マウスCD3e抗体(1μg/mL)及び抗マウスCD28抗体(2.5μg/mL)を6 well plateに添加し、4℃にて一晩静置固相化したplateをPBSで3回洗浄後、細胞懸濁液 5mL(1.5×107個)を播種し、37℃、5% CO2インキュベーターにて24時間培養した。
細胞を回収し、96穴V底マイクロプレート(ヌンク)に移し、遠心分離(2000×rpm、2分、4℃)し上清除去後、1wellあたり200μLの染色用バッファー(0.5% BSA含有PBS)に懸濁した。200μLの染色用バッファーで細胞を2回洗浄後、100倍希釈したA2 MAGE-A4 tetramer 30μLを加え、30分暗所で静置した。染色用バッファーで細胞を2回洗浄後、各抗体のメーカーが推奨する使用濃度に従い、細胞懸濁液にAPC標識抗マウスCD4抗体及びAPC/Cyanine7標識抗マウスCD8抗体を添加混合後、4℃の暗所にて15分間静置した。150μLの染色用バッファーを加え、遠心分離して上清を除去後、さらに200μLの染色用バッファーで細胞を2回洗浄後、バッファー200μLに再懸濁を行い、丸底ポリスチレンチューブ(BDバイオサイエンス)へ移した。細胞はフローサイトメーターLSRFortessa Cell Analyzer(BDバイオサイエンス)及び付属の解析ソフトウェア(FACSDiva)を用いて解析した(図17-5)。
3-1 レトロウイルスの作製
1-1(1)で調製したmMAGE#17 zG CARウイルスベクタープラスミドの代わりに1-2(1)で調製したmNGFR共発現MAGE-A4ウイルスベクタープラスミド、もしくは1-2(2)で調製したHHD-A2ウイルスベクタープラスミドを用いたこと以外は2-1に記載と同じ方法でレトロウイルスを作製した。
T75培養フラスコ(コーニング)を用いて、マウス大腸がん細胞株MC38及びCT26細胞株を10%FBS含有RPMI1640培地中で培養した。培養した細胞は0.5%トリプシン含有PBSを用いて剥離し、10%FBS含有RPMI1640培地に懸濁した。懸濁液を遠心分離(400×g、5分、4℃)した後に上清を除いた。その後、10%FBS含有RPMI1640培地を用いて2回洗浄し、3.3×105個/mLの濃度で10%FBS含有RPMI1640培地に懸濁した。
HHD-A2:メーカーが推奨する使用濃度に従い、細胞懸濁液にPE標識抗HLA-A2抗体を添加し、4℃の暗所にて15分間静置した。150μLの染色用バッファーを加え、遠心分離して上清を除去後、さらに200μLの染色用バッファーで細胞を2回洗浄後、バッファー200μLに再懸濁を行い、丸底ポリスチレンチューブ(BDバイオサイエンス)へ移した。細胞はフローサイトメーターFACS LSRFortessa Cell Analyzer(BDバイオサイエンス)及び付属の解析ソフトウェア(FACSDiva)を用いて解析した(図17-6)。
3-2で作製した標的細胞(1. MC38細胞、2. HHD-A2導入MC38細胞、3. HHD-A2導入MC38細胞(MAGE-A4 CD8 エピトープペプチド(配列番号9)10μMパルス)、4. HHD-A2及びMAGE-A4導入MC38細胞)を5×104個/100μlの濃度で10% FBS含有RPMI1640培地に懸濁し、同じく10% FBS含有RPMI1640培地に懸濁したmMAGE zG CAR T細胞(1×105個/100μl)又はCAR遺伝子非導入細胞をプレートに添加し、24時間共培養後、培養上清を回収した。CT26細胞についても同様にmMAGE zG CAR T細胞と共培養後、培養上清を回収した。回収した培養上清は下記に示すELISA法により、IFN-γ産生について評価した。
4-1 分子量10kのHAナノゲル、ペプチドMAGE-A4 29merを用いた複合体の作製
ヒアルロン酸ナノゲル(HANG)
国際公開第2010/053140号の実施例1及び2に記載のとおりに、コレステリル基が導入されてなるヒアルロン酸ナノゲル(重量平均分子量が10kDa、コレステリル基の導入率が44%)を合成した。
上記で得たヒアルロン酸ナノゲルを用いて、ペプチド-ヒアルロン酸ナノゲル複合体の調製を次の通り行った。MAGE-A4ペプチドはユーロフィンから購入した。配列は次のとおりである:AMEGDSASEEEIWEELGVMGVYDGREHTV(配列番号8)。当該配列は、CD8陽性細胞傷害性T細胞認識エピトープ配列としてGVYDGREHTV(配列番号9)を含む。
ヒアルロン酸ナノゲルの凍結乾燥体を秤量し、5mg/mLの濃度となるように注射用水を添加し、一晩攪拌して十分に溶解させた。一方、別の容器にて、ペプチドの濃度が50mg/mLとなるようにジメチルスルホキシドへ溶解させた。溶解を確認した後に、室温で、上記ヒアルロン酸ナノゲル水溶液と1mol/Lの水酸化ナトリウム水溶液を容量比100:2の割合で混合した。続いて上記ヒアルロン酸ナノゲル水溶液とペプチドのジメチルスルホキシド溶液を容量比102:1の割合で混合し、室温で24時間攪拌することで、ペプチド-ヒアルロン酸ナノゲル複合体を含む組成物を得た。その後、固体の精製スクロース(富士フイルム和光社製、製造専用、製品番号198-18385)をペプチド-ヒアルロン酸ナノゲル複合体を含む組成物が10質量%スクロースとなるように添加して、1時間以上攪拌した。続いて、10質量%スクロース含有25mMリン酸緩衝液(pH 7.4)にてペプチドの理論濃度が0.3 mg/mLとなるように希釈し、0.2μmPES(ポリエーテルスルフォン)(Pall社製、アクロディスクシリンジフィルター、25mmΦ)で滅菌ろ過し、ペプチド-ヒアルロン酸ナノゲル複合体を含む組成物を得た。この時のペプチド-ヒアルロン酸ナノゲル複合体を含む組成物中のペプチド濃度を逆相クロマトグラフィー分析により定量し、ペプチド濃度が250ug/mLとなるように濃度調整し、投与溶液とした。
以下に示す長鎖ペプチド抗原を用いた他は上記4-1に記載の方法で調製した。ペプチド濃度が250ug/mLとなるように濃度調整し、投与溶液とした。
長鎖ペプチド抗原 MAGE-A4 20mer
MAGE-A4 20merはユーロフィンから購入した。配列は次の通りである:GVYDGREHTVGVYDGREHTV(配列番号10)。当該配列は、CD8陽性細胞傷害性T細胞認識エピトープ配列として下記MAGEA4(アミノ酸配列:GVYDGREHTV(配列番号9)を含む。
HLA-A2 Tgマウスの右側前背部側に3-2で調製したMAGE-A4/HHD-A2発現MC38細胞を5×106個/匹で皮下移植する。腫瘍移植後5日及び10日目に、2-2で調製したキメラ抗原受容体発現リンパ球をPBSに3×106個/200μLの濃度で懸濁後、マウス尾静脈内より200μL輸注する。4-1、4-2で調製した長鎖ペプチド抗原搭載HAナノゲルがんワクチンは腫瘍移植後4、9(及び14)日目に、長鎖ペプチド抗原搭載HAナノゲルがんワクチン50μgをPBSに溶解した50μgのCpGオリゴDNAと共にマウスの右側後背部に皮下投与する。その後、経時的に腫瘍面積を測定する。
Claims (31)
- 疎水性基が導入されてなるヒアルロン酸誘導体及び抗原を含有する、前記抗原に対する免疫受容体を発現するリンパ球との併用投与に用いるための医薬組成物。
- 前記疎水性基がステリル基である、請求項1に記載の医薬組成物。
- 前記ヒアルロン酸誘導体と前記抗原とが複合体を形成している、請求項1又は2に記載の医薬組成物。
- 前記抗原ががん抗原である、請求項1~3のいずれかに記載の医薬組成物。
- 前記抗原が抗原ペプチド又は抗原タンパク質である、請求項1~4のいずれかに記載の医薬組成物。
- 前記抗原がCD8陽性細胞傷害性T細胞認識エピトープ及び/又はCD4陽性ヘルパーT細胞認識エピトープを含む、請求項5に記載の医薬組成物。
- 前記免疫受容体がT細胞受容体又はキメラ抗原受容体である、請求項1~6のいずれかに記載の医薬組成物。
- さらにアジュバントを含有する、或いはアジュバントとの併用投与に用いるための、請求項1~7のいずれかに記載の医薬組成物。
- がんの予防又は治療用である、請求項1~8のいずれかに記載の医薬組成物。
- 抗原に対する免疫受容体を発現するリンパ球を含有する、疎水性基が導入されてなるヒアルロン酸誘導体及び前記抗原を含有する組成物との併用投与に用いるための医薬組成物。
- 疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物と、前記がん抗原に対する免疫受容体を発現するリンパ球とを含む、がん治療キット。
- 疎水性基が導入されてなるヒアルロン酸誘導体及び抗原を含有する、T細胞活性化ワクチン。
- がん抗原に対する免疫受容体を発現するT細胞と、医薬組成物とを含むがん治療キットであり、
前記医薬組成物は、疎水性基が導入されてなるヒアルロン酸誘導体と、前記がん抗原に由来する抗原ペプチド又は抗原タンパクと、アジュバントとを含有し、
前記ヒアルロン酸誘導体が式(I):
[式中、R1、R2、R3、およびR4は、それぞれ独立に、水素原子、C1-6アルキル、ホルミルおよびC1-6アルキルカルボニルから選択され;
R5は、水素原子、ホルミル、またはC1-6アルキルカルボニルであり;
Zは、直接結合、または2~30個の任意のアミノ酸残基からなるペプチドリンカーを表し;X1は、以下の式:
-NRb-R、
-NRb-COO-R、
-NRb-CO-R、
-NRb-CO-NRc-R、
-COO-R、
-O-COO-R、
-S-R、
-CO-Ya-S-R、
-O-CO-Yb-S-R、
-NRb-CO-Yb-S-R、および
-S-S-R、
により表される基から選択される疎水性基であり;
Ra、RbおよびRcは、それぞれ独立に、水素原子、C1-20アルキル、アミノC2-20アルキルおよびヒドロキシC2-20アルキルから選択され、ここで当該基のアルキル部分は、-O-および-NRf-から選択される1~3個の基が挿入されていてもよく;
Rfは、水素原子、C1-12アルキル、アミノC2-12アルキルおよびヒドロキシC2-12アルキルから選択され、当該基のアルキル部分は-O-および-NH-から選択される1~2個の基が挿入されていてもよく;
Rは、ステリル基であり;
Yは、C2-30アルキレン、または-(CH2CH2O)m-CH2CH2-であり、ここで、当該アルキレンは、-O-、-NRg-および-S-S-から選択される1~5の基が挿入されていてもよく;
Rgは、水素原子、C1-20アルキル、アミノC2-20アルキルまたはヒドロキシC2-20アルキルから選択され、当該基のアルキル部分は-O-および-NH-から選択される1~3個の基が挿入されていてもよく;
Yaは、C1-5アルキレンであり;
Ybは、C2-8アルキレンまたはC2-8アルケニレンであり;
mは、1~100から選択される整数である]
で表される繰り返し単位を1以上含む、がん治療キット。 - 前記ヒアルロン酸誘導体のグルクロン酸部分のカルボキシル基の疎水性基による修飾率が5~50%である、請求項13に記載のがん治療キット。
- Zが直接結合であり、YがC2-12アルキレンであり、X1が-NH-COO-Rであり、且つRがコレステリル基である、請求項14に記載のがん治療キット。
- 前記ヒアルロン酸誘導体の重量平均分子量が5,000~2,000,000である、請求項13に記載のがん治療キット。
- 前記医薬組成物が前記がん抗原と前記ヒアルロン酸誘導体との複合体を含有する、請求項13に記載のがん治療キット。
- 前記抗原ペプチドがCD8陽性細胞傷害性T細胞認識エピトープおよび/またはCD4陽性ヘルパーT細胞認識エピトープを2つ以上含む、請求項13に記載のがん治療キット。
- 前記抗原ペプチドが、2種以上のCD8陽性細胞傷害性T細胞認識エピトープ、又は1種以上のCD8陽性細胞傷害性T細胞認識エピトープ及び1種以上のCD4陽性ヘルパーT細胞認識エピトープを含む長鎖ペプチド抗原である、請求項19に記載のがん治療キット。
- 低免疫原性のがん、転移性のがん、又は腫瘍内T細胞数が少ないがんに対するT細胞輸注療法に用いられる、請求項13に記載のがん治療キット。
- 前記医薬組成物は前記T細胞を投与する前に投与される、請求項13に記載のがん治療キット。
- 前記医薬組成物が1回投与された後に前記T細胞が1回輸注されることを投与回数の単位とした際に、1単位又は2単位の投与がなされた後に、前記医薬品組成物が少なくとも1回投与される、請求項22に記載のがん治療キット。
- 前記医薬組成物は少なくとも2回皮下又は静脈内投与され、前記T細胞は前記医薬品組成物の投与1回目の後且つ前記医薬品組成物の投与2回目の前に輸注される、請求項22に記載のがん治療キット。
- がんを有する対象に、疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物と、がん抗原に対する免疫受容体を発現するリンパ球とを、併用投与することを含む、がんを治療する方法。
- がん治療における使用のための、前記がん治療キットが、疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物と、前記がん抗原に対する免疫受容体を発現するリンパ球とを含む、がん治療キット。
- がん抗原に対する免疫受容体を発現するリンパ球を投与するがん治療における使用のための、疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物。
- 疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物を投与するがん治療における使用のための、がん抗原に対する免疫受容体を発現するリンパ球を含有する医薬組成物。
- がん治療キットの製造のための、疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物と、前記がん抗原に対する免疫受容体を発現するリンパ球との、組合せの使用。
- がん抗原に対する免疫受容体を発現するリンパ球と併用投与するためのがん治療剤の製造のための、疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物の使用。
- 疎水性基が導入されてなるヒアルロン酸誘導体及びがん抗原を含有する医薬組成物と併用投与するためのがん治療剤の製造のための、がん抗原に対する免疫受容体を発現するリンパ球の使用。
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KR20240016309A (ko) | 2024-02-06 |
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