Modulators of the cancer immune microenvironment and their preventive, diagnostic, and therapeutic uses.

Abstract

The present invention aims to identify regulatory factors of the cancer immune microenvironment and to provide methods for the therapeutic use of these regulatory factors. [Solution] Specifically, the present invention is an inhibitor of the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor immune microenvironment (TIME), comprising as an active ingredient a substance that suppresses or inhibits the function of immunomodulatory factors released from tumor dead cells. More specifically, the inhibitor comprises, for example, a substance that suppresses or inhibits the function of TCTP (translationally controlled tumor protein).

Description

Technical Field

[0001] The present invention relates to a regulator of the cancer immune microenvironment and its preventive, diagnostic and therapeutic uses. More specifically, the present invention relates to a functional inhibitor of TCTP (translationally controlled tumor protein), a therapeutic agent or therapeutic composition for cancer containing the substance, and the preventive, diagnostic and therapeutic uses of the substance for cancer.

[0002] The immune response mechanism against cancer has been debated for a long time, and various research reports have been conducted. In recent years, due to research, the mechanism and the like have been gradually understood, and "cancer immunotherapy" has attracted attention. Currently, the main immunotherapies actually used for cancer treatment include "checkpoint inhibitory antibody therapy" and "CAR-T cell therapy". However, although the effects of both have been confirmed to have a certain effect in certain types of cancer, the current situation is that they are not sufficient therapies for the treatment of many other cancers.

[0003] Both of the above two therapies aim to treat cancer by activating lymphocytes (which attack cancer). On the other hand, in vivo, a suppression mechanism of the immune function that attacks cancer cells, called the tumor immune microenvironment (TIME), is known (Non-Patent Document 1 and Non-Patent Document 2). The cancer immune microenvironment is also called the "cancer nest", and it is known that a mechanism exists there to suppress the immune response against cancer, creating an environment favorable for the growth of cancer cells. Therefore, elucidating the interaction between cancer cells and immune system cells and the interaction between related molecules in TIME is expected to lead to the development of new immunotherapies.

[0004] Incidentally, myeloid-derived suppressor cells (MDSCs) play an important role in the immunosuppressive function associated with tumor progression. MDSCs are a population of progenitor cells present in the bone marrow that have different degrees of differentiation before differentiating into granulocytes, dendritic cells, and macrophages. Among the cell populations that make up MDSCs, two types of cell populations have been identified and are attracting attention in mice and humans. One of these two types of cells is polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC), which has phenotypic and morphological characteristics similar to neutrophils, and the other is monocytic myeloid-derived suppressor cell (M-MDSC), which is similar to monocytes and is known to differentiate into tumor-associated macrophages (TAMs) (Non-Patent Literature 3). Regarding the relationship between MDSCs and cancer, it has been reported that in malignant tumors, the expression level of MDSCs increases and accumulates in TIME, and that this is directly related to the clinical stage of cancer, the amount of metastatic tumors, and the prognosis of cancer. Since MDSCs actually suppress the activity and proliferation of lymphocytes (Non-Patent Literature 4), it is thought that they play a role in promoting tumor progression. However, the mechanism by which TIME accumulates MDSCs remains unclear. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Binnewies et al., Nature Medicine 24, 541-550, doi:10.1038 / s41591-018-0014-x (2018). [Non-Patent Document 2] Munn et al., Current opinion in immunology 39, 1-6, doi:10.1016 / j.coi.2015.10.009 (2016). [Non-Patent Document 3] Kumar et al., Trends in immunology 37, 208-220, doi:10.1016 / j.it.2016.01.004 (2016). [Non-Patent Document 4] Fleming et al., Frontiers in immunology 9, 398, doi:10.3389 / fimmu.2018.00398 (2018). [Non-Patent Document 5] Hangai et al., Proceedings of the National Academy of Sciences of the United States of America 113, 3844-3849, doi:10.1073 / pnas.1602023113 (2016). [Non-Patent Document 6] Katoh et al., Cancer cell 24, 631-644, doi:10.1016 / j.ccr.2013.10.009 (2013). [Non-Patent Document 7] Hsu et al., Nature 445, 785-788, doi:10.1038 / nature05528 (2007). [Non-Patent Document 8] Cans et al., Proceedings of the National Academy of Sciences of the United States of America 100, 13892-13897, doi:10.1073 / pnas.2335950100 (2003). [Non-Patent Document 9] Amson et al., Nature medicine 18, 91-99, doi:10.1038 / nm.2546 (2011). [Non-Patent Document 10] Gong et al., Nature reviews. Immunology 20, 95-112, 250 doi:10.1038 / s41577-019-0215-7 (2020). [Non-Patent Document 11] Fujita et al., FEBS letters 582 1055-1060, doi:10.1016 / j.febslet.2008.02.055 (2008). [Non-Patent Document 12] Hiraoka et al., British journal of cancer. 103, 1057-1065, (2010). [Non-Patent Document 13] Marjou et al., Genesis. 39(3), 186-93, (2004). [Non-Patent Document 14] Zhang et al., The Journal of Gene Medicine. 7(3), 354-65, (2005). [Non-Patent Document 15] Schroder et al., Scientific Reports. 8(1), 13399, (2018). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In view of the above circumstances, the present invention aims to solve the problem of identifying regulatory factors of the cancer immune microenvironment and providing a method for therapeutic use by inhibiting said regulatory factors. [Means for solving the problem]

[0007] It is known that many tumor cells die, primarily through necrosis, during proliferation due to the introduction of mutations or changes in the surrounding environment (e.g., hypoxia) (Non-Patent Literature 12). The present inventors formulated a working hypothesis that molecules released from dead tumor cells (hereinafter also referred to as "dead tumor cells") function as immunomodulators of MDSCs during TIME, and as a result of diligent research, they discovered for the first time that Translationally controlled tumor protein (hereinafter referred to as TCTP), released from dead tumor cells, is a novel immunomodulator that controls the function and dynamics of MDSCs during TIME. Specifically, the inventors have revealed that extracellular TCTP induces the expression of CXCL1 family chemokines, primarily by acting on M-MDSCs. CXCL1 family chemokines recruit PMN-MDSCs to TIME, placing TIME in a highly immunosuppressive state. The inventors have shown that administration of an anti-TCTP monoclonal antibody or a TCTP function inhibitor inhibits the induction of PMN-MDSCs into TIME, thereby suppressing tumor growth. This invention addresses the unresolved question of how tumors construct and regulate TIME by identifying TCTP and elucidating its novel function, and is completed by developing a method to inhibit it.

[0008] The present invention is as follows (1) to (13). (1) An inhibitor of the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor immune microenvironment (TIME), containing as an active ingredient a substance that suppresses or inhibits the function of immunomodulatory factors released from tumor dead cells. (2) The inhibitor according to (1) above, wherein the myeloid-derived immunosuppressor cells are polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). (3) The inhibitor according to (1) or (2) above, wherein the immunomodulatory factor is TCTP (translationally controlled tumor protein). (4) The inhibitor described in (3) above, wherein the substance that suppresses or inhibits the function of TCTP is an antibody. (5) The inhibitor described in (3) above, wherein the substance that suppresses or inhibits the function of TCTP is dihydroartemisinin (DHA). (6) A therapeutic agent or therapeutic composition for cancer, comprising any of the inhibitors described in (1) to (5) above as an active ingredient. (7) The therapeutic agent or therapeutic composition according to (6) above, wherein the cancer is colorectal cancer, malignant melanoma, or fibrosarcoma. (8) A method for diagnosing cancer or a method for assisting in diagnosis, comprising measuring the amount of TCTP mRNA or TCTP protein present in a sample derived from a subject. (9) The method according to (8) above, wherein the sample is blood or tissue. (10) An antibody or antigen-binding fragment characterized in that the amino acid sequence of CDR (complementarity determining region) 1 to 3 satisfies any of the following (A), (B), or (C). (A) Heavy chain CDR1 containing the amino acid sequence represented by Sequence ID No. 1, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 2, Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 3, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 5, and It has a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 6. (B) Heavy chain CDR1 containing the amino acid sequence represented by Sequence ID No. 7, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 9, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 10, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 11, and It has a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 12. (C) Heavy chain CDR1 containing the amino acid sequence represented by Sequence ID No. 13, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 14, Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 15, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 16, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 17, and It has a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 18. (11) An antibody that suppresses or inhibits the function of TCTP, wherein the antibody or its antigen-binding fragment competitively inhibits the binding of the antibody described in (10) above to TCTP. (12) The antibody or antigen-binding fragment thereof according to (10) or (11) above, characterized in that it is a humanized antibody. (13) An antigen-binding fragment according to any one of (10) to (12) above, characterized in that it is Fab, Fab', F(ab')2, Fv, a single-chain antibody, scFv, an scFv dimer, or dsFv. In this specification, the symbol "~" indicates a numerical range that includes the values ​​to its left and right. [Effects of the Invention]

[0009] The present invention provides novel cancer therapeutics and novel cancer treatments. [Brief explanation of the drawing]

[0010] [Figure 1]Investigation of the effects of molecules released from dead tumor cells on immune cells. (a) Representative examples of hematoxylin / eosin (H&E) staining and TUNEL staining of SL4 cells are shown. An enlarged image of the area within the frame in the upper figure is shown in the lower figure. Arrowheads indicate necrotic lesions. Scale bar is 100 μm. (b) PECs (2 × 10⁵ cells) were stimulated with the supernatant of dead cells of SL4 cells (2 × 10⁶ cells) for 2 hours, and changes in gene expression within the PECs were analyzed by microarray (n=2). The figure shows a volcano plot, with genes showing altered expression indicated in red or green. (c) Results of quantifying expressed Cxcl1 mRNA, Cxcl2 mRNA, Tnf mRNA, and Il1b mRNA by RT-qPCR after stimulating PECs (2 × 10⁵ cells) with the supernatant of dead cells of SL4 cells (2 × 10⁶ cells) for 2 hours (n=3). Data are shown as mean ± standard error (sem). [Figure 2]Investigation of the effects of TCTP on tumor growth (1). (a) Results of quantifying expressed Cxcl1 mRNA, Cxcl2 mRNA, Tnf mRNA, and Il1b mRNA by RT-qPCR after stimulating PEC (2 × 10⁵ cells) with 5 nM or 15 nM recombinant TCTP for 2 hours are shown (n=3). (b) Serum from mice without tumors (NTB; n=4) and mice with SL4 cell tumors (day 0, n=4) was collected on day 13 (n=6) and day 21 (n=7) after tumor transplantation, and the amount of TCTP in the serum was quantified by immunoblotting. (c) TCTP WT SL4 cells (TCTP-expressing SL4 cells) and TCTP KO SL4 cells (TCTP-deficient SL4 cells) (2 × 10⁵ cells each) were transplanted into C57BL / 6 mice by subcutaneous injection (WT #1 and KO #1; n=4, WT #2; n = 6, KO #2; n = 5). Tumor volume was then measured daily. (d) TCTP WT B16F10 cells (TCTP-expressing B16F10 cells) and TCTP KO B16F10 cells (TCTP-deficient B16F10 cells) (1 × 10⁵ cells each) were transplanted into C57BL / 6 mice by subcutaneous injection (n=7). Tumor volume was then measured daily. (e) TCTP WT Meth-A induced sarcoma cells (hereinafter, Meth-A cells) and TCTP KO Meth-A cells (1 × 10⁵ cells) were transplanted into C57BL / 6 mice by subcutaneous injection (n=7). Tumor volume was then measured daily. (f) Immunoblotting was performed on TCTP protein and β-actin in intestinal epithelial cells of tamoxifen-treated TCTPflox / flox, Apc+ / Δ716, villin-Cre ERT2 mice (KO) or untreated mice (WT). For (c)~(e), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired two-dided Student's t-test. NS indicates no significant difference. Data are expressed as mean ± standard error (sem). [Figure 3]Investigation of the effects of TCTP on tumor growth (2). (a) Apoptosis (without serum and with adriamycin) or necrosis (freeze-thaw) was induced in SL4 cells. Culture supernatant was collected and the amount of TCTP protein was measured by immunoblotting (n=3). (b) Serum from mice with B16F10 tumors (left) or Meth-A tumors (right) was collected at 0 days (NTB; n=3) and 21 days (NTB; n=3) after subcutaneous transplantation of tumor cells, and the amount of TCTP protein was measured by immunoblotting. (c) Protein expression of TCTP in wild-type (WT) or TCTP gene-inactive SL4 cells, B16F10 cells, and Meth-A cells was investigated. Whole cell lysates were prepared for each cell line, and immunoblotting analysis was performed for TCTP and β-actin. (d) The change in the number of TCTP WT SL4 cells and TCTP KO SL4 cells grown in vitro over time is shown (n=3). (e) TCTP WT SL4 cells and TCTP KO SL4 cells (2 × 10⁵ cells) were cultured under standard conditions (20% O₂, 10% FBS), low serum conditions (0% O₂, 1% FBS), or hypoxic conditions (1% O₂, 10% FBS). The number of cells was counted 72 hours after culturing. (f) TCTP WT SL4 cells, TCTP KO SL4 cells, and TCTP KO SL4 cells transduced with TCTP cDNA (TCTP transduced) (2 × 10⁵ cells each) were subcutaneously transplanted into C57BL / 6 mice (n=5), and the volume of the resulting tumors was measured over time. (g) Time course of the number of TCTP WT Meth-A cells and TCTP KO Meth-A cells grown in vitro (n=3). (h) Time course of the number of TCTP WT B16F10 cells and TCTP KO B16F10 cells grown in vitro (n=3). For (b), *P<0.05, unpaired two-tailed Student's t-test. For (f), Repeated measures one-way ANOVA with Tukey's multiple comparisons test. Data are presented as mean ± standard error (sem). [Figure 4]Investigation of the effects of TCTP on tumor growth (3) (a) The total number of intestinal polyps, the number of intestinal polyps with a diameter of less than 1.0 mm, the number of intestinal polyps with a diameter of 1.0-2.0 mm, and the number of intestinal polyps with a diameter greater than 2.0 mm are shown for 10-week-old TCTPflox / flox, Apc+ / Δ716 mice (WT; n = 6) or TCTPflox / flox, Apc+ / Δ716, villin-Cre ERT2 mice (KO). (b) Representative examples of hematoxylin / eosin staining of intestinal polyps that developed in 10-week-old TCTPflox / flox, Apc+ / Δ716 mice (WT) or TCTPflox / flox, Apc+ / Δ716, villin-Cre ERT2 mice (KO) are shown. The scale bar is 200 μm. Arrowheads indicate tumors. (c) TCTP WT cells or TCTP KO cells were irradiated with a lethal dose (100 Gy) of X-rays and mixed with TCTP WT cells (2 × 10⁵ cells). The cell mixture was transplanted subcutaneously into C57BL / 6 mice (n=6). Tumor volume was measured over time. For (a), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired two-dided Student's t-test. For (c), repeated measures one-way ANOVA with Tukey's multiple comparisons test. NS indicates no significant difference. Data are presented as mean ± standard error (sem). [Figure 5]Investigation of extracellular TCTP function (1). (a) Culture supernatants of TCTP KO SL4 cells (Mock) and IL-2ss-TCTP SL4 cells (IL-2ss-TCTP) were collected, and the amount of TCTP protein was measured by immunoblotting (n=3). (b) The number of TCTP KO SL4 cells, IL-2ss-TCTP SL4 cells, and WT-TCTP SL4 cells in culture was counted over time (n=4). (c) TCTP KO SL4 cells, IL-2ss-TCTP SL4 cells, or WT-TCTP SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=4), and tumor volume was measured over time. (d) WT-TCTP SL4 cells or TCTP KO SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=4). 21 days post-transplant, the tumor was excised and a lysate of the entire tumor was prepared. The protein levels of CXCL1 (left, n=6) and CXCL2 (right, WT n=7, KO n=6) in the prepared lysate were measured by ELISA. (e) TCTP KO SL4 cells (Mock) or IL-2ss-TCTP SL4 cells (2 × 10⁵ cells) were subcutaneously transplanted into C57BL / 6 mice. 21 days post-transplant, the tumor was excised and a lysate of the entire tumor was prepared. The protein levels of CXCL1 (left, n=7) and CXCL2 (right, n=10) in the prepared lysate were measured by ELISA. For (c), *P<0.05, **P<0.01, repeated measures one-way ANOVA with Dunnett's multiple comparisons test. For (d) and (e), unpaired two-dided Student's t-test. ND indicates not detected. The data is presented as the mean ± standard error (SEM) of the values. [Figure 6]Investigation of extracellular TCTP function (2). (a) TCTP and nuclei (DAPI) were stained in TCTP KO SL4 cells (left figure), WT-TCTP cells (TCTP KO cells expressing WT TCTP cDNA; middle figure), and IL-2ss-TCTP SL4 cells (TCTP secretory cells expressing IL-2ss-TCTP cDNA; right figure). (b) PECs (2 × 10⁵ cells) were stimulated for 2 hours with the culture supernatant (Mock) of TCTP KO SL4 cells or the culture supernatant (IL2ss-TCTP) of IL-2SS-TCTP SL4 cells. Subsequently, expressed Cxcl1 and Cxcl2 mRNA were quantified by RT-qPCR (n=3). (c) WT SL4 cells or TCTP KO SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. 21 days post-transplant, the tumor was excised and a lysate of the entire tumor was prepared. The protein levels of G-CSF (left) and GM-CSF (right) in the prepared lysate were measured using a cytometric bead assay (CBA). (d) TCTP WT B16F10 cells (WT) or TCTP KO B16F10 cells (KO) were subcutaneously transplanted into C57BL / 6 mice. 17 days post-transplant, single-cell suspensions were prepared from the tumor and flow cytometry analysis was performed (n=4). The percentage of PMN-MDSC or M-MDSC in the CD45+ cell population is shown. (e) TCTP WT Meth-A cells (WT; n=3) and TCTP KO Meth-A cells (KO; n=4) were subcutaneously transplanted into C57BL / 6 mice. 20 days post-transplant, single-cell suspensions were prepared from the tumor and flow cytometry analysis was performed. The proportion of PMN-MDSCs or M-MDSCs in the CD45+ cell population is shown in percentage. (f) TCTP KO SL4 cells were transplanted subcutaneously into C57BL / 6 mice. At 1, 4, 7, 10, and 13 days post-transplant, polymorphonuclear cell lineage myeloid-derived immunosuppressive cells (PMN-MDSCs) or PBS derived from the spleen and bone marrow of mice with SL4 tumors were injected into the peritoneal cavity. Tumor volume was measured over time. PBS: n=5, PMN-MDSC: n=4. (g) TCTP KO SL4 cells and IL-2ss-TCTP SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice, respectively.Anti-Ly6G antibody or control IgG was administered intraperitoneally every two days (n=5). (Left figure) Tumor volume was measured over time. (Right) The proportion of PMN-MDSCs in the CD45+ cell population is shown in %. (h) TCTP WT SL4 cells (WT) and TCTP KO SL4 cells (KO) were transplanted subcutaneously into C57BL / 6 mice (n=3). Eighteen days post-transplant, single-cell suspensions were prepared from the tumors and flow cytometry analysis was performed. The mean fluorescence intensity (MFI) of markers (CD69, CD107a) indicating NK cell activation was determined. For (b~f, h), *P < 0.05, *P < 0.01, ****P < 0.0001, unpaired two-sided Student's t-test. For (g), repeated measures one-way ANOVA with Tukey's multiple comparisons test. NS indicates no statistically significant difference. Data are presented as the mean ± standard error (SEM) of the values. [Figure 7]Investigation of the immunosuppressive function of TCTP during TIME. (a, b) WT SL4 cells or TCTP KO SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. 19 days post-transplant, single-cell suspensions were prepared from the tumors and flow cytometry analysis was performed (n=4). (a) (Left figure) Representative plots are shown. (Right figure) The relative abundance of PMN-MDSCs (CD11b+Ly6C+Ly6G+ cells) and M-MDSCs (CD11b+Ly6Chigh Ly6G- cells) in the CD45+ cell population is shown in %. (b) The relative abundance of TAMs (CD11b+F4 / 80+ cells) and DCs (CD11b-CD11c+ cells) in the CD45+ cell population is shown in %. (c) TCTP KO SL4 cells or IL-2ss-TCTP SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. 19 days post-transplant, single-cell suspensions were prepared from the tumors and flow cytometry analysis was performed (n=4). (a) (Left figure) Representative plots are shown. (Right figure) Percentages of PMN-MDSCs and M-MDSCs in the CD45+ cell population are shown. (d) (Left figure) Representative examples of hematoxylin / eosin (H&E) staining and Ly6G staining of colorectal cancer tissue from TCTPflox / flox, Apc+ / Δ716 mice (WT) and TCTPflox / flo, Apc+ / Δ716, Villin-CreERT2 mice (KO) are shown. Scale bars represent 100 μm. (Right figure) The results of quantifying the number of Ly6G+ cells in a 90,000 μm2 area of ​​the field of view of WT tumor tissue and KO tumor tissue (n=4). The area enclosed by the dashed line is the tumor. (e) Inhibitory effect of CD11b+Ly6C+Ly6G+ cells isolated from the spleen of mice with SL4 tumors on T cell proliferation. T cells were stimulated with anti-CD3 / CD28 in the presence of CD11b+Ly6C+Ly6G+ cells from mice with tumors (left) or mice without tumors (right). The proportion of T cells that proliferated was determined by dilution of CFSE (carboxyfluorescein diacetate succinimimidyl ester) (n=3). For (a~d), *P < 0.05, *P < 0.01, ****P < 0.0001, unpaired two-sided Student's t-test. NS indicates no significant difference.The data is presented as the mean ± standard error (SEM) of the values. [Figure 8]Investigation of the effects of TCTP on the number of T cells and NK cells and their antitumor activity. (a) WT SL4 cells or TCTP KO SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. 19 days post-transplant, single-cell suspensions were prepared from the tumors and analyzed by flow cytometry (n=7 for WT, n=6 for KO). (b) The relative abundance of CD8+ T cells (CD3ε+NK1.1-CD8+ cells) and NK cells (CD3ε-NK1.1+ cells) in the CD45+ cell population is shown in %. (c) TCTP KO cells were introduced with Mock-IRES-GFP vector (Mock) or IL-2ss-TCTP-IRES-GFP vector (IL-2ss-TCTP). Subsequently, GFP-positive cells were isolated by cell sorting. Mock or IL-2ss-TCTP cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. 19 days post-transplantation, single-cell suspensions were prepared from the tumors and flow cytometry analysis was performed (n=4). The relative abundance of CD8+ T cells (CD3ε+NK1.1-CD8+ cells) and NK cells (CD3ε-NK1.1+ cells) in the CD45+ cell population is shown in %. (c and d) WT SL4 cells or TCTP KO (a) SL4 cells (2 × 10⁵ cells) were subcutaneously transplanted into C57BL / 6 mice. (c) Anti-Asialo GM1 antibody (WT n=4, KO n=6) or control IgG (WT n=5, KO n=6) was administered intraperitoneally on day 1 before transplantation and on days 3, 7, 11, and 15 after transplantation. (d) Anti-CD8α (n=5) or control IgG (n=6) was administered intraperitoneally on day 1 before transplantation and on days 2, 5, 8, and 11 after transplantation. (e) WT SL4 cells or TCTP KO SL4 cells (2 × 10⁵ cells) were subcutaneously transplanted into WT mice (WT n=5, KO n=6) or RAG1 KO mice (n=3). Anti-Asialo GM1 antibody or control IgG was administered intraperitoneally on day 1 before transplantation and on days 3, 7, and 11 after transplantation. For (a~e), *P < 0.05, ****P < 0.0001, unpaired two-sided Student's t-test. Data are presented as mean ± standard error (SEM). [Figure 9]Investigation of the effects of TCTP on immune checkpoint molecule expression and angiogenesis. (a and b) WT SL4 cells and TCTP KO SL4 cells (2 × 10⁵ cells) were subcutaneously transplanted into C57BL / 6 mice. 21 days post-transplantation, single-cell suspensions were prepared from the tumors and performed flow cytometry analysis. The mean fluorescence intensity (MFI) of PD-L1 (a) and PD-1 (b) was determined. (c) (Left figure) Representative CD31 stained images of TCTP WT SL4 tumors or TCTP KO SL4 tumors subcutaneously transplanted into C57BL / 6 mice are shown. Scale bar is 100 μm. (Right figure) Quantification of CD31-positive regions. NS indicates no significant difference. For (c), unpaired two-sided Student's t-test. Data are shown as mean ± standard error (sem). [Figure 10]Identification of cells targeted by TCTP and TCTP receptors (1). (a) SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=3). 21 days post-transplantation, single-cell suspensions were prepared from the tumors. Various immune cells (PMN-MDSC, M-MDSC, TAM, DC, T cells, B cells, and NK cells) were isolated, and the Cxcl1 mRNA of each cell was quantified by qRT-PCR. (b) TCTP WT SL4 cells (2 × 10⁵ cells) and TCTP KO SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=3). 21 days post-transplantation, single-cell suspensions were prepared from the tumors. Subsequently, M-MDSCs were isolated, and the Cxcl1 mRNA of each cell was quantified by qRT-PCR. (c) Peritoneal exudative cells (PECs) from mice of various genotypes (WT (wild type), MyD88 KO (MyD88-deficient), IPS-I KO (IPS-I-deficient), STING KO (STING-deficient)) were stimulated with recombinant TCTP. Cxcl1 mRNA was quantified by qRT-PCR (n=3). (d) PECs from mice of various genotypes (WT, TLR2 KO (TLR2-deficient), TLR4 KO (TLR4-deficient)) were stimulated with recombinant TCTP. Cxcl1 mRNA was quantified by qRT-PCR (n=3). For (b), *P < 0.05, **P < 0.01, unpaired two-sided Student's t-test. For (a), repeated measures one-way ANOVA with Dunnett's multiple comparisons test. Data are shown as mean ± standard error (sem). [Figure 11]Identification of cells targeted by TCTP and TCTP receptors (2). (a) PEC (2 × 10⁵ cells) or SL4 cells (1 × 10⁵ cells) were stimulated with recombinant TCTP for 2 hours. Cxcl1 mRNA was then quantified by RT-qPCR (n=3). (b) Cxcl1 mRNA in TCTP WO SL4 cells and TCTP KO SL4 cells was quantified by RT-qPCR. n=3. (c) SL4 cells (2 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=3). 21 days post-transplantation, single-cell suspensions were prepared from the tumors. Various immune cells (PMN-MDSC, M-MDSC, TAM, DC, T cells, B cells, and NK cells) were then isolated, and Cxcl2 mRNA in each cell was quantified by RT-qPCR. (d) PEC (2 × 10⁵ cells) were stimulated with recombinant IL-1α for 2 hours. Subsequently, Cxcl1 mRNA was quantified by RT-qPCR (n=3). (e) Immunoprecipitation assay for TCTP-TLR2 binding. HEK293T cells transiently expressing TLR2-YFP and Flag-TCTP were immunoprecipitated with anti-GFP antibody and then analyzed with anti-Flag antibody (upper figure). TLR2 and TCTP were detected in the total cell lysate (lower figure). (f) A luciferase reporter construct containing a multimerized NKκB binding motif and expression vectors for various proteins were transfected into HEK293T cells. 24 hours after transfection, HEK293T cells (2 × 10⁴ cells) were seeded in 96-well plates and stimulated with recombinant TCTP or agonists for each TLR (Pam3CSK4 300 ng / ml, poly I:C 100 μg / ml, poly U 10 μg / ml, CpG-M 1 μM). Six hours after stimulation, cell lysates were extracted and luciferase assays were performed. (g and h) SL4 cells (g) or IL-2ss-TCTP cells (h) (2 × 10⁵ cells) were subcutaneously transplanted into WT mice (n=7 for SL4, n=6 for IL-2ss-TCTP) or TLR2 KO mice (n=5 for SL4, n=6 for IL-2ss-TCTP). Tumor volume was measured over time. (i) IL-2ss-TCTP SL4 cells (2 × 10⁵ cells) were subcutaneously transplanted into C57BL / 6 mice, and tumor volume was subsequently measured over time.After tumor transplantation, O-vanillin (50 mg / kg) was administered orally every two days. For (b, f, g~i), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired two-sided Student's t-test. For (c), repeated measures one-way ANOVA with Dunnett's multiple comparisons test. NS indicates no significant difference. Data are presented as mean ± standard error (sem). [Figure 12]Investigation of the effects of TCTP neutralizing antibodies and TCTP inhibitors on tumor growth (1). (a) Whole cell lysates of TCTP WT SL4 cells (WT) or TCTP KO SL4 cells (KO) were prepared and the results of 55F3E5 (55F3) monoclonal antibody immunoblotting are shown. (b) PEC (2 × 10⁵ cells) were stimulated with the supernatant of dead cells of SL4 cells containing 55F3 antibody or control IgG. After 2 hours of stimulation, expressed Cxcl1 mRNA was quantified by RT-qPCR. n=3, Sup: culture supernatant. (c) TCTP WT SL4 cells or TCTP KO SL4 cells (1 × 5¹⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. After transplantation, DHA (50 mg / kg) or DMSO was administered intraperitoneally daily (n=6). (d) (Left figure) Comparison of TCTP expression levels in the stromal region of normal colon tissue and the stromal region of colorectal cancer tissue. (Right figure) Comparison between normal colonic crypts and tumor lesions. (e) Co-expression plots of CD8A, GZMB, PRF1 or CD69 and TCTP mRNA in the TCGA colorectal cancer dataset (n=382). (f) Co-expression plots of cytolytic activity (defined as the geometric mean of GZMA mRNA and PRF1 mRNA expression levels) and TCTP mRNA in the TCGA colorectal cancer dataset (n=382). Spearman r: Spearman rank correlation coefficients. *P < 0.05, **P < 0.01. For (b), repeated measures one-way ANOVA with Dunnett's multiple comparisons test. For (c), repeated measures one-way ANOVA with Tukey's multiple comparisons test. For (d), unpaired two-sided Student's t-test. NS indicates no statistically significant difference. Data are presented as mean ± standard error (SEM). [Figure 13]Investigation of the effects of TCTP neutralizing antibodies and TCTP inhibitors on tumor growth (2). (a) SL4 cells (1 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. Monoclonal antibody against TCTP (55F3) (n=5) or control IgG (n=6) was administered intraperitoneally every 1 to 2 days after transplantation (200 μg / mouse). Tumor volume was measured over time. (b) On day 13 after transplantation, single-cell suspensions were prepared from the tumors and flow cytometry analysis was performed (n=5). The vertical axis shows the ratio of PMN-MDSCs to CD45+ cells. (c) SL4 cells (1 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=6). DHA (50 mg / kg) was administered intraperitoneally daily after transplantation. Tumor volume was measured over time. (d) SL4 cells (1 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice (n=6). 55F3 antibody or control IgG (200 μg / mouse each) was administered intraperitoneally daily starting 1 day post-transplantation. Ten days after transplantation, anti-PD-1 monoclonal antibody (100 μg) or PBS was administered. Tumor volume was measured daily. (e) SL4 cells (1 × 10⁵ cells) were transplanted subcutaneously into C57BL / 6 mice. After transplantation, DMSO, DHA, DHA and anti-PD-1 antibody (n=5 each) or anti-PD-1 antibody (n=6) were administered. DHA (50 mg / kg) or DMSO was administered intraperitoneally daily after transplantation. Six days after transplantation, anti-PD-1 antibody (100 μg) or PBS was administered. (f) The amount of TCTP in serum from healthy individuals (those without cancer) (n=5) and colorectal cancer patients (n=14) was quantified by immunoblotting. (g) (Left figure) Representative examples of staining images of human colorectal cancer tissue (CRC) and normal colorectal tissue (Normal) with hematoxylin and anti-TCTP antibody are shown. Scale bar is 100 μm. (Right figure) The relative staining intensity with anti-TCTP antibody was semi-quantified and the staining intensity of colorectal cancer tissue and normal colorectal tissue was compared (n=45). (h) The expression levels of TCTP in T1 / 2 (n=9) and T3 / 4 (n=35) colorectal cancer tissue were compared. (i) Human colorectal cancer tissue was stained with anti-CD15 antibody and anti-TCTP antibody. The number of CD15+ cells present in a 40,000 μm2 area of ​​the field of view was counted and the relative staining intensity with anti-TCTP antibody was semi-quantified (n=27).(k) Co-expression plot of CD15 and TCTP. Spearman correlation coefficient is shown. (j) FPKM (Fragments Per Kilobasse of transcript per Million mapped reads) of TCTP stratified by DNA copy number for CRC patients (n=376) obtained from the TCGA database. Deep (n=4) or shallow (n=14) patients have deletions of 2 or 1 alleles of the TCTP gene, respectively. Gain (n=220) or amplification (n=16) patients have acquired 1 or more alleles of the TCTP gene, respectively. Diploid, n=122. (k) Kaplan-Meier graph of TCGA data showing survival of colon cancer patients with amplified TCTP alleles (n=19) or without amplified TCTP alleles (n=520). (l) Summary diagram of the present invention. For (a-c, f, h), *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired two-sided Student's t-test. Log-rank test (g). For (d, e), repeated measures one-way ANOVA with Dunnett's multiple comparisons test. For (j), repeated measures one-way ANOVA with Tukey's multiple comparisons test. For (g), paired two-sided t-test. Data are presented as mean ± standard error (sem). [Modes for carrying out the invention]

[0011] Embodiments of the present invention will be described below. A first embodiment of the present invention is an inhibitor of the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor immune microenvironment (TIME), comprising as an active ingredient a substance that suppresses or inhibits the function of immunomodulatory factors released from tumor dead cells (hereinafter also referred to as "the inhibitor according to this embodiment"). Here, the cancer immunomicroenvironment (hereinafter also referred to as "TIME") refers to the site of interaction between cancer cells and non-cancer cells, mainly immune cells, within a tumor, and is defined as the microenvironment inside a tumor that has an environment favorable to cancer survival, including high immunosuppression (Non-Patent Literature 1). Myeloid-derived immunosuppressive cells (also referred to as "MDSCs") are immature myeloid cells that appear in chronic inflammation such as infections and cancer, and possess potent immunosuppressive activity. Monocyte-derived myeloid immunosuppressive cells (also referred to as "M-MDSCs") and polymorphonuclear cell-derived myeloid immunosuppressive cells (also referred to as "PMN-MDSCs") constitute the main subpopulations, and their morphology and cell surface markers are similar to monocytes and neutrophils, respectively (Non-Patent Literature 3). Accumulation of MDSCs in TIME means that MDSCs such as PMN-MDSCs are localized within or near TIME. Here, immunomodulatory factors are factors that promote or suppress the immune response and affect the function of the immune system.

[0012] The inventors have found that when TCTP (translationally controlled tumor protein), a molecule released from tumor-dead cells, acts on hematopoietic cells in TIME, cytokines such as CXCL1 and CXCL2 are significantly released, particularly from M-MDSCs. Furthermore, the inventors have found that CXCL1 and CXCL2 guide PMN-MDSCs into or near the cancer immune microenvironment via the CXCR2 receptor on PMN-MDSCs. PMN-MDSCs induced in or near the cancer immune microenvironment are known to suppress the attack of cancer cells by immune cells such as T cells and NK cells (Non-Patent Literature 3). This analysis has newly revealed that the TCTP-(M-MDSC)-CXCL1 / 2-(PMN-MDSC) pathway leads to the accumulation of PMN-MDSCs in TIME, suppressing the antitumor activity of immune cells and promoting tumor growth. Therefore, inhibiting the accumulation of MDSCs (e.g., PMN-MDSCs) in TIME can promote the accumulation and activation of T cells and NK cells, ultimately inhibiting or suppressing tumor growth.

[0013] To inhibit PMN-MDSC accumulation in TIME, one possible approach is to block the TCTP-(M-MDSC)-CXCL1 / 2-(PMN-MDSC) pathway. Methods for blocking this pathway include, for example, the use of substances that suppress or inhibit the function of TCTP (for human TCTP, NCBI accession number; cDNA sequence: CCDS9397.1, amino acid sequence: NP_003286.1). Here, one example of TCTP's function is that it binds to its receptor (e.g., TLR2) and induces the release of cytokines (e.g., CXCL1 / 2) from cells (e.g., M-MDSCs). Substances that suppress or inhibit the function of TCTP include, but are not limited to, antibodies, peptide aptamers, etc. that suppress or inhibit the function of TCTP, or substances that degrade or induce the degradation of TCTP, such as dihydroartemisinin (DHA) and sertraline, or substances that suppress or inhibit the expression of TCTP, such as siRNA and miRNA. Furthermore, substances that suppress or inhibit the function of TCTP also include substances that inhibit the binding of TCTP to its receptor (TLR2), such as TLR2 antagonists. Such inhibitors may be substances that interact with TCTP or its receptor (TLR2), or degrade either of them. The inhibitor according to this embodiment may include a substance that suppresses or inhibits the function of TCTP described above.

[0014] A second embodiment of the present invention is an antibody that suppresses or inhibits the function of TCTP (hereinafter also referred to as "the anti-TCTP antibody according to this embodiment"). In this specification, the term "antibody" is not particularly limited in terms of its preparation method or structure, and includes all antibodies that bind to a desired antigen with desired properties, such as monoclonal antibodies, polyclonal antibodies, or nanoantibodies. If the anti-TCTP antibody according to this embodiment is a polyclonal antibody, it can be prepared, for example, by injecting a mixture of antigen and adjuvant into an immunized animal (not limited to, but such as, rabbits, goats, sheep, chickens, guinea pigs, mice, rats, or pigs). Typically, the antigen and / or adjuvant are injected subcutaneously or intraperitoneally into the immunized animal multiple times. Examples of adjuvants, not limited to, include complete Freund's and monophosphoryl lipid A synthesis-trehalose dicolinomycolate (MPL-TMD). After immunization with the antigen, the anti-TCTP antibody can be purified from the serum of the immunized animal by a standard method (for example, by using Sepharose containing Protein A).

[0015] Furthermore, if the anti-TCTP antibody according to this embodiment is a monoclonal antibody, it can be produced, for example, as follows. In this specification, "monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies (a population of antibodies in which the amino acid sequences of the heavy and light chains constituting the antibody are identical), and is not interpreted to mean that the antibody is produced by a specific method (for example, the hybridoma method). Examples of methods for producing monoclonal antibodies include the hybridoma method (Kohler and Milstein, Nature 256 495-497 1975) or the recombinant method (U.S. Patent No. 4,816,567). Alternatively, the anti-TCTP antibody according to this embodiment may be isolated from a phage antibody library (e.g., Clackson et al., Nature 352 624-628 1991; Marks et al., J.Mol.Biol. 222 581-597 1991, etc.). More specifically, when preparing using the hybridoma method, the preparation method includes, for example, the following four steps: (i) immunizing an immunized animal with an antigen, (ii) recovering monoclonal antibody-secreting (or potentially secreting) lymphocytes, (iii) fusing the lymphocytes with immortalized cells, and (iv) selecting cells that secrete the desired monoclonal antibody. Suitable immunized animals include, for example, mice, rats, guinea pigs, hamsters, and rabbits. After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol or electrofusion to establish hybridoma cells. For example, rat or mouse myeloma cell lines are used as fusion cells. After cell fusion, the cells are grown in a suitable medium containing a substrate that inhibits the growth or survival of unfused lymphocytes and immortalized cell lines. Conventional techniques use parent cells lacking the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, aminopterin is added to a medium (HAT medium) that inhibits the growth of HGPRT-deficient cells and allows hybridoma growth. From the hybridomas thus obtained, hybridomas that produce the desired antibody can be selected, and the target monoclonal antibody can be obtained from the medium in which the selected hybridomas grow, according to conventional methods. The hybridomas prepared in this way can be cultured in vitro, or in vivo in the ascites fluid of mice, rats, guinea pigs, hamsters, etc., and the target antibody can be prepared from the culture supernatant or ascites fluid.

[0016] Nanoantibodies are polypeptides consisting of the variable domain of the heavy chain of a heavy chain antibody (VHH). While antibodies in humans and other animals typically consist of heavy and light chains, camelid animals such as llamas, alpacas, and camels produce single-chain antibodies (heavy chain antibodies) composed solely of the heavy chain. Like conventional antibodies with both heavy and light chains, heavy chain antibodies can recognize and bind to target antigens. The variable domain of a heavy chain antibody is the smallest unit possessing binding affinity to the antigen, and this variable domain fragment is called a "nanoantibody." Nanoantibodies exhibit high heat resistance, digestion resistance, and room-temperature stability, and can be easily prepared in large quantities using genetic engineering techniques. Nanoantibodies can be produced, for example, as follows: Camelid animals are immunized with an antigen, the presence or absence of the target antibody is detected in the collected serum, and cDNA is produced from RNA derived from peripheral blood lymphocytes of immunized animals in which the desired antibody titer is detected. A DNA fragment encoding VHH is amplified from the obtained cDNA and inserted into a phagemide to prepare a VHH phagemide library. The desired nanoantibodies can be produced from the prepared VHH phagemide library after several screenings.

[0017] The anti-TCTP antibody according to this embodiment may be a recombinant antibody. Recombinant antibodies are not limited to, but examples include humanized antibodies and chimeric antibodies with human antibodies. A chimeric antibody is, for example, an antibody in which a variable region and a constant region derived from different animal species are linked (for example, an antibody in which the variable region of a rat-derived antibody is bound to a constant region derived from a human) (e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855 1984), and can be easily constructed by genetic engineering.

[0018] Humanized antibodies are antibodies that have a human-derived sequence in their framework region (FR) and a complementation-determining region (CDR) derived from a sequence of another animal species (e.g., mouse). Humanized antibodies can be produced by first transplanting the CDR from the variable region of an antibody derived from another animal species—in this case, mouse—into the variable region of a human antibody, then reconstituted the heavy and light chain variable regions, and finally ligating these humanized reconstituted human antibody variable regions to the constant region of a human antibody. Such methods for producing humanized antibodies are well known in this field (e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86, 10029-10033 1989).

[0019] The antigen-binding fragment of the anti-TCTP antibody according to this embodiment is an antibody fragment that is a part of the anti-TCTP antibody according to this embodiment and binds to TCTP. Examples of fragments include Fab, Fab', F(ab')2, Fv (variable fragment of antibody), single-chain antibodies (heavy chain, light chain, heavy chain variable region, light chain variable region, and nano-antibody, etc.), scFv (single chain Fv), diabody (scFv dimer), dsFv (disulfide-stabilized Fv), and peptides containing at least a portion of the CDR of the anti-TCTP antibody according to this embodiment.

[0020] Fab is an antibody fragment that possesses antigen-binding activity, obtained by treating an antibody molecule with the proteolytic enzyme papain, in which approximately half of the N-terminal side of the heavy chain and the entire light chain are linked by a disulfide bond. Fab can be produced by obtaining the fragment by treating the antibody molecule with papain, or by constructing a suitable expression vector into which DNA encoding Fab is inserted, introducing this vector into a suitable host cell (e.g., mammalian cells such as CHO cells, yeast cells, insect cells, etc.), and then expressing Fab within the cell.

[0021] F(ab')2 is an antibody fragment with antigen-binding activity, obtained by treating an antibody molecule with the protease pepsin, and is slightly larger than fragments in which the Fab is linked via a disulfide bond in the hinge region. In addition to obtaining the fragment by treating the antibody molecule with pepsin, F(ab')2 can also be produced by linking the Fab via thioether or disulfide bonds, and furthermore, it can be produced by genetic engineering techniques, similar to Fab.

[0022] Fab' is an antibody fragment with antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F(ab')2 described above. Like Fab, Fab' can also be produced by genetic engineering techniques.

[0023] scFv is an antibody fragment having antigen-binding activity, which is a VH-linker-VL or VL-linker-VH polypeptide formed by linking one heavy chain variable region (VH) and one light chain variable region (VL) using an appropriate peptide linker. scFv can be produced by obtaining cDNA encoding the heavy chain variable region and light chain variable region of an antibody and using genetic engineering techniques.

[0024] A diabody is an antibody fragment in which scFv is dimerized and possesses bivalent antigen-binding activity. The bivalent antigen-binding activity may be the same antigen-binding activity or one antigen-binding activity may be different. A diabody can be produced by obtaining cDNA encoding the heavy chain variable region and light chain variable region of the antibody, constructing cDNA encoding scFv by linking the heavy chain variable region and light chain variable region with a peptide linker, and then using genetic engineering techniques.

[0025] dsFv refers to a polypeptide in which one amino acid residue in each of the heavy chain variable region and light chain variable region is replaced with a cysteine ​​residue, and these polypeptides are linked together via disulfide bonds between the cysteine ​​residues. The amino acid residue to be substituted for the cysteine ​​residue can be selected based on the prediction of the antibody's three-dimensional structure. dsFv can be produced by obtaining the cDNA encoding the heavy chain variable region and light chain variable region of the antibody, constructing the DNA encoding the dsFv, and then using genetic engineering techniques.

[0026] Peptides containing CDRs are constructed to include at least one region of the heavy chain or light chain CDRs (CDR1-3). Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker. To produce a CDR-containing peptide, DNA encoding the heavy chain or light chain CDR of the antibody is constructed and inserted into an expression vector. There are no particular limitations on the type of vector, and it can be appropriately selected depending on the type of host cell to which it will be introduced. These can be produced by introducing them into a suitable host cell (e.g., mammalian cells such as CHO cells, yeast cells, insect cells, etc.) for expression as antibodies. CDR-containing peptides can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

[0027] Human antibodies (fully human antibodies) generally have the same structure as human antibodies in the hypervariable region (the antigen-binding site in the V region), the rest of the V region, and the constant region. Human antibodies can be easily produced by those skilled in the art using known techniques. Human antibodies can be obtained, for example, by methods using human antibody-producing mice that have human chromosome fragments containing the genes for the H and L chains of human antibodies (e.g., Tomizuka et al., Proc. Natl. Acad. Sci. USA, 97, 722-727 2000.) or by methods of obtaining human antibodies derived from phage displays selected from human antibody libraries (see, for example, Siriwardena et al., Opthalmology, 109, 427-431 2002.).

[0028] A multispecific antibody can be constructed using the antigen-binding fragment of the anti-TCTP antibody according to this embodiment. Multispecificity means having binding specificity to two or more antigens, and examples include monoclonal antibodies or proteins containing antigen-binding fragments that have binding specificity to two or more antigens. This can be carried out by those skilled in the art using known techniques. Methods for constructing multispecific antibodies include techniques for constructing asymmetric IgG by performing protein engineering operations to form heterodimers between two different antibody heavy chain molecules, and techniques for linking low molecular weight antigen-binding fragments obtained from antibodies, or linking them to other antibody molecules. For specific examples of construction methods, see, for example, the following literature: Kontermann et al., Drug Discovery Today, 20, 838-847 2015.

[0029] Examples of the anti-TCTP antibody and its antigen-binding fragment according to this embodiment include an antibody and its antigen-binding fragment characterized in that the amino acid sequence of CDR (complementarity determining region) 1 to 3 satisfies any of the following (A), (B), or (C). (A) CDR of 55F3 antibody The heavy chain CDR1 amino acid sequence is GYSIASDYAWN (SEQ ID NO: 1), The heavy chain CDR2 amino acid sequence is YINYSGSTGYNPSLKS (SEQ ID NO: 2), The heavy chain CDR3 amino acid sequence is FEAGY (SEQ ID NO: 3), The light chain CDR1 amino acid sequence is KASQDINRYLS (SEQ ID NO: 4) The light chain CDR2 amino acid sequence is RANRLVD (SEQ ID NO: 5), and The light chain CDR3 amino acid sequence contains LQYNEFPLT (SEQ ID NO: 6). (B) CDR of 44E1 antibody The heavy chain CDR1 amino acid sequence is GYTFTDHAIH (SEQ ID NO: 7), The heavy chain CDR2 amino acid sequence is YISPGNGDLKYNEKFKG (SEQ ID NO: 8), The heavy chain CDR3 amino acid sequence is GWTL (SEQ ID NO: 9), The light chain CDR1 amino acid sequence is KSSQSLLYRSNQKNYLV (SEQ ID NO: 10) The light chain CDR2 amino acid sequence is WAFTRES (SEQ ID NO: 11), and The light chain CDR3 amino acid sequence has QQHYSYPWT (SEQ ID NO: 12). (C) CDR of 51A9 antibody The heavy chain CDR1 amino acid sequence is GYSITSDYAWN (SEQ ID NO: 13), The heavy chain CDR2 amino acid sequence is YINYSGSTGYNPSLKS (SEQ ID NO: 14), The heavy chain CDR3 amino acid sequence is FEAGY (SEQ ID NO: 15), The light chain CDR1 amino acid sequence is KASQDINSYLS (SEQ ID NO: 16) The light chain CDR2 amino acid sequence is RANRLVD (SEQ ID NO: 17), and The light chain CDR3 amino acid sequence contains LQYYEFPLT (SEQ ID NO: 18).

[0030] Furthermore, the anti-TCTP antibody and its antigen-binding fragment according to this embodiment includes an antibody comprising any of the heavy chain variable regions containing the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and an antibody comprising any of the light chain variable regions containing the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and their antigen-binding fragments, as well as antibodies comprising amino acid sequences having about 70% or more, preferably about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, more preferably about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, most preferably about 99% or more amino acid sequence identity with each amino acid sequence of the heavy chain variable region and / or light chain variable region constituting these antibodies, and antibodies that inhibit the binding of TCTP to its receptor and their antigen-binding fragments.

[0031] Variable heavy chain region of 55F3 antibody MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTATGYSIASDYAWNWIRQFPGNKLEWMGYINYSGSTGYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARFEAGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT (Sequence ID 19) Variable heavy chain region of 44E1 antibody MEWSWVFLFFLSVTTGVHSEVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWAKQKPEQGLEWIGYISPGNGDLKYNEKFKGKATLTTDKSSSTAYMQLNSLTSEDSAVYFCKSGWTLWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT (Sequence ID 20) Variable heavy chain region of 51A9 antibody MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTATGYSITSDYAWNWIRQFPGNKLEWMGYINYSGSTGYNPSLKSRISITRDTSKNKFFLQLNSVTTEDTATYYCARFEAGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT (Sequence ID 21)

[0032] Variable region of the light chain of the 55F3 antibody MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMSASLGERVTITCKASQDINRYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYGDMGIYYCLQYNEFPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLN (Sequence ID 22) Light chain variable region of 44E1 antibody MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSPVVSVGEKVTMSCKSSQSLLYRSNQKNYLVWYQLKPGQSPKLLIYWAFTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQHYSYPWTFGGGTKLEVKRADAAPTVSIFPPSSEQLTSGGASVVCFLN (Sequence ID 23) Variable region of the light chain of the 51A9 antibody MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTFTCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCLQYYEFPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLN (Sequence ID 24).

[0033] Furthermore, the anti-TCTP antibody according to this embodiment also includes an antibody (hereinafter also referred to as "competitive antibody according to this embodiment") that competitively inhibits the binding of an antibody to TCTP and suppresses or inhibits the function of TCTP, characterized in that the amino acid sequence of CDR (complementarity determining region) 1 to 3 satisfies any of (A), (B), or (C) above. The competitive antibody according to this embodiment can be prepared and obtained by competitive experiments, which are well known to those skilled in the art. Specifically, when the binding of a first anti-TCTP antibody (the anti-TCTP antibody according to this embodiment) to TCTP is competitively inhibited by a second anti-TCTP antibody, it is determined that the first anti-TCTP antibody and the second anti-TCTP antibody are substantially the same or bound to an extremely nearby antigen site. If the second anti-TCTP antibody suppresses or inhibits the function of TCTP, then the second anti-TCTP antibody is a competitive antibody according to this embodiment and is included in the anti-TCTP antibody according to this embodiment. As a method for the above competitive experiment, for example, a method using Fab fragments, etc., is commonly performed in the art. See, for example, WO95 / 11317, WO94 / 07922, WO2003 / 064473, WO2008 / 118356, and WO2004 / 046733. Furthermore, whether or not the function of TCTP is suppressed or inhibited can be easily confirmed by the methods disclosed in the examples.

[0034] Furthermore, the anti-TCTP antibody according to this embodiment also includes an antibody that binds to EDGVTPYMIFFKDGLEMEKC (SEQ ID NO: 25), a partial peptide of TCTP, and suppresses or inhibits the function of TCTP.

[0035] A third embodiment of the present invention is a therapeutic agent or therapeutic composition for cancer, comprising the inhibitor of the present invention as an active ingredient, wherein the therapeutic agent or therapeutic composition (hereinafter referred to as "therapeutic agent, etc.") (therapeutic agent, etc. according to the embodiment of the present invention). As a cancer treatment drug according to embodiments of the present invention, the active ingredient (for example, a substance that suppresses or inhibits the function of TCTP) itself may be administered, but generally, it is preferable to administer it in the form of a therapeutic composition containing one or more active ingredients as well as one or more pharmaceutical additives. The active ingredient of the therapeutic drug of the present invention may include multiple different inhibitors of the present invention. Furthermore, the therapeutic drug of the present invention may also contain other known components such as anticancer drugs or immune checkpoint inhibitors.

[0036] The dosage forms of therapeutic drugs, etc., according to embodiments of the present invention include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections. These preparations are prepared according to conventional methods. In the case of liquid preparations, they may be dissolved or suspended in water or other suitable solvents at the time of use. Tablets and granules may also be coated by well-known methods. In the case of injections, the active ingredient is dissolved in water to prepare the preparation, but it may also be dissolved in physiological saline or glucose solution as needed, and buffers and preservatives may be added.

[0037] Preparations for oral or parenteral administration are provided in any formulation form. For example, oral therapeutic agents or therapeutic compositions can be prepared in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or liquids; and parenteral therapeutic agents or therapeutic compositions can be prepared in the form of injections for intravenous, intramuscular, or subcutaneous administration, drips, transdermal agents, transmucosal agents, nasal sprays, inhalants, suppositories, etc. Injectable agents and drips, for example, can also be prepared in powder form, such as lyophilized form, and dissolved in an appropriate aqueous medium such as physiological saline at the time of use.

[0038] The type of pharmaceutical additive used in the manufacture of therapeutic drugs, etc., according to embodiments of the present invention, the ratio of pharmaceutical additives to the active ingredient, or the method of manufacturing the pharmaceutical or pharmaceutical composition can be appropriately selected by those skilled in the art depending on the form. As pharmaceutical additives, inorganic or organic substances, or solid or liquid substances can be used, and generally, they can be blended in amounts ranging from 1% to 90% by weight relative to the weight of the active ingredient. Specifically, examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resin, methylcellulose, gelatin, acacia gum, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, beegum, titanium dioxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, refined lanolin, glycerol gelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, and water.

[0039] To manufacture solid dosage forms for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, and anhydrous silicic acid to form a powder, or, if necessary, binders such as sucrose, hydroxypropylcellulose, and polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose and carboxymethylcellulose calcium are added and the mixture is wet or dry granulated to form granules. To manufacture tablets, these powders and granules can be compressed as is, or with lubricants such as magnesium stearate and talc added. These granules or tablets can be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate and methacrylate-methyl methacrylate polymer to form enteric-coated preparations, or coated with ethylcellulose, carnauba wax, or hydrogenated oil to form sustained-release preparations. To manufacture capsules, the powder or granules can be filled into hard capsules, or the active ingredient can be used as is, or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin membrane to form soft capsules.

[0040] To manufacture the injectable preparation, the active ingredient is dissolved in distilled water for injection along with pH adjusters such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, monohydrogen phosphate, and dihydrogen dihydrogen phosphate, as needed, and isotonic agents such as sodium chloride and glucose. The mixture is then sterile filtered and filled into ampoules, or mannitol, dextrin, cyclodextrin, gelatin, etc., are added and the mixture is vacuum freeze-dried to produce an injectable preparation ready for use. Alternatively, the active ingredient can be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc., to produce an emulsion for injection.

[0041] To manufacture rectal administration preparations, the active ingredient may be humidified and dissolved with cocoa butter, triglycerides, diglycerides, and monoglycerides of fatty acids, and a suppository base such as polyethylene glycol, poured into a mold, and cooled; or the active ingredient may be dissolved in polyethylene glycol, soybean oil, etc., and then coated with a gelatin film.

[0042] The dosage and frequency of administration of therapeutic agents, etc., according to embodiments of the present invention are not particularly limited and can be appropriately selected at the discretion of a physician depending on the purpose of preventing the worsening or progression of the target disease and / or treatment, the type of disease, the patient's weight and age, and other conditions. Generally, the daily oral dose for adults is approximately 0.01 to 1000 mg (weight of the active ingredient), and it can be administered once a day, in several divided doses, or every few days. When used as an injectable preparation, it is desirable for adults to receive a daily dose of 0.001 to 100 mg (weight of the active ingredient) continuously or intermittently.

[0043] The therapeutic agents according to embodiments of the present invention may be prepared as sustained-release formulations, such as implantable tablets and delivery systems encapsulated in microcapsules, using a carrier that can prevent immediate removal from the body. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used as such carriers. Such materials can be readily prepared by those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Liposomes, though not limited to those described above, can be prepared as lipid compositions containing phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanol (PEG-PE), passed through a filter of an appropriate pore size to a size suitable for use, and purified by reverse-phase evaporation.

[0044] The therapeutic agents, etc., according to embodiments of the present invention may be provided in the form of a kit along with instructions on how to administer them. The drugs contained in the kit are supplied in a container made of a material that effectively maintains the activity of the constituent components of the therapeutic agent, etc., for a long period of time, does not adsorb to the inside of the container, and does not alter the constituent components. For example, the sealed glass ampoule may contain a buffer sealed in the presence of a neutral, unreactive gas such as nitrogen gas. Furthermore, the kit may include an instruction manual. This instruction manual may be printed on paper or stored on an electronically readable medium such as a CD-ROM or DVD-ROM and supplied to the user.

[0045] A third embodiment of the present invention is a method for preventing or treating cancer, comprising administering a therapeutic agent or the like according to an embodiment of the present invention (the second embodiment of the present invention) to a patient. Here, "treatment" means preventing or mitigating the progression and worsening of the disease in mammals suffering from cancer. "Prevention" means preventing the onset of cancer in mammals at risk of developing it. The "mammals" targeted for prevention or treatment refer to any animal classified as a mammal, and are not particularly limited, but include, for example, humans, pet animals such as dogs, cats, rabbits, and ferrets, and livestock animals such as cows, pigs, sheep, and horses. Humans are a particularly preferred "mammal."

[0046] Cancers (malignant tumors / malignant neoplasms) that can be targeted by the prevention or treatment method of the present invention include, for example, hepatocellular carcinoma, cholangiocarcinoma, renal cell carcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, malignant melanoma, fibrosarcoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma, lymphangiosarcoma, malignant meningioma, non-Hodgkin lymphoma, and Hodgkin's lymphoma. This includes lymphoma, leukemia, brain tumors, epithelial cell neoplasms (epithelial carcinomas), basal cell carcinoma, adenocarcinoma, lip cancer, oral cancer, gastrointestinal cancers such as esophageal cancer, small intestine cancer and stomach cancer, colon cancer, rectal cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancers such as squamous cell carcinoma and basal cell carcinoma, prostate cancer and renal cell carcinoma, as well as other known cancers that affect epithelial, mesenchymal or blood cells throughout the body.

[0047] A fourth embodiment of the present invention is a method for diagnosing cancer or a method for assisting in diagnosis, the method comprising measuring the amount of TCTP mRNA or TCTP protein present in a sample derived from a subject. In subjects who have developed cancer, the amount of TCTP protein or TCTP mRNA in tumor tissue or blood is significantly increased compared to healthy individuals (those confirmed not to have cancer) (see, for example, Figure 2b). Therefore, if the amount of TCTP mRNA or TCTP protein in a sample derived from a subject is significantly higher than in a sample from a healthy individual, it can be concluded that the subject may have developed some form of cancer. In this embodiment, the subject-derived sample can be, without limitation, blood (including blood-derived components such as serum), tissue suspected of being malignant tumor, etc. A person skilled in the art can easily select an appropriate method for measuring the amount of TCTP protein in the sample; for example, immunological assays such as ELISA (enzyme-linked immunosorbent assay) or Western blotting can be used. Furthermore, a person skilled in the art can easily select an appropriate method for measuring the amount of TCTP mRMA in the sample; for example, real-time PCR (RT-qPCR) can be used.

[0048] Where this specification is translated into English and contains the singular words "a," "an," and "the," it shall be assumed that they also include the plural, not just the singular, unless the context clearly indicates otherwise. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Examples]

[0049] 1. Method 1-1. Mouse I purchased the C57BL / 6 mouse and BALB / c mouse from CLEA Japan. Δ716 The mice were prepared according to previously reported instructions (19) and used in the background of C57BL / 6 mice. TCTP flox mice were provided by Dr. Hsin-Fang Yang-Yen of Academia Sinica (Republic of China) and used in the background of C57BL / 6 mice. Villin-CreERT2 mice were provided by Dr. Sylvie Robine of the Curie Institute (France) and used in the background of C57BL / 6 mice. MyD88 and IPS-I deficient mice were provided by Dr. Shizuo Akira of Osaka University and used in the background of C57BL / 6 mice. TLR2 and TLR4 deficient mice were purchased from Oriental Bio Service Co., Ltd. and used in the background of C57BL / 6 mice. STING deficient mice were provided by Dr. Glen N. Barber of the University of Miami (USA) and used in the background of C57BL / 6 mice. RAG1 knockout mice were provided by Dr. Shinsuke Taki of Shinshu University. Unless otherwise specified, male and female animals (6-12 weeks old) were used in this example. All animal experiments were conducted with the approval of the Animal Experiment Committee of the University of Tokyo.

[0050] 1-2.Cells RAW264.7 cells, mouse melanoma cell line B16F10 cells, mouse fibroblast cell line Meth-A cells, and HEK293T cells were obtained from the RIKEN BioResource Research Center (Japan). The mouse colorectal cancer cell line SL4 was donated by Dr. T. Irimura (Juntendo University). RAW264.7 cells, B16F10 cells, and HEK293T cells were maintained in DMEM (Nacalai Tesque) supplemented with 10% FBS (HyClone). Meth-A cells were maintained in RPMI (Nacalai Tesque) supplemented with 10% FBS. SL4 cells were maintained in DMEM-F12 (Gibco) supplemented with 10% FBS. For the preparation of peritoneal exudate cells (PECs), 2 ml of 4% thioglycolate (DIFCO solution) was administered into the peritoneal cavity of mice. After 4 days, the peritoneal cavity was washed with PBS and the PECs were collected. The cells were incubated overnight in RPMI with 10% FBS in a petri dish. After incubation, the cells were washed with RPMI medium and used in subsequent experiments.

[0051] 1-3. Flow cytometry analysis and fluorescence-activated cell sorting of tumor-infiltrating cells Tumor-infiltrating cells were collected after subcutaneous transplantation of tumors into mice and analyzed by flow cytometry. The excised tumor was finely chopped and treated with collagenase (0.75 mg / ml, cat# 11088882001, Roche), DNase I (40 μg / ml, cat# 11284932001, Roche), and dispase (0.5 mg / ml, cat# 17105041, ThermoFisher Scientific), followed by vigorous mixing (180 rpm, 37°C, 1 hour). The resulting cell suspension was passed through a cell strainer (cat# 352340, Falcon) and treated with RBC lysis buffer (cat# 00-4333-57, Invitrogen). After washing with PBS, the cells were first incubated with anti-CD16 / 32 antibody (clone 93, cat# 101302, BioLegend) on ice for 5 minutes. Subsequently, the cells were incubated with PFE (2% FBS and 1 mM of EDTA). Cells were stained in PBS (containing [specific substance]) on ice for 20 minutes and analyzed using LSR Fortessa (BD Biosciences). Cell sorting of tumor-infiltrating cells was performed using FACSAria II (BD Biosciences). The obtained data were analyzed using FlowJo software (BD BioSciences).

[0052] The following antibodies were used for flow cytometry analysis or fluorescence-activated cell sorting: Ly6C-AF488 (clone HK1.4, cat# 128022, BioLegend), NK1.1-FITC (clone PK136, cat# 553164, PharMingen), CD11b-PE (clone M1 / 70, cat# 101208, BioLegend), CD3ε-PE (clone 145-2C11, cat# 12-0031-81, eBioscience), CD11c-APC (clone N418, cat# 17-0114-81, eBioscience), B220-APC (clone RA3-6B2, cat# 20-0452-U025, TONBO) biosciences), F4 / 80-PerCP / Cy5.5(clone BM8, cat# 123128, BioLegend), CD8α-PerCP / Cy5.5(clone 53-6.7, cat# 100734, BioLegend), Ly6G-PE / Cy7(clone 1A8, 127618, BioLegend), CD4-PE / Cy7 (clone GK1.5, cat# 100422, BioLegend), CD45.2-Pacific Blue (clone 104, cat# 109820, BioLegend), anti-CD16 / 32 (clone 93, cat# 101302, BioLegend), PD-1-APC (clone 29F.1A12, cat# 135209, BioLegend), PD-L1-APC (clone 10F.9G2, cat# 124311, BioLegend).

[0053] 1-4. Preparation of culture supernatant The culture supernatant was prepared according to previously reported information (Non-Patent Literature 5). SL4 cells were incubated overnight with indomethacin (10 μM) to remove PGE2 (prostaglandin E2), and 10 8The cells were suspended in PBS at a concentration of cells / ml and subjected to five freeze-thaw cycles using a 37°C incubator and liquid nitrogen. The cells were then centrifuged at 8,000 × g for 5 minutes to remove necrotic cell fragments. The culture supernatant was obtained by filtration through a 0.45 μm membrane filter (Millipore).

[0054] 1-5. Reagents LPS(O55:B5) was purchased from SIGMA-Aldrich (cat# L2637). Oligonucleotides were purchased from FASMAC. Indomethacin was purchased from WAKO (cat# 093-02473). Recombinant IL-1α was purchased from Peprotech (cat# 211-11A). Recombinant TCTP was purchased from ORIGENE (cat# TP301664). TUNEL staining was performed using the in situ Apoptosis Detection Kit (cat# MK500, TaKaRa). The endotoxin level of recombinant TCTP was assayed using the LAL Endotoxin Assay Kit, Chromogenic, ToxinSensor (cat# L00350, Genscript), and it was confirmed that the endotoxin level was < 0.1 EU / μg.

[0055] 1-6. Identification of TCTP The culture supernatant of necrotic cells was first treated with DNase I solution (0.5 U / μl; TaKaRa Bio), RNase A (0.25 mg / ml; MACHEREY-NAGEL), or PBS, and incubated at 37°C for 30 minutes. For proteinase K treatment, the culture supernatant was treated with proteinase K (100 μg / ml; TaKaRa Bio) at 37°C for 1 hour. Subsequently, the samples treated with proteinase K were treated with APMSF (5 mM; Nacalai Tesque) on ice for 20 minutes to inactivate proteinase K. Since cytokine mRNA induction was abolished by proteinase K treatment alone, it was suggested that the activator is a protein. Next, the culture supernatant was subjected to ion-exchange chromatography (using either a Hitrap Capto S column (cat# 17544105, GE Healthcare) or a Hitrap Capto Q column (cat# 11001302, GE Healthcare). The Hitrap Capto S or Hitrap Capto Q column was first washed with 1 ml of DDW and then equilibrated with 30 ml of PBS. Then, 5 ml of SL4 cell culture supernatant was charged and washed with 5 ml of PBS. The flow-through was collected and PEC was added. RT-qPCR analysis revealed that the flow-through from the Hitrap Capto S column induced cytokine mRNA, while the flow-through from the Hitrap Capto Q column did not. This suggests that the target molecule binds to the Hitrap Capto Q column.

[0056] Based on the above results, the following purification procedure combining ion exchange chromatography and size exclusion chromatography was performed. First, a Hitrap Capto S column was washed with 1 ml of DDW and then equilibrated with 30 ml of PBS. 5 ml of SL4 cell culture supernatant was charged into the column and then washed with 5 ml of PBS. The flow-through was collected and charged into a Hitrap Capto Q column, which had been equilibrated in the same manner as the Hitrap Capto S column. Next, the column was washed with 20 ml of PBS and eluted with 5 ml of 0.4 M NaCl. The eluate was concentrated using Spin-X UF 10k MWCO (cat# CLS431488, MERCK). Finally, size exclusion chromatography was performed using a Superdex 200 Increase 10 / 300 GL column connected to an AKTA purifier (GE Healthcare). The column was equilibrated with twice the volume of PBS and the concentrated eluate was charged into the column. Using an AC-5700P MicroCollector (ATTO), eluted droplets (18 drops) were continuously collected into the wells of a 48-well plate. Each fraction was added to PEC or RAW264.7 cells, and the amounts of Cxcl1, Cxcl2, Tnf, and Il1b mRNA were determined by RT-qPCR. Fractions 18 to 27 were stained with silver using SilverQuest (cat# LC6070, invitrogen), and the darkest band corresponding to cytokine induction activity was subjected to LC-MS (liquid chromatography-mass spectrometry).

[0057] 1-7. RT-qPCR Total RNA from tissues or cells was extracted using NucleoSpin RNA II (MACHEREY NAGEL) and reverse transcribed using PrimeScript RT Master Mix (TaKaRa Bio). RT-qPCR reactions were performed using TB Green Premix Ex Taq. TMThe analysis was performed using II (TaKaRa Bio) with either LightCycler 480 (Roche Life Science) or LightCycler 96 (Roche Life Science). The obtained values ​​were normalized to the expression level of Gapdh mRNA. The primers used had the sequences shown below. Gapdh Forward; 5'-ctcatgaccacagtccatgc-3' (SEQ ID NO: 26) Reverse; 5'-cacattgggggtaggaacac-3' (SEQ ID NO: 27) Tnf Forward; 5'-tcataccaggagaaagtcaacctc-3' (SEQ ID NO: 28) Reverse; 5'-gtatatgggctcataccagggttt-3' (Sequence ID 29) Cxcl1 Forward; 5'-agaccatggctgggattcac-3' (SEQ ID NO: 30) Reverse; 5'-agcttcagggtcaaggcaag-3' (SEQ ID NO: 31) Cxcl2 Forward; 5'-tccagagcttgagtgtgacg-3' (Sequence ID 32) Reverse; 5'-tcagttagccttgcctttgttc-3' (SEQ ID NO: 33) Cxcr2 Forward; 5'- gacaccctcatgagaaccaagc-3' (SEQ ID NO: 34) Reverse; 5'- gttaaggcagctgtggaggaag-3' (SEQ ID NO: 35) Il1b Forward; 5'-gtggaccttccaggatgagg-3' (Sequence ID 36) Reverse; 5'-cggagcctgtagtgcagttg-3' (Sequence ID 37)

[0058] 1-8. CRISPR / Cas9 TCTP KO SL4 cells, TCTP KO B16F10 cells, and TCTP KO Meth-A cells were generated by CRISPR / Cas9 genome editing using the CRISPR design tool (http: / / www.genome-engineering.org, accessed May 2017). The genomic sequence of the TCTP gene was targeted (5'- CGGGCGGAAAAGGCCGACGC-3' (SEQ ID NO: 38) and 5'- AGGCCCGCCATTTCCCGCGC-3' (SEQ ID NO: 39)). The first exon of the TCTP gene was flanked by the two sequences, SEQ ID NO: 38 and SEQ ID NO: 39. Oligonucleotides corresponding to these guide sequences were cloned into the BbsI sites of pSpCas9(BB)-2A-GFP (PX458) (Addgene) and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene), respectively. Both expression vector constructs were introduced into SL4 cells. Cells into which the construct was introduced were selected with puromycin, and then GFP-expressing cells were subjected to single-cell sorting using FACSAria II (BD Biosciences) or SH800S (SONY).

[0059] 1-9. Immunoblotting Immunoblotting analysis was performed according to a previous report (Inoue et al., Nature 434, 243-249, doi:10.1038 / nature03308 (2005)). β-Actin (cat# A5441) was purchased from Sigma-Aldrich. TCTP antibodies (cat# ab133568 and cat# ab37506) were purchased from abcam. The TCTP antibody (cat# 5128S) was purchased from Cell Signaling Technology. β-Actin was used as a loading control. To determine the amount of TCTP in serum, serum was subjected to immunoblotting analysis with the TCTP antibody (ab133568). Recombinant TCTP (cat# TP301664, OriGene) was used for the measurement of the amount of TCTP. Anti-rabbit IgG-HRP (cat# NA934V, GE healthcare) or anti-mouse IgG-HRP (cat# NA931V, GE healthcare) was used as a secondary antibody. Immunoblotting signals were detected with a FUSION Solo S (Vilber-Lourmat) and analyzed with FUSION Capt Advanced software (Vilber-Lourmat).

[0060] 1-10. In vitro cell growth analysis WT SL4 cells and TCTP KO SL4 cells (2.5×10 5 cells), B16F10 cells (1.2×10 5 cells) or Meth-A cells (2.5×10 5 cells) were seeded into each well of a 6-well plate. Every 3 days, one-eighth of the cells were passaged into a new 6-well plate. Cell numbers were calculated on days 3, 6, and 9.

[0061] 1-11. Subcutaneous tumor growth assay Subcutaneously in mice, 2×10 5 SL4 cells, 1×10 5 B16F10 cells or 5×10 5 Meth-A cells were transplanted. Tumor volume was calculated as average volume = πab 2The calculation was performed using the formula / 6 (where a and b are the major and minor axes, respectively).

[0062] 1-12. Apc + / Δ716 Colon cancer model TCTP flox / flox Apc + / Δ716 Mouse or TCTP flox / flox Apc + / Δ716 Villin-CreERT2 mice were treated with 4 mg / mouse tamoxifen once a week starting at 5 weeks of age. Intestinal tumors in these mice were analyzed at 10 weeks of age using a stereoscope (Leica S9 D, Leica).

[0063] 1-13. Establishment of TCTP-introduced strains The encoding DNA of the human IL-2 peptide (MYRMQLLSCIALSLALVTNS: SEQ ID NO: 40) was fused to the 5' end of mouse TCTP cDNA. Each cDNA fragment of IL-2ss-TCTP and WT TCTP was inserted into a pMXs-IRES-GFP retroviral expression vector. Retroviral vector introduction into TCTP KO SL4 cells was performed according to previously reported procedures (Chiba et al., Elife. 2014 Aug 22;3:e04177. doi: 10.7554 / eLife.04177.). Subsequently, GFP-positive cells were sorted using a Cell Sorter SH800S (SONY). Mock cells or IL-2ss-TCTP-transformed cells were sorted into 2 × 10⁶ cells. 6 The cells were identified by performing the immunoblotting analysis described above on the culture supernatant collected from 60 mm culture dishes on which the cells were seeded.

[0064] 1-14. Enzyme-linked immunosorbent assay (ELISA) The tumor was excised 21 days after transplantation and finely chopped in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM Na3VO4, 1 mM APMSF). The chopped material was then incubated on ice for 30 minutes, the lysate was centrifuged, and the supernatant was collected for ELISA. Mouse CXCL1 and CXCL2 production during TIME was quantified using an ELISA kit (R&D Systems) according to the accompanying instructions.

[0065] 1-15. Evaluation of the immunosuppressive activity of PMN-MDSCs T cells were collected from the splenocytes of tumor-free mice using the Pan T cell isolation kit II (Miltenyi Biotec). The prepared cells were then processed using CellTrace. TM CFSE staining was performed using the CFSE Cell Proliferation Kit (Thermo Fischer Scientific) according to the attached instructions. PMN-MDSCs were SL cells (2 × 10⁶). 5 Splenocytes from mice that underwent subcutaneous transplantation of (Phaseol) cells were collected 17-19 days after transplantation using anti-Ly6-G microbeads (Miltenyi Biotec). Neutrophils were collected from tumor-free C57BL / 6 mice using the same method as for PMN-MDSCs. CFSE-labeled 1×10⁶ cells were collected. 5 T cells of cells, and 5 × 10 4 cells, 2.5 x 10 4 Cells or 1.25 × 10 4 PMN-MDSC cells were seeded into 96-well plates. T cell proliferation was induced for 3 days using Dynabeads Mouse T-Activator CD3 / CD28 (Thermo Fischer Scientific), and then flow cytometry analysis was performed to evaluate the CFSE dilution rate in the T cells.

[0066] 1-16. CD8 in vivo + Removal of T cells, NK cells, and PMN-MDSCs For NK cell removal, anti-asialo GM1 (cat# 014-09801, WAKO) or rat control IgG (cat# 31933, Thermofisher) was administered intraperitoneally on days 1, 3, 7, 11, and 15 after tumor cell transplantation (200 μg / mouse). CD8 + For T cell removal, anti-CD8α (clone 2.43, cat# BE0061, Bio X Cell) or rat control IgG (cat# 31933, Thermofisher) was administered intraperitoneally on days 1, 3, 7, and 11 after tumor cell transplantation (100 μg / mouse). NK cells and CD8 + For T cell removal, anti-asialo GM1 (cat# 014-09801, WAKO) or rat control IgG (cat# 31933, Thermofisher) was administered intraperitoneally to RAG1 KO mice (200 μg / mouse). For PMN-MDSC removal, anti-anti-Ly6G antibody (cat# BE0075-1, Bio X Cell) or rat control IgG was administered intraperitoneally on days 1, 3, 5, 7, 9, and 11 after tumor cell transplantation.

[0067] 1-17. Treatment with dihydroartemisinin (DHA) or o-vanillin DHA (Selleck, TX, USA) was dissolved in DMSO and administered intraperitoneally (50 mg / kg) daily from day 1 after tumor transplantation. In the case of combined administration of anti-PD-1 antibody and DHA, DHA administration was discontinued on day 6. O-vanillin (cat# 120804-10G, Merck) was first dissolved in DMSO to a concentration of 500 mg / ml, and then diluted with PBS to a final concentration of 50 mg / ml. The diluted solution was administered orally every two days from day 1 after tumor transplantation (50 mg / kg).

[0068] 1-18. Production of monoclonal antibodies 1-18-1. Antibody production Mouse monoclonal antibodies against TCTP were produced by Monoclonal Antibody Laboratory Co., Ltd. (http: / / www.monoclo.com; Nagano, Japan) using a standard hybridoma method. BALB / c mice were immunosuppressed with a synthetic peptide (EDGVTPYMIFFKDGLEMEKC: SEQ ID NO: 25), which is a partial sequence of human TCTP. Antibody screening was performed based on immunoblotting and immunoprecipitation analyses against TCTP. As a result, antibodies 55F3, 44E1, and 51A9 were obtained. For antibody treatment, 200 μg of 55F3 or control IgG (cat# 0107-01, SouthernBiotech) was administered intraperitoneally to each mouse.

[0069] 1-18-2. Determination of the amino acid sequences of the variable regions of antibody heavy and light chains. The amino acid sequences of the heavy and light chains of the prepared monoclonal antibodies were determined by referring to PLoS ONE e0218717,14: 2019. Total RNA was extracted from hybridoma cells, and cDNA was synthesized using RT (reverse transcription) primers (mIGK RT, mIGHG RT, Template-switch oligo F) specific to the variable regions of the antibody heavy and light chains. Subsequently, PCR reactions were performed using the synthesized cDNA as a template and then with specific primers (ISPCR, mIGK PCR, mIGHG PCR). The resulting cDNA was TA cloned (pTA2 vector, TOYOBO) and sequenced. The detailed experimental conditions are as follows:

[0070] (1) cDNA preparation Primers used in the reverse transcription reaction Template-switch oligo F:5'-aagcagtggtatcaacgcagagtacatGGG-3' (G is riboguanine) (SEQ ID NO: 41) mIGK RT:5'-ttgtcgttcactgccatcaatc-3'(SEQ ID NO: 42) mIGL RT:6'-ggggtaccatctaccttccag-3'(SEQ ID NO: 43) mIGHG RT:5'-agctgggaaggtgtgcacac-3'(SEQ ID NO: 44) Reaction solution (I) (cDNA synthesis reaction) 2 μL 50 ng / μL Total RNA 1 μL 10 μM reverse transcription primer (mIGK RT, mIGL RT, or mIGHG) 1 μL 10 mM dNTPs. The above was mixed in an 8-well strip tube. Reaction solution (II) (cDNA synthesis reaction) 2.2 μL H2O 2 μL 5×SMARTScribe buffer 1 μL 20 mM DTT 0.3μL 100μM template-switch oligo F. 0.5μL 100U / μL SMARTScribe Reverse Transcriptase The above was mixed in an 8-well strip tube. Reaction solution (I) was heat-treated in a thermal cycler at 72°C for 3 minutes. After heat treatment, 6 μL of reaction solution (II) was added to reaction solution (I), and the mixture was incubated in a thermal cycler at 42°C for 60 minutes. Subsequently, the reaction was stopped by heat treatment at 70°C for 5 minutes.

[0071] (2) Determination of the amino acid sequences of the variable regions of the antibody heavy chain and light chain. Using the synthesized cDNA as a template, a PCR reaction was performed using the primers shown below. The resulting amplification product was used to determine the DNA sequences of the variable regions of the antibody heavy and light chains, and then the resulting amino acid sequences were determined. Primer ISPCR F:5'-aagcagtggtatcaacgcagag-3' (SEQ ID NO: 45) mIGK PCR: 5'-acattgatgtctttggggtagaag-3' (SEQ ID NO: 46) mIGL PCR: 5'-atcgtacacaccagtgtggc-3' (SEQ ID NO: 47) mIGHG PCR: 5'-gggatccagagttccaggtc-3' (SEQ ID NO: 48) Reaction solution 25μL 2× PCR buffer for KOD FX 10 μL 2 mM dNTPs 3μL Synthesized cDNA from the RT reaction 2.5μL 10μM universal forward primer ISPCR 2.5 μL 10 μM reverse PCR primer (mIGK PCR, mIGL PCR, or mIGHG PCR) 6 μL H2O 1 μL KOD FX (1 U / μL) PCR cycle After reacting at 98°C for 30 seconds, 98℃, 15 seconds, 63-57.5°C, 30 seconds (decreasing the temperature by 0.5°C in each cycle) 72℃, 30 seconds, The above reaction is repeated for 10 cycles, and then, 98℃, 15 seconds, 56℃, 30 seconds, 72℃, 30 seconds, After performing the above reaction 15 times, The reaction was carried out at 72°C for 7 minutes, and then allowed to stand at 4°C.

[0072] The determined amino acid sequences of the variable regions of the antibody heavy and light chains were compared with antibody sequencing databases (http: / / www.abybank.org / kabat / and http: / / www.bioinf.org.uk / abs / info.html#cdrid, etc.) to identify amino acid sequences corresponding to CDRs according to Kabat's definition.

[0073] 1-19. Analysis of data obtained from colorectal cancer patients. The TDGA dataset from 640 colorectal cancer patients was downloaded from cBioPortal (https: / / www.cbioportal.org / , accessed December 2019), and GISTIC 2.0 analysis was performed on the cBioportal platform.

[0074] 1-20. Pathological analysis SL4 tumors and APCs Δ716 The colon of mouse tumors was preserved in PBS containing 4% paraformaldehyde and embedded in paraffin. Hematoxylin / eosin (H&E) staining, TUNEL staining, and 3,3'-diaminobenzidine (DAB) staining for CD31 (cat# ab28364, abcam) or Ly6G (cat# 127601, Biolegend) were performed at the Pathology Core Laboratory of the Institute of Medical Science, University of Tokyo. Sample preparation and analysis of formalin-fixed paraffin-embedded tissue from the colon of colorectal cancer patients and normal colonic epithelium from the same patients were performed at Kanazawa University. This analysis was approved by the Human Genome / Gene Analysis Research Ethics Committee of Kanazawa University (2016-086-433), and written informed consent was obtained from the patients.

[0075] TCTP (cat# 133568, abcam) or CD15 (cat# M363129, Dako) was used for DAB staining. Briefly, paraffin-embedded samples were deparaffinized by two incubations in xylene, dehydrated with an ethanol series, and rehydrated in PBS. Epitope activation by heat treatment was performed at 121°C for 10 minutes using an antigen retrieval reagent at pH 9 (cat# 415211, NICHIREI BIOSCIENCES INC). To inactivate endogenous peroxidase, REAL Peroxidase-Blocking solution (cat# S2023, DAKO) was used according to the instructions for use, followed by blocking with 1% BSA / TBST at room temperature for 30 minutes. Sections were incubated with TCTP antibody or CD15 antibody at room temperature for 1 hour. Primary antibody detection was performed using Histofine simple stain MAX-PO(MULTI) (cat# 424151, NICHIREI BIOSCIENCES INC.) with DAB (cat# 415171, NICHIREI BIOSCIENCES INC.) as the substrate. Samples were counterstained with hematoxylin (at no. 415081, NICHIREI BIOSCIENCES INC.). Quantification of signal intensity from TCTP was performed using ImageJ Fiji (Bio Protoc. 2019 Dec 20;9(24):e3465.) as previously reported. The mean intensity in normal colonic mucosa was set to 1. Quantification of CD31-positive regions was performed using a BZ-9000 microscope and cell counting software BZ-H4C (Keyence).

[0076] 1-21. Microarrays PEC is a cell that has undergone necrosis (2 × 10 6Cells were stimulated with the culture supernatant of SL4 cells. After a 2-hour incubation, total RNA was extracted and used for analysis using a Clariom S Array (Thermo Fisher Scientific). Volcano plots were created using Transcriptome Analysis Console (TAC) software v4.0 (Thermo Fisher Scientific). Microarray data were registered in the Gene Expression Omnibus (GEO) database (accession No. GSE150465).

[0077] 1-22.Immunostaining Cells were cultured overnight in a 35 mm glass-bottom dish (MATSUNAMI), washed with PBS, and fixed with 4% paraformaldehyde / PBS for 15 minutes. After washing with PBS, membrane permeabilization was performed with 0.5% Triton X-100 / PBS for 15 minutes, followed by blocking with 3% BSA / PBS. Next, the cells were incubated with anti-TCTP antibody (ab37506) for 2 hours, washed, and treated with secondary antibody (Alexa Fluor 594 Goat anti-rabbit IgG, cat# A-11012, Invitrogen) for immunostaining. After washing with PBS, nuclear counterstaining was performed using VECTASHIELD Hard Set Mounting Medium with DAPI (cat# H-1500, VECTOR LABORATORIES, INC), and analysis was immediately performed using a C2si confocal microscopy system (NIKON) equipped with an ECLIPSE Ti microscope (NIKON).

[0078] 1-23. Adoptive transfer of PMN-MDSC Bone marrow cells and spleen cells are SL4 cells (2 × 10⁶ 5 Ly-6G cells were collected from mice (with tumors) 17 days after subcutaneous transplantation. +Cells were isolated using the anti-Ly6-G MicroBead Kit, mouse (Miltenyi Biotec). TCTP KO SL4 cells (2 × 10⁻¹⁶) 5 In mice that underwent subcutaneous transplantation of cells (Ly-6G) at days 1, 4, 7, 10, and 13, isolated Ly-6G cells were administered. + cells (4×10 6 The cells were injected intravenously.

[0079] 1-24. Immunoprecipitation HEK293T cells (5 × 10 6 After seeding, HEK293T cells were transiently transduced using X-tremeGENE9 (Roche Life Science) with either pCXNII-FLAG-hTCTP (2 μg) alone, or pCXNII-FLAG-hTCTP (2 μg) and pcDNA3.1-hTLR2-YFP (2 μg). The pcDNA3.1-hTLR2-YFP vector was provided by Dr. Douglas Golenbock. Subsequently, cell lysates were prepared using a lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF). Immunoprecipitation was performed on 1 mg of cell lysate using 1 μg of anti-GFP antibody (598; MBL) and 30 μl of Dynabeads Protein G for immunoprecipitation (Thermo Fisher Scientific). Immunoblotting was then performed on the immunoprecipitation using either anti-GFP antibody or anti-FLAG M2 antibody (Sigma Aldrich).

[0080] 1-25. Luciferase Reporter Assay Mouse TLR3, TLR7, TLR9, and human CD14 cDNA were cloned into the pCXNII-HA vector. Human TLR2 cDNA construct (pcDNA3.1-hTLR2-YFP), mouse TLR3 cDNA construct (pCXNII-HA-mTLR3), mouse TLR7 cDNA construct (pCXNII-HA-mTLR7), or mouse TLR9 cDNA construct (pCXNII-HA-mTLR9) were transfused into HEK293T cells along with an NFκB luciferase reporter (pNFκB-Luc(Stratagene)). For TLR2 stimulation, the human CD4 cDNA construct (pCXNII-HA-hCD14) was co-transfused. 24 hours after transfusion, 2 × 10⁶ cells were transfused. 4 Cells were seeded in 96 welldishes. After 24 hours, cells were treated with recombinant TCTP or each TLR agonist. Subsequently, luciferase activity was measured using the Pikka Gene Dual Assay kit (cat# PD-11, TOYO B-Net) and a MicroLumat Plus LB96V (Berthold Technologies) according to the instructions for use.

[0081] 1-26. Determination of G-CSF and GM-CSF The quantification of G-CSF (cat# 560152, BD Biosciences) and GM-CSF (cat# 558347, BD Biosciences) was performed using a cytometric bead assay (CBA) according to the attached instructions for use.

[0082] 1-27. Induction of cell death To induce apoptosis, serum starvation and Adriamycin treatment were performed. For serum starvation, SL4 cells (5 × 10) were treated. 4Cells were seeded in 48 welldishes, and after 12 hours, the medium was replaced with serum-free medium. After 72 hours of serum starvation, the culture supernatant was collected. For Adriamycin treatment, SL4 cells were seeded in the same manner as in serum starvation. After 12 hours, the cells were treated with a medium containing Adriamycin (50 μM) for 24 hours, and the culture supernatant was collected. To induce necrosis, SL4 cells (1 × 10⁶ 7 The cells were suspended in 1 ml of PBS. Freeze-thaw cycles were performed as described in "1-4. Preparation of Culture Supernatant" above. As a control, supernatant of SL4 cells incubated in PBS for the same amount of time as the freeze-thaw cycle was used. Hypoxia treatment (1% O2, 5% CO2) was performed using a multi-gas incubator (MCO-5MUV-PJ, Panasonic).

[0083] 1-28. Determination of TCTP in human serum Serum samples from colorectal cancer patients and non-cancerous patients were obtained from Bionbank Japan. This study was approved by the Ethics Committee of the University of Tokyo (20-239). TCTP was quantified by immunoblotting.

[0084] 1-29. Administration of tumor-dead cells to a subcutaneous tumor model TCTP WT cells or TCTP KO cells on a 10 cm dish were irradiated with a lethal dose of X-rays (100 Gy). TCTP WT SL4 cells (2 × 10⁻¹⁶) 5 The cells were mixed with an equal amount of the above-mentioned TCTP WT cells or TCTP KO cells and subcutaneously transplanted into C57BL / 6 cells.

[0085] 1-30. Statistical Processing Sample size and statistical tests were performed as described in the figure captions. Unless otherwise specified, data are presented as mean ± standard error (SEM). Dunnett multiple comparison after one-way ANOVA or Tukey's multiple comparison after one-way ANOVA, Spearman correlation coefficient calculation, log-rank test, and two-dided Student's t-test were performed using Prism 8.0 (GraphPad Software). All alpha levels were defined as 0.05, with p<0.05 considered statistically significant.

[0086] 2.Results 2-1. Identification of tumor cell-derived TCTP as an immunomodulatory factor Since a considerable number of tumor cells die (mostly due to necrosis) during tumor growth, we hypothesized that the environment of necrotic cells functions as an immunomodulator for MDSCs (myeloid-derived suppressor cells) within the tumor immune microenvironment (TIME). As an approach to identify immunomodulators derived from tumor dead cells, we first used a culture supernatant model of necrotic SL4 cells (mouse colorectal cancer cell line) (Non-patent Literature 5) (Figure 1a). When the SL4 cell culture supernatant was exposed to mouse PEC, the expression of several cytokine mRNAs was induced. These mRNAs were analyzed by microarray (Figure 1b) and RT-qPCR (Figure 1c). Among the cytokines whose mRNA expression was confirmed, Cxcl1 and Cxcl2 It is known to control the migration of hematopoietic cells such as MDSCs, Cxcl1 and Cxcl2 We focused on this. CXCR2, a receptor common to CXCL1 and CXCL2, is known to be important for PMN-MDSC migration in mice (Non-Patent Literature 6).

[0087] Among chemokines derived from tumor dead cells, Cxcl1 and Cxcl2 To identify the molecules that induce expression of [substance name], we attempted to isolate the target molecule from the culture supernatant of SL4 cells using ion exchange chromatography and size exclusion chromatography. The culture supernatant of SL4 cells was passed through an anion exchange column (Hitrap Q) and a cation exchange column (Hitrap S). The flow-through from the Hitrap S column was collected and charged onto the Hitrap Q column. The fraction bound to Hitrap Q was collected and charged onto a size exclusion column (Superdex 200). Each of the resulting fractions was subjected to PEC (Periodic Emission Control). Cxcl1 , Cxcl2 , Il1b ;2×10 5 Cells) or RAW264.7 cells ( Tnf ;2×10 5 The solution was added to the cells and incubated for 2 hours. Cxcl1 , Cxcl2 , Tnf and Il1b The mRNA levels were quantified by RT-qPCR. SDS-PAGE and silver staining were performed on the peak expression fractions of each mRNA, and liquid chromatography-mass spectrometry (LC-MS) was performed on the protein with the highest expression level. This revealed that TCTP (Tumor cell-derived translationally controlled tumor protein) is a candidate inducer for each of the above mRNAs. Although TCTP has been reported to be present in the cytoplasm of eukaryotic cells (Non-Patent Documents 7, 8, and 9), the function of TCTP present extracellularly was previously unknown.

[0088] After stimulating PEC with recombinant TCTP, the expressed mRNA was quantified by RT-qPCR. Cxcl1 and Cxcl2 and other cytokines ( Tnf and Il1bThe mRNA of ) was observed to be induced (Figure 2a). These results confirmed that TCTP has an immune-activating function. Interestingly, it has been reported that TCTP mRNA expression levels are elevated in tumor samples (Du et al., Oncotarget 8, 101922-101935, doi:10.18632 / oncotarget.21747 (2017)). However, it was not clear whether TCTP plays a role in tumor progression, and if so, how it plays a role.

[0089] TCTP was abundant in the culture supernatant of dead tumor cells, whereas living tumor cells released very little TCTP (Figure 3a). Furthermore, as shown in Figure 2b, the amount of TCTP protein in the serum increased over time in C57BL / 6 mice subcutaneously transplanted with SL4 cells. This result suggests that TCTP is released from dying tumor cells in vivo. Similarly, elevated levels of TCTP protein were observed in the serum of mice with B16F10 melanoma and Meth-1 fibrosarcoma tumors (Figure 3b).

[0090] 2-2. Contribution of tumor-derived TCTP to tumor growth Based on the above results, we investigated the effect of TCTP on tumor growth formed by TCTP-expressing SL4 cells and TCTP-deficient SL4 cells. Whole cell lysates were prepared from TCTP-expressing SL4 cells (TCTP WT SL4:WT) and TCTP-deficient SL4 cells (TCTP KO SL4:KO), and immunoblotting was performed for TCTP and β-actin to confirm the deficiency of TCTP in TCTP KO SL4 cells (Figure 3c). The proliferation rates of TCTP WT SL4 cells and TCTP KO SL4 cells were similar in vitro under standard, hypoxic, and low serum conditions (Figures 3d and e). In contrast, when TCTP WT SL4 cells and TCTP KO SL4 cells were transplanted into C57BL / 6 mice by subcutaneous injection, and the volume of tumors generated in vivo was measured over time, it was found that the growth of tumors derived from TCTP KO SL4 cells was significantly slower than that of tumors derived from TCTP WT SL4 cells (Figure 2c). Furthermore, it was confirmed that the tumor growth rate in vivo was restored when the TCTP gene was reintroduced into TCTP KO SL4 cells to re-express the TCTP protein (Figure 3f). In addition, the TCTP gene was disrupted in B16F10 cells and Meth-A cells (Figure 3c), and the same investigations were performed. As a result, similar to SL4 cells, the in vitro growth rates of wild-type and TCTP gene-deficient strains were similar (Figures 3g and h), but the growth of tumors transplanted into living mice was significantly slower in tumors derived from the TCTP gene-deficient strain than in tumors derived from the wild-type strain (Figures 2d and e). These results indicate that TCTP promotes tumor growth in vivo.

[0091] We investigated whether tumor-derived TCTP promotes tumor growth. Administration of TCTP WT SL4 cells irradiated with a lethal dose of X-rays (dead WT cells) promoted the growth of TCTP WT SL4 cell tumors, but administration of TCTP KO SL4 cells irradiated with a lethal dose of X-rays (dead KO cells) did not promote tumor growth (Figure 4c). Furthermore, in order to clarify the role of TCTP in tumor progression, the tumor suppressor is Apc Genetic deletion mutations ( Apc Δ716 A model of colorectal tumors promoted by ) was investigated in the presence or absence of the TCTP gene (Figure 2f). TCTP gene expression can be regulated by tamoxifen. flox / flox Apc + / Δ716 We used villin-Cre ERT2 mice (Figure 2f). In these mice, tamoxifen administration specifically caused a deficiency of the TCTP gene in intestinal epithelial cells. As a result, when comparing the total number of tumors that developed in the intestines of tamoxifen-treated mice (TCTP-deficient mice) and untreated mice (mice retaining the TCTP gene), no significant difference was observed between the two (Figure 4a, Total). On the other hand, the number of tumors with a diameter exceeding 2.0 mm was significantly reduced in TCTP-deficient mice (Figure 4a, <2.0 mm). This result indicates that TCTP does not affect the incidence rate of tumors, but promotes tumor growth, which is consistent with the experimental results using the cancer cell transplantation mouse model mentioned earlier. In other words, TCTP does not affect the incidence rate of tumors, but rather promotes the growth of cancers that have already developed.

[0092] 3. Examination of the role of extracellular TCTP Next, we investigated whether extracellularly released TCTP promotes tumor growth. To induce extracellular secretion of TCTP, we used a retroviral gene transfer vector to express cDNA encoding a chimeric TCTP protein fused with a human IL-2 signaling sequence in TCTP KO SL4 cells (IL-2ss-TCTP SL4 cells). These cells were then grown in cell culture medium. In vitro, TCTP protein was detected in the culture supernatant (Figure 5a) but not intracellularly (Figure 6a). Next, we stimulated PEC with the culture supernatant of TCTP KO SL4 cells and IL-2SS-TCTP SL4 cells to investigate cytokine induction. As a result, stimulation with the culture supernatant of IL-2ss-TCTP SL4 cells resulted in... Cxcl1 and Cxcl2 Elevated mRNA expression levels were observed (Figure 6b, IL2ss-TCTP). The proliferation rate of IL-2ss-TCTP SL4 cells in vitro was similar to that of TCTP KO SL4 cells (Figure 5b), but the proliferation rate of tumors derived from IL-2ss-TCTP SL4 cells in vivo was significantly faster than that of tumors derived from TCTP KO SL4 cells (Figure 5c). These results indicate that TCTP released extracellularly functions as an immunomodulator and as a factor that promotes tumor growth in vivo.

[0093] Based on the results so far, it is possible that TCTP released from dying tumor cells induces CXCL1 and CXCL2, which in turn attract PMN-MDSCs to TIME, suppressing the antitumor immune response in TIME and promoting tumor growth. In fact, when comparing the amount of CXCL1 in TCTP KO SL4 tumors and TCTP WT SL4 (TCTP-expressing SL4) tumors, CXCL1 expression was significantly higher in TCTP WT SL4 tumors (Figure 5d). Similar results were observed for CXCL2 (Figure 5d). Furthermore, the amount of CXCL1 / 2 in IL-2ss-TCTP tumors was also significantly higher than that in TCTP KO SL4 tumors (Figure 5e). These results indicate that extracellular TCTP induces these chemokines within TIME.

[0094] Next, tumor-infiltrating immune cells were prepared from TCTP WT SL4 tumors and TCTP KO SL4 tumors and analyzed by flow cytometry. As shown in Figure 7a, CD11b, representing mouse PMN-MDSCs, was found within the TCTP KO SL4 tumor. + Ly6C low Ly6G + A dramatic decrease in the number of cells was confirmed. On the other hand, CD11b representing M-MDSC + Ly6C high Ly6G - Cells and CD11b+ Ly6C-Ly6G - F4 / 80 + Myeloid cells such as tumor-associated macrophages (TAMs) did not show such dramatic changes (Figures 7a and b). While the amount of PMN-MDSCs was elevated in IL-2ss-TCTP tumors compared to TCTP KO tumors, there was little difference in the number of M-MDSCs (Figure 7c). It is noteworthy that there was no significant difference in the expression levels of G-CSF and GM-CSF, which promote MDSC proliferation, between WT-type tumors and TCTP KO-type tumors (Figure 6c). This result indicates that G-CSF and GM-CSF are not the primary cause of the decrease in PMN-MDSC cell count in TCTP KO tumors. Similarly, a decrease in PMN-MDSC cell count was also observed in B16F10 and Meth-A tumors (Figures 6d and e). Interestingly, ly6G, representing PMN-MDSCs, was also observed. + The significant decrease in cells is due to the aforementioned TCTP, a tumor transplantation model. flox / flox Apc + / Δ716 This was also observed in de novo tumors in villin-Cre ERT2 mice (Figure 7d).

[0095] CD11b (see above) + Ly6C low Ly6G + To confirm that the cells actually represent PMN-MDSCs, we used CD11b derived from mice with SL4 tumors. + Ly6C low Ly6G + CD11b derived from mice without cells or SL4 tumors. + Ly6C low Ly6G + The cells were subjected to an in vitro T cell proliferation assay. As shown in Figure 7e, CD11b cells were isolated from mice with SL4 tumors. + Ly6C low Ly6G + When T cells were stimulated with an anti-CD3 / CD28 antibody in the presence of cells, and T cell proliferation was observed, CD11b + Ly6Clow Ly6G + T cell proliferation was suppressed in a manner dependent on the cell abundance. In contrast, CD11b derived from tumor-free mice showed the same effect. + Ly6C low Ly6G + Similar experiments were conducted using cells, but no inhibitory effect on T cell proliferation was observed. Furthermore, adoptive immunization with PMN-MDSCs isolated from mice with SL4 tumors significantly increased the proliferation of TCTP KO SL4 tumors (Figure 6g). These results suggest that the growth retardation of TCTP KO tumors is induced by PMN-MDSCs, supporting the existence of a TCTP-PMN-MDSC pathway that induces the progression of immunosuppressive TIME.

[0096] The results of the MDSC adoptive immunization described above suggest that antitumor lymphocytes remain activated even in mice with TCTP KO tumors. As shown in Figure 8b, CD8 during TIME + The number of T cells was significantly increased in mice with TCTP KO SL4 tumors compared to mice with TCTP WT SL4 tumors. On the other hand, in IL-2ss-TCTP tumors, CD8 was higher than in TCTP KO tumors. + The number of cells was low. In addition, tumor NK cell activation markers (CD69 and CD107a) were also elevated in mice with TCTP KO SL4 tumors compared to mice with TCTP WT SL4 tumors (Figure 6h). Therefore, these results suggest that CD8 + This suggests that both T cells and NK cells are involved in inducing an immune response against tumors. In particular, the proliferation of TCTP KO SL4 tumors is associated with CD8 + Removing either T cells or NK cells partially restored the tumor (Figures 8c and d), while removing both cells promoted tumor growth (Figure 8e). These observations suggest that the deficiency in the MDSC repertoire is due to CD8 + It is possible to explain that both T cells and NK cells remain active against TCTP KO tumors during TIME. On the other hand, in WT tumors, CD8+ The elimination of T cells and NK cells did not affect tumor growth. This result suggests that these ejector cells are already dysfunctional due to the more potent MDSC repertoire (Figures 8c-e). Of particular note are the expression levels of PD-L1 on TAM, MDSCs, and stromal cells, and CD8 in TCTP WT and TCTP KO tumors. + There was essentially no difference in the expression level of PD-1 on T cells (Figures 9a and b). Furthermore, endothelial cells (CD31) in TCTP KO tumors + The cell density was comparable to that in TCTP WT tumors (Figure 9c). Based on these results, PMN-MDSCs, which are taken up by TCTP released from tumor cells during TIME, are CD8 + This suggests that it suppresses the antitumor immune action of T cells and NK cells, thereby promoting tumor growth in at least some cases.

[0097] 4. Identification of cells and receptors on which TCTP acts. Based on previous results, it is thought that the TCTP-CXCL1 / 2-PMN-MDSC pathway is involved in the suppression of antitumor immunity in the TIME (oncology immune microenvironment). Therefore, we next investigated which cell types are factors in chemokine induction in TIME. We selected a subset of immune cells from the TIME of TCTP WT SL4 tumors and TCTP KO SL4 tumors and examined the mRNA expression levels of chemokines. D11b + Ly6C high Ly6G cells (M-MDSCs) were the most abundant Cxcl1 mRNA was expressed (Figure 10a). Of particular note here is the M-MDSC isolated from the TCTP KO SL4 tumor. Cxcl1 The mRNA expression level is significantly reduced (Figure 10b). In this respect, in vitro, SL4 tumor cells do not respond to TCTP. Cxcl1 mRNA expression was not induced (Figure 11a). Furthermore, in vitro, in TCTP WT SL cells and TCTP KO SL4 cells... Cxcl1 mRNA expression levels were comparable (Figure 11b). Therefore, it is thought that tumor cells themselves hardly induce CXCL1 in vivo in response to TCTP stimulation. Furthermore, the receptor genes for the CXCL1 / 2 chemokines are Cxcr2 mRNA expression was significantly higher in PMN-MDSCs than in any other cell type (Figure 11c). These results suggest that TCTP acts on M-MDSCs, inducing the expression of CXCL1 / 2 chemokines, and that stimulation by these CXCL1 / 2 chemokines leads to the migration of CXCR2-expressing PMN-MDSC cells to TIME, promoting tumor growth through suppression of the anti-tumor immune system.

[0098] Next, we investigated whether TCTP-induced chemokines activate specific receptors. First, we examined the involvement of adapter molecules that commonly act on several innate immune receptors. As shown in Figure 10c, TCTP-induced chemokine induction was abolished in PECs lacking the MyD88 gene, but this abolition did not occur in PECs lacking the IPSI / MAVS or STING gene. MyD88 is a common adapter molecule for Toll-like receptors (TLRs). Among Toll-like receptors, TLR2 and TLR4 are known to recognize multiple proteins released from dead cells (Non-Patent Literature 10). Therefore, PECs prepared from wild-type (WT) mice, TLR2-deficient (TRL2 ​​KO) mice, and TLR4-deficient (TLR4 KO) mice were stimulated with recombinant TCTP. Cxcl1 mRNA expression levels were measured. As a result, TCTP Cxcl1 mRNA induction is Tlr2 -It disappeared in the missing PEC, Tlr4 In the PEC-deficient, it did not disappear (Figure 10d). In this experiment, Tlr2 - Recombinant IL-1α in deficient PEC Cxcl1Since mRNA induction occurred normally, it was suggested that IL-1α is not involved in TLR2-mediated tumor growth (Figure 11d). The interaction between TCTP and TLR2 was confirmed by co-immunoprecipitation of epitope-tagged TCTP and TLR2 (Figure 11e). This result supports the role of TLR2 as a receptor for signals that induce CXCL1 / 2 expression via TCTP. Furthermore, as shown by the luciferase reporter assay, none of TLR3, TLR7, and TLR9, which signal via the MyD88 pathway, were activated by recombinant TCTP (Figure 11f).

[0099] Tlr2 It is noteworthy that SL4 tumors transplanted into knockout mice proliferate more slowly than SL4 tumors transplanted into wild-type mice (Figure 11g). Furthermore, Tlr2 IL-2-ss-TCTP tumors transplanted into knockout mice also showed slower growth (Figure 11h). Furthermore, the selective TLR2 inhibitor O-vanillin suppressed the growth of IL-2ss-TCTP tumors (Figure 11i). These results indicate that persistent, weak signaling from tumor-derived TCTP (rather than transient, strong signals from infection, etc.) is important for inducing and maintaining a well-developed TIME.

[0100] 5. Effects of TCTP inhibition on tumor growth Next, we investigated the effects of TCTP inhibitory antibodies and inhibitors on tumor growth. Monoclonal antibodies (55F3, 44E1, 51A9) against the human TCTP peptide (SEQ ID NO: 25) were produced. The peptide of SEQ ID NO: 25 consists of the C-terminal 20 residues of the human TCTP protein. First, we confirmed that the 55F3 antibody (55F3) exhibits species cross-reactivity with mouse TCTP (Figure 12a). When PECs were stimulated with the culture supernatant of SL4 cells containing 55F3 or control IgG, the PECs stimulated with the culture supernatant of SL4 cells containing 55F3 showed a significant difference in the results. Cxcl1mRNA expression was suppressed (Figure 12b). Next, SL4 cells were transplanted into C57BL / 6 mice by subcutaneous injection, and then 55F3 or control IgG (200 μg / mouse) was administered intraperitoneally every 1 to 2 days after transplantation, and tumor volume was measured. As a result, administration of 55F3 reduced the proliferation rate of SL4 tumor cells (Figure 13a) and decreased the amount of PMN-MDSC during TIME (Figure 6b). This result indicates that inhibiting the TCTP-CXCL1 / 2-MDSC pathway suppresses tumor growth. Furthermore, we investigated the effects of dihydroartemisinin (DHA), which is known to bind to TCTP and promote its degradation by the proteasome (Non-Patent Literature 11). As shown in Figure 6c, intraperitoneal administration of DHA was found to inhibit the proliferation of SL4 tumors. On the other hand, this inhibitory effect on tumor proliferation by DHA was not observed in TCTP knockout tumors (Figure 12c), indicating that TCTP is indeed the target in inhibiting tumor proliferation. These results suggest that TCTP inhibitors (TCTP antagonists) are effective as cancer treatment agents.

[0101] We investigated the effects of combining TCTP function inhibition with PD-1 immune checkpoint inhibition on tumor growth. The PD-1 immune checkpoint antibody used was clone RMP1-14 (BioLegend). SL4 cells were transplanted into C57BL / 6 mice via subcutaneous injection, and then 55F3 or DHA was administered intraperitoneally daily starting 1 day post-transplant. Ten days after transplantation, an anti-PD-1 monoclonal antibody was administered, and tumor volume was measured. The results showed that co-administration of 55F3 or DHA with a PD-1 antagonist antibody improved tumor growth inhibition compared to 55F3 or DHA alone (Figures 13d and e).

[0102] 6. Involvement of TCTP in human cancer To investigate the role of TCTP in human cancer, we measured the amount of TCTP protein in serum from human colorectal cancer (CRC) patients. Similar to the mouse model (Figures 2b and 3g), the amount of TCTP protein in serum samples from cancer patients was higher than in control samples (Figure 13f). Furthermore, the amount of TCTP protein was higher in CRC tissue compared to normal colorectal tissue (Figure 13g). In addition, elevated TCTP expression correlated with cancer progression (Figure 13h). Elevated TCTP protein levels were observed in tumor lesions but not in the stromal region (Figure 12d). This indicates that TCTP expression is selectively increased in tumor cells. Furthermore, when CRC tissue was stained with an antibody against CD15, a human PMN-MDSC marker, the levels of TCTP protein and CD15 were found to be correlated. + The number of cells showed a positive correlation (Figure 13i). These results suggest that the TCTP-OMN-MDSC pathway also functions in human cancer.

[0103] Furthermore, data from The Cancer Genome Atlas (TCGA) were analyzed. Figure 13j shows the mRNA levels of TCTP, classified by DNA copy number, for colorectal cancer (CRC) patients (n=376) obtained from the TCGA database. Patients classified as "deep" or "shallow" had deletions of 1 or 2 alleles of the TCTP gene, respectively, while patients classified as "gain" or "amplification" acquired 1 or more alleles of the TCTP gene, respectively. This analysis indicates that TCTP gene amplification was observed in approximately 5% of colorectal cancer patients, and that TCTP mRNA expression levels correlated with TCTP gene copy number (Figure 13j). In addition, TCTP expression levels were negatively correlated with cytotoxic T cell or NK cell markers (i.e., CD8A, GZMB, PRF1, and CD69) (Figure 12e). This negative correlation was also observed for cytolytic responses of cytotoxic T cells and NK cells (defined as the geometric mean of GAMA mRNA and PRF1 mRNA expression levels, respectively) (Figure 12f). Notably, progression-free survival (PFS) was significantly lower in patients with amplified TCTP genes (Figure 13k).

[0104] Based on the above findings, the function of TCTP in tumor growth is summarized in Figure 13.TCTP is released from dead tumor cells, binds to the TLR2 receptor on M-MDSCs, and induces CXCL1 / 2 chemokines.It is concluded that these chemokines recruit PMN-MDSCs to TIME, blunting the anti-tumor immune response and further promoting tumor growth. [Industrial applicability]

[0105] This invention provides a drug for treating cancer and a method for treating cancer, etc. Therefore, this invention is expected to have great potential for use in the medical field.

Claims

1. An anti-TCTP antibody or its antigen-binding fragment characterized in that the amino acid sequence of CDR (complementarity determining region) 1 to 3 satisfies any of the following (A), (B), or (C). (A) Heavy chain CDR1 containing the amino acid sequence represented by Sequence ID No. 1, Heavy chain CDR2 containing the amino acid sequence represented by Sequence ID No. 2, Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 3, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 5, and It has a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO:

6. (B) Heavy chain CDR1 containing the amino acid sequence represented by Sequence ID No. 7, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 9, Light chain CDR1 containing the amino acid sequence represented by Sequence ID No. 10, A light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 11, and It has a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO:

12. (C) Heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 13, Heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 14, Heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 15, Light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 16, A light chain CDR2 containing the amino acid sequence represented by Sequence ID No. 17, and It has a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO:

18.

2. An anti-TCTP antibody that suppresses or inhibits the function of TCTP, wherein the anti-TCTP antibody or its antigen-binding fragment competitively inhibits the binding of the anti-TCTP antibody described in claim 1 to TCTP.

3. The anti-TCTP antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that it is a humanized antibody.

4. Fab, Fab', F (ab') 2 The antigen-binding fragment according to any one of claims 1 to 3, characterized in that it is Fv, a single-chain antibody, scFv, an scFv dimer, or dsFv.

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