Composition for suppressing accumulation of th1-treg in tumors

Abstract

A composition for suppressing the accumulation of helper T1 regulatory T cells in tumors, said composition containing an antibody that recognizes platelet factor 4 proteins as an active component. A drug composition for the treatment or prevention of cancer.

Description

Composition for suppressing accumulation of Th1-Treg in tumors

[0001] The present invention relates to a composition for suppressing the accumulation of T helper type 1 regulatory T cells (Th1-Treg) in tumors, and more particularly to a composition for suppressing said accumulation and activating anti-tumor immunity by using as an active ingredient an antibody that recognizes platelet factor 4 (PF4) protein or a functional fragment thereof. Furthermore, the present invention relates to a pharmaceutical composition for treating or preventing cancer, which contains as an active ingredient an antibody that recognizes PF4 protein or a functional fragment thereof. The present invention also relates to an antibody that recognizes PF4 protein or a functional fragment thereof.

[0002] The tumor microenvironment (TME) is composed of various types of cells, such as tumor cells, cancer stem cells, fibroblasts, innate immune cells, adaptive immune cells, and angiogenic cells. Among the adaptive immune cells in the TME, regulatory T cells (Tregs) are known to suppress antitumor immunity and promote tumor growth. Tregs are CD4+ T cells that play an important role in maintaining immune homeostasis. ＋ This is a special subset of T cells that expresses the Treg-specific transcription factor Foxp3.

[0003] Tumor-infiltrating Tregs are further polarized to the T helper 1 (Th1) type and express not only Foxp3 but also the Th1 lineage-determining transcription factor T-bet (encoded by the Tbx21 gene) (Non-patent Documents 1 and 2). ＋ T-bet ＋ These cells are also called Th1-type Tregs (Th1-Tregs) (Non-Patent Document 3). The present inventors have demonstrated that Th1-Tregs accumulate at higher levels in tumors compared to non-tumor tissues, and furthermore, that selective depletion of Th1-Tregs can reactivate anti-tumor immunity and inhibit tumor growth (Non-Patent Document 4). Thus, Th1-Tregs play a role in promoting tumors.

[0004] Immunotherapy has already been established as one of the standard treatments for tumors. Antibody preparations called checkpoint inhibitors are used as a means to activate this anti-tumor immunity, and neutralizing antibodies targeting PD-1 (and its ligand PD-L1) and CTLA4, which are brake molecules in the immune system, are widely used. However, one of the problems with these checkpoint inhibitors is that they can cause autoimmune diseases as a side effect.

[0005] As described above, the present inventors have demonstrated that the removal of Th1-Tregs, a subset of Tregs that act as brakes on immune cells, can potently activate anti-tumor immunity. Furthermore, they have found that this activation is not associated with autoimmune disease (Non-Patent Document 4). Although this suggests that selective removal of Th1-Tregs could be a novel cancer immunotherapy, inhibitors of Th1-Treg accumulation in tumor tissues that could achieve this goal have not yet been identified.

[0006] Furthermore, tumor-associated macrophages (TAMs) are innate immune cells found in the TME (Non-Patent Documents 5 and 6). It has been reported that TAMs recruit Tregs to the TME and suppress the function of anti-tumor T cells by secreting various cytokines and chemokines (Non-Patent Documents 7-9), suggesting a high correlation between TAMs and Tregs. Therefore, if the substances in the TME (e.g., cells such as TAMs, factors present in the TME, etc.) that contribute to the high abundance of Th1-Tregs in tumors were identified, it would be possible to suppress the accumulation of the above-mentioned Th1-Tregs by targeting these substances. However, nothing has been clarified about such target substances.

[0007] Kachler, K. et al. , Oncoimmunology 7, e1456612, doi:10.1080 / 2162402X. 2018.1456612 (2018). Santegoets, S. J. et al. , J Immunother Cancer 7, 14, doi:10.1186 / s40425-019-0497-0 (2019). Koch, M. A. et al. , Nat Immunol 10, 595-602, doi:10.1038 / ni. 1731 (2009). Okamoto, M. et al. , Cell Rep 42, 112813, doi:10.1016 / j. celrep. 2023.112813 (2023). Pittet, M. J. et al. , Nat Rev Clin Oncol 19, 402-421, doi:10.1038 / s41571-022-00620-6 (2022). Cassetta, L. &Pollard, J. W. , Nat Rev Cancer 23, 238-257, doi:10.1038 / s41568-022-00547-1 (2023). Qian, B. Z. &Pollard, J. W. , Cell 141, 39-51, doi:10.1016 / j. cell. 2010.03.014 (2010). Mantovani, A. et al. , Cancer Metastasis Rev 25, 315-322, doi:10.1007 / s10555-006-9001-7 (2006). Curiel, T. J. et al. , Nat Med 10, 942-949, doi:10.1038 / nm1093 (2004).

[0008] The present invention has been made in view of the problems associated with the prior art, and aims to provide a composition that identifies a substance that induces Th1-Treg in tumor tissue and targets the substance, thereby suppressing the accumulation of Th1-Treg in tumor tissue and thereby enabling the activation of anti-tumor immunity.

[0009] As a result of extensive research to achieve the above object, the present inventors have discovered a TAM (Arg1) that expresses arginase 1 (Arg1). ＋Furthermore, we have found that the Arg1 TAM is important for the high proportion of Th1-Tregs present in the TME. ＋ We also demonstrated that selective depletion of TAMs suppresses tumor growth and reduces the Th1-Treg ratio in the TME. ＋ The present inventors have also demonstrated that TAMs secrete platelet factor 4 (PF4) and polarize Tregs to Th1-Tregs in a CXCR3-dependent manner. Furthermore, they have successfully produced a monoclonal antibody specific to PF4 (anti-PF4 mAb (#6-1-5)), which has been shown to suppress the accumulation of Th1-Tregs in the TME and tumor growth, thereby completing the present invention.

[0010] That is, the present invention provides the following aspects.

[0011] [1] A composition for suppressing the accumulation of helper T1 regulatory T cells (Th1-Treg) in tumors, comprising as an active ingredient an antibody or a functional fragment thereof that recognizes platelet factor 4 (PF4) protein.

[0012] [2] The composition described in [1], which is a pharmaceutical composition for treating or preventing cancer.

[0013] [3] An antibody or functional fragment thereof recognizing PF4 protein, having the characteristics described in (1) or (2) below: (1) A heavy chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 7 to 9, and a light chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 15 to 17; (2) A heavy chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 10 to 12, and a light chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequence set forth in SEQ ID NO: 18, an amino acid sequence consisting of Leu-Ala-Ser, and the amino acid sequence set forth in SEQ ID NO: 19.

[0014] [4] The antibody or functional fragment thereof according to [3], wherein the heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 6, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 6, and at least one of the amino acid sequences of SEQ ID NO: 6, in which one or several amino acids have been substituted, deleted, added, and / or inserted; and the light chain variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 14, an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 14, and at least one of the amino acid sequences of SEQ ID NO: 14, in which one or several amino acids have been substituted, deleted, added, and / or inserted.

[0015] [5] A composition for suppressing the accumulation of Th1-Treg in a tumor, comprising the antibody or functional fragment thereof according to [3] or [4] as an active ingredient.

[0016] [6] The composition described in [5], which is a pharmaceutical composition for treating or preventing cancer.

[0017] The present invention also provides the following aspects.

[0018] [7] Use of an antibody that recognizes PF4 protein or a functional fragment thereof for producing a composition for suppressing the accumulation of Th1-Treg in a tumor, or a pharmaceutical composition for treating or preventing cancer.

[0019] [8] Use of the antibody or functional fragment thereof according to [3] or [4] for producing a composition for suppressing the accumulation of Th1-Treg in a tumor, or a pharmaceutical composition for treating or preventing cancer.

[0020] [9] Use of an antibody that recognizes PF4 protein or a functional fragment thereof for suppressing the accumulation of Th1-Treg in tumors, or for treating or preventing cancer.

[0021]

[10] Use of the antibody or functional fragment thereof according to [3] or [4] for suppressing the accumulation of Th1-Treg in a tumor, or for treating or preventing cancer.

[0022]

[11] An antibody or a functional fragment thereof that recognizes PF4 protein, for suppressing the accumulation of Th1-Treg in tumors, or for treating or preventing cancer.

[0023]

[12] The antibody or functional fragment thereof according to [3] or [4], for suppressing the accumulation of Th1-Treg in tumors or for treating or preventing cancer.

[0024]

[13] A method for suppressing the accumulation of Th1-Treg in tumors or treating or preventing cancer, comprising administering to a subject an effective amount of an antibody that recognizes PF4 protein or a functional fragment thereof.

[0025]

[14] A method for suppressing the accumulation of Th1-Treg in a tumor or treating or preventing cancer, comprising administering to a subject an effective amount of the antibody or functional fragment thereof according to [3] or [4].

[0026] The amino acid sequence of SEQ ID NO: 6 represents the amino acid sequence of the heavy chain variable region of anti-PF4 mAb (#6-1-5) (encoded by the DNA sequence of SEQ ID NO: 5); the amino acid sequences of SEQ ID NOs: 7 to 9 represent the amino acid sequences of complementarity determining regions (CDRs) 1 to 3 identified by Kabat from the amino acid sequence of the heavy chain variable region; the amino acid sequences of SEQ ID NOs: 10 to 12 represent the amino acid sequences of CDRs 1 to 3 identified by IMGT from the amino acid sequence of the heavy chain variable region; the amino acid sequence of SEQ ID NO: 14 represents the amino acid sequence of the light chain variable region of anti-PF4 mAb (#6-1-5) (encoded by the DNA sequence of SEQ ID NO: 13); and the amino acid sequences of SEQ ID NOs: 15 to 17 represent the amino acid sequences of CDRs 1 to 3 identified by Kabat from the amino acid sequence of the light chain variable region; The amino acid sequence of SEQ ID NO: 18, the amino acid sequence consisting of Leu-Ala-Ser (an amino acid sequence consisting of leucine, alanine, and serine in order from the N-terminus), and the amino acid sequence of SEQ ID NO: 19 each represent the amino acid sequences of CDR1 to CDR3 identified by IMGT from the amino acid sequence of the light chain variable region.

[0027] According to the present invention, it is possible to suppress the accumulation of Th1-Treg in tumor tissue, activate anti-tumor immunity, and ultimately treat or prevent cancer. Furthermore, according to the present invention, by selectively suppressing Th1-Treg, cancer immunotherapy that is not accompanied by the onset of autoimmune diseases becomes possible.

[0028] Arg1, produced using VeDTR ＋ Tumor-specific macrophages (Arg1 ＋ This figure shows an overview of TAM (yellow fluorescent protein)-targeted mice. The upper part shows an overview of the process for generating Cx3cr1-Cre / Arg1-Flp / VeDTR (LF) mice, which enable cross-expression of Cx3cr1-dependent and Arg1-dependent YFP (yellow fluorescent protein) and DTR (diphtheria toxin receptor). The lower part shows an overview of cross-expression of Cre / Flp-dependent YFP and DTR in Cx3cr1-Cre / Arg1-Flp / VeDTR (LF) mice. As shown in the figure, expression of Cre recombinase is controlled by the promoter of the Cx3cr1 gene. Expression of Flp recombinase is controlled by the promoter of the Arg1 gene. Cx3cr1 and Arg1 are marker genes for TAM. When Cre recombinase and Flp recombinase are expressed, two transcription termination sequences located between the sequences recognized by these recombinases are removed, allowing the expression of DTR and YFP downstream. Furthermore, when DT (diphtheria toxin) is administered to the mice, death is induced in cells expressing DTR in a Cre / Flp-dependent manner. YFP expression in macrophages of each tissue (brain, lung, liver, spleen, or tumor) of Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice (n=5) subcutaneously inoculated with MC38 cells (a mouse colon cancer cell line) was measured. ＋Graphs showing the percentage of cells. In Figures 1B to 1F, data are presented as mean ± SEM and are pooled from two to three independent experiments. Statistical analysis was performed using one-way analysis of variance followed by Dunnett's multiple comparison test (Figure 1B) and two-tailed Student's t-test (Figures 1D to 1F). ** indicates P<0.01, *** indicates P<0.001, *** indicates P<0.0001, and ns indicates not significant. Photographs showing the results of immunohistochemical analysis of tissue sections (tumor, spleen, or liver sections) from Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice subcutaneously inoculated with MC38 cells. Representative images of tissue sections stained with anti-CD11b antibody, anti-F4 / 80 antibody, or anti-DTR antibody, or a composite of these, are shown. Intratumoral CD45 expression in Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice subcutaneously inoculated with MC38 cells and treated with PBS or DT (n=6 per group). ＋ CD11b ＋ Ly6G － The results of flow cytometry analysis of TAM cells (YFP in TAM cells) ＋ 1 is a graph showing the percentage of CD4+ cells in the tumor-infiltrating Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice subcutaneously inoculated with MC38 cells and treated with PBS or DT. 2 is a graph showing the percentage of CD4+ cells in the tumor-infiltrating Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice subcutaneously inoculated with MC38 cells and treated with PBS or DT. 3 is a graph showing the percentage of CD4+ cells in the tumor-infiltrating Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice subcutaneously inoculated with MC38 cells and treated with PBS or DT (each group, n=3). ＋ Flow cytometry analysis of T cells (tumor-infiltrating CD4 ＋Figures 2A-2E show graphs showing the percentage of Foxp3 and T-bet double-positive T cells in MC38 cells. In Figures 2A-2E, n=3 results are shown for each group. Data are presented as mean ± SEM and are pooled from 2-3 independent experiments. Statistical analysis was performed using a two-tailed Student's t-test (Figures 2A, D, and E). * indicates P<0.05, ** indicates P<0.01, and ns indicates not significant. CD45 expression in Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice transplanted with MC38 cells ＋ CD11b － This is a dot plot showing the results of CyTOF analysis of cells, in which 19,356 CD45 ＋ CD11b － The t-SNE plot of cells is shown in Figure 2B. The tSNE plot and contour plot are shown for the results shown in Figure 2B, divided into mice in the PBS state and mice in the DT state. The area surrounded by the dashed line in the figure indicates a cell population with a statistical difference. The CD4 ＋ Foxp3 ＋ T-bet ＋ Graph showing the results of flow cytometry analysis of tumor-infiltrating T cells derived from Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice transplanted with MC38 cells. ＋ (left side of the figure) and CD8 ＋ (Right side of the figure) The percentage of IFN-γ and TNF-α double-positive T cells is shown. RFP derived from MC38-bearing Arg1-RFP mice ＋ or RFP － CD45 ＋ CD11b ＋ Ly6G － Cell (Arg1 ＋ TAM or Arg1 － YFP in the spleen of MC38-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice directly co-cultured with TAM － CD4 ＋CD25 ＋ Graph showing the results of flow cytometry analysis of cells. YFP pooled from three independent experiments. ＋ The percentage of cells is shown. Data are presented as mean ± SEM and are pooled from 2 to 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test (Figures 3A and 3J), two-way ANOVA test (Figures 3B, 3D, and 3F), and two-tailed Student's t-test (Figures 3G to 3I). * indicates P<0.05, ** indicates P<0.01, and ns indicates not significant. RFP derived from Arg1-RFP mice containing MC38 or B16F10 ＋ or RFP － CD45 ＋ CD11b ＋ Ly6G － Cell (Arg1 ＋ TAM or Arg1 － YFP in the spleens of Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice indirectly co-cultured with TAM in B16F10-containing transwell plates. － CD4 ＋ CD25 ＋ Graph showing the results of flow cytometry analysis of cells. YFP pooled from three independent experiments. ＋ The percentage of cells derived from Arg1-RFP mice bearing MC38 or B16F10 (n=3 for each group) is shown. ＋ or RFP － CD45 ＋ CD11b ＋ Ly6G － Cell (Arg1 ＋ TAM or Arg1 － Figure 1 shows genes that are differentially expressed between MC38 mice and TAMs. ＋ 145 up-regulated genes (fold change > 1.5 and p value < 0.05) in TAM and RFP from B16F10 mice ＋The genes overlapped with the 82 up-regulated genes in TAM (fold change > 1.5 and p value < 0.05). YFP derived from the spleen of B16F10-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice stimulated with each protein (rPF4 or rPPBP) shown in the figure. － CD4 ＋ CD25 ＋ Graph showing the results of flow cytometry analysis of cells. YFP pooled from three independent experiments. ＋ Figure 1 shows the percentage of cells in the TAM and SPM. Figure 2 shows t-SNE plots showing Pf4 mRNA expression in TAM and SPM. Figure 3 shows graphs showing the results of quantitative RT-PCR (q-PCR) analysis of Pf4 mRNA levels in macrophages of each tissue in wild-type mice bearing MC38. Macrophages in each tissue were identified by the expression of cell surface markers (brain (microglia): Cx3cr1 ＋ , P2RY12 ＋ , Lung (alveolar macrophages): CD11c ＋ , SiglecF ＋ , liver (Kupffer cells): CD11b ｌｏｗ , F4 / 80 ＋ , splenic macrophages: CD11b ＋ , Ly6G － , tumor macrophages: CD11b ＋ , Ly6G － ). RFP from MC38-bearing Arg1-RFP mice (n=3) － TAM or RFP ＋ TAM (CD45 ＋ , CD11b ＋ , Ly6G － 1 is a graph showing the results of measuring the PF4 concentration in the culture supernatant of B16F10-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice by ELISA. － CD4 ＋ CD25 ＋ Carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4 cells co-cultured with PF4-stimulated cells ＋1 is a graph showing the results of flow cytometry analysis of T cells (Tconv). YFP pooled from three independent experiments. ＋ The percentage of YFP cells derived from the spleen of B16F10-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice stimulated with PF4 is shown. － CD4 ＋ CD25 ＋ 1 is a graph showing the results of flow cytometry analysis of the expression (mean fluorescence intensity (MFI)) of each surface marker (CD25, GITR, or CTLA-4) in cells. － CD4 ＋ CD25 ＋ 1 is a graph showing the results of flow cytometry analysis of cells stimulated with PF4, AMG487, or PF4 and AMG487. ＋／＋ Mouse or Arg1-RFP / Pf4 －／－ RFP in tumors of mice (n=4 per group) － or RFP ＋ CD45 ＋ CD11b ＋ Ly6G － 4A and 4B are graphs showing the results of measuring the concentration of PF4 in the cell culture supernatant by ELISA. Data are shown as mean ± SEM and are pooled from 2 to 3 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test (FIG. 4A) and two-tailed Student's t-test (FIGS. 4B to 4E). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates not significant. MC38-loaded Arg1-RFP / Pf4 ＋／＋ Mouse or Arg1-RFP / Pf4 －／－ 1 is a graph showing the change in tumor volume over time in mice. ＋／＋ Mouse or Arg1-RFP / Pf4 －／－1 is a graph showing the change in tumor volume over time in mice. ＋／＋ Mouse or Arg1-RFP / Pf4 －／－ In mice (n=4-5 per group), CD4 derived from each tissue (tumor or spleen) was ＋ T cells were analyzed by flow cytometry (CD4 ＋ 1 is a graph showing the percentage of Foxp3 and T-bet double positive cells in T cells. ＋／＋ Mouse or Arg1-RFP / Pf4 －／－ In mice (n=4-5 per group), CD4 derived from each tissue (tumor or spleen) was ＋ T cells were analyzed by flow cytometry (CD4 ＋ 1 is a graph showing the percentage of Foxp3 and T-bet double positive cells in T cells. YFP T cells derived from the spleens of B16F10-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice stimulated with PF4 or anti-PF4 monoclonal antibody (mAb). － CD4 ＋ CD25 ＋ Graph showing the results of flow cytometry analysis of cells. YFP pooled from three independent experiments. ＋The percentage of cells is shown. In the figure, "#6-1-5" indicates the anti-PF4 mAb developed by the present inventors, and "com" indicates a commercially available anti-PF4 mAb. Data are presented as mean ± SEM and are pooled from two to three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test (Figure 5A) and two-tailed Student's t-test (Figures 5B-5G). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and ns indicates not significant. This graph shows the time course of tumor volume in B16F10- or MC38-bearing wild-type mice injected with anti-PF4 mAb or its isotype control mAb. mAb injections were initiated 3 days after tumor inoculation. B16F10-implanted wild-type mice (n=3) were injected with anti-PF4 mAb or its isotype control mAb, and CD4 cells derived from each tissue (tumor or spleen) were detected. ＋ T cells were analyzed by flow cytometry (CD4 ＋ MC38-engrafted wild-type mice (n=3) were injected with anti-PF4 mAb or its isotype control mAb, and CD4 cells derived from each tissue (tumor or spleen) were analyzed. ＋ T cells were analyzed by flow cytometry (CD4 ＋ B16F10-engrafted wild-type mice (n=3-5) were injected with anti-PF4 mAb or its isotype control mAb, and CD4 cells derived from each tissue (tumor or spleen) were analyzed. ＋ T cells or CD8 ＋ 1 is a graph showing the results of flow cytometry analysis of T cells (the proportion of IFN-γ and TNF double-positive cells in each T cell group). MC38-engrafted wild-type mice (n=3-5) were injected with anti-PF4 mAb or its isotype control mAb, and CD4 cells derived from each tissue (tumor or spleen) were analyzed. ＋ T cells or CD8 ＋1 is a graph showing the results of flow cytometry analysis of T cells (the percentage of IFN-γ and TNF double-positive cells in each T cell group). －／－ Figure 6B shows a graph depicting the time course of tumor volume in mice. Injection of each mAb was initiated 3 days after tumor inoculation. Figure 6C shows a dot plot depicting the results of a flow cytometric test of the affinity of anti-PF4 mAb (#6-1-5) for mouse PF4, mouse CXCL9, mouse CXCL10, CXCL11, or human PF4. Data are representative of three independent experiments. Figure 6D shows a graph depicting the time course of tumor volume in wild-type mice bearing B16F10 or MC38 tumors injected with anti-PF4 mAb or its isotype control mAb. Injection of each mAb was initiated 10 days after tumor inoculation. Data are presented as mean ± SEM and are pooled from three independent experiments. Statistical analysis was performed using a two-tailed Student's t-test (Figures 6B-6D). * indicates P<0.05, ** indicates P<0.01, and ns indicates no significance. CD4 in B16F10 transplanted Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice (n=4-5) ＋ CD45 ＋ 1 is a graph showing the results of flow cytometry analysis of cells. ＋ YFP in T cells ＋ The percentage of CD4+ cells in MC38-implanted Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice (n=4-5) is shown. ＋ CD45 ＋ 1 is a graph showing the results of flow cytometry analysis of cells. ＋ YFP in T cells ＋Figure 7 shows the percentage of CD4+ cells in wild-type mice injected with anti-PF4 mAb or its isotype control mAb. The injection of each mAb was initiated on day 3 after tumor inoculation. Data are shown as mean ± SEM and are pooled from three independent experiments. Statistical analysis was performed using a two-tailed Student's t-test (Figures 7A-7D). ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001, and ns indicates not significant. Splenic CD4+ cells were measured in wild-type mice (n=6-8 per group) 12 days after the first injection of anti-PF4 mAb or its isotype control mAb. ＋ T cells or CD8 ＋ CD44 on T cells ｈｉｇｈ or CD62L ｌｏｗ 1 is a graph showing the results of flow cytometry quantification of the percentage of cells. 2 is a graph showing the time course of body weight in Foxp3-Cre / VeDTR(ΔFRT) mice injected with PBS or DT. 3 is a graph showing the splenic CD4 ＋ T cells or CD8 ＋ CD44 on T cells ｈｉｇｈ or CD62L ｌｏｗ1 is a graph showing the results of quantification of the proportion of PF4 cells by flow cytometry. This is a Kaplan-Meier curve showing the survival time of patients with high PF4 expression and patients with low PF4 expression, based on TCGA (The Cancer Genome Atlas) pan-cancer data analysis. Statistical analysis was performed using the log-rank test. ***P<0.0001. This is a graph showing the results of quantification of the proportion of PF4 cells by flow cytometry. This is a Kaplan-Meier curve showing the survival time of patients with low PF4 expression and low CD11b (ITGAM) expression, patients with low PF4 expression and high CD11b expression, patients with high PF4 expression and low CD11b expression, and patients with high PF4 expression and high CD11b expression, based on TCGA pan-cancer data analysis. Statistical analysis was performed using the log-rank test. ***P<0.0001. 9A is a graph showing the results of differential analysis of the deuteration level in a complex of anti-PF4 mAb (#6-1-5) and a mouse PF4 peptide fragment by hydrogen-deuterium exchange mass spectrometry (HDX-MS). In the figure, the vertical axis indicates the difference in deuteration level, and the horizontal axis indicates the number of the mouse PF4 peptide fragment. This figure shows the position of each peptide fragment shown in FIG. 9A in mouse PF4. In the figure, the numbers (23 to 33) shown on the left side are the numbers of each peptide fragment and correspond to the horizontal axis of FIG. 9A. The numbers (62 to 105) shown at the top indicate the numbers of the amino acid positions in mouse PF4. This is a heat map showing the deuteration level in a complex of anti-PF4 mAb (#6-1-5) and a mouse PF4 peptide fragment. In the figure, "mP4" and "mPF4 + mAb (#6-1-5)" indicate the results of analyzing only the antigen sample and the antigen-antibody complex sample, respectively, and "Difference (complex-antigen)" indicates the difference in the analytical results between the two samples. This graph shows the results of differential analysis of the deuteration level in a complex of anti-PF4 mAb (com-anti-PF4) and mouse PF4 peptide fragments by HDX-MS. In the figure, the vertical axis indicates the difference in deuteration level, and the horizontal axis indicates the number of the mouse PF4 peptide fragment. This figure shows the position in mouse PF4 of each peptide fragment shown in Figure 10A. In the figure, the numbers (1 to 6) shown on the left side are the numbers of each peptide fragment and correspond to the horizontal axis of Figure 9A. The numbers (31 to 64) shown at the top indicate the numbers of the amino acid positions in mouse PF4.1 is a heat map showing the deuteration levels in a complex between anti-PF4 mAb (com-anti-PF4) and a mouse PF4 peptide fragment. In the figure, "mP4" and "mPF4+com-anti-PF4" represent the results of analyzing an antigen sample alone and an antigen-antibody complex sample, respectively, and "Difference (complex-antigen)" represents the difference in the analysis results between the two samples.

[0029] As shown in the Examples below, the present inventors have demonstrated that antibodies that recognize platelet factor 4 protein can suppress the accumulation of T helper type 1 regulatory T cells in tumors and activate anti-tumor immunity, thereby suppressing tumor growth. Thus, the present invention relates to a composition for suppressing the accumulation of T helper type 1 regulatory T cells in tumors, which comprises an antibody that recognizes platelet factor 4 protein or a functional fragment thereof as an active ingredient.

[0030] (Platelet Factor 4 Protein) In the present invention, the target "platelet factor 4 (PF4, PF-4)" is a humoral factor (cytokine) belonging to the CXC chemokine family, and is also called chemokine (C-X-C motif) ligand 4 (CXCL4) or SCYB4. If it is derived from a human, it is typically a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (a protein encoded by the DNA sequence set forth in SEQ ID NO: 1). More specifically, examples include a protein identified by UniProt ID: P02776, and a protein consisting of the amino acid sequence set forth in NCBI Reference Sequence: NP_002610 or NP_001350281 (a protein encoded by a DNA sequence specified by NCBI Reference Sequence: NM_002619 or NM_001363352). A typical example of a protein derived from a mouse is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 (a protein encoded by a DNA sequence set forth in SEQ ID NO: 3). More specifically, examples include a gene encoding a protein identified by UniProt ID: Q9Z126 and a protein consisting of the amino acid sequence set forth in NCBI Reference Sequence: NP_064316 (a protein encoded by a DNA sequence identified by NCBI Reference Sequence: NM_019932). However, the amino acid sequence of a protein can mutate in nature (i.e., non-artificially) due to mutations in the DNA sequence (gene) that encodes it. Therefore, in the present invention, such natural mutants can also be targets.

[0031] (Anti-PF4 Antibody) In the present invention, the term "antibody recognizing PF4 protein" refers to an antibody that recognizes PF4 protein, i.e., an antibody having binding activity to PF4 protein. The antibody according to the present invention may be any antibody that recognizes PF4 protein, but is preferably one that can inhibit binding of PF4 protein to its receptor, CXCR3 protein (i.e., has neutralizing activity). From this perspective, the antibody according to the present invention is more preferably an antibody that has binding activity to a peptide containing a binding site or binding groove for CXCR3 protein. With respect to the amino acid sequence of SEQ ID NO: 2, for example, an antibody having binding activity to a peptide consisting of the amino acid sequence of positions 32 to 101 is included, an antibody having binding activity to a peptide consisting of the amino acid sequence of positions 44 to 82 is preferred, and as shown in the Example (anti-PF4 mAb (#6-1-5)) described below, an antibody having binding activity to a peptide consisting of the amino acid sequence of positions 68 to 82 is even more preferred. The proline at position 68 and the isoleucine at position 82 of the human PF4 protein (amino acid sequence set forth in SEQ ID NO: 2) correspond to the alanine at position 72 and the isoleucine at position 86, respectively, of the mouse PF4 protein (amino acid sequence set forth in SEQ ID NO: 4). Antibodies having binding activity to the binding groove of the CXCR3 protein (at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of positions 44, 45, 47, 50, 54, 73, 76, 80, and 82) are more preferred, and antibodies having binding activity to at least one amino acid (e.g., 1, 2, 3, or 4) of positions 73, 76, 80, and 82 are even more preferred. On the other hand, it is even more desirable for the antibodies of the present invention to lack binding activity to a peptide consisting of the amino acid sequence of positions 32 to 51. The "position" in the amino acid sequence set forth in SEQ ID NO: 2 refers to the order in which the first methionine is placed, with the first position being 1 (the same applies hereinafter).

[0032] As described above, the antibody of the present invention preferably has neutralizing activity. Here, "neutralizing activity" includes not only the activity of inhibiting the binding of PF4 protein and CXCR3 protein, but also the activity of inhibiting downstream signal transduction induced by said binding. Examples of such downstream signal transduction include activation of G protein (α, β, γ subunits), PLCβ, or IP3, increase in intracellular calcium ions, activation of PI3 kinase or JAK kinase, and the STAT pathway.

[0033] As shown in the Examples below (anti-PF4 mAb (#6-1-5)), preferred antibodies of the present invention are those that recognize amino acids conserved between humans and mice in the PF4 protein. Examples of such antibodies include antibodies that have binding activity to at least one amino acid selected from positions 34 to 39, 41 to 48, 53 to 64, 66, 67, 71 to 85, and 87 to 100, more preferably antibodies that have binding activity to at least one amino acid selected from positions 71 to 85 and 87 to 100, and even more preferably antibodies that have binding activity to at least one amino acid selected from positions 71 to 82 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). Furthermore, from the viewpoint of having binding activity toward a peptide containing the binding site or binding groove for the CXCR3 protein, preferred antibodies have binding activity toward at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the amino acids at positions 44 to 48, 53 to 64, 66, 67, and 71 to 82, more preferred antibodies have binding activity toward at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the amino acids at positions 44, 45, 47, 54, 73, 76, 80, and 82, and even more preferred antibodies have binding activity toward at least one (e.g., 1, 2, 3, or 4) of the amino acids at positions 73, 76, 80, and 82. On the other hand, it is more desirable for the antibodies according to the present invention not to have binding activity toward a peptide consisting of the amino acid sequence of positions 32 to 51.

[0034] The antibody of the present invention is preferably an antibody having specific binding activity to the PF4 protein. Examples of such antibodies include antibodies that do not bind to at least one molecule of the PF4 protein family (e.g., CXCL9, CXCL10, CXCL11, and other ligands of the CXCR3 protein other than PF4). Those skilled in the art can analyze whether an antibody has such binding activity using immunological techniques. For example, in antigen analysis by flow cytometry, if the peak or dot plot derived from the test antibody is shifted toward a stronger fluorescence intensity compared to the peak or dot plot derived from its isotype control antibody, it can be determined that the test antibody (substantially) binds to the antigen. On the other hand, if no such shift is observed, it can be determined that the test antibody (substantially) does not bind to the antigen.

[0035] The binding affinity of the antibody of the present invention is Ｄ (dissociation constant) is 10 －７ Preferably, it is 10 －８ It is more preferable that K is equal to or less than K. Ｄ is calculated from ka (association rate constant) and kd (dissociation rate constant) (K Ｄ = kd / ka). Ka and kd are rate constants in the binding / dissociation reaction between two molecules, and can be determined, for example, by measurement using surface plasmon resonance (SPR). SPR measurement of the binding between an antibody and an antigen is well known, and a person skilled in the art can determine the k and k of an antibody based on such well-known techniques, and further, K Ｄ can be calculated.

[0036] The antibody of the present invention is also preferably an antibody that does not have the ability to activate platelets. Here, the platelet activation ability includes, for example, the ability to enhance the expression of a platelet activation marker, more specifically, the ability to enhance the expression of at least one platelet activation marker selected from β-TG, CD62P (P-selectin), CD63, PAC-1, annexin V, sCD40L, sP-selectin, sGPV, sGPIb (glycocalicin), and PDMP.

[0037] The term "antibody" as used herein includes all classes and subclasses of immunoglobulins. "Antibody" includes polyclonal and monoclonal antibodies. A "polyclonal antibody" is an antibody preparation containing different antibodies directed against different epitopes. A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. In contrast to polyclonal antibodies, monoclonal antibodies recognize a single determinant on an antigen. The antibody of the present invention is preferably a monoclonal antibody. The antibody of the present invention is an antibody that has been separated and / or recovered (i.e., isolated) from components of its natural environment.

[0038] The antibody according to the present invention is not particularly limited in terms of origin, type, shape, etc., as long as it can recognize the PF4 protein. Specific examples include antibodies derived from non-human animals (e.g., antibodies derived from mice, rabbits, rats, and camelids (VHH (variable domain of heavy chain antibodies) antibodies, also known as nanobodies)), antibodies derived from humans, chimeric antibodies, and humanized antibodies. When the antibody according to the present invention is administered to humans as a therapeutic or prophylactic agent, chimeric antibodies, humanized antibodies, or human antibodies are preferred from the viewpoint of reducing side effects.

[0039] In the present invention, an example of a "non-human animal-derived antibody" is anti-PF4 mAb (#6-1-5) as shown in the Examples below. Antibodies according to the present invention also include: antibodies that contain the heavy chain variable region and light chain variable region of anti-PF4 mAb (#6-1-5); and antibodies that contain heavy chain complementarity-determining regions (CDRs) 1 to 3 determined from the heavy chain variable region of anti-PF4 mAb (#6-1-5) and light chain CDRs 1 to 3 determined from the light chain variable region of anti-PF4 mAb (#6-1-5). Regarding such antibodies, the method for determining CDRs based on the amino acid sequences of the variable regions is not particularly limited, and examples include known numbering schemes such as Kabat, Chothia, IMGT, and Aho.

[0040] More specifically, such antibodies according to the present invention include the antibodies described in any one of (1) to (3) below. The amino acid sequences of SEQ ID NOs: 6 to 13 and 14 to 19 and the amino acid sequence consisting of Leu-Ala-Ser for anti-PF4 mAb (#6-1-5) are as described above. (1) An antibody that recognizes PF4 protein, having a heavy chain variable region comprising CDR1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 7 to 9, and a light chain variable region comprising CDR1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 15 to 17. (2) An antibody that recognizes PF4 protein, having a heavy chain variable region comprising CDR1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 10 to 12, and a light chain variable region comprising CDR1 to 3 each comprising the amino acid sequence set forth in SEQ ID NO: 18, an amino acid sequence consisting of Leu-Ala-Ser, and the amino acid sequence set forth in SEQ ID NO: 19. (3) An antibody whose heavy chain variable region comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 6, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 6, and an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any of the amino acid sequences set forth in SEQ ID NO: 6, An antibody that recognizes PF4 protein, wherein the light chain variable region comprises one amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 14, an amino acid sequence having 80% or more homology to the amino acid sequence set forth in SEQ ID NO: 14, and an amino acid sequence in which one or several amino acids have been substituted, deleted, added, and / or inserted in at least any one of the amino acid sequences set forth in SEQ ID NO: 14.

[0041] In the present invention, the number of amino acids to be substituted, deleted, added, and / or inserted, i.e., modified (the several amino acids), is not particularly limited as long as the antibody has activity equivalent to that of the antibody before modification, but is preferably 10 amino acids or less, more preferably 5 amino acids or less, and most preferably 3 amino acids or less (e.g., 2 amino acids or less, 1 amino acid or less). The amino acid modification is preferably a conservative substitution. In the present invention, "conservative substitution" means substitution with another similar amino acid residue (having a chemically similar side chain). Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention pertains. For example, amino acids can be classified into acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxyl groups (serine, threonine), amino acids containing sulfur (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).

[0042] Furthermore, antibodies containing a variable region containing an amino acid sequence that, after modification, has 80% or more identity or homology at the amino acid sequence level with a variable region consisting of the above-mentioned specific amino acid sequence are also included in the antibodies of the present invention, as long as they have activity equivalent to that of the antibody before modification. Such identity or homology should be at least 80%, but is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more). "Identity" refers to the percentage of sites in which the amino acid types are identical between the amino acid sequences being compared, and "homology" refers to the percentage of sites in which similar amino acids are identical, plus the percentage of sites in which similar amino acids are identical. Sequence homology or identity can be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing an amino acid sequence using BLASTP, the parameters are, for example, score = 50 and wordlength = 3. Furthermore, when analyzing an amino acid sequence using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known.

[0043] From the viewpoint of minimizing the effect on antigen-binding ability, such modifications in the antibody variable region are preferably made to a region other than the CDR, i.e., the FR. More specifically, the antibody described in (3) above preferably further has the characteristic of (1) or (2) above. The antigen-binding activity can be evaluated by analysis using, for example, a flow cytometer, ELISA, Western blotting, immunoprecipitation, or the like.

[0044] Furthermore, the antibodies of the present invention contain signal sequences at the N-terminus of the amino acid sequences of SEQ ID NOs: 6 and 14. The variable regions of the antibody described in (3) above may be those excluding the signal sequence, and may further have the characteristics of (1) or (2) above.

[0045] In the present invention, a "chimeric antibody" refers to an antibody in which the variable region of one antibody is linked to the constant region of a heterologous antibody. Chimeric antibodies can be obtained, for example, by immunizing a mouse with an antigen, excising the antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, ligating it to an antibody constant region (constant region) gene derived from human bone marrow, incorporating it into an expression vector, and introducing it into a host for production (e.g., JP-A-8-280387, U.S. Pat. No. 4,816,397, U.S. Pat. No. 4,816,567, U.S. Pat. No. 5,807,715).

[0046] The constant region of a chimeric antibody is usually derived from a human antibody. For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε can be used as the heavy chain constant region. Furthermore, Cκ and Cλ can be used as the light chain constant region. The amino acid sequences of these constant regions and the nucleotide sequences encoding them are known. Furthermore, to improve the stability of the antibody itself or the stability of antibody production, one or several amino acids in the human antibody constant region can be substituted, deleted, added, and / or inserted.

[0047] In the present invention, a "humanized antibody" refers to an antibody in which the gene sequence of the antigen-binding region (CDR) of a non-human antibody has been grafted onto a human antibody gene (CDR grafting). Methods for producing such antibodies include known overlap extension PCR methods (see, for example, EP 239400, EP 125023, WO 90 / 07861, and WO 96 / 02576). The variable region of an antibody typically consists of three CDRs sandwiched between four FRs. CDRs essentially determine the binding specificity of an antibody. While the amino acid sequences of CDRs are highly diverse, the amino acid sequences constituting FRs often show high homology or identity even between antibodies with different binding specificities. Therefore, it is generally believed that CDR grafting can transfer the binding specificity of one antibody to another. Furthermore, from the viewpoint of maintaining the function of the CDR, when grafting a non-human CDR onto a human FR, a human FR having high homology or identity to the non-human animal-derived FR is selected. That is, since the amino acids in the CDR not only recognize the antigen but also coordinate with the amino acids in the FR adjacent to the CDR and are involved in maintaining the loop structure of the CDR, it is preferable to use a human FR having an amino acid sequence having high homology or identity to the amino acid sequence of the FR adjacent to the CDR to be grafted.

[0048] A search for known human FRs that are highly homologous or identical to non-human animal-derived FRs can be performed, for example, using an antibody-specific search system available on the Internet (http: / / www.bioinfo.org.uk / abysis / ). Mutations can be introduced into sequences other than the CDRs of the non-human antibody so that they match the sequence of the human FR obtained in this manner. Alternatively, if a gene (cDNA) encoding the amino acid sequence of the human FR obtained by the search is available, the non-human CDRs can be introduced into that sequence. Introduction of mutations, etc., can be performed using techniques known in the art, such as nucleic acid synthesis and site-directed mutagenesis.

[0049] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody thus prepared, it is possible to suitably select FRs of a human-derived antibody that form a good antigen-binding site when linked via the CDRs. Furthermore, if necessary, amino acid residues in the FRs can be substituted so that the CDRs of the humanized antibody form a suitable antigen-binding site, according to the method described in Sato, K. et al., Cancer Res, 1993, 53, 851-856, etc., and mutant FR sequences with desired properties can be selected by measuring and evaluating the antigen-binding activity of mutant antibodies with the amino acid substitutions.

[0050] In the present invention, a "human antibody" is an antibody in which all regions are derived from humans. In producing human antibodies, it is possible to use transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies upon immunization. Techniques for producing human antibodies are known (e.g., Nature, 1993, vol. 362, pp. 255-258; Intern. Rev. Immunol, 1995, vol. 13, pp. 65-93; J. Mol. Biol, 1991, vol. 222, pp. 581-597; Nature Genetics, 1997, Vol. 15, pp. 146-156; Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, pp. 722-727; JP-A Nos. 10-146194, 10-155492, Japanese Patent Nos. 2938569, 11-206387, JP-A Nos. 8-509612 and 11-505107).

[0051] Furthermore, antibodies that recognize the PF4 protein include antibodies whose amino acid sequence has been modified without reducing desired activities (such as binding activity to the PF4 protein, neutralizing activity, activity to suppress accumulation of T helper 1-type regulatory T cells in tumors, and / or other biological properties). Amino acid sequence variants can be produced by introducing mutations into DNA encoding the antibody chain or by peptide synthesis. The site at which the amino acid sequence of the antibody is modified may be the constant region of the heavy or light chain of the antibody, or may be the variable region (framework region and CDRs), as long as the antibody has activity equivalent to that of the antibody before modification. Although modification of amino acids other than those in the CDRs is thought to have a relatively small effect on the binding affinity to the antigen, currently, techniques are known in which antibodies with enhanced affinity to the antigen are obtained by modifying amino acids in the CDRs (PNAS, 2005, Vol. 102, pp. 8466-8471; Protein Engineering, Design & Selection, 2008, Vol. 21, pp. 485-493; WO2002 / 051870; J. Biol. Chem., 2005, Vol. 280, pp. 24880-24887; Protein Engineering, Design & Selection, 2008, Vol. 21, pp. 345-351). Currently, it is also possible to model antibodies with enhanced antigen affinity by utilizing an integrated computational chemistry system (e.g., Molecular Operating Environment, manufactured by CCG, Canada) (see, for example, http: / / www.rsi.co.jp / kagaku / cs / ccg / products / application / protein.html). Furthermore, as described in Protein Eng Des Sel. 2010 Aug;23(8):643-51, there are known examples in which CDR1 of the heavy chain variable region and CDR3 of the light chain variable region are not involved in antigen affinity. Similarly, Molecular Immunology 44:1075-1084 (2007) reports that in most antibodies, CDR2 of the light chain variable region is not involved in the affinity to the antigen.Thus, the affinity of an antibody to an antigen can be equivalently exerted without requiring all of CDRs 1 to 3 from each of the heavy chain variable region and the light chain variable region. In fact, Biochem Biophys Res Commun. 2003 Jul 18; 307(1): 198-205, J Mol Biol. 2004 Jul 9; 340(3): 525-42, and J Mol Biol. 2003 Aug 29; 331(5): 1109-20 have reported examples in which antigen affinity was maintained by retaining at least one CDR of the original antibody.

[0052] Furthermore, in the present invention, for the purpose of increasing antibody stability, deamidation may be suppressed by substituting the deamidated amino acid or the amino acid adjacent to the deamidated amino acid with another amino acid. Furthermore, antibody stability can also be increased by substituting glutamic acid with another amino acid. The present invention also provides antibodies stabilized in this manner.

[0053] In the present invention, the term "functional fragment" of an antibody refers to a portion (partial fragment) of the antibody that recognizes PF4. Specific examples include Fab, Fab', F(ab')2, variable region fragments (Fv), disulfide-linked Fv, single-chain variable region fragments (single-chain Fv, scFv), sc(Fv)2, and polymers thereof.

[0054] "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain. It can be obtained by papain digestion of an antibody or by recombinant methods. "Fab'" differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the antibody hinge region. "F(ab')2" refers to a bivalent antigen-binding fragment of an immunoglobulin consisting of both light chains and part of both heavy chains.

[0055] A "variable region fragment (Fv)" is the smallest antibody fragment that has a complete antigen recognition and binding site. Fv is a dimer in which a heavy chain variable region and a light chain variable region are tightly linked by non-covalent bonds. A "single-chain variable region fragment (single-chain Fv, scFv)" contains an antibody's heavy chain variable region and light chain variable region, and these regions are present in a single polypeptide chain. An "sc(Fv)2" is a single chain formed by linking two heavy chain variable regions and two light chain variable regions with a linker or the like.

[0056] The antibodies of the present invention can be produced by the hybridoma method or by recombinant DNA technology. A representative example of the hybridoma method is the Kohler and Milstein method (Kohler & Milstein, Nature, 256:495 (1975)). The antibody-producing cells used in the cell fusion step in this method include spleen cells, lymph node cells, peripheral blood leukocytes, etc., of an animal immunized with an antigen (PF4 protein, its partial peptides, proteins in which the PF4 protein or a partial peptide thereof is fused with an Fc protein or the like, or cells expressing these, etc.). Antibody-producing cells obtained by reacting an antigen in a culture medium with the above-mentioned cells or lymphocytes, etc., previously isolated from an unimmunized animal, can also be used. The animal used is not particularly limited, and examples include non-human animals (mice, rats, hamsters, rabbits, monkeys, goats, etc.). However, as shown in the Examples below, PF4-deficient non-human animals are preferred because, in principle, it is possible to produce antibodies against the functional site of PF4, i.e., the binding site with CXCR3. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be derived from different animal species as long as they are fusible, but are preferably derived from the same animal species. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells. Subsequent screening can yield hybridomas that produce monoclonal antibodies that recognize the PF4 protein. Monoclonal antibodies that recognize the PF4 protein can be obtained by culturing hybridomas or from the ascites fluid of a mammal administered with the hybridoma.

[0057] The recombinant DNA method involves cloning DNA encoding the antibody of the present invention from hybridomas, B cells, or the like, incorporating it into an appropriate vector, and then introducing it into host cells (e.g., mammalian cell lines such as HEK cells, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme A. M. et al., Eur. J. Biochem. 192:767-775 (1990)). In expressing DNA encoding the antibody of the present invention, DNA encoding the heavy chain and DNA encoding the light chain may be separately incorporated into expression vectors and used to transform host cells, or DNA encoding the heavy chain and DNA encoding the light chain may be incorporated into a single expression vector and used to transform host cells (see WO 94 / 11523). The antibody of the present invention can be obtained in a substantially pure and homogeneous form by culturing the host cells and isolating and purifying it from within the host cells or from the culture medium. Antibody isolation and purification can be performed using methods commonly used for purifying polypeptides. By using transgenic animal production techniques to produce transgenic animals (such as cows, goats, sheep, and pigs) incorporating antibody genes, it is possible to obtain large quantities of monoclonal antibodies derived from the antibody genes from the milk of the transgenic animals.

[0058] (Composition) The composition of the present invention is a composition for suppressing the accumulation of T helper T1 regulatory T cells in a tumor. "T helper T1 regulatory T cells (Th1-Treg)" refers to CD4-positive cells that are positive for at least Foxp3 and T-bet. In addition, suppression of Th1-Treg accumulation in a tumor refers to, for example, a state in which the ratio of Th1-Treg to all Treg cells (CD4-positive cells that are positive for at least Foxp3) in a tumor is 40% before administration, but is reduced to 30% or less, preferably 20% or less, and more preferably 10% or less after administration of the composition of the present invention.

[0059] The compositions of the present invention can also be in the form of, for example, pharmaceutical compositions or reagents used for research purposes (eg, in vitro or in vivo experiments).

[0060] More specifically, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating or preventing cancer. Examples of "cancers" that are the subject of the present invention include solid tumors, and more specifically, examples include colorectal cancer (e.g., colon cancer), melanoma, pancreatic cancer, lymphoma, lung cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, gastric cancer, uterine cancer, ovarian cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumors, bone and soft tissue tumors, retroperitoneal tumors, and angiolymphangiosarcomas. Furthermore, such cancers may be primary or metastatic.

[0061] In the present invention, "treatment" includes suppressing the accumulation of Th1-Treg in tumors, and more specifically, suppressing (blocking or delaying) tumor growth, etc. Furthermore, "prevention" includes preventing, delaying, or reducing the risk of cancer recurrence, etc.

[0062] The "composition" of the present invention may contain other pharmacologically acceptable components in addition to an antibody that recognizes PF4 protein. Examples of such other components include carriers, emulsifiers, humectants, pH buffers, culture media, excipients, disintegrants, buffers, isotonicity agents, suspending agents, solubilizers, soothing agents, stabilizers, preservatives, and antiseptics. More specifically, in the case of liquid preparations such as injections, examples of pharmacologically acceptable components include aqueous solutions (e.g., physiological saline, water for injection, phosphate buffer, aqueous glucose solution, aqueous glycerol solution), and aluminum hydroxide. In the case of lyophilized preparations, examples include, but are not limited to, sugars (e.g., mannitol, lactose, saccharose), albumin, and the like. Furthermore, the pharmaceutical composition of the present invention may be in the form of a kit in which the above components can be mixed prior to administration. Furthermore, when used as an injection, the composition may be in the form of a syringe.

[0063] The pharmaceutical composition of the present invention may contain only an antibody that recognizes the PF4 protein as an active ingredient, or may contain the molecule and at least one therapeutic or preventive agent for cancer (hereinafter also simply referred to as "other therapeutic agent, etc."). The antibody that recognizes the PF4 protein and the other therapeutic agent, etc. may be administered separately. Such combined use can enhance the therapeutic or preventive effect on cancer. The "other therapeutic agent" to be used in combination is not particularly limited, and examples thereof include immune checkpoint inhibitors (anti-PD-1 antibodies, etc.), gemcitabine, S-1, erlotinib, 5-FU, leucovorin, irinotecan (CPT-11), oxaliplatin, abraxane, carboplatin, paclitaxel, pemetrexed, sorafenib, vinblastine, LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), and agents described in International Publication No. WO2003 / 038043, but are not limited thereto as long as they have antitumor activity.

[0064] The product (drug, reagent, etc.) of the composition of the present invention or its instruction manual may bear a label indicating that the composition is used for suppressing the accumulation of Th1-Treg in tumors or for the treatment or prevention of cancer. Here, "bearing a label on the product or instruction manual" means that the label is affixed to the product itself, container, packaging, etc., or to an instruction manual, package insert, promotional material, other printed matter, etc. that discloses information about the product.

[0065] The composition of the present invention may also be in the form of a kit. For example, an antibody that recognizes the PF4 protein and the other pharmacologically acceptable components described above typically exist as two or more substances, but can be mixed or otherwise prepared into a single composition before administration to a subject. When used as an injection, the kit may be in the form of a syringe.

[0066] <Therapeutic and Preventive Methods> The present invention also provides a method for suppressing the accumulation of Th1-Treg in tumors or for treating or preventing cancer, which comprises administering to a subject an effective amount of an antibody that recognizes the PF4 protein.

[0067] The "subject" of treatment, etc., in the present invention is not particularly limited and may be not only humans but also non-human animals. Non-human animals are not particularly limited and may include various livestock, poultry, pets, laboratory animals, etc. More specifically, vertebrates, preferably mammals, more preferably primates (humans, monkeys, chimpanzees, orangutans, gorillas, etc.), ungulates (cattle, horses, sheep, goats, etc.), and rodents (mice, rats, guinea pigs, etc.), are included. However, the subject of the present invention is usually humans, and is not limited by gender, age, race, etc. More specifically, examples include humans with cancer, humans at risk of cancer, humans at risk of cancer recurrence, and humans with cancer recurrence.

[0068] The method of administration of the composition of the present invention is not particularly limited and varies depending on the type of the antibody or composition, the age, weight, sex, health condition, etc. of the subject, but can be administered by any of the following administration routes: parenteral administration (e.g., intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, or administration by infusion) or oral administration. Alternatively, the composition can be administered directly (locally) to the target tissue (tumor tissue).

[0069] When administering the composition of the present invention, the dosage can be appropriately selected by a person skilled in the art depending on the age, body weight, degree of progression of symptoms, health condition, type of antibody that recognizes PF4 protein, dosage form of the composition, administration method, etc. of the subject. For example, the dosage of the pharmaceutical composition of the present invention (equivalent to the amount of active ingredient) is 0.1 to 10 mg / kg body weight.

[0070] The administration schedule of the composition of the present invention may be adjusted appropriately depending on the various factors mentioned above, and may be a single administration, or multiple administrations continuously or periodically. Furthermore, the extent of cancer may be monitored after administration, and the timing of administration may be determined based on the results. Administration may be discontinued depending on the degree of recovery, but from the viewpoint of preventing recurrence, administration may be continued without discontinuation. Note that "continuous" may mean daily administration or continuous administration at intervals.

[0071] The therapeutic methods of the present invention may also be used in combination with other known cancer therapeutic methods. Examples of such other therapeutic methods include radiation therapy and surgical therapy. Other examples include chemotherapy and immunotherapy using the aforementioned other therapeutic agents. "Radiotherapy" is not particularly limited, and examples of the type of radiation used to irradiate cancer include X-rays, electron beams, proton beams, heavy particle beams, alpha rays, beta rays, gamma rays, and neutron beams. It may also be external or internal radiation. More specific examples include three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), stereotactic radiotherapy (SRT), particle beam therapy (proton beam therapy, heavy particle beam therapy), image-guided radiation therapy (IGRT), brachytherapy (interstitial irradiation, intracavitary irradiation), and nuclear medicine therapy. "Surgical treatment" is not particularly limited, and is the resection of tissue, including tumor tissue.

[0072] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. The reference numbers cited below in the description of these examples correspond to the reference numbers listed in the <References> section below. The examples were carried out using the materials and methods shown below.

[0073] (Cell lines) Mouse colon cancer cell line MC38 was cultured in RPMI1640 (Nacalai Tesque) containing 10% heat-inactivated FBS (Gibco), 100 U / ml penicillin, and 0.1 mg / ml streptomycin (Nacalai Tesque). Mouse melanoma cell line B16F10 was cultured in DMEM (Nacalai Tesque) containing 10% heat-inactivated FBS (Gibco), 100 U / ml penicillin, and 0.1 mg / ml streptomycin (Nacalai Tesque). Cells were maintained at 37°C and 5% CO. ２ were cultured under

[0074] (Mice) C57BL / 6NCrSlc (C57BL / 6N; CD45.2) mice were purchased from Japan SLC, Inc. For information on Foxp3-Cre mice, RAG2-deficient mice, VeDTR(LF) mice, Foxp3-Cre / VeDTR(ΔFRT) mice, Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice, and congenic CD45.1 mice, see reference 6. All animal experiments were performed with the approval of the Animal Research Committee of the Research Institute for Microbial Diseases, Osaka University.

[0075] (Reagents) anti-mouse / human CD11b (M1 / 70), anti-mouse CD45.2 (104), anti-mouse CD4 (GK1.5), anti-mouse CD8a (53-6.7), anti-mouse / human B220 (RA3-6B2), anti-mouse CD11c (N418), anti-mouse Ly6G (1A8), anti-mouse SiglecF (E50-2440), anti-mouse CX3CR1 (SA011F11), anti-mouse P2RY12 (S16007D), anti-mouse CD25 (PC61), anti-mouse Foxp3 (MF-14), Anti-mouse T-bet (4B10), anti-mouse IFN-γ (XMG1.2), anti-mouse TNF-α (MP6-XT22), anti-mouse CD62L (MEL-14), anti-mouse / human CD44 (17A2), anti-mouse CD80 (16-10A1), anti-mouse CD206 (MMR), anti-mouse Nos2 (W16030C), anti-mouse CD152 (UC10-4B9), anti-mouse CD357 / GITR (DTA-1), and anti-mouse F4 / 80 (BM8) were purchased from BioLegend. Anti-mouse / human Arg1 (A1exF5) was purchased from Invitrogen. Rabbit polyclonal anti-mCherry and nor-NOHA were purchased from Abcam. Goat polyclonal anti-human HB-EGF (DTR), anti-CXCL7 / PPBP (159742), and anti-CXCL4 / PF4 antibodies (140910) were purchased from R&D Systems. DAPI was purchased from Nacalai Tesque. For antibodies used in CyTOF analysis, see reference 6. Recombinant mouse IFN-γ, human TGF-β1, and mouse IL-2 were obtained from Pepro Tech, and AMG487 was purchased from Selleck Chemicals.

[0076] (Generation of Cx3cr1-Cre mice, Arg1-RFP mice, Arg1-Flp mice, and PF4-deficient mice) T7-transscribed products Cx3cr1_gRNA1, Arg1_gRNA2, Pf4_gRNA3 gRNA, and Pf4_gRNA4 were amplified using KOD FX NEO (Toyobo) and the following primers. Cx3cr1_gRNA1 5'-TTAATACGACTCACTATAGGatgggtctctcctgctctgaGTTTTAGAGAGCTAGAAATAGCAAGTTAAAAT-3' (SEQ ID NO: 20); Arg1_gRNA2 5'-TTAATACGACTCACTATAGGtggcgcattcacagtcacttGTTTTAGAGCTAGAAATAGCAAGTTAAAT-3' (SEQ ID NO: 21); Pf4_gRNA3 5'-TTAATACGACTCACTATAGGccttcccctcggaaggcaggGTTTTAGAGCTAGAAATAGCAAGTTAAAAAT-3' (SEQ ID NO: 22); Pf4_gRNA4 5'-TTAATACGACTCACTATAGGggttggaagaagggaagagaGTTTTAGAGCTAGAAATAGCAAGTTAAAAAT-3' (SEQ ID NO: 23). Then, using the T7-transcribed products and MEGAshortscript (Life Technologies), Cx3cr1_gRNA1, Arg1_gRNA2, Pf4_gRNA3 gRNA, and Pf4_gRNA4 were generated.

[0077] Cas9 mRNA was produced by in vitro transcription (IVT) using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) and template DNA. The template DNA was amplified by PCR using pEF6-hCas9-Puro and a primer set (T7Cas9_IVT_F and Cas9_R), and gel-purified.

[0078] The synthesized gRNA and Cas9 mRNA were purified using a MEGAclear kit (Life Technologies).

[0079] To obtain Cx3cr1-Cre, Arg1-RFP, Arg1-Flp, or PF4-deficient mice, CD45.2 C57BL / 6N female mice (6 weeks old) were superovulated and mated with CD45.2 C57BL / 6N stud males. Fertilized one-cell embryos were collected from the oviduct and injected into the pronucleus or cytoplasm with 100 ng / μl of Cas9 mRNA, 50 ng / μl of gRNA, and 50 ng / ml of each targeting vector (for Cx3cr1-Cre, Arg1-RFP, or Arg1-Flp mice). Live injected embryos were transferred into the oviducts of pseudopregnant ICR females 0.5 days postcoitum. Male pups carrying the mutation were bred with CD45.2 C57BL / 6N female mice to test for germline transmission.

[0080] To generate Cx3cr1-Cre mice, a 1.4 kb fragment containing exon 2 and the 3' untranslated sequence of the Tbx21 gene was amplified by PCR using the following primers and cloned into the pBluescript vector. Cx3cr1_LA_F 5'-gaattcGTGTCACCATTAGTCTGGGCGTCTCTCTCTCTCTCT-3' (SEQ ID NO: 24); Cx3cr1_LA_R 5'-ctcGAGCAGGAGAGACCCATCTCCCTCGCT-3' (SEQ ID NO: 25); Cx3cr1 _RA_F 5'-agatctAGGGGTCTCCCCCGACCCTAGCTCC-3' (SEQ ID NO: 26); Cx3cr1_RA_R 5'-gcggccgcTCCATGGTAAGGCGAGTCAGCA-3' (SEQ ID NO: 27). An artificially synthesized P2A peptide sequence containing mammalian codon-optimized Cre recombinase cDNA, provided by FASMAC, was inserted immediately before the stop codon in exon 2 of Cx3cr1. The targeting vector was gel-purified and co-injected into embryos with Cx3cr1_gRNA1 and Cas9 mRNA.

[0081] To generate Arg1-RFP or Arg1-Flp mice, a 1.4 kb fragment containing exon 8 and the 3' untranslated sequence of the Arg1 gene was amplified by PCR using the following primers and cloned into a pBluescript vector. Arg1_LA_F 5'-gaattcCTCCATGACTGAAGTAGACAAGCT-3' (SEQ ID NO: 28); Arg1_LA_R 5'-ctcgagCTTAGGTGGTTTAAGGTAGTCAGT -3' (SEQ ID NO: 29); Arg1 _RA_F 5'-ggatccCTGTGAATGCGCCACATGAAAACC -3' (SEQ ID NO: 30); Arg1_RA_R 5'-gcggccgcCTTGTCTCATTACAGAGCCAAG-3' (SEQ ID NO: 31). The artificially synthesized P2A peptide sequence containing mCherry or mammalian codon-optimized Flp recombinase cDNA (for Arg1-RFP or Arg1-Flp mice, respectively) was inserted immediately before the stop codon of exon 8 of Arg1. The targeting vector was gel-purified and injected into embryos together with Arg1_gRNA2 and Cas9 mRNA.

[0082] (Mouse tumor model) Tumor cells were injected subcutaneously into 7-11 week old gender-matched mice. 1.0 x 10 in PBS ６ MC38 cells or 3.0 × 10 ５ B16F10 cells were injected subcutaneously into the backs of mice. For diphtheria toxin (DT) treatment, tumor-bearing mice were injected i.p. with 100 ng DT (Millipore) in 200 μL of PBS. DT injections were initiated 10 days after tumor inoculation and continued daily. Tumor volume was measured using digital calipers (minor diameter). ２ The tumor volume was calculated using the formula: x long diameter x 0.52. ３ If the tumor density exceeded 100%, the mice were euthanized within 24 hours. Tumor-bearing mice were analyzed 15-21 days after tumor inoculation without specific endpoints.

[0083] (Cell preparation from mice) To isolate splenocytes, spleens were crushed in PBS using a syringe plunger on a 70 μm cell strainer. After centrifugation at 2000 rpm for 5 minutes, the cells were collected and suspended in ACK buffer and kept at room temperature for 2 minutes to lyse red blood cells. After ACK treatment, the cells were washed and filtered through a 40 μm cell strainer.

[0084] For brain cell isolation, brains were crushed in HBSS using a syringe plunger on a 70 μm cell strainer. The cells were centrifuged at 2,000 rpm for 5 minutes, collected, and then treated with ACK buffer. The cells were then suspended in 40% Percoll (Sigma-Aldrich) in HBSS and centrifuged at 2,380 × g for 20 minutes to remove floating debris. The pellet was washed with HBSS.

[0085] To digest the lungs and liver, each tissue was cut into small pieces (approximately 3 x 3 mm) using a laser in HBSS. Collagenase D (1 mg / mL), dispase II (80 μg / mL), and DNase I (20 μg / mL) were then added and incubated at 37°C for 60 minutes with shaking. After digestion, the cells were treated with ACK buffer, placed in 40% Percoll (Sigma-Aldrich) in HBSS, and centrifuged at 2,380 x g for 20 minutes to remove floating debris. The pellet was washed with HBSS.

[0086] Tumors were digested using a mouse Tumor Dissociation Kit (Miltenyi) and a gentleMACS Octo Dissociator with Heaters (Miltenyi). After digestion, cells were treated with ACK buffer and depleted of dead cells using a dead cell removal kit (Miltenyi). The prepared cells were suspended in a buffer appropriate for each experiment.

[0087] (Flow cytometry and cell sorting) For surface staining, cells were blocked with anti-CD16 / 32 (Biolegend) for 15 minutes on ice. Cells were stained with each antibody in the presence of anti-CD16 / 32 for 15 minutes on ice. For intranuclear staining, Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) was used according to the manufacturer's instructions. For TNF-α and IFN-γ staining, cells were stimulated with 50 ng / mL PMA (Nacalai Tesque) and 1 μg / mL ionomycin (Nacalai Tesque) in the presence of 1 μL / mL GolgiStop reagent (BD Biosciences) at 37°C for 4 hours. After stimulation, cells were stained for surface markers, followed by fixation, permeabilization, and staining with cytokine-specific antibodies using a Cytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences) according to the manufacturer's instructions. A FACS Aria III (BD Biosciences) was used for data acquisition and cell sorting. Acquired data were analyzed using FlowJo (BD Biosciences).

[0088] Single-cell RNA library construction and sequencing. Cells were stained with cell hashing antibodies (TotalSeq-C0301 to -C0306 (BioLegend)). Single-cell suspensions were processed with a 10x Genomics Chromium Controller according to the protocol outlined in the Chromium Single Cell 5' Reagent Kit v2 User Guide. Chromium Next GEM Single Cell 5' Kit v2, Chromium Next GEM Single Cell KChip Kit, Dual Index Kit TT Set A, 5' Feature Barcode Kit, and Dual Index Kit TN Set A were used in this process. Approximately 16,500 live cells per sample were loaded into a Chromium Controller according to the manufacturer's recommendations to generate 10,000 single-cell gel-bead emulsions for library preparation and sequencing. The encapsulated single cells and barcoded bead (GEM) droplets were then subjected to reverse transcription in a Veriti thermal cycler (Thermo Fisher Scientific) to generate mRNA-derived cDNA and HTO-derived cDNA tagged with cell barcodes and unique molecular indices (UMIs). The cDNA was then amplified and single-cell libraries were generated according to the manufacturer's protocol. Quantification was performed using the Agilent Bioanalyzer DNA High Sensitivity Assay (Agilent, DNA High Sensitivity Kit). The amplified cDNA was then enzymatically fragmented, end-repaired, and polyA-tagged. Amplified cDNA was cleaned up / size selected using SPRIselect magnetic beads (Beckman-Coulter, SPRIselect). Next, Illumina sequencing adapters were ligated to the size-selected fragments and cleaned up using SPRIselect magnetic beads. Finally, sample indexes were selected, amplified, and double-sided size selected using SPRIselect magnetic beads. Functional barcode libraries were constructed using cDNA from HTOs to hash cells.The quality of the final library was assessed using the Agilent Bioanalyzer DNA High Sensitivity Assay. Samples were then sequenced on a NovaSeq 6000 platform in 28+90 base paired-end mode. The resulting raw reads were processed using cellranger 6.0.0 (10x Genomics). Data analysis was performed using SeqGeq (BD Life Sciences). After quality control and normalization, PCA and unsupervised cell clustering were performed using the Seurat plugin.

[0089] (In vitro culture and stimulation of Tregs) Spleen YFP － Treg cells (YFP － CD4 ＋ CD25 ＋ Cells were selected from Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice and stimulated for 3 days in RPMI 1640 medium containing mIL-2 (20 ng / ml) and hTGF-β (5 ng / ml) with pre-fixed α-CD3 (5 μg / ml) and α-CD28 (2 μg / ml) in the presence or absence of recombinant mouse PF4 protein (5 μg / ml), PPBP (5 μg / ml), AMG487 (0.1 μM), anti-PF4 mAb (#6-1-5; 3 μg / ml), com-anti-PF4 (3 μg / ml), anti-PPBP (3 μg / ml), and nor-NOHA (10 μM).

[0090] (Co-culture experiment) YFP-Treg (YFP derived from tumor-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice) － CD4 ＋ CD25 ＋ Spleen cells, 5.0 × 10 ４ cells) and TAM (RFP in tumors of Arg1-RFP mice － or RFP ＋ CD45 ＋ CD11b ＋ Ly6G － cells, 5.0 x 10 ４Cells) were stimulated with pre-fixed α-CD3 (5 μg / ml) and α-CD28 (2 μg / ml) in RPMI 1640 medium containing mIL-2 (20 ng / ml) and hTGF-β (5 ng / ml) in a 96-well culture plate and directly co-cultured for 3 days. After 3 days of co-culture, YFP was detected using flow cytometry. ＋ The percentage of cells was analyzed.

[0091] In a Transwell indirect co-culture system of Tregs and TAMs, Tregs (1.0 × 10 ５ cells) were seeded in a 24-well culture plate supplemented with RPMI 1640 medium containing mIL-2 and hTGF-β, and stimulated with pre-immobilized α-CD3 and α-CD28. ５ Cells) were seeded onto the upper Transwell insert (pore size 0.4 μm, Corning) of the 24-well plate. After 3 days of co-culture, YFP was detected using flow cytometry. ＋ The percentage of cells was analyzed.

[0092] Splenic macrophages (CD45 from wild-type mice) ＋ CD11b ＋ Ly6G － Spleen cells, 1.0 × 10 ５ cells) and B16F10 cells (5.0 × 10 ５ In the Transwell coculture system with B16F10 cells, splenic macrophages were seeded in a 24-well culture plate, and B16F10 cells were seeded on the top of the 24-well plate in a Transwell insert (0.4 μm pore size, Corning). After 24 hours of coculture, splenic macrophages were collected and subjected to PCR.

[0093] (In vitro culture and stimulation of splenic macrophages) Splenic macrophages (CD45 ＋ CD11b ＋ Ly6G － Spleen cells, 5.0 × 10 ４Cells) were seeded into 96-well round-bottom culture plates supplemented with RPMI 1640 containing 25 mM lactic acid (Nacalai Tesque) or low-pH RPMI 1640. Low-pH RPMI 1640 was prepared by adding hydrochloric acid (Nacalai Tesque).

[0094] (RNA-seq) For RNA-seq analysis, total RNA was extracted from cells using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Full-length cDNA was generated using the SMART-Seq HT Kit (Takara Bio) according to the manufacturer's instructions. Illumina libraries were prepared using the Nextera DNA Library Preparation Kit (Illumina) according to the SMARTer kit's instructions. Sequencing was performed using an Illumina NovaSeq 6000 sequencer (Illumina) in 101-base single-end mode. Bowtie2 ver. 2.2.3 and SAMtools ver. Sequencing reads were mapped to the mouse reference genome sequence (mm10) using TopHat v2.0.13 combined with 0.1.19. Fragments per kilobase of exon per million mapped fragments (FPKMs) were calculated using Cufflinks ver. 2.2.1. Volcano plots and heat maps were generated using GraphBio (http: / / www.graphbio1.com / en / ).

[0095] (In vitro Treg suppression assay) YFP-Tregs were isolated from Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice and cultured without stimulation or stimulated with PF4 (5 μg / ml). After 3 days, Tregs were collected and washed twice with PBS to prepare PF4-stimulated or unstimulated Tregs. Splenic naive CD4 ＋ T cells (CD4 ＋ CD45 ＋ CD62L ｈｉｇｈ CD44 ｌｏｗ CD25 －CFSE-labeled T cells (5.0 × 10 ４ cells) were cultured in a 1000-well plate with PF4-stimulated or unstimulated Tregs (5.0 × 10 ４ Treg-treated Tconv cells were cultured in the presence of Dynabeads mouse T-activating factor CD3 / CD28 (Gibco) and mIL-2 (20 ng / ml). After 3 days of culture, the CFSE intensity in Tconv cells was measured using flow cytometry, and the suppressive activity (%) was calculated using the following formula: 100 × (1-(CFSE in Tconv treated with Treg)) ｌｏｗ Frequency) / (CFSE in Tconv alone ｌｏｗ CD45.1 congenic mice were used to distinguish Tconv (CD45.1) from Treg (CD45.2).

[0096] (Quantitative RT-PCR) For quantitative PCR, total RNA was extracted using an RNeasy Mini Kit (Qiagen). The RNA was then reverse-transcribed using a Verso cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's instructions. Quantitative PCR was performed using GoTaq qPCR Master Mix (Promega) and CFX Connect (Bio-Rad). mRNA expression was normalized to that of β-actin mRNA.

[0097] (Immunohistochemical staining) Tumors, spleens, and livers were fixed in 4% PFA for 2 hours at 4°C. After overnight immersion in 30% sucrose at 4°C, they were embedded in FSC22 frozen section medium (Leica) and 10 μm sections were prepared using CM1860 UV (Leica). The sections were blocked with 0.1% BSA and 1% mouse serum for 1 hour and then incubated with antibodies overnight at 4°C. Slides were washed three times with PBS and then revealed using secondary antibodies at room temperature for 2 hours. Immediately after staining, images were photographed using an FV3000 (Olympus).

[0098] (ELISA) The concentrations of PF4, CCL2 and IL-10 were measured using ELISA kits from R&D Systems according to the manufacturer's instructions.

[0099] (Determination of Lactate Concentration) Tumors and spleens were excised and homogenized. To measure the lactate concentration, an L-lactate assay kit (Abcam) was used according to the manufacturer's instructions. Lactate concentrations (nmol / g) were calculated as the mean and SEM.

[0100] (CyTOF Cell Staining) First, each sample was barcoded with a different metal-conjugated α-CD45 antibody in CyFACS buffer (PBS containing 0.1% BSA, 2 mM EDTA, and 0.01% sodium azide) along with anti-CD16 / 32 (Biolegend). After barcoding, the samples were washed twice with CyFACS buffer and pooled. The pooled samples were stained with a metal-conjugated antibody cocktail against surface proteins in CyFACS buffer for 45 minutes at room temperature. Next, the samples were washed twice with CyFACS buffer and stained with PBS containing zirconium(IV) chloride for 5 minutes at room temperature to distinguish live and dead cells. Then, they were fixed and permeabilized using Foxp3 / transcription factor buffer set according to the manufacturer's instructions. Subsequently, the samples were stained with a metal-conjugated antibody cocktail against intracellular proteins for 45 minutes at 4°C. The stained samples were then washed twice in CyFACS buffer and once in PBS and fixed overnight in 2% formaldehyde (Invitrogen) in PBS with Cell-ID Intercalator-103Rh (Fluidigm) at 4°C.

[0101] (CyTOF Analysis) Prior to data acquisition, fixed samples were washed once with CyFACS buffer and twice with cell acquisition solution (CAS) (Fluidigm). The washed samples were suspended in CAS containing 15% EQ Four Elements Calibration Beads (Fluidigm) and filtered. Data were acquired using a Helios mass cytometer (Fluidigm). For data analysis, live singlet cell gating and sample debarcoding were performed using FlowJo, and subsequence analysis was performed using Cytobank ver. 9.1 (Beckman Coulter). The vi-SNE and FlowSOM algorithms in Cytobank were used for t-SNE2D mapping and clustering, respectively.

[0102] (Generation of Monoclonal Antibody (Anti-PF4 mAb)) PF4-deficient mice were immunized with recombinant mouse PF4 protein emulsified with TiterMax Gold adjuvant (TiterMax). Lymph nodes from the immunized mice were harvested, homogenized into single-cell suspensions, and fused to U3P1 cells with PEG (Hampton Research). The fused hybridoma cells were seeded into 96-well plates containing RPMI 1640 containing BM Condimed H1 (Roche). After 24 hours, HAT supplement (Thermo Scientific) was added to the culture supernatant. After ~10 days of culture, primary screening of the supernatant was performed using PF4-coupled latex beads.

[0103] (Determination of affinity of anti-PF4 mAb) Antibody affinity was detected using antigen coated latex beads. To prepare antigen-coupled latex beads, latex beads (Invitrogen) and antigen protein (mouse PF4, mouse CXCL9, mouse CXCL10, mouse CXCL11, or human PF4, recombinant protein, Biolegend) were mixed and incubated at room temperature with gentle mixing. To detect antibodies with affinity to the antigen, the antigen-coupled latex beads were incubated with anti-PF4 mAb for 15 minutes. A secondary antibody was used to detect antibodies bound to the antigen-coupled latex beads.

[0104] Human Correlation Study: Using publicly available human cancer datasets from The Cancer Genome Atlas (TCGA) (www.cancergenome.nih.gov) and UCSC Xena (https: / / xenabrowser.net / ), we correlated PF4 and ITGAM mRNA expression levels in tumors with patient survival.

[0105] (Epitope Mapping by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)) To identify the epitope of anti-PF4 mAb (#6-1-5), the antibody and purified PF4 protein were prepared in PBS to final concentrations of 4.6 mg / mL (32 μM) and 0.49 mg / mL (60 μM), respectively. To identify the epitope of anti-PF4 mAb (com-anti-PF4), the anti-PF4 mAb (com-anti-PF4) and purified PF4 protein were prepared in PBS to final concentrations of 4.4 mg / mL (30.2 μM) and 0.49 mg / mL (60 μM), respectively. The purified PF4 protein used was Recombinant Mouse CXCL4 (PF-4) (carrier-free) manufactured by Biolegend.

[0106] In HDX-MS analysis, each protein solution was ２The deuterium-labeled samples were diluted 10-fold with PBS in 0°C. The diluted solutions were then incubated at 10°C. Deuterium-labeled samples were quenched by approximately 2-fold dilution with a quenching buffer consisting of 8 M urea and 1 M tris(2-carboxyethyl)phosphine hydrochloride at pH 3.0. All dilutions were performed using an HDx-3PAL (LEAP Technologies). After quenching, the solutions were subjected to online pepsin digestion, followed by LC / MS analysis using an UltiMate 3000 RSLC nano (Thermo Fisher Scientific) interfaced with a Q Exactive plus mass spectrometer (Thermo Fisher Scientific). Online pepsin digestion was performed using a protease type XIII / pepsin column (w / w, 1:1; 2.1 × 30 mm; NovaBioAssays, Inc.) with formic acid solution (pH 2.5) at 8°C for 3 minutes at a flow rate of 50 μL / min. After pepsin digestion, the sample was desalted and analyzed using an Acclaim PepMap 100 C18 LC column (1.0 × 5 mm; Thermo Fisher Scientific) and a Hypersil GOLD column (1.0 × 50 mm; Thermo Fisher Scientific). The mobile phases used were 0.1% formic acid (Buffer A) and 90% acetonitrile with 0.1% formic acid (Buffer B). Deuterated peptides were eluted with a gradient of 10-90% buffer B for 9 minutes at a flow rate of 45 μL / min.

[0107] Mass spectrometer conditions were as follows: electrospray voltage, 3.8 kV; positive ion mode; sheath and auxiliary nitrogen flow rates, 20 and 2 arbitrary units, respectively; ion transfer tube temperature, 275°C; auxiliary gas heater temperature, 100°C; data-dependent acquisition was performed using a normalized collision energy of 27 arbitrary units.

[0108] MS and MS / MS spectra were analyzed using database search with Proteome Discoverer 2.2 (Thermo Fisher Scientific). Deuteration levels in peptide fragments were analyzed by comparing the spectra of deuterated and non-deuterated samples using HDExaminer software 2.0 (Sierra Analytics).

[0109] Statistical Analysis: All quantitative data are presented as mean ± standard error of the mean (SEM) and were analyzed using GraphPad software (Prism v10.0.3). Two-tailed Student's t-test was used to compare two data sets. One- or two-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's multiple comparison test was used to detect differences in results between groups. A p-value of <0.05 was considered statistically significant.

[0110] Below, we present the results obtained using the above materials and methods.

[0111] Example 1: Development of a tumor-associated macrophage (TAM)-specific depletion system using VeDTR Previously, the present inventors have demonstrated that depletion of T helper T1 Tregs (Th1-Tregs), a subset of regulatory T cells (Tregs) that act as brakes on immune cells, can induce potent antitumor immune activation. Furthermore, they have found that this activation is not associated with autoimmune disease (Reference 6). On the other hand, TAMs are innate immune cells found in the tumor microenvironment (TME) (References 3 and 5). It has been reported that TAMs secrete various cytokines and chemokines to recruit Tregs to the TME and suppress the function of antitumor T cells (References 21-23), suggesting a high correlation between TAMs and Tregs.

[0112] Therefore, if the substances in the TME (for example, cells such as TAM, factors present in the TME, etc.) that contribute to the high abundance of Th1-Treg in tumors are identified, it will be possible to suppress the accumulation of Th1-Treg in tumors by targeting these substances.

[0113] To clarify whether TAMs are involved in the high abundance of Th1-Tregs in the TME, we attempted to develop a genetic system to deplete TAMs (Figure 1A). TAMs are well known to express arginase I (Arg1) (References 21, 24, 25). Furthermore, Arg1 has been shown to be present only in tumors and not in other non-tumor tissues (Reference 24).

[0114] First, to confirm that Arg1 gene expression is specific to TAMs, knock-in mice expressing red fluorescent protein (RFP) under the control of the endogenous Arg1 gene (not shown in the figure) were generated. In these knock-in mice, RFP expression was detected and compared in various tissue macrophages, including microglia (brain macrophages), alveolar macrophages (lung macrophages), Kupffer cells (liver macrophages), and splenic macrophages (SPM), in addition to TAMs. As a result, significantly higher RFP expression was observed in TAMs compared to other tissue macrophages.

[0115] Arg1 is also known as a marker for M2 macrophages (References 26 and 27). ＋ TAM and Arg1 － The gene expression of TAMs was compared by bulk RNA-seq analysis. Although not shown in the figure, Arg1 ＋ In TAMs, M2 macrophage signature genes such as Vegfa and Folr2 (References 28, 29) were found to be highly expressed. In contrast, Arg1 －TAMs were highly expressed with M1 macrophage signature genes (references 30-32) encoding the inflammatory cytokine IL12, CCR7, chemokine receptors, and MHCII (encoded by H2-Oa). ＋ Although TAMs were confirmed to be M2-like, Arg1 alone could not be used to specifically mark TAMs because it is highly expressed in the liver in a macrophage-independent manner.

[0116] Therefore, we decided to use the VeDTR mouse system, which allows cross-gene expression of yellow fluorescent protein (YFP) and diphtheria toxin receptor (DTR) (Reference 6). ＋ In the VeDTR system, TAMs need to be specifically characterized by two different genes. On the other hand, Cx3cr1 was selected as the second marker because it is a well-known macrophage / monocyte marker gene and is widely used in Cre-driver mice (Reference 33).

[0117] Specifically, although not shown in the figure, Cx3cr1-Cre knock-in mice were generated by genome editing. Furthermore, Cx3cr1-Cre / VeDTR(ΔFRT) mice were generated. Note that most CD11b ＋ In cells, all Cx3cr1 ＋ It was found that the cells expressed YFP. ＋ CD45 ＋ Single-cell RNA-seq (scRNA-seq) analysis using 1000 cells revealed that TAMs and splenic macrophages (SPMs) clustered clearly in the t-SNE plot. As previously reported (Reference 34), several non-CD11b TAMs, such as B cells, T cells, NK cells, and dendritic cells, were clustered. ＋ The Arg1 gene was also detected in YFP+ cells of Cx3cr1-Cre / VeDTR(ΔFRT) mice. As expected, Arg1 expression was only in a subset of TAMs, but was barely detectable in SPMs or other non-macrophage populations.

[0118] To utilize the Arg1 gene in the VeDTR system, we generated knock-in mice expressing Flp recombinase under the control of the endogenous Arg1 gene (Figure 1A). These mice were then crossed with Cx3cr1-Cre and VeDTR(LF) mice. VeDTR(LF) mice require both Cre and Flp recombinase to express YFP and DTR under the control of the endogenous Cx3cr1 and Arg1 genes (Figure 1A). We analyzed YFP expression in various tissue macrophages in the resulting Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice. YFP was detected exclusively in TAMs compared with other tissue macrophages (Figure 1B). Furthermore, strong DTR expression was detected in TAMs, but was barely detectable in SPMs or non-macrophage liver tissues (Figure 1C). This suggests that TAMs are specifically marked in Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) tumor-bearing mice.

[0119] When DT was injected, YFP ＋ CD11b ＋ The cells were depleted in the tumor (Fig. 1D). ＋ The growth of tumors (MC38 colon adenocarcinoma and B16F10 melanoma) in TAM-depleted Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice was significantly inhibited compared with that in non-depleted control mice (Figures 1E and 1F). ＋ Depletion of TAMs has been shown to reduce tumor growth.

[0120] Example 2: Arg1 ＋ Depletion of TAMs reduces the Th1-Treg ratio in the TME. ＋ We analyzed the effect of TAM depletion on the Th1-Treg status of the TME. In particular, Arg1 TAMs were found in MC38 and B16F10 tumors. ＋ After TAM depletion, Foxp3 ＋ T-bet ＋ CD4 ＋A significantly reduced T cell ratio was detected (Fig. 2A).

[0121] Arg1 ＋ TAM depletion leads to CD11b － CD45 ＋ The effects on lymphocyte populations (non-macrophage populations) were analyzed unbiasedly using mass cytometry (CyTOF). ＋ T-bet ＋ The proportion of cells is Arg1 ＋ After TAM depletion, CD11b specifically and significantly decreased, whereas the proportions of other populations were not statistically changed (Figures 2B-2D). － Arg1 to Th1-Treg ratio in cells ＋ This suggests a specific effect of TAM depletion.

[0122] Arg1 ＋ CD11b in the TME after TAM depletion ＋ CD45 ＋ The state of the cells was evaluated by CyTOF. As a result, although not shown in the figure, Arg1 ＋ Depletion of TAMs leads to Ly6G ＋ CD11b ＋ In addition to cells, CD80 ｈｉｇｈ or iNOS ＋ CD11b ＋ Furthermore, flow cytometry and immunohistochemistry revealed that the proportion of Arg1 cells was significantly increased. ＋ We observed an increase in CD80 and iNOS and a decrease in CD206 after TAM depletion.

[0123] Considering the decreased ratio of pro-tumor Th1-Treg and M2 macrophage markers (CD206) and the increased ratio of anti-tumor M1 macrophage markers (CD80 and iNOS), Arg1 ＋ Depletion of TAMs may lead to antitumor immunity. To investigate this possibility, we investigated the CD4 receptors involved in the production of antitumor cytokines such as tumor necrosis factor α (TNF-α) and interferon gamma (IFN-γ). ＋ The production from T cells was assessed by flow cytometry.＋ TNF-α in tumors from TAM-depleted Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice ＋ / IFN-γ ＋ CD4 ＋ The proportion of T cells was much higher than that in non-depleted control mice (Fig. 2E).

[0124] From the above results, Arg1 ＋ It is suggested that TAM depletion reduces the proportion of Th1-Treg and M2-like TAM in tumors, while increasing the proportion of M1-like TAM, leading to anti-tumor immunity.

[0125] Example 3: Arg1 ＋ TAMs secrete PF4, which polarizes Tregs into Th1-Tregs. Th1-Tregs are highly accumulated in tumors compared to other non-tumor tissues (Reference 6). ＋ Macrophages are more abundant in tumors than in non-tumor tissues (Fig. 1B). ＋ Depletion of TAMs leads to a decrease in tumor Th1-Tregs (Figure 2).

[0126] Arg1 in MC38-bearing Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice ＋ The localization of TAM and Treg was examined by immunohistochemistry. Although not shown in the figure, most of the Foxp3 ＋ CD4 ＋ The cells are DTR in the tumor. ＋ associated with Treg and Arg1 cells in the TME. ＋ It is suggested that there is a close interaction with TAM.

[0127] For Th1-Treg to accumulate in the TME, Treg must be polarized to Th1-Treg somewhere. In this regard, the present inventors have proposed that Arg1 is involved in Treg polarization to Th1-Treg in the TME. ＋ We hypothesized that TAM is involved. To test this possibility, we ＋We examined whether Tregs become Th1-Tregs when co-cultured with TAMs. Specifically, we investigated the YFP expression in the spleens of non-Th1-Treg (tumor-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice. － CD4 ＋ CD25 ＋ T cells (hereinafter referred to as “YFP”) － Tregs were isolated. － Treg and Arg1 － TAM or Arg1 ＋ To monitor Th1-Treg polarization, YFP expression was detected by co-culture with TAM. － Treg and Arg1 － In co-culture with TAM, YFP expression in Tregs was observed, whereas Arg1 ＋ This was not observed in co-culture with TAM (Fig. 3A).

[0128] Arg1 ＋ We analyzed the molecular mechanism by which TAM polarizes Tregs to Th1-Tregs. ＋ We tested whether direct contact between TAM and non-Th1 Treg is required for in vitro Th1 Treg polarization by TAM. ＋ TAM and YFP － Even when Tregs were physically separated by a filter, YFP was observed after co-culture. － YFP expression was observed in Tregs (Fig. 3B). This suggests that Arg1 is essential for Th1-Treg generation. ＋ This suggests the presence of a humoral factor derived from TAM. ＋ YFP expression was detected in non-Th1 Tregs, regardless of whether the TAMs were derived from either MC38 colorectal tumor or B16F10 melanoma-bearing mice (Figure 3B). ＋ TAM-induced Th1-Treg polarization was significantly suppressed by Arg1 in both tumor models. ＋ This suggests that it may depend on factors common to TAMs but may be independent of tumor antigens.

[0129] To investigate these common humoral factors, bulk RNA-seq was used to identify Arg1 from B16F10 or MC38 colorectal tumor-bearing mice. ＋ TAM and Arg1 － The gene expression profiles of TAM and TAM were compared. ＋ TAM and Arg1 － Ten genes showing differential gene expression patterns between TAMs and TAMs were identified (Fig. 3C). Among these were two genes encoding humoral factors, platelet factor 4 (PF4, also known as CXCL4) and platelet-promoting protein 7 (PPBP, also known as CXCL7). Arg1 was also included as a candidate humoral factor because it mediates arginine depletion during culture (Fig. 3C).

[0130] The roles of PF4, PPBP, and Arg1 in Th1-Treg polarization were evaluated. Although not shown in the figure, in co-culture with Arg1, treatment with nor-NOHA (Arg1 inhibitor) or anti-PPBP neutralizing antibody inhibited Arg1. ＋ Co-culture of TAM with non-Th1 Tregs did not affect Th1 Treg conversion. ＋ When non-Th1 Tregs were stimulated with PF4 or PPBP without TAM, it was surprisingly found that PF4, but not PPBP, induced YFP expression in non-Th1 Tregs, suggesting that PF4 induces polarization toward Th1 Tregs (Fig. 3D).

[0131] In addition, injection of recombinant PF4 protein caused inflammation and inhibited angiogenesis (References 43, 53-58), suggesting that PF4 acts as an antitumor factor. ＋ CD11b ＋ It has also been reported to be produced by myeloid cells and to suppress tumor metastasis (Reference 59).

[0132] In the scRNA-seq data, PF4 mRNA was detected in TAMs but not in SPMs (Fig. 3E). When PF4 mRNA expression was detected in macrophages from various tissues, the PF4 mRNA level was detected in TAMs much higher than in other tissue macrophages (Fig. 3F). Furthermore, Arg1 － PF4 is not expressed in TAM, and Arg1 ＋ Since Arg1 is specifically expressed in TAMs (Fig. 3G), Th1-Treg polarization via PF4 secretion in tumors is thought to be related to Arg1 ＋ This suggests that this is a specific function of TAM.

[0133] PF4-induced Th1-Tregs then react with conventional CD4 ＋ We evaluated whether PF4 functionally inhibits T cell proliferation. As a result, PF4-stimulated Th1-Tregs showed a greater effector CD4 T cell proliferation than unstimulated non-Th1-Tregs. ＋ PF4 strongly suppressed T cell proliferation (Fig. 3H), indicating that PF4-induced Th1-Tregs have a suppressive function.

[0134] We analyzed the expression of CD25, GITR, and CTLA-4 (References 35, 36), which play important roles in regulating Treg function. The results showed that, although there was no significant difference in the surface expression of GITR, the expression of CD25 and CTLA-4 was increased in PF4-stimulated Tregs compared with unstimulated Tregs (Fig. 3I). This suggests that PF4 enhances their suppressive ability.

[0135] We analyzed the molecular mechanism by which PF4 stimulates the polarization of non-Th1 Tregs to Th1 Tregs. CXCR3 is the human receptor for PF4 (Reference 37). We found that treatment with a CXCR3 inhibitor (AMG487) significantly inhibited the PF4-induced conversion of non-Th1 Tregs to Th1 Tregs (Figure 3J). This indicates that PF4 stimulates Th1 Treg polarization via CXCR3.

[0136] From the above results, Arg1 ＋It is suggested that TAMs secrete PF4 and polarize Tregs to Th1-Tregs in a CXCR3-dependent manner.

[0137] Previous studies have shown that CXCR3 is important for Treg accumulation and immunosuppression in tumors, and here, the importance of CXCR3 was suggested in the interaction between Tregs expressing CXCL9, another CXCR3 ligand, and DC1 in tumors (Reference 7). Meanwhile, this example revealed that the importance of CXCR3 in Tregs is crucial for Th1-Treg polarization itself. Arg1, which produces PF4, ＋ Given that macrophages are particularly prominent in tumors, Tregs may be involved in the regulation of PF4-producing Arg1 ＋ They are thought to be in close contact with TAMs and may differentiate into Th1-Tregs in the TME in a CXCR3-dependent manner. CXCL9 and PF4 are both CXCR3 ligands, but CXCL9 is important for antitumor immunity and is often associated with a good prognosis in human cancer patients (References 46-50). Therefore, PF4 and CXCL9 may play distinct roles in the TME.

[0138] Next, Arg1 ＋We further analyzed the mechanism by which TAMs express PF4. Although not shown in the figure, it is noteworthy that coculture of SPM with B16F10 melanoma cells enhanced PF4 mRNA expression. Tumor tissues are known to contain high concentrations of lactate due to the Warburg effect (Reference 38). Indeed, both B16F10 melanoma and MC38 colorectal tumor tissues exhibited significantly higher lactate concentrations than the spleens of the same tumor-bearing mice. Because lactate stimulates Arg1 expression in macrophages (Reference 25), we used quantitative RT-PCR to assess whether it also induces Pf4 expression. Although not shown in the figure, lactate stimulation alone enhanced Pf4 mRNA expression in SPM. On the other hand, low pH alone did not induce Pf4 mRNA expression. These findings suggest that lactate-induced PF4 expression in macrophages is independent of acid-induced low pH. Th2 cytokines, such as IL-4 and IL-13, have been shown to be involved in the polarization of M2-like macrophages in the TME (Reference 39). However, Pf4 mRNA expression levels were not increased in SPM stimulated with IL-4 / IL-13. In contrast, IL-4 / IL-13, lactate, and low pH conditions all strongly stimulated Arg1 mRNA expression in SPM (References 25, 39, and 40). This suggests differential determinants of Pf4 and Arg1 mRNA expression in macrophages. Thus, PF4 induction in TAMs may be dependent on tumor-specific conditions, such as high lactate concentrations.

[0139] Example 4: Genetic inactivation of PF4 reduces intratumoral Th1-Treg and tumor growth. We investigated whether PF4 contributes to the high ratio of Th1-Treg in the TME in vivo. Specifically, PF4-deficient mice were generated by CRISPR / Cas9 genome editing. Arg1 ＋ TAMs were isolated from both wild-type and PF4-deficient mice in the Arg1-RFP strain background, and PF4 protein levels were detected by ELISA. ＋ High PF4 protein levels were observed in TAMs, but Arg1 in PF4-deficient mice.＋ PF4 protein was not detected in TAMs (Figure 4A). Comparison of tumor growth between wild-type and PF4-deficient mice revealed delayed tumor growth in PF4-deficient mice (Figures 4B and 4C). Flow cytometry analysis of the Th1-Treg ratios in tumors and spleens of PF4-deficient mice revealed that the ratios of Th1-Tregs in tumors were significantly lower than those in wild-type mice (Figures 4D and 4E). In contrast, the Th1-Treg ratios in spleens were comparable between tumor-bearing wild-type and PF4-deficient mice (Figures 4D and 4E). These results suggest that PF4 is specifically involved in promoting intratumoral Th1-Treg accumulation and tumor growth.

[0140] Example 5: Antibody-mediated neutralization of PF4 reduces the Th1-Treg ratio in tumors and tumor growth. As described above, genetic inactivation of PF4 reduced the Th1-Treg ratio in tumors and slowed tumor growth (Figure 4). PF4 binds to human CD4 ＋ It has previously been reported that PF4 stimulates T cells to express Foxp3 and T-bet (Reference 41). Therefore, we next tested whether neutralization of systemic PF4 with a PF4-specific antibody could be a potential cancer immunotherapy strategy. However, a commercially available anti-PF4 (com-anti-PF4) monoclonal antibody (mAb) only marginally neutralized PF4-induced Treg polarization toward Th1 Tregs (Figure 5A).

[0141] Therefore, we decided to generate a new anti-PF4 monoclonal antibody. PF4-deficient mice were immunized with recombinant PF4 protein, and hybridoma-fused B cells were isolated and screened. As a result, we successfully isolated an anti-PF4 mAb (#6-1-5) that neutralized the PF4-induced conversion of Tregs to Th1-Tregs (Fig. 5A). This anti-PF4 mAb (#6-1-5) was specific for PF4 but did not recognize any of the other CXCR3 ligands, CXCL9, CXCL10, or CXCL11 (Reference 42) (Fig. 6A).

[0142] Next, we evaluated the immunotherapeutic effect of anti-PF4 mAb (#6-1-5) on B16F10 melanoma and MC38 colorectal tumors. Ten days after tumor inoculation, anti-PF4 mAb (#6-1-5) was administered simultaneously with DT administration to Cx3cr1-Cre / Arg1-Flp / VeDTR(LF) mice (Figure 1h). As a result, anti-PF4 mAb (#6-1-5)-treated mice significantly reduced B16F10 melanoma growth. A trend toward reduced MC38 colorectal tumor growth was also observed (Figure 6B).

[0143] Administration of anti-PF4 mAb (#6-1-5) on day 3 after tumor inoculation dramatically inhibited the growth of both tumors (Figure 5B). Comparison of the Th1-Treg ratio in tumors from anti-PF4 mAb-treated and untreated mice revealed that the Th1-Treg ratio in tumors from anti-PF4 mAb-treated mice was significantly reduced compared with that in control mice treated with an isotype control mAb (Figures 5C and 5D). Meanwhile, the Th1-Treg ratio in the spleen was comparable in both conditions (Figures 5C and 5D), reminiscent of the phenotype of PF4-deficient mice (Figures 4D and 4E).

[0144] YFP in tumors and spleens in MC38 or B16F10-bearing Foxp3-Cre / Tbx21-Flp / VeDTR(LF) mice ＋ When the ratio of Th1-Treg cells was examined, anti-PF4 mAb treatment reduced the Th1-Treg ratio in the tumor, but not in the spleen (Fig. 6C and 6D). ＋ / TNF-α ＋ CD4 ＋ T cells or CD8 ＋ The proportion of T cells was significantly increased compared to that of control mice (Figs. 5E and 5F).

[0145] PF4 has been reported to inhibit angiogenesis (Reference 43). Therefore, it is possible that anti-PF4 mAb suppresses tumor growth through factors other than immunity, such as angiogenesis. Therefore, to evaluate whether the anti-tumor effect induced by anti-PF4 mAb is immune-dependent, we evaluated the effect of administering anti-PF4 mAb to RAG2-deficient mice, which are completely deficient in T cells and B cells. As a result, tumor growth inhibition by anti-PF4 mAb was completely lost in RAG2-deficient mice (Figure 5G). This indicates that tumor growth delay mediated by anti-PF4 mAb (#6-1-5) is dependent on lymphocytes (and therefore immunity) but not on other non-immune effects.

[0146] Regarding safety, administration of anti-PF4 mAb (#6-1-5) did not result in weight loss. Furthermore, administration of this antibody did not result in abnormal activation of immune cells (Figs. 7A to 7D). Note that such abnormal activation of immune cells is due to CD44 ｈｉｇｈ CD62L ｌｏｗ CD4 ＋ or CD8 ＋ This includes an increase in the proportion of T cells, which is strongly and rapidly induced by total Treg depletion but not after selective Th1-Treg depletion (Reference 6). Therefore, PF4 neutralization-dependent Th1-Treg depletion may be safer than total Treg depletion with respect to autoimmunity.

[0147] From the perspective of the relationship between human cancer and immunity, we analyzed the correlation between the expression level of PF4 and the prognosis of various cancer patients using the Cancer Genome Atlas (TCGA) database. ｈｉｇｈ The survival probability of patients with pan-tumor is PF4 ｌｏｗ It was revealed that the PF4 level was significantly lower than that of patients with tumors (Fig. 8A). ｈｉｇｈ In the group, CD11b ｈｉｇｈ The survival rate of cancer patients is higher with CD11b ｌｏｗ The survival probability of cancer-bearing patients was lower than that of PF4 (Fig. 8B). ＋Higher numbers of TAMs may be associated with poorer prognosis in humans. Given that anti-PF4 mAb (#6-1-5) also recognizes human PF4 protein (Fig. 8A), neutralization of PF4 using humanized anti-PF4 mAb (#6-1-5) through selective depletion of Th1-Tregs may be useful in cancer immunotherapy strategies for PF4-high cancer patients.

[0148] Example 6: Anti-PF4 mAb (#6-1-5) and com-anti-PF4 recognize different sites on PF4. We attempted to identify the epitope sites of anti-PF4 mAb (#6-1-5) with neutralizing activity and com-anti-PF4 without neutralizing activity using hydrogen-deuterium exchange mass spectrometry (HDX-MS).

[0149] First, mouse PF4 as an antigen and anti-PF4 mAb (#6-1-5) were prepared at the concentrations shown below. <Preparation of antigen-only sample> Antigen 1.4 mg / mL (170 μM): 35 μL (final concentration: 0.49 mg / mL, 60 μM) PBS 65 μL Total 100 μL <Preparation of antigen-antibody complex sample> Antigen 1.4 mg / mL (170 μM): 35 μL (final concentration: 0.49 mg / mL, 60 μM) Antibody 9.2 mg / mL (63.4 μM): 50 μL (final concentration: 32 μM) PBS 15 μL Total 100 μL.

[0150] HDX-MS was performed under two conditions: PF4 alone and PF4 and anti-PF4 mAb (#6-1-5), and the degree of hydrogen-deuterium substitution was compared. As a result, as shown in Figures 9A to 9C, a large amount of hydrogen-deuterium substitution was observed from alanine 72 to isoleucine 86 (A72-I86) of mouse PF4, demonstrating that the A72-I86 site is the epitope site of anti-PF4 mAb (#6-1-5) with neutralizing activity.

[0151] Next, the epitope site of com-anti-PF4, which has no neutralizing activity, was identified using HDX-MS in the same manner as described above. Each sample was prepared at the following concentrations. <Preparation of antigen-only sample> Antigen 1.4 mg / mL (170 μM): 35 μL (final concentration: 0.49 mg / mL, 60 μM) PBS 65 μL Total 100 μL <Preparation of antigen-antibody complex sample> Antigen 1.4 mg / mL (170 μM): 35 μL (final concentration: 0.49 mg / mL, 60 μM) Antibody 8.8 mg / mL (60.4 μM): 50 μL (final concentration: 30.2 μM) PBS 15 μL Total 100 μL.

[0152] As a result, as shown in Figures 10A to 10C, a large amount of hydrogen-deuterium substitution was observed in the region from serine 32 to histidine 55 (S32-H55), indicating that the epitope site of com-anti-PF4, which has no neutralizing activity, is S32-H55, and that this site is different from the epitope site A72-I86 of anti-PF4 mAb (#6-1-5) which has neutralizing activity.

[0153] The serine at position 32, the histidine at position 55, the alanine at position 72, and the isoleucine at position 86 of the mouse PF4 protein (amino acid sequence described in SEQ ID NO: 4) correspond to the serine at position 30, the arginine at position 51, the proline at position 68, and the isoleucine at position 82 of the human PF4 protein (amino acid sequence described in SEQ ID NO: 2), respectively.

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[0155] As described above, the present invention makes it possible to suppress the accumulation of Th1-Treg in tumor tissue, activate anti-tumor immunity, and ultimately treat or prevent cancer. Furthermore, the present invention makes it possible to perform cancer immunotherapy without the onset of autoimmune disease by selectively suppressing Th1-Treg. Therefore, the present invention is useful in the development of anti-cancer agents, cancer treatment, etc.

Claims

1. A composition for suppressing the accumulation of helper T1 regulatory T cells (Th1-Treg) in tumors, comprising as an active ingredient an antibody or a functional fragment thereof that recognizes platelet factor 4 (PF4) protein.

2. The composition according to claim 1, which is a pharmaceutical composition for treating or preventing cancer.

3. An antibody or functional fragment thereof that recognizes PF4 protein, having the characteristics described in (1) or (2) below: (1) A heavy chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 7 to 9, and a light chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 15 to 17; (2) A heavy chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequences set forth in SEQ ID NOs: 10 to 12, and a light chain variable region comprising complementarity determining regions 1 to 3 each comprising the amino acid sequence set forth in SEQ ID NO: 18, an amino acid sequence consisting of Leu-Ala-Ser, and the amino acid sequence set forth in SEQ ID NO: 19.

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