Pharmaceutical composition for preventing or treating cancer

Abstract

The present invention addresses the problem of clarifying the relationship between the onset and progression of cancer and macrophages and providing a novel pharmaceutical composition for preventing or treating cancer. The present invention provides: a pharmaceutical composition for preventing or treating cancer, said pharmaceutical composition containing, as an active ingredient, a substances having an ability to kill CD206-positive M2 macrophages; an agent for killing fibroblasts around a tumor tissue; an agent for promoting the infiltration of cytotoxic T cells into a tumor tissue; an agent for inhibiting the expression of a gene, said gene inhibiting the infiltration of cytotoxic T cells into a tumor, in fibroblasts; an agent for promoting the proliferation of cytotoxic T cells; an agent for enhancing the cytotoxic activity of cytotoxic T cells; and an agent for enhancing the expression of a gene that relates to the cytotoxicity of cytotoxic T cells.

Description

Pharmaceutical composition for preventing or treating cancer

[0001] The present invention relates to pharmaceutical compositions for preventing or treating cancer, which contain, as an active ingredient, a substance capable of killing CD206-positive M2 macrophages.

[0002] There are a variety of anticancer drugs based on various mechanisms that are used in cancer treatment, but no definitive anticancer drug has yet been developed, and the development of more powerful anticancer drugs and cancer treatment adjuvants is desired. In addition, there are cases where resistance to anticancer drugs develops, making the drugs ineffective, and so the development of new anticancer drugs is required.

[0003] Macrophages are one of the main cells in the cancer microenvironment. Some macrophages contribute to the proliferation and metastasis of cancer cells (Non-Patent Document 1). Reducing macrophages, which protect cancer cells, is thought to be beneficial for cancer treatment, but this has not yet been adopted as a general treatment.

[0004] Macrophages have been broadly classified into M1 and M2 macrophages using cell surface markers for analysis. Although there are reports suggesting that M2 macrophages function in protecting cancer cells, macrophages are diverse cells, and it is now thought that simply classifying them into two types is inappropriate and that they should be analyzed in more detail.

[0005] CD206 is one of the markers for M2 macrophages. However, whether CD206-positive M2 macrophages are actually related to the onset and progression of cancer, and what role they play, have not been fully investigated in vivo, and the molecular mechanism underlying this has remained unclear.

[0006] Nat Rev Immunol. 19(6): 369-382, 2019

[0007] In view of the above circumstances, an objective of the present invention is to clarify the relationship between the onset and progression of cancer and macrophages, and to provide a novel pharmaceutical composition for preventing or treating cancer.

[0008] The present inventors have conducted extensive research to clarify the relationship between macrophages and cancer onset and progression, and to develop novel pharmaceutical compositions for the prevention or treatment of cancer. Specifically, using a unique mouse model, they investigated the properties and role of CD206-positive M2 macrophages in cancer onset and progression. As a result, they demonstrated that CD206-positive M2 macrophages contribute to the promotion of cancer onset and progression in an in vivo model. They also demonstrated that depletion of CD206-positive M2 macrophages can induce anti-cancer responses. Furthermore, they found that depletion of CD206-positive M2 macrophages can promote various anti-tumor immune responses. Based on these findings, the present invention was completed.

[0009] That is, the gist of the present invention is as follows: [1] An agent for killing fibroblasts around tumor tissue, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages. [2] An agent for promoting cytotoxic T cell infiltration into tumor tissue, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages. [3] The agent for promoting cytotoxic T cell infiltration into tumor tissue according to [2], wherein the promotion of cytotoxic T cell infiltration into tumor tissue is based on a decrease in fibroblasts around the tumor tissue. [4] An agent for suppressing the expression of one or more genes selected from Acta2, CXCL12, and CXCL14 in fibroblasts around tumor tissue, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages. [5] An agent for promoting the proliferation of cytotoxic T cells, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages. [6] An agent for enhancing the cytotoxic activity of cytotoxic T cells, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages. [7] An agent for enhancing the expression of one or more genes selected from TNFα, IFNγ, and Gzmb, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages.

[0010] [8] A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a substance capable of killing CD206-positive M2 macrophages. [9] The pharmaceutical composition for preventing or treating cancer according to [8], wherein the substance capable of killing CD206-positive M2 macrophages is an anti-CD206 antibody or a functional fragment thereof.

[10] The pharmaceutical composition for preventing or treating cancer according to [9], wherein the antibody is an antibody selected from the following: (a) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 27, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 28, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 29, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 30, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31; (b) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 37, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 38, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 39, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40, a light chain LCDR2 consisting of the amino acid sequence represented by LMS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 41; (c) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 42, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 43, (d) an antibody comprising a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 44, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 45, a light chain LCDR2 consisting of the amino acid sequence represented by RAS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 46; (d) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 47, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 48, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 49, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 50, a light chain LCDR2 consisting of the amino acid sequence represented by FAS, and(e) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 52, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 53, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 54, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 55, a light chain LCDR2 consisting of the amino acid sequence represented by AAT, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 56; (f) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 57, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 58, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 59, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 60, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 61; (g) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 62, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 63, and a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 64, (h) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 67, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 68, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 69, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 70, a light chain LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 71; (i) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 77, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 78, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 79, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 80,(j) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 82, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 83, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 84, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 85, a light chain LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: RAN, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 86; and (k) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 87, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 88, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 89, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 90, a light chain LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 91.

[11] The pharmaceutical composition for preventing or treating cancer according to [9] or

[10] , wherein the antibody is selected from the following: (A) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; (B) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity;(C) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206; (D) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206; (E) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206; (F) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; (G) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity;(H) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; (I) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; (J) a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; (K) an antibody comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity; and (K) an antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having CD206-binding activity.

[12] A method for preventing or treating cancer, comprising the step of administering a substance capable of killing CD206-positive M2 macrophages to a subject in need of such prevention or treatment.

[13] Use of a substance capable of killing CD206-positive M2 macrophages for preventing or treating cancer.

[14] Use of a substance capable of killing CD206-positive M2 macrophages for the manufacture of a pharmaceutical composition for preventing or treating cancer.

[15] A method for killing fibroblasts surrounding tumor tissue, comprising the step of administering to a subject a substance capable of killing CD206-positive M2 macrophages.

[16] Use of a substance capable of killing CD206-positive M2 macrophages for killing fibroblasts surrounding tumor tissue.

[17] Use of a substance capable of killing CD206-positive M2 macrophages for producing an agent for killing fibroblasts surrounding tumor tissue.

[18] A method for promoting the infiltration of cytotoxic T cells into tumor tissue, comprising the step of administering to a subject a substance capable of killing CD206-positive M2 macrophages.

[19] Use of a substance capable of killing CD206-positive M2 macrophages for promoting the infiltration of cytotoxic T cells into tumor tissue.

[20] Use of a substance capable of killing CD206-positive M2 macrophages for producing an agent for promoting the infiltration of cytotoxic T cells into tumor tissue.

[21] A method for suppressing the expression of one or more genes selected from Acta2, CXCL12, and CXCL14 in fibroblasts surrounding tumor tissue, comprising the step of administering to a subject a substance capable of killing CD206-positive M2 macrophages.

[22] Use of a substance capable of killing CD206-positive M2 macrophages for suppressing the expression of one or more genes selected from Acta2, CXCL12, and CXCL14 in fibroblasts surrounding tumor tissue.

[23] Use of a substance capable of killing CD206-positive M2 macrophages for the manufacture of an agent for suppressing the expression of one or more genes selected from Acta2, CXCL12, and CXCL14 in fibroblasts surrounding tumor tissue.

[24] A method for promoting the proliferation of cytotoxic T cells, comprising the step of administering to a subject a substance capable of killing CD206-positive M2 macrophages.

[25] Use of a substance capable of killing CD206-positive M2 macrophages for promoting the proliferation of cytotoxic T cells.

[26] Use of a substance capable of killing CD206-positive M2 macrophages for the manufacture of an agent for promoting the proliferation of cytotoxic T cells.

[27] A method for enhancing the cytotoxic activity of cytotoxic T cells, comprising the step of administering to a subject a substance capable of killing CD206-positive M2 macrophages.

[28] Use of a substance capable of killing CD206-positive M2 macrophages for enhancing the cytotoxic activity of cytotoxic T cells.

[29] Use of a substance capable of killing CD206-positive M2 macrophages for producing an agent for enhancing the cytotoxic activity of cytotoxic T cells.

[30] A method for enhancing the expression of one or more genes selected from TNFα, IFNγ, and Gzmb, comprising the step of administering to a subject a substance capable of killing CD206-positive M2 macrophages.

[31] Use of a substance capable of killing CD206-positive M2 macrophages for enhancing the expression of one or more genes selected from TNFα, IFNγ, and Gzmb.

[32] Use of a substance capable of killing CD206-positive M2 macrophages for producing an agent for enhancing the expression of one or more genes selected from TNFα, IFNγ, and Gzmb.

[0011] The present invention clarifies the relationship between macrophages and the onset and progression of cancer and provides a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a substance capable of removing CD206-positive M2 macrophages. The present invention also provides an agent for killing fibroblasts surrounding tumor tissue, an agent for promoting the infiltration of cytotoxic T cells into tumor tissue, an agent for suppressing the expression of genes in fibroblasts that suppress the infiltration of cytotoxic T cells into tumors, an agent for promoting the proliferation of cytotoxic T cells, an agent for enhancing the cytotoxic activity of cytotoxic T cells, and an agent for enhancing the expression of genes related to the cytotoxicity of cytotoxic T cells, each of which comprises as an active ingredient a substance capable of removing CD206-positive M2 macrophages.

[0012] Figure 1 shows the results of evaluating the effect of CD206-positive M2 macrophage depletion on tumors. The left panel shows the effect of CD206-positive M2 macrophage depletion on tumor incidence. The middle panel shows the effect of CD206-positive M2 macrophage depletion on tumor weight. The right panel shows the effect of CD206-positive M2 macrophage depletion on tumor volume. Figure 2 shows the results of evaluating the effect of CD206-positive M2 macrophage depletion on CD8-positive T cell counts. The two panels on the left show the results of immunohistochemical (IHC) staining of tumor CD8-positive T cells (photographs used as drawing substitutes). The right panel shows the results of flow cytometry (FACS) analysis of tumor CD8-positive T cells. Figure 3 shows the results of evaluating the effect of CD206-positive M2 macrophage depletion on CD8-positive T cell cytotoxicity (assessed by qPCR of genes related to cytotoxicity). Figure 4 shows the results of evaluating the effect of CD206-positive M2 macrophage depletion on the number of fibroblasts surrounding tumor tissue. The two left figures show the results of immunohistochemical (IHC) staining of tumor CD8-positive T cells and fibroblasts (photographs used as drawing substitutes). The two right figures show the results of analyzing tumor CD8-positive T cells and fibroblasts. Figure 5 shows the results of evaluating the effect of fibroblasts surrounding tumor tissue on the inhibitory activity of CD8-positive T cells in tumor infiltration (evaluation by qPCR of genes related to CD8-positive T cell infiltration). Figure 6 shows the results of flow cytometric detection of CD206-positive M2 macrophages using the anti-CD206 antibody used in the present invention. Figure 7 shows the results of evaluating the CDC activity of the anti-CD206 antibody used in the present invention against CD206-positive cells. Figure 8 shows the results of evaluating the ADCC activity of the anti-CD206 antibody used in the present invention against CD206-positive cells. Figure 9 shows the results of evaluating the cytotoxicity of the anti-CD206 antibody used in the present invention against CD206-positive M2 macrophages in biological tissues (qPCR). Figure 10 shows the results of evaluating the cytotoxicity of the anti-CD206 antibody used in the present invention against CD206-positive M2 macrophages in biological tissues (FACS).

[0013] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In this specification, when a numerical range is expressed as "lower limit to upper limit," the upper limit may be "equal to or less than" or "less than," and the lower limit may be "equal to or greater than" or "more than."

[0014] <Pharmaceutical composition for preventing or treating cancer> One aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, a substance capable of killing CD206-positive M2 macrophages (hereinafter, may be referred to as the "pharmaceutical composition of the present invention for preventing or treating cancer").

[0015] As described above, the present inventors have been investigating new pharmaceutical compositions for the prevention or treatment of cancer, and have demonstrated in an in vivo model that CD206-positive M2 macrophages contribute to the promotion of cancer onset and progression. They have also found that tumor development can be suppressed and tumors can be reduced by removing CD206-positive M2 macrophages. Based on these findings, the present invention has devised a pharmaceutical composition for the prevention or treatment of cancer.

[0016] Furthermore, the present inventors have demonstrated that depletion of CD206-positive M2 macrophages can promote antitumor immune responses by: 1) increasing the number of CD8-positive T cells; 2) enhancing the cytotoxicity of CD8-positive T cells; 3) reducing fibroblasts that function in a cancer-protective manner (particularly, reducing fibroblasts that surround cancer cells and prevent the infiltration of cytotoxic T cells into cancer tissues promotes the migration of cytotoxic T cells into cancer tissues); and 4) reducing the cancer-protective function of fibroblasts. Such methods are believed to be beneficial in cancer treatment. That is, the present invention further provides: 1) means for controlling the number and cytotoxicity of CD8-positive T cells that attack tumors; and 2) means for controlling the number and properties of fibroblasts that inhibit the infiltration of CD8-positive T cells into tumors, as described below.

[0017] <<Substances Capable of Killing CD206-Positive M2 Macrophages>> The substances capable of killing CD206-positive M2 macrophages used in the present invention may be any substances that act on CD206-positive M2 macrophages to kill (or damage) them and eliminate them ("elimination" does not mean complete elimination but also includes partial elimination to reduce their numbers), such as substances that act directly on CD206-positive M2 macrophages to kill (or damage) them, or substances that act indirectly on CD206-positive M2 macrophages to kill (or damage) them. Substances capable of killing CD206-positive M2 macrophages may be used singly or in combination of two or more.

[0018] Specific examples of substances capable of killing CD206-positive M2 macrophages used in the present invention include low-molecular-weight compounds, anti-CD206 antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and functional fragments thereof, as well as siRNAs (short interfering RNAs), shRNAs (short hairpin RNAs), and antisense oligonucleotides. Of these substances, preferred are anti-CD206 antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and functional fragments thereof, more preferred are anti-CD206 antibodies or functional fragments thereof, and particularly preferred are anti-CD206 antibodies.

[0019] <<Anti-CD206 Antibody>> The anti-CD206 antibody used in the present invention may be any antibody capable of binding to CD206 on CD206-positive M2 macrophages and killing them. Specifically, the CD206-positive M2 macrophage-killing activity may be one or more activities selected from CDC activity and ADCC activity. The anti-CD206 antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. In the present invention, a monoclonal antibody is preferred. Furthermore, the anti-CD206 antibody used in the present invention may be a CD206 monospecific antibody or a multispecific antibody that recognizes CD206 and multiple other antigens, but is preferably a CD206 monospecific antibody.

[0020] The anti-CD206 antibodies used in the present invention may be produced by standard methods used in molecular biology. Examples include polyclonal and monoclonal antibodies obtained by immunizing mammals such as mice with the CD206 protein or a partial fragment thereof (e.g., the epitope fragments described above), chimeric and humanized antibodies produced using genetic recombination techniques, and human antibodies produced using human antibody-producing transgenic animals. When administering the antibodies of the present invention to humans as pharmaceuticals, humanized or human antibodies are preferred in terms of side effects. Furthermore, the anti-CD206 antibodies used in the present invention can also be produced by the ISAAC method described below.

[0021] <<Anti-CD206 Antibodies or Functional Fragments thereof>> Specific examples of anti-CD206 antibodies used in the present invention include the antibodies (a) to (k) and (A) to (K) described below.

[0022] The anti-CD206 antibody used in the present invention can be obtained, for example, by administering an antigen to an animal that produces an antibody against a specific antigen, immunizing the animal, recovering the antibody-producing cells, and then screening using the ISAAC (ImmunoSpot Array Assay on a Chip) method. The ISAAC method is a method for rapidly and comprehensively screening antigen-specific antibody-secreting cells (ASCs) using a microwell array chip. This method uses a microwell array chip with multiple wells (approximately 10-15 μm in diameter) large enough to accommodate a single antibody-producing cell, with the chip surface surrounding the wells coated with anti-immunoglobulin antibodies (or the antigen from which the antibody is to be obtained). Splenic lymphocytes containing antibody-producing cells are collected from an animal that produces an antibody against a specific antigen, and the cells are seeded individually into the wells of the microwell array chip. The chip is immersed in culture medium and cultured under conditions that allow the secreted antibodies to diffuse from the wells into the coating layer. The antibodies secreted from the antibody-producing / secreting cells bind to the anti-immunoglobulin antibodies (or antigens) surrounding the wells. Next, the antigen (or antibody against the antigen) labeled with a fluorescent substance is added to the microwell array chip. The labeled antigen (or antibody against the antigen) binds to the anti-immunoglobulin antibody (or antigen) surrounding the well, and the signal emitted from the labeling substance identifies which well contains cells that produce and secrete antigen-specific antibodies. These cells are then collected using a microcapillary under a fluorescent microscope. DNA is extracted from the cells, and the antibody cDNA is amplified by RT-PCR or other methods, and the antibody gene is cloned. The gene can be used to produce the desired antibody as a recombinant antibody.

[0023] CD206, also known as the mannose receptor, is a C-type lectin receptor with a single transmembrane domain and is a surface antigen specific to M2 macrophages. The anti-CD206 antibody used in the present invention can be prepared as a recombinant mouse anti-human CD206 monoclonal antibody by, for example, collecting lymphocytes from the spleen of a CD206-deficient mouse administered with a CD206 antigen, and then is subjected to the above-described ISAAC to obtain cDNA for a mouse antibody against CD206.

[0024] The ISAAC method can be performed according to the descriptions in JP 2009-34047 A, JP 2014-73100 A, JP 2014-162772 A, Aishun Jin et al., Nature Medicine, Vol. 15, No. 9, September 2009, pp. 1088-1093, and Aishun Jin et al., Nature Protocols, Vol. 6, No. 5, 2011, pp. 668-676.

[0025] The following 11 mouse anti-human anti-CD206 monoclonal antibodies were obtained by the ISAAC method as anti-CD206 antibodies for use in the present invention: Nos. 10, 27, 35, 40, 44, 47, 54, 59, 72, 73, and 83.

[0026] Monoclonal antibodies having the above-mentioned specific sequences can also be obtained using techniques commonly used in the art, such as chemical synthesis and genetic recombination, based on the sequence information.

[0027] The anti-CD206 antibodies used in the present invention include not only the monoclonal antibodies but also recombinant antibodies artificially modified to reduce heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods. As used herein, the terms "antibodies No. 10, 27, 35, 40, 44, 47, 54, 59, 72, 73, and 83" include chimeric and humanized antibodies produced based on the sequences of the antibodies, unless otherwise inconsistent.

[0028] The anti-CD206 antibodies used in the present invention also include shuffled antibodies containing the H chain or L chain of any of antibodies Nos. 10, 27, 35, 40, 44, 47, 54, 59, 72, 73, and 83.

[0029] The anti-CD206 antibodies used in the present invention can be produced by inserting DNA encoding the antibody heavy chain and DNA encoding the light chain into an expression vector, transforming host cells with the vector, and culturing the host cells. In this case, the DNA encoding the heavy chain and DNA encoding the light chain may be inserted into the same expression vector and used to transform host cells, or the DNA encoding the heavy chain and DNA encoding the light chain may be inserted into separate vectors and used to transform host cells. In this case, DNA encoding the heavy chain variable region and DNA encoding the light chain variable region may be inserted into a vector already containing DNA encoding the heavy chain constant region and light chain constant region of a specific isotype. The vector may also contain DNA encoding a signal peptide that promotes antibody secretion from host cells. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are ligated in-frame. The signal peptide is removed during antibody production, allowing the antibody to be obtained as a mature protein.

[0030] The DNA and amino acid sequences of the heavy chain variable regions, the DNA and amino acid sequences of the light chain variable regions, the amino acid sequences of the CDRs (complementarity determining regions) (HCDR1, HCDR2, and HCDR3) of the heavy chain variable regions, and the amino acid sequences of the CDRs (complementarity determining regions) (LCDR1, LCDR2, and LCDR3) of the light chain variable regions of the 11 anti-CD206 monoclonal antibodies are shown below as sequences or SEQ ID NOs.

[0031] (Antibody No. 10) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (a) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 27, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 28, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 29, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 30, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31.

[0032] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (A) an antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0033] (Antibody No. 27) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (b) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 37, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 38, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 39, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40, a light chain LCDR2 consisting of the amino acid sequence represented by LMS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 41.

[0034] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (B) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0035] (Antibody No. 35) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (c) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 42, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 43, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 44, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 45, a light chain LCDR2 consisting of the amino acid sequence represented by RAS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 46.

[0036] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (C) An antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0037] (Antibody No. 40) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (d) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 47, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 48, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 49, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 50, a light chain LCDR2 consisting of the amino acid sequence represented by FAS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 51.

[0038] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (D) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 9 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0039] (Antibody No. 44) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (e) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 52, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 53, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 54, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 55, a light chain LCDR2 consisting of the amino acid sequence represented by AAT, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 56.

[0040] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (E) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0041] (Antibody No. 47) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (f) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 57, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 58, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 59, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 60, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 61.

[0042] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (F) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0043] (Antibody No. 54) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (g) An antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 62, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 63, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 64, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 65, a light chain LCDR2 consisting of the amino acid sequence represented by KAS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 66.

[0044] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (G) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0045] (Antibody No. 59) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (h) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 67, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 68, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 69, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 70, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 71.

[0046] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (H) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 17 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0047] (Antibody No. 72) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (i) an antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 77, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 78, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 79, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 80, a light chain LCDR2 consisting of the amino acid sequence represented by ATS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 81.

[0048] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (I) an antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 22 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0049] (Antibody No. 73) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (j) An antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 82, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 83, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 84, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 85, a light chain LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: RAN, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 86.

[0050] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (J) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 25 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 26 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0051] (Antibody No. 83) An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (k) An antibody comprising a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 87, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 88, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 89, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 90, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 91.

[0052] An antibody or functional fragment thereof that specifically binds to CD206, wherein the antibody is one of the following antibodies: (K) An antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 25 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 26 or an amino acid sequence having 90% or more sequence identity with said amino acid sequence, and having binding activity to CD206.

[0053] In the above 11 types of anti-CD206 monoclonal antibodies, the heavy chain variable regions include not only heavy chain variable regions consisting of the amino acid sequences represented by the respective SEQ ID NOs, but also heavy chain variable regions consisting of amino acid sequences with one or several, for example, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid deletion, substitution, or addition, of the amino acid sequences, and consisting of proteins with the activity of an antibody heavy chain variable region, i.e., binding activity to human CD206. Similarly, the light chain variable regions include not only light chain variable regions consisting of the amino acid sequences represented by the respective SEQ ID NOs, but also light chain variable regions consisting of amino acid sequences with one or several, for example, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid deletion, substitution, or addition, of the amino acid sequences, and consisting of proteins with the activity of an antibody light chain variable region, i.e., binding activity to human CD206.

[0054] Although such substitutions, deletions, and additions may be introduced into the CDR, they are preferably introduced into regions other than the CDR. Furthermore, to maintain the properties of the present invention, the amino acid substitutions are preferably conservative. Here, conservative substitution refers to replacing an amino acid residue with another chemically similar amino acid residue without substantially altering the activity of the peptide. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue with the same charge. Examples of functionally similar amino acids that can be substituted in this way include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0055] Such amino acid sequences in which one or several amino acids have been deleted, substituted, or added include those that have, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of each SEQ ID NO when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, default, i.e., initial setting parameters).

[0056] A protein having such an amino acid sequence in which one or several amino acids are deleted, substituted or added is substantially identical to a protein having the amino acid sequence of each SEQ ID NO.

[0057] Furthermore, DNA encoding the amino acid sequences represented by the above-mentioned respective SEQ ID NOs: 1 and 2, when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., default, i.e., initial setting parameters), includes DNA consisting of a base sequence that has, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the DNA sequence, and that encodes a protein having the activity of an antibody heavy chain variable region or light chain variable region, i.e., binding activity to human CD206.

[0058] Furthermore, DNA encoding the heavy chain variable region or light chain variable region of an anti-CD206 antibody also includes DNA that can hybridize under stringent conditions with DNA consisting of a sequence complementary to the DNA encoding the amino acid sequence represented by each of the above SEQ ID NOs, and that encodes a protein having the activity of the heavy chain variable region or light chain variable region of an antibody, i.e., the activity of binding to human CD206. Those skilled in the art can determine the stringent conditions as appropriate.

[0059] Furthermore, a human anti-human CD206 monoclonal antibody that specifically binds to human CD206 and contains the heavy chain variable region and light chain variable region is composed of the heavy chain variable region and heavy chain constant region and the light chain variable region and light chain constant region. The heavy chain constant region is composed of three domains, C H 1. C H 2 and C H The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. The light chain constant region is composed of one domain, C L The light chain constant region is either a kappa or lambda constant region.

[0060] DNA encoding an antibody can be obtained as DNA encoding a heavy chain and DNA encoding a light chain by ligating DNA encoding a heavy chain variable region to DNA encoding a heavy chain constant region, and then ligating DNA encoding a light chain variable region to DNA encoding a light chain constant region.

[0061] The anti-CD206 antibodies used in the present invention also include functional fragments of antibodies or modified versions thereof. For example, functional fragments of antibodies are antibody fragments capable of specifically binding to antigens. Functional fragments include Fab, F(ab'), Fv, Fab / c having one Fab and an intact Fc, and single-chain Fv (scFv) in which the Fv of the H chain or L chain is linked via an appropriate linker. Polynucleotides include both DNA and RNA.

[0062] The anti-CD206 antibodies bind to CD206, preferably to the extracellular domain of CD206 present on the cell membrane surface of M2 macrophages, and have a cytocidal effect on CD206-positive M2 macrophages. Specifically, the cytocidal effect may be one or more activities selected from CDC activity and ADCC activity. Thus, the above-mentioned anti-CD206 antibodies have a cytocidal effect against CD206-positive M2 macrophages and can remove CD206-positive M2 macrophages from biological tissues and the like. They also have anticancer effects, killing fibroblasts around tumor tissue, promoting the infiltration of cytotoxic T cells into tumor tissue, inhibiting the expression of genes in fibroblasts that suppress the infiltration of cytotoxic T cells into tumors, promoting the proliferation of cytotoxic T cells, enhancing the cytotoxic activity of cytotoxic T cells, and enhancing the expression of genes associated with the cytotoxicity of cytotoxic T cells, and can be used as the active ingredient of pharmaceutical compositions for the prevention or treatment of cancer, agents for killing fibroblasts around tumor tissue, agents for promoting the infiltration of cytotoxic T cells into tumor tissue, agents for inhibiting the expression of genes in fibroblasts that suppress the infiltration of cytotoxic T cells into tumors, agents for promoting the proliferation of cytotoxic T cells, agents for enhancing the cytotoxic activity of cytotoxic T cells, and agents for enhancing the expression of genes associated with the cytotoxicity of cytotoxic T cells.

[0063] Any of the 11 mouse anti-human CD206 monoclonal antibodies can be used as an active ingredient in the pharmaceutical compositions for preventing or treating cancer, including agents for killing fibroblasts around tumor tissue, agents for promoting cytotoxic T cell infiltration into tumor tissue, agents for suppressing the expression of genes in fibroblasts that suppress the infiltration of cytotoxic T cells into tumors, agents for promoting the proliferation of cytotoxic T cells, agents for enhancing the cytotoxic activity of cytotoxic T cells, and agents for enhancing the expression of genes related to the cytotoxicity of cytotoxic T cells, all of which have the ability to kill CD206-positive M2 macrophages. Among these, antibodies Nos. 10, 35, 54, and 83, which strongly bind to CD206, are preferred, but not limited to these. These antibodies may be used alone or in combination of two or more.

[0064] The pharmaceutical compositions for cancer prevention or treatment of the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical compositions for cancer prevention or treatment of the present invention may contain active ingredients other than the active ingredient, a substance capable of killing CD206-positive M2 macrophages, as long as the effects are not impaired. The pharmaceutical compositions for cancer prevention or treatment of the present invention may contain carriers, diluents, and excipients commonly used in the pharmaceutical field in addition to the active ingredient, a substance capable of killing CD206-positive M2 macrophages. For example, lactose, magnesium stearate, etc. are used as carriers and excipients for tablets. Aqueous solutions for injection include physiological saline, PBS, and isotonic solutions containing glucose and other adjuvants, which may be used in combination with appropriate solubilizers, such as alcohol, polyalcohols such as propylene glycol, and nonionic surfactants. Oily solutions include sesame oil, soybean oil, etc., and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The pharmaceutical composition for preventing or treating cancer of the present invention can be administered in various forms, including oral administration using tablets, capsules, granules, powders, syrups, etc., or parenteral administration using injections, drip infusions, suppositories, etc.

[0065] The dosage varies depending on symptoms, age, body weight, etc., but typically, for oral administration, the adult dose is, for example, about 0.01 mg to 1000 mg per day, which can be administered once or in divided doses. For parenteral administration, for example, about 0.01 mg to 1000 mg per dose can be administered by subcutaneous injection, intramuscular injection, or intravenous injection.

[0066] Cancers that can be prevented or treated include, but are not limited to, lung cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, colon cancer, biliary tract cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, testicular cancer, skin cancer, bone tumors, osteosarcomas, soft tissue tumors, pharyngeal cancer, head and neck cancer, and childhood cancer.

[0067] The subjects for prevention or treatment in the present invention (preferably mammals, particularly humans) are subjects who have been diagnosed as having a high probability of developing cancer or subjects who have developed cancer, and the pharmaceutical composition for preventing or treating cancer of the present invention can be administered to these subjects.

[0068] <Agent for killing fibroblasts around tumor tissue> Another aspect of the present invention relates to an agent for killing fibroblasts around tumor tissue (hereinafter sometimes referred to as "the agent for killing fibroblasts around tumor tissue of the present invention"), which comprises as an active ingredient a substance capable of killing CD206-positive M2 macrophages.

[0069] As used herein, fibroblasts surrounding tumor tissue can be rephrased as fibroblasts localized in the tumor microenvironment, or fibroblasts localized in the tumor microenvironment that can confer a protective function to the tumor, etc. Alternatively, fibroblasts surrounding tumor tissue may be fibroblasts that are present so as to surround the tumor tissue.

[0070] As described above, the present inventors have demonstrated that depletion of CD206-positive M2 macrophages can reduce tumor-protective fibroblasts localized around tumor tissue. This reduction in tumor-protective fibroblasts is believed to enhance antitumor immune responses. In particular, depletion of fibroblasts surrounding tumor tissue facilitates the entry of antitumor drugs, substances, and cells into tumor tissue, thereby promoting tumor killing.

[0071] The optional ingredients, dosage, target cancer, target of prevention or treatment, etc., as explained in the above section <Pharmaceutical composition for preventing or treating cancer> can also be applied to the agent for killing fibroblasts around tumor tissue of the present invention. Furthermore, the agent for killing fibroblasts around tumor tissue of the present invention can also be used as a reagent, and the production and use of the reagent can be carried out based on conventional methods in the field of molecular biology.

[0072] <Agent for promoting cytotoxic T cell infiltration into tumor tissue> Another aspect of the present invention relates to an agent for promoting cytotoxic T cell infiltration into tumor tissue, which comprises, as an active ingredient, a substance capable of killing CD206-positive M2 macrophages (hereinafter, may be referred to as "the agent for promoting cytotoxic T cell infiltration into tumor tissue of the present invention").

[0073] The term "cytotoxic T lymphocyte or cytotoxic T cell (CTL)" is used herein interchangeably with the term "CD8-positive T cell" and refers to a subgroup of T lymphocytes that can recognize non-self cells (e.g., tumor / cancer cells, virus-infected cells) and induce the death of such cells.

[0074] As described above, the inventors have demonstrated that depletion of CD206-positive M2 macrophages can reduce the infiltration-inhibitory function of fibroblasts localized around tumor tissue, which inhibits the infiltration of cytotoxic T cells into tumors. Reducing the infiltration-inhibitory function of fibroblasts and their cancer-protective function is believed to enhance antitumor immune responses. In one embodiment, the promotion of cytotoxic T cell infiltration into tumor tissue is based on a reduction in fibroblasts around tumor tissue. A reduction in fibroblasts around tumor tissue is believed to facilitate the infiltration (introduction / invasion) of cytotoxic T cells into tumor tissue. In one embodiment, the promotion of cytotoxic T cell infiltration into tumor tissue is based on the suppression of expression of genes (e.g., one or more genes selected from Acta2, CXCL12, and CXCL14) in fibroblasts around tumor tissue that suppress the infiltration of cytotoxic T cells into tumors. Here, suppression of gene expression can be assessed by measuring the expression or secretion level of the protein encoded by the gene, the expression level of the gene (mRNA or cDNA), or the like. Furthermore, it is believed that the agent of the present invention facilitates the introduction of anticancer agents into tumor tissue, and the present invention can also be used as an adjuvant for cancer treatment.

[0075] The optional ingredients, dosage, target cancer, and target of prevention or treatment described above in the section <Pharmaceutical composition for cancer prevention or treatment> can also be applied to the agent for promoting cytotoxic T cell infiltration into tumor tissue of the present invention. Furthermore, the agent for promoting cytotoxic T cell infiltration into tumor tissue of the present invention can also be used as a reagent, and the production and use of the reagent can be carried out based on conventional methods in the field of molecular biology.

[0076] <Expression inhibitor of genes that suppress the infiltration of cytotoxic T cells into tumors in fibroblasts> Another aspect of the present invention relates to an expression inhibitor of one or more genes selected from Acta2, CXCL12, and CXCL14 in fibroblasts surrounding tumor tissue, which comprises as an active ingredient a substance capable of killing CD206-positive M2 macrophages (hereinafter, sometimes referred to as "the expression inhibitor of genes that suppress the infiltration of cytotoxic T cells into tumors in fibroblasts of the present invention").

[0077] The present inventors have demonstrated that the promotion of cytotoxic T cell infiltration into tumor tissues can be achieved by depleting CD206-positive M2 macrophages, which can suppress the expression of genes (e.g., one or more genes selected from Acta2, CXCL12, and CXCL14) that suppress the infiltration of cytotoxic T cells into tumors in fibroblasts surrounding tumor tissues. Suppression of the expression of these genes is thought to contribute to a decrease in the infiltration inhibitory function of fibroblasts localized around tumor tissues that inhibits the infiltration of cytotoxic T cells into tumors.

[0078] The optional ingredients, dosage, target cancer, and target of prevention or treatment described above in the section <Pharmaceutical composition for preventing or treating cancer> can also be applied to the inhibitor of gene expression that suppresses tumor infiltration of cytotoxic T cells in fibroblasts of the present invention. Furthermore, the inhibitor of gene expression that suppresses tumor infiltration of cytotoxic T cells in fibroblasts of the present invention can also be used as a reagent, and the production and use of the reagent can be carried out based on standard methods in the field of molecular biology.

[0079] <Agent for promoting proliferation of cytotoxic T cells> Another aspect of the present invention relates to an agent for promoting proliferation of cytotoxic T cells (hereinafter, sometimes referred to as "agent for promoting proliferation of cytotoxic T cells"), which comprises, as an active ingredient, a substance capable of killing CD206-positive M2 macrophages.

[0080] As described above, the present inventors have demonstrated that the number of CD8+ T cells that attack tumors can be increased by removing CD206+ M2 macrophages. It is believed that an increase in the number of CD8+ T cells enhances antitumor immune responses. Furthermore, it is believed that an increase in the number of cytotoxic T cells not only enhances antitumor immune responses, but also enhances immune responses based on the cytotoxic activity of cytotoxic T cells, such as antiviral and antibacterial immune responses. Therefore, the cytotoxic T cell proliferation promoter of the present invention can be used as a therapeutic agent or adjuvant for diseases that can be treated by the cytotoxic activity of cytotoxic T cells.

[0081] The optional ingredients, dosages, target cancers, targets for prevention or treatment, etc., as explained above in the section <Pharmaceutical composition for cancer prevention or treatment> can also be applied to the cytotoxic T cell proliferation promoter of the present invention. Furthermore, the cytotoxic T cell proliferation promoter of the present invention can also be used as a reagent, and the production, use, etc. of the reagent can be carried out based on conventional methods in the field of molecular biology.

[0082] <Agent for enhancing the cytotoxic activity of cytotoxic T cells> Another aspect of the present invention relates to an agent for enhancing the cytotoxic activity of cytotoxic T cells (hereinafter, sometimes referred to as the "agent for enhancing the cytotoxic activity of cytotoxic T cells of the present invention"), which comprises, as an active ingredient, a substance capable of killing CD206-positive M2 macrophages.

[0083] As described above, the inventors have demonstrated that depletion of CD206-positive M2 macrophages can enhance the cytotoxic activity of cytotoxic T cells that attack tumors. It is believed that enhancing the cytotoxic activity of cytotoxic T cells enhances antitumor immune responses. Furthermore, enhancing the cytotoxic activity of cytotoxic T cells is believed to enhance not only antitumor immune responses but also immune responses based on the cytotoxic activity of cytotoxic T cells, such as antiviral and antibacterial immune responses. Therefore, the cytotoxic activity enhancers of the present invention can be used as therapeutic agents or adjuvants for diseases that can be treated with the cytotoxic activity of cytotoxic T cells. In one embodiment, the enhancement of the cytotoxic activity of cytotoxic T cells is based on enhanced expression of genes related to the cytotoxicity of cytotoxic T cells (e.g., one or more genes selected from TNFα, IFNγ, and Gzmb). Here, suppression of gene expression can be evaluated by measuring the expression or secretion level of the protein encoded by the gene, the expression level of the gene (mRNA or cDNA), or the like.

[0084] The optional ingredients, dosages, target tumors, targets for prevention or treatment, etc., as explained above in the section <Pharmaceutical composition for preventing or treating cancer> can also be applied to the enhancer of cytotoxic activity of cytotoxic T cells of the present invention. Furthermore, the enhancer of cytotoxic activity of cytotoxic T cells of the present invention can also be used as a reagent, and the production, use, etc. of the reagent can be carried out based on conventional methods in the field of molecular biology.

[0085] <Agent for enhancing the expression of genes associated with the cytotoxicity of cytotoxic T cells> Another aspect of the present invention relates to an agent for enhancing the expression of one or more genes selected from TNFα, IFNγ, and Gzmb, which comprises, as an active ingredient, a substance capable of killing CD206-positive M2 macrophages (hereinafter, may be referred to as "an agent for enhancing the expression of genes associated with the cytotoxicity of cytotoxic T cells of the present invention").

[0086] As described above, the present inventors have demonstrated that depletion of CD206-positive M2 macrophages can enhance the expression of genes related to the cytotoxicity of cytotoxic T cells (e.g., one or more genes selected from TNFα, IFNγ, and Gzmb). Enhanced expression of these genes is thought to contribute to the enhanced cytotoxic activity of cytotoxic T cells that attack tumors.

[0087] The optional ingredients, dosages, target tumors, targets for prevention or treatment, etc., as explained above in the section <Pharmaceutical composition for preventing or treating cancer> can also be applied to the enhancer for expression of genes associated with the cytotoxicity of cytotoxic T cells of the present invention. Furthermore, the enhancer for expression of genes associated with the cytotoxicity of cytotoxic T cells of the present invention can also be used as a reagent, and the production, use, etc. of the reagent can be carried out based on conventional methods in the field of molecular biology.

[0088] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples as long as it does not depart from the gist of the invention.

[0089] Example 1: Examination of the effect of CD206-positive M2 macrophages on tumors. Analysis of tumors in CD206-positive M2 macrophage reporter mice that develop breast cancer revealed that CD206-positive M2 macrophages are distributed mainly around tumors and adjacent to blood vessels. Based on these results, CD206-DTR / PyMT mice (mice that develop breast cancer and in which CD206-positive macrophages can be reduced by administration of diphtheria toxin) were generated as follows, CD206-positive M2 macrophages were removed, and the effect of CD206-positive M2 macrophages on tumors was examined.

[0090] (CD206-DTR / PyMT mice) PyMT mice (purchased from The Jackson Laboratory), which spontaneously develop breast cancer, were crossed with CD206-DTR mice, which can eliminate CD206-positive cells (Nat. Commun. 8, 554 1-15 (2017)), to produce mice that develop breast cancer and can eliminate CD206-positive cells (CD206-DTR / PyMT mice).

[0091] Methods: We evaluated tumor incidence, weight, and volume in CD206-DTR / PyMT mice after depletion of CD206-positive macrophages. Diphtheria toxin was administered intraperitoneally to CD206-DTR / PyMT mice and control PyMT mice at a dose of 3 ng / g of mouse body weight (n = 19, n = 17). The presence or absence of tumors was confirmed by palpation of the mouse mammary glands. Tumor-free rates (number of mice with tumors / total number of mice in each group) were calculated using the JMP15 analysis software (SAS Institute) to assess tumor incidence after depletion of CD206-positive macrophages. After 9 weeks, CD206-DTR / PyMT mice and control PyMT mice were euthanized, and tumor weight and volume after depletion of CD206-positive macrophages were assessed. The total weight of all tumors was measured using a digital weighing scale (A&D Company). For volumetric evaluation, the size of all tumors was measured using a Digital Caliper DN-100 (Niigata Seiki). The volumetric calculation model was m1 × m1 × m2 × 0.5236 (m1: the shorter axis, m2: the longer axis) and the analysis software JMP15 (SAS Institute).

[0092] Results: The results are shown in Figure 1. Reducing CD206-positive M2 macrophages suppressed tumor development and reduced tumor weight and volume. This demonstrated that CD206-positive M2 macrophages contribute to the promotion of cancer development and progression in an in vivo model. We also demonstrated that reducing CD206-positive M2 macrophages can be expected to have a tumor-reducing effect.

[0093] Example 2: Examination of the effect of CD206-positive M2 macrophages on CD8-positive T cells The effect of CD206-positive M2 macrophages in the cancer microenvironment was examined as follows. Specifically, the effect of CD206-positive M2 macrophages on CD8-positive T cells was examined.

[0094] 2-1: Study of the effect on the number of CD8+ T cells. Methods: CD8+ T cells from the tumors excised in Example 1 were analyzed by immunohistochemistry (IHC) staining and flow cytometry (FACS). Specifically, tumors were stained with anti-CD8 antibody (53-6.7 (Biolegend)), 647-labeled anti-CD206 antibody (MCA2235 (Biorad)), secondary antibody (Life Technologies), and DAPI (counterstain), and observed under a fluorescence microscope (Leica TCS SP5 (Leica Microsystems)). Additionally, tumor CD8+ T cells were stained with APC-labeled anti-CD8 antibody (53-6.7 (Biolegend)) and analyzed using a FACSAria® (manufactured by Becton, Dickinson and Company Japan).

[0095] [Analysis of CD8-positive T cells] 1. Collect tumor and homogenize. 2. Treat with collagenase for 30 minutes. 3. Filter through a 100 μm filter. 4. Destroy red blood cells with lysing buffer. 5. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. 6. Resuspend cells in buffer. 7. Add APC-labeled anti-CD8 antibody and incubate for 30 minutes. 8. Remove dead cells with 7AAD. 9. Analyze CD8-positive T cells using FACSAria.

[0096] Results: The results are shown in Figure 2. By reducing CD206-positive macrophages, the CD8-positive T cells in the tumor increased. Generally, CD8-positive T cells are cytotoxic cells that attack tumors, and the greater their number, the more likely it is that tumor growth will be suppressed.

[0097] <2-2: Examination of the effect on the cytotoxicity of CD8-positive T cells> Method: Genes related to the cytotoxicity of CD8-positive T cells from the tumors excised in Example 1 were confirmed by qPCR. CD8-positive T cells from the tumors were collected using a CD8+ T cell isolation kit (Miltenyi Biotec).

[0098] [qPCR] 1. mRNA was prepared from tumor CD8-positive T cells using the RNeasy kit (Qiagen). 2. cDNA was prepared using Prime Script® RT master mix (TAKARA) in a Veriti® 96-Well Thermal Cycler (Applied Biosystems). 3. qPCR was performed using TB Green Fast qPCR mix (TAKARA) and target gene primers (Invitrogen) on an MX3000p (Agilent Technologies).

[0099] Results: The results are shown in Figure 3. Depletion of CD206-positive macrophages increased the expression of genes related to the cytotoxicity of CD8-positive T cells (TNFα, IFNγ, Gzmb). This change is thought to be due to upregulation of the cytotoxicity of CD8-positive T cells.

[0100] Example 3: Examination of the effect of CD206-positive M2 macrophages on peritumoral fibroblasts The effect of CD206-positive M2 macrophages in the cancer microenvironment was examined as follows. Specifically, the effect on peritumoral fibroblasts was examined.

[0101] <3-1: Examination of the effect on the number of fibroblasts surrounding tumor tissue> (PDGFRα-GFP-CreERT2 / CD206-DTR mice) PDGFRα-GFP-CreERT2 mice (provided by a collaborator) were crossbred with CD206-DTR mice, and breast cancer was transplanted to produce mice in which CD206-positive cells were eliminated and PDGFRα-positive cells could be tracked with GFP (PDGFRα-GFP-CreERT2 / CD206-DTR mice).

[0102] Methods: PDGFRα-GFP-CreERT2 / CD206-DTR mice bearing breast tumors and control PDGFRα-GFP-CreERT2 mice bearing breast tumors were intraperitoneally injected with diphtheria toxin at a dose of 20 ng / g of mouse body weight every 2 days starting 7 weeks after tumor inoculation (PDGFRα-GFP-CreERT2 / CD206-DTR mice, n=3; PDGFRα-GFP-CreERT2 mice, n=3). Five days after the start of diphtheria toxin administration, the mice were euthanized and tumors were excised. Fibroblasts from the excised tumors were analyzed by immunohistochemistry (IHC). Specifically, cells were stained with anti-CD8 antibody (53-6.7, Biolegend), 647-labeled anti-CD206 antibody (MCA2235, Biorad), secondary antibody (Life Technologies), and DAPI counterstain, and observed under a fluorescence microscope (Leica TCS SP5, Leica Microsystems). The number of CD8+ cells and the GFP area index (GFP-positive area / GFP-positive area + GFP-negative area) were calculated using the image analysis software ImageJ (NIH) and statistical analysis software JMP15 (SAS Institute), respectively, to assess the number of CD8+ T cells and fibroblasts.

[0103] Results: The results are shown in Figure 4. By reducing CD206-positive macrophages, CD8-positive T cells within the tumor increased and GFP-labeled PDGFRα-positive fibroblasts around the tumor decreased. This suggests that the reduction in fibroblasts promotes the infiltration of CD8-positive T cells into the tumor.

[0104] <3-2: Examination of the effect of fibroblasts surrounding tumor tissue on the inhibitory activity of CD8-positive T cells infiltrating into tumors> Method: Genes that suppress the infiltration of CD8-positive T cells into tumors were identified by qPCR in fibroblasts from the tumors excised in Example 1.

[0105] [Fibroblast recovery] 1. Collect tumor and homogenize. 2. Treat with collagenase for 30 minutes. 3. Filter through a 100 μm filter. 4. Destroy red blood cells with lysing buffer. 5. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. 6. Resuspend cells in buffer. 7. Recover PDGFRα-positive fibroblasts using the PDGFRα Micro Bead Kit (Miltenyi Biotec).

[0106] [qPCR] 1. Prepare mRNA from tumor CD8-positive T cells using the RNeasy kit (Qiagen). 2. Prepare cDNA using Prime Script RT master mix (TAKARA) in a Veriti 96-Well Thermal Cycler (Applied Biosystems). 3. Perform qPCR using TB Green Fast qPCR mix (TAKARA) and target gene primers (Invitrogen) on an MX3000p (Agilent Technologies).

[0107] Results: The results are shown in Figure 5. Reducing CD206-positive macrophages reduced the expression of genes (CXCL12, CXCL14, and Acta2) in tumor fibroblasts that suppress the infiltration of CD8-positive T cells into tumors. This change is thought to facilitate the infiltration of CD8-positive T cells into tumors.

[0108] These results demonstrate that CD206-positive M2 macrophages contribute to the development and progression of cancer, and that reducing CD206-positive M2 macrophages can have an anticancer effect. These results suggest that substances capable of eliminating CD206-positive M2 macrophages can be used as active ingredients in pharmaceutical compositions for the prevention or treatment of cancer. Furthermore, these results suggest that substances capable of eliminating CD206-positive M2 macrophages can be used as active ingredients in fibroblast-killing agents around tumor tissue, promoters of cytotoxic T cell infiltration into tumor tissue, inhibitors of the expression of genes that suppress the infiltration of cytotoxic T cells into tumors in fibroblasts, promoters of cytotoxic T cell proliferation, enhancers of the cytotoxic activity of cytotoxic T cells, and enhancers of the expression of genes related to the cytotoxicity of cytotoxic T cells.

[0109] An example of a substance capable of removing CD206-positive M2 macrophages is an anti-CD206 antibody. Examples of anti-CD206 antibodies capable of removing CD206-positive M2 macrophages are shown below.

[0110] (Antigen) The extracellular domain of mouse CD206 antigen (64 to 4161 of SEQ ID NO: 118) was expressed in cultured cells (FreeStyle (registered trademark) 293-F cells), and the purified protein was used as an antigen. (CD206-deficient mice) B6.129P2-Mrc1 <tm1mnz> / J mice were purchased from Jackson Laboratory.

[0111] Example 4: Production of anti-CD206 monoclonal antibodies. CD206-deficient mice were immunized with a mixture of CD206 antigen and adjuvant. Two weeks after the first antibody administration, the mice received another dose of CD206 antigen, and one week later, the spleens were harvested. Spleen cells were isolated and used to obtain CD206-specific antibodies using the ISAAC method. The remaining cells were stored at -80°C. Lymphocytes producing antibodies that bind to CD206 were first screened using the ISAAC method from the spleen cells, yielding 90 clones. Antibody cDNAs were cloned and transfected into HEK293F cells to obtain antibody-producing cells, and 13 mouse anti-human CD206 monoclonal antibodies were produced.

[0112] The anti-CD206 antibodies prepared as described above and a comparative anti-CD206 antibody (commercially available anti-CD206 antibody, RATANTI MOUSE CD206 (BIO-RAD)) were labeled with the Allophycocyanin Labeling Kit - NH2 (detected with APC) (DOJINDO). Cultured HEK293T cells were transfected with CD206 and Venus (detected with FITC) (prepared by the Department of Molecular Neuroscience, University of Toyama), and the binding ability of the antibodies to CD206-positive M2 macrophages was detected by flow cytometry. The results are shown in Figure 6. Eleven antibodies (Nos. 10, 27, 35, 40, 44, 47, 54, 59, 72, 73, and 83) were selected as the final antibodies that clearly detected CD206-positive M2 macrophages compared to the comparative anti-CD206 antibody.

[0113] Furthermore, ELISA was performed using a plate immobilized with CD206 antigen (prepared by the Department of Molecular Neuroscience, University of Toyama). Binding was observed with the anti-CD206 antibody of the present invention, which was also observed with an anti-CD206 control antibody (prepared by the Department of Immunology, University of Toyama). Therefore, it was demonstrated that the anti-CD206 antibody of the present invention has the ability to bind to CD206.

[0114] The nucleotide sequences of the heavy and light chain variable regions and each CDR of 13 types of antibodies were analyzed. The amino acid sequences of the heavy and light chain variable regions and each CDR of the antibodies obtained based on these nucleotide sequences are shown in Table 1 and the Sequence Listing below.

[0115]

[0116] Example 5 Evaluation of Complement-Dependent Cytotoxicity (CDC) Activity of Anti-CD206 Antibodies The CDC activity of the anti-CD206 antibodies prepared in Example 4 against CD206-positive cells was evaluated as follows: CD206-positive fractions were collected using a FACSAria (registered trademark) (manufactured by Nippon Becton Dickinson Co., Ltd.), and a CDC assay was performed. [CDC assay] 1. Collect the spleen from one mouse. 2. Homogenize using RPMI1640. 3. Place a 70μm Cell Strainer in a 50mL Falcon® tube and collect the isolated splenocytes. 4. Centrifuge at 1,500 rpm for 5 minutes at 4℃. 5. Discard the supernatant and resuspend in 1x Lysis Buffer. 6. Incubate at RT for 10 minutes. 7. Centrifuge at 1,500 rpm for 5 minutes at 4℃ and discard the supernatant. 8. Add 7.5μL of CD3e-PE (for T cells), 5μL of CD19-APCCY7 (for B cells), and 5μL of NK1.1-FITC (for NK cells), and stain on ice for 40 minutes. 9. Add 200μL of FACS buffer and centrifuge at 1,500 rpm for 3 minutes (in a 96-well plate). 10. Discard the supernatant (repeat steps 10 and 11 three times). 11. Use a FACSAria to collect stained cells such as T cells and the unstained CD206-positive fraction. 12. Centrifuge at 1,500 rpm for 5 minutes at 4°C and discard the supernatant. 13. Plate the collected cells onto a 96-well plate. 14. Add anti-CD206 antibody or anti-CD206 control antibody (Hel2, Hel6, Hel10 (Department of Immunology, University of Toyama), rCD206 (BIO-RAD), MR (Abcam)). 15. Add complement (Low Tox-M Rabbit complement (Cedarlane Laboratories Ltd.)) (10% Comp). 16. Incubate at 37°C for 1 hour. 17. Stain with secondary antibody to identify CD206-positive cells (APC) and dead cells (7AAD). 18. Check the percentage of CD206-positive cells and CD206-positive and 7AAD-positive cells by FACS

[0117] The results are shown in Figure 7. In wells to which the anti-CD206 antibody prepared in Example 4 was added, the percentage of live CD206-positive cells decreased, and the percentage of dead CD206-positive cells increased. This demonstrates that the anti-CD206 antibody prepared in Example 4 has CDC activity against CD206-positive cells.

[0118] Example 6 Evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-CD206 antibody The ADCC activity of the anti-CD206 antibody prepared in Example 4 against CD206-positive cells was evaluated as follows: Specifically, the CD206-positive fraction was collected using a FACSAria and subjected to an ADCC assay. [ADCC assay] 1. Collect the spleen from one mouse. 2. Homogenize using RPMI1640. 3. Place a 70μm cell strainer in a 50mL Falcon tube and collect the isolated splenocytes. 4. Centrifuge at 1,500 rpm for 5 minutes at 4℃. 5. Discard the supernatant and resuspend in 1x Lysis Buffer. 6. Incubate at RT for 10 minutes. 7. Centrifuge at 1,500 rpm for 5 minutes at 4℃ and discard the supernatant. 8. Add 7.5μL of CD3e-PE, 5μL of CD19-APCCY7, and 5μL of NK1.1-FITC and stain on ice for 40 minutes. 9. Add 200μL of FACS buffer and centrifuge at 1,500 rpm for 3 minutes (96-well plate). 10. Discard the supernatant (repeat steps 10 to 11 three times). 11. Use FACSAria to collect stained cells such as T cells and the unstained CD206-positive fraction. 12. Centrifuge at 1,500 rpm for 5 minutes at 4°C and discard the supernatant. 13. Seed the collected cells in a 96-well plate. 14. Add anti-CD206 antibody or anti-CD206 control antibody. 15. Incubate at 37°C for 12 hours. 16. Stain CD206-positive cells (APC) and dead cells (7AAD) with the secondary antibody. 17. Check the percentage of CD206-positive cells and CD206-positive and 7AAD-positive cells using FACS.

[0119] The results are shown in Figure 8. In the wells to which the anti-CD206 antibody prepared in Example 4 was added, the percentage of live CD206-positive cells decreased and the percentage of dead CD206-positive cells increased. This demonstrates that the anti-CD206 antibody prepared in Example 4 has ADCC activity against CD206-positive cells.

[0120] Example 7: Evaluation of the cytotoxic activity of anti-CD206 antibodies against CD206-positive M2 macrophages in biological tissues The cytotoxic activity of the anti-CD206 antibodies prepared in Example 4 against CD206-positive M2 macrophages in biological tissues was evaluated as follows. Test antibodies diluted in PBS or control PBS were intraperitoneally administered twice every 48 hours to C57BL / 6J mice (CLEA Japan, Inc.) at a concentration of 1 μg / g mouse body weight (No. 47, n=4; No. 83, n=5). Four days (96 hours) after administration, spleens, visceral fat, and subcutaneous fat were collected from the mice, and RNA was extracted. qPCR was performed on the F4 / 80, CD11c, and CD206 genes as follows. Spleen, visceral fat (epididymal white adipose tissue [eWAT]), and inguinal subcutaneous fat (inguinal white adipose tissue [iWAT]) cells were collected from mice and subjected to FACS analysis as described below. The qPCR results shown in Figure 9 confirmed a trend toward a decrease in CD206-positive M2 macrophages in the visceral fat of the anti-CD206 antibody-administered group (indicated by the arrow). Furthermore, the FACS analysis results shown in Figure 10 confirmed a significant decrease in CD206-positive M2 macrophages in the visceral fat and subcutaneous fat of the anti-CD206 antibody-administered group. These results demonstrate that the anti-CD206 antibody prepared in Example 4 has a cytocidal effect against CD206-positive M2 macrophages, particularly in tissues where CD206-positive M2 macrophages accumulate.

[0121] [qPCR] 1. Collect spleen, visceral fat, and subcutaneous fat from mice. 2. After homogenizing the tissues, extract RNA using the Qiagen RNA extraction kit. 3. Measure RNA concentration. 4. Prepare cDNA using the TaKaRa Prime Script (registered trademark) ET reagent kit. (Reverse transcription reaction) 37℃, 15 minutes (Reverse transcription reaction) 85℃, 5 seconds (Heat inactivate reverse transcriptase) 4℃, infinity 5. Real Time PCR (SYBR) (1) Prepare ice and sample (cDNA). (2) Remove the control and test primers from the -20℃ freezer and place them on aluminum foil. (3) Remove the master mix from the -20℃ freezer and place it on ice. (4) Prepare Micro Amp tubes and lids. (5) Prepare light yellow tubes for the number of primers and write the names of the primers on the lids. (6) Prepare the master mix and primer mix in the light yellow tubes. Master Mix 12.5μL (7.5) x number of samples = μL H2O, PCR Grade 9μL (5.4) x number of samples = μL Primer FW 0.5μL (0.4) x number of samples = μL Primer RV 0.5μL (0.4) x number of samples = μL ROX Reference Dye II 0.5μL (0.3) x number of samples = μL *Pipette the primers into the tube containing the Master Mix. Mix well and spin down. (7) Dispense 23μL of the mix into each Micro Amp tube. (8) After dispensing 23μL of mix, add 2μL of sample each to make a total of 25μL. (7) 23 μL + (8) 2 μL = 25 μL (9) Put the lid on the Micro Amp, check that there are no bubbles and that the lid is tightly closed, then spin it down to let the liquid drip down the side.(10) Set in the real-time quantitative PCR system (MX3000P (Agilent Technologies)). 6. Analyze the results of the MX3000P.

[0122] (Krebs-Hense-leit-HEPES (KRHAG) buffer) Saline, 5% BSA in saline, 1M KCl, 1M CaCl2, 1M KH2PO4, 1M MgSO4, 200mM Hepes, 200mM glucose (collagenase) 18mg in 9mL KRHAG (take a portion in advance and store at -30℃, then add immediately before use)

[0123] [FACS analysis] (Recovery of adipose tissue stromal vascular fraction (SVF)) 1. Recover white adipose tissue (WAT) 2. Mince 3. Place in collagenase at 37°C, 27 times / min 4. Tap at 20 and 40 minutes 5. Place on ice at 45 minutes 6. Add 4 mL of KRHAG and strain through a stocking 7. 1,500 rpm, 5 minutes, 4°C 8. Aspirate the supernatant. Transfer the pellet to a 2 mL tube, leaving 2 mL. 9. 1500 rpm 5 min 4℃ 10. Aspirate the supernatant and add 2 mL of KRHAG. 11. 1500 rpm 5 min 4℃ 12. Aspirate the supernatant and add 2 mL of KRHAG. 13. 1500 rpm 5 min 4℃ 14. Aspirate the supernatant and add 150 μL of FACS buffer. 15. Lyse 1.35 mL of Lysis Buffer. 16. Protect from light 15 min 4℃ 17. 1500 rpm 5 min 4℃ 18. Discard the supernatant. FACS Buffer 1 mL

[0124] (Recovery of spleen cells) 1. Recover spleen. 2. Homogenize. 3. 1,500 rpm, 5 min, 4°C. 4. Aspirate supernatant and add 150 μL of FACS buffer. 5. Lyse with 1.35 mL of Lysis Buffer. 6. Protect from light for 15 min, 4°C. 7. 1,500 rpm, 5 min, 4°C. 8. Discard supernatant. Add 1 mL of FACS Buffer.

[0125] (Staining) 1. FC Block: Add 2% FCS / PBS (1:100) and then heat for 15 minutes at 4°C. 2. CD45-PECy7: Add 2% FCS / PBS (1:400), F4 / 80-APCCy7: Add 2% FCS / PBS (1:80), CD11c-PE: Add 2% FCS / PBS (1:50), CD206-APC: Add 2% FCS / PBS (1:80) and then heat for 30 minutes at 4°C. 3. Add 200 μL of 2% FCS / PBS. 1,500 rpm, 5 minutes at 4°C. 4. Discard the supernatant and add 200 μL of 2% FCS / PBS. 5. 1,500 rpm, 5 minutes at 4°C. 6. Discard the supernatant and add 200 μL of 2% FCS / PBS. 7. Measure by FACS (7AAD should be added immediately before measurement). Add 1 μL)

[0126] The present invention can be used as a pharmaceutical composition for preventing or treating cancer.

[0127] <Explanation of the Sequence Listing> SEQ ID NO: 1: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 10) SEQ ID NO: 2: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 10) SEQ ID NO: 3: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 11) SEQ ID NO: 4: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 11) SEQ ID NO: 5: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 27) SEQ ID NO: 6: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 27) SEQ ID NO: 7: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 35) SEQ ID NO: 8: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 35) SEQ ID NO: 9: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 40) SEQ ID NO: 10: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 40) SEQ ID NO: 11: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 44) SEQ ID NO: 12: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 44) SEQ ID NO: 13: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 47) SEQ ID NO: 14: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 47) SEQ ID NO: 15: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 54) SEQ ID NO: 16: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 54) SEQ ID NO: 17: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 59) SEQ ID NO: 18: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 59) SEQ ID NO: 19: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 64) SEQ ID NO: 20: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 64) SEQ ID NO: 21: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 72) SEQ ID NO: 22: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 72) SEQ ID NO: 23: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 73) SEQ ID NO: 24: Amino acid sequence of the light chain variable region of anti-CD206 antibody (No. 73) SEQ ID NO: 25: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No. 83) SEQ ID NO: 26: Amino acid sequence of the heavy chain variable region of anti-CD206 antibody (No.SEQ ID NO: 27: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 10) SEQ ID NO: 28: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 10) SEQ ID NO: 29: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 10) SEQ ID NO: 30: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 10) SEQ ID NO: 31: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 10) SEQ ID NO: 32: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 11) SEQ ID NO: 33: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 11) SEQ ID NO: 34: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 11) SEQ ID NO: 35: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 11) SEQ ID NO: 36: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 11) SEQ ID NO: 37: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 27) SEQ ID NO: 38: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 27) SEQ ID NO: 39: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 27) SEQ ID NO: 40: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 27) SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 27) SEQ ID NO: 42: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 35) SEQ ID NO: 43: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 35) SEQ ID NO: 44: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 35) SEQ ID NO: 45: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 35) SEQ ID NO: 46: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 35) SEQ ID NO: 47: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 40) SEQ ID NO: 48: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 40) SEQ ID NO: 49: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 40) SEQ ID NO: 50: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 40) SEQ ID NO: 51: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 40) SEQ ID NO: 52: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 44) SEQ ID NO: 53: Amino acid sequence of HCDR3 of anti-CD206 antibody (No.SEQ ID NO: 54: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 44) SEQ ID NO: 55: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 44) SEQ ID NO: 56: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 44) SEQ ID NO: 57: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 47) SEQ ID NO: 58: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 47) SEQ ID NO: 59: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 47) SEQ ID NO: 60: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 47) SEQ ID NO: 61: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 47) SEQ ID NO: 62: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 54) SEQ ID NO: 63: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 54) SEQ ID NO: 64: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 54) SEQ ID NO: 65: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 54) SEQ ID NO: 66: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 54) SEQ ID NO: 67: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 59) SEQ ID NO: 68: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 59) SEQ ID NO: 69: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 59) SEQ ID NO: 70: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 59) SEQ ID NO: 71: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 59) SEQ ID NO: 72: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 64) SEQ ID NO: 73: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 64) SEQ ID NO: 74: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 64) SEQ ID NO: 75: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 64) SEQ ID NO: 76: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 64) SEQ ID NO: 77: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 72) SEQ ID NO: 78: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 72) SEQ ID NO: 79: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 72) SEQ ID NO: 80: Amino acid sequence of LCDR1 of anti-CD206 antibody (No.SEQ ID NO: 81: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 72) SEQ ID NO: 82: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 73) SEQ ID NO: 83: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 73) SEQ ID NO: 84: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 73) SEQ ID NO: 85: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 73) SEQ ID NO: 86: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 73) SEQ ID NO: 87: Amino acid sequence of HCDR1 of anti-CD206 antibody (No. 83) SEQ ID NO: 88: Amino acid sequence of HCDR2 of anti-CD206 antibody (No. 83) SEQ ID NO: 89: Amino acid sequence of HCDR3 of anti-CD206 antibody (No. 83) SEQ ID NO: 90: Amino acid sequence of LCDR1 of anti-CD206 antibody (No. 83) SEQ ID NO: 91: Amino acid sequence of LCDR3 of anti-CD206 antibody (No. 83) SEQ ID NO: 92: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 10) SEQ ID NO: 93: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 10) SEQ ID NO: 94: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 11) SEQ ID NO: 95: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 11) SEQ ID NO: 96: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 27) SEQ ID NO: 97: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 27) SEQ ID NO: 98: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 35) SEQ ID NO: 99: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 35) SEQ ID NO: 100: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 40) SEQ ID NO: 101: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 40) SEQ ID NO: 102: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 44) SEQ ID NO: 103: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 44) SEQ ID NO: 104: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 47) SEQ ID NO: 105: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No.SEQ ID NO: 106: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 54) SEQ ID NO: 107: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 54) SEQ ID NO: 108: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 59) SEQ ID NO: 109: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 59) SEQ ID NO: 110: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 64) SEQ ID NO: 111: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 64) SEQ ID NO: 112: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 72) SEQ ID NO: 113: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 72) SEQ ID NO: 114: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 73) SEQ ID NO: 115: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 73). SEQ ID NO: 116: Nucleotide sequence encoding the heavy chain variable region of anti-CD206 antibody (No. 83). SEQ ID NO: 117: Nucleotide sequence encoding the light chain variable region of anti-CD206 antibody (No. 83). SEQ ID NO: 118: Nucleotide sequence of the mouse CD206 coding region.

Claims

1. A fibroblast killer around a tumor tissue, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

2. An agent for promoting infiltration of cytotoxic T cells into a tumor tissue, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

3. The agent for promoting infiltration of cytotoxic T cells into a tumor tissue according to claim 2, wherein the promotion of infiltration of cytotoxic T cells into the tumor tissue is based on a decrease in fibroblasts around the tumor tissue.

4. An inhibitor of the expression of one or more genes selected from Acta2, CXCL12, and CXCL14 in fibroblasts around a tumor tissue, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

5. An agent for promoting the proliferation of cytotoxic T cells, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

6. An agent for enhancing the cytotoxic activity of cytotoxic T cells, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

7. An enhancer of the expression of one or more genes selected from TNFα, IFNγ, and Gzmb, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

8. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a substance having the ability to kill CD206-positive M2 macrophages.

9. The pharmaceutical composition for preventing or treating cancer according to claim 8, wherein the substance having the ability to kill CD206-positive M2 macrophages is an anti-CD206 antibody or a functional fragment thereof.

10. The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the antibody is an antibody selected from the following: (a) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 27, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 28, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 29, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 30, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and an antibody comprising a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 31; (b) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 37, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 38, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 39, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 40, An antibody comprising a light chain LCDR2 consisting of an amino acid sequence represented by LMS, and a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 41; (c) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 42, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 43, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 44, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 45, a light chain LCDR2 consisting of the amino acid sequence represented by RAS, and an antibody comprising a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 46; (d) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 47, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 48, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 49, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 50, a light chain LCDR2 consisting of the amino acid sequence represented by FAS, and an antibody comprising a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 51; (e) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 52, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 53, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 54, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 55, a light chain LCDR2 consisting of the amino acid sequence represented by AAT, and an antibody comprising a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 56; (f) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 57, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 58, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 59, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 60, a light chain LCDR2 consisting of the amino acid sequence represented by YTS, and an antibody comprising a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 61; (g) a heavy chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 62, a heavy chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 63, a heavy chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 64, a light chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 65, a light chain LCDR2 consisting of the amino acid sequence represented by KAS, and an antibody comprising a light chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 66; The heavy-chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 67, the heavy-chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 68, the heavy-chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 69, the light-chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 70, the light-chain LCDR2 consisting of the amino acid sequence represented by YTS, and an antibody comprising the light-chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 71; (i)The heavy-chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 77, the heavy-chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 78, the heavy-chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 79, the light-chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 80, the light-chain LCDR2 consisting of the amino acid sequence represented by ATS, and an antibody comprising the light-chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 81; (j)The heavy-chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 82, the heavy-chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 83, the heavy-chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 84, the light-chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 85, the light-chain LCDR2 consisting of the amino acid sequence represented by RAN, and an antibody comprising the light-chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 86; and (k)The heavy-chain HCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 87, the heavy-chain HCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 88, the heavy-chain HCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 89, the light-chain LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 90, the light-chain LCDR2 consisting of the amino acid sequence represented by YTS, and an antibody comprising the light-chain LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:

91.

11. The pharmaceutical composition for preventing or treating cancer according to claim 9 or 10, wherein the antibody is an antibody selected from the following: (A)A heavy-chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence and having binding activity to CD206, and an antibody comprising a light-chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence and having binding activity to CD206. (B) a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 5 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and an antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (C) a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and an antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (D) a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 9 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and an antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (E) a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and an antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 12 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (F) a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 13 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and a heavy chain variable region, and an antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 14 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (G) A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and An antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (H) A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 17 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and An antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (I) A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 21 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and An antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 22 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; (J) A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and An antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 24 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206; and (K) A heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 25 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206, and An antibody comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 26 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence, and having a binding activity to CD206.