WO2019059267A1 - Immunosuppressive myeloid cell marker - Google Patents

Abstract

An object of the present invention is to provide a technique for identifying immunosuppressive myeloid cells. Immunosuppressive myeloid cells can be identified by recognizing CD206 in myeloid cells.

Description

Immunosuppressive myeloid cell marker

The present invention relates to techniques for identifying immunosuppressive myeloid cells. In particular, the present invention relates to a marker for immunosuppressive myeloid cells, a drug and method for identifying immunosuppressive myeloid cells, a method of screening a substance targeting immunosuppressive myeloid cells, and immunosuppressive myeloid cells. It also relates to targeted cancer treatment.

Bone marrow-derived immunosuppressive cells (MDSC, myeloid-derived suppressor cells) are myeloid cells whose main action is to suppress T cell proliferation. It is believed that MDSC acts to promote tumor growth by strongly suppressing the antitumor immune response (Non-patent Document 1). Peripheral blood MDSCs are reported to be more frequent in patients with poor prognosis cancer and those who are not effective in immunotherapy using immune checkpoint inhibitors etc. MDSCs are important targets for immunotherapy for cancer It is considered to be a cell group. That is, if a drug that specifically removes MDSC or a drug that inhibits the immunosuppressive function of MDSC can be developed, it is expected that anti-tumor immunity is activated to bring about a therapeutic effect on cancer.

MDSCs are immunosuppressed by inactivating T cells or inducing regulatory T cells (Treg) through the production of arginase, reactive oxygen species, nitric oxide, TGF-β, IL-10, etc. It is considered to express a function (Non-patent Document 1).

MDSC is a diverse cell population, and it is assumed that the monocyte-based MDSC (M-MDSC, Monocytic-MDSC) and the granulocyte-based MDSC (G-MDSC, Granulocytic-MDSC) are present (Non-patent Document 1). Although no definite marker for identifying MDSC has been determined, Non-patent Document 1 proposes that in human and mouse, a cell group expressing a specific cell surface molecule is regarded as M-MDSC and G-MDSC. ing. For human MDSCs, Lineage marker (CD3, CD14, CD19, CD56) negative, HLA-DR negative, CD11b positive, CD33 positive cells can be regarded as MDSC as a trait common to M-MDSC and G-MDSC. is there.

M-MDSCs differentiate into macrophages as the differentiation stage further progresses. Macrophages that infiltrate and accumulate in tumors and are affected by the tumor microenvironment are called tumor-associated macrophages (TAM, tumor-associated macrophages), and through the production of angiogenic factors and cell growth factors, and suppression of antitumor immunity, It is believed to act as a tumor promoting agent (Non-patent Document 2). Macrophages are roughly divided into M1 macrophages and M2 macrophages mainly because of differences in activation mode, and it has been hypothesized that TAM is biased to M2 macrophages. However, at present, TAM is a heterogeneous cell population, and it has been recognized that macrophages in vivo can not simply be classified and interpreted as M1 / M2 (Non-patent Document 3). Among TAMs, macrophages and MDSC, which exhibit T cell proliferation inhibitory activity directly in particular, have high functionality in common with marker molecules, and it is considered difficult to accurately distinguish them. Therefore, in the present specification, those cell groups are collectively referred to as "immunosuppressive myeloid cells".

The challenge in targeting immunosuppressive myeloid cells as a therapeutic target is the lack of specific markers. Human CD11b is also expressed on neutrophils, and human CD33 is a molecule widely expressed on myeloid cells. Therefore, for example, when developing antibody drugs specific to these, side effects such as neutropenia It may occur. If marker molecules with high specificity for human immunosuppressive myeloid cells can be found, they have high utility in therapeutic applications.

CD206 is also known as a mannose receptor. It is a C-type lectin receptor having a single transmembrane structure. Among the sugar chains expressed by cells such as macrophages and dendritic cells and possessed by foreign pathogens, they bind to mannose, N-acetylglucosamine and fucose, and the antigen is taken into the cell via endocytosis and phagocytosis By being involved in antigen presentation, it contributes to both innate immunity and acquired immunity to play a role in host infection protection (Non-patent Document 4). CD206 is known to be expressed in M2 macrophages (Non-patent Document 1). However, there is no information on whether CD206 expression correlates with immunosuppressive effects.

As a diagnostic agent targeting CD206, there is an imaging reagent (trade name: Lymphoseek) for detecting metastatic lymph nodes in breast cancer and melanoma patients. This imaging reagent is obtained by isotope labeling of mannose binding dextran having binding activity to the mannose receptor, but it is unclear whether it exhibits an antitumor effect.

Also, a complex in which an anti-CD206 antibody that binds to a mannose receptor expressed in dendritic cells and β-human chorionic gonadotropin (βhCG) are linked has been reported. The effect of facilitating induction of antigen-specific cytotoxic T cells to βhCG has been shown by promoting the efficiency of antigen presentation by dendritic cells (Patent Document 1). However, it is unclear whether the antibody exhibits an immunomodulatory function on immunosuppressive myeloid cells.

In addition, among drugs developed for targeting CD206, those targeting immunosuppressive myeloid cells have not been reported.

International Publication WO2006 / 073493

Nature Reviews Immunology, 12, p. 253-268 (2012) Journal of Clinical Investigation, 125 (9), p. 3365-3376 (2015) Cancer Research, 76, p. 6439-6442 (2016) Journal of Leukocyte Biology, 92 (6), p. 1177-1186 (2012)

The object of the present invention relates to providing a technique for identifying immunosuppressive myeloid cells. In particular, the object of the present invention is to use a drug for identifying immunosuppressive myeloid cells, a method for identifying immunosuppressive myeloid cells, a method for screening a drug targeting immunosuppressive myeloid cells, and an immunosuppressive myeloid cell The goal is to provide T cell proliferation agents targeting cancer and treatment techniques for cancer.

As a result of intensive studies on the above problems, the present inventors have found that myeloid cells having CD206 suppress T cell proliferation, and based on this, the present invention has been completed.

The present invention includes, but is not limited to, the following inventions.
(1) An agent for identifying an immunosuppressive myeloid cell, comprising a substance that recognizes CD206.
(2) The agent according to (1), wherein the substance is an antibody or a functional fragment thereof.
(3) A medicament for treating cancer comprising a substance that recognizes CD206 on immunosuppressive myeloid cells.
(4) The medicine according to (3), wherein the substance is an antibody or a functional fragment thereof.
(5) The medicament according to (3) or (4) for promoting proliferation of T cells.
(6) The medicine according to any one of (3) to (5), which has a damaging effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity.
(7) The medicament according to any one of (3) to (6), further comprising a drug.
(8) The medicine according to (7), wherein the drug is a cytocidal agent.
(9) The medicament according to any one of (3) to (8), which is administered in combination with an immune checkpoint inhibitor.
(10) An anti-CD206 antibody or functional fragment thereof for promoting proliferation of T cells.
(11) The antibody or functional fragment thereof according to (10), which has a damaging effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity.
(12) The antibody or functional fragment thereof according to any of (10) or (11), which is linked to a drug.
(13) The antibody or functional fragment thereof according to (12), wherein the drug is a cytocidal agent.
(14) A T cell proliferation promoter comprising a substance that recognizes CD206 on immunosuppressive myeloid cells.
(15) The T cell proliferation promoter according to (14), wherein the substance is an antibody or a functional fragment thereof.
(16) The T cell proliferation promoting agent according to (15), wherein the antibody or the functional fragment thereof has a damaging effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity.
(17) The T cell proliferation promoter according to (15) or (16), wherein the antibody or a functional fragment thereof is linked to a cytocidal agent.
(18) The T cell proliferation promoter according to any of (14) to (17), which is administered in combination with an immune checkpoint inhibitor.
(19) A method for identifying an immunosuppressive myeloid cell, comprising the step of recognizing or detecting CD206 on myeloid cells.
(20) The method according to (17), wherein the step of recognizing or detecting is performed using an antibody against CD206 or a functional fragment thereof.
(21) A method for selecting immunosuppressive myeloid cells, comprising the steps of: identifying immunosuppressive myeloid cells by recognizing or detecting CD206 on myeloid cells; and selecting the identified immunosuppressive myeloid cells. .
(22) A method for screening a cancer therapeutic drug, comprising the step of selecting a substance that recognizes CD206 on immunosuppressive myeloid cells.
(23) A diagnostic agent for evaluating the presence of immunosuppressive myeloid cells in a tissue, comprising a substance that recognizes CD206 on immunosuppressive myeloid cells.

According to the present invention, immunosuppressive myeloid cells can be identified. Furthermore, according to the present invention, it becomes possible to screen for pharmaceuticals targeting immunosuppressive myeloid cells. Furthermore, the present invention is effective in the treatment of cancer that targets immunosuppressive myeloid cells.

FIG. 1 is a graph showing experimental results according to Example 2, and shows CD206 expression in CD33 positive myeloid cells derived from ascites fluid of ovarian cancer patients. FIG. 2 is a graph showing the experimental results according to Example 3, and shows that CD33 positive CD206 positive cells derived from ascites fluid of ovarian cancer patients suppress T cell proliferation in a cell number dependent manner. The T cell proliferative response was measured by detecting the fluorescence of CFSE which is halved with each T cell division. FIG. 3 is a graph showing the results of an experiment according to Example 4, and shows that CD33 positive cells derived from PBMC suppress T cell proliferation in a cell number dependent manner. As a control, CD33 positive cells immediately after thawing frozen PBMC were separated and used. FIG. 4 is a graph showing the results of experiments according to Example 5, and shows CD206 expression of MDSCs derived from PBMC. In the histogram on the right end, the solid line shows the result of anti-CD206 antibody, and gray shows the result of isotype control antibody. FIG. 5 is a graph showing the results of an experiment according to Example 6, and shows that among the MDSCs derived from PBMC, CD206 strongly positive cells strongly suppress T cell proliferation. FIG. 6 is a graph showing the results of an experiment relating to Example 7, wherein tumor CT 26. FIG. 16 shows that intravenous administration of saporin-conjugated anti-CD206 antibody inhibited tumor growth in a mouse model implanted subcutaneously with WT cells.

In the present specification, the "substance" that recognizes CD206 is not particularly limited as long as it recognizes CD206, and examples include an antibody against CD206 or a functional fragment thereof, a ligand or a compound thereof.

In the present specification, the "agent" for identifying an immunosuppressive myeloid cell comprising a substance that recognizes CD206 refers to the identification of an immunosuppressive myeloid cell containing a substance that recognizes CD206, as described later. For example, cell markers and reagents may be used. The agent may contain other components besides the substance that recognizes CD206.

As used herein, the term "drug" is used to mean a drug such as a cell killing agent or an anticancer agent which is used together with a substance that recognizes CD206 on immunosuppressive myeloid cells, as described later. The agent that recognizes CD206 on the immunosuppressive myeloid cells may or may not be conjugated to the drug.

In one embodiment, the present invention is an agent for identifying an immunosuppressive myeloid cell, which comprises a substance that recognizes CD206 of myeloid cells. The present inventors conducted intensive studies on myeloid cells that suppress T cell proliferation and found that CD206 in myeloid cells serves as a marker for immunosuppressive myeloid cells. The invention also relates to methods of identifying immunosuppressive myeloid cells comprising recognizing CD206 on or in immunosuppressive myeloid cells.

Bone marrow-derived immunosuppressive cells (MDSCs) are myeloid cells whose main action is to suppress T cell proliferation. MDSC is a diverse cell population, and it is believed that monocyte-based M-MDSC and granulocyte-based G-MDSC exist, but so far no clear marker has been found to distinguish MDSC. In the above-mentioned Non-Patent Document 1 (Nature Reviews Immunology, 12, p. 253-268, 2012), a group of cells showing the expression mode of cell surface molecules shown in Table 1 for humans and mice is M-MDSC and G. -It is proposed to consider it as MDSC. For human MDSCs, Lineage marker (CD3, CD14, CD19, CD56) negative, HLA-DR negative, CD11b positive, CD33 positive cells can be regarded as MDSC as a trait common to M-MDSC and G-MDSC. is there.

M-MDSCs differentiate into macrophages as the differentiation stage further progresses. Macrophages that infiltrate and accumulate in the tumor and are affected by the tumor microenvironment are called tumor-associated macrophages (TAMs) and act as tumor-promoting agents through the production of angiogenic factors and cell growth factors, and suppression of anti-tumor immunity. It is believed that.

The present inventors compare the gene expression difference of CD33 positive myeloid cells in the peritoneal fluid of ovarian cancer patient ascites and CD33 positive myeloid cells in peripheral blood mononuclear cells (hereinafter referred to as PBMC) by means of Microarray analysis. , CD206 mRNA was found to be highly expressed in the patient group.

The amino acid and nucleotide sequences of human CD206 are available by reference to GenBank Accession Nos. NP_002429.1 and NM_002438.3 (nucleotide sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2).

Human CD206, also called mannose receptor, is a type I single chain transmembrane glycoprotein having a multilectin receptor structure. In general, CD206 is considered to play an important role in intracellular uptake of proteins containing sugar chains, and the CD206 antigen is expressed not only in macrophages but also in various cells such as immature dendritic cells and endothelial cells. It is known. Although not expressed on lymphocytes and monocytes, expression of CD206 is enhanced during differentiation from monocytes to macrophages. It is known that bone marrow-derived myeloid cells have cells that suppress immunity, but according to our studies, CD206 on myeloid cells is a good marker as to whether it is an immunosuppressive myeloid cell or not. It was found to be That is, it was confirmed that myeloid cells having CD206 on the cell surface have an activity to suppress the proliferation of immune cells such as T cells.

In one embodiment, the invention relates to a method of identifying an immunosuppressive myeloid cell comprising recognizing or detecting CD206 on an immunosuppressive myeloid cell.

The recognition or detection can use an antibody against CD206 or a functional fragment thereof. The method for detecting a molecule of interest using an antibody or a functional fragment thereof can be performed using methods well known to those skilled in the art, and is detected by, for example, flow cytometry or immunohistochemical analysis. Here, a functional fragment of an antibody means an antibody fragment whose function is maintained, for example, a functional fragment of an antibody against CD206 means an antibody fragment whose function of recognizing CD206 is maintained.

In identifying immunosuppressive myeloid cells, a substance that recognizes CD206 can be used. As such a substance, in a preferred embodiment, a ligand for CD206 such as an antibody or a functional fragment thereof can be used. Here, the term "ligand" as used herein means any molecule that can specifically bind to a marker on the cell surface, and includes, for example, any compound, antibody and the like. When it is an antibody, it includes not only whole antibodies but also antibody fragments such as scFv and domain antibodies. The ligand preferably includes an antibody or an antibody fragment thereof.

In the present invention, antibodies may be prepared by known methods. For example, an antibody against CD206 immunizes a non-human animal with a target antigen, and the lymph fluid, lymph tissue, blood cell sample or bone marrow-derived cells are collected from the immunized animal and known methods (eg Kohler and Milstein, Nature) (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N. Y. (1980)), and antibody-producing cells producing an antibody against CD206 A hybridoma can be established by fusing with myeloma cells to obtain a monoclonal antibody. Specific examples of such methods are described in WO 2009/48072 (published April 16, 2009) and WO 2010/17011 (published October 14, 2010).

The antibodies of the present invention include, in addition to monoclonal antibodies, genetically modified antibodies, such as Chimeric antibodies and Humanized antibodies, which are artificially modified for the purpose of reducing heterologous antigenicity to human etc. And human antibodies. These antibodies can be produced using known methods.

Examples of chimeric antibodies include antibodies in which the variable region of the antibody and the constant region are heterologous to each other, such as a chimeric antibody in which the variable region of a mouse- or rat-derived antibody is conjugated to a constant region derived from human (Proc. Natl. Acad) Sci.U.S.A., 81, 6851-6855, (1984)).

As a humanized antibody, an antibody in which only a CDR is incorporated into a human-derived antibody (see Nature (1986) 321, p. 522-525), and by CDR grafting, amino acid residues of some frameworks in addition to the CDR sequences. The group may also be an antibody (International Publication WO 90/07861) grafted to a human antibody.

The antibodies of the present invention can further include human antibodies. For example, an anti-CD206 human antibody means a human antibody having only the gene sequence of an antibody derived from a human chromosome. The anti-CD206 human antibody is a method using a human antibody-producing mouse having a human chromosomal fragment containing heavy and light chain genes of human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133). Kuroda, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. Al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. .69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et. Al., roc.Natl.Acad.Sci.USA (2000) 97, can be obtained by reference.) The p.722-727 like.

In such a human antibody-producing mouse, specifically, the loci of endogenous immunoglobulin heavy and light chains are disrupted, and instead, human artificial chromosome (HAC) vector and mouse artificial chromosome (Mouse artificial) A transgenic animal in which human immunoglobulin heavy chain and light chain loci have been introduced via a vector such as a chromosomal (MAC) vector or the like is used to create knockout animals and transgenic animals, and these animals are crossed with each other. Can be produced.

Furthermore, eukaryotic cells are transformed with a cDNA encoding each of heavy and light chains of such human antibody, preferably a vector containing the cDNA, by genetic recombination technology to produce a recombinant human monoclonal antibody. This antibody can also be obtained from the culture supernatant by culturing transformed cells. Here, as a host, for example, eukaryotic cells, preferably CHO cells, mammalian cells such as lymphocytes and myelomas can be used.

Also, a method for obtaining a phage display-derived human antibody selected from a human antibody library (Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et. Al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 (3), p. 427-431, etc.) are also known.

For example, a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of human antibody is expressed on the phage surface as a single chain antibody (scFv) and phages that bind to the antigen are selected. -1116) can be used. By analyzing the gene of the selected phage by binding to the antigen, the DNA sequence encoding the variable region of human antibody binding to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is clarified, a human antibody can be obtained by preparing an expression vector having the sequence and introducing it into a suitable host for expression (WO 92/01047, WO 92 / 20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).

Substances that recognize specific proteins on immunosuppressive myeloid cells include, for example, ligands such as antibodies or functional fragments thereof that can bind to proteins. An agent that recognizes a particular protein on an immunosuppressive myeloid cell may comprise more than one ligand for another marker / receptor. In a preferred embodiment, for example, a ligand for CD33 may be included.

In one embodiment, when identifying immunosuppressive myeloid cells according to the present invention, it is preferable to further recognize CD33 on myeloid cells.

In one embodiment, the invention is a diagnostic for evaluating the presence of immunosuppressive myeloid cells, which comprises a substance that recognizes CD206 on immunosuppressive myeloid cells.

In another aspect, the invention is a method of selecting immunosuppressive myeloid cells, the method comprising identifying and identifying immunosuppressive myeloid cells by recognizing CD206 on the immunosuppressive myeloid cells. Selecting the selected immunosuppressive myeloid cells.

As described above, since immunosuppressive myeloid cells are involved in the suppression of proliferation of T cells which are immune cells, they suppress the proliferation suppression of T cells by controlling the immunosuppressive myeloid cells, and thus an antitumor immune response is achieved. The activation can kill cancer cells. The present invention can be used in the treatment of any tumor, including but not limited to melanoma, cancer in the prostate, intestine, breast and lung. The invention can also be used in the treatment of leukemia and lymphoma.

The invention can be used to treat tumors in any mammal, but is particularly suitable for treating tumors in humans. The term tumor as used herein refers to benign and malignant solid tumors as well as solid and non-solid cancers. The site of the tumor or cancer is not particularly limited, and examples include ovary, brain, breast, uterus, endometrium, pancreas, kidney, peritoneum and lung.

In one embodiment, the invention is a medicament for treating cancer comprising a substance that recognizes CD206 on immunosuppressive myeloid cells. As a substance that recognizes CD206 on immunosuppressive myeloid cells, as described above, ligands such as antibodies and functional fragments thereof can be used.

In a preferred embodiment, the medicament according to the present invention has a T cell proliferation promoting action, and the proliferation of T cells which are immune cells is promoted by inhibiting the function of the immunosuppressive myeloid cells.

In a preferred embodiment, the medicament according to the present invention has a cytotoxic effect on immunosuppressive myeloid cells, for example, antibody-dependent-cellular cytotoxicity (hereinafter referred to as ADCC) activity, complement dependence Immune cells by damaging immunosuppressive myeloid cells, such as by Complement-dependent cytotoxicity (hereinafter referred to as CDC activity) and / or antibody-dependent cellular phagocytosis (hereinafter referred to as ADCP) activity T cell proliferation is promoted. Antibody-dependent cellular cytotoxicity (ADCC) activity can be measured by immune cells, antibodies and ⁵¹ Cr-labeled ⁵¹ Cr release assay methods of measuring cell death caused when contacting the target cells with the effector activity . Complement dependent cytotoxicity (CDC) activity can be evaluated by measuring cell death that occurs when contacting complement contained in blood, antibody and target cells. Antibody-dependent cellular phagocytosis (ADCP) activity can be measured by dual fluorescence labeling of phagocytosis caused by contacting a phagocytotic immune cell, an antibody and a target cell.

In a preferred embodiment, the medicament according to the present invention may further comprise not only substances that recognize CD206 on immunosuppressive myeloid cells, but also drugs such as cytocidal agents and anticancer agents. The ligand for CD206 and the drug may or may not be conjugated.

When the ligand for CD206 is an antibody, a drug, for example, a drug having a cytotoxic activity, may optionally be linked to the antibody through a linker and used as an antibody drug complex. In this case, the antibody is preferably an antibody having internalization activity. The linker for binding the antibody and the drug through the linker is not particularly limited as long as it binds the anti-CD206 antibody and the drug in such a form that the activity of the drug is maintained (Protein Cell, 14 October 2016, Kyoji Tuchikama et al. (PROTEIN CELL. 2018 Jan; 9 (1): 33-46, Kyoji Tsuchikama et al.)).

An antibody-drug complex is one in which an antibody and a drug are linked by a linker, and is also referred to as ADC (Antibody-drug conjugates). Multiple drug linkers can be linked to the antibody, for example, an ADC with two drug linkers bound, an ADC with four drug linkers linked, an ADC with six drug linkers linked, eight drug linkers linked ADCs and the like can be suitably used, and mixtures of these can also be used. Here, the ratio of drug to antibody is also called DAR (Drug to Antibody Ratio), and means the average number of drugs bound to one antibody molecule. In the present invention, DAR can be, for example, 1 to 10, preferably 2 to 9, and more preferably 3 to 8.

The drug used for ADC is not particularly limited, as long as it has a substituent or a partial structure capable of binding to the linker structure. In the drug of ADC, part or all of the linker is cleaved in the cell to produce a cytotoxic effect and the like. In ADC, a drug is linked to an antibody via a linker, but a drug linker including this drug and a linker moiety may be referred to as a drug.

The linker used for ADC is not particularly limited, and any of a cleavable linker and a non-cleavable linker can be used. In the present invention, a linker having an N-substituted maleimidyl group can be mentioned as a particularly preferred example. The cleavable linker can include, for example, a peptide linker that is cleaved by an intracellular protease such as lysosomal protease and endosomal protease.

The ADC according to the present invention can also be produced using a drug linker intermediate. That is, for example, the carboxyl group of the linker compound and the amino group of the drug are reacted using a condensing agent or the like to obtain a drug linker intermediate, and this intermediate is reacted with an interchain thiol of an antibody for use can do. In a preferred embodiment, the drug linker intermediate according to the present invention has an N-substituted maleimidyl group, but is not limited thereto, and preferably used as long as it has a functional group that allows the reaction of the antibody with interchain thiol to proceed. be able to. In the present invention, the ADC can be produced, for example, by reducing the disulfide of an antibody to convert it into a thiol group, and then reacting the antibody having a thiol group with a drug linker.

Generally, there are four interchain disulfides in the antibody and the disulfide is composed of two thiol groups, so when producing ADCs using thiol groups, the drug antibody ratio (DAR) is 2, 4, 6 or It will be eight. In the present invention, the heavy chain-light chain thiol may be linked to a drug linker, and the heavy chain-heavy chain thiol may be linked to a drug linker. ADCs are manufactured under defined reaction conditions such as the amounts of starting materials and reagents to be reacted so that the number of drugs bound is controlled, but are usually obtained as a mixture in which different numbers of drugs are bound. . Therefore, unless otherwise stated, DAR is described as the average value of the number of drugs bound to one antibody molecule.

The ADC can be used alone or in combination with other therapies in adjuvant therapy, and can be combined with surgery, radiation therapy, hormonal therapy and the like. Furthermore, it can also be used as a drug for drug therapy in neoadjuvant therapy. Also, the ADC according to the present invention may be administered locally as a systemic therapy to patients. In a preferred embodiment of the present invention, parenteral administration can be carried out, and parenteral routes of administration may include intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes, and examples of administration methods include: Infusion, bolus injection and the like can be mentioned.

As the drug, not only anticancer agents, but also antibacterial, antiprotozoa, other active substances having antiviral or antifungal activity and the like can be suitably used. In a preferred embodiment, the drug is a cytotoxic agent comprising a cytocidal agent. Specifically, as drugs, for example, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, thiotepa, busulfan, cytoxin, paclitaxel, doxetaxel, taxotere, methotrexate, cisplatin, vinblastine, bleomycin, etoposide , Ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamycin, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP -16, chlorambucil and melphalan can be mentioned. When a cytocidal agent is used as the drug, for example, saporin, curcin, crotin, gelonin, miterin, restrictocin, phenomycin, enomycin, trichothecene, diphtheria A chain, non-binding active fragment of diphtheria toxin, cholera toxin, botulinum toxin Exotoxin A chain, ricin A chain, abrin A chain, modexin A chain, alpha-sarcin, aleurites foody protein, diantin protein, calicheamicin, maytansinoid, palytoxin, CC1065, etc. .

The anti-CD206 antibody of the present invention can also be administered two, three or more other therapeutic agents depending on the therapeutic purpose, and those other therapeutic agents can be simultaneously administered by encapsulating them in the same formulation. can do. The other therapeutic agent and the anti-CD206 antibody can also be administered simultaneously by encapsulating them in the same formulation. Alternatively, the anti-CD206 antibody and the other therapeutic agent can be enclosed in separate formulations and administered simultaneously. Additionally, the other drug and the anti-CD206 antibody can be administered separately in tandem. That is, after administration of another therapeutic agent, a therapeutic agent containing an anti-CD206 antibody or an antigen-binding fragment of the antibody as an active ingredient is administered, or an anti-CD206 antibody or an antigen-binding fragment of the antibody is contained as an active ingredient Other therapeutic agents may be administered after administering the therapeutic agent.

Other therapeutic agents can include, for example, immune checkpoint inhibitors such as anti-PD-1 antibody and anti-PD-L1 antibody. Here, an immune checkpoint inhibitor refers to an agent that inhibits signal transduction by the immune checkpoint by inhibiting the binding of the immune checkpoint and its ligand. As the immune checkpoint and its ligand, for example, PD-1, PD-L1, PD-L2, CTLA-4, TIM3, LAG3, BTNL2, B7-H3, B7-H4, CD48, CD80, BT80, BTLA, 2B4, CD160 , CD60, CD86, VISTA, etc., but it is not limited thereto. Preferred examples of the immune checkpoint inhibitor include, for example, Pembrolizumab (CAS number: 1374853-91-4), PD-1 antibody such as Nivolumab (CAS number: 946414-94-4), and Atezolizumab (CAS number: 1380723). PD-L1 antibodies such as -44-3), Avelumab (CAS number: 1537032-82-8) and Durvalumab (CAS number: 1428935-60-7) can be mentioned. In addition, as other therapeutic agents that can be used in combination in the present invention, for example, chemotherapeutic agents such as angiogenesis inhibitors, antimetabolites, plant alkaloids, platinum preparations and the like can be suitably used.

The present invention provides a pharmaceutical composition comprising a therapeutically and / or prophylactically effective amount of an anti-CD206 antibody and additives such as a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Also provide goods. The pharmaceutical composition may further contain an active ingredient other than the anti-CD206 antibody.

The pharmaceutical composition of the present invention changes or holds pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, dissolution rate, sustained release rate, absorption rate, permeability. It can contain substances for formulation of The addition amount of the substance for these preparations is preferably 0.01 to 100 times the weight of the anti-CD206 antibody. The composition of a suitable pharmaceutical composition in a preparation can be appropriately determined by those skilled in the art according to the disease to be applied, the administration route of application, and the like.

The excipient or carrier in the pharmaceutical composition may be liquid or solid. Suitable excipients and carriers may be water or saline for injection, artificial cerebrospinal fluid, or other substances commonly used for parenteral administration. Neutral saline or saline containing serum albumin can also be used as a carrier. The pharmaceutical composition can include Tris buffer of pH 7.0-8.5, acetate buffer of pH 4.0-5.5, citrate buffer of pH 3.0-6.2. These buffers can also include sorbitol and other compounds. The pharmaceutical composition of the present invention can include a pharmaceutical composition containing an anti-CD206 antibody, and a pharmaceutical composition containing an anti-CD206 antibody and at least one anti-tumor therapeutic agent, and the pharmaceutical composition of the present invention is selected It is prepared as a lyophilizate or liquid as a drug having a composition and required purity. Pharmaceutical compositions comprising the anti-CD206 antibody, and pharmaceutical compositions comprising the anti-CD206 antibody and at least one anti-cancer drug therapeutic agent can also be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical composition of the present invention can be prepared for parenteral administration or can be prepared for oral gastrointestinal absorption. The composition and concentration of the formulation can be determined by the method of administration. When the antibody of the present invention is administered to humans, about 0.1 to 100 mg / kg may be administered one or more times in 1 to 180 days. However, since the dose and the number of doses should generally be determined in consideration of the patient's sex, weight, age, symptoms, severity, side effects, etc., they are not limited to the above dose or usage. I assume.

Examples of the form of the pharmaceutical composition of the present invention include an injection containing a drop, a suppository, a nasal agent, a sublingual agent, a percutaneous absorption agent and the like. The administration route is an oral route or a parenteral route, and a parenteral route includes, for example, intravenous, intraarterial, intramuscular, intrarectal, transmucosal, intradermal and the like.

In addition, on the basis of the present invention, cancer therapeutic agents can be screened. That is, in one embodiment, the present invention is a method for screening a cancer therapeutic drug, which comprises the step of selecting a substance that recognizes CD206 on immunosuppressive myeloid cells.

The type of test substance to be subjected to screening is not particularly limited, and may be either an organic compound or an inorganic compound. As the organic compound, in addition to low molecular weight organic compounds, screening can be performed using any organic compound such as amino acids, oligopeptides, polypeptides, saccharides, lipids, nucleic acids and the like as a test substance.

The present invention will be described in more detail by way of specific examples, but the present invention is not limited to the following examples. In the present specification, concentration and the like are on a weight basis unless otherwise stated, and a numerical range is described as including its end points.

Example 1 Gene Expression Analysis of Ascites Fluid Myeloid Cells in Ovarian Cancer Patients
Microarray analysis was used to compare gene expression differences between CD33 positive myeloid cells in the ascites fluid of ovarian cancer patients and CD33 positive myeloid cells in peripheral blood mononuclear cells (hereinafter referred to as PBMC) in healthy individuals.

From cells collected from ascites fluid of 6 ovarian cancer patients, CD33 positive cells were separated by MACS method. In addition, PBMC were separated from fresh peripheral blood of 10 healthy individuals by Ficoll density gradient centrifugation, and CD33 positive cells were further separated by MACS. For MACS method, CD33 microbeads (Miltenyi Biotec) and an automatic magnetic cell separator (autoMACS, Miltenyi Biotec) were used.

RNA was extracted from the separated CD33-positive cells, and after quality confirmation, comprehensive gene expression analysis (Affymetrix GeneChip (trademark) Array expression analysis) was performed using Total RNA. Analysis of gene expression differences between ovarian cancer patients and healthy individuals showed that, in the patient group, the mRNA expression of MRC1, which is a gene encoding the CD206 protein, was 24 times higher than in the healthy group. Here, the numerical value (24 times) of the gene expression difference was calculated by dividing the average value of mRNA expression level of patient group by the average value of mRNA expression level of healthy subject group.

(Example 2. Analysis of protein expression in ascites fluid myeloid cells of ovarian cancer patients)
Whether the MRC1 gene found in Example 1 was expressed at the protein level in ascites fluid of ovarian cancer patients CD33 positive myeloid cells was analyzed by flow cytometry.

Ascites fluid cells collected from ovarian cancer patients were stained with APC-Cy7-labeled anti-CD33 antibody and APC-labeled anti-CD206 antibody, and dead cells were further stained with 7-AAD. The flow cytometer (MACS Quant) was used to analyze CD33 and CD206 expression in living cells.

The results are shown in FIG. We confirmed the expression of CD206 in CD33 positive myeloid cells of ovarian cancer patients ascites fluid. It was also shown that all cells expressing CD206 simultaneously express CD33.

(Example 3. Immunosuppressive function of CD206 positive cells of ascites fluid myeloid cells in ovarian cancer patients)
Whether there is a relationship between the immunosuppressive activity of CD33 positive myeloid cells in the ascites fluid of ovarian cancer patients and the expression of CD206, the inhibitory activity on T cell proliferation was examined as an index.

Ascites fluid of ovarian cancer patients, CD33 positive myeloid cells were separated into a CD206 positive fraction and a CD206 negative fraction. After staining the cells recovered from ascites fluid of ovarian cancer patients in the same manner as in Example 2, the cell suspension was stained using a cell sorter (FACS ARIA IIu, Becton Dickinson) to CD33 positive CD206 positive cells and CD33 positive CD206 negative cells Separated. In addition, CD3 positive T cells were separated from healthy human peripheral blood PBMC by MACS method, and the separated T cells were labeled with a fluorescent dye CFSE. The T cell proliferative response can be measured by detecting by flow cytometry the fluorescence of CFSE which is halved with every cell division.

In a 96-well round bottom plate, CD33 positive CD206 positive cells or CD33 positive CD206 negative cells were seeded at 2 × 10 ⁴ cells / well, 4 × 10 ⁴ cells / well, or 8 × 10 ⁴ cells / well . Thereto, CFSE-labeled CD3 positive T cells were added at 1.6 × 10 ⁵ cells / well. Furthermore, Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher Scientific) was added to 4 × 10 ⁴ beads / well. The medium used was RPMI 1640 adjusted to 5% human type AB serum, Penicillin / Streptomycin, 1 × MEM NEAA, 1 mM Sodium Pyruvate, and 5 mM Hepes (hereinafter referred to as “co-culture medium”).

After culturing the plate for 72 hours at 37 ° C., 5% CO ² , cells were recovered. The recovered cells were stained with PE labeled anti-CD3 antibody, nuclear staining reagent DAPI. It measured using the flow cytometer (MACSQuant), and analyzed using FlowJo (Tree Star). DAPI negative cells were gated as live cells, and the ratio of the division peak was quantified among the histogram peaks of CFSE (FITC) in CD3 positive T cells, and the inhibitory activity on T cell proliferation was compared.

The results are shown in FIG. CD33 positive CD206 positive cells (CD33 ⁺ CD206 ⁺ ) strongly suppress the proliferative response of T cells, compared to CD33 positive CD206 negative cells (CD33 ⁺ CD206 ⁻ ) The ratio of dividing cells was 45.3% at 8: 1, 27.7% at 4: 1, and 8.67% at 2: 1 ). This result indicates that CD33 positive CD206 positive cells are involved in T cell suppression, and removal of CD33 positive CD206 positive cells makes it possible to release T cell suppression.

Example 4 MDSC Preparation by Induction from PBMCs
Frozen PBMC (Cellular Technology Limited) were thawed, and CD3 positive cells and CD3 negative cells were separated by MACS method. CD3 positive cells were cryopreserved again at -80.degree. C. using Servanker (Nippon Zenyaku Kogyo Co., Ltd.). CD3 negative cells were prepared by adding 20% of culture supernatant of human renal cell carcinoma cell line 786-O (ATCC Inc. CRL-1932) and co-culture medium (hereinafter referred to as induction medium) to which 10 ng / mL human GM-CSF was added. The cells were cultured at 37 ° C., 5% CO ² for 6 to 8 days. After culture, adherent cells were detached using a cell dispersive reagent Detachin (Genlantis) and recovered together with floating cells. After washing, a part of the cells was used for expression analysis of surface markers, and the rest were separated CD33 positive cells in the same manner as in Example 3.

The expression analysis of the surface marker used FITC-labeled anti-CD3 antibody, FITC-labeled anti-CD19 antibody and FITC-labeled anti-CD56 antibody as Lineage markers. In addition, PerCP-Cy5.5-labeled anti-HLA-DR antibody, Pacific Blue-labeled anti-CD11b antibody and PE-labeled anti-CD33 antibody were used. Cells were stained with these antibodies, and dead cell staining was performed using LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific). Stained cells were measured using a flow cytometer (FACS Canto II, Becton Dickinson) and analyzed using FlowJo. In the cells after induction culture, it was confirmed that there were cells expressing Lineage negative HLA-DR negative and weak positive CD11b positive CD33 positive MDSC markers.

Next, it was examined whether CD33 positive cells contained in the cells after induction culture had inhibitory activity on T cell proliferation. As control cells for CD33 positive cells after induction, CD3 negative CD33 positive cells separated by MACS immediately after thawing frozen PBMC were used (referred to as untreated CD33 positive cells). The CD3 positive T cells stored by selvan were thawed and CFSE labeled. In a 96-well round bottom plate, CD33 positive cells were seeded at 4 × 10 ⁴ cells / well, 8 × 10 ⁴ cells / well, or 1.6 × 10 ⁵ cells / well. Thereto, CFSE-labeled CD3 positive T cells were added to give 1.6 × 10 ⁵ cells / well. Furthermore, Dynabeads human T-Activator CD3 / CD28 was added to 3.2 × 10 ⁴ beads / well. After culturing the plate for 72 hours at 37 ° C., 5% CO ² , cells were recovered. Dead cells were stained using the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit. Aqua (Amcyan) negative cells were gated as live cells, and the ratio of the division peak was quantified among the histogram peaks of CFSE labeled CD3 positive T cells to compare the inhibitory activity on T cell proliferation reaction.

As apparent from the results shown in FIG. 3, compared to untreated CD33 positive cells, CD33 positive cells after induction suppress T cell proliferation, and the suppression activity is the number of CD33 positive myeloid cells to be cocultured. (The proportion of dividing cells was 81.0% at a T cell: CD33 positive ratio of 8: 1, 66.5% at 4: 1, 39.0% at 2: 1) . From this, CD33 positive myeloid cells obtained by induction culture of cancer culture supernatant and GM-CSF from PBMC contain cells expressing MDSC markers and have T cell proliferation inhibitory activity. It was shown that MDSC having an immunosuppressive function could be induced.

(Example 5. CD206 expression analysis in MDSC derived from PBMC)
CD206 expression in MDSCs derived from PBMC was analyzed. In the same manner as in Example 4, the surface markers of cells obtained by culturing CD3 negative cells separated from commercially available frozen PBMC in the presence of cancer cell culture supernatant and GM-CSF are analyzed by flow cytometry. did. In addition to FITC-labeled anti-CD3 antibody, FITC-labeled anti-CD19 antibody, FITC-labeled anti-CD56 antibody, PerCP-Cy5.5-labeled anti-HLA-DR antibody, Pacific Blue-labeled anti-CD11b antibody and PE-labeled anti-CD33 antibody, APC-labeled anti-CD206 antibody Stained using Dead cell staining was also performed using the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit.

As a result of analysis, Lineage negative HLA-DR negative and weak positive CD11b positive CD33 positive MDSC obtained by culturing in the presence of cancer cell culture supernatant and GM-CSF (Lineage ^- HLA-DR- ^{/ low} CD11b ⁺ CD33 ⁺ In the -gated fraction, expression of CD206 was observed (lower right in FIG. 4). On the other hand, expression of CD206 was not observed in untreated CD33 positive cells having no immunosuppressive activity.

Example 6 Relationship Between CD206 Expression and Immunosuppressive Function of Myeloid Cells Containing MDSC Derived from PBMC
The suppressive activity on T cell proliferation was examined as an indicator whether the immunosuppressive activity of CD33 positive myeloid cells containing MDSCs derived from PBMC is related to the expression of CD206.

Cells induced by the method described in Example 4 are stained with a PE-labeled anti-CD33 antibody and an APC-labeled anti-CD206 antibody, and using a cell sorter (FACS Aria IIu), CD33 positive CD206 strongly positive cells, CD33 positive CD206 weak Positive cells and CD33 positive CD206 negative cells were separated. CD3 positive T cells were CFSE labeled in the same manner as in Example 3 and Example 4. In a 96-well round bottom plate, CD33 positive CD206 strongly positive cells, CD33 positive CD206 weakly positive cells or CD33 positive CD206 negative cells were seeded at 8 × 10 ⁴ cells / well. Thereto, CFSE-labeled CD3 positive T cells were added to give 1.6 × 10 ⁵ cells / well. Furthermore, Dynabeads human T-Activator CD3 / CD28 was added to 3.2 × 10 ⁴ beads / well. The plates were cocultured for 72 hours at 37 ° C., 5% CO ² . The cells were collected and dead cell staining was performed using LIVE / DEAD Fixable Aqua Dead Cell Stain Kit. Aqua (Amcyan) negative cells were gated as live cells, and the ratio of the division peak was quantified among the CFSE (FITC) positive CD3 positive T cell histogram peaks, and the inhibitory activity on T cell proliferation was compared.

The results are shown in Figure 5, CD33-positive CD206-negative cells ^- compared to (CD33 ⁺ CD206, the proportion 86.4% of dividing cells), CD33-positive CD206 strongly positive cells (CD33 ⁺ CD206 ^high, the proportion of dividing cells 9.19% very strongly suppressed the proliferation of T cells. Although CD33 positive CD206 weakly positive cells (CD33 ⁺ CD206 ^low , percentage of dividing cells 64.6%) showed T cell growth inhibitory activity, T cell proliferation inhibitory activity is weak when compared to CD33 positive CD206 strongly positive cells The

From the above, it was suggested that the CD206 expression intensity in CD33 positive MDSCs is correlated with the immunosuppressive activity, and it was shown that CD206 can be an excellent surface marker of inhibitory myeloid cells.

This indicates that, similar to the findings obtained in Example 3, CD33 positive CD206 positive cells are involved in T cell suppression, and T cell suppression can be released by removing CD33 positive CD206 positive cells. It shows.

Example 7 Antitumor Effect of Antibody Drug Targeting CD206
From the findings obtained in Examples 1 to 6, it was suggested that a drug targeting CD206 may release the immunosuppressive state and contribute to the improvement of the pathological condition. Therefore, the anti-tumor effect was examined using an anti-CD206 antibody.

An antibody drug complex was prepared in which a cytocidal drug was linked to an anti-CD206 antibody, using a CD206 positive cell as an antibody model capable of selectively removing it. Although any compound may be sufficient as a cytocidal agent, saporin was used. Saporin is a toxin contained in the seeds of Saponaria officinalis and causes cell death by directly inactivating ribosomes. Therefore, the complex in which the antibody and saporin are bound can be taken into the cell via the antigen on the target cell membrane and induce cell death.

Efficacy can be evaluated by administering this antibody drug complex to a tumor-bearing mouse model. Mouse colon cancer cell line CT26. Evaluation was performed in a model in which WT (ATCC CRL-2638) was subcutaneously implanted in BALB / c mice. CT 26. In tumors in WT tumor-bearing mice, it was confirmed that CD206 positive G-MDSC and CD206 positive M-MDSC were present. 5 × 10 ⁵ CT26. ⁵ mice / mouse in the right axilla of BALB / c mice (11 females / group). WT cells were implanted subcutaneously. 10 μg of saporin-conjugated anti-CD206 antibody was intravenously administered per mouse for 4 days from day 4 after transplantation and further for 4 days from day 10 after transplantation. The control group received vehicle (phosphate buffered saline). The tumor diameter was measured over time to calculate the tumor volume, and the average value and standard error of the tumor volume were calculated for each group. As a result, on the final day of antibody administration on the 13th day after transplantation, the tumor volume was smaller in the antibody administration group compared to the control group, and the tumor growth inhibitory effect was observed by administration of the saporin-conjugated anti-CD206 antibody (FIG. 6) ). The induction of cell death in CD206-positive MDSCs is thought to release the immunosuppression by MDSCs and to show an antitumor effect.

Thus, if it is possible to selectively target and kill CD206-positive inhibitory myeloid cells containing MDSCs or alter their suppressive function, immunosuppression by inhibitory myeloid cells containing MDSCs is released. It is thought that anti-tumor immunity in the tumor microenvironment is activated by, for example, an increase in infiltrating T cells and anti-tumor macrophages in the tumor, leading to anti-tumor effects such as tumor growth inhibition and survival effect.

Claims (23)

An agent for identifying an immunosuppressive myeloid cell, comprising a substance that recognizes CD206. The agent according to claim 1, wherein the substance is an antibody or a functional fragment thereof. A medicament for treating cancer, comprising a substance that recognizes CD206 on immunosuppressive myeloid cells. The medicine according to claim 3, wherein the substance is an antibody or a functional fragment thereof. The medicine according to claim 3 or 4 for promoting T cell proliferation. The pharmaceutical composition according to any one of claims 3 to 5, which has a damaging effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity. The medicament according to any one of claims 3 to 6, further comprising a drug. The medicine according to claim 7, wherein the drug is a cytocidal agent. The medicament according to any one of claims 3 to 8, which is administered in combination with an immune checkpoint inhibitor. Anti-CD206 antibody or functional fragment thereof for promoting proliferation of T cells. The antibody or a functional fragment thereof according to claim 10, which has a damaging effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity. 12. The antibody or functional fragment thereof according to any of claims 10 or 11 linked to a drug. The antibody or functional fragment thereof according to claim 12, wherein the drug is a cytocidal agent. A T cell proliferation promoter comprising a substance that recognizes CD206 on immunosuppressive myeloid cells. The T cell proliferation promoter according to claim 14, wherein the substance is an antibody or a functional fragment thereof. The T cell proliferation promoter according to claim 15, wherein the antibody or the functional fragment thereof has a damaging effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity. The T cell proliferation promoter according to claim 15 or 16, wherein the antibody or a functional fragment thereof is linked to a cytocidal agent. The T cell proliferation promoter according to any one of claims 14 to 17, which is administered in combination with an immune checkpoint inhibitor. A method of identifying an immunosuppressive myeloid cell, comprising the step of recognizing or detecting CD206 on myeloid cells. The method according to claim 19, wherein the step of recognizing or detecting is performed using an antibody against CD206 or a functional fragment thereof. Identifying immunosuppressive myeloid cells by recognizing or detecting CD206 on myeloid cells;
Selecting the identified immunosuppressive myeloid cells;
A method of selecting an immunosuppressive myeloid cell, comprising A method of screening a cancer therapeutic drug, comprising the step of selecting a substance that recognizes CD206 on an immunosuppressive myeloid cell. A diagnostic agent for evaluating the presence of immunosuppressive myeloid cells in a tissue, comprising a substance that recognizes CD206 on immunosuppressive myeloid cells.

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