JP2021028293A - Immunosuppressive myeloid cell marker - Google Patents

Abstract

To provide a technique for identifying immunosuppressive myeloid cells.SOLUTION: In the present invention, immunosuppressive myeloid cells can be identified by recognizing CD206 in myeloid cells.SELECTED DRAWING: None

Description

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

Bone marrow-derived immunosuppressive cells (MDSCs, myeloid-deved supressor cells) are myeloid cells whose main action is to suppress T cell proliferation. MDSC is considered to act promotely on tumor growth by strongly suppressing the antitumor immune response (Non-Patent Document 1). It has been reported that peripheral blood MDSCs are high in cancer patients with poor prognosis and cancer patients for whom immunotherapy using immune checkpoint inhibitors is difficult to respond, and MDSCs are important targets for immunotherapy for cancer. It is thought to be a group of cells. 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 antitumor immunity will be activated to bring about a therapeutic effect on cancer.

MDSC suppresses immunosuppression by inactivating T cells and inducing regulatory T cells (Treg) through the production of arginase, reactive oxygen species, nitric oxide, TGF-β, IL-10, etc. It is considered to exhibit a function (Non-Patent Document 1).

MDSC is a diverse cell population, and it is said that monocyte-based MDSC (M-MDSC, Monoactive-MDSC) and granulocyte-based MDSC (G-MDSC, Granulocytic-MDSC) exist (Non-Patent Document 1). Although a clear marker for identifying MDSC has not been determined, Non-Patent Document 1 proposes that a group of cells expressing a specific cell surface molecule in human and mouse are regarded as M-MDSC and G-MDSC. ing. Regarding human MDSC, Lineage marker (CD3, CD14, CD19, CD56) negative, HLA-DR negative, CD11b positive, and CD33 positive cells can be regarded as MDSC as traits common to M-MDSC and G-MDSC. is there.

M-MDSC differentiates into macrophages as the differentiation stage progresses. Macrophages that infiltrate and accumulate in tumors and are affected by the tumor microenvironment are called tumor-associated macrophages (TAMs), which are produced through the production of angiogenic factors and cell growth factors and the suppression of antitumor immunity. It is considered to act in a tumor-promoting manner (Non-Patent Document 2). Macrophages are roughly classified into M1 macrophages and M2 macrophages mainly based on the difference in activation mode, and the hypothesis that TAM is biased to M2 macrophages has been proposed. However, at present, it is recognized that TAM is a heterogeneous cell population, and macrophages in vivo cannot be simply classified into M1 / M2 and interpreted (Non-Patent Document 3). Among TAMs, macrophages and MDSCs, which directly exert a T cell proliferation inhibitory effect, have high functional and marker molecules in common, and it is considered that it is difficult to strictly distinguish them. Therefore, in the present specification, these cell groups are collectively referred to as "immunosuppressive myeloid cells".

The challenge in targeting immunosuppressive myeloid cells as therapeutic targets is the lack of specific markers. Since human CD11b is also expressed on neutrophils and human CD33 is also a molecule widely expressed in myeloid cells, for example, when an antibody drug specific to these is developed, side effects such as neutropenia may occur. It can occur. If a marker molecule having high specificity for human immunosuppressive myeloid cells can be found, it will be highly useful in practical application of treatment.

CD206 is also known as the mannose receptor. It is a C-type lectin receptor having a single transmembrane structure. It is expressed in cells such as macrophages and dendritic cells, and among the sugar chains possessed by foreign pathogens, it binds to mannose, N-acetylglucosamine, and fucose, and takes up antigens into the cells via endocytosis and fagocytosis. By participating in antigen presentation, it contributes to both innate immunity and acquired immunity and plays a role in host infection protection (Non-Patent Document 4). CD206 is known to be expressed on 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 patients with breast cancer or melanoma. This imaging reagent isotope-labeled with mannose-binding dextran having mannose-binding activity, but it is unknown whether or not it exhibits an antitumor effect.

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

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

International release WO2006 / 073493

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

An object of the present invention relates to providing a technique for identifying immunosuppressive myeloid cells. In particular, an object of the present invention is a drug for identifying immunosuppressive myeloid cells, a method for identifying immunosuppressive myeloid cells, a method for screening a drug targeting immunosuppressive myeroid cells, and immunosuppressive myeroid cells. The purpose is to provide therapeutic technology for cancers targeted at.

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

The present invention includes, but is not limited to, the following inventions.
(1) A drug for identifying immunosuppressive myeloid cells, which comprises a substance that recognizes CD206.
(2) The drug according to (1), wherein the substance is an antibody or a functional fragment thereof.
(3) A method for identifying immunosuppressive myeloid cells, which comprises a step of recognizing or detecting CD206 on myeroid cells.
(4) The method according to (4), wherein the recognition or detection step is performed using an antibody against CD206 or a functional fragment thereof.
(5) A method for selecting immunosuppressive myeloid cells, which comprises a step of identifying immunosuppressive myeloid cells by recognizing or detecting CD206 on myeroid cells; and a step of selecting the identified immunosuppressive myeroid cells; ..
(6) A method for screening a cancer therapeutic agent, which comprises a step of selecting a substance that recognizes CD206 on immunosuppressive myeloid cells.
(7) A diagnostic agent for evaluating the presence of immunosuppressive myeloid cells in a tissue, which comprises a substance that recognizes CD206 on immunosuppressive myeroid cells.
(8) A drug for treating cancer, which comprises a substance that recognizes CD206 on immunosuppressive myeloid cells.
(9) The medicament according to (8), wherein the substance is an antibody or a functional fragment thereof.
(10) The medicament according to (8) or (9), which has a T cell proliferation promoting action.
(11) The medicament according to any one of (8) to (10), which has an inhibitory effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity.
(12) The medicament according to any one of (9) to (11), further comprising a drug.
(13) The medicament according to (12), wherein the drug is a cell-killing drug.
(14) An anti-CD206 antibody or a functional fragment thereof exhibiting a T cell proliferation promoting action.
(15) The antibody or functional fragment thereof according to (14), which has an inhibitory effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity.
(16) The antibody according to any one of (14) or (15) or a functional fragment thereof linked to a drug.
(17) The antibody or functional fragment thereof according to (16), wherein the drug is a cell-killing drug.

According to the present invention, immunosuppressive myeloid cells can be identified. Further, according to the present invention, it becomes possible to screen a drug targeting immunosuppressive myeloid cells. Furthermore, the present invention is effective in treating cancers targeting immunosuppressive myeloid cells.

FIG. 1 is a graph showing the experimental results according to Example 2, showing the expression of CD206 in CD33-positive myeloid cells derived from ascites of an ovarian cancer patient. FIG. 2 is a graph showing the experimental results according to Example 3, showing that CD33-positive and CD206-positive cells derived from ascites of an ovarian cancer patient suppress T cell proliferation in a cell number-dependent manner. The T cell proliferation reaction was measured by detecting the fluorescence of CFSE, which is halved each time T cells divide. FIG. 3 is a graph showing the experimental results according to Example 4, showing 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 PBMCs were separated and used. FIG. 4 is a graph showing the experimental results according to Example 5, showing the CD206 expression of MDSC derived from PBMC. In the rightmost histogram, the solid line shows the results of the anti-CD206 antibody, and the gray shows the results of the isotype control antibody. FIG. 5 is a graph showing the experimental results according to Example 6, showing that CD206 strongly positive cells strongly suppress T cell proliferation in MDSCs derived from PBMC.

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

In the present specification, the "agent" for identifying an immunosuppressive myeloid cell containing a substance that recognizes CD206 is an identification of an immunosuppressive myeroid cell containing a substance that recognizes CD206, as will be described later. Examples of drugs used in the above are cell markers and reagents. The drug may contain other components in addition to the substance that recognizes CD206.

As described herein, the term "drug" as used herein means a drug such as a cell-killing drug or an anticancer drug used together with a substance that recognizes CD206 on immunosuppressive myeloid cells. The substance that recognizes CD206 on immunosuppressive myeloid cells and the drug may or may not be bound.

In one embodiment, the invention is an agent for identifying immunosuppressive myeloid cells, comprising a substance that recognizes CD206 of myeroid cells. The present inventors diligently studied myeloid cells that suppress the proliferation of T cells, and found that CD206 in myeroid cells serves as a marker for immunosuppressive myeroid cells. The present invention also relates to methods for identifying immunosuppressive myeloid cells, including recognizing CD206 on or in immunosuppressive myeroid 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 said that monocyte-based M-MDSC and granulocytic-based G-MDSC are present, but so far no clear marker has been found to identify MDSC. In Non-Patent Document 1 (Nature Reviews Immunology, 12, p.253-268, 2012), the cell groups showing the expression patterns of the cell surface molecules shown in Table 1 are referred to as M-MDSC and G for humans and mice. -It is advocated to consider it as an MDSC. Regarding human MDSC, as traits common to M-MDSC and G-MDSC, Lineage marker (CD3, CD14, CD19, CD56) negative, HLA-DR negative, CD11b positive, and CD33 positive cells can be regarded as MDSC. is there.

M-MDSC differentiates into macrophages as the differentiation stage progresses. Macrophages that infiltrate and accumulate in tumors and are affected by the tumor microenvironment are called tumor-related macrophages (TAMs) and act to promote tumors through the production of angiogenic factors and cell growth factors and suppression of antitumor immunity. It is believed that.

The present inventors compared the gene expression difference between CD33-positive myeloid cells in the ascites of ovarian cancer patients and CD33-positive myeroid cells in healthy human peripheral blood mononuclear cells (hereinafter referred to as PBMC) by 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_0024383. (Nucleotide sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2).

Human CD206, also called a mannose receptor, is a type I single-chain transmembrane glycoprotein with a multilectin receptor structure. It is generally considered that CD206 plays an important role in intracellular uptake of proteins containing sugar chains, and 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 in lymphocytes and monocytes, the expression of CD206 is enhanced in the process of differentiation from monocytes to macrophages. It has been known that bone marrow-derived myeloid cells include cells that suppress immunity, but according to the study by the present inventors, a good marker of whether CD206 or the like on myeloid cells is an immunosuppressive myeroid cell or not. It was found that That is, it was confirmed that myeloid cells having CD206 on the cell surface have an activity of suppressing 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 the immunosuppressive myeroid cell.
Antibodies to CD206 or functional fragments thereof can be used for the above recognition or detection. A method for detecting a target molecule using an antibody or a functional fragment thereof can be carried out by a method well known to those skilled in the art, and is detected by, for example, flow cytometry or an immunohistochemical analysis method. Here, the functional fragment of an antibody means an antibody fragment in which the function is maintained, and for example, a functional fragment of an antibody against CD206 means an antibody fragment in which the 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 ligand means an arbitrary molecule capable of specifically binding to a marker on the cell surface in the present specification, and examples thereof include an arbitrary compound and an antibody. When it is an antibody, it contains not only the whole antibody but also antibody fragments such as scFv and domain antibodies. Preferred examples of the ligand include an antibody or an antibody fragment thereof.

In the present invention, the antibody may be prepared by a known method. For example, an antibody against CD206 immunizes a non-human animal with a target antigen, collects lymph fluid, lymphoid tissue, blood cell sample or cells derived from bone marrow from the animal after immunization, and uses a known method (for example, Kohler and Milstein, Nature). (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Lymph Press, NY (1980)) with antibody-producing cells that produce antibodies against CD206. By fusing with myeloma cells, a hybridoma can be established and a monoclonal antibody can be obtained. Specific examples of such methods are described in WO2009 / 48072 (published April 16, 2009) and WO2010 / 117011 (published October 14, 2010).

In addition to the monoclonal antibody, the antibody of the present invention includes a recombinant antibody artificially modified for the purpose of reducing heterologous antigenicity to humans, for example, a chimeric antibody, a humanized antibody, and the like. , Human antibodies, etc. are also included. These antibodies can be produced using known methods.

Examples of the chimeric antibody include an antibody in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is bonded to a human-derived constant region (Proc. Natl. Acad). (See Sci. USA, 81, 6851-6855, (1984)).

As humanized antibodies, an antibody in which only CDR is incorporated into a human-derived antibody (see Nature (1986) 321, p.522-525), and amino acid residues of some frameworks in addition to the CDR sequence by the CDR transplantation method. As the group, an antibody transplanted into a human antibody (International Publication No. WO90 / 07861 pamphlet) can be mentioned.

Further, as the antibody of the present invention, a human antibody can be mentioned. For example, the 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 the genes of the heavy chain and the light chain of the human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133). -143; Kuroiwa, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. Al., Animal Cell Technology: Basic and Applied Aspects vol. .69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kruwer Academic Publicers, 1999; Tomizuka, K. et. Al., Proc. Natl. Acad. 97, p.722-727, etc.).

Specifically, in such human antibody-producing mice, the loci of the endogenous immunoglobulin heavy chain and light chain are disrupted, and instead, a human artificial chromosome (HAC) vector or a mouse artificial chromosome (Human artificial chromosome) (HAC) vector or mouse artificial chromosome (Mousse artificial) By producing a knockout animal and a transgenic animal, and crossing these animals with a genetically modified animal into which the human immunoglobulin heavy chain and light chain loci have been introduced through a vector such as an artificial chromosome (MAC) vector. Can be created.

In addition, a recombinant human monoclonal antibody is produced by transforming eukaryotic cells with a cDNA encoding each of the heavy chain and the light chain of such a human antibody, preferably a vector containing the cDNA, by a gene recombination technique. This antibody can also be obtained from the culture supernatant by culturing the transformed cells. Here, as the host, for example, eukaryotic cells, preferably CHO cells, mammalian cells such as lymphocytes and myeloma can be used.

In addition, a method for obtaining a human antibody derived from a phage display 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 Antibody and Genetics (2002), 1 (2), p.189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 (3), p. 427-431 etc.) is also known. For example, a phage display method (Nature Biotechnology (2005), 23, (9), p.1105) in which the variable region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage to select a phage that binds to an antigen. -1116) can be used. By analyzing the gene of the phage selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of scFv that binds to the antigen is clarified, a human antibody can be obtained by preparing an expression vector having the sequence, introducing it into an appropriate host and expressing it (WO92 / 01047, WO92 /). 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 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 capable of binding to proteins or functional fragments thereof. A substance that recognizes a particular protein on immunosuppressive myeloid cells may contain multiple ligands 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 by the present invention, it is preferable to further recognize CD33 on the myeloid cells.
In one embodiment, the invention is a diagnostic agent for assessing the presence of immunosuppressive myeloid cells, which comprises a substance that recognizes CD206 on immunosuppressive myeloid cells.

In another aspect, the present invention is a method for selecting immunosuppressive myeloid cells, which identifies and identifies immunosuppressive myeloid cells by recognizing CD206 on immunosuppressive myeroid cells. Includes the selection of immunosuppressive myeloid cells.

As described above, immunosuppressive myeloid cells are involved in the suppression of T cell proliferation, which is an immune cell. Therefore, by controlling immunosuppressive myeloid cells, the suppression of T cell proliferation is released and an antitumor immune response is induced. By activating it, cancer cells can be killed. The present invention can be used in the treatment of melanoma and any tumor including, but not limited to, cancers in the prostate, intestine, breast and lung. The present invention can also be used in the treatment of leukemia and lymphoma.

Although the present invention can be used to treat tumors in any mammal, it is particularly suitable for treating tumors in humans. The term tumor means benign and malignant solid tumors as well as solid and non-solid cancers herein. The site of the tumor or cancer is not particularly limited, and examples thereof 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, a ligand such as an antibody or a functional fragment thereof can be used.

In a preferred embodiment, the medicament according to the present invention has a T cell proliferation promoting action, and inhibition of the action of immunosuppressive myeloid cells promotes the proliferation of T cells, which are immune cells.

In a preferred embodiment, the medicament according to the present invention has cellular cytotoxicity on immunosuppressive myeloid cells, for example, antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity. It is an immune cell by damaging immunosuppressive myeloid cells by sex cell injury (Complement-dependent cytotoxicity (hereinafter, CDC activity) and / or antibody-dependent cellular cytotoxicity (hereinafter, ADCP) activity) and / or antibody-dependent cell phagocytosis (ADCP) activity. Proliferation of T cells is promoted. Antibody-dependent cellular cytotoxicity (ADCC) activity can be measured by the method of ^{the 51} Cr release assay, which measures cell death resulting from contact of ^{immune cells with effector activity, antibodies and 51 Cr-labeled target cells.} .. Complement-dependent cytotoxicity (CDC) activity can be assessed by measuring cell death that occurs when complement, antibodies, and target cells contained in blood are brought into contact. Antibody-dependent cellular cytotoxicity (ADCP) activity can be measured by double fluorescent labeling for phagocytosis caused by contacting immune cells, antibodies and target cells with phagocytosis.

In a preferred embodiment, the medicament according to the present invention may further contain a drug such as a cell killing agent or an anticancer agent as well as a substance that recognizes CD206 on immunosuppressive myeloid cells. The ligand for CD206 and the drug may or may not be bound.

When the ligand for CD206 is an antibody, a drug, for example, a drug having a cytotoxic effect may be bound to the antibody through a linker, if necessary, and used as an antibody-drug conjugate. In this case, the antibody is preferably an antibody having an internalizing activity. The linker for binding the antibody to the drug through the linker is not particularly limited as long as it is a linker that binds the anti-CD206 antibody to the drug in a form in which the activity of the drug is maintained (Protein Cell, 14 October 2016, Kyoji Tsuchikama et al.).

As the drug, not only an anticancer agent but also an antibacterial, antigenic living animal, an antiviral or other agent having antifungal activity and the like can be preferably used. In a preferred embodiment, the drug is a cytotoxic agent, including a cell killing agent. Specifically, as drugs, for example, doxorubicin, epirubicin, 5-fluorouracil, citocin arabinoside, cyclophosphamide, thiotepa, busulfan, cytoxin, paclitaxel, doxetaxel, taxotere, methtrexate, cisplatin, vincristine, bleomycin, etoposide. , Ifosfamide, mitomycin C, mitoxanthrone, vincristine, vinorerbin, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamycin, 6-thioguanine, 6-mercaptopurine, actinomycin D, VP -16, chlorambucil, melfaran and the like can be mentioned. When using a cell-killing drug as a drug, for example, saporin, curcin, crotin, geronin, mitogerin, restrictocin, phenomycin, enomycin, trichotesene, diphtheria A chain, unbound active fragment of diphtheria toxin, cholera toxin, botulinum toxin , Extoxin A chain, ricin A chain, abrin A chain, modelin A chain, alpha-salcin, alleurites fordy protein, diphtheria protein, calikeamycin, maytansinoid, paritoxin, CC1065 and the like. ..

The anti-CD206 antibody of the present invention may be administered with a few or more other therapeutic agents depending on the purpose of treatment, and these other therapeutic agents may be administered simultaneously by encapsulation in the same formulation. can do. Other therapeutic agents and anti-CD206 antibodies can also be administered simultaneously by encapsulation in the same formulation. In addition, the anti-CD206 antibody and other therapeutic agents can be encapsulated in separate preparations and administered at the same time. In addition, other drugs and anti-CD206 antibody can be administered separately before and after. That is, after administration of another therapeutic agent, an anti-CD206 antibody or a therapeutic agent containing an antigen-binding fragment of the antibody is administered as an active ingredient, 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 the therapeutic agent is administered.

The present invention comprises a therapeutic and / or prophylactically effective amount of an anti-CD206 antibody and additives such as pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. We also provide things. The pharmaceutical composition may further contain an active ingredient other than the anti-CD206 antibody.

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

Excipients and carriers in the pharmaceutical composition may be liquid or solid. Suitable excipients and carriers may be water for injection, saline, artificial cerebrospinal fluid or other substances commonly used for parenteral administration. A neutral saline solution or a saline solution containing serum albumin can also be used as a carrier. The pharmaceutical composition can include a Tris buffer of pH 7.0-8.5, an acetic acid buffer of pH 4.0-5.5, and a citrate buffer of pH 3.0-6.2. These buffers can also contain sorbitol and other compounds. Examples of the pharmaceutical composition of the present invention include a pharmaceutical composition containing an anti-CD206 antibody and a pharmaceutical composition containing an anti-CD206 antibody and at least one antitumor therapeutic agent, and the pharmaceutical composition of the present invention was selected. Prepared as lyophilized or liquid as a drug with composition and required purity. The pharmaceutical composition containing the anti-CD206 antibody and the pharmaceutical composition containing the anti-CD206 antibody and at least one anticancer drug therapeutic agent can also be molded as a lyophilized product using a suitable excipient such as sucrose.

The pharmaceutical composition of the present invention can be prepared for parenteral administration or for oral gastrointestinal absorption. The composition and concentration of the preparation 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 once or multiple times every 1 to 180 days. However, the dose and frequency of administration should generally be determined in consideration of the patient's gender, weight, age, symptoms, severity, side effects, etc., and are not limited to the above doses and usages. And.

Examples of the form of the pharmaceutical composition of the present invention include injections containing infusions, suppositories, nasal preparations, sublingual preparations, transdermal absorbents and the like. The route of administration is an oral route or a parenteral route, and examples of the parenteral route include intravenous, intraarterial, intramuscular, rectal, intramucosal, and intradermal routes. In addition, screening for a cancer therapeutic agent can also be performed based on the present invention. That is, in one embodiment, the present invention is a method for screening a cancer therapeutic agent, which comprises a step of selecting a substance that recognizes CD206 on immunosuppressive myeloid cells.

The type of the test substance to be subjected to the 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, any organic compound such as amino acids, oligopeptides, polypeptides, saccharides, lipids, and nucleic acids can be screened as a test substance.

The present invention will be described in more detail with reference to specific examples, but the present invention is not limited to the following examples. In this specification, unless otherwise specified, the concentration and the like are based on weight, and the numerical range is described as including the end points thereof.

(Example 1. Gene expression analysis of ascites myeloid cells of ovarian cancer patients)
Differences in gene expression between CD33-positive myeloid cells in the ascites of ovarian cancer patients and CD33-positive myeloid cells in healthy peripheral blood mononuclear cells (hereinafter referred to as PBMC) were compared by microarray analysis.

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

RNA was extracted from the isolated CD33-positive cells, and after quality confirmation, comprehensive gene expression analysis (Affymetrix GeneChip ™ Array expression analysis) was performed using Total RNA. When the gene expression difference between ovarian cancer patients and healthy subjects was analyzed, the mRNA expression of MRC1, which is a gene encoding the CD206 protein, was 24 times higher in the patient group 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 in the patient group by the average value of mRNA expression in the healthy subject group.

(Example 2. Protein expression analysis in ascites myeloid cells of ovarian cancer patients)
The MRC1 gene found in Example 1 was analyzed by flow cytometry to see if it was expressed at the protein level in ascites CD33-positive myeloid cells of ovarian cancer patients.

Ascites 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 expression of CD33 and CD206 in living cells was analyzed using a flow cytometer (MACS Quant).

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

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

CD33-positive myeloid cells in the ascites of ovarian cancer patients were separated into a CD206-positive fraction and a CD206-negative fraction. After staining the cells collected from the ascites of an ovarian cancer patient in the same manner as in Example 2, the cell suspension was subjected to CD33-positive CD206-positive cells and CD33-positive CD206-negative cells using a cell sorter (FACS ARIA IIu, Becton Dickinson). Was separated. In addition, CD3-positive T cells were isolated from healthy human peripheral blood PBMC by the MACS method, and the isolated T cells were labeled with the fluorescent dye CFSE. The T cell proliferation reaction can be measured by detecting the fluorescence of CFSE, which is halved each time the cell divides, by the flow cytometry method.

CD33-positive CD206-positive cells or CD33-positive CD206-negative cells were seeded on a 96-well round bottom plate at 20000 cells / well, 40,000 cells / well, or 80,000 cells / well. CFSE-labeled CD3-positive T cells were added thereto at 160000 cells / well. In addition, Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher Scientific) was added to 40,000 beads / well. As the medium, RPMI 1640 adjusted to 5% human AB type serum, Pencillin / Streptmycin, 1X MEM NEAA, 1 mM Sodium Pyrubate, and 5 mM Hepes (hereinafter referred to as co-culture medium) was used.

After culturing the plate for 72 hours under the conditions of 37 ° C. and 5% CO ^{2, cells were collected.} The recovered cells were stained with PE-labeled anti-CD3 antibody and nuclear staining reagent DAPI. The measurement was performed using a flow cytometer (MACSQuant), and the analysis was performed using FlowJo (Tree Star). DAPI-negative cells were gated as living cells, the proportion of division peaks among the histogram peaks of CFSE (FITC) in CD3-positive T cells was quantified, and the inhibitory activity on the T cell proliferation reaction was compared.

The results are shown in FIG. CD33-positive CD206-negative cells ^(CD33 + CD206 ^-) as compared to, CD33-positive CD206 positive cells ^(CD33 + CD206 ⁺⁾ is strongly inhibited proliferation response of T cells, CD33-positive CD206 positive cells that inhibitory effect of co-culturing It was number-dependent (the ratio of dividing cells was 45.3% when the ratio of T cells: CD33-positive and CD206-positive cells was 8: 1, 27.7% when 4: 1 and 8.67% when 2: 1. ). This result indicates that 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.

(Example 4. MDSC preparation by induction from PBMC)
Frozen PBMC (Cellular Technology Limited) was thawed, and CD3 positive cells and CD3 negative cells were separated by the MACS method. CD3 positive cells were cryopreserved again at -80 ° C using Cerbanker (Nippon Zenyaku Kogyo Co., Ltd.). For CD3 negative cells, use a co-culture medium (hereinafter referred to as induction medium) to which 20% of the culture supernatant of human renal cell carcinoma strain 786-O (ATCC CRL-1932) and 10 ng / mL of human GM-CSF / mL are added. The cells were cultured at 37 ° C. ^{under 5% CO 2} conditions for 6 to 8 days. After culturing, the adherent cells were detached using a cell dispersion reagent Detachin (Genlantis) and collected together with the floating cells. After washing, some of the cells were used for expression analysis of surface markers, and the rest were isolated from CD33-positive cells by the same method as in Example 3.

For the expression analysis of the surface markers, FITC-labeled anti-CD3 antibody, FITC-labeled anti-CD19 antibody and FITC-labeled anti-CD56 antibody were used 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. The cells were stained with these antibodies and further stained with dead cells using the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Stained cells were measured using a flow cytometer (FACS Canto II, Becton Dickinson) and analyzed using FlowJo. It was confirmed that cells expressing Lineage-negative HLA-DR-negative and weakly-positive CD11b-positive and CD33-positive MDSC markers were present in the cells after the induction culture.

Next, it was examined whether the CD33-positive cells contained in the cells after the induction culture had an inhibitory activity on the T cell proliferation reaction. As control cells for the induced CD33-positive cells, CD3-negative CD33-positive cells isolated by the MACS method immediately after thawing frozen PBMCs were used (referred to as untreated CD33-positive cells). CD3-positive T cells stored in a cell bunker were thawed and CFSE labeled. CD33 positive cells were seeded on 96-well round bottom plates to 40,000 cells / well, 80,000 cells / well, or 1600,000 cells / well. To that, CFSE-labeled CD3-positive T cells were added to 160000 cells / well. In addition, Dynabeads human T-Activator CD3 / CD28 was added to 32000 beads / well. After culturing the plate for 72 hours under the conditions of 37 ° C. and 5% CO ^{2, cells were collected.} Dead cells were stained with a LIVE / DEAD Fixable Aqua Dead Cell Stein Kit. Aqua (Amchan) -negative cells were gated as living cells, and the proportion of division peaks among the histogram peaks of CFSE-labeled CD3-positive T cells was quantified to compare the inhibitory activity on the T cell proliferation reaction.

As is clear from the results shown in FIG. 3, the induced CD33-positive cells suppress the proliferation of T cells as compared with the untreated CD33-positive cells, and the inhibitory activity is the number of CD33-positive myeloid cells co-cultured. (The ratio of dividing cells was 81.0% for T cells: post-induction CD33 positive ratio, 66.5% for 4: 1 and 39.0% for 2: 1). .. From this, the CD33-positive myeloid cells obtained by inducing culture from PBMC in the presence of cancer culture supernatant and GM-CSF 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. Analysis of CD206 expression in MDSC derived from PBMC)
CD206 expression in MDSCs derived from PBMCs was analyzed. Analysis of cell surface markers obtained by culturing CD3 negative cells isolated from commercially available frozen PBMC in the presence of cancer cell culture supernatant and GM-CSF by the same method as in Example 4 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 Was stained with. In addition, dead cells were stained using LIVE / DEAD Fixable Aqua Dead Cell Stein Kit.

As a result of the analysis, Lineage-negative HLA-DR negative and weakly positive CD11b-positive CD33-positive MDSC (Lineage ^- HLA-DR- ^{/ low} CD11b ⁺ CD33 ^{+) obtained by culturing in the presence of cancer cell culture supernatant and GM-CSF.} The expression of CD206 was observed in -gated fraction) (lower right of FIG. 4). On the other hand, no expression of CD206 was 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)
Whether there is a relationship between the immunosuppressive activity of CD33-positive myeloid cells containing MDSC derived from PBMC and the expression of CD206 was examined using the inhibitory activity on T cell proliferation as an index.

The cells induced by the method described in Example 4 were stained with PE-labeled anti-CD33 antibody and APC-labeled anti-CD206 antibody, and CD33-positive CD206 strongly positive cells and CD33-positive CD206 weak were used using a cell sorter (FACS Aria IIu). Positive cells and CD33-positive and CD206-negative cells were isolated. CD3-positive T cells were CFSE-labeled in the same manner as in Example 3 and Example 4. CD33-positive CD206 strong-positive cells, CD33-positive CD206-weak-positive cells, or CD33-positive CD206-negative cells were seeded on a 96-well round bottom plate at 80,000 cells / well. To that, CFSE-labeled CD3-positive T cells were added to 160000 cells / well. In addition, Dynabeads human T-Activator CD3 / CD28 was added to 32000 beads / well. The plates were co-cultured for 72 hours under 37 ° C. and 5% CO ^{2 conditions.} Cells were harvested and stained with dead cells using the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit. Aqua (Amchan) -negative cells were gated as living cells, and the proportion of division peaks among the CFSE (FITC) -positive CD3-positive T cell histogram peaks was quantified to compare the inhibitory activity on T cell proliferation.

The results are shown in FIG. 5. Compared with CD33-positive CD206-negative cells (CD33 ⁺ CD206 ⁻ , proportion of dividing cells 86.4%), CD33-positive CD206 strongly positive cells (CD33 ⁺ CD206 ^high , proportion of dividing cells) 9.19%) very strongly suppressed the proliferation of T cells. CD33-positive CD206 weakly positive cells (CD33 ⁺ CD206 ^low , proportion of dividing cells 64.6%) showed T cell proliferation inhibitory activity, but T cell proliferation inhibitory activity was weaker than that of CD33 positive CD206 strongly positive cells. It was.

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

This means that, as in 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. Shown.

(Example 7. Antitumor effect of antibody drug targeting CD206)
From the findings obtained in Examples 1 to 6, it was suggested that the drug targeting CD206 may release the immunosuppressive state and contribute to the improvement of the pathological condition. Therefore, the antitumor effect of the anti-CD206 antibody will be examined.

As an antibody model capable of selectively removing CD206-positive cells, an antibody-drug conjugate in which a cell-killing drug is bound to an anti-CD206 antibody is prepared. The cell killing agent can be any compound, for example saporin. Saporin is a toxin contained in the seeds of Saponaria officinalis, which causes cell death by directly inactivating ribosomes. Therefore, the complex in which the antibody and saporin are bound can be taken up into the cell via the antigen on the target cell membrane and induce cell death.

Drug efficacy can be evaluated by administering this antibody-drug conjugate to a cancer-bearing mouse model. For example, mouse colon cancer cell line CT26. Evaluation is performed in a model in which WT (ATCC CRL-2638) is subcutaneously transplanted into BALB / c mice. CT26. It was confirmed that CD206-positive G-MDSC and CD206-positive M-MDSC were present in the tumors in the WT cancer-bearing mice. When the saporin-binding anti-CD206 antibody is administered, the immunosuppression by MDSC is released by removing these MDSCs, and an antitumor effect is observed.

In this way, if CD206-positive inhibitory myeloid cells containing MDSC can be selectively targeted and killed or their inhibitory function can be changed, the immunosuppression by the inhibitory myeloid cells containing MDSC is released. Therefore, it is considered that antitumor immunity in the tumor microenvironment is activated by increasing intratumoral infiltrating T cells and antitumor macrophages, leading to antitumor effects such as tumor regression and survival prolongation.

Claims (17)

A drug for identifying immunosuppressive myeloid cells, which comprises a substance that recognizes CD206. The agent according to claim 1, wherein the substance is an antibody or a functional fragment thereof. A method for identifying immunosuppressive myeloid cells, which comprises the step of recognizing or detecting CD206 on myeloid cells. The method of claim 4, wherein the recognition or detection step is performed using an antibody against CD206 or a functional fragment thereof. The step of identifying immunosuppressive myeloid cells by recognizing or detecting CD206 on myeroid cells, and
The process of selecting the identified immunosuppressive myeloid cells and
Methods for selecting immunosuppressive myeloid cells, including. A method for screening a cancer therapeutic agent, which comprises a step of selecting a substance that recognizes CD206 on immunosuppressive myeloid cells. A diagnostic agent for assessing the presence of immunosuppressive myeloid cells in tissues, comprising a substance that recognizes CD206 on immunosuppressive myeroid cells. A drug for treating cancer, which comprises a substance that recognizes CD206 on immunosuppressive myeloid cells. The medicament according to claim 8, wherein the substance is an antibody or a functional fragment thereof. The medicament according to claim 8 or 9, which has a T cell proliferation promoting action. The medicament according to any one of claims 8 to 10, which has an inhibitory effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity. The medicament according to any one of claims 8 to 11, further comprising a drug. The medicament according to claim 12, wherein the drug is a cell-killing drug. An anti-CD206 antibody or a functional fragment thereof that promotes T cell proliferation. The antibody or functional fragment thereof according to claim 14, which has an inhibitory effect on immunosuppressive myeloid cells by ADCC activity, CDC activity and / or ADCP activity. The antibody or functional fragment thereof of any of claims 14 or 15, linked to a drug. The antibody or functional fragment thereof according to claim 16, wherein the drug is a cell-killing drug.

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