Composition for inhibiting muscle tissue loss

Abstract

[Problem] The purpose of the present invention is to provide a composition for suppressing loss of muscle tissue. [Solution] This composition is for suppressing loss of muscle tissue in a subject having monocytes with promoted expression of CD38 and contains a compound for inhibiting the activity of the IL-36 receptor.

Description

[Technical Field]

[0001] The present invention relates to a composition for inhibiting muscle tissue loss. [Background technology]

[0002] Cachexia is a complex metabolic disorder syndrome that accompanies various diseases. Cachexia is particularly prevalent in 50-80% of all cancer patients, and is directly linked to a decreased tolerance to treatment and a decline in the patient's quality of life. As the disease progresses, cancer patients gradually experience weight loss, malnutrition, and wasting, and this state in cancer patients is specifically called "cancer cachexia." Because cancer cachexia patients often also suffer from anorexia, it is sometimes referred to as cancer anorexia-cachexia syndrome (CACS).

[0003] Although the diagnostic criteria for cachexia are unclear, one of its characteristics is weight loss, especially loss of muscle mass. Weight loss is expressed in terms of lean body mass (LBM). While weight loss due to normal starvation maintains LBM, cachexia reduces LBM, and in this respect cachexia differs from starvation.

[0004] Although cachexia has been suggested to be indirectly involved in approximately 20% of cancer deaths (Non-Patent Document 1), the pathogenesis of the condition has not been fully elucidated. For example, a relationship between systemic inflammation and metabolic abnormalities has been suggested, but the detailed molecular mechanism of the pathogenesis remains unknown.

[0005] Therefore, elucidating the mechanism of cancer cachexia and developing new treatments is an urgent issue common to many cancer research fields.

[0006] To date, a treatment using anamorelin has been proposed as a treatment for cancer cachexia. Anamorelin is a ghrelin-like substance that acts to increase appetite. This method attempts to treat cancer cachexia primarily by increasing the patient's appetite (Patent Documents 1, 2, and 3). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Josep MAet al.,Nature Reviews Cancers,2014(14),754-762 [Patent documents]

[0008] [Patent Document 1] Patent No. 6109925 specification [Patent Document 2] Patent No. 6356907 specification [Patent Document 3] Special Publication No. 2017-526695 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a composition for inhibiting the loss of muscle tissue. [Means for solving the problem]

[0010] Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic tumor caused by abnormalities in hematopoietic stem cells. It is an intractable blood cancer characterized by an increase in inflammatory monocytes and dysplasia of myeloid cells. CMML is known to be characterized by cachexia, which is relatively rare in blood cancers. The only curative treatment for CMML is hematopoietic stem cell transplantation.

[0011] The inventors have previously succeeded in establishing a disease model mouse that faithfully reproduces the pathology of CMML using the novel NUP98-HBO1 fusion gene identified from CMML patients (Hayashi, et al. Blood Advances. 2019).

[0012] Based on the idea that an increase in inflammatory monocytes specific to CMML might be involved in the onset of cachexia, which is also prominent in CMML, the inventors conducted extensive research using NUP98-HBO1 CMML model mice to elucidate the mechanism of cachexia. Interestingly, we found that the increased inflammatory monocytes in these model mice exhibited a different gene expression pattern than those in wild-type mice (Figures 1-3). Furthermore, a comparison of NUP98-HBO1 CMML model mice revealed differences in the gene expression patterns of inflammatory monocytes depending on whether or not cachexia developed (Figure 5).

[0013] Based on these differences between wild-type and NUP98-HBO1 CMML model mice, we identified a gene that was specifically overexpressed in inflammatory monocytes from cachectic NUP98-HBO1 CMML mice (Fig. 6A). The identified gene encoded a cell surface antigen (CD38), and flow cytometry analysis confirmed that it was highly expressed on the surface of inflammatory monocytes from cachectic mice (Fig. 6B).

[0014] Furthermore, the inventors identified IL1F9 (IL-36γ) as a candidate humoral factor involved in cachexia and confirmed that IL1F9 (IL-36γ) derived from monocytic cells induces muscle atrophy (Figures 7 and 8).

[0015] As a result of further intensive research, the present inventors confirmed that the induction of cachexia-associated CD38-positive monocytes is not limited to CMML, a blood cancer, but also appears in a cachexia model mouse with solid tumors (specifically, breast cancer) in accordance with the progression of the disease (Figs. 10B and 10C). The present invention was made based on the above findings.

[0016] That is, the present invention provides: [1] A composition for inhibiting muscle tissue loss in a subject having monocytes with increased CD38 expression, comprising: a composition comprising a compound that inhibits the activity of the IL-36 receptor; [2] The composition according to [1], wherein the subject is a mammal; [3] The composition according to [1] or [2], wherein the mammal is selected from the group consisting of humans, monkeys, mice, rats, cows, horses, sheep, dogs, and cats; [4] The composition according to [2] or [3], wherein the mammal is a human; [5] The composition according to any one of [1] to [4], wherein the subject having monocytes with increased CD38 expression is suffering from at least one disease selected from the group consisting of cancer, inflammatory disease, autoimmune disease, and chronic infectious disease; [6] The composition according to [5], wherein the cancer is at least one of blood cancer and solid cancer; [7] The blood cancer is at least one selected from the group consisting of malignant lymphoma, multiple myeloma, leukemia, and myeloproliferative neoplasms; The composition according to [6], wherein the solid cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, malignant melanoma, lung cancer, pancreatic cancer, gastric cancer, and lung cancer; [8] The blood cancer is chronic myelomonocytic leukemia; The composition according to [6] or [7], wherein the solid cancer is breast cancer; [9] The composition according to any one of [1] to [8], wherein the subject has a cancer-related symptom;

[10] The composition according to [9], wherein the cancer-related symptom is cancer cachexia;

[11] The compound that inhibits the activity of the IL-36 receptor is CD38 antagonists, IL-36γ antagonist and IL-36 receptor antagonist The composition according to any one of [1] to

[10] , which is at least one selected from the group consisting of:

[12] The compound that inhibits the activity of the IL-36 receptor is ·Anti-CD38 antibody, Anti-IL-36γ antibody and Anti-IL-36 receptor antibody The composition according to any one of [1] to

[11] , wherein the composition is at least one selected from the group consisting of:

[13] The composition according to any one of [1] to

[12] , which is a pharmaceutical composition for treating at least one selected from the group consisting of cancer, inflammatory diseases, autoimmune diseases, and chronic infectious diseases;

[14] The composition according to any one of [1] to

[13] , which is a pharmaceutical composition for treating cancer cachexia;

[15] A composition that inhibits IL-36γ-mediated activation of the IL-36 receptor, comprising: the IL-36γ is produced by monocytes expressing CD38; a composition comprising a compound that inhibits the activity of the IL-36 receptor;

[16] A composition that inhibits the IL-36 receptor activation pathway, comprising: the IL-36 receptor activation pathway comprises monocytes expressing CD38 and the IL-36 receptor; a composition comprising a compound that inhibits the activity of the IL-36 receptor;

[17] The composition according to

[16] , wherein the IL-36 receptor activation pathway comprises IL-36γ production by CD38-expressing monocytes and IL-36γ binding to the IL-36 receptor;

[18] The compound that inhibits the activity of the IL-36 receptor is CD38 antagonists, IL-36γ antagonist and IL-36 receptor antagonist The composition according to any one of

[15] to

[17] , which is at least one selected from the group consisting of: Regarding. [Effects of the Invention]

[0017] According to the present invention, a composition that inhibits the loss of muscle tissue can be provided. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the novel fusion gene NUP98-HBO1 identified from a patient with chronic myelomonocytic leukemia (CMML). [Figure 2A] Figure 2A shows that a characteristic increase in inflammatory monocytes was observed in CMML model mice obtained by bone marrow transplantation (BMT) of cells carrying the novel fusion gene NUP98-HBO1 into wild-type mice. [Figure 2B] Figure 2B shows that CMML model mice obtained by bone marrow transplantation (BMT) of cells carrying the novel fusion gene NUP98-HBO1 into wild-type mice showed a significant increase in white blood cells (WBC), macrocytic anemia (high mean corpuscular volume (MCV) and decreased Hb), and decreased platelets (PLTS). [Figure 2C] Figure 2C shows that significant megakaryocytic dysplasia was observed in CMML model mice obtained by bone marrow transplantation (BMT) of cells carrying the novel fusion gene NUP98-HBO1 into wild-type mice. [Figure 2D]Figure 2D shows that in CMML model mice obtained by bone marrow transplantation (BMT) of cells carrying the novel fusion gene NUP98-HBO1 into wild-type mice, the increase in bone marrow blasts was less than 20%. [Figure 2E] Figure 2E shows that the gene group whose expression was increased when NUP98-HBO1 was introduced into human umbilical cord blood hematopoietic stem / progenitor cells (NUP98-HBO1-induced genes) was significantly increased in the CMML patient group (Figure 2E). [Figure 3A] FIG. 3A shows that the CMML mice established by the present inventors showed signs of cachexia (progressive weight loss and skeletal muscle atrophy), a characteristic symptom of CMML. [Figure 3B] FIG. 3B shows that the CMML mice established by the present inventors showed signs of cachexia (progressive weight loss and skeletal muscle atrophy), a characteristic symptom of CMML. [Figure 4A] Figure 4A shows that whole bone marrow cells from NUP98-HBO1 mice and control mice, or Ly6C-positive inflammatory monocytes collected by cell sorting, were co-cultured with C2C12 cells differentiated into myofibroblasts. [Figure 4B] Figure 4B shows the results of measuring the width of C2C12 myofiber cells after 72 hours of co-culture with a culture insert inserted, without direct cell contact. [Figure 5] FIG. 5 shows the specific gene expression patterns in monocytes from cachexia-affected CMML mice. [Figure 6A] Figure 6A shows the results of RNA-Seq analysis of gene expression in monocytes from CMML mice with cachexia. Among genes encoding cell surface antigens, several genes were found to be upregulated only in CMML mice with cachexia. [Figure 6B] Figure 6B shows the results of flow cytometry analysis of the actual protein expression of some of the candidate genes shown to be upregulated in Figure 6A. It was revealed that CD38 expression was upregulated in inflammatory monocytes from CMML mice with cachexia. [Figure 7A] Figure 7A shows the results of RNA-Seq analysis of gene expression in monocytes from CMML mice with cachexia. Among the genes encoding humoral factors, we focused on IL1F9 (IL-36γ), which was upregulated only in CMML mice with cachexia. [Figure 7B] FIG. 7B shows NFκB signaling involving IL1F9 (IL-36γ) and its receptors (IL-1Rrp2 and IL-1RAcP). [Figure 8] FIG. 8 shows the levels of IL-36γ in peripheral blood (plasma) of cachectic CMML mice, as measured by ELISA. [Figure 9] FIG. 9 shows the results of an investigation into the muscle atrophy induction of monocyte-derived IL-36γ. [Figure 10A] FIG. 10A shows subcutaneous implantation of 4T1 cells into wild-type BALB / c mice. [Figure 10B] FIG. 10B shows the results of measuring the body weight of 4T1 cell-transplanted mice. [Figure 10C] FIG. 10C shows the results of flow cytometry measurement of the proportion of CD38-positive population among peripheral blood monocytes (CD115-positive) in 4T1 cell-transplanted mice. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will now be described in more detail.

[0020] A first aspect of the present invention relates to a composition for inhibiting muscle tissue loss in a subject having monocytes with elevated CD38 expression, which comprises a compound that inhibits the activity of the IL-36 receptor.

[0021] As used herein, a "subject having monocytes with increased CD38 expression" refers to a subject in which the expression level of CD38 on the surface of monocytes has been measured to be elevated compared to that of a healthy subject. The elevated CD38 expression level can be detected by methods known in the art, such as flow cytometry, immunocytostaining, real-time RT-PCR, etc. In a "subject having monocytes with increased CD38 expression," the expression level of CD38 on the surface of monocytes is elevated, for example, by more than 1.0-fold, preferably 1.2-fold or more, more preferably 1.5-fold or more, even more preferably 2.0-fold, even more preferably 3.0-fold or more, even more preferably 4.0-fold or more, even more preferably 5.0-fold or more, even more preferably 6.0-fold or more, and even more preferably 7.0-fold or more, compared to that of a healthy subject.

[0022] A "subject" is a vertebrate, preferably a mammal. As used herein, the term "mammal" refers to any animal classified as a mammal, including, but not limited to, humans, monkeys, mice, rats, cows, horses, sheep, dogs, and cats. As used herein, a preferred mammal is a human.

[0023] "Muscle tissue loss" refers to a decrease in at least one of the volume and weight of muscle tissue, and "inhibiting muscle tissue loss" refers to maintaining or increasing at least one of the volume and weight of muscle tissue. Muscle tissue volume and weight are measured according to methods known in the art. Whether "muscle tissue loss" in a subject is inhibited can be evaluated by methods known in the art, such as measuring the width of muscle tissue, measuring the weight of muscle tissue, or measuring the volume of muscle tissue. For example, muscle tissue width is measured by taking images while observing cell morphology with an inverted microscope and measuring the width of muscle fiber cells in the images.

[0024] The term "compound that inhibits the activity of IL-36 receptor" is used in the broadest sense and includes compounds involved in the pathway that activates the IL-36 receptor (hereinafter also referred to as the "IL-36 receptor activation pathway") and compounds that inhibit one or more biological activities of cells. As used herein, the "IL-36 receptor activation pathway" refers to a pathway that includes CD38-expressing monocytes and the IL-36 receptor, and preferably includes CD38-expressing monocytes, IL-36γ, and the IL-36 receptor. More specifically, the "IL-36 receptor activation pathway" refers to a pathway in which CD38-expressing monocytes produce IL-36γ and the produced IL-36γ binds to the IL-36 receptor.

[0025] Specifically, the "IL-36 receptor" is IL-1Rrp2 (also referred to as "1L-1RL2" or "1L1RL2"). As shown in Figure 7B, IL-36 receptor ligands (IL-36α, IL-36β, and IL-36γ) bind to the IL-36 receptor, and the ligand-bound IL-36 receptor forms a heterodimer with the IL-36 receptor accessory protein (IL-1RAcP), thereby initiating an intracellular signaling cascade (see BLOOD, 7 APRIL 2011, VOLUME 117, NUMBER 14).

[0026] As mentioned above, the present inventors found that CD38 expression was elevated on the surface of inflammatory monocytes in mice with symptoms of cancer cachexia, and further found that IL1F9 (IL-36γ) produced by these inflammatory monocytes induced muscle atrophy. Specifically, IL1F9 (IL-36γ) binds to the transmembrane IL-36 receptor, inducing muscle atrophy via intracellular signaling (see Figure 7).

[0027] These findings suggest that inhibiting IL-36 receptor activation via the binding of IL-36γ to the IL-36 receptor can inhibit signaling that induces muscle atrophy and prevent muscle tissue loss in subjects with symptoms of cancer cachexia. Furthermore, the findings of the present inventors demonstrate the usefulness of targeting the pathway that leads to IL-36 receptor activation (the "IL-36 receptor activation pathway") in treating subjects with cachexia. Therefore, the terms "compounds involved in the pathway that leads to IL-36 receptor activation" and "cells involved in the pathway that leads to IL-36 receptor activation" include, for example, CD38, monocytes expressing CD38 on their surface, IL-36 receptor ligands (IL-36α, IL-36β, and IL-36γ, particularly IL-36γ), IL-36 receptors, and cells bearing IL-36 receptors.

[0028] "Compounds that inhibit the activation of IL-36 receptor" include compounds that inhibit at least one activity of the above-mentioned compounds and cells, and include, for example, CD38 antagonists, IL-36γ antagonists, and IL-36 receptor antagonists.

[0029] As used herein, the term "antagonist" is used in the broadest sense: an "antagonist" is a molecule that partially or completely blocks, inhibits, neutralizes, prevents, and / or interferes with the biological activity of a target molecule, regardless of the underlying mechanism of action.

[0030] In the context of a "CD38 antagonist," the "biological activity" of the target molecule "CD38" refers to, for example, the ability of CD38 to bind to a ligand and to induce the production of IL-36α, IL-36β, and IL-36γ, particularly IL-36γ, in monocytes expressing CD38 on their surface. CD38 antagonists can be identified, for example, based on their ability to specifically recognize and bind to CD38 and induce antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cellular phagocytosis (ADCP) activity through the activation of immune system cells, as well as their ability to inhibit or block IL-36γ production by CD38-expressing monocytes. For example, CD38 antagonists can be identified by incubating monocytes expressing CD38 on their surface with and without a test compound, further incubating with effector cells or serum containing complement, and measuring the level of a substance, such as LDH, released into the culture supernatant upon cytotoxicity. If the concentration of the substance released upon cytotoxicity is higher in the presence of the test compound than in the absence of the test compound, the test compound is a CD38 antagonist. Alternatively, CD38 antagonists can be identified by incubating monocytes expressing CD38 on their surface with and without a test compound and monitoring the IL-36γ level in the cell culture supernatant, for example, by ELISA. If the IL-36γ level is lower in the presence of the test compound than in the absence of the test compound, the test compound is a CD38 antagonist. Alternatively, real-time RT-PCR can be used to monitor IL-36γ mRNA expression in CD38-expressing tissues before and after treatment with the test compound. A decrease in IL-36γ mRNA levels in the presence of the test compound indicates that the compound is a CD38 antagonist.

[0031] Examples of "CD38 antagonists" include, but are not limited to, neutralizing antibodies against the entire native CD38 polypeptide or a native CD38 polypeptide subunit, immunoadhesins comprising a CD38 subunit fused to an immunoglobulin constant region sequence, small molecules, aptamers that bind to the entire native CD38 polypeptide or a native CD38 polypeptide subunit, nucleic acid molecules (e.g., siRNAs, antisense oligonucleotides, decoys, ribozymes) that can inhibit at least one of the translation and transcription of the gene encoding the entire native CD38 polypeptide or a subunit thereof, and the like.

[0032] The amino acid sequence of a native CD38 polypeptide can be obtained from databases known in the art. Examples of the amino acid sequence of a native CD38 polypeptide include SEQ ID NO: 1 (Human CD38, NCBI Reference Sequence: NP_001766.2) and SEQ ID NO: 2 (Mus musculus CD38, NCBI Reference Sequence: NP_031672.2) shown below. Therefore, CD38 antagonists include antibodies and fragments thereof that specifically recognize polypeptides having such sequences, as well as antibodies and fragments thereof that specifically recognize polypeptides having a certain degree of sequence identity with the sequences (e.g., 80% or more, 85% or more, or 90% or more sequence identity).

[0033] [Table 1]

[0034] Furthermore, examples of the nucleotide sequences of genes encoding native CD38 polypeptides include SEQ ID NO: 3 (Human CD38; NCBI Reference Sequence: NM_001775.4 (Coding sequence, CDS)) and SEQ ID NO: 4 (Mus musculus CD38; NCBI Reference Sequence: NM_007646.5 (Coding sequence, CDS)). Therefore, CD38 antagonists include: a gene encoding a polypeptide having the amino acid sequence as described above; A gene encoding a polypeptide having a certain sequence identity (e.g., 80% or more, 85% or more, or 90% or more sequence identity) with the amino acid sequence described above; A gene having the above-mentioned base sequence, A gene having a base sequence that has a certain degree of sequence identity (e.g., 80% or more, 85% or more, or 90% or more sequence identity) with the above base sequence and nucleic acid molecules capable of inhibiting at least one of the translation and transcription of a gene.

[0035] [Table 2]

[0036] "CD38 antagonists" may include those known in the art. For example, Japanese Patent No. 5843884, Japanese Patent No. 5926568, Japanese Patent No. 6148696, Japanese Patent No. 6494053, Japanese Translation of PCT International Publication No. 2017-507953, Japanese Translation of PCT International Publication No. 2017-528642, Japanese Translation of PCT International Publication No. 2018-502834, Japanese Translation of PCT International Publication No. 2018-520101, Japanese Translation of PCT International Publication No. 2018-537525, Japanese Translation of PCT International Publication No. 2019-503167, Japanese Translation of PCT International Publication No. 2019-527678, Japanese Translation of PCT International Publication No. 2019-516396, US Patent Application Publication No. 2002 / 164788, etc. Anti-CD38 antibodies and fragments thereof, as well as commercially available products and investigational drugs having the function of anti-CD38 antibodies, are mentioned. Examples of commercially available products and investigational drugs include daratumumab, isatuximab, MOR-202, and Darazlex (registered trademark) (Janssen Pharmaceuticals, Inc.), a human anti-CD38 monoclonal antibody.

[0037] In the context of an "IL-36γ antagonist," the "biological activity" of the target molecule, IL-36γ, is, for example, the ability to bind to the IL-36 receptor. IL-36γ antagonists may be identified, for example, based on their ability to specifically recognize and neutralize IL-36γ, or their ability to specifically recognize and / or neutralize IL-36γ and inhibit or block the binding of IL-36γ to the IL-36 receptor. IL-36γ antagonists can be identified, for example, by adding a test compound to the culture supernatant of IL-36γ-producing cells and monitoring the IL-36γ level in the cell culture supernatant, for example, by ELISA. If the IL-36γ level is lower in the presence of the test compound compared to the absence of the test compound, the test compound is an IL-36γ antagonist.

[0038] Examples of "IL-36γ antagonists" include, but are not limited to, neutralizing antibodies against the whole native IL-36γ polypeptide or a subunit of a native IL-36γ polypeptide, immunoadhesins comprising an IL-36γ subunit fused to an immunoglobulin constant region sequence, small molecules, aptamers that bind to the whole native IL-36γ polypeptide or a subunit of a native IL-36γ polypeptide, and nucleic acid molecules (e.g., siRNAs, antisense oligonucleotides, decoys, ribozymes) that can inhibit at least one of the translation and transcription of the gene encoding the whole native IL-36γ polypeptide or a subunit.

[0039] The sequence of native IL-36γ polypeptide can be obtained from databases known in the art. Examples of native IL-36γ polypeptide sequences include SEQ ID NO: 5 (Human IL1F9; NCBI Reference Sequence: NP_062564.1) and SEQ ID NO: 6 (Mus musculus IL1F9; NCBI Reference Sequence: NP_705731.2). Therefore, IL-36γ antagonists include antibodies and fragments thereof that specifically recognize polypeptides having such sequences, as well as antibodies and fragments thereof that specifically recognize polypeptides having a certain degree of sequence identity with the sequences (e.g., 80% or more, 85% or more, or 90% or more sequence identity).

[0040] [Table 3]

[0041] Furthermore, examples of the nucleotide sequence of the gene encoding the native IL-36γ polypeptide include SEQ ID NO: 7 (Human IL1F9; NCBI Reference Sequence: NM_019618.4 (Coding sequence, CDS)) and SEQ ID NO: 8 (Mus musculus IL1F9; NCBI Reference Sequence: NM_153511.3 (Coding sequence, CDS)). Therefore, IL-36γ antagonists include: a gene encoding a polypeptide having the amino acid sequence as described above; A gene encoding a polypeptide having a certain sequence identity (e.g., 80% or more, 85% or more, or 90% or more sequence identity) with the amino acid sequence described above; A gene having the above-mentioned base sequence, A gene having a base sequence that has a certain degree of sequence identity (e.g., 80% or more, 85% or more, or 90% or more sequence identity) with the above base sequence and nucleic acid molecules capable of inhibiting at least one of the translation and transcription of a gene.

[0042] [Table 4]

[0043] As used herein, the terms "IL-36γ," "IL-36G," "IL36γ," "IL36G," "IL1F9," and "IL-1F9" are synonymous and are used interchangeably.

[0044] In the context of an "IL-36 receptor antagonist," the "biological activity" of the target molecule, IL-36 receptor, is, for example, signal transduction mediated by IL-36 ligands (particularly IL-36γ). An "IL-36 receptor antagonist" inhibits the binding of IL-36 receptor ligands (α, β, and γ) to the IL-36 receptor.

[0045] An "IL-36 receptor antagonist" can be identified, for example, based on its ability to act as a competitive inhibitor of IL-36γ binding to the IL-36 receptor, its ability to inhibit or block the binding of IL-36γ to the IL-36 receptor, its binding affinity to a specific IL-36 receptor epitope, its inhibition of the binding of the IL-36 receptor to its ligand, its neutralization or inhibition of IL-36 receptor activity in vivo (e.g., IC 50 ), as well as cross-reactivity (eg, cross-reactivity with homologs or orthologs of the IL-36 receptor protein, or with other proteins or tissues).

[0046] In one aspect, the IL-36 receptor antagonist of the present invention exhibits one or more of the following biological activities: inhibition of the interaction between the IL-36 receptor and at least one of IL-36α, IL-36β, and IL-36γ, inhibition of intracellular signals mediated by the IL-36 receptor, and inhibition of cross-reactivity and activity with human and non-human primate IL-36 receptors.

[0047] Other biological properties or characteristics of receptor antagonists recognized in the art include, for example, avidity, selectivity, solubility, folding, immunotoxicity, and expression. These properties or characteristics can be observed, measured, and / or assessed using techniques known in the art, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORET M), KINEXAT M, in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and / or secretion assays, and signal transduction and immunohistochemistry assays.

[0048] IL-36 receptor antagonists can be identified, for example, by incubating cells expressing IL-36 receptor in the presence and absence of a test compound and monitoring the activity level of the IL-36 receptor signaling pathway in the cells, for example, the expression of mRNA of NF-κB signaling-related genes, using, for example, real-time RT-PCR. If the expression of mRNA of NF-κB signaling-related genes is lower in the presence of the test compound compared to the absence of the test compound, the test compound is an IL-36 receptor antagonist.

[0049] Examples of "IL-36 receptor antagonists" include, but are not limited to, neutralizing antibodies against the entire native IL-36 receptor polypeptide or a natural IL-36 receptor polypeptide subunit, immunoadhesins comprising an IL-36 receptor subunit fused to an immunoglobulin constant region sequence, small molecules, aptamers that bind to the entire native IL-36 receptor polypeptide or a natural IL-36 receptor polypeptide subunit, and nucleic acid molecules (e.g., siRNAs, antisense oligonucleotides, decoys, ribozymes) that can inhibit at least one of the translation and transcription of the gene encoding the entire native IL-36 receptor polypeptide or a subunit.

[0050] The sequence of a native IL-36 receptor polypeptide can be obtained from databases known in the art. Examples of native IL-36 receptor polypeptide sequences include SEQ ID NO: 9 (Human IL1RL2; NCBI Reference Sequence: NP_003845.2) and SEQ ID NO: 10 (Mus musculus IL1RL2; NCBI Reference Sequence: NP_573456.1), as shown below. Therefore, IL-36 receptor antagonists include antibodies and fragments thereof that specifically recognize polypeptides having such sequences, as well as antibodies and fragments thereof that specifically recognize polypeptides having a certain degree of sequence identity with the sequences (e.g., 80% or more, 85% or more, or 90% or more sequence identity).

[0051] [Table 5]

[0052] Furthermore, examples of the nucleotide sequences of the genes encoding the native IL-36 receptor polypeptide include SEQ ID NO: 11 (Human IL1RL2; NCBI Reference Sequence: NM_003854.4 (Coding sequence, CDS)) and SEQ ID NO: 12 (Mus musculus IL1RL2; NCBI Reference Sequence: NM_133193.4 (Coding sequence, CDS)). Therefore, IL-36γ antagonists include: a gene encoding a polypeptide having the amino acid sequence as described above; A gene encoding a polypeptide having a certain sequence identity (e.g., 80% or more, 85% or more, or 90% or more sequence identity) with the amino acid sequence described above; A gene having the above-mentioned base sequence, A gene having a base sequence that has a certain degree of sequence identity (e.g., 80% or more, 85% or more, or 90% or more sequence identity) with the above base sequence The present invention includes nucleic acid molecules capable of inhibiting at least one of the translation and transcription of a gene.

[0053] [Table 6]

[0054] As used herein, with respect to "IL-36 receptor," the terms "IL-1Rrp2," "1L-1RL2," and "1L1RL2" are synonymous and are used interchangeably.

[0055] The term "IL-36 receptor antagonist" may include those known in the art, such as antibodies and fragments thereof described in Japanese Patent No. 6289375, JP 2018-093885 A, and JP 2018-512157 A, molecules described in JP 2017-114829 A, commercially available products, and investigational drugs. An example of an investigational drug is the anti-IL-36 receptor monoclonal antibody BI655130 (Boehringer Ingelheim).

[0056] In one embodiment, the IL-36 receptor antagonist of the present invention is an anti-IL-36 receptor antibody. Anti-IL-36 receptor antibodies have high molecule / cell binding ability. For example, IL-1F5 is known as an IL-36 receptor antagonist.

[0057] As used herein, the term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies (e.g., neutralizing antibodies and agonistic antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. Monoclonal antibodies also include chimeric antibodies and fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies are antibodies in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies from another species, as well as fragments of such antibodies, or in antibodies belonging to another antibody class or subclass (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ). Monoclonal antibodies also include "humanized" antibodies or fragments containing minimal sequence derived from non-human immunoglobulin, such as, but not limited to, Fv, Fab, Fab', F(ab')2, and other antigen-binding subsequences of antibodies. In one embodiment, a humanized antibody can be a human immunoglobulin (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) with the desired specificity, affinity, or capacity. Examples of non-human species include mouse, rat, or rabbit. In one example, Fv and FR residues of the human immunoglobulin are replaced by the corresponding residues in the non-human species. Humanized antibodies can also contain residues that are not found in the CDR or framework sequences introduced into the recipient antibody. These modifications are made with the intent of improving antibody performance. Generally, a humanized antibody will comprise substantially all of one or more, typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.Humanized antibodies may include primatized antibodies, as described in detail in documents known in the art, such as Jones et al., Nature, 321:522-525 (1986) and Reichmann et al., Nature, 332:323-329 (1998).

[0058] "Antibody fragments" include portions of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from single-chain antibody molecules and antibody fragments.

[0059] The term "monoclonal antibody" refers to a substantially homogeneous antibody, i.e., an antibody obtained from a population in which the individual antibodies comprising the population are identical except for minor naturally occurring mutations that may be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0060] The term "antisense oligonucleotide" is defined as a nucleic acid molecule capable of inhibiting the transcription or translation, or both, of a target gene in a sequence-specific manner. The term "antisense" indicates that the nucleic acid is complementary to the coding ("sense") gene sequence of the target gene. Antisense oligonucleotides hybridize to nascent mRNA in an antiparallel orientation via Watson-Crick base linkages. By binding to the target mRNA template, antisense oligonucleotides inhibit successful translation of the encoded protein. This term specifically includes antisense agents called "ribozymes," which are designed to induce catalytic cleavage of target RNA by adding a sequence with natural self-splicing activity (see, e.g., Warzocha et al., Leuk. Lymphoma 24:267-281

[1997] ).

[0061] Another embodiment of the present invention relates to a composition that inhibits the production of IL-36γ, comprising a CD38 antagonist. As described above, monocytes that express CD38 produce IL-36γ, and therefore, a composition that comprises a CD38 antagonist can inhibit the production of IL-36γ.

[0062] Yet another embodiment of the present invention relates to a composition for inhibiting IL-36 receptor activity, comprising at least one of a CD38 antagonist, an IL-36γ antagonist, and an IL-36 receptor antagonist. As described above, IL-36 receptor activation is mediated by one or more of CD38, IL-36γ, and IL-36 receptor, and therefore a composition comprising one or more of a CD38 antagonist, an IL-36γ antagonist, and an IL-36 receptor antagonist can inhibit IL-36 receptor activity.

[0063] Another embodiment of the present invention relates to a composition for suppressing muscle tissue loss, comprising a compound that inhibits IL-36 receptor activity. As described above, the present inventors have found that muscle atrophy is induced through intracellular signaling mediated by IL-36 receptor activation ( FIG. 7B ). Therefore, inhibiting IL-36 receptor activity inhibits intracellular signaling, thereby suppressing muscle atrophy (i.e., muscle tissue loss).

[0064] In one embodiment, a "subject" "having monocytes with increased CD38 expression" means that the subject is suffering from cancer and / or has a cancer-related symptom, where "cancer-related symptom" includes cancer cachexia, weight loss, anorexia, and muscle weakness, and the cancer-related symptom is preferably cancer cachexia.

[0065] That is, a second aspect of the present invention is a pharmaceutical composition for treating cancer, particularly a pharmaceutical composition for treating cancer-related symptoms.

[0066] "Cancer" includes both blood cancers and solid cancers. Examples of "blood cancers" include, but are not limited to, malignant lymphoma, multiple myeloma, leukemia (including chronic and acute), and myeloproliferative neoplasms. Examples of "solid cancers" include, but are not limited to, breast cancer, colorectal cancer, malignant melanoma, bone tumors, soft tissue tumors, brain tumors, tongue cancer, pharyngeal cancer, esophageal cancer, thyroid cancer, lung cancer, gastric cancer, ovarian cancer, cervical cancer, uterine cancer, renal cell cancer, prostate cancer, bladder cancer, skin cancer, liver cancer, and pancreatic cancer. In a preferred embodiment, the "blood cancer" is chronic myelomonocytic leukemia. In another preferred embodiment, the "solid cancer" is breast cancer.

[0067] On the other hand, even in diseases and disorders other than cancer, subjects may have monocytes that express CD38 and may exhibit or be expected to exhibit a decrease in muscle tissue.

[0068] That is, in another embodiment, when a "subject" "has monocytes with elevated CD38 expression," it means that the subject is suffering from an inflammatory disease. "Inflammatory disease" refers to a pathological condition that causes inflammation, which is typically caused by neutrophil chemotaxis.

[0069] "Inflammatory diseases" include, but are not limited to, inflammatory skin diseases (e.g., psoriasis and dermatitis), generalized sclerosis, systemic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, surgical tissue reperfusion injury and myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery, stenosis after percutaneous transluminal coronary angioplasty, ischemic reperfusion disease (e.g., stroke and abdominal aortic aneurysm), cerebral edema secondary to stroke, cranial trauma, hypovolemic shock, asphyxia, and adult respiratory distress syndrome. Acute lung injury, Behçet's disease, dermatomyositis, polymyositis, multiple sclerosis (MS), dermatitis, meningitis, encephalitis, uveitis, osteoarthritis, lupus nephritis, joint inflammatory conditions (e.g., rheumatoid arthritis (RA), rheumatoid spondylitis, gouty arthritis), Sjögren's syndrome, vasculitis, diseases with leukocyte extravasation, central nervous system (CNS) inflammatory diseases, sepsis or multiple organ injury syndrome secondary to trauma, alcoholic hepatitis, bacterial pneumonia, antigen-antibody complex-mediated diseases including nephritis, sepsis, Sarcoidosis, abnormal immune response to tissue transplants, abnormal immune response to organ transplants, pleuritis, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), lung inflammation including cystic fibrosis, chronic inflammation, acute inflammation, autoimmune diabetes, asthma, systemic lupus erythematosus, adult respiratory distress syndrome, chronic pulmonary inflammatory disease, graft-versus-host disease, Alzheimer's disease, fever, and other inflammation-related diseases, disorders, and abnormal conditions.

[0070] In yet another embodiment, a "subject" having "monocytes with elevated CD38 expression" means that the subject suffers from an autoimmune disease. Examples of "autoimmune diseases" include, but are not limited to, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, hyperthyroidism, autoimmune adrenal insufficiency, autoimmune hemolytic anemia, multiple sclerosis, psoriatic arthritis, Sjögren's syndrome, polymyositis, dermatomyositis, myasthenia gravis, idiopathic thrombocytopenic purpura, autoimmune optic neuropathy, and scleroderma. The "autoimmune disease" is preferably at least one selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, and psoriasis.

[0071] In yet another embodiment, the term "subject" having "monocytes with increased CD38 expression" means that the subject is suffering from a chronic infection. Examples of "chronic infections" include, but are not limited to, HIV infection, tuberculosis, malaria, leprosy, and syphilis. The "chronic infection" is preferably at least one selected from the group consisting of HIV infection, tuberculosis, and malaria.

[0072] The term "treatment" refers to both therapeutic treatment and prophylactic measures, the purpose of which is to prevent or slow (alleviate) undesirable physiological changes or disorders. In the context of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms and the extent of disease, whether detectable or not, stabilization of the disease state (i.e., not worsening), delaying or slowing the progression of disease, improvement or palliation of the disease state, or remission (including partial and total remission). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Subjects in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0073] The pharmaceutical composition according to the present invention may typically contain pharmaceutically acceptable additives, such as excipients, binders, lubricants, solvents, diluents, stabilizers, isotonicity agents, etc., which are known in the art. Examples of excipients include starch, lactose, methylcellulose, crystalline cellulose, synthetic aluminum silicate, etc.; binders include hydroxypropyl cellulose, polyvinylpyrrolidone, etc.; lubricants include talc, magnesium stearate, calcium stearate, etc.; and isotonicity agents include sodium chloride, glucose, glycerol, mannitol, etc.

[0074] The pharmaceutical composition of the present invention can be formulated into, for example, tablets, powders, granules, capsules, injections, liquids, suppositories, etc., and can be administered, for example, orally, intravenously, subcutaneously, intraperitoneally, or intramuscularly. The dosage of the pharmaceutical composition of the present invention varies depending on the age, weight, medical condition, etc. of the subject.

[0075] The compositions of the present invention may be used as a combination drug in combination with an additional pharmaceutical composition.

[0076] Regarding the combination, the components of each pharmaceutical composition (i.e., the first active ingredient and the second active ingredient) may be administered together, sequentially, or separately in one combined unit dosage form or in two separate unit dosage forms. A therapeutically effective amount of each active ingredient of the combination of the present invention may be administered simultaneously, separately, or in any order, sequentially, or consecutively. In the combination, the first active ingredient and the second active ingredient may be present in multiple units. The combination also includes, for example, a kit containing each active ingredient and instructions for use. [Example]

[0077] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples in any way.

[0078] The samples and animals used in the examples were prepared and measurements were carried out according to the procedures described below.

[0079] [Cell culture] Plat-E packaging cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (FBS) (Atlas Biological), 1% penicillin-streptomycin (P / S) (Nacalai Tesque), 1 ng / ml puromycin, and 10 ng / ml blastcidine at 37°C and 5% CO2. For subculture, cells were detached using 0.25 w / v% trypsin in 1 mmol / L EDTA·4Na solution (containing phenol red) (Fujifilm Wako Pure Chemical Industries). C2C12 cells were cultured in DMEM containing 10% FBS and 1% P / S. For myofiber differentiation, the medium was replaced with DMEM containing 2% horse serum (Thermo Fisher Scientific) and 1% P / S. 4T1 and THP1 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% P / S. Plat-E cells were kindly provided by Dr. Toshio Kitamura of the Institute of Medical Science, University of Tokyo. C2C12 cells were purchased from RIKEN BRC, and 4T1 cells were purchased from ATCC.

[0080] [Vector Plasmid] Total RNA was extracted from mouse hematopoietic cells using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized using RevertraAce (Toyobo). Using this cDNA as a template, the ORF sequence of Il1f9 (Il36g) was amplified by PCR. The primers used are shown below.

[0081] [Table 7]

[0082] The PCR product was purified and subcloned into the pCR Blunt II TOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific). The Il1f9 ORF sequence was amplified by PCR using this as a template, and restriction enzyme (XhoI, NotI) recognition sequences were added. The primers used are shown below.

[0083] [Table 8]

[0084] The PCR product was digested with restriction enzymes and inserted into the multi-cloning site of the pMYs-IRES-GFP retroviral vector using Ligation High ver. 2 (Toyobo). The pMYs-NUP98-HBO1-IRES-GFP vector was described in Hayashi Y, et al. Blood Adv. 2019; 3(7): 1047-1060.

[0085] [Retrovirus production] Plat-E cells were passaged onto 10 cm dishes the day before, reaching a cell density of 70-80%, and then transfected with a retroviral vector using FuGENE-HD (Promega). OPTI-MEM (Thermo Fisher Scientific) was used as the serum-free medium for transfection. The medium was replaced 24 hours after transfection, and the Plat-E culture supernatant containing the virus particles was collected 48 hours after transfection. The supernatant was passed through a 0.45 μm PVDF filter (Merck), mixed with 4x PEG buffer (3:1 ratio), mixed by inversion, and left overnight at 4°C. The cells were then centrifuged at 1800 g and 4°C for 45 minutes and resuspended in 2 ml of medium to obtain a concentrated virus solution.

[0086] 〔mouse〕 Wild-type C57BL / 6 mice (7 weeks old, female) and BALB / c mice (7 weeks old, female) were purchased from Tokyo Experimental Animals. Animal experiments were approved by the Animal Experimentation Committee of Tokyo University of Pharmacy and Life Sciences. Experiments were conducted in accordance with the Act on the Welfare and Management of Animals, the Standards for the Care and Management of Laboratory Animals and the Reduction of Pain, the Basic Guidelines for the Conduct of Animal Experiments in Research Institutions, the Guidelines for the Proper Conduct of Animal Experiments prepared by the Science Council of Japan, and the Tokyo University of Pharmacy and Life Sciences Animal Experimentation Regulations established based on these. Only necessary experiments were conducted, with careful consideration given to reducing animal pain, reducing the number of animals, and considering alternative experimental methods.

[0087] [Collection and culture of mouse hematopoietic stem and progenitor cells] Wild-type C57BL / 6 mice (7 weeks old, female) were intraperitoneally administered 150 mg / kg 5-fluorouracil (5FU) (Kyowa Hakko Kirin). 96 hours later, the mice were euthanized under inhalation anesthesia with isoflurane (Fujifilm Wako Pure Chemical Industries). All subsequent procedures were performed on ice. Femurs and tibias were collected and flushed into a 50 ml centrifuge tube with 2% FBS / phosphate-buffered saline (PBS) using a 21G needle (Terumo) and a 1 ml syringe (Terumo). The bone marrow cell suspension was centrifuged (400 g, 4°C, 5 minutes), the supernatant was aspirated, and the cells were resuspended in Pharm Lyse (BD Biosciences) to lyse and remove mature red blood cells. After centrifugation (400 g, 4°C, 5 minutes), the supernatant was aspirated and the cells were resuspended in 2% FBS / PBS for washing. The cells were centrifuged again (400×g, 4°C, 5 min) and resuspended in bone marrow cell culture medium. They were then cultured overnight at 37°C in 5% CO2. The bone marrow cell culture medium was Iscove's Modified Dulbecco's Medium (IMDM) (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 20% FBS (Equitech-Bio), 1% penicillin-streptomycin, 50 ng / ml mouse stem cell factor (SCF), 50 ng / ml mouse interleukin 6 (IL-6), 50 ng / ml mouse thrombopoietin (TPO), and 50 ng / ml mouse FMS-like tyrosine kinase 3 (FLT3) ligand (FLT3-L) (Peprotech).

[0088] [Gene transfer into mouse hematopoietic stem and progenitor cells and cell lines] Concentrated virus solution was added to a non-treated 6-well plate coated with RetroNectin (Takara) according to the product manual, and centrifuged (2000g, 32°C, 2 hours) to allow viral particles to bind to RetroNectin. The virus solution was aspirated and washed with PBS, after which a cultured mouse bone marrow cell suspension or THP1 cells were added. The plates were then cultured at 37°C in the presence of 5% CO2 for 72 hours, allowing the virus to infect the mouse bone marrow cells or THP1 cells (gene transfer).

[0089] [Mouse bone marrow transplantation] The transfected mouse bone marrow cells were collected and centrifuged (400 g, 4°C, 5 minutes). The supernatant was aspirated and resuspended in PBS (200 μL of PBS per recipient mouse). The cell suspension for transplantation was kept on ice until immediately before transplantation. Wild-type C57BL / 6 mice (7 weeks old, female) were irradiated with a sublethal dose (6-7 Gray), and the cell suspension for transplantation was injected into the tail vein (bone marrow transplantation). A 27G insulin syringe (Terumo) was used for injection.

[0090] [Creation of a solid tumor subcutaneous transplant model] 1 × 10 per mouse 4 4T1 cells were resuspended in 200 μL of PBS and subcutaneously implanted using a 27G insulin syringe. Mice were injected subcutaneously with the same volume of PBS as control mice.

[0091] [Peripheral blood collection] Under sufficient anesthesia (intraperitoneal administration of a triple anesthetic mixture consisting of medetomidine hydrochloride (0.3 mg / kg) (Nippon Zenyaku Kogyo), midazolam (4 mg / kg) (Sandoz), and butorphanol tartrate (5 mg / kg) (Meiji Seika Pharma) or inhalation of isoflurane (Fujifilm Wako Pure Chemical Industries)), 50–100 μL of peripheral blood was collected from the orbital venous plexus of the transplanted mice using a heparinized blood collection capillary tube (Drummond Scientific Company). The collected peripheral blood was placed in a 1.5 ml tube coated with ethylenediaminetetraacetic acid (EDTA), mixed well, and then placed on ice.

[0092] [Complete blood count measurement] Peripheral complete blood count was measured using a multiparameter automated hematology analyzer pocH (registered trademark)-100iV Diff (Sysmex).

[0093] [Peripheral blood smear] A few μL of peripheral blood was placed on a glass slide to prepare a peripheral blood smear. After thorough drying with cold air, the slide was stained with Diff-Quick (simple Wright-Giemsa staining) (Sysmex), and cell morphology was observed using an upright microscope, Nikon Eclipse Ni (Nikon).

[0094] [ELISA] Il1f9 (Il36g) levels in mouse plasma were measured using a Mouse Interleukin-36 gamma ELISA Kit (Mybiosource).

[0095] [Bone marrow cell collection] Mice were euthanized by cervical dislocation under sufficient inhalation anesthesia with isoflurane (Fujifilm Wako Pure Chemical Industries). All subsequent procedures were performed on ice. Femurs and tibias were collected and flushed with 2% FBS / PBS in a 15 ml centrifuge tube or a 5 ml FACS tube using a 21G needle (Terumo) and a 1 ml syringe (Terumo). The bone marrow cell suspension was centrifuged (400 g, 4°C, 5 min), the supernatant was aspirated, and the cells were resuspended in 500 μL of Pharm Lyse (BD Biosciences) to lyse and remove mature red blood cells. The total volume was adjusted to 5 ml with 2% FBS / PBS and centrifuged (400 g, 4°C, 5 min), after which the supernatant was aspirated and resuspended in an appropriate volume of 2% FBS / PBS for use in experiments.

[0096] [Bone marrow cell cytospin preparation] 3 × 10 5Bone marrow cells were resuspended in 100 μL of FBS and cytospin preparations were prepared on glass slides using a Cytotec AutoSmear 2500 (Sakura Finetech Japan). After thorough drying with cold air, the samples were stained with Diff-Quick (simple Wright-Giemsa staining) (Sysmex), and cell morphology was observed using an upright microscope, Nikon Eclipse Ni (Nikon).

[0097] [C2C12 co-culture experiment] C2C12 cells were seeded in a 6-well plate and differentiated into myofibers. Cell culture inserts (ThinCert (0.4 μm), Greiner) were inserted, and mouse whole bone marrow cells (2 × 10 6 cells) or mouse inflammatory monocytes (2-4 × 10 5 Co-culture was performed with 100 (100) of the 100 cells / ml ...

[0098] [Measurement of muscle fiber cell width] Cell morphology was observed under an inverted microscope (Olympus CKX31, Olympus), and images were taken with a Nikon 1 J5, and the width of C2C12 myofiber cells was measured using ImageJ software.

[0099] [Flow cytometry] The FACS buffer used was 2% FBS / PBS supplemented with 2 mM EDTA. For peripheral blood analysis, 10 μL of peripheral blood was hemolyzed with 1 mL of Pharm Lyse (BD Biosciences) (on ice for 10 minutes), washed with FACS buffer, and resuspended in 150 μL of FACS buffer. For bone marrow cell analysis, 1–2 × 10 cells were used. 6Bone marrow cells were resuspended in 200 μL of FACS buffer per sample. Cell surface antigens were stained with anti-mouse Gr-1 (RB6-8C5), anti-mouse CD115 (AFS98), anti-mouse c-Kit (2B8), anti-mouse Ly6C (HK1.4), anti-mouse B220 (RA3-6B2), and anti-mouse CD38 (90) antibodies (Biolegend) for 30 minutes on ice in the dark. After staining, cells were washed with a sufficient amount of FACS buffer (centrifugation: 400 x g, 4°C, 3 minutes) and then resuspended in 300 μL of FACS buffer. The cell suspension was transferred to a FACS tube through a 40-100 μm filter immediately before analysis. Dead cells were labeled with 7-Aminoactinomycin D (7AAD) (Biolegend). FACS Canto (Beckton Dickinson) was used for flow cytometry analysis, and FlowJo (Tree Star) was used for data analysis.

[0100] [RNA-Seq] Ly6C in mouse bone marrow was analyzed using a cell sorter (SH800, Sony). high CD115 + Inflammatory monocytes were collected, and total RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA samples were submitted to GeneNex for RNA sequencing (Novaseq, paired-end, 150 bp, 6 G / sample). Principal component analysis (PCA) and data analysis were performed using AltAnalyze software (v. 2.1.0-Win64).

[0101] [Gene Set Enrichment Analysis (GSEA)] Microarray data from human umbilical cord blood CD34+ cells were analyzed, and 434 genes whose expression levels were increased by NUP98-HBO1 at least two-fold over those of control were identified as the NUP98-HBO1-induced gene cluster. Using GSEA (v3.0), we analyzed the dynamics of the NUP98-HBO1-induced gene cluster in gene expression data from publicly available databases of CMML (E-MTAB-1044) and MDS patients (GSE43399 and GSE19429). The following parameters were used for the analysis: 1,000 gene_set permutations, Signal2Noise ranking metric, and descending order real mode for gene sorting.

[0102] <1> CMML model mouse using the novel NUP98-HBO1 fusion gene We cloned the novel fusion gene NUP98-HBO1 (Figure 1) identified from CMML patient cells and created a retroviral vector (pMYs-NUP98-HBO1-IRES-GFP). Mouse bone marrow hematopoietic stem and progenitor cells were infected with the retrovirus to transduce the NUP98-HBO1 gene, and the cells were then transplanted into wild-type mice by bone marrow transplantation (BMT).

[0103] After BMT, mice showed a significant increase in white blood cells (WBC), macrocytic anemia (high MCV, decreased Hb), decreased platelets (PLTS), and a characteristic increase in inflammatory monocytes (Figures 2A and 2B). The bone marrow blast count was less than 20%, and megakaryocytic dysplasia was prominent (Figures 2C and 2D).

[0104] The gene group whose expression was increased when NUP98-HBO1 was introduced into human umbilical cord blood hematopoietic stem / progenitor cells (NUP98-HBO1-induced genes) was significantly increased in the CMML patient group (Figure 2E).

[0105] These results indicate the establishment of a model that faithfully reproduces the diverse clinical findings and gene expression patterns of CMML using the novel fusion gene NUP98-HBO1 (Hayashi Y, et al. Blood Adv. 2019; 3(7): 1047-1060).

[0106] <2> Development of cancer cachexia in NUP98-HBO1 CMML model mice The CMML mice established by the present inventors showed cachexia, a characteristic symptom of CMML, specifically skeletal muscle atrophy (Fig. 3A) and progressive weight loss (Fig. 3B).

[0107] <3> Induction of skeletal muscle atrophy by inflammatory monocytes characteristic of CMML Whole bone marrow cells from NUP98-HBO1 mice and control mice, or Ly6C-positive inflammatory monocytes collected by cell sorting, were co-cultured with C2C12 cells differentiated into myofibrocytes (Figure 4A). Culture inserts were inserted and the cells were co-cultured for 72 hours without direct cell-to-cell contact, and the width of the C2C12 myofibrocytes was measured. The measurement results are shown in Figure 4B.

[0108] The results in Figure 4B show that the width of co-cultured C2C12 myofibers was reduced in NUP98-HB01 mice compared with control mice, indicating that inflammatory monocytes, characteristic of NUP98-HB01 mice, are involved in inducing muscle tissue atrophy.

[0109] <4> Differential gene expression in inflammatory monocytes of CMML mice Bone marrow cells were collected from NUP98-HBO1 mice (with cachexia), NUP98-HBO1 mice (without cachexia), and control mice, and CD115-positive Ly6C-positive inflammatory monocytes were isolated using a cell sorter. RNA was extracted from these cells, RNA-Sequencing was performed, and the data were analyzed using AltAnalyze (Figures 6A and 7A). Principal component analysis suggested that specific gene expression changes occurred in inflammatory monocytes from CMML mice that developed cachexia (Figure 5).

[0110] <5> Identification of genes upregulated in monocytes from cachectic CMML mice Based on the RNA-Seq data analysis (Figure 6A), we focused on genes encoding cell surface antigens that were upregulated only in CMML mice with cachexia. The actual protein expression of several candidate genes was analyzed by flow cytometry. Results revealed that CD38 expression was upregulated on inflammatory monocytes from CMML mice with cachexia (Figure 6B). The CD38 gene expression level was 7.68-fold higher in CMML mice with cachexia compared with control mice. Furthermore, flow cytometry analysis of protein expression revealed that CD38 expression was almost absent on the surface of monocytes from control mice, whereas CD38 expression was confirmed on the surface of more than 50% of monocytes from CMML mice with cachexia.

[0111] Based on the results of RNA-Seq data analysis (Figure 7A), we next focused on IL1F9 (IL36G), a gene encoding humoral factors whose expression was elevated only in CMML mice with cachexia. Figure 7B shows NFκB signaling involving IL1F9 (IL-36γ) and its receptors (IL-1Rrp2 and IL-1RAcP).

[0112] Based on these results, the present inventors hypothesized that IL1F9 (IF36G) might be involved in the development of cachexia in subjects with inflammatory monocytes that highly express CD38.

[0113] <6> Verification of the involvement of IL1F9 (IF36G) in the development of cachexia To verify the above hypothesis, the present inventors conducted the following test.

[0114] <6-1>IL36G levels in peripheral blood (plasma) of cachexia-infected CMML mice The IL36G levels in the peripheral blood (plasma) of CMML mice with cachexia were measured using ELISA, and it was confirmed that the IL36G levels in the peripheral blood (plasma) of CMML mice with cachexia were elevated compared to control mice (Figure 8).

[0115] <6-2> Verification of muscle atrophy induction by monocytic cell-derived IL36G To further investigate whether monocytic cell-derived IL36G can induce muscle atrophy, we retrovirally transfected THP1 cells with the Il36g gene and then collected GFP-labeled transfected cells using a cell sorter. The medium of C2C12 cells differentiated into myofibers was replaced with the culture supernatant of IL36G-overexpressing THP1 cells, and the width of C2C12 cell myofibers was measured 48 hours later. Culture supernatant of THP1 cells transfected with an empty vector served as a control. The results are shown in Figure 9. Figure 9 shows that the width of C2C12 cell myofibers was reduced in the Il36g gene-transfected samples compared to the control samples, suggesting that monocytic cell-derived IL36G induces muscle atrophy.

[0116] <7> Confirmation of increased monocytic expression of CD38 in solid tumor models 4T1 cells, a mouse breast cancer cell line, were subcutaneously transplanted into wild-type BALB / c mice (Figure 10A). The weight of the transplanted mice was measured periodically, and weight changes compared to day 0 were recorded (Figure 10B). Flow cytometry was also used to measure the proportion of CD38-positive populations among peripheral blood monocytes (CD115-positive) of the mice (Figure 10C).

[0117] The results in Figures 10B and 10C show that CD38-positive monocytes are induced even in breast cancer, a solid cancer. These results confirm that the induction of cachexia-associated CD38-positive monocytes is not limited to CMML, a blood cancer, but also appears in solid tumor cachexia model mice in line with the progression of the disease. [Industrial Applicability]

[0118] The compositions of the present invention can be used to suppress muscle tissue loss in subjects with monocytes that express CD38. It is also suggested that existing pharmaceuticals containing as active ingredients compounds involved in the pathway that activates the IL-36 receptor and compounds that inhibit one or more biological activities of the cells can be used to suppress muscle tissue loss.

Claims

1. 1. A composition for inhibiting muscle tissue loss in a subject having monocytes with elevated CD38 expression, comprising: a compound that inhibits the activity of the IL-36 receptor, The compound that inhibits the activity of the IL-36 receptor is IL-36γ antagonists and ・IL-36 receptor antagonist At least one selected from the group consisting of: the IL-36γ antagonist is selected from the group consisting of a neutralizing antibody against the whole native IL-36γ polypeptide or a subunit of the native IL-36γ polypeptide, and a nucleic acid molecule capable of inhibiting at least one of the translation and transcription of a gene encoding the whole native IL-36γ polypeptide or a subunit of the native IL-36γ polypeptide; the IL-36 receptor antagonist is selected from the group consisting of a neutralizing antibody against the whole native IL-36 receptor polypeptide or a subunit of the native IL-36 receptor polypeptide, and a nucleic acid molecule capable of inhibiting at least one of the translation and transcription of a gene encoding the whole native IL-36 receptor polypeptide or a subunit of the native IL-36 receptor polypeptide; the subject has a cancer-related symptom; The composition, wherein the cancer-related symptom is cancer cachexia.

2. A composition for inhibiting muscle tissue loss in a subject having monocytes with increased CD38 expression, comprising: a compound that inhibits the activity of the IL-36 receptor, The compound that inhibits the activity of the IL-36 receptor is IL-36γ antagonists and ・IL-36 receptor antagonist At least one selected from the group consisting of: the IL-36γ antagonist is an anti-IL-36γ antibody; the IL-36 receptor antagonist is an anti-IL-36 receptor antibody; the subject has a cancer-related symptom; The composition, wherein the cancer-related symptom is cancer cachexia.

3. The IL-36γ antagonist is an antibody or an antigen-binding fragment thereof that specifically recognizes an IL-36γ polypeptide having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6; The composition according to claim 1 or 2, wherein the IL-36 receptor antagonist is an antibody or an antigen-binding fragment thereof that specifically recognizes an IL-36 receptor polypeptide having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO:

10.

4. The composition according to any one of claims 1 to 3, wherein the subject is a mammal.

5. The composition according to any one of claims 1 to 4, wherein the mammal is selected from the group consisting of humans, monkeys, mice, rats, cows, horses, sheep, dogs and cats.

6. The composition according to any one of claims 1 to 5, wherein the subject having monocytes with increased CD38 expression is suffering from cancer.

7. The composition of claim 6 , wherein the cancer is at least one of a blood cancer and a solid cancer.

8. the blood cancer is at least one selected from the group consisting of malignant lymphoma, multiple myeloma, leukemia, and myeloproliferative neoplasm; The composition according to claim 7, wherein the solid cancer is at least one selected from the group consisting of breast cancer, colon cancer, malignant melanoma, lung cancer, pancreatic cancer, gastric cancer, and lung cancer.

9. the blood cancer is chronic myelomonocytic leukemia; The composition of claim 7 or 8, wherein the solid cancer is breast cancer.

10. The composition according to any one of claims 1 to 9, which is a pharmaceutical composition for treating at least one selected from the group consisting of cancer, inflammatory diseases, autoimmune diseases, and chronic infectious diseases.

11. The composition according to any one of claims 1 to 10, which is a pharmaceutical composition for treating cancer cachexia.

12. 1. A composition for inhibiting IL-36γ-mediated activation of IL-36 receptor in a subject with cancer cachexia, comprising: the IL-36γ is produced by monocytes expressing CD38; the composition comprises a compound that inhibits the activity of an IL-36 receptor; The compound that inhibits the activity of the IL-36 receptor is IL-36γ antagonists and ・IL-36 receptor antagonist At least one selected from the group consisting of: the IL-36γ antagonist is selected from the group consisting of a neutralizing antibody against the whole native IL-36γ polypeptide or a subunit of the native IL-36γ polypeptide, and a nucleic acid molecule capable of inhibiting at least one of the translation and transcription of a gene encoding the whole native IL-36γ polypeptide or a subunit of the native IL-36γ polypeptide; The composition, wherein the IL-36 receptor antagonist is selected from the group consisting of a neutralizing antibody against the entire native IL-36 receptor polypeptide or a subunit of the native IL-36 receptor polypeptide, and a nucleic acid molecule capable of inhibiting at least one of the translation and transcription of a gene encoding the entire native IL-36 receptor polypeptide or a subunit of the native IL-36 receptor polypeptide.

13. A composition for inhibiting IL-36γ-mediated activation of IL-36 receptor in a subject with cancer cachexia, comprising: the IL-36γ is produced by monocytes expressing CD38; the composition comprises a compound that inhibits the activity of an IL-36 receptor; The compound that inhibits the activity of the IL-36 receptor is IL-36γ antagonists and ・IL-36 receptor antagonist At least one selected from the group consisting of: the IL-36γ antagonist is an anti-IL-36γ antibody; The composition, wherein the IL-36 receptor antagonist is an anti-IL-36 receptor antibody.

14. A composition for inhibiting the IL-36 receptor activation pathway in a subject with cancer cachexia, comprising: the IL-36 receptor activation pathway comprises a monocyte expressing CD38 and an IL-36 receptor; the composition comprises a compound that inhibits the activity of an IL-36 receptor; The compound that inhibits the activity of the IL-36 receptor is IL-36γ antagonists and ・IL-36 receptor antagonist At least one selected from the group consisting of: the IL-36γ antagonist is selected from the group consisting of a neutralizing antibody against the whole native IL-36γ polypeptide or a subunit of the native IL-36γ polypeptide, and a nucleic acid molecule capable of inhibiting at least one of the translation and transcription of a gene encoding the whole native IL-36γ polypeptide or a subunit of the native IL-36γ polypeptide; The composition, wherein the IL-36 receptor antagonist is selected from the group consisting of a neutralizing antibody against the entire native IL-36 receptor polypeptide or a subunit of the native IL-36 receptor polypeptide, and a nucleic acid molecule capable of inhibiting at least one of the translation and transcription of a gene encoding the entire native IL-36 receptor polypeptide or a subunit of the native IL-36 receptor polypeptide.

15. A composition for inhibiting the IL-36 receptor activation pathway in a subject with cancer cachexia, comprising: the IL-36 receptor activation pathway comprises a monocyte expressing CD38 and an IL-36 receptor; the composition comprises a compound that inhibits the activity of an IL-36 receptor; The compound that inhibits the activity of the IL-36 receptor is IL-36γ antagonists and ・IL-36 receptor antagonist At least one selected from the group consisting of: the IL-36γ antagonist is an anti-IL-36γ antibody; The composition, wherein the IL-36 receptor antagonist is an anti-IL-36 receptor antibody.

16. The composition according to claim 14 or 15, wherein the IL-36 receptor activation pathway comprises IL-36γ production by CD38-expressing monocytes and IL-36γ binding to the IL-36 receptor.

17. The IL-36γ antagonist is an antibody or an antigen-binding fragment thereof that specifically recognizes an IL-36γ polypeptide having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6; The composition according to any one of claims 12 to 16, wherein the IL-36 receptor antagonist is an antibody or an antigen-binding fragment thereof that specifically recognizes an IL-36 receptor polypeptide having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10.

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