WO2023287096A1

WO2023287096A1 - Composition for inhibiting m2 polarization of macrophages and treating cancer, comprising humanized monoclonal antibody specific to chi3l1

Info

Publication number: WO2023287096A1
Application number: PCT/KR2022/009655
Authority: WO
Inventors: 홍진태; 유지은; 김기천
Original assignee: 충북대학교 산학협력단
Priority date: 2021-07-13
Filing date: 2022-07-05
Publication date: 2023-01-19
Also published as: KR20240010079A; KR20230011064A

Abstract

The present invention relates to a method for inhibiting M2 polarization of macrophages, comprising a humanized monoclonal antibody specific to Chi3L1, and a composition therefor. In addition, the present invention relates to: a composition for preventing or treating cancer or inhibiting cancer metastasis comprising, as an active ingredient, a composition for inhibiting M2 polarization of macrophages; a composition for diagnosing cancer; and a kit therefor, and relates to: a method for screening for candidate substances preventing or treating cancer or inhibiting cancer metastasis; and a composition and a kit therefor. A composition for inhibiting M2 polarization of macrophages, of the present invention, effectively inhibits M2 polarization of macrophages and STAT6 phosphorylation and has the excellent effect of inhibiting tumor growth and metastasis, and thus can be variously used as compositions for preventing and treating cancer and inhibiting cancer metastasis. In addition, a screening method and a composition or kit therefor, of the present invention, are used, and thus therapeutic candidate substances capable of effectively preventing and treating cancer and inhibiting cancer growth and metastasis can be easily and significantly selected.

Description

Composition for Inhibiting M2 Polarization of Macrophages and Treating Cancer Containing a CHI3L1-specific Humanized Monoclonal Antibody

The present invention relates to a method for inhibiting M2 polarization of macrophages, including a humanized monoclonal antibody specific for Chi3L1, and a composition thereof. In addition, the present invention relates to a composition for preventing, treating, or inhibiting metastasis of cancer, a composition for diagnosing cancer, and a kit comprising the composition for inhibiting M2 polarization of macrophages as an active ingredient, and a candidate material for preventing, treating, or inhibiting cancer metastasis It relates to a screening method and its composition and kit.

Chi3L1 (chitinase 3-like 1), also known as YKL-40, is a 40 kDa secretory glycoprotein and belongs to the chitinase-like proteins (CLPs). Chi3L1 is a highly conserved chitin-binding protein and its three-dimensional crystal structure shows a typical folding structure of chitinase family proteins, but its chitinase activity is characterized by deletion due to essential amino acid residues in the enzyme domain . Chi3L1 is known to be overexpressed in cancer or tumor-related macrophages, and is known to be highly expressed in the serum of patients with metastatic cancer or chronic inflammatory diseases.

Chi3L1 together with IL13R2 can regulate apoptosis in a TMEM219-dependent manner, and in rheumatoid arthritis, when Chi3L1 is reduced using shRNA or miRNA, it is confirmed that a therapeutic effect can be expected by reducing IL-18 production through the PI3K/AKT pathway It has been confirmed that the proteasome inhibitor, Bortezomib, uses this mechanism.

Korean Registered Patent Publication No. 10-2142499 discloses a humanized monoclonal antibody that specifically binds to Chi3L1 protein, but its mechanism of action and efficacy have not been specifically identified. Research on this is still incomplete. Accordingly, the present inventors identified for the first time that a humanized monoclonal antibody specific for Chi3L1 inhibits macrophage M2 polarization and STAT6 phosphorylation, and a composition for inhibiting macrophage M2 polarization and STAT6 phosphorylation, prevention of cancer, A composition for treatment, inhibition of metastasis or diagnosis and a composition for screening cancer drugs have been invented.

One object of the present invention is to provide a composition for inhibiting M2 polarization of macrophages, comprising a monoclonal antibody specifically binding to Chi3L1 as an active ingredient.

Another object of the present invention is to provide a method for inhibiting M2 polarization of macrophages, comprising contacting cells expressing Chi3L1 protein with a composition containing the monoclonal antibody as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing, treating or inhibiting metastasis of cancer, a composition for diagnosing cancer, and a kit for diagnosing cancer comprising the composition for inhibiting M2 polarization of macrophages as an active ingredient.

Another object of the present invention is to provide a screening method for cancer prevention, treatment or metastasis inhibitory candidates using the efficacy of a monoclonal antibody that specifically binds to Chi3L1 to inhibit M2 polarization of macrophages, and a composition and kit capable of implementing the same will be.

Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific description described below. In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the invention described in this application. Also, such equivalents are intended to be included in this invention.

One aspect of the present invention,

A composition for inhibiting M2 polarization of macrophages, comprising a monoclonal antibody specifically binding to Chi3L1 as an active ingredient, wherein the monoclonal antibody comprises the following regions: to provide:

(1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; And a heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and

CDR1 consisting of the amino acid sequence of SEQ ID NO: 11; CDR2 consisting of the amino acid sequence of SEQ ID NO: 12; And a light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, or

(2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 8; CDR2 consisting of the amino acid sequence of SEQ ID NO: 9; And a heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; and

CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 consisting of the amino acid sequence of SEQ ID NO: 15; And a light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 16.

In one embodiment, the monoclonal antibody comprises (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3.

In another embodiment, the monoclonal antibody comprises (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4.

The composition for inhibiting STAT6 phosphorylation of the present invention may be used as a reagent composition.

Another aspect of the present invention is a method for inhibiting M2 polarization of macrophages, comprising the step of contacting cells expressing Chi3L1 protein with a composition for inhibiting M2 polarization of macrophages containing the monoclonal antibody as an active ingredient. to provide.

Another aspect of the present invention provides a pharmaceutical composition for preventing, treating, or inhibiting metastasis of cancer, comprising the composition for inhibiting M2 polarization of macrophages as an active ingredient.

Another aspect of the present invention provides a composition for diagnosing cancer comprising the composition for inhibiting M2 polarization of macrophages as an active ingredient.

Another aspect of the present invention provides a kit for diagnosing cancer comprising the composition for inhibiting M2 polarization of macrophages as an active ingredient.

As used herein, the term "antibody" includes an immunoglobulin molecule immunologically reactive with a specific antigen, and is a concept that includes both polyclonal antibodies and monoclonal antibodies. The term also includes forms produced by genetic engineering, such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

As used herein, the term "monoclonal antibody" is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which typically include different antibodies directed against different epitopes (epitopes), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture. .

Typically, an immunoglobulin has a heavy chain and a light chain, each comprising a constant region and a variable region (the regions are also known as “domains”). The light chain variable region and the heavy chain variable region include three variable regions called "complementarity determining regions" (hereinafter referred to as "CDRs") and four "framework regions". The CDR mainly serves to bind to an antigenic epitope. The CDRs of each chain are typically called CDR1, CDR2, CDR3 sequentially starting from the N-terminus, and can also be identified by the chain in which a particular CDR is located.

As used herein, "heavy chain" refers to a full-length heavy chain and fragments thereof comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3 comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. I mean everything. The term "light chain" may refer to both a full-length light chain including a variable region domain VL and a constant region domain CL including an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen, and fragments thereof.

As used herein, the term "variable" is used to indicate that the sequence of a specific region is different between antibodies and is used to indicate the binding specificity of each specific antibody to a specific antigen. The variability of an antibody may be concentrated in the CDRs rather than evenly distributed throughout the variable domains of the antibody. These CDRs may have a characteristic sequence for each monoclonal antibody, and some or all of the CDRs may interact so that one monoclonal antibody recognizes a specific epitope.

As used herein, the term "phage display technology" refers to a technology for selecting antibodies exhibiting antigen-binding ability by selecting only phages exhibiting significant binding ability with a target antigen from a phage library. In the above technology, “panning” refers to the application of peptides having the property of binding to target molecules (antibodies, enzymes, cell surface receptors, etc.) from a phage library displaying peptides on the surface of the phages. It refers to the process of selecting only expressing phages. By repeating this process 3 to 10 times, antibodies exhibiting significant binding ability to the target antigen can be selected, and the selected antibodies can be prepared as humanized monoclonal antibodies.

The monoclonal antibody of the present invention may include variants of the amino acid sequence described in the attached Sequence Listing to the extent that it can specifically bind to Chi3L1. For example, changes may be made to the amino acid sequence of a monoclonal antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the monoclonal antibody. Such amino acid variations may be made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents. In introducing mutations, the hydropathy index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Lysine (-3.9); and arginine (-4.5) hydrophobic amino acid index can be considered in conferring the interactive biological function of the protein, and can retain similar biological activity when substituted with an amino acid having a similar hydrophobicity index. When a mutation is introduced with reference to the hydrophobicity index, substitution may be made between amino acids exhibiting a difference in hydrophobicity index, preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

In addition, equivalent or similar biological activities can be exhibited by substitution between amino acids having similar hydrophilicity values. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); Asphaltate (+3.0±1); glutamate (+3.0 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); Alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4). When a mutation is introduced by referring to the hydrophilicity value, substitution may be made between amino acids exhibiting a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5. Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H Neurath, RLHill, The Proteins, Academic ress, New York, 1979). For example, typically the amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro , Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly can be exchanged. Considering the mutations having the above-mentioned biologically equivalent activity, the monoclonal antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to include a sequence showing substantial identity with the sequences described in the sequence listing. The above substantial identity is at least 61% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It may mean a sequence exhibiting homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means exhibiting non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffer, a preservative, a pain reliever, a solubilizer, an isotonic agent, a stabilizer, a base, an excipient, a lubricant, and the like.

In addition, the pharmaceutical composition of the present invention is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can be used Furthermore, it may be used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or skin external preparations in the form of gels. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include, for example, tablets, pills, powders, granules, capsules, etc., and these solid preparations contain one or more excipients, for example, starch, calcium carbonate, sucrose Alternatively, it may be prepared by mixing lactose, gelatin, and the like. In addition to the excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include, for example, suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin as diluents. .

Formulations for parenteral administration include, for example, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "administration" used in the present invention means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration may be administered, but is not limited thereto.

In one embodiment, the composition for inhibiting M2 polarization of macrophages may be used to inhibit M2 polarization of macrophages in a subject or cells isolated from the subject. The composition inhibits M2 polarization of macrophages by specifically binding to Chi3L1.

The composition for inhibiting M2 polarization of macrophages of the present invention effectively inhibits the polarization of macrophages from M1 to M2, prevents T cell activity from being inhibited, and suppresses tumor proliferation, invasion, angiogenesis, etc. It can effectively act in the prevention, treatment and inhibition of metastasis of

The composition for inhibiting M2 polarization of macrophages of the present invention can inhibit phosphorylation of STAT6. STAT6 is phosphorylated and activated by M2 polarization of macrophages, and since such phosphorylated STAT6 can increase gene expression involved in cancer metastasis by being distributed in the nucleus, the composition of the present invention prevents or prevents cancer growth and metastasis. can be used for treatment. The composition is administered to a subject or treated with cells isolated from the subject, administered for the purpose of treating a disease of the subject, administered to a subject for the purpose of in vivo or in vitro testing, or treated with cells isolated from the subject. It can be. The "individual" may mean a mammal, including, for example, humans, rats, mice, livestock, and the like. When the composition is directly administered to a subject for the purpose of treatment or the like, the mammal may mean excluding humans.

Another aspect of the present invention,

1) contacting a candidate substance to cells expressing the Chi3L1 protein;

2) measuring the level of inhibition of macrophage M2 polarization in the cells of step 1);

3) A method for screening a candidate for preventing, treating or inhibiting cancer metastasis, comprising comparing the level of inhibition in step 2) with the level of inhibition in cells not treated with the candidate, wherein step 2) Measuring the level of inhibition of M2 polarization of macrophages provides a screening method that measures the expression level of phosphorylated STAT6 protein.

The cancer prevention, treatment or metastasis suppression candidate may target the Chi3L1 protein. The Chi3L1 protein may have a nucleotide sequence encoding it defined by NCBI Gene ID: 1116, and a precursor thereof may be defined by an amino acid sequence of NP_001267.2. The cells expressing the Chi3L1 protein may be isolated from an individual, or may be engineered to overexpress the Chi3L1 protein by transfecting a gene into mammalian cells. Cells expressing the Chi3L1 protein may be cancer cells.

The step of treating the cells with the candidate substance is to bring the cell into contact with the candidate substance, and the candidate substance may be directly contacted by treating the cultured cells, or cells isolated from an individual who has received the candidate substance may be obtained. there is. The "contact" means that a specific substance is bound to the cell membrane or cell wall or injected into the cell, and the contacting step is, for example, added to a medium containing the cell, or injected into the cell. It can be directly injected into the containing tissue, or injected into the blood when applied to a subject to reach the desired cells. When contacting the cell with the candidate material includes administering the candidate material to a subject, the subject may be a mammal other than a human.

The present inventors identified for the first time the efficacy of Chi3L1 humanized monoclonal antibody to inhibit M2 polarization and STAT6 phosphorylation of macrophages and to use it to screen candidates for cancer prevention, treatment, cancer diagnosis or metastasis inhibition. .

The screening method of the present invention measures the expression level of CD206, Arg-1 or a combination thereof in the cells of step 1), and CD206 (Cluster of Differentiation 206), Arg-1 (Arginase 1) in the cells Alternatively, a step of selecting a candidate substance that reduces the expression level of a protein that is a combination thereof may be further included. The candidate material may reduce the expression level of CD206, Arg-1 or a combination thereof by 30% or more compared to the candidate material untreated group.

In addition, the candidate material may not affect the expression level of CD86 (Cluster of Differentiation 86), iNOS (inducible nitric oxide synthase), or a combination thereof. The "having no effect" means that the degree of increase or decrease in the expression level of the protein is not significant and cannot be determined to be fluctuating, for example, when the degree of change in protein expression level is less than 30% that can mean

The screening method of the present invention is an invention devised by evaluating the effects of animal experiments and the expression level of factors involved in candidate materials targeting the Chi3L1 protein that specifically bind to Chi3L1 and consequently have tumor metastasis inhibitory effects. Therefore, in order to discover candidates that show similar or superior effects to existing drugs with a higher significance, it is necessary to evaluate the expression of CD86 and iNOS, which are M1 polarization factors, and CD206 and Arg-1, which are M2 polarization factors, differently in a predictable range. may be desirable.

In one embodiment of the present invention, the present inventors found that a humanized monoclonal antibody targeting Chi3L1 is important in cancer. In order to determine whether the main factors inducing polarization of M1 and M2 macrophages are affected, Thp-1 macrophages treated with Chi3L1 humanized monoclonal antibody (H1) were used to induce polarization with A549 conditioned medium. The mRNA expression levels of M1 and M2 polarization markers were confirmed by RT-qPCR. As a result, it was confirmed that the mRNA expression of the M2 polarization markers CD206 and Arg-1 significantly decreased when the H1 antibody was treated, whereas the mRNA expression of the M1 polarization markers CD86 and iNOS did not change. Accordingly, it was confirmed that the expression level change or change of CD206, Arg-1, CD86 or iNOS can be used as an indicator for screening candidates for cancer prevention, treatment or metastasis inhibition.

Step 1) of the present invention may include measuring phosphorylated STAT6 protein present in the cell nucleus. STAT6 can increase the expression of genes involved in cancer metastasis by being phosphorylated and distributed in the nucleus. Therefore, measuring the expression level of the phosphorylated STAT6 protein distributed in the nucleus may be more preferable for predicting the cancer metastasis inhibitory effect of the candidate substance. The candidate material can reduce the expression level of phosphorylated STAT6 while not changing or reducing the total expression level of the STAT6 protein, and more preferably specifically reduce the expression level of phosphorylated STAT6 in the nucleus. can More specifically, the candidate material may reduce the expression level of phosphorylated STAT6 in the nucleus by 50% or more, more preferably by 70% or more.

Step 1) may further include separating the nucleus from the cell in order to measure the phosphorylated STAT6 protein present in the nucleus of the cell.

In the screening method, cells expressing the Chi3L1 protein are contacted with a candidate material, and then MMP13 (Matrix Metalloproteinase 13), MMP9, MMP2, CDK6 (Cyclin-dependent kinase6), CDK4, CDK2, CyclinE, CyclinD1, and Measuring the expression level of one or more proteins selected from the group consisting of proliferating cell nuclear antigen (PCNA), and any one or more proteins selected from the group consisting of MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, and PCNA A step of selecting a candidate material that reduces the expression level of may be further included.

The selected candidate substance may reduce the expression level of one or more proteins selected from the group consisting of MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, and PCNA by 50% or more compared to the candidate substance non-treated group. there is.

The selected candidate material may reduce the expression level of MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, and PCNA proteins.

In one embodiment of the present invention, the present inventors measured the expression levels of related proteins using tumor tissues obtained from lung cancer-induced mice in order to identify factors involved in reducing cancer growth of candidate substances. As a result, it was confirmed that the change in the expression level of MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, Cyclin E, Cyclin D1 or PCNA can be used as an indicator to screen candidates for cancer prevention, treatment or metastasis inhibition.

The method for detecting the expression level of the protein of the present invention can be appropriately performed by a person skilled in the art using a known technique, for example, high performance liquid chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC/MS), Enzyme-Linked ImmunoSorbent Assay (ELISA), Protein Immunoprecipitation, Immunoelectrophoresis, Western Blot ( Western Blot), protein immunostaining, confocal microscopy, etc., but are not limited thereto.

The Chi3L1, CD206, Arg-1, CD86, iNOS, MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, PCNA, and STAT6 proteins may be endogenous, exogenous, or a combination thereof. there is.

Candidates selected by the screening method may exhibit similar or superior cancer treatment or metastasis inhibitory effects to existing cancer prevention, treatment, or metastasis inhibitory drugs.

As used herein, the term "candidate for preventing, treating, or inhibiting metastasis of cancer" is a substance expected to be able to prevent or treat cancer or inhibit metastasis of cancer, which directly or indirectly improves or inhibits cancer. Any substance expected to improve or inhibit metastasis can be used without limitation, and includes all substances expected to be therapeutic, such as compounds, genes, or proteins.

Another aspect of the present invention, a cell expressing the Chi3L1 protein; And a composition for screening a candidate for preventing, treating or inhibiting cancer, comprising a composition for measuring the M2 polarization level of macrophages, wherein the composition for measuring the M2 polarization level of macrophages binds specifically to phosphorylated STAT6 protein It provides a composition for screening, which includes a compound or polypeptide that does.

Another aspect of the present invention, a cell expressing the Chi3L1 protein; And a kit comprising a composition for measuring the M2 polarization level of macrophages, wherein the composition for measuring the M2 polarization level of macrophages comprises a compound or polypeptide that specifically binds to phosphorylated STAT6 protein, prevention of cancer, Kits for screening therapeutic or metastasis inhibitory candidates are provided.

The compound or polypeptide that specifically binds to the phosphorylated STAT6 protein may be a synthesized compound, aptamer, antibody, or fragment thereof, and the composition or kit visualizes the compound or polypeptide that specifically binds to the phosphorylated STAT6 protein It may further include a marker for

In addition to the STAT6 protein, the composition or kit may include any one or more proteins selected from the group consisting of Chi3L1, CD206, Arg-1, CD86, iNOS, MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, and PCNA It may further include a marker for confirming.

The composition or kit comprises a substrate, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of antibodies, polypeptides, or combinations thereof capable of specifically binding to these proteins. can include As the substrate, a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and glass slide glass may be used, and the coloring enzyme may be peroxidase, alkaline phosphatase ( alkaline phosphatase) may be used, FITC, RITC, etc. may be used as the fluorescent substance, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), and the like may be used.

A composition or kit of the present invention may further comprise a composition, solution or device with one or more other components suitable for screening.

"Cancer" in the compositions, screening methods, and kits of the present invention includes, for example, breast cancer, colorectal cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, head and neck cancer , colon cancer, ovarian cancer, rectal cancer, colorectal cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone marrow cancer, but are not limited thereto. don't In one embodiment, the cancer may be lung cancer.

Terms not otherwise defined in the present invention are interpreted to have meanings commonly used in the technical field to which the present invention belongs. In addition, the expression "or" described in the present invention may be interpreted as a concept including "and" unless otherwise stated.

By using the composition for inhibiting M2 polarization of macrophages according to one aspect of the present invention, M2 polarization of macrophages is inhibited and STAT6 phosphorylation is inhibited in a subject or cells isolated from the subject, thereby preventing, treating or metastasizing cancer in the subject can be used to suppress In addition, the composition for inhibiting M2 polarization of macrophages can be used to diagnose cancer, and can be provided as a kit for the purpose of diagnosing cancer.

Using the composition, screening method, or kit according to one aspect of the present invention, it is possible to easily and significantly select therapeutic candidates capable of effectively inhibiting the prevention, treatment, growth, or metastasis of cancer.

Candidates selected according to one aspect of the present invention have effects similar to or superior to those of therapeutic agents that have been confirmed to have excellent cancer growth inhibition and metastasis inhibition effects through animal models. It is economical because the effect and mechanism can be predicted without conducting an experiment.

Figure 1A is a graph showing the expression level of Chi3L1 protein present in the serum of cancer patients, and Figure 1B is a graph showing the expression of Chi3L1 protein present in cancer tissues of cancer patients.

Figure 2A is a result of confirming the tumor growth reduction effect of the candidate material as tumor volume in a cancer mouse model, and Figure 2B is a graph showing the result of confirming as tumor weight.

Figure 3 confirms the factor involved in reducing the tumor growth of the candidate material by protein expression level.

Figure 4A is the result of confirming the cancer metastasis inhibitory effect of the candidate substance as the number of tumor nodes, and Figure 4B is a graph showing the result of confirming the tumor surface area.

Figure 5 confirms the mRNA expression levels of factors (CD86, iNOS, CD206, Arg-1) involved in suppressing cancer metastasis, which are M2 polarization markers of macrophage candidates.

6A is a graph confirming the effect of the candidate substance on the phosphorylation of STAT6 protein by protein expression level, and FIG. 6B is a graph confirming the protein expression level in and outside the nucleus.

7A is an image and a graph showing the effect of a candidate material on phosphorylation of STAT6 protein in a cancer mouse model as the amount of phosphorylated STAT6 protein expression in tumor tissue, and FIG. 7B is a graph showing the amount of phosphorylated STAT6 protein expression in cancer metastatic tissue. It is an image and a graph to represent.

Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for explaining the present invention, and the scope of the present invention is not limited in any way by these.

[실시예 1] Chi3L에 특이적으로 결합하는 항체의 제조: 파지 디스플레이 라이브러리 패닝(Phage display library panning)[Example 1] Preparation of antibodies that specifically bind to Chi3L: Phage display library panning

Antibodies against Chi3L1 were selected through phage display technology, and antibodies exhibiting significant binding ability with Chi3L1 were selected. The ability to bind to hChi3L1 was confirmed by ELISA against hChi3L1 phage. Briefly, 30 ng of hChi3L1 antigen was immobilized per well, and the antigen-immobilized wells were blocked with MPBS (5% skim milk in PBS), and then hChi3L1 phage and mChi3L1 phage were added and reacted. In order to detect bound phage, after washing with PBST (0.05% Tween20 in PBS) 4 times, Anti-M13-HRP (1:5,000) was added and reacted. Finally, after washing four times with PBST, a color reaction was induced by adding TMB (3,3',5,5'-Tetramethylbenzidine) substrate, and the reaction was terminated by adding 2N H ₂ SO ₄ . The absorbance of the well was measured.

As a result, 7 clones having hChi3L1 antibodies were selected from the Fab-I library and the Fab-II library, and 3A10-F1(H1), which showed the lowest EC50 value, was selected and further experiments were performed.

In addition, antibodies showing significant binding ability to mChi3L1 were selected, and binding ability to mChi3L1 was confirmed by ELISA on mChi3L1 phage. Simply, 30 ng of mChi3L1 antigen was immobilized per well, and the wells to which the antigen was fixed were plated in MPBS (5 After blocking treatment with % skim milk in PBS), hChi3L1 phage and mChi3L1 phage were added and reacted. In order to detect the bound phage, after washing with PBST (005% Tween20 in PBS) 4 times, Anti-M13-HRP (1:5,000) was added and reacted. Finally, after washing four times with PBST, TMB (3,3',5,5'-Tetramethylbenzidine) substrate was added to induce a color reaction, and 2N H2SO4 was added to terminate the reaction, and the absorbance of each well was measured at a wavelength of 450 nm. did As a result, 6 antibodies were selected from the Fab-II library, but no clones were formed from the Fab-I or scFv libraries. Among them, the antibody with the lowest EC50 value was 4E3-F2 (M3).

Sequence information of the selected 3A10-F1 (H1) antibody and 4E3-F2 (M3) antibody are as follows.

[실시예 2] 폐암 성장 감소 효과의 확인[Example 2] Confirmation of lung cancer growth reduction effect

(1) 폐암 환자에서 Chi3L1의 발현 확인(1) Verification of Chi3L1 expression in lung cancer patients

To confirm whether the expression of Chi3L1 is actually increased in lung cancer patients, serum, non-lung cancer tissues and lung cancer tissues of lung cancer patients were collected from Chungbuk National University Hospital Human Resource Bank, Keimyung University Dongsan Hospital Human Resource Bank, and Ajou University Human Resource Bank. Twenty tissue and serum samples were provided, respectively. Then, the Chi3L1 level in serum was quantified using the Chi3L1 ELISA kit using patient serum samples. AUC-ROC curve analysis was performed using the results, which are graphs with TPR (= sensitivity) and FPR (= 1-specificity) as x and y axes, respectively. Sensitivity, specificity, accuracy, etc. are used to evaluate the accuracy of the test in predicting the dichotomous outcome. In the present invention, sensitivity refers to the ratio of positive days among people susceptible to Chi3L1, and specificity refers to the ratio of negative days among people without sensitivity to Chi3L1. The cut-off value is the criterion for dividing test results into negative and positive, and the lower the cut-off value, the higher the sensitivity and the lower the specificity. AUC (area under the curve) is the area under the ROC curve.

The experimental results are shown in FIG. 1 . As a result of ROC curve analysis of Chi3L1 protein expression levels in serum of lung cancer patients, sensitivity of 85%, specificity of 85%, cut-off value of 11.74ng/ml, and AUC of 0.9533 were shown (FIG. 1A). As a result of ROC curve analysis of the Chi3L1 protein expression level present in tissues, a sensitivity of 78.26%, a specificity of 78.26%, a cut off value of 100ng/ml, and an AUC value of 0.8625 were shown (FIG. 1B). Therefore, it was confirmed that Chi3L1 is a key regulator of human lung cancer development.

(2) In vivo 폐암 성장 감소 효과의 확인(2) Confirmation of in vivo lung cancer growth reduction effect

In order to confirm whether the Chi3L1 humanized antibody selected in Example 1 exhibits a tumor growth reducing effect, the tumor growth reducing effect of H1, a Chi3L1 humanized monoclonal antibody showing the highest affinity in the Fab phage display library, was confirmed.

LLC (lewis lung carcinoma) lung cancer cell line was subcutaneously administered to 8-week-old C57BL/6 mice at 3 * 10 ⁵ cells/200 μl, and the next day, Chi3L1 humanized monoclonal antibody H1 was administered at 0.5 mg per kg twice a week for 4 weeks. After intravenous administration with a body weight, the size was measured using Vernier calipers to measure the tumor size, and the formula is as follows; (A *B ² ) ² , where A is the long axis and B is the short axis, the muscles and dermis surrounding the tumor were separated and discharged.

As a result, as shown in FIG. 2, the size of the tumor tissue in the control group not administered with the H1 antibody was 1.57±0.59 cm ² , and in the case of the mouse treated with the H1 antibody, the size of the tumor was greatly reduced to 0.58±0.31 cm ² (FIG. 2A). In addition, as a result of measuring the weight of the tumor tissue, the weight (0.56±0.30 g) was decreased in the mice intravenously injected with H1 compared to the control group (1.08±0.36 g) (FIG. 2B).

(3) 폐암 성장 감소에 관여하는 인자의 확인(3) Identification of factors involved in reducing lung cancer growth

In order to identify factors involved in the reduction of lung cancer growth by the Chi3L1 humanized antibody selected in Example 1, the expression level of the involved proteins was measured using the tissues obtained in Example 2-(2).

Specifically, the tumor tissue collected for Western blotting was crushed to about 1 mm ³ and cultured in a buffer containing 50 mM Tris-Hcl (pH7.6), 0.1% Triton X-100, 0.25 mM NaCl, and 2 mM EDTA. treated for 1 hour. Then, the dissolved supernatant proteins were separated by centrifugation, and the extracted proteins were quantified using the Bradford protein assay, and 10 μg of protein was separated by size through SDS-PAGE, transferred to a PVDF membrane, and then blocked. Primary antibodies specific for each factor to be identified were reacted at 4°C for one day. After that, the reaction was performed at room temperature for 45 minutes using a secondary antibody binding to the primary antibodies, and the result of the experiment was confirmed using an ECL detection kit. Antibodies used for Western blot were commercially available.

As a result, as shown in FIG. 3, it was confirmed that the cell transfer factor proteins MMP13, MMP9, and MMP2 and the cell-cycle related proteins CDK6, CDK4, CDK2, Cyclin E, Cyclin D1, and PCNA were all reduced by 50% or more.

[실시예 3] 폐암 전이 억제 효과의 확인[Example 3] Confirmation of lung cancer metastasis inhibitory effect

(1) 폐암 전이 억제 효과 확인(1) Confirmation of lung cancer metastasis inhibitory effect

It was confirmed that the Chi3L1 humanized antibody selected in Example 1 not only reduces lung cancer growth, but also has an effect of inhibiting lung cancer metastasis.

Specifically, A549 lung cancer cell line was administered to 8-week-old C57BL/6 mice by tail vein at 1 * 10 ⁷ cells/200 μl, and the next day, H1 antibody was administered at 0.5 mpk (mg per kg body weight) twice a week for 3 weeks. After intravenous administration, they were sacrificed on the 8th week of the experiment, and after acquiring lung cancer tissues, the number of tumor nodes and the surface area of metastatic tumor lungs were measured. The number of tumor nodes in lung tissue and the surface area of metastatic tumor lung were measured by Hematoxylin & Eosin staining and quantified as a percentage of the total lung surface area.

As a result, as shown in FIG. 4, when the H1 antibody was treated, the number of tumor nodes metastasized to the lungs was significantly reduced by more than 50% on average (FIG. 4A), and the surface area of the tumor lung was also significantly reduced by more than 50% on average compared to the control group. can be confirmed (Fig. 4B).

(2) M1, M2 대식세포의 분극 유도 인자 발현 조절 효과 확인(2) Confirmation of the effect of regulating the expression of polarization-inducing factors in M1 and M2 macrophages

Chi3L1 humanized monoclonal antibody H1 is important in cancer In order to determine whether the main factors inducing polarization of M1 and M2 macrophages are affected, Thp-1 macrophages treated with the Chi3L1 humanized monoclonal antibody H1 were used to induce polarization with A549 conditioned medium to induce polarization in M1 and M2 macrophages. The mRNA expression level of the M2 polarization marker was confirmed by RT-qPCR.

Specifically, Thp-1 macrophages were cultured, treated with Chi3L1 humanized monoclonal antibody, polarized with A549 conditioned medium, and then RNA was extracted. CD86, iNOS, Arg-1 and CD206 were selected to analyze the expression of genes related to macrophage differentiation with the synthesized cDNA. A mixture of cDNA, primers for each gene, and SYBR Green PCR Master mix was dispensed into a 96 well-plate. To normalize mRNA expression levels between samples, GAPDH was co-amplified. It was amplified and analyzed using the ABI PRISM 7700 Sequence Detection system. The primer sequences for each gene used in the experiment are shown in Table 2 below.

As a result, as shown in FIG. 5 , it was confirmed that the mRNA expression of CD206 and Arg-1, which are M2 polarization markers, significantly decreased when the H1 antibody was treated. On the other hand, the mRNA expression of CD86 and iNOS, which are M1 polarization markers, did not change.

[실시예 4] 폐암 세포 내 STAT6 인산화 억제의 확인[Example 4] Confirmation of inhibition of STAT6 phosphorylation in lung cancer cells

Using the Chi3L1 humanized antibody selected in Example 1, it was confirmed whether STAT6 phosphorylation could be utilized as an indicator of a therapeutic agent that inhibits lung cancer metastasis.

Specifically, after culturing Thp-1 macrophages in the same manner as in Example 3-(2), treating H1 and inducing polarization, Thp-1 macrophages are harvested and antibodies specific to each protein to be identified. Western blotting was performed as in Example 2-(3) using Antibodies used for Western blot were commercially available.

As a result, as shown in FIG. 6, it was confirmed that the expression of Try641 phosphorylated STAT6, an activated form of STAT6, was reduced when the Chi3L1 humanized monoclonal antibody H1, a candidate material of Example 1, was treated (FIG. 6A). STAT6 activation by M2 polarization results in Try641 phosphorylation, which translocates into the nucleus and activates gene transcription. As a result of Western blotting after separating into the cytoplasm and nucleus, it was confirmed that the expression of Try641 phosphorylated STAT6 in the nucleus was significantly reduced when the cells were treated with the Chi3L1 humanized monoclonal antibody H1 (FIG. 6B).

In order to more clearly confirm the cell test results, Western blotting was performed using tumors and lung tissues obtained from the lung cancer growth and metastasis models of Example 2-(2) and Example 3-(1). As a result, as shown in FIG. 7 , it was confirmed that phosphorylation of STAT6 was significantly reduced in both the growth and metastasis models of lung cancer treated with the Chi3L1 humanized monoclonal antibody H1.

Claims

A composition for inhibiting M2 polarization of macrophages, comprising a monoclonal antibody that specifically binds to Chi3L1 as an active ingredient, wherein the monoclonal antibody comprises the following regions: Composition:

(1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; And a heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and CDR1 consisting of the amino acid sequence of SEQ ID NO: 11; CDR2 consisting of the amino acid sequence of SEQ ID NO: 12; And a light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, or

(2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 8; CDR2 consisting of the amino acid sequence of SEQ ID NO: 9; And a heavy chain variable region comprising CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; and CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 consisting of the amino acid sequence of SEQ ID NO: 15; And a light chain variable region comprising a CDR3 consisting of the amino acid sequence of SEQ ID NO: 16.
The composition for inhibiting M2 polarization of macrophages according to claim 1, wherein the monoclonal antibody comprises the following regions:

(a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1; and

(b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3.
The composition for inhibiting M2 polarization of macrophages according to claim 1, wherein the monoclonal antibody comprises the following regions:

(a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; and

(b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4.
The composition for inhibiting M2 polarization of macrophages according to claim 1, wherein the composition inhibits phosphorylation of STAT6.
A method of inhibiting M2 polarization of macrophages, comprising contacting cells expressing Chi3L1 protein with the composition of any one of claims 1 to 4.
A pharmaceutical composition for preventing, treating or inhibiting metastasis of cancer, comprising the composition of any one of claims 1 to 4 as an active ingredient.
A composition for diagnosing cancer comprising the composition of any one of claims 1 to 4 as an active ingredient.
A kit for diagnosing cancer comprising the composition of any one of claims 1 to 4 as an active ingredient.
1) contacting a candidate substance to cells expressing the Chi3L1 protein;

2) measuring the level of inhibition of macrophage M2 polarization in the cells of step 1);

3) A method for screening a candidate for preventing, treating or inhibiting cancer metastasis, comprising comparing the level of inhibition in step 2) with the level of inhibition in cells not treated with the candidate, wherein step 2) The screening method of measuring the level of inhibition of M2 polarization of macrophages is to measure the expression level of phosphorylated STAT6 protein.
According to claim 9,

The screening method measures the expression level of CD206, Arg-1 or a combination thereof in the cells of step 1), and the candidate reducing the expression of CD206, Arg-1 or a combination thereof in the cells. Which further comprises the step of screening the substance, the screening method.
According to claim 9,

The candidate material does not affect the expression level of CD86, iNOS or a combination thereof, the screening method.
According to claim 9,

The step 1) is to measure the expression level of the phosphorylated STAT6 protein present in the nucleus of the cell, the screening method.
According to claim 9,

In the cells of step 1), the expression level of one or more proteins selected from the group consisting of MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, and PCNA was measured, and the MMP13, MMP9, MMP2, CDK6, CDK4, CDK2, CyclinE, CyclinD1, and screening method further comprising the step of selecting a candidate material that reduces the expression level of any one or more proteins selected from the group consisting of PCNA.
According to claim 9,

The screening method, wherein the cells expressing the Chi3L1 protein of step 1) are cancer cells.
cells expressing the Chi3L1 protein; And a composition for screening a candidate for preventing, treating or inhibiting cancer, comprising a composition for measuring the M2 polarization level of macrophages, wherein the composition for measuring the M2 polarization level of macrophages binds specifically to phosphorylated STAT6 protein A composition for screening comprising a compound or polypeptide that
A kit for screening a candidate for preventing, treating, or inhibiting cancer metastasis, comprising the composition of claim 15.