Novel use of CXCL5 inhibitor and novel antibody or antigen-binding fragment thereof or organic compound usable as CXCL5 inhibitor

Abstract

The present invention relates to a novel use of a CXCL5 inhibitor and a novel antibody or antigen-binding fragment thereof usable as a CXCL5 inhibitor. The novel use of a CXCL5 inhibitor and the novel antibody or antigen-binding fragment thereof usable as a CXCL5 inhibitor of the present invention enable the treatment or prevention of cancer cachexia or muscle disease, enable cancer cachexia or muscle disease to be diagnosed with high efficiency, and enable a drug for the treatment or prevention of cancer cachexia or muscle disease to be screened with high efficiency.

Description

Novel uses of CXCL5 inhibitors and novel antibodies or their antigen-binding fragments or organic compounds available as CXCL5 inhibitors

[0001] The present invention relates to a novel use of a CXCL5 inhibitor and a novel antibody or its antigen-binding fragment that can be used as a CXCL5 inhibitor.

[0002] Cancer cachexia is a multi-organ metabolic syndrome defined as a condition in which a cancer patient loses more than 5% of their body weight within six months. Cancer cachexia impedes the effectiveness of chemotherapy and immunotherapy for cancer patients; it can occur in up to 80% of cancer patients depending on the type of cancer and is known to account for about 25% of the causes of death in cancer patients.

[0003] In patients with cancer cachexia, the catabolism of muscle proteins caused by tumor-derived inflammatory cytokines such as tumor necrosis factor (TNF-α), interferon-γ, interleukin-1, and interleukin-6 occurs actively, and these factors are also referred to as tumor-derived cachexokines that induce cancer cachexia.

[0004] Treatment methods for such cancer cachexia have not been clearly established, and treatments used include the administration of fluids that are excreted in large quantities in urine, adrenocortical steroid preparations that cause immunosuppression, and megestrol acetate preparations that have limited symptom relief and carry a risk of thromboembolism. However, these treatment methods have the problem that their therapeutic effects on cancer cachexia are unclear or they cause side effects.

[0005] The present invention aims to provide a novel use for CXCL5 inhibitors.

[0006] In addition, the present invention aims to provide a novel antibody or its antigen-binding fragment, or an organic compound, that can be used as a CXCL5 inhibitor.

[0007] In addition, the present invention aims to provide a diagnostic kit capable of diagnosing cancer cachexia or muscle disease with high efficiency, or a method for providing information necessary for such diagnosis.

[0008] In addition, the present invention aims to provide a drug screening method capable of screening drugs for the treatment or prevention of cancer cachexia or muscle disease with high efficiency.

[0009] 1. A pharmaceutical composition for the treatment or prevention of cancer cachexia comprising a CXCL5 inhibitor as an active ingredient.

[0010] 2. A pharmaceutical composition for the treatment or prevention of cancer cachexia, wherein the CXCL5 inhibitor comprises one or more selected from the group consisting of a CXCL5 neutralizing antibody or its antigen-binding fragment, dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

[0011] 3. A pharmaceutical composition for the treatment or prevention of cancer cachexia, wherein the CXCL5 neutralizing antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and a light chain variable region comprising LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO. 8.

[0012] 4. A pharmaceutical composition for the treatment or prevention of cancer cachexia, wherein, in 3 above, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 2.

[0013] 5. A pharmaceutical composition for the treatment or prevention of muscle diseases comprising a CXCL5 inhibitor as an active ingredient.

[0014] 6. In the above 5, the above muscle disease is a pharmaceutical composition for treating or preventing muscle disease, wherein the muscle disease is a muscle disease related to cancer cachexia.

[0015] 7. A pharmaceutical composition for the treatment or prevention of muscle diseases, wherein the CXCL5 inhibitor comprises one or more selected from the group consisting of a CXCL5 neutralizing antibody or its antigen-binding fragment, dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

[0016] 8. A pharmaceutical composition for the treatment or prevention of muscle diseases, wherein the CXCL5 neutralizing antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and a light chain variable region comprising LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO. 8.

[0017] 9. A pharmaceutical composition for treating or preventing muscle diseases, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 2.

[0018] 10. An antibody or its antigen-binding fragment that specifically binds to CXCL5, comprising: a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and a light chain variable region comprising LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO. 8.

[0019] 11. An antibody or its antigen-binding fragment that specifically binds to CXCL5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 2.

[0020] 12. A kit for diagnosing cancer cachexia or muscle disease comprising, as an active ingredient, a preparation capable of detecting the expression level of CXCL5 protein or a gene encoding said protein.

[0021] 13. A method for providing information necessary for diagnosing cancer cachexia or muscle disease, comprising: a step of measuring the expression or activity level of a CXCL5 protein, or the expression level of a gene encoding said protein, in a biological sample isolated from an individual suspected of cancer cachexia; and a step of comparing the expression level or activity level of said CXCL5 protein, or the expression level of said gene encoding said protein, with the expression level or activity level of a CXCL5 protein in a control sample, or the expression level of said gene encoding said CXCL5 protein in the control sample, respectively.

[0022] 14. A drug screening method for the prevention or treatment of cancer cachexia or muscle disease comprising: a step of treating cells expressing CXCL5 with a candidate substance; and a step of comparing a treatment group treated with the candidate substance with a control group.

[0023] 15. A drug screening method for the prevention or treatment of cancer cachexia or muscle disease, wherein, in the above 14, the step of comparing the treatment group and the control group is characterized by comparing the expression or activity level of the CXCL5 protein in the control group and the treatment group, or the expression level of the gene encoding the protein.

[0024]

[0025] Figure 1 is a schematic diagram of the experimental process and a diagram showing the results of observing the root canal diameter according to the experiment.

[0026] Figure 2 is a diagram showing a cytokine array.

[0027] Figure 3 is a diagram showing the results of the cytokine array analysis.

[0028] Figure 4 is a diagram showing the experimental results verifying whether CXCL5 is a direct causative factor in causing muscle atrophy.

[0029] Figure 5 is a diagram showing experimental results indicating that the muscle atrophy response induced by CXCL5 can be alleviated by a CXCL5-specific neutralizing antibody.

[0030] Figures 6 to 9 show the results of a fibroblast (CAF) co-transplantation experiment.

[0031] Figures 10 and 11 are drawings showing the results of the histological analysis of the gastrocnemius muscle.

[0032] Figure 12 is a diagram showing the results of real-time polymerase chain reaction (qPCR) analysis of tumor tissue.

[0033] Figures 13 to 15 show the results of an experiment in which human colon cancer cells and cancer-associated fibroblasts (CAFs) were transplanted together into immunodeficient mice and treated with CXCL5 neutralizing antibodies, and the effect on tumor size was analyzed.

[0034] Figure 16 is a diagram showing that CXCL5 neutralizing antibodies prevent weight loss.

[0035] Figure 17 is a diagram showing that CXCL5 neutralizing antibody improves muscle performance.

[0036] FIGS. 18 to 22 are the results of an experiment in which human colon cancer cells and cancer-associated fibroblasts (CAF) were transplanted into immunodeficient mice and treated with a CXCL5 neutralizing antibody, and the figures show the recovery of the weight and diameter of the tibialis anterior (TA) muscle and the decrease in CXCR2, the receptor for CXCL5, following antibody treatment.

[0037] Figure 23 is a graph showing the staining intensity of MyHC2 by the chemicals used in the experiment.

[0038] Figure 24 is a diagram showing the results of observing the staining intensity of MyHC2 and the size of the root canal by the drugs used in the experiment using a fluorescence microscope.

[0039]

[0040] The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the claims. Throughout the specification, the same reference numerals refer to the same components.

[0041]

[0042] The present invention relates to a novel use of a CXCL5 inhibitor. Specifically, the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer cachexia or muscle disease comprising a CXCL5 inhibitor as an active ingredient.

[0043] CXCL5 (Uniprot: P42830, 1995-11-01 v1) is a protein also known as neutrophil activation peptide 78 (ENA-78).

[0044] The inventors have completed the present invention by discovering that such CXCL5 can induce cancer cachexia. Since cancer cachexia caused by CXCL5 can lead to weight loss and muscle disease in cancer patients, it is possible to treat or prevent cancer cachexia or muscle disease by using a CXCL5 inhibitor.

[0045] CXCL5 inhibitors may include any substance that inhibits the expression, activity, or action of such CXCL5 proteins, thereby preventing the CXCL5 proteins from functioning. For example, CXCL5 inhibitors may include any one selected from the group consisting of transcription inhibitors or translation inhibitors that prevent the expression of a gene encoding the CXCL5 protein, activity inhibitors that inhibit the activation of the CXCL5 protein, and action inhibitors that inhibit the action of the CXCL5 protein.

[0046] In other words, CXCL5 inhibitors can treat or prevent cancer cachexia and / or muscle disease caused by CXCL5.

[0047] At this time, the muscle disease may be a muscle disease resulting from decreased muscle function, muscle atrophy, muscle wasting, or muscle degeneration, and specifically, it may be any one selected from atony, muscular atrophy, muscular dystrophy, myotonia, rigid spine syndrome, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Pompe disease, Canavan disease, dystonia, muscle degeneration, myasthenia, sarcopenia, and muscle fatigue.

[0048] In a pharmaceutical composition for the treatment or prevention of cancer cachexia according to one embodiment of the present invention, the CXCL5 inhibitor may comprise one or more selected from the group consisting of a CXCL5 neutralizing antibody or its antigen-binding fragment, dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

[0049] An antibody is a glycoprotein in which a heavy chain and a light chain are interconnected through bonds such as disulfide bonds, and which possesses the ability to bind to antigens. An antigen-binding fragment of an antibody refers to an active fragment capable of specifically binding to a specific antigen by including at least a portion of the antibody, specifically, at least a portion of the complementarity determining site (CDR) capable of specifically binding to a specific antigen. The heavy chain complementarity determining site and the light chain complementarity determining site are abbreviated as HCDR and LCDR, respectively.

[0050] The heavy and light chains each contain a variable region and a constant region; the variable region contains a complementation determining site and binds specifically to antigens, while the constant region performs auxiliary functions, such as binding to cells other than antigens or activating complement.

[0051] A neutralizing antibody refers to an antibody that directly and specifically binds to a specific protein and neutralizes its function by blocking its interaction with a receptor. CXCL5 neutralizing antibodies are neutralizing antibodies that specifically bind to CXCL5 and may include, for example, LY-3041658, MAB654, and AF254.

[0052] In a pharmaceutical composition for treating or preventing cancer cachexia according to one embodiment of the present invention, the CXCL5 neutralizing antibody may include: a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and a light chain variable region comprising LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO. 8.

[0053] In addition, in a pharmaceutical composition for treating or preventing cancer cachexia according to one embodiment of the present invention, the heavy chain variable region may include the amino acid sequence of SEQ ID NO. 1, and the light chain variable region may include the amino acid sequence of SEQ ID NO. 2.

[0054] Sequence numbers 1 to 8 are shown in Table 1 below.

[0055] SEQ ID NO: Amino Acid SequenceRemarks1QVQLVQSGAEVKKPGASVKVSCKASGYEFTSYWIHWVRQAPGQGLEWMGNISPNSGSANYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGPYSYYPSREYYGSDLWGQGTLVTVSSVH2EIVLTQSPATLSLSPGERATLSCRASQSISNNLHWYQQKPG QAPRLLIYYTSRSVSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGQNNWEIPEVFGGGTKVEIKVL3GYEFTSYWIHHCDR14NISPNSGSANYNEKFKSHCDR25EGPYSYYPSREYYGSDLHCDR36RASQSISNNLHLCDR17YTSRSVSLCDR28GQNNEWPEVLCDR3

[0056] The above antibody and its antigen-binding fragment can be manufactured by methods commonly used in the field of antibody synthesis. For example, the above antibody and its antigen-binding fragment can be manufactured through monoclonal antibody culture technology using a hybridoma, phage library technology, etc. Phage display technology includes a technology for introducing a phagemid vector containing a scFv library into a host cell, and then infecting a helper phage to recover phage particles on which scFv is expressed on the surface.

[0057] Additionally, CXCL5 inhibitors may include, for example, any one selected from the group consisting of dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

[0058] A pharmaceutical composition for the treatment or prevention of cancer cachexia according to one embodiment of the present invention may be administered orally or parenterally. For example, it may be administered orally via a tablet, locally, intravenously, or subcutaneously, intracerebral or intrathecally, or nasogastric or rectal.

[0059] In addition, the pharmaceutical composition of the present invention can be formulated with pharmaceutically acceptable additives and manufactured in the form of injections, suppositories, powders, nasal drops, granules, tablets, etc.

[0060] Pharmaceutically acceptable additives may be selected by taking into account various factors well known to those skilled in the art. For example, pharmaceutically acceptable additives may be selected by taking into account the formulation, concentration, stability, and bioavailability of the pharmaceutical composition, the disease or condition to be treated, the patient's age, weight, etc., and the route of administration of the composition.

[0061] In one embodiment, pharmaceutically acceptable additives that may be used for oral or parenteral administration may include fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents, or excipients. Disintegrants may be sodium starch glycolate, calcium anhydrous phosphate, or a combination thereof. Binders may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by adding at least one excipient, for example, microcrystalline cellulose, lactose, low-substituted hydroxycellulose, starch, calcium carbonate, sucrose, and / or gelatin, to the active ingredient. Additionally, lubricants such as magnesium and talc may be used. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various additives, such as humectants, sweeteners, flavorings, and / or preservatives. Formulations for parenteral administration include injectable liquids, suspensions, emulsions, lyophilized preparations, nasal irrigators, and suppositories. Injectable liquids, suspensions, and emulsions may be prepared by mixing the active ingredient with water, a non-aqueous solvent, or a suspending agent; non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerol, and / or gelatin may be used as bases for suppositories. Parenteral administration can be performed via subcutaneous, intravenous, or intramuscular injection. In addition, transdermal administration is possible, and external preparations may be external liquids, creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, drug-containing bandages, lotions, or combinations thereof.

[0062] One embodiment of the present invention, for use in treating or preventing cancer cachexia, may be for use in manufacturing a formulation for treating, inhibiting, improving, and / or preventing cancer cachexia of an active ingredient.

[0063] The pharmaceutical composition for the treatment or prevention of muscle diseases according to the present invention comprises a CXCL5 inhibitor as an active ingredient. Below, any description that overlaps with the description of the pharmaceutical composition for the treatment or prevention of cancer cachexia comprising the aforementioned CXCL5 inhibitor as an active ingredient is omitted.

[0064] CXCL5 inhibitors can treat or prevent muscle diseases caused by CXCL5.

[0065] In a pharmaceutical composition for treating or preventing muscle disease according to one embodiment of the present invention, the muscle disease may be a muscle disease related to cancer cachexia. However, it is not limited thereto.

[0066] Muscle disease may be induced by cancer cachexia caused by CXCL5, and a pharmaceutical composition for treating or preventing muscle disease according to one embodiment of the present invention can treat or prevent cancer cachexia-related muscle disease by including a CXCL5 inhibitor as an active ingredient.

[0067] In a pharmaceutical composition for treating or preventing muscle diseases according to one embodiment of the present invention, the CXCL5 inhibitor may comprise one or more selected from the group consisting of a CXCL5 neutralizing antibody or its antigen-binding fragment, dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

[0068] In a pharmaceutical composition for treating or preventing muscle diseases according to one embodiment of the present invention, the CXCL5 neutralizing antibody may include: a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and a light chain variable region comprising LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO. 8.

[0069] In a pharmaceutical composition for treating or preventing muscle diseases according to one embodiment of the present invention, the heavy chain variable region may include the amino acid sequence of SEQ ID NO. 1, and the light chain variable region may include the amino acid sequence of SEQ ID NO. 2.

[0070] A pharmaceutical composition for the treatment or prevention of muscle diseases according to one embodiment of the present invention may be administered orally or parenterally. For example, it may be administered orally via a tablet, topically, intravenously, or subcutaneously, intracerebral or intrathecally, or nasogastric or rectal.

[0071] In addition, the pharmaceutical composition of the present invention can be formulated with pharmaceutically acceptable additives and manufactured in the form of injections, suppositories, powders, nasal drops, granules, tablets, etc.

[0072] One embodiment of the present invention, for use in treating or preventing muscle diseases, may be for manufacturing a protein supplement of an active ingredient, a muscle-strengthening agent, or a preparation for treating, inhibiting, improving, and / or preventing muscle loss, muscle wasting, muscle degeneration, muscle disease, or muscle damage.

[0073]

[0074] A pharmaceutical composition according to one embodiment of the present invention may contain 0.001 to 99.99 weight% of an active ingredient, preferably 0.001 to 80 weight%.

[0075] A pharmaceutical composition according to one embodiment of the present invention may contain 0.1 to 20 μM of an active ingredient.

[0076] The active ingredient included in the pharmaceutical composition according to one embodiment of the present invention may be administered at a dose of 0.1 mg / kg to 50 mg / kg when administered orally or parenterally.

[0077] The dosage can be appropriately adjusted according to the subject's body weight, health status, metabolism, etc.

[0078]

[0079] Administration may be performed once a day or divided into several times. However, the scope of the present invention is not limited by the above dosage and frequency of administration.

[0080] Meanwhile, when administered to animals other than humans, it goes without saying that the dosage may vary by animal species based on the calculation of the Human Equivalent Dose (HED) using the widely known body surface area-based conversion method (see USFDA. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteer. Rockville, MD: US Food and Drug Administration; 2005.).

[0081]

[0082] The treatment or prevention method of the present invention may include the step of administering the active ingredient, the pharmaceutical composition for treating or preventing cancer cachexia, or the pharmaceutical composition for treating or preventing muscle disease to a subject (animal including humans or animal excluding humans) who is suffering from or appears likely to suffer from cancer cachexia or muscle disease.

[0083] The treatment or prevention method of the present invention may include a step of inhibiting CXCL5 in the body of the subject. For example, the step of inhibiting CXCL5 may include a step of inhibiting the expression of a gene encoding CXCL5, a step of inhibiting the activity of an expressed CXCL5 protein, or a step of inhibiting the action of an activated CXCL5 protein.

[0084] The above subject may be a mammal.

[0085] The above subject may be an animal that requires administration of the above active ingredient.

[0086] The above active ingredient may be present in an effective amount.

[0087]

[0088] The antibody or antigen-binding fragment thereof that specifically binds to CXCL5 according to the present invention comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and a light chain variable region comprising LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO. 8.

[0089] According to one embodiment of the present invention, the heavy chain variable region of an antibody or its antigen-binding fragment that specifically binds to CXCL5 may include the amino acid sequence of SEQ ID NO. 1, and the light chain variable region may include the amino acid sequence of SEQ ID NO. 2.

[0090] The antibody or its antigen-binding fragment that specifically binds to CXCL5 of the present invention may be a neutralizing antibody or its antigen-binding fragment that specifically binds to CXCL5.

[0091]

[0092] The kit for diagnosing cancer cachexia or muscle disease according to the present invention comprises, as an active ingredient, a preparation capable of detecting the expression level of the CXCL5 protein or the gene encoding said protein.

[0093] The kit for diagnosing cancer cachexia or muscle disease according to the present invention refers to a kit capable of diagnosing cancer cachexia or muscle disease or predicting the prognosis through biological samples collected from individuals who have not been diagnosed with cancer cachexia or from patients with cancer cachexia or muscle disease.

[0094] The kit for diagnosing cancer cachexia or muscle disease according to the present invention comprises a preparation capable of detecting the expression level of CXCL5 protein as an active ingredient. Here, the expression level of CXCL5 protein may include the absolute amount, concentration, degree of activation, and / or degree of effect of CXCL5 protein, and the active ingredient may include an ELISA preparation capable of measuring the absolute amount and concentration of CXCL5 protein, a preparation containing a specific substrate capable of measuring the degree of activation of CXCL5 protein, and / or a preparation containing a neutralizing antibody capable of specifically binding to CXCL5 protein.

[0095] Alternatively, the kit for diagnosing cancer cachexia or muscle disease according to the present invention comprises, as an active ingredient, a preparation capable of detecting the expression level of a gene encoding the CXCL5 protein. The expression level of the gene encoding the CXCL5 protein may include the degree of transcription of the gene encoding the CXCL5 protein and / or the absolute amount and / or concentration of mRNA, which is a transcription product, and the active ingredient may include an RT-PCR preparation capable of measuring the amount of gene transcription and the absolute amount and concentration of mRNA.

[0096]

[0097] The method for providing information necessary for diagnosing cancer cachexia or muscle disease according to the present invention comprises: a step of measuring the expression or activity level of a CXCL5 protein, or the expression level of a gene encoding said protein, in a biological sample isolated from an individual suspected of having cancer cachexia or muscle disease; and a step of comparing the expression level or activity level of said CXCL5 protein, or the expression level of said gene encoding said protein, with the expression level or activity level of a CXCL5 protein of a control sample, or the expression level of said gene encoding said CXCL5 protein, respectively.

[0098] The step of measuring the expression or activity level of the CXCL5 protein may be a step of measuring the expression or activity level of the CXCL5 protein through methods such as ELISA. However, it is not limited thereto.

[0099] The step of measuring the expression level of the gene encoding the CXCL5 protein may be a step of measuring the expression level of the gene encoding the CXCL5 protein through methods such as RT-PCR. However, it is not limited thereto.

[0100] The control sample is isolated from an individual that does not suffer from cancer cachexia or muscle disease. Since the CXCL5 protein is expressed and active within the normal range in the control sample, information necessary for diagnosing cancer cachexia or muscle disease in an individual can be provided by comparing the expression level or activity level of the CXCL5 protein, or the expression level of the gene encoding said protein, between the biological sample isolated from an individual suspected of cancer cachexia or muscle disease and the biological sample isolated from the control sample.

[0101] The diagnostic method of the present invention may include a method for diagnosing cancer cachexia or muscle disease or predicting the prognosis through a biological sample taken from a patient suspected of suffering from cancer cachexia or muscle disease.

[0102] The diagnostic method of the present invention may include a method for measuring the degree of expression of a gene encoding CXCL5 protein, the degree of expression of CXCL5 protein, or the level of activity of CXCL5 protein through the biological sample.

[0103]

[0104] The drug screening method for the prevention or treatment of cancer cachexia or muscle disease according to the present invention comprises the steps of: treating cells expressing CXCL5 with a candidate substance; and comparing a treatment group treated with the candidate substance with a control group.

[0105] The drug screening method for the prevention or treatment of cancer cachexia or muscle disease according to the present invention is a method for screening drugs for the prevention or treatment of cancer cachexia or muscle disease by treating cells expressing CXCL5 with a candidate substance and determining whether CXCL5 is inhibited.

[0106] In this case, the control group is the group not treated with the candidate substance. That is, by comparing whether CXCL5 is inhibited between the group not treated with the candidate substance and the group treated with the candidate substance, drugs for the prevention or treatment of cancer cachexia or muscle disease can be screened.

[0107] In a drug screening method for the prevention or treatment of cancer cachexia or muscle disease according to one embodiment of the present invention, the step of comparing the treatment group and the control group is characterized by comparing the expression or activity level of the CXCL5 protein in the control group and the treatment group, or the expression level of the gene encoding the protein.

[0108]

[0109] The descriptions of the composition, use, method, and kit of the present invention described above may be interpreted by referring to each description to the extent that they are not contradictory.

[0110]

[0111] Hereinafter, in order to specifically explain the novel use of a CXCL5 inhibitor according to one embodiment of the present invention and the novel antibody or its antigen-binding fragment that can be used as a CXCL5 inhibitor, examples will be described in detail.

[0112]

[0113] I. Confirmation of Action of CXCL5 Neutralizing Antibody

[0114] 1. Preparation of CXCL5 neutralizing antibody and verification of action

[0115] cell culture

[0116] C2C12 mouse myoblasts were purchased from Coram Biotech (South Korea). Myoblasts were cultured in DMEM, 10% FBS, and 1% penicillin streptomycin (PenStrep) (GM). When the myoblasts reached approximately 90% density, they were cultured in differentiation medium (DMEM, 2% horse serum (HS), PenStrep) for 72 hours to differentiate into myotubes. Myotubes were defined as elongated cells containing three or more nuclei.

[0117] Patient-derived colon CAF (CAF115) was purchased from Neuromics (USA) and cultured in DMEM+F12 (3:1), 10% fetal bovine serum (FBS), and 1% penicillin streptomycin (PenStrep). Human colorectal cancer cell line (HCT 116) and CCD-18Co normal human colorectal fibroblasts (NF) were purchased from the American Type Culture Collection (USA). HCT 116 cells were cultured in DMEM, 10% FBS, and 1% penicillin streptomycin (GM). CCD-18Co cells were cultured in DMEM+F12 (3:1), 10% fetal bovine serum (FBS), and 1% penicillin streptomycin (PenStrep).

[0118] Conditioned media (CM) was harvested by culturing HCT 116, CCD-18Co, and cancer-associated fibroblasts (CAF) in serum-free medium for 24 hours when they reached approximately 100% density. Conditioned media for cancer-associated fibroblasts (CAF) was harvested after culturing for 24 hours in a mixture of 50% HCT 116 Conditioned media and 50% F-medium. When preparing Conditioned media for normal fibroblasts, cancer-associated fibroblasts, and cancer cells, the Conditioned media was diluted with serum-free medium at a 1:1 ratio, and horse serum (HS) was added to achieve a final concentration of 7% to create a composition identical to that of the myotube differentiation medium. The Conditioned media was applied to differentiated myotubes for 72 hours. The CXCL5 neutralizing antibody MAB-254 was purchased from R&D Systems (USA), and the CXCR2 antagonist SSB225005 was obtained from MedChemExpress (New Jersey, USA).

[0119] Human-derived myoblasts were purchased from Thermo-Fisher Scientific (USA). The myoblasts were thawed in a water bath, centrifuged at 180g for 5 minutes at room temperature, and washed with 20mL of differentiation medium. Then, the myoblasts were resuspended in differentiation medium, seeded into a 12-well plate (density = 4.8 x 10⁴ cells / well), and used for the experiment after 48 hours.

[0120] Root canal diameters were measured using ImageJ 1.52 software (NIH, USA) with images captured by a differential interference contrast (DIC) microscope (Olympus CKX41). At the end of the experiment, five photographs were taken from each well, and the diameters of 30 root canals were measured from each image (Fig. 1A).

[0121]

[0122] CAF isolation from patient's colorectal cancer tissue

[0123] Patient-derived cancer-associated fibroblasts (CAF-HS2) were isolated from tumor tissue obtained from patients undergoing tumor resection surgery for colorectal cancer (provided by Chonnam National University College of Medicine, Gwangju, South Korea (IRB approval number: CNUHH-2021-217)). The donated tumor tissue was dissected under sterile conditions, and cancer-associated fibroblasts (CAF) were harvested using disintegration buffers (collagenase A (Roche, USA) and dispase II (Wako, Japan)). Prior to co-culture and transplantation studies, the cancer-associated fibroblasts (CAF) were tested three times for Mycoplasma infection using a Mycoplasma removal reagent kit (Capricorn, Germany). The fibroblast origin of the cancer-associated fibroblasts (CAF) was confirmed by Vimentin staining.

[0124]

[0125] Immunocytochemical analysis

[0126] To measure root canal diameter and differentiation and fusion indices, C2C12 root canals and human donor skeletal muscle root canals were visualized using myosin heavy chain immunocytochemistry. Root canals were fixed with 3.7% formaldehyde solution, permeated through the cell membrane using 0.2% Triton X-100, and blocked with 1% bovine serum albumin (BSA) dissolved in PBST (PBS + 0.2% Tween 20). The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. Root canals were counterstained with 1 μg / mL of DAPI dissolved in PBS.

[0127]

[0128] Western Blotting

[0129] To harvest the protein lysate, the cells were rinsed with phosphate-buffered saline (PBS) to remove the culture medium, and cold lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, USA) was added to the cells cultured in a 10 cm dish. The cell lysate was harvested with a cell scraper and transferred to a microcentrifuge tube. The tube was incubated on ice for 15 minutes, followed by centrifugation at 13,000 rpm for 15 minutes. The supernatant was then transferred to a new 1.5 mL tube.

[0130] The excised tissue was finely chopped with dissecting scissors, transferred to a microcentrifuge tube, and cultured with cold lysis buffer. The culture and centrifugation steps were performed in the same manner as for the cell lysate.

[0131] Protein lysates harvested from cells or tissues were quantified using Bradford reagent (Bio-Rad, USA). Proteins were separated by electrophoresis via 10–12% SDS-PAGE, transferred to PVDF membranes (Merck, Germany), and blocked with a solution of 5% bovine serum albumin (BSA) dissolved in TBST. Primary antibody incubation was performed overnight at 4°C. Secondary antibody incubation was conducted for 30 minutes at room temperature (RT). Protein band density measurements were performed using ImageJ 1.52 software. The primary and secondary antibodies used in this study are listed in Tables 2 and 3.

[0132] Primary antibodyCloneCompanyCat. NoDilutionGAPDHMonoclonalSantacruzSc-3650621:1000MYH2MonoclonalSantacruzSc-530951:1000Atrogin-1MonoclonalAbcamAb1683721:1000MuRF-1MonoclonalSantacruzSc-3986081:500Phoshpo-p44 / 42 MAPK (ERK1 / 2) (Thr202 / Tyr204)MonoclonalCST9101S1:1000p44 / 42 MAPK (ERK1 / 2)MonoclonalCST9102S1:1000Phospho-PI3 Kinase p85 (Tyr458) / p55 (Tyr199) (E3U1H)MonoclonalCST17366S1:1000PI3 Kinase p85α (6G10)MonoclonalCST13666S1:1000Phospho-AKT ( Ser473 )MonoclonalCST9271S1:1000AKTMonoclonalCST9272S1:1000P-IκBα (Ser32 / 36)MonoclonalCST9246S1:1000IκBα (44D4)MonoclonalCST4812S1:1000NF-κB p65 (D14E12)MonoclonalCST8242S1:1000Puromycin (12D10)MonoclonalMerckMABE-3431:2000Alpha-tubulinPolyclonalInvitrogenPA5-294441:1000VimentinMonoclonalCST5741S1:100CytokeratinPolyclonalAbcamAb93771:100CXCR2PolyclonalInvitrogenPA5-1009511:1000LamininPolyclonalAbcamAb115751:1000Mouse IgG1 Isotype controlMonoclonalR&D systemsMAB002120 μg / kgHuman CXCL5 / ENA-78 AntibodyMonoclonalR&D systemsMAB254-500120 μg / kg

[0133] Secondary antibodyConjugated useCompanyCat. NoDilutionHorse anti-Mouse IgG HRPHRPCST#70761:10000Goat anti-Rabbit IgG HRPHRPCST#70741:10000Alexa FluorTM 488 Goat anti-mouse IgG(H+L)Alexa Fluor 488InvitrogenA110011:200Goat anti-rabbit IgG H+L ChainAntibody DyLight® 488 ConjugatedDylight 488BethylA120-101D21:1000Goat anti-rabbit IgG H+L ChainAntibody DyLight® 594 ConjugatedDylight 594BethylA120-101D4-41:1000Goat anti-mouse IgG H+L ChainAntibody DyLight® 594 ConjugatedDylight 594BethylA90-116D41:200

[0134] Messenger RNA (mRNA) expression levels of the gene of interest were measured using a StepOnePlus real-time polymerase chain reaction system (Applied Biosystems, UK). AccuPower RT PreMix (Bionia, South Korea) was used to synthesize complementary DNA (cDNA) from total RNA. Real-time polymerase chain reaction (qPCR) was performed according to the manufacturer's instructions, using 20 μL of 2X Power SYBR Green PCR Master Mix (Engenomics, South Korea) containing 200 nM target primers and 1 μL of cDNA, and was conducted in three replicates. The primers used in this study are listed in Table 4. A specific cDNA sample was included in each run to serve as a reference between runs. GAPDH expression was used as the normalization standard when measuring the expression levels of the gene of interest.

[0135] Primer namePrimer sequence서열번호Size (bp)Accession numberAtrogin-1ForwardCAGAGAGCTGCTCCGTCTCA9178NM_026346ReverseACGTATCCCCCGCAGTTTC10MuRF-1ForwardCCGAGTGCAGACGATCATCTC11198NM_001039048ReverseTGGAGGATCAGAGCCTCGAT12MyoGForwardAGCGCAGGCTCAAGAAAGTG13181NM_031189ReverseCCGCCTCTGTAGCGGAGAT14GAPDHForwardCTCCACTCACGGCAAATTCA15120NM_001289726ReverseGCCTCACCCCATTTGATGTT16Dkk2ForwardCGCAACCATGGTCACTATTCC17150NM_020265ReverseGAAGTGGCGAGCACAACAAA18Col6a5ForwardTGTAGTGGTCGGGTTCGACAT19250NM_001167923ReverseACGGACTGTGACGTGCAACA20Pf4ForwardGTTTCTGCCAGCGGTGGTT21100NM_019932ReverseATGGATCCCAGAGGAGATGGT22Dusp2ForwardCGAGGCGGTTTCAAAAGCT23110NM_010090ReverseACCCTCGGGTCAGAGTTGCT24GatmForwardAGCTACAGCTTCCTCCCGAAA25150NM_025961ReverseAATGGTGGCACACAGGCATT26Sphk1ForwardGCCAGTGCCTTCTCATTGGA27150NM_001172472ReverseCCCGCACGTACGTAGAACAGA28Serpina3nForwardGGCTCAACCAGCCAAAGGA29150NM_009252ReverseGACGAGGCTGCTGGAAGTCT30Cxcl1 (human)ForwardCACTCAAGAATGGGCGGAAA31104NM_001511.4ReverseCCCTTCTGGTCAGTTGGATTTG32Cxcl2 (human)ForwardCTCAAGAATGGGCAGAAAGCTT33101NM_002089.4ReverseCCTTCTGGTCAGTTGGATTTGC34Cxcl3 (human)ForwardCCGAAGTCATAGCCACACTCAA35101NM_002090.3ReverseGCTCCCCTTGTTCAGTATCTTTTC36Cxcl5 (human)ForwardTTCATCCCAAAATGATCAGTAATCTG37100NM_002994.5ReverseCAAATTTCCTTCCCGTTCTTCA38Cxcl6 (human)ForwardTTGCACTTGTTTACGCGTTACG39103NM_002993.4ReverseGGCTACCACTTCCACCTTGGA40Cxcl7 (human)ForwardATGCTGAACTCCGCTGCAT41100NM_002704.3ReverseTGGTTGCAATGGGTTCCTTT42Cxcl8 (human)ForwardTCAGAGACAGCAGAGCACACAA43100NM_000584.4ReverseGGCCAGCTTGGAAGTCATGT44IL-6 (human)ForwardAGTGGCTGCAGGACATGACA45104NM_000600.5ReverseTCTGAGGGTGCCCATGCTACA46GAPDH (human)ForwardCTGCACCACCAACTGCTTAGC47107NM_002046.7ReverseTCTTCTGGGTGGCAGTGATG48.

[0136] ELISA analysis

[0137] Duoset for Human CXCL5 / ENA-78 (Catalog No.: DY008) and Human IL-6 (Catalog No.: DY206) ® The ELISA development system kit was purchased from R&D Systems (USA). The enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions.

[0138]

[0139] Cytokine antibody array

[0140] Cytokine levels were measured in HCT 116 CM, cancer-associated fibroblast (CAF) CM, and cancer-associated fibroblast (CAF) conditioned medium (CCM) using the human cytokine antibody array C3 (Raybiotech, USA) according to the manufacturer's instructions. Quantification of cytokine levels was performed using ImageJ 1.52 software.

[0141]

[0142] Protein synthesis measurement

[0143] Surface translation detection (SUnSET) assay was used to evaluate protein synthesis. In summary, 1 μg / mL puromycin was added to the myotube culture medium, and cell lysates were harvested after 10 minutes. The lysates were then treated for Western blotting using the anti-puromycin 12D10 antibody (MABE343, Sigma-Aldrich, USA).

[0144]

[0145] animal research

[0146] Animal research was conducted under the guidance of the Institute Guidelines on the Care and Use of Laboratory Animals and was approved by the Gwangju Institute of Science and Technology Animal Care and Use Committee (Research Approval No. GIST-2021-098). As the animal research was approved by the relevant ethics committee, it was conducted in accordance with the ethical standards specified in the 1964 Helsinki Declaration and subsequent amendments. The animals were purchased from Orient Bio (South Korea).

[0147]

[0148] Humanized Model of Colorectal Cancer-Inducing Cachexia

[0149] 1x10 on the right flank of 10-week-old female NOD-SCID mice 6 Canisters of HCT 116-Luc2 cells (ATCC, VA, USA) alone or 1 x 10⁶ 6 Dogs with 116 HCT cells and 2x106 Canine human cancer-associated fibroblasts (CAFs) were subcutaneously xenografted (n=7 per group). To inhibit CXCL5, 120 μg / kg of neutralizing antibodies (R&D Systems, MAB-254, clone 33160, USA) and IgG1 monoclonal antibodies (R&D Systems, MAB-002, clone 11711, USA) were administered via intraperitoneal (IP) injection starting 3 days prior to transplantation. Evidence of neutralization was provided by the supplier and the study of Chen et al., which demonstrated that these antibodies have a neutralizing effect in a hindlimb ischemia model. Tumor growth in tumor-bearing mice was measured using the IVIS system (Caliper IVIS Kinetic In Vivo Optical Imaging System, USA). Luciferin (Promega, USA) was dissolved in phosphate-buffered saline (DPBS) and injected at a concentration of 6 mg per mouse.

[0150]

[0151] Hanging tolerance test

[0152] Hanging tolerance was tested by dropping the mice from a height of 50 cm onto a cushioned surface before sacrifice. Hanging tolerance was recorded once for each mouse.

[0153]

[0154] Muscle resection and histology

[0155] Mice were anesthetized by intraperitoneal injection of 22 mg / kg ketamine (Yuhan, South Korea) and 10 mg / kg xylazine (Bayer, South Korea) before sacrifice, or by 2.5% isoflurane (Hana, South Korea) starting 3 minutes before sacrifice and continuing during the process. The quadriceps femoris, gastrocnemius, tibialis anterior (TA), or soleus muscles were resected and weighed. The muscles were fixed overnight in 3.7% paraformaldehyde at 4°C, embedded in paraffin solution, and stored at 4°C. Paraffin section preparation and hematoxylin-eosin (H&E) staining were performed at the Gwangju Institute of Science and Technology Animal Research Facility using an H&E kit (Merck, Germany). Muscle fiber cross-sectional area was measured using ImageJ 1.52 software (NIH, USA). 100 muscle fibers were measured from 5 captured images per muscle. For immunohistochemistry, excised muscles were sequentially embedded in 10%, 20%, and 30% sucrose solutions at 4°C at 24-hour intervals, then embedded in Cryo-OCT blocks at 4°C for 24 hours and stored at -80°C. Muscle sections were obtained using a CM 1860 cryosection machine (Leica) and mounted with a DAPI mount (Invitrogen).

[0156]

[0157] Immunohistochemistry

[0158] Skeletal muscle sections were prepared using anti-laminin antibodies (Abcam, UK) and anti-CXCR2 (Invitrogen, USA). Counterstaining was performed with a 1 μg / mL DAPI solution. Tibialis anterior (TA) muscle was sectioned to a thickness of 10 μM to measure the muscle fiber cross-sectional area (CSA) and the ratio of CXCR2-positive fibers. Sections were visualized using a fluorescence microscope (Leica DM 2500). CSA and muscle fiber distribution were measured using ImageJ 1.48 software (NIH, USA).

[0159]

[0160] In vivo transcriptome analysis (RNA-Seq)

[0161] RNA samples were harvested from the quadriceps of treated mice. Transcriptome analysis (RNA-Seq) was performed by Macrogen (South Korea). Sample quality control (QC) was performed using FastQCv0.11.7 (http: / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Trimmomatic 0.38 software (http: / www.usadellab.org / cms / ?page=trimmomatic) was used with various parameters for the Illumina paired-end or single-end trimming process. HISAT2 version 2.1.0, Bowtie2 2.3.4.1 (https: / ccb.jhu.edu / software / hisat2 / index.shtml) provided sequencing read mapping to the Hierarchical Graph FM Index (HGFM). The potential transcriptome combination tool used was StringTie version 2.1.3b (https: / ccb.jhu.edu / software / stringtie / ).

[0162]

[0163] Cytokine-processed cachexia model

[0164] Recombinant CXCL5 40 ng / kg and IL-6 80 ng / kg were administered intraperitoneally to 10-week-old female C57BL / 6 mice three times a week for 4 weeks. 72 hours prior to cytokine administration, mice were pretreated with IgG1 and CXCL5 neutralizing antibodies at a dose of 120 μg / kg. For 4 weeks, the neutralizing antibodies were co-administered with the cytokines three times a week.

[0165]

[0166] Preparation and immunohistochemical analysis of patient colon tumor tissue

[0167] Colorectal tumor tissues were provided by Chonnam National University College of Medicine (Gwangju, South Korea) (IRB approval number: CNUHH-2021-217). The donor's age, sex, and stage are presented in Table 5. Tissues were fixed in 3.7% paraformaldehyde at 4°C for 24 hours for paraffin sections. Slide sections were deparaffinized with xylene and sequentially diluted ethanol, and membranes were permeated with 0.1% Triton-X; blocking and antibody treatment were performed using a solution of 3% bovine serum albumin (BSA) dissolved in 0.05% Triton-X. Prolong Gold anti-fading reagent containing DAPI (Thermofisher, USA) was used for mounting. Immunofluorescence images were acquired using a DMI 3000 B microscope and analyzed using ImageJ 1.52 software.

[0168]

[0169] NumberSexAgeStage1Female8632Male7113Male712

[0170] Statistical Analysis: Statistical significance was determined using Student's t-test. A p-value of less than 0.05 was considered significant. Unless otherwise noted, all presented data represent at least three experimental repetitions, and error bars in the graphs represent the standard deviation or standard error as specified.

[0171]

[0172] 2. Experimental Results

[0173] To identify in vitro factors inducing muscle atrophy, differentiated C2C12 myotubes were treated with conditioned media (CM) derived from normal fibroblasts (NF), CM derived from cancer-associated fibroblasts (CAF), and conditioned media from fibroblasts treated with conditioned media derived from cancer cells (CAF CCM, CCM being Complete Culture Media), respectively. As a result, unlike the experimental groups treated with NF CM or CAF CM, it was confirmed that the thickness of C2C12 myotubes significantly decreased only in the experimental group treated with CAF CCM (Figs. 1B and C).

[0174] Based on the above results, a cytokine array analysis was performed on HCT 116 (Human Colon Tumor 116) CM, CAF CM, and CAF CCM to identify the key factors causing the reduction in root canal thickness (Fig. 2). The analysis confirmed that the expression of the cytokine CXCL5 was significantly increased in CAF CCM compared to other experimental groups (Fig. 3).

[0175] To verify whether CXCL5 is a direct causative factor in causing muscle atrophy, differentiated C2C12 myoducts were treated with recombinant CXCL5 protein, and as a result, it was confirmed that the thickness of C2C12 myoducts was significantly reduced by treatment with recombinant CXCL5 (Fig. 4).

[0176] Furthermore, in the experimental group treated with CAF CCM and CXCL5 neutralizing antibody together, the decrease in root canal thickness, i.e., muscle atrophy observed in the group treated with CXCL5 alone, was significantly alleviated, confirming that the muscle atrophy response induced by CAF CCM can be alleviated by the CXCL5-specific neutralizing antibody (Fig. 5).

[0177] Based on the results of animal experiments, it was verified that cancer-associated fibroblasts (CAFs) promote cancer cachexia in a humanized mouse model, and that CXCL5 neutralizing antibodies improve cancer cachexia.

[0178] Specifically, co-transplantation of cancer-associated fibroblasts (CAFs) with cancer cells did not affect tumor formation, as there was no significant effect on total light intensity or resected tumor mass in IVIS results (Figs. 6–8). On the other hand, co-transplantation of cancer-associated fibroblasts (CAFs) significantly reduced body weight excluding the tumor (Fig. 9). Histological analysis of the gastrocnemius muscle revealed that co-transplantation of cancer-associated fibroblasts (CAFs) reduced body weight excluding the tumor and induced muscle fiber atrophy, indicated by a significantly reduced cross-sectional area (CSA) (Figs. 10–11). Real-time polymerase chain reaction (qPCR) analysis of the tumor tissue showed that the presence of cancer-associated fibroblasts (CAFs) upregulated the expression of CXCL5 during tumor formation, but did not significantly affect the expression of CXCL1, 2, 3, 6, 7, 8, and IL-6 (Fig. 12).

[0179] Specifically, human colorectal cancer cells and cancer-associated fibroblasts (CAFs) were co-transplanted into immunodeficient mice and treated with CXCL5 neutralizing antibodies. In vivo imaging system (IVIS) analysis revealed that CXCL5 neutralization did not have a significant effect on tumor progression or tumor mass (Figs. 13–15). CXCL5 neutralizing antibodies prevented body weight loss (Fig. 16). Neutralization improved muscle performance by increasing the time to fall in the hanging tolerance test (Fig. 17). Evaluation of the tibialis anterior (TA) muscle (composed mainly of fast-twitch fibers that are preferentially lost in cancer cachexia) showed that CXCL5 neutralizing antibodies increased muscle mass (Fig. 18) and cerebrovascular area (CSA) (Figs. 19–20). Immunohistochemical analysis of the tibialis anterior (TA) muscle revealed that CXCR2 expression was upregulated in tumor-carrying mice and downregulated by CXCL5 neutralizing antibodies (Figs. 21–22).

[0180]

[0181] II. Confirmation of Action of CXCL5 Inhibitor

[0182] 1. Cell Culture and CM Collection Methods

[0183] cell culture

[0184] Patient-derived cancer-associated fibroblasts (CAF115, CAF-HS2) were cultured in DMEM+F12 (3:1), 10% FBS, and 1% penicillin streptomycin (GM). Human colorectal cancer cell lines (HCT 116) were cultured in DMEM, 10% FBS, and 1% penicillin streptomycin (GM).

[0185] C2C12 mouse myoblasts were cultured in DMEM, 10% FBS, and 1% penicillin streptomycin (GM), and differentiation was induced with DMEM, 2% horse serum, and 1% penicillin streptomycin (DM). To form myotubes, the cells were treated with differentiation medium for 72 hours.

[0186]

[0187] Conditioned Media Collection Method

[0188] To collect Conditioned Media (CM), HCT116 (colon cancer) cells were cultured in 100 mm culture dishes until 80–90% confluency was achieved. Subsequently, the medium was removed, washed twice with 1X PBS, and replaced with serum-free medium. After 48 hours, the collected medium was centrifuged at 1,500 rpm for 3 minutes at 4 ℃ and filtered through a 0.2 μm syringe filter.

[0189]

[0190] 2. Analysis Method

[0191] Induction of CXCL5 expression from CAF and Enzyme-Linked Immunosorbent Assay (ELISA) analysis

[0192] An ELISA was performed to quantify the CXCL5 expression concentration of CAF. 15x10 3 CAF115, 1x10 4HS2 cells were seeded into 96-well plates and cultured for 24 hours. After removing the medium, each drug (5 μM) was diluted in a 1:1 mixture of serum-free DMEM (Dulbecco's Modified Eagle's Medium, DM) + F12 and cancer CM, and treated for 24 hours. Subsequently, the culture supernatant was collected, and a CXCL5-specific ELISA was performed to measure human CXCL5 concentrations. The culture supernatant and 1X diluent reagent were diluted in a 1:1 ratio and treated on plates conjugated with Capture ab (R&D Systems, MAB654), followed by incubation at 4°C for 24 hours. Human CXCL5 protein (R&D Systems, 254-XB) was used to construct the standard curve. Subsequently, the samples were treated with Detection ab (R&D Systems, BAF254) and reacted for 2 hours. After adding a chromogenic substrate (TMB substrate, R&D Systems, DY999B), the color reaction was confirmed. The absorbance of each sample was calculated by subtracting a 570 nm correction value from the value measured at 450 nm using an ELISA plate reader (VersaMax, Molecular Devices). Result analysis was performed using GraphPad Prism 7 software.

[0193]

[0194] Analysis of CXCL5-induced inhibition of root canal formation and muscle atrophy

[0195] 20x10 stabilized for 24 hours in DMEM, 10% FBS, and 1% penicillin streptomycin 4C2C12 myoblasts were seeded into 12-well plates. Recombinant CXCL5 20 ng / mL (R&D systems, 254-XB) was administered during differentiation induction with 2% horse serum DMEM for 72 hours. Each drug (10 μM) was administered concurrently with CXCL5. The results of the treatment are shown in Table 2 and Figure 23.

[0196] Differentiated myocanal ducts were fixed with a 4% formaldehyde solution. Cells were permeated and blocked with 0.3% Triton-X and 5% BSA solutions, respectively. To measure the intensity of fluorescence for comparison of muscle differentiation maturity, myosin heavy chains were stained with a specific antibody (Santa Cruz SC-53095). The myosin heavy chain antibody was exposed to the environment at a 1:200 ratio and conjugated with Alexa 488 (Thermo, A11001) for 1 hour. The stained myocanal ducts were examined using a fluorescence microscope (Leica DMI 3000B; Leica). The results of the fluorescence microscopy observation are shown in Figure 24.

[0197]

[0198] 3. Experimental Results

[0199] The experimental results are shown in Table 2. Indicated in the left column of Table 2 a The CXCL5 Inhibition rate measured by ELISA (%) represents the percentage of the inhibitory effect of the drug on the expression level of secreted CXCL5 induced in CAFs treated with cancer CM (conditioned media) (quantified by CXCL5-specific ELISA), and is indicated in the right column of Table 2. b Relative MyHC2 staining intensity (fold change) indicates the effect of the drug on myotube formation in myoblasts treated with CXCL5 (quantified by measuring MyHC2 staining intensity). The drugs indicated in bold can be seen as having a therapeutic effect on cancer cachexia.

[0200] Treatment a CXCL5 Inhibition rate measured by ELISA (%) b Relative MyHC2 staining intensity (fold change)CXCL5 alonen.t.1.0 (control)Cancer CM alone0 (control)ntDicoumarol32.611.13Aceclofenac29.001.33Creatine Monohydrate52.301.50Ethamsylate16.041.74Quercetin Dihydrate40.171.14Meclofenamate sodium17.851.56Clonixin33.441.78Teriflunomide26.421.82Tiratricol43.061.29

[0201] Confirmation of drug-induced inhibitory effect on CXCL5 expression in CAF indicated in the left column of Table 2 a The value shown in the CXCL5 Inhibition rate measured by ELISA (%) indicates that the drug can reduce the concentration of CXCL5 secreted from CAF cells.

[0202] The expression of CXCL5 in CAF was increased by colon cancer CM (conditioned media) and effectively inhibited by drug treatment, which was expected to have an inhibitory effect.

[0203] As shown in the left column of Table 2, all nine compounds tested in CAF showed inhibitory effects: Creatine Monohydrate (52.30%), Tiratricol (43.06%), Clonixin (33.4%), Dicoumarol (32.61%), Quercetin Dihydrate (27.98%), Teriflunomide (26.42%), Aceclofenac (18.55%), Meclofenamate sodium (11.87%), Ethamsylate (6.26%).

[0204]

[0205] Confirmation of the effect of a drug alleviating CXCL5-induced inhibition of root canal formation and atrophy

[0206] The values ​​corresponding to Relative MyHC2 staining intensity (fold change) are shown on the right side of Table 2, and this was graphed and presented as Figure 23. In the graph of Figure 23, the x-axis represents the type of drug used, and the y-axis represents the value corresponding to Relative MyHC2 staining intensity (fold change).

[0207] Figure 24 shows the results of fluorescence microscopy observation, illustrating how antibodies bind to the location of myosin heavy chain protein and emit fluorescence according to the maturity of muscle differentiation. Referring to Figure 24, in a model of myotube formation inhibition and muscle atrophy induced by recombinant CXCL5, treatment with recombinant CXCL5 alone was found to reduce the expression of myosin heavy chain protein (MyHC2), which indicates the maturity of muscle differentiation, by approximately 40% compared to normal myotubes (control group not treated with CXCL5).

[0208] Therefore, CXCL5 can be a target for the treatment or prevention of cancer cachexia or muscle disease, and can be used for the diagnosis of cancer cachexia or muscle disease, or for screening drugs effective against cancer cachexia or muscle disease.

[0209] On the other hand, among the compounds used in the experiment, nine drugs—Dicoumarol (1.13 times), Aceclofenac (1.33 times), Creatine Monohydrate (1.50 times), Ethamsylate (1.74 times), Quercetin Dihydrate (1.14 times), Meclofenamate sodium (1.56 times), Clonixin (1.78 times), Teriflunomide (1.82 times), and Tiratricol (1.29 times)—were found to increase the staining intensity of MyHC2 (Fig. 23), and are considered to have the effect of restoring the formation of root canals that was inhibited by CXCL5 treatment. In addition, these drugs appear to have an effect of inhibiting muscle atrophy by restoring the diameter of the root canals that had atrophied due to CXCL5 (Fig. 24).

[0210] In conclusion, drugs such as dicoumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol, which reduce the expression of CXCL5—a muscle atrophy-inducing factor secreted by CAF—promote the formation of root canals and inhibit root canal atrophy induced by CXCL5, can be seen as having preventive and therapeutic effects against cancer cachexia.

[0211]

[0212] Referring to the experiment above, it can be confirmed that the use and method of the present invention has the effect of inhibiting muscle atrophy by restoring the formation of root canals that were inhibited by CXCL5 through a reduction in CXCL5 expression and / or inhibition of the action of the CXCL5 protein, and by restoring the diameter of the root canals. In other words, it can be confirmed that the use and method of the present invention can exhibit a therapeutic or preventive effect against cancer cachexia and muscle diseases, and that the method and kit of the present invention can diagnose cancer cachexia and muscle diseases with high efficiency through the CXCL5 expression and / or activation level.

Claims

1. A pharmaceutical composition for the treatment or prevention of cancer cachexia comprising a CXCL5 inhibitor as an active ingredient.

2. In Claim 1, The above CXCL5 inhibitor is a pharmaceutical composition for the treatment or prevention of cancer cachexia comprising one or more selected from the group consisting of a CXCL5 neutralizing antibody or its antigen-binding fragment, dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

3. In Claim 2, The above CXCL5 neutralizing antibody or its antigen-binding fragment comprises a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and A pharmaceutical composition for the treatment or prevention of cancer cachexia comprising a light chain variable region including LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO.

8.

4. In Claim 3, A pharmaceutical composition for the treatment or prevention of cancer cachexia, wherein the heavy chain variable region of the above CXCL5 neutralizing antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO. 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO.

2.

5. A pharmaceutical composition for the treatment or prevention of muscle diseases comprising a CXCL5 inhibitor as an active ingredient.

6. In Claim 5, The above-mentioned muscle disease is a pharmaceutical composition for the treatment or prevention of muscle disease, which is a muscle disease related to cancer cachexia.

7. In Claim 5, The above CXCL5 inhibitor is a pharmaceutical composition for the treatment or prevention of muscle diseases comprising one or more selected from the group consisting of a CXCL5 neutralizing antibody or its antigen-binding fragment, dicumarol, aceclofenac, creatine monohydrate, ethamsylate, quercetin dihydrate, meclofenamate sodium, clonixin, teriflunomide, and tiratricol.

8. In Claim 7, The above CXCL5 neutralizing antibody or its antigen-binding fragment comprises a heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and A pharmaceutical composition for treating or preventing muscle diseases comprising a light chain variable region including LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO.

8.

9. In Claim 8, A pharmaceutical composition for treating or preventing muscle diseases, wherein the heavy chain variable region of the above CXCL5 neutralizing antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO. 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO.

2.

10. A heavy chain variable region comprising HCDR1 of SEQ ID NO. 3, HCDR2 of SEQ ID NO. 4, and HCDR3 of SEQ ID NO. 5; and An antibody or its antigen-binding fragment that specifically binds to CXCL5, comprising a light chain variable region including LCDR1 of SEQ ID NO. 6, LCDR2 of SEQ ID NO. 7, and LCDR3 of SEQ ID NO.

8.

11. In Claim 10, An antibody or its antigen-binding fragment that specifically binds to CXCL5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO.

2.

12. A kit for diagnosing cancer cachexia or muscle disease comprising, as an active ingredient, a preparation capable of detecting the expression level of CXCL5 protein or a gene encoding said protein.

13. A step of measuring the expression or activity level of the CXCL5 protein, or the expression level of the gene encoding said protein, in a biological sample isolated from an individual suspected of cancer cachexia; and A method for providing information necessary for diagnosing cancer cachexia or muscle disease, comprising the step of comparing the expression level or activity level of the CXCL5 protein, or the expression level of the gene encoding the protein, with the expression level or activity level of the CXCL5 protein of a control sample, or the expression level of the gene encoding the CXCL5 protein of the control sample, respectively.

14. A step of treating cells expressing CXCL5 with a candidate substance; and A drug screening method for the prevention or treatment of cancer cachexia or muscle disease comprising the step of comparing a treatment group treated with the above candidate substance with a control group.

15. In Claim 14, A drug screening method for the prevention or treatment of cancer cachexia or muscle disease, characterized in that the step of comparing the treatment group and the control group involves comparing the expression or activity level of the CXCL5 protein, or the expression level of the gene encoding the protein, in the control group and the treatment group.

Images