Use of cathepsin l inhibitor in combination administration

Abstract

In the present invention, it has been confirmed that cathepsin L (CTSL) is a dual-action target capable of simultaneously inhibiting tumor progression and CD8⁺ T cell-mediated muscle wasting. That is, pharmacological inhibition of CTSL not only alleviated muscle wasting induced by anti-PD-L1, but also further inhibited tumor growth through down-regulation of BNIP3. As a result, the present invention demonstrates that CTSL acts as a dual-action target that enhances anticancer efficacy while alleviating muscle-specific irAEs, which suggests a new strategic approach that can overcome the clinical limitations of ICIs and improve therapeutic efficacy.

Description

Uses of concomitant administration of cathepsin L inhibitors

[0001] The present invention relates to the use of cathepsin L inhibitors in combination.

[0002] Immune checkpoint inhibitors (ICIs) are monoclonal antibodies (mAbs) that target programmed cell death protein 1 (PD-1) or its ligand PD-L1, and are therapeutic agents that induce anticancer effects by activating the immune system in various cancers, including non-small cell lung cancer (NSCLC). When the immune checkpoint protein PD-L1 on the surface of cancer cells interacts with PD-1 on CD8+ T cells, T cell activity is suppressed, creating an immunosuppressive environment; however, ICIs block this PD-L1 / PD-1 interaction, thereby restoring the cytotoxic activity of CD8⁺ T cells and enhancing the anticancer immune response to inhibit tumor growth.

[0003] However, such immune activation has limitations in that it can induce immune-related adverse events (irAEs) affecting various organs, including the lungs, colon, liver, and skin. In particular, skeletal muscle is attracting attention as a new target for muscle-specific irAEs, such as myositis and muscle atrophy associated with anti-PD-1 (αPD-1) or anti-PD-L1 (αPD-L1) therapy. These muscle-specific irAEs not only significantly reduce the quality of life for cancer patients but also require immunosuppressive therapy for symptom relief, which can impede the anticancer efficacy of ICIs. Therefore, there is an urgent need to develop new therapeutic strategies that can prevent or alleviate muscle-specific irAEs while maintaining anticancer immunity.

[0004] Cachexia occurs in approximately 80% of terminal cancer patients and is a malignant wasting syndrome characterized by weight loss, systemic inflammation, and the rapid loss of fat and skeletal muscle. It directly contributes to about 20% of cancer deaths and is associated with poor responsiveness to ICI, whereas obesity is often associated with a better prognosis after ICI treatment. However, the correlation between cancer cachexia and immunotherapy has not yet been clearly established.

[0005]

[0006] One object of the present invention is to provide a pharmaceutical composition and a treatment method for the prevention or treatment of cancer, cachexia, and muscle atrophy, comprising a cathepsin L inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0007] Another object of the present invention is to provide a pharmaceutical composition and a treatment method for preventing or treating side effects caused by the administration of an anticancer agent comprising a cathepsin L inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

[0008]

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010]

[0011] Various embodiments of the present invention are described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to make the present invention unnecessary or obscure. Reference throughout this specification to one embodiment implies that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the present invention. Accordingly, the circumstances of the embodiments expressed at various locations throughout this specification do not necessarily represent the same embodiment of the present invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0012]

[0013] Cathepsin L (CTSL) is a cysteine ​​protease expressed in most tissues, primarily found within lysosomes, although some are also found in endosomes or secretory forms within the extracellular matrix. This enzyme is primarily activated in acidic environments with a pH of 4–6. In various cancers, CTSL is overexpressed to promote angiogenesis, invasion, and metastasis, and higher expression levels are associated with poor clinical prognosis. Conversely, however, CTSL may inhibit tumor formation by regulating growth signaling pathways, promoting oncogene-induced senescence, and participating in the remodeling of chromatin. Furthermore, CTSL is known to maintain the cytotoxic activity of cytotoxic T cells and NK cells, induce the development of NKT cells and CD4+ T cells, and regulate immune function through Major Histocompatibility Complex II (MHC-II). In skeletal muscle, it exacerbates cancer-associated muscle loss by promoting protein degradation dependent on autophagy. In summary, CTSL demonstrates that it performs context-dependent, conflicting functions in tumor progression, immune regulation, and muscle homeostasis.

[0014] In the present invention, “immune-related adverse events (irAEs)” are unique side effects that occur during treatment with immune checkpoint inhibitors (ICIs), and are caused by the drug excessively activating immune responses not only against tumor cells but also against normal cells. Unlike the dose-dependent toxicity commonly seen in conventional cytotoxic anticancer drugs, this is characterized by being caused by an autoimmune mechanism.

[0015] irAEs can occur in almost any organ and tissue of the body, and the skin, gastrointestinal tract, liver, lungs, endocrine system, kidneys, musculoskeletal system, and nervous system are reported as major target organs. The most common symptoms include rash, itching, diarrhea, colitis, elevated liver enzymes, and thyroid dysfunction, but rarely, life-threatening severe adverse reactions such as pneumonia, myocarditis, neuritis, nephritis, pancreatitis, and adrenal insufficiency may also occur.

[0016] The onset varies from weeks to months after the start of treatment, and in some cases, it may occur with a delay even after the treatment has ended. Since early detection and appropriate management have a decisive impact on the prognosis, it is common practice to immediately discontinue treatment if an immune-related adverse reaction is suspected and to administer steroids or immunosuppressants depending on the severity of the disease. Furthermore, in some patients, irAEs may not fully recover even after immunomodulatory treatment, potentially leaving chronic sequelae such as endocrine dysfunction.

[0017] Skeletal muscle wasting is a major characteristic of cachexia and is caused by reduced protein synthesis, activation of proteasome or autophagy pathways, apoptosis, and unregulated mitophagy. Tumor-infiltrating immune cells also contribute to muscle wasting. Macrophages, neutrophils, and CD8+ T cells infiltrate the skeletal muscle of tumor mice to promote muscle loss; in particular, CD8+ T cells are reported to be involved in wasting associated with viral infections, although this phenomenon has been observed in adipose tissue rather than skeletal muscle. However, considering that ICI activates cytotoxic CD8+ T cells and that skeletal muscle can be a target of irAE, there is a possibility that ICI treatment may exacerbate cachexia in the form of muscle-associated irAE.

[0018] In this invention, it was confirmed that ICI treatment is associated with weight loss in cancer patients. In a tumor-bearing (TB) mouse model induced using lung cancer cells, anti-PD-L1 treatment effectively inhibited the growth of lung tumors but exacerbated muscle wasting by promoting the infiltration of cytotoxic CD8⁺ T cells into skeletal muscle. These results suggest that cachexia may manifest as a form of muscle-associated irAE induced by CD8+ T cells.

[0019] However, while depleting CD8+ T cells alleviates muscle wasting, there is a limitation in that it simultaneously inhibits the anticancer efficacy of ICI.

[0020] Therefore, the inventors sought to identify drug targets capable of alleviating cachexia and enhancing the tumor-suppressing effects of ICI. Through integrated transcriptome analysis, it was confirmed that cathepsin L (CTSL) is a dual target that enhances anticancer activity in response to anti-PD-L1 therapy while simultaneously alleviating cachexia. Furthermore, increased CTSL expression was correlated with poor prognosis in lung cancer patients, and its clinical relevance was proven by the significant elevation of CTSL expression in the skeletal muscles of cachexic patients.

[0021] Cachexia is a multifactorial syndrome resulting from complex interactions among various organs, including tumors, skeletal muscle, the liver, adipose tissue, and the central nervous system; therefore, understanding these inter-organ interactions is essential. Growth / differentiation factor-15 (GDF-15), one of the key regulators of this axis, is known to play multiple roles in cachexia. GDF-15 suppresses appetite via central nervous system pathways, promotes muscle wasting by inducing metabolic abnormalities, and simultaneously weakens anti-cancer immunity by preventing CD8+ T cells from infiltrating tumors. These mechanisms demonstrate that cachexia is a systemic disease in which the metabolic, immune, and nervous systems are closely intertwined.

[0022] The tumor-skeletal muscle axis is central to cancer cachexia. Tumors accelerate muscle wasting, and the presence or absence of cachexia is closely associated with poor clinical prognosis in cancer patients. In particular, skeletal muscle acts not merely as a passive target organ, but as an active endocrine organ that maintains systemic homeostasis by secreting myokines. Therefore, this bidirectional interaction between cancer and skeletal muscle suggests that muscle health must be considered when designing anticancer treatment strategies.

[0023] In this invention, the inventors observed that anti-PD-L1 therapy induces weight loss in cancer patients and exacerbates cachexia in a TB mouse model, despite its tumor-suppressing effects. Notably, cytotoxic CD8+ T cells accumulated in the skeletal muscle of TB mice treated with anti-PD-L1. This raises the possibility that muscle wasting may be an irAE induced by ICI. Supporting this, myositis and myasthenia gravis, conditions involving T-cell infiltration in the muscle, have been reported as irAEs in patients treated with ICI. Even in the absence of ICI, TB mice showed an increase in CD8+ T cells in the skeletal muscle, indicating that these cells may contribute to muscle wasting as part of a more widespread cachexia syndrome.

[0024] In other words, immune cells can be involved in the development and progression of cachexia. For example, macrophages can induce skeletal muscle wasting by interacting with pancreatic cancer cells and secreting TNF-like weak inducer of apoptosis (TWEAK). Additionally, macrophages, neutrophils, and CD8+ T cells can infiltrate the skeletal muscle of tumor-affected mice and contribute to muscle loss. This immune system acts as a key regulator coordinating inter-organ signaling by secreting cytokines and chemokines. Therefore, profiling immune cells is essential for understanding cachexia as an inter-organ syndrome. Indeed, results of single-cell RNA sequencing (scRNA-seq) performed simultaneously on tumor and skeletal muscle tissues revealed differences in immune cell composition and transcriptomic responses between the two tissues, suggesting the complexity of tissue-specific immune regulatory mechanisms.

[0025] As a result of the experiment of the present invention, T cells expressing cytotoxicity-related genes (e.g., Cd8a, Prf1, Nkg7, Gzma, and Grzmb) were dominant in skeletal muscle but not in cancer tissue. These T cells, verified by IHC and flow cytometry, were identified as CD49a+ CD8+ tissue-resident memory T cells.

[0026] This invention suggests that concomitant administration of anticachemic agents may be necessary when using ICIs to prevent CD8+ T cell-mediated muscle wasting. Transcriptome data obtained from tumors and muscles of TB mice were utilized to address the conflicting roles of CD8+ T cells in tumors and muscles. Recent advancements in omics technology are making significant contributions to understanding cachexia as a complex syndrome involving inter-organ interactions. For example, RNA-seq data analysis of 12 different cancer types identified 25 tumor-specific cachexia-inducing factors associated with patient survival and prognosis, while integrated transcriptome analysis of skeletal muscle identified muscle atrophy target genes, including DNA damage-inducing transcript 4 (DDIT4) and CCN1 / Cyr61. In this study, transcriptome data from lung tissue, lung tumors, and skeletal muscle of normal (CON) and lung cancer (TB) mice were compared with data obtained from the muscles of CD8+ T cell depletion group mice. This approach was able to identify CTSL, which can simultaneously inhibit cancer growth and cachexia while reversing side effects mediated by muscle-infiltrating CD8+ T cells.

[0027] CTSL is a cysteine ​​protease expressed in various tissues and is known to be overexpressed in human cancer tissues, contributing to extracellular matrix (ECM) remodeling and tumor progression. While CTSL promotes tumor formation, it induces skeletal muscle atrophy under systemic debilitating conditions such as fasting or denervation. Furthermore, CTSL plays an essential role in maintaining the cytotoxicity of immune cells. Genetic or pharmacological blockade of CTSL reduces the cytotoxic capacity of CD8+ T cells and NK cells by regulating the expression of cytotoxic genes such as perforin.

[0028] Along with clinical correlations (Fig. 6), the present invention suggests that CTSL inhibition is a potential therapeutic strategy capable of alleviating muscle wasting induced by CD8+ T cells while simultaneously enhancing the anticancer immunity of CD8+ T cells. Thus, CTSL blockade provides strong evidence supporting that it can prevent muscle-related irAEs while simultaneously enhancing the anticancer efficacy of ICIs.

[0029] In conclusion, cachexia can be suggested as one of the irAEs resulting from the abnormal accumulation of CD8+ T cells in skeletal muscle. CTSL can be a promising candidate for simultaneously controlling cancer and cachexia and can be used alone or in combination with ICI.

[0030]

[0031] In one embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer comprises a cathepsin L inhibitor as an active ingredient, wherein a subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredient.

[0032] In the present invention, the "intended individual" or "object" refers to an individual that has developed a disease or is highly likely to develop a disease, and may be a mammal including humans, and may be selected from the group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be a human, but is not limited thereto.

[0033] In this invention, “cathepsin L” is a cysteine ​​protease primarily found in lysosomes and is an enzyme that plays a crucial role in protein degradation. This enzyme regulates intracellular protein metabolism and is essential for the physiological function and survival of cells. Cathepsin L breaks down proteins into amino acids to supply nutrients required by cells and contributes to the removal of damaged proteins. Furthermore, cathepsin L is involved in various physiological processes and plays a particularly important role in immune responses. It contributes to the activation and regulation of immune cells to control inflammatory responses, supports defense mechanisms against infection, plays a significant role in the process of apoptosis, and contributes to maintaining normal cell growth and development. However, excessive activation of cathepsin L can be associated with various diseases. In this invention, increased expression of cathepsin L was observed in cancer cells.

[0034] In the present invention, a “cathepsin L inhibitor” is a substance that inhibits the activity of cathepsin L, a proteolytic enzyme, and plays an important role in various physiological and pathological processes. By inhibiting the activity of cathepsin L, an anticancer effect can be expected, which can contribute to inhibiting the growth and metastasis of cancer cells. Furthermore, cathepsin L inhibitors can help treat various inflammatory diseases by regulating inflammatory responses, and can exhibit a protective effect on nerve cells in neurodegenerative diseases. The cathepsin L inhibitor provided in the present invention is not particularly limited as long as it is a substance capable of inhibiting the expression of the cathepsin L protein or gene.

[0035] As used in the present invention, the term "pharmaceuticalally acceptable salt" refers to a salt prepared according to methods conventional in the art, and such methods are known to those skilled in the art. Specifically, the pharmaceutically acceptable salt includes, but is not limited to, salts derived from the following inorganic acids, organic acids, and bases that are pharmacologically or physiologically acceptable. Examples of suitable acids may include hydrochloric acid, bromic acid, hydrobromide, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include, but are not limited to, alkali metals, e.g., sodium or potassium, and alkaline earth metals, e.g., magnesium.

[0036] In another embodiment of the present invention, a pharmaceutical composition is provided in which the cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine. Any type of inhibitor capable of inhibiting cathepsin L is not limited, including various small molecules, antibodies, miRNAs, antisense nucleotides, etc.

[0037] In another embodiment of the present invention, the cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Provides a pharmaceutical composition containing N2O5.

[0038] In another embodiment of the present invention, a pharmaceutical composition is provided in which the Z-Phe-Tyr-CHO(C26H26N2O5) is represented by the following chemical formula 1:

[0039] [Chemical Formula 1]

[0040]

[0041] In another embodiment of the present invention, a pharmaceutical composition is provided in which the cancer is gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.

[0042] In one embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of cachexia comprises a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredients.

[0043] In the present invention, the term "cachexia" refers to a syndrome of severe systemic debilitation that appears in the terminal stages of diseases such as cancer, tuberculosis, diabetes, and AIDS. The main symptoms include a decrease in appetite and a decline in body weight and physical strength due to muscle and fat loss; the condition progresses to a state where weight decreases despite normal food intake. Cachexia can reduce the effectiveness of disease treatment and shorten the patient's life expectancy. Cancer is a disease in which abnormal cells grow uncontrollably; cancer cells divide more rapidly than normal cells and possess the characteristic of being able to infiltrate surrounding tissues or metastasize to distant sites. On the other hand, cachexia is a syndrome that appears in the terminal stages of various diseases such as cancer, tuberculosis, diabetes, and AIDS, and is caused by systemic inflammatory responses and metabolic changes. Cachexia is characterized by weight loss, decreased muscle mass, fatigue, and a general state of debilitation. Cachexia is more than just simple weight loss; it has a severe impact on the body's nutritional status and metabolism and can lower the patient's quality of life.

[0044] Cachexia is driven by various conditions such as altered energy balance, increased production of pro-cachexia cytokines and factors, and atrophy. The most well-known cachexia-inducing factors are myostatin, activin, GDF15 (growth differentiation factor 15), TWEAK (tumor necrosis factor-like weak inducer of apoptosis), and inflammatory cytokines such as interferon γ (IFNγ), tumor necrosis factor α (TNFα), interleukin 1α and β (IL-1α and IL-1β), and interleukin (IL)-6. These factors induce catabolism by inducing proteolysis through the activation of the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (ALS). Under physiological conditions, serine / threonine-protein kinase (AKT) phosphorylates FoxO3, leading to cytoplasmic localization. Under cachexia conditions, AKT activity is inhibited by the influence of inflammatory cytokines or reduced levels of insulin-like growth factor 1 (IGF1). Reduced AKT activity causes the nuclear translocation of FoxO3a protein following dephosphorylation, enabling the transcription of Murf-1 and Atrogin-1. The general convergence step regulating inflammatory cytokines is associated with nuclear factor kappa B (NF-κB), a common transcription factor that mediates cellular responses to various stimuli such as lipopolysaccharides, reactive oxygen species (ROS), and various cytokines. It has been demonstrated that TNF-α acts as an upstream element of a general pathway that upregulates inflammatory cytokines through NF-κB to produce catabolic cytokines.

[0045] In the present invention, the cachexia may include, for example, cancer cachexia, AIDS cachexia, chronic obstructive pulmonary disease cachexia, multiple sclerosis cachexia, or congestive heart failure cachexia, but is not limited thereto; specifically, it may be cancer cachexia.

[0046] In the present invention, the cancer that can cause cachexia is not limited in type and may include melanoma, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colorectal cancer (=colon cancer), rectal cancer, anal cancer, hepatocellular carcinoma, cholangiocarcinoma, gallbladder cancer, pancreatic cancer, lung cancer (non-small cell lung cancer, small cell lung cancer), thymic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, sarcoma, gastrointestinal stromal tumor, cancer of unknown primary site, mesothelioma, neuroendocrine tumor, skin cancer, blood cancer, etc., and more preferably may be a digestive organ cancer such as stomach cancer, colorectal cancer, rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, or lung cancer or breast cancer.

[0047] In another embodiment of the present invention, a pharmaceutical composition is provided in which the cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine. Any type of inhibitor capable of inhibiting cathepsin L is not limited, including various small molecules, antibodies, miRNAs, antisense nucleotides, etc.

[0048] In another embodiment of the present invention, the cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Provides a pharmaceutical composition containing N2O5.

[0049] In another embodiment of the present invention, a pharmaceutical composition is provided in which the Z-Phe-Tyr-CHO(C26H26N2O5) is represented by the following chemical formula 1:

[0050] [Chemical Formula 1]

[0051]

[0052] In another embodiment of the present invention, the pharmaceutical composition is provided for improving and treating one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

[0053] In one embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease comprises a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredients.

[0054] In another embodiment of the present invention, a pharmaceutical composition is provided in which the cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine. Any type of inhibitor capable of inhibiting cathepsin L is not limited, including various small molecules, antibodies, miRNAs, antisense nucleotides, etc.

[0055] In another embodiment of the present invention, the cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Provides a pharmaceutical composition containing N2O5.

[0056] In another embodiment of the present invention, a pharmaceutical composition is provided in which the Z-Phe-Tyr-CHO(C26H26N2O5) is represented by the following chemical formula 1:

[0057] [Chemical Formula 1]

[0058]

[0059] In another embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein the muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, muscle loss, and sarcopenia.

[0060] The "muscle disease" of the present invention may be one or more muscle diseases selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, and senile sarcopenia, but is not limited thereto. Specifically, it may be a disease such as senile muscle atrophy, muscle disease caused by cancer and chronic disease, or muscle atrophy caused by muscle disuse. More specifically, it may include senile muscle atrophy or muscular atrophy caused by cancer, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia, senile sarcopenia, and muscle loss. In particular, in the present invention, the muscle disease may be caused by cancer or a chronic disease.

[0061] In another embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein the muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused or damaged muscle.

[0062] Muscle disorders, particularly muscle loss, are one of the most significant characteristics of cachexia. This is known to be caused by increased protein catabolism and decreased protein synthesis resulting from the excessive activity of various cytokines. Cachexia involves symptoms that include muscle loss (sarcopenia), so there is significant overlap between the two. While the majority of patients with cachexia also have muscle loss (sarcopenia), not all patients exhibiting muscle loss display symptoms of cachexia. Clinically speaking, muscle loss (sarcopenia) can be described as a prodromal symptom of cachexia. Among the causes of weight loss in surgical patients, sarcopenia commonly occurs in elderly patients, whereas cachexia shows a higher disease association. In sarcopenia, basal metabolic rate decreases because fat increases while muscle decreases; however, in cachexia, both muscle and fat decrease, yet basal metabolic rate is often observed to increase.

[0063] In another embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein the muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused muscle, or muscle damage.

[0064] In another embodiment of the present invention, a pharmaceutical composition is provided in which the immunotherapeutic agent is one or more selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0065] The pharmaceutical composition of the present invention can be prepared by methods commonly used in the art to which the present invention belongs. The pharmaceutical composition of the present invention can be prepared as an oral or parenteral formulation, preferably as an injectable formulation which is a parenteral formulation, and can be administered via the dermal, intramuscular, peritoneal, intravenous, subcutaneous, nasal, or epidural routes.

[0066] The pharmaceutical composition of the present invention may be administered to an individual in an immunologically effective amount. The “immunologically effective amount” refers to a sufficient amount to produce a preventive effect against tuberculosis and an amount that does not cause side effects or severe or excessive immune responses. The precise dosage concentration varies depending on the specific immunogen to be administered and can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the age, weight, health, gender, sensitivity of the individual to drugs, route of administration, and method of administration of the vaccinated person, and may be administered one to several times.

[0067] In addition, the composition provided in the present invention may be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0068] The "prevention" of the present invention may include, without limitation, any act that can block, suppress, or delay symptoms caused by cancer, cachexia, or muscle disease using the composition of the present invention.

[0069] The "treatment" and "improvement" of the present invention may include, without limitation, any act that enables the improvement or benefit of symptoms caused by acid-fast bacteria, particularly non-tuberculous acid-fast bacteria, by using the composition of the present invention.

[0070] In the present invention, the pharmaceutical composition may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is intended for humans.

[0071] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, it may include bases, excipients, lubricants, preservatives, etc. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0072] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0073] The routes of administration of the pharmaceutical composition according to the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0074] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0075] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0076] A food composition containing the composition of the present invention as an active ingredient can be manufactured in the form of various food products, such as beverages, chewing gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts that have almost no toxicity or side effects, it can be used safely even when taken for a long period for preventive purposes.

[0077] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0078] Here, when the above food composition is prepared in the form of a beverage, there are no special limitations other than containing the above food composition in the indicated proportions, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in ordinary beverages. That is, as natural carbohydrates, it may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the above flavoring agents include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0079] In addition, the food composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0080] These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0081]

[0082] In the first embodiment of the present invention, a pharmaceutical composition is provided for treating or preventing drug side effects caused by the administration of an anticancer drug comprising a cathepsin L inhibitor as an active ingredient.

[0083] The diseases and side effects that may occur due to the administration of anticancer drugs are highly diverse and vary depending on the type of drug, dosage, patient age, and health status. Generally, because anticancer drugs attack rapidly dividing cells, they affect normal cells as well as cancer cells, potentially causing various diseases. For example, anemia, leukopenia (neutropenia), and thrombocytopenia may occur due to bone marrow suppression, which can lead to infections, sepsis, and a tendency to bleed. Additionally, nausea, vomiting, diarrhea, stomatitis, and esophagitis resulting from damage to the gastrointestinal mucosa are commonly reported. Hepatotoxicity or nephrotoxicity may occur, potentially leading to diseases such as drug-induced hepatitis or acute renal failure. Cardiotoxic anticancer drugs (e.g., doxorubicin) can cause cardiomyopathy and heart failure, while pulmonary toxic drugs (e.g., bleomycin) can cause interstitial pneumonia or pulmonary fibrosis. Some drugs may also induce neurotoxicity, resulting in peripheral neuropathy, paresthesia, and motor disorders. Changes in the skin and nails, hair loss, and photosensitivity are also relatively common side effects. In the case of drugs that affect the immune system, autoimmune diseases (e.g., thyroiditis, adrenal insufficiency, skin rashes, colitis, etc.) may occur, and hormonal abnormalities may lead to amenorrhea, infertility, and osteoporosis. Furthermore, long-term use of anticancer drugs carries the potential to induce secondary malignancies (e.g., leukemia).

[0084] In a second embodiment of the present invention, a pharmaceutical composition is provided in which the anticancer agent is an immune checkpoint inhibitor (ICI).

[0085] In the third embodiment of the present invention, a pharmaceutical composition is provided in which, in any one of the first and second embodiments, the immune checkpoint inhibitor is one or more selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.

[0086] In the fourth embodiment of the present invention, a pharmaceutical composition is provided in which, in any one of the first to third embodiments, the drug side effect resulting from the administration of the anticancer agent is one or more diseases selected from the group consisting of infectious diseases, anemia, hemorrhagic diseases, liver and kidney diseases, heart diseases, lung diseases, neurological diseases, gastrointestinal diseases, autoimmune diseases, endocrine disorders, and secondary cancers.

[0087] In the fifth embodiment of the present invention, a pharmaceutical composition is provided in which, in any one of the first to fourth embodiments, the cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine.

[0088] In the sixth embodiment of the present invention, in any one of the first to fifth embodiments, the cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Provides a pharmaceutical composition containing N2O5.

[0089] In the seventh embodiment of the present invention, in any one of the first to sixth embodiments, the Z-Phe-Tyr-CHO(C 26 H 26 The present invention provides a pharmaceutical composition in which N2O5) is represented by the following chemical formula 1.

[0090] [Chemical Formula 1]

[0091]

[0092] In the eighth embodiment of the present invention, a pharmaceutical composition for cancer prevention or treatment comprising a cathepsin L inhibitor as an active ingredient is provided, wherein a subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredient.

[0093] In the ninth embodiment of the present invention, a pharmaceutical composition is provided in which, in the eighth embodiment, the cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine.

[0094] In the 10th embodiment of the present invention, in any one of the 8th to 9th embodiments, the cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Provides a pharmaceutical composition containing N2O5.

[0095] In the 11th embodiment of the present invention, in any one of the 8th to 10th embodiments, the Z-Phe-Tyr-CHO(C 26 H 26 The present invention provides a pharmaceutical composition in which N2O5) is represented by the following chemical formula 1:

[0096] [Chemical Formula 1]

[0097]

[0098] In the 12th embodiment of the present invention, a pharmaceutical composition is provided in which, in any one of the 8th to 11th embodiments, the cancer is gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.

[0099] In the 13th embodiment of the present invention, a pharmaceutical composition for treating or preventing cachexia caused by the administration of an anticancer drug comprising a cathepsin L inhibitor as an active ingredient is provided, in any one of the 8th to 12th embodiments.

[0100] In the 14th embodiment of the present invention, in any one of the 8th to 13th embodiments, the pharmaceutical composition is provided to improve and treat one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

[0101] In the 15th embodiment of the present invention, in any one of the 8th to 14th embodiments, the pharmaceutical composition is provided to improve and treat one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

[0102] In the 16th embodiment of the present invention, a pharmaceutical composition for treating or preventing muscle disease caused by the administration of an anticancer drug is provided, comprising a cathepsin L inhibitor as an active ingredient.

[0103] In the 17th embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein, in the 16th embodiment, the muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, muscle loss, and sarcopenia.

[0104] In the 18th embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein in any one of the 16th to 18th embodiments, the muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused muscle, or muscle damage.

[0105] In the 19th embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of cachexia comprises a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredients.

[0106] In the 20th embodiment of the present invention, the pharmaceutical composition is provided such that, in the 19th embodiment, the pharmaceutical composition improves and treats one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

[0107] In the 21st embodiment of the present invention, in any one of the 19th to 20th embodiments, the pharmaceutical composition is provided to improve and treat one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

[0108] In the 22nd embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease comprises a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredients.

[0109] In the 23rd embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein, in the 22nd embodiment, the muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, muscle loss, and sarcopenia.

[0110] In the 24th embodiment of the present invention, a pharmaceutical composition for improving or treating a muscle disease is provided, wherein in any one of the 22nd to 23rd embodiments, the muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused muscle, or muscle damage.

[0111]

[0112] It has been confirmed that cathepsin L (CTSL) inhibition not only alleviates muscle wasting but also enhances the anticancer efficacy of immune checkpoint inhibitor therapy. Therefore, when cathepsin L inhibitors are administered in combination with immune checkpoint inhibitors, muscle-related irAEs can be prevented or treated, and in particular, weight and muscle tissue loss resulting from the administration of immune checkpoint inhibitors can be prevented or improved.

[0113]

[0114] Figure 1a shows the tumor volume and body weight excluding tumor (BW) 13 weeks after cancer administration in CT26 mice.

[0115] Fig. 1b shows Kras in male C57BL / 6 mice IKKαLCells were injected intratracheally (it), and the groups were divided into an IgG antibody or anti-PD-L1 treatment group, a group administered anti-PD-L1 and anti-CD8a in combination, and an anti-CD8 antibody treatment group, and administered intraperitoneally (ip) three times a week. In any group, the animals were sacrificed when a weight loss of 10% or more compared to IBW was reached, and a schematic diagram of this is shown.

[0116] Figures 1c to 1f show lung size (Figure 1c), lung cancer area (Figure 1d), body weight change (Figure 1e), and gastrocnemius (GA) and tibial (TA) muscle weights in TB versus CON mice (Figure 1f), respectively.

[0117] Figures 1g and 1h show representative H&E staining results of TA muscle (Figure 1g). The average muscle cross-sectional area CSA per mouse (Figure 1h) is shown.

[0118] Figure 1i shows the rate of CD3+ CD8+ cells within CD45+ infiltrating tumors and GA muscle through flow cytometry analysis.

[0119] Figure 1j shows the results of the J principal component analysis (PCA) of the TA muscle transcriptome.

[0120] Figures 1k to 1m are the top five gene ontologies (GOs) for differentially expressed genes (DEGs) in muscle: IgG vs CON (K); αPD-L1 vs IgG (L); αCD8a vs IgG (M).

[0121] Figure 1n shows the WB analysis results of MuRF-1, LC3B-I / II, and Vinculin proteins in GA muscle.

[0122] Figure 10 shows the results of measuring grip strength before mouse sacrifice.

[0123] Figure 1p is Kras IKKαLThis is the result of measuring the diameter of the tube after co-culturing CD8+ T cells derived from the spleen of CON or TB mice in cell condition medium (CM).

[0124] Figure 1q shows the WB analysis results of C2C12 myotube protein after co-culture.

[0125] Figure 2a shows the results of scRNA-seq analysis using CD45+ cells (isolated from three animals) isolated from lung tumors or GA muscles of TB mice.

[0126] Figures 2b and 2c show the relative proportions of each CD45+ immune cluster in a lung tumor (Figure 2b) and GA muscle (Figure 2c), respectively.

[0127] Figure 2d shows the results of UMAP expression for selected T cell-associated and cytotoxic genes in CD45+ cells isolated from tumor or muscle tissue.

[0128] Figures 2e and 2f are the top 5 GO / Kyoto Encyclopedia of Genes and Genomes (KEGG) results for genes upregulated in classical T cells (Figure 2e) and classical T cells (Figure 2f), respectively, relative to CD49a+CD8+ T cells. Figures 2g and 2h are the ratios (n = 6) of CD49a+CD8+ T cells (Figure 2g, CD45+CD3+CD49a+) and CD8+ T cells (Figure 2h, CD45+CD3+CD8+) within CD45+ infiltrating tumor and GA muscle tissue via flow cytometry.

[0129] Figure 2i shows the qRT-PCR results of Itga1, Prf1, Gzma, and Gzmb in CD8+ T cells co-cultured with C2C12 myotubes in the presence or absence of CM as described in Figure 1q.

[0130] Figure 3a is a Venn diagram of genes upregulated in muscle (TA, GA) and lung tumors in TB mice and CON mice, and genes downregulated in TA in the αCD8a group and IgG group.

[0131] Figure 3b is the result of the PPI network analysis of the 55 commonly changed DEGs in Figure 3a.

[0132] Figure 3c is a list of genes altered in various cancer cachexia mouse models using a public database.

[0133] Figure 3d shows representative WB results for tumors and GA muscle. These results were obtained using Gapdh (lung tissue) and vinculin (muscle tissue) as loading controls.

[0134] Figures 3e to 3g show the changes in gene expression neighboring Ctsl as a result of PPI network analysis of lung tissue (Figure 3e) and muscle tissue (Figures 3f, g), respectively.

[0135] Figure 3h shows the correlation coefficients between the MMP7, MMP13, and CTSL genes in the lungs of TB mice and CON mice (refer to the results in Figure 3e).

[0136] Figure 3i shows the top 5 KEGG paths for upregulated Ctsl neighbors in the PPI network analysis (see results in Figure 3f).

[0137] Figure 3j shows the top 5 KEGG paths for downregulated Ctsl neighbors in the PPI network analysis (see results in Figure 3g).

[0138] Figure 3k is a Kaplan-Meier plot of TCGA LUAD patients classified according to CTSL expression levels (high, red; low, black).

[0139] Figures 3l and 3m show the results of the analysis of the correlation between CTSL and CD274 expression in TCAG LUAD (L, n = 541; log2(TPM+1)) and GSE283829 NSCLC (M, n = 27; log2(read count+1)).

[0140] Figure 3n shows the results of CTSL mRNA expression in skeletal muscle of NSCLC cachexia patients (CAC) and healthy control groups (CON).

[0141] Figures 4a to 4d show the lung size (Fig. 4a), lung cancer area (Fig. 4b), BW change (Fig. 4c), and GA and TA muscle weight (Fig. 4d), respectively, of a mouse.

[0142] Figure 4e is a representative image of the H&E and Masson trichrome staining results using TA muscle.

[0143] Figure 4f shows the results of muscle CSA quantification.

[0144] Figure 4g shows the results of measuring grip strength before mouse sacrifice.

[0145] Figure 4h shows the results of measuring the concentration of CTSL using tumor and TA muscle tissue.

[0146] Figures 4i and 4j show the percentage of CD45+ designated immune subsets identified by flow cytometry using tumors (Figure 4i) and GA muscle (Figure 4j).

[0147] Figure 4k is the correlation between CTSL and HLA-B expression in human skeletal muscle.

[0148] Figure 41 shows the correlation between Ctsl and H2-D1 expression in lung tumors and muscle.

[0149] Figure 4m shows the results of MHC-I protein expression analysis using WB performed on GA muscle.

[0150] Figure 4n is the result of a WB analysis confirming MHC-I expression in C2C12 myotubes after injecting the corresponding siRNA into the cell to suppress Ctsl expression using CTSL si-RNA.

[0151] Figure 4o shows the results of measuring the c2C12 tube diameter after injecting CTSL siRNA into the cell and co-culturing it with TB mouse spleen CD8+ T cells under CM medium conditions.

[0152] Figure 5a is a scatter plot of genes ranked according to Ctsl expression and Pearson correlation in lung tumors and skeletal muscle (TB mouse vs. CON mouse).

[0153] Figure 5b is a comparative ranking of the correlation coefficients between lung tumors and muscles for neighbors of Ctsl in the PPI network (see Figure 3e).

[0154] Figure 5c shows the correlation between Ctsl and Bnip3 expression in lung tumors and muscle (log2TPM+1).

[0155] Figure 5d is a three-way Venn diagram of neighbors of Ctsl that are upregulated in lung tumors, upregulated in muscle (TB mouse vs. CON mouse), and downregulated in muscle (αCD8a mouse vs. IgG mouse).

[0156] Figure 5e is a subnetwork of Ctsl-Bnip3 neighbors upregulated in the tumor.

[0157] Figure 5f is a bar graph of the densely packed KEGG pathway between genes.

[0158] Figure 5g is a sub-network of upregulated Ctsl-Bnip3 neighbors and downregulated Ctsl-Bnip3 neighbors in the muscle.

[0159] Figure 5h is a bar graph of the KEGG pathway densely packed among genes.

[0160] Figure 6a shows the human homolog network of CTSL-BNIP3 adjacent genes in TCGA LUAD.

[0161] Figure 6b shows the results of immunofluorescence analysis of CTSL (green) and BNIP3 (red) using LUAD tissue microarray: normal adjacent, n = 22; tumor core, n = 128. The bar graph shows the ratio of BNIP3-high / -low within CTSL-high and CTSL-low tumors.

[0162] Figure 6c shows the Kaplan-Meier overall survival rate of TCGA LUAD classified by summing CTSL and BNIP3 expression (CTSL-BNIP3-high, red; CTSL-BNIP3-low, black).

[0163] Figure 6d is a GSEA plot of ALONSO_METASTASIS_UP for the CTSL-BNIP3 high and low groups in TCGA LUAD (left) and TB mice (right).

[0164] Figure 6e shows representative WBs of Ctsl, N-Cad, Gapdh, and Bnip3 proteins in TB mouse muscle.

[0165] Figure 6f is the WB of the indicated protein in A549 cells treated with or not treated with CTSL inhibitor.

[0166] Figures 6g and 6h show the results of migration (Figure 6g) and infiltration (Figure 6h) analysis for A549 cells treated with or not treated with CTSL inhibitor, respectively. The bar graphs represent the quantified migration % (Figure 6g) and infiltrated cells (Figure 6h).

[0167] Figures 6i and 6j are the WB of the indicated proteins after injecting si-CTSL (Figure 6i) or si-BNIP3 (Figure 6j) into A549 cells, respectively.

[0168] Figures 6k and 6l are images (left) showing the migration of A549 cells after injecting si-CTSL (Figure 6k) or si-BNIP3 (Figure 6l) into the cells, and a graph (right) quantifying the migration percentage, respectively.

[0169] Figure 7a shows the human homolog network of adjacent genes to CTSL-BNIP3.

[0170] Figure 7b is the GSEA for the DEG of the GTEx muscle.

[0171] Figure 7c is a representative GSEA plot for WP_PROTEASOME_DEGRADATION (left) and WP_ELECTRON_TRANSPORT_CHAIN_OXPHOS_SYSTEM_IN_MITOCHONDRIA (right) in muscles with high and low CTSL-BNIP3.

[0172] Figure 7d shows representative WBs of Ctsl, Bnip3, and vinculin proteins in the muscle of TB mice.

[0173] Figure 7e shows the WB and quantitative analysis results of Bnip3 in C2C12 myotubes 24 hours after injecting si-Ctsl or si-Cont into the cells.

[0174] Figure 7f shows the C2C12 tube diameter after 72 hours of co-culture with CD8+ T cells in CM following injection into cells with si-Bnip3 or si-Cont for 24 hours.

[0175] Figures 7g to 7i show the relative mtDNA copy numbers in GA muscle of TB mice (Fig. 7g, n = 8); and the results of comparing C2C12 myotubes co-cultured with CD8+ T cells treated with CTSL inhibitors under conditions including or excluding CM (Figs. 7h, 7i).

[0176] Figures 7j and 7k show the C2C12 tube diameter (Figure 7j) and mtDNA copy number (Figure 7k) after co-culturing with CD8+ T cells treated with mouse recombinant Gzma (10 ng / ml) under conditions with or without CM.

[0177]

[0178] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0179]

[0180] [Experimental Method]

[0181] 1. Ethical Statement

[0182] This study was approved by the Institutional Review Boards of Yonsei University Dental Hospital (IRB# 2-2024-0048) and Pusan ​​National University Hospital (PNUH) (IRB# 2306-016-128). Written informed consent for the use of study data was obtained from all participants, and the Declaration of Helsinki was complied with. All mouse procedures in this study were approved by the Yonsei University Institutional Animal Care and Use Committee (IACUC; protocol# 2023-0074) and were conducted in accordance with international guidelines.

[0183]

[0184] 2. Analysis using patient data

[0185] Electronic health records (EHRs) were collected from two tertiary hospitals in Korea: Yonsei University Severance Hospital and PNUH. Patients diagnosed with various cancers who received αPD-1 (pembrolizumab, nivolumab), αPD-L1 (atezolizumab, duvalumab), or other anticancer agents (gemcitabine, docetaxel, doxorubicin, pemetrexed, bevacizumab, trastuzumab) were included. The index date was defined as the first day of drug administration. Pre-treatment weight was measured 30 days prior to the index date, and post-treatment weight was defined as the lowest weight recorded within 18 months after the index date. Differences in pre- and post-treatment weight were analyzed using a paired t-test with a statistical significance of p < 0.05, and recruitment criteria based on gender were not applied.

[0186]

[0187] 3. Establishment of a mouse model using orthotopic tumor transplantation

[0188] The lung tumor-bearing (TB) mouse model is Kras IKKαL It was established using cells. Cells were maintained in RPMI1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% antibiotic-antimicrobial mixture (Gibco), and 100 mg / L normosin (InvivoGen, San Diego, CA). Seven-week-old male C57BL / 6 mice were randomly assigned to treatment groups to ensure comparable mean body weights. Each mouse was placed in 50 μL phosphate-buffered saline (PBS) at a rate of 5 x 10⁶ 6 Kras IKKαL Cells were administered intratracheally (it), and body weight and food intake were monitored weekly. In the case of the LLC model using Lewis Lung Carcinom cells, Kras IKKαL Just like a cell, 5 x 10 6LLC cells were injected into the trachea through the mouse airway. Mice were sacrificed when their initial body weight (IBW) decreased by 10% according to human evaluation criteria approved by the IACUC and standard cachexia thresholds in rat studies.

[0189]

[0190] 4. αPD-L1 and αCD8a treatment in TB mice

[0191] Three days after tumor cell injection, mice were administered 10 mg / kg of the anti-mouse PD-L1 antibody (αPD-L1, clone 10F.9G2™, BE0101, BioXCell, Lebanon, NC, USA) via intraperitoneal injection three times a week. At this time, some groups were co-administered with 10 mg / kg of the anti-mouse CD8 antibody (αCD8a, clone 2.43, BE0061, BioXCell), while the remaining groups were administered either the αPD-L1 or αCD8a antibody alone. The control group was administered 10 mg / kg of isotype IgG (mouse IgG2b, clone LTF-2, BE0090, BioXCell).

[0192]

[0193] 5. αPD-L1 and CTSLi combination therapy in TB mice

[0194] From the third day after tumor cell injection, mice were treated as follows.

[0195] The group was divided into two groups: one receiving the combination of the cathepsin L (CTSL) inhibitor Z-Phe-Tyr-CHO (23249, Cayman, Ann Arbor, MI, USA) 0.5 mg / kg and αPD-L1 10 mg / kg, and the other receiving each drug alone; the drugs were administered intraperitoneally three times a week. The control group received an equal amount of isotype IgG.

[0196]

[0197] 6. Combination therapy with αPD-L1 and TNF blockade in TB mice

[0198] After the injection of tumor cells, mice were treated as follows starting from the 3rd day.

[0199] The subjects were divided into a group administered 6.2 mg / kg of anti-mouse TNF-α antibody (αTNF-α, clone XT3.11, BE0058, BioXCell) and 10 mg / kg of αPD-L1 in combination, and a group administered each drug alone, and administered intraperitoneally three times a week. The control group was administered an equal amount of isotype IgG.

[0200]

[0201] 7. Grip Strength Test

[0202] Forelimb grip strength was evaluated using a digital grip strength meter (JD-A-22, Jeungdo Bio & Plant, Seoul, Korea). Each mouse was gently placed on a mesh bar to allow it to grasp the bar with its forelimbs. Once the grip was secured, the tail was gently pulled back from the horizontal plane to release the bar. Maximum force (in Newtons N) was recorded in meters. The average of 10 attempts per mouse was calculated as absolute grip strength. The Newton (N) values ​​were converted to grams (1 N = 101.97 g) and then normalized to the IBW (g) of each animal.

[0203]

[0204] 8. Histology, measurement of lung cancer area and muscle cross-sectional area

[0205] Tissue samples were collected, fixed in 10% neutral buffered formalin (NBF) for 24 hours, and dehydrated (70% ethanol: muscle / lung, 20% sucrose: WAT). Paraffin-containing tissues (muscle, lung) or frozen-sectioned tissues (adipose tissue) were analyzed at the Avison Biomedical Research Center (ABMRC) at Yonsei University. Subsequently, hematoxylin and eosin (H&E) or Masson's trichrome staining was performed for morphological analysis and evaluation of collagen fibrosis. Additionally, to perform immunohistochemistry (IHC), samples were incubated overnight at 4°C using a primary antibody (1:100), and the amount of the target protein was determined after incubation with a secondary antibody at room temperature for 1 hour the following day. The antibodies are listed in Table 1. For IHC quantification, positively stained cells were counted in three randomly selected fields per section of each mouse using a microscope (Leica DMI1, Leica Biosystems, Wetzlar, Germany). To evaluate lung cancer area, ImageJ software (National Institutes of Health, Bethesda, USA) was used to express the lung area occupied by the tumor as a percentage of the total lung area of ​​the H&E-stained sections. To measure muscle cross-sectional area (CSA), H&E-stained muscle sections were measured using ImageJ software. The average CSA of fibers per field (three fields per section of each mouse) was analyzed, and the average CSA for each mouse was calculated.

[0206]

[0207] WB, IHC, IF 항체 목록AntibodyCat#VendorAtrogin-1sc-166806Santa Cruz Biotechnology (Dallas, TX, USA)MuRF-1sc-398608Santa Cruz BiotechnologyCTSLsc-390385Santa Cruz BiotechnologyMHC-I (for tissues)sc-59199Santa Cruz BiotechnologyN-cadherinsc-59987Santa Cruz BiotechnologyGAPDH97166Cell Signaling Technology (Danvers, MA, USA)Vinculin13901Cell Signaling TechnologyMHC-I (for cell lines)36923Cell Signaling TechnologyBnip3 (for mouse)3769Cell Signaling TechnologyBNIP3 (for human)44060Cell Signaling TechnologyLC3B2775Cell Signaling TechnologyAnti-mouse IgG7076Cell Signaling TechnologyAnti-rabbit IgG7074Cell Signaling TechnologyFoxp3ab215206Abcam (Cambridge, UK)CD8a14-0081-82Invitrogen (Waltham, MA, USA)Anti-rat IgG31470Invitrogen

[0208] 9. 인간 LUAD 조직 마이크로어레이 면역형광 염색

[0209] The human LUAD tissue microarray (LC1504) was purchased from US Biomax, Inc. (Rockville, MD, USA). Tissue sections were inoculated with primary antibodies against CTSL (1:100) and BNIP3 (1:100) and incubated overnight at 4°C. The following day, after washing, the sections were stained with fluorescently conjugated secondary antibodies at room temperature for 1 hour. The antibodies are listed in Table 1. Finally, the sections were stained with diamidino-2-phenylindole (DAPI) (P36962, Invitrogen), which can stain the nucleus, and then mounted.

[0210] Fluorescence images were acquired using a ZEISS LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany). For quantitative analysis, the mean fluorescence intensity (MFI) of each marker was measured in all 150 tissue cores (22 adjacent normal lung tissues; 128 LUAD malignant and tumor-adjacent lung tissues, corresponding to 50 cases) using ZEISS ZEN 3.11 software (Carl Zeiss). The overall mean MFI for all cores was 5.50549 for CTSL and 15.1949 for BNIP3. Cores were classified based on MFI: CTSL MFI³ 5.50549 was classified as CTSL-high, CTSL MFI < 5.50549 as CTSL-low, BNIP3 MFI³ 15.1949 as BNIP3-high, and BNIP3 MFI < 15.1949 as BNIP3-low. Then, the proportions of BNIP3-high and BNIP3-low cores within the CTSL-high and CTSL-low groups were calculated and displayed as a bar graph. Fisher's exact test was used to evaluate the statistical association between CTSL and BNIP3 expression levels.

[0211]

[0212] 10. Separation of tissue-infiltrating CD45+ cells

[0213] After finely chopping the isolated tissue, it was enzymatically digested in RPMI1640 medium containing 0.3 mg / ml Liberase TL (5401119001, Sigma-Aldrich, St. Louis, MO) and 0.4 μg / ml Deoxyribonuclease I (DN25, Sigma-Aldrich) by gentle stirring at 37°C for 2 hours. After enzymatic digestion, the cells were lysed into single cells using 18G and 21G syringes (Korea Vaccine Co., Ltd., Seoul, Korea) and passed through a 100 μm cell filter (9310, SPL Life Sciences, Pocheon, Korea) to obtain a final single-cell suspension. Red blood cells were removed using ammonium-chloride-potassium (ACK) lysis buffer (A10492-01, Gibco), and CD45+ immune cells were selected as positive using magnetic beads (130-052-301, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Subsequently, flow cytometry or scRNA-seq was performed using the isolated CD45+ cells.

[0214]

[0215] 11. Flow cytometry

[0216] CD45+ cells were counted using the LUNA-II™ The Ultimate Brightfield Cell Counter (Logos Biosystems, Anyang, Korea), and a total of 1 x 10⁶ 6 (muscle) or 5 x 10 7CD45+ cells from the lung were stained with fluorescent conjugated antibodies. The following antibodies (listed in Table 2) were used: for lymphocytes, CD45 (1:100), CD3 ε (1:100), CD8α (1:200), CD49a (1:100), NK1.1 (1:100), CD4 (1:100), Foxp3 (1:100); for bone marrow-derived suppressor cells (MDSCs), CD45 (1:100), CD11b (1:200), Ly6G (1:100); all purchased from BioLegend (San Diego, California, USA). After surface staining, Foxp3 was detected by intracellular staining using the Foxp3 / Transcription Factor Staining Buffer Set (00-5521-00, San Diego, California, eBioscience) according to the manufacturer's instructions. Data were measured using the BD FACS Symphony A5 (Franklin Lakes, New Jersey, USA) and analyzed with FlowJo™ Software (BD Biosciences).

[0217]

[0218] List of antibodies for flow cytometry analysis.AntibodyClone #FluorophoreCat#VendorCD45S18009FPerCP-Cyanine5.5157208BioLegendCD3ε17A2APC Cy7100222BioLegendCD8aS18018EPE162304BioLegendCD49aHMα1APC142606BioLegendCD4GK1.5Alexa Fluor 700100430BioLegendFoxp3MF-14Alexa Fluor 488126406BioLegendCD11bM1 / 70APC-Cy7101226BioLegendLy6G1A8Alexa Fluor 700127622BioLegend

[0219] 12. Western Block

[0220] The cells or tissues used in the experiment were lysed on ice for 1 hour using RIPA buffer (9806, Cell Signaling Technology, Danvers, MA, USA) containing 1 mM phenylmethylsulfonylfluoride (PMSF, PS2064, Gabiochem, Norcross, GA, USA) and a protease inhibitor cocktail (11836170001, Roche, Basel, Switzerland). The concentration of the protein obtained by centrifuging the lysate at 15,000 xg for 15 minutes was measured. Proteins (30-50 µg) were mixed with protein sample buffer (1610737, Bio-Rad Laboratories, Hercules, CA, USA) and heated at 95 °C for 5 minutes, after which they were separated by 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA, USA), blocked with 5% skim milk powder, treated with the primary antibody, and incubated overnight at 4°C. The following day, the secondary antibody was applied, and proteins were detected using a chemiluminescent reagent (Biomax, Seoul, Korea) and visualized using an image analyzer (Amersham, GE healthcare, Little Chalfont, UK). The antibodies are presented in Table 1. Band intensity was quantified using ImageJ software.

[0221]

[0222] 13. Enzyme Immunoassay for CTSL

[0223] CTSL concentrations in tissue lysates were measured using the Mouse Cathepsin L-ELISA Kit (NBP2-89172, Novus Biologicals, Centennial, CO, USA) according to the manufacturer's protocol. Tissue lysates were placed on antibody-coated plates and incubated at 37°C for 2 hours. Subsequently, the plates were washed with a wash solution and incubated with a biotinylation detection antibody at 37°C for 1 hour. After washing again, HRP-conjugated streptavidin was added at 37°C for 30 minutes. After the final wash step, the substrate solution was added to each plate and incubated for 15 minutes. Absorbance was measured at a wavelength of 450 nm using a microplate reader. CTSL concentrations were determined using a standard curve and normalized to the initial tissue mass.

[0224]

[0225] 14. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

[0226] RNA was extracted from tissues and cells using the TRIzol reagent (15596026; Invitrogen). cDNA was synthesized using a cDNA synthesis kit (25081, iNtRON Biotechnology, Seongnam, Korea). qRT-PCR was performed to confirm target gene expression after amplification using Power SYBR™ Green PCR Master Mix (4367659, Applied Biosystems, Waltham, MA) on a QuantStudio3 Real-time PCR System (Applied Biosystems) according to the manufacturer's protocol. Gene expression levels were normalized to the housekeeping gene Gapdh or Vinculin. Data analysis was performed using the ΔΔ changesCT method, and results were expressed as fold-changes in mRNA expression compared to control samples.

[0227]

[0228] 15. DNA Extraction and mtDNA Copy Number Measurement

[0229] Genomic DNA was extracted from cells or tissues using the AccuPrep Genomic DNA Extraction Kit (Bioneer) according to the manufacturer's instructions. To quantify the mtDNA copy number, qRT-PCR was performed using primers specific to mtDNA (Cytochrome c oxidase subunit 2, Cox2). As a reference, the ribosomal protein S18 (Rsp18) gene was amplified using the primers listed in Table 3. The amount of mtDNA was adjusted to the amount of nuclear DNA.

[0230]

[0231] 16. In vitro co-culture of C2C12 myoducts and spleen-derived T cells

[0232] C2C12 myoblasts (4 × 10 4 Cells (CRL-1772, ATCC, Manassas, Virginia, USA) were plated in 6-well plates and cultured in DMEM medium (Gibco) containing 10% FBS and a 1% antibiotic-antifungal mixture at 37°C and 5% CO2. To induce myotube differentiation, cells were cultured in differentiation medium (DM, DMEM medium containing 2% horse serum) for 5 days to promote the formation of multinucleated myotubes. After differentiation, C2C12 myotubes were subjected to Kras to stimulate muscle wasting conditions. IKKKαLCells were cultured with or without cell-derived conditioned media (CM, diluted 1:4 in DM). CD8+ T cells were isolated from the spleen and lymph nodes of CON or TB mice. After tissue degradation and erythrocyte removal, CD8+ T cells were selected as positive using the EasySep Mouse CD8α Positive Selection Kit (18953, STEMCEL Technologies, Vancouver, Canada) according to the manufacturer's protocol. For co-culture with CD4+ T cells, CD4+ T cells were selected as positive using the EasySep Mouse CD4+ T Cell Isolation Kit (19852, STEMCEL Technologies). Isolated CD8+ or CD4+ T cells (4 × 10⁶ 5 Cells (wells) were co-cultured with C2C12 myocanal in DM or CM for 96 hours. The myocanal was imaged using an ECLISPET Ts2 LED microscope (Tokyo Nikon Corporation, Japan), and the myocanal diameter was measured using ImageJ software.

[0233]

[0234] 17. C2C12 siRNA Knockdown Analysis

[0235] After differentiation, C2C12 myotubes were injected into the cells with 20 nM siRNA for Ctsl (SR411229, OriGene Technologies, Rockville, MD, USA) or Bnip3 (SR404627, OriGene Technologies) using Lipofectamine RNAiMax (13778150, Invitrogen) for 24 hours. Subsequently, C2C12 myotubes were co-cultured with splenic CD8+ T cells in the presence of DM or CM for an additional 72 hours.

[0236]

[0237] 18. Confirmation of the effect of recombinant Granzyme A on C2C12 root canal formation

[0238] Differentiated C2C12 myotubes were treated with mouse recombinant granzyme A (Gzma; 10 ng / ml, HY-P76377, MedChemExpress, Monmouth Junction, USA) for 96 hours under DM or CM conditions.

[0239] At this time, some groups were treated with a CTSL inhibitor (1 μg / ml), while the remaining groups were set as a control group without the inhibitor for comparative analysis.

[0240]

[0241] 19. Analysis of cell invasiveness

[0242] Cell invasion was evaluated using a 6.5 mm Transwell chamber with an 8.0 µm porous polycarbonate membrane (Corning Coster, Lowell, MA, USA). The lower and upper surfaces of the membrane were coated with 10 µl of gelatin (1 mg / ml distilled water) and 40 µl of Matrigel (1 mg / ml PBS), respectively. Cells (A549, 1 × 10⁶) 5 Dog cells / wells; Kras IKKαL , 5 × 10 4 Canine cells / well) were cultured in medium containing 5% FBS with or without 10 µg / ml CTSL inhibitor. They were cultured for 24 hours, and after 24 hours, invasive cells at the bottom of the membrane were stained with Mayer's hematoxylin (Cancer Diagnostics Inc., Durham, NC, USA). Non-invasive cells at the top of the membrane were removed using a cotton swab. The number of invasive cells was counted using a light microscope, and the invasion rate was calculated by comparing the number of invasive cells to the control group.

[0243]

[0244] 20. Cell migration analysis

[0245] Cell migration was evaluated using Culture-Insert (EBDI, Gräfelfing, Germany). After creating a 500 µm cell-free space using the insert, cells (7 × 10⁶ 3 Cells / wells were prepared, and after all cells were well attached, the inserts were removed to maintain a 500 µm spacing. Subsequently, the cells were treated with a CTSL inhibitor (10 µg / ml) and 6 hours (Kras IKKαL Cells were cultured for 24 hours (A549 cells) or 24 hours. To suppress gene expression, 20 nM siRNA was injected into CTSL (SR319840, Origin Technologies) or BNIP3 (SR319494, Origin Technologies) cells using Lipofectamine RNAiMax according to the manufacturer's protocol, and the cells were cultured for 48 hours. The degree of cell migration was evaluated by measuring the change in distance between the initial and final cell intervals using ImageJ software.

[0246]

[0247] 21. Bulk RNA-seq

[0248] RNA-seq was performed on the following samples: TA muscle (n = 5), IgG (n = 4), αPD-L1 (n = 3) or αCD8a (n = 3) from CON mice, GA muscle (n = 3) from CON mice, lung tissue (n = 3) from TB mice, and lung tumors (n = 5) from TB mice. Approximately 10 µg of total RNA was isolated from each sample using the TRIzol reagent. Then, mRNA libraries were prepared using the MGIEasy RNA Directional Library Prep Kit (MGI Tech Co., Ltd., Shenzhen, China) and sequenced on an MGISEQ-2000 system (MGI Tech Co., Ltd.) to generate 100 bp paired reads. The quality of the FASTQ files was evaluated using FastQC (v.11.9), and reads containing adapter sequences, low-quality bases, and excessively ambiguous bases were removed using TrimGalore (v.6.6). Trimmed reads were aligned to the Musculus reference genome (GRCM39) using the STAR aligner (v.7.9a) 123. Transcriptome abundance was quantified for each gene using RSEM (v.3.3) and reported as reads per million or transcriptomes (TPM).

[0249]

[0250] 22. Differential Gene Expression Analysis and Functional Annotations

[0251] The following criteria were applied to identify differential gene expression between sample groups (e.g., TB vs. CON) using DESeq2 (v4.3.1): αPD-L1 vs. IgG: p-value <0.005; αCD8a vs. IgG or IgG vs. CON: False positive rate (FDR) adjusted p-value <0.01, absolute log2 fold change (FC) > 1; TB vs. CON: FDR adjusted p-value <0.01, absolute log2 FC > 1.5. Functional enhancement analysis was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

[0252]

[0253] 23. Single Transcriptome Analysis (scRNA-seq)

[0254] Single-cell suspensions derived from lung tumors and skeletal muscle of TB mice were isolated using the CD45+ cell isolation protocol. A total of 4 × 10⁶ cells were extracted from skeletal muscle. 6 Collect CD45+ cells and 2 × 10⁶ from lung tumors 8 CD45+ cells were obtained. scRNA-seq was performed using the BGI MGISEQ-2000 platform at GENINUS Inc. (Seoul, Korea). A barcode library for scRNA-seq was generated using the Chromium single-cell 3' v2 reagent kit (PleasantOn, California, USA, 10 X Genomics) and sequenced on the Illumina NovaSeq 6000 platform (San Diego, California, USA).

[0255]

[0256] 24. scRNA-seq data analysis

[0257] Sequencing reads were aligned to the Musculus reference genome (GRCm39) using CellRanger (v7.0.1). The gene barcode matrix was analyzed using the Seurat R package (v5.0.3). Quality control filters were applied to exclude cells expressing fewer than 300 or more than 4,000 genes to avoid low-quality cells and potential doublets. Cells with high mitochondrial gene expression (>30%) were also discarded as they were likely damaged. To mitigate batch effects, the lung and GA muscle tissue datasets were integrated using Seurat's standard integration pipeline. After quality control, 11,348 of the original 14,028 cell barcodes were retained for downstream analysis. Data normalization was performed using the NormalData function, which log-normalizes gene expression values, scales them by 10,000, and applies a log transform. Principal Component Analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) were used for dimensionality reduction and visualization of distinct cell populations. The optimal number of principal components (PCs) was determined using ElbowPlot, and 24 PCs were selected for further analysis. Clustering was performed using the FindNeighbors and FindClusters functions to group cells based on similar gene expression profiles. Marker genes for each cluster were identified using the FindAlmarkers function, which selects genes with a minimum log2FC of 0.25 (FDR-adjusted p-value <0.05) and detects them in at least 25% of the cells within the cluster. Cell type annotations were GPT-4o, GPTCelltype(v1.0.1), and Azimuth(azimuth.hubmapconsortium.It was assigned based on well-established marker genes from literature and resources such as org / ).

[0258]

[0259] 25. Gene Network Analysis

[0260] Protein-protein interaction (PPI) data from the STRING database (v12.0) for Mus musculus 71 were utilized to identify functional gene modules within a given gene set. STRING integrates various sources of evidence, including co-expression, co-occurrence, literature mining, pathway databases, and experimental data. Therefore, the links in the resulting network represent predicted or known functional associations rather than direct physical interactions.

[0261] To identify densely connected sub-networks, the Walktrap community detection algorithm (cluster_walktrap) implemented in the iGraph R package (v2.0.3) was applied. Functional enhancement analysis of selected gene modules (e.g., Community 1 in Fig. 3b) was performed using the enrichKEGG function of the ClusterProfiler R package (v4.10.1).

[0262] For network visualization, igraph objects were converted to a Cytoscape-compatible format using the createNetworkFromIgraph function from the RCy3 R package (v2.26.0) and rendered in Cytoscape (v3.10.2). Unless otherwise specified, interaction pairs with intermediate confidence (joint score > 500) were retained for PPI network construction. To map human gene expression data (TCGA or GTEx) to mouse-derived networks, mouse gene symbols were converted to human orthologs using the Homologen R package (v1.4.68.19.3.27).

[0263]

[0264] 26. Analysis of Open Transcriptome Data

[0265] To explore gene expression patterns in various cachexia models, publicly available mouse transcriptome data were searched in the GEO database (ncbi.nlm.nih.gov / geo / ) using the keyword 'cachexia' to obtain four relevant datasets: GSE107470, GSE51931, GSE144567, and GSE114820. Considering the technical variability between datasets (microarray and RNA-seq), each dataset underwent independent preprocessing suitable for its respective platform. For RNA-seq data, raw FASTQ files were retrieved from the European Nucleotide Archive (ENA, ebi.ac.uk / ena / ) and processed according to the RNA-seq preprocessing protocol described earlier. Differential gene expression between the two groups was evaluated using the Wald test in the R package DESeq2. For microarray data, normalized gene expression profiles for each sample were obtained using the R package GEO Query (v2.72.0). Differential gene expression between the two groups was evaluated using a t-test implemented in the ebayes function of the R package limma (v3.60.4). To assess the clinical relevance of CTSL expression in human cancer cachexia, human muscle transcriptome data were retrieved from the public repositories, the GEO database (GSE133523) and EMBL-EBI ArrayExpress (E-MTAB-12781). FASTQ files were preprocessed by trimming low-quality reads and adapter sequences, mapping the cleaned reads to the human reference genome (GRCh38), and quantifying gene expression in the TPM using RSEM (v1.3.3) for individual samples.

[0266]

[0267] 27. TCGA Transcriptome Data Analysis

[0268] RNA-seq data of tumor tissues covering 33 cancer types were retrieved from the TCGA legacy gene expression dataset using the GDCquery function within the R package TCGAbiolinks (v2.32.0). This dataset contains read counts and TPM values ​​for 19,969 protein-coding genes across individual patient samples. For survival analysis, patients were stratified into two groups based on CTSL expression levels (high and low). To estimate the difference in overall survival of reads between groups, Kaplan-Meier (K–M) survival curves were generated using the Survivit function from the R package SurvivalRate (v3.6.4), and statistical significance was assessed using the log-rank test. A Cox proportional hazards regression model was constructed using the coxph function in R, and hazard ratios (HRs) were derived to estimate the corresponding relative hazards.

[0269] To investigate transcriptional changes specifically associated with the CTSL-BNIP3 axis in the LUAD cohort, tumor samples were stratified according to the expression levels of CTSL and BNIP3. For each gene, samples were ranked according to the expression levels of CTSL and BNIP3, respectively. The top 50% of samples for each gene were classified as CTSL-high and BNIP3-high, while the bottom 50% were designated as CTSL-low and BNIP3-low. The intersection of the CTSL-high and BNIP3-high subsets defined the CTSL-BNIP3-high group, whereas the overlap between CTSL-low and BNIP3-low samples defined the CTSL-BNIP3-low group. Differential gene expression analysis between the CTSL-BNIP3-high and CTSL-BNIP3-low groups was performed using the DESeq2 package in R, followed by pathway enrichment analysis using GSEA with a selected gene set from the WikiPathways database.

[0270]

[0271] 29. Analysis of Genotype-Tissue Expression (GTEx) Data

[0272] Human skeletal muscle RNA-seq data were obtained from the Genotype-Tissue Expression (GTEx) project (v8 release) via the GTEx portal (gtexportal.org / ). A total of 803 skeletal muscle samples from healthy individuals were downloaded along with their corresponding gene expression quantification files (in TPM and read count formats). Similar to TCGA LUAD analysis, samples were stratified based on CTSL and BNIP3 expression levels. For each gene, the top 50% and bottom 50% of samples were classified as high and low, respectively. Crosses between CTSL-high and BNIP3-high samples were defined as the CTSL-BNIP3-high group, and crosses between CTSL-low and BNIP3-low samples constituted the CTSL-BNIP3-low group. Differential gene expression analysis between the two groups was performed using DESeq2, followed by the identification of enriched biological pathways via GSEA using the WikiPathways gene set.

[0273]

[0274] [Experimental Results]

[0275] 1. αPD-L1 inhibits tumor formation while exacerbating muscle wasting.

[0276] To evaluate whether ICIs contribute to cancer cachexia, we analyzed weight changes in cancer patients treated with ICIs and compared them with patients who received other cachexia-inducing treatments, such as doxorubicin and pemetrexid, at Yonsei University Severance Hospital. Patients treated with ICIs containing αPD-1 and αPD-L1 showed weight loss similar to patients who received other chemotherapy.

[0277] To determine whether the observed body weight changes were induced by the ICI treatment itself, we reanalyzed a preclinical subcutaneous tumor model in which tumor growth was effectively inhibited by αPD-L1 treatment. Interestingly, mice responding to αPD-L1 treatment showed a decrease in body weight despite the tumors being almost completely gone, whereas the untreated tumor-bearing control group showed an increase in net body weight regardless of tumor size (Fig. 1A). These data indicate that αPD-L1 can directly exacerbate cachexia as an irAE, regardless of its anticancer efficacy.

[0278] To verify this in vivo, a tumor-bearing (TB) mouse model was established by allogeneic orthogenetic transplantation of lung cancer cells into male C57BL / 6 mice. This model is one that has already been established in previous studies, and Kras IKKαL Cells were injected into the intratrachea to establish the system (Fig. 1B), and the tumor forms an immunosuppressive tumor microenvironment (TME) characterized by low CD8+ T cell and high regulatory T cell (Treg) infiltration. Therefore, this model is considered a suitable system for immune checkpoint inhibitor (ICI) research.

[0279] After the injection of tumor cells, mice were treated as follows starting from the 3rd day.

[0280] The subjects were divided into a group administered anti-mouse CD8 antibody and αPD-L1 in combination and a group administered each drug alone, and received intraperitoneal administration three times a week. When all treatment groups first reached a 10% weight loss from baseline, all groups were sacrificed on the same day.

[0281] Surprisingly, αPD-L1-treated TB mice reached this threshold earliest, despite having much smaller tumors and lower lung weights than the IgG controls (Fig. 1C-E). At the time of final evaluation, the αPD-L1 group showed a decrease in muscle fiber cross-sectional area (CSA) compared to the IgG group, along with severe loss of epididymal WAT (eWAT), gastrocnemius (GA), and tibialis anterior (TA) muscles (Fig. 1E-H, blue versus red). In contrast, the IgG group maintained body weight close to baseline despite larger tumors, and both muscle and fat weights were higher compared to the αPD-L1 group. A comparison of food intake between the TB groups showed no significant difference, and normal mice treated with αPD-L1 also showed no significant difference between the TB groups. Therefore, the possibility of anorexia or apparent toxicity regarding the body weight loss caused by αPD-L1 could be ruled out.

[0282] Next, we examined CD8+ T cells in lung tumors of TB mice. As expected, we confirmed that αPD-L1 increased CD8+ T cells in lung tumors and lowered Foxp3+ Tregs, thereby reversing the immunosuppressive state (Fig. 1I). Conversely, CD8+ T cell infiltration within the muscles of TB mice increased compared to CON mice (Fig. 1I, red vs. black), and was further amplified after αPD-L1 treatment (Fig. 1I, blue vs. red). This suggests a role for CD8+ T cells in muscle wasting. To test causality, we depleted CD8+ T cells using an anti-CD8α (αCD8a) antibody. As expected, CD8+ T cell depletion eliminated the anticancer efficacy of αPD-L1 (Fig. 1C, D, yellow vs. blue), but reversed body weight, fat, or muscle loss caused by αPD-L1 (Fig. 1E-H, yellow vs. blue) (Fig. 2B, yellow vs. blue). Depletion of CD8+ T cells reduces muscle wasting, however. Importantly, when only CD8+ T cells are depleted without αPD-L1, body weight is maintained and fat and muscle loss is prevented, while tumor size does not change (Fig. 1C-I, purple versus red), which highlights that it is an important mediator of cancer-induced muscle atrophy.

[0283] RNA-seq was performed on skeletal muscle tissue to identify the underlying molecular mechanisms of change. Principal component analysis (PCA) revealed that the IgG and αPD-L1 groups clustered distinctly apart from CON mice; interestingly, the αCD8a-alone group showed a tendency for muscle transcriptome patterns to shift back toward CON mice (Fig. 1J). Analysis of related physiological pathways revealed that genes and pathways involved in ECM tissue were downregulated in the IgG-treated groups compared to CON mice, while autophagy-related pathways were upregulated (Fig. 1K). Additionally, the αPD-L1-treated group showed a slight increase in gene pathways related to muscle protein degradation compared to the IgG-treated group (Fig. 1L), and CD8⁺ T cell depletion reversed these transcriptional changes (Fig. 1M). Notably, αPD-L1 was found to significantly increase gene pathways related to leukocyte migration and chemotaxis (Fig. 1L). Consistent with these transcriptome changes, IgG mice upregulated the specific muscle ligase MuRF-1 and the autophagy marker LC3B-II, both of which were inhibited by CD8+ T cell depletion (Fig. 1N). Functionally, the grip strength of TB mice was also reduced (Fig. 10, red vs. black), further reduced by αPD-L1 (Fig. 10, blue vs. red) (Fig. 10, purple vs. red), but restored by CD8+ T cell depletion (Fig. 10, purple vs. red). These results support the requirement of cytotoxic CD8+ T cells for cancer-related muscle wasting and deterioration caused by PD-L1 blockade.

[0284] To verify this directly, Kras IKKαLSplenic CD8+ T cells from CON or TB mice were co-cultured with differentiated C2C12 myotubes in the presence of cellular conditioning medium (CM) (not shown). As a result, CM treatment alone reduced myotube diameter, and the addition of CD8+ T cells further exacerbated muscle atrophy (Fig. 1p). On the other hand, CD4+ T cells did not affect muscle atrophy regardless of the presence of CM. Western blot analysis confirmed that CM increased the expression of the muscle-specific ubiquitin ligase atrogin-1 and the autophagy marker LC3B-II (Fig. 1q). In particular, co-culture with CD8+ T cells further increased the expression of atrogin-1 (Fig. 1q) and exacerbated muscle atrophy (Fig. 1p, blue versus red). Therefore, cytotoxic CD8+ T cells can directly induce muscle wasting and suggest the possibility that they may be the underlying mechanism of muscle-specific irAE observed with αPD-L1 treatment.

[0285]

[0286] 2. Cytotoxic CD49a+ CD8+ T cells accumulating in the skeletal muscle of TB mice

[0287] To analyze the immune environment of tumors and muscles in cachexia, single-cell RNA sequencing (scRNA-seq) was performed on CD45+ cells isolated from lung tumors and skeletal muscles of TB mice exhibiting cachexia characteristics. At this time, the patterns of muscle loss and body weight reduction observed in αPD-L1-treated TB mice were similar (Figs. 1E and F), and Tnf-α and Il-6, the major cytokines of cachexia, increased and grip strength decreased in the lungs, muscles, eWAT, and liver of TB mice. Similar results were obtained when LLC (Lewis Lung Carcinoma, LLC) cells, a cell line commonly used in cachexia mouse models, were injected intratracheally, which validates our model.

[0288] Of the 14,028 captured CD45+ cells, 11,348 single-cell transcripts passed quality control for analysis. We identified eight major immune communities with distinct histological biases (Figs. 2A–C). Lung tumors were dominated by neutrophils and macrophages, followed by classical T cells and B cells (Fig. 2B). Surprisingly, skeletal muscle exhibited selective abundance of CD49a+CD8+ T cells, a unique T cell subset (Fig. 2C). This CD49a+CD8+ T cell population formed its own transcriptional cluster distinct from classical T cells (Fig. 2A). While both subsets expressed T cell receptor (TCR) related genes, the CD49a+CD8+ subset displayed increasingly heterogeneous Cd3 and Cd8 expression and muscle-rich features including Nr3c1, Nrp1, Entpd1, Cd200, Ly6c1, Zbtb16, and Itga1 (Fig. 2D). In particular, Itga1, which labels tissue-resident memory CD8+ T cells (TRM), was highly and extensively expressed in the population, and Ly6c1 and Zbtb16 were associated with tissue homing and innate T-cell-like identity (Fig. 2D).

[0289] Transcriptome analysis revealed that, unlike classical T cells (rich in genes related to leukocyte activation and TCR signaling), CD49a+ CD8+ T cells upregulated genes for epithelial migration and Rap1 signaling (Figs. 2E and F). Given that Rap1 enhances integrin-dependent tissue homing, this may strengthen the identity of TRM-like cytotoxic T cell subsets in skeletal muscle.

[0290] Next, flow cytometry analysis confirmed that CD49a+ CD8+ T cells were significantly abundant in the muscles of TB mice but decreased in lung tumors. In lung tumors, Tregs and myeloid-derived suppressor cells (MDSCs) were increased compared to CON mice (Fig. 2G). In muscles, most CD8+ T cells co-expressed CD49a, whereas in tumors, only some expressed CD49a (Fig. 2G, H). Similarly, in a cachexia model using LLC cells, CD49a+ CD8+ T cells were found to be abundant in muscles.

[0291] Finally, CD8+ T cells co-cultured with C2C12 myotubes under CM conditions were isolated, and the gene expression of Itga, Prf1, Gzma, and Gzmb was examined. The results verified that they were upregulated, leading to increased cytotoxicity (Fig. 2D). Considering the novel association between TRM cells and irAE, our data suggest that CD49a+ CD8+ TRM-like cells contribute to cancer-induced muscle wasting.

[0292]

[0293] 3. Discovery of CTSL, a Target for Controlling Tumor Formation and T-Cell-Mediated Muscle Loss

[0294] It was confirmed that cytotoxic CD8+ T cells act as a significant cause of cancer-related muscle loss. However, their depletion eliminates anti-cancer immunity (Fig. 1C), and selectively targeting CD49a+ CD8+ T cells remains a technically challenging task. To identify targets capable of simultaneously inhibiting tumor progression and muscle atrophy, an integrated transcriptome analysis of tumors and skeletal muscle was performed in TB mice.

[0295] We identified 55 genes that are upregulated in both tumors and muscles of cachexic mice and downregulated in muscles upon CD8⁺ T cell depletion (Fig. 3A). The protein–protein interaction (PPI) network revealed a densely linked "Community 1" rich in genes related to proteasomes and autophagy, which was a central process of muscle catabolism (Fig. 3B). These genes were consistently upregulated not only in our mouse model but also in various cancer cachexia models (Fig. 3C).

[0296] Within this core cluster, Ctsl could be considered a particularly promising target due to its high and consistent upregulation across the dataset (Fig. 3C). Western blot confirmed that Ctsl expression was increased in both tumors and muscles of the CON mouse group compared to the TB mouse group (Fig. 3D). In muscles, Ctsl levels were further elevated by αPD-L1 treatment and decreased by CD8+ T cell depletion, whereas in tumors, only CD8+ T cell depletion reduced Ctsl expression (Fig. 3D). This pattern implies that CTSL is important in both tumor progression and T cell-mediated muscle wasting, highlighting its dual-action therapeutic target.

[0297] The results of the analysis of PPI network pathways co-expressed with Ctsl are shown (Figs. 3E-J). In tumors, Ctsl was co-upregulated with matrix metalloproteinases Mmp7 and Mmp13, which induce ECM remodeling and metastasis (Figs. 3E and H). On the other hand, in skeletal muscle, it was co-expressed with genes involved in autophagy, apoptosis, and lysosomal pathways (Figs. 3F and I). These muscle-related co-expression signs were reversed by CD8+ T cell depletion (Figs. 3G and J), suggesting that CTSL may contribute to T cell-mediated muscle wasting.

[0298] To confirm clinical relevance, CTSL expression was analyzed in a human cancer dataset. Analysis of the Cancer Genome Atlas (TCGA) data revealed that CTSL was widely expressed across various cancer types, showing particularly prominent expression in non-small cell lung cancer (NSCLC and LUSC).

[0299] High CTSL expression showed a significant correlation with reduced overall survival in LUAD, LUSC, and pan-cancer cohorts (Fig. 3K). CTSL levels showed a strong correlation with CD274 (PD-L1) expression across tumors (Fig. 3L), and this correlation was also confirmed in an independent NSCLC dataset (Fig. 3M). Furthermore, reanalysis of human muscle transcriptome data revealed that CTSL was significantly upregulated in the muscles of cachexic NSCLC patients compared to healthy individuals (Fig. 3N), demonstrating that CTSL is involved in human cachexia. Collectively, these results suggest that CTSL can serve as a clinically important therapeutic target for inhibiting tumor progression and T-cell-mediated muscle wasting.

[0300]

[0301] 4. Confirmed that CTSL inhibitors prevent muscle wasting induced by αPD-L1 and further suppress tumors.

[0302] Based on the previous results, to directly test whether targeting CTSL could enhance the efficacy of αPD-L1 and prevent muscle-specific toxicity, a CTSL inhibitor (CTSLi) was administered to TB mice either alone or in combination with αPD-L1 (Fig. 4A). The combination therapy further reduced the lung tumor burden compared to αPD-L1 monotherapy (Figs. 4B and C), indicating that it exhibited additional anticancer activity through distinct but complementary mechanisms. Importantly, when administered alone or in combination with αPD-L1, CTSLi significantly prevented the body weight loss, fat depletion, and muscle wasting observed with αPD-L1 (Figs. 4D-H). Furthermore, the combination therapy significantly reduced the RNA levels of the muscle cachexia-associated cytokines Tnf-α and Il-6, supporting its ability to inhibit muscle wasting induced by αPD-L1.

[0303] Next, we investigated the association between CTSL and cytotoxic T cells. Ctsl concentrations in TB mice were increased in both tumors and muscles and were effectively reduced by CTSLi (Fig. 4I). In skeletal muscle, Ctsl levels showed a strong correlation with the infiltration of CD8+ and CD49a+ CD8+ T cells, both of which were amplified by αPD-L1 (Fig. 4I-K, blue vs. red) and reversed by CTSLi (Fig. 4I-K, yellow vs. blue). On the other hand, in the lungs, CTSLi slightly increased CD8+ T cell infiltration when administered in combination with αPD-L1 (Fig. 4J, K, yellow vs. blue), suggesting that CTSL regulates immune cell dynamics differently in muscle and tumors.

[0304] These contrasting effects were consistent with the tissue-specific immune contexture in TB mice. Immunosuppressive MDSCs and Tregs were abundant in the tumor, while an immunostimulatory characteristic with increased CD8⁺ T cells was observed in the muscle (Figs. 4J and K). Interestingly, CTSL inhibitors (CTSLi) inversely regulated the frequencies of CD8⁺ T cells and Tregs in the tumor and muscle (Figs. 4J and K), confirming that the immunomodulatory action is context-dependent and varies depending on the tissue environment.

[0305] MHC-I molecules, which are crucial for antigen recognition by CD8+ T cells via TCRs, are generally under-expressed in healthy muscle but are upregulated under inflammatory conditions, leading to CD8+ T cell-mediated tissue damage. Accordingly, we investigated the association between CTSL and MHC-I expression in skeletal muscle. Analysis of the public human muscle dataset revealed that CTSL expression showed a significant correlation with HLA-B (encoding MHC-I) expression levels in both healthy individuals and cachexic patients (Fig. 4L). Additionally, in a TB mouse model, CTSL expression in muscle showed a positive correlation with H2-D1 (a mouse MHC-I homolog), whereas this correlation was not observed in tumors (Fig. 4M).

[0306] MHC-I expression in the skeletal muscle of TB mice was increased at both transcriptome and protein levels and was further upregulated by αPD-L1 (Fig. 4N). To rule out other effects of CTSL inhibitors, CTSL gene knockdown was performed in C2C12 myotubes: siRNA-mediated CTSL inhibition reduced MHC-I expression at both protein and mRNA levels and protected against CD8+ T cell-induced atrophy (Fig. 4O, P). Collectively, these results demonstrate that CTSL inhibition can separate anti-cancer immunity from muscle-specific irAE. In other words, CTSL inhibition implies that it enhances the anti-cancer efficacy of αPD-L1 while suppressing muscle wasting induced by cytotoxic T cells through MHC-I-dependent mechanisms.

[0307]

[0308] 5. BNIP3 as a mediator mediating tumorigenesis and muscle wasting induced by CTSL expression

[0309] To elucidate the mechanism by which CTSL induces both tumor progression and muscle wasting, we focused on key interactors of CTSL in an integrated PPI network analysis (Fig. 5A). Among these candidates, Bnip3 was found to be the most consistently correlated with CTSL in both tumors and muscles (Figs. 5B, C). Importantly, Bnip3 was the only direct CTSL interaction partner closely co-regulated with CTSL in both tumors and muscles, and its expression decreased in muscles upon CD8+ T cell depletion (Fig. 5D). Furthermore, as previously confirmed in Fig. 3B, it was identified as a key member of Community 1.

[0310] BNIP3 acts as a mitophagy receptor to regulate intracellular mitochondrial quality control and energy homeostasis by removing damaged mitochondria. However, its function varies depending on the tissue context; in tumors, BNIP3 plays a role in promoting cancer cell survival, metabolic adaptation, and metastasis. In contrast, in skeletal muscle, BNIP3 contributes to atrophy by inducing autophagy and / or mitophagy. Therefore, it was confirmed that the Ctsl-Bnip3 network exhibits tissue-specific expression patterns: in tumors, Bnip3 was co-expressed with genes involved in mitochondrial function (e.g., Bid, Bok, Pgam5, Ppif, Pmaip1, Vdac1) and energy metabolism (e.g., Bid, Dddh, Ldha, Ppif, Pgk1, Phb2, Tpi1) (Fig. 5E, F). In contrast, in muscle, it was co-expressed with autophagy and mitophagy regulators (e.g., Akt1, Bnip3l, Foxo3, Sqstm1, Map1lc3b, Mtor, Npc1), but this pattern was reversed by CD8+ T cell depletion (Fig. 5G, H). These contrasting co-expression modules suggest that BNIP3 acts as a context-dependent effector of CTSL signaling in tumors and muscle.

[0311]

[0312] 6. Contributes to enhancing tumor metastasis potential through BNIP3 upregulation

[0313] To evaluate the clinical relevance of the CTSL-BNIP3 network in LUAD, we first analyzed TCGA data and observed that most CTSL-BNIP3 network genes were significantly upregulated in normal tissues corresponding to tumors (Fig. 6A). This was also confirmed in our TB model (Fig. 5E). Immunofluorescence staining of human LUAD tissue microarrays confirmed that BNIP3 expression was significantly higher in CTSL-high-expression tumors than in CTSL-low-expression tumors (Fig. 6B) (Fig. 6B). Interestingly, when LUAD patients were classified according to CTSL and BNIP3 expression levels, the CTSL-BNIP3 high-expression subgroup had a significantly lower overall survival rate compared to the CTSL-only high-expression group (Fig. 3K, HR = 1.53; p = 0.0035) (Fig. 6C, HR = 1.9; log-rank p = 0.0013), demonstrating a stronger prognostic predictive power than CTSL alone. As a result of functional enhancement analysis, metastasis-related genes were significantly enhanced in the CTSL-BNIP3 high-expression group in both the TCGA cohort (Fig. 6D, left), the independent verification cohort, and tumors of TB mice (Fig. 6D, right). These results support a model in which CTSL and BNIP3 act cooperatively to promote the metastasis program in LUAD.

[0314] In TB mice, Ctsl and Bnip3 were upregulated along with N-cadherin (N-cad), an EMT-related gene indicating epithelial-to-mesenchymal transition (EMT), in lung tumors (Fig. 6E). Administration of αPD-L1 alone had a negligible effect on N-cadherin expression, and no changes were observed in the expression levels of CTSL and BNIP3 (Fig. 6E).

[0315] This indicates that the CTSL-BNIP3 axis promotes the EMT process independently of αPD-L1 signaling. Consistently, BNIP3 and N-cadherin expression are reduced upon treatment with CTSL inhibitors in human LUAD A549 cells, and A549 and Kras IKKαL Cell migration and invasion were significantly inhibited (Figs. 6G and H). Additionally, genetic knockdown of CTSL in A549 cells reduced both BNIP3 and N-cadherin expression (Fig. 6I), while knockdown of BNIP3 did not affect CTSL expression but reduced N-cadherin (Fig. 6J). Functionally, CTSL or BNIP3 knockdown significantly inhibited the motility of A549 cells (Figs. 6K and L). Collectively, these results suggest that CTSL promotes the motility and invasiveness of LUAD cells through a BNIP3-dependent EMT program (Figs. 6G and H). This suggests that targeting CTSL may act complementarily with αPD-L1 therapy to further enhance anticancer efficacy.

[0316]

[0317] 7. CTSL causes mitochondrial dysfunction and muscle wasting through BNIP3.

[0318] To investigate the involvement of the CTSL-BNIP3 axis in muscle physiology, skeletal muscle transcriptome data from 803 healthy individuals in the GTEx RNA-seq database were analyzed. Despite being examined in non-bronchial tissues, this large cohort showed a strong positive correlation between CTSL and BNIP3 expression. In particular, individuals with high co-expression of CTSL and BNIP3 exhibited muscle gene expression profiles very similar to those of TB mice (Figs. 5G, 7A). Functional analysis revealed significant changes in biological processes related to muscle metabolism and protein homeostasis in the CTSL-BNIP3-high expression group (Fig. 7B). Specifically, this group showed upregulation of proteasome degradation-related genes and downregulation of mitochondrial oxidative phosphorylation genes (Fig. 7C), suggesting that the CTSL–BNIP3 axis may induce mitochondrial dysfunction and contribute to muscle wasting.

[0319] Consistent with results obtained from the human dataset, the expression of Ctsl and Bnip3 in TB mouse muscle was upregulated (Fig. 7D), and importantly, when Bnip3 expression was further increased by αPD-L1, administration of a CTSL inhibitor reversed this increase (Fig. 7D). Furthermore, knockdown of Ctsl reduced Bnip3 expression in C2C12 myotubes (Fig. 7E), and muscle atrophy induced by CD8⁺ T cells was significantly alleviated in C2C12 myotubes with knockdown of Bnip3 (Fig. 7F). These results suggest that the CTSL–BNIP3 axis is involved in T-cell-mediated muscle wasting, and that the mechanism may be mediated through the promotion of mitochondrial dysfunction.

[0320] Mitophagy is a process that regulates the number of mitochondrial DNA (mtDNA) copies, which is essential for maintaining muscle function. The inventors observed a significant decrease in the number of mtDNA copies in the muscles of TB mice, and this decrease was exacerbated by αPD-L1 treatment but restored by a CTSL inhibitor (CTSLi) (Fig. 7G). In particular, the number of mtDNA copies (Fig. 7G) showed an inverse relationship with Ctsl expression and the degree of CD8⁺ T-cell infiltration (Fig. 4I–K), suggesting an association between cytotoxic T-cell activity and mitochondrial loss.

[0321] In addition, in co-culture experiments of C2C12 myotube cells and CD8⁺ T cells, CD8⁺ T cells reduced both myotube diameter and mtDNA copy number, but these two indicators were restored by treatment with CTSL inhibitor (Fig. 7H). Furthermore, siRNA knockdown of ctsl or Bnip3 in the presence of CD8⁺ T cells similarly restored mtDNA copy number, supporting the CTSL–BNIP3 axis acting as a key pathway for T cell-mediated mitochondrial dysfunction (Fig. 7I).

[0322] To determine how the CTSL-BNIP3 axis and CD8+ T cells are associated with mitochondrial dysfunction, we investigated the role of the cytotoxic factor granzyme A (GZMA), known to induce mitochondrial damage. When C2C12 myotube cells were treated with recombinant GZMA, both myotube diameter and mtDNA copy number decreased even in the absence of CD8⁺ T cells, and this effect was abolished by treatment with the CTSL inhibitor (CTSLi) (Fig. 7J, K). Furthermore, when combining the results of a single-cell analysis in which the expression of Gzma, Gzmb, and Prf1 was confirmed in CD49a⁺ CD8⁺ T cells (Fig. 2D), we can suggest the possibility that GZMA plays a role in promoting mtDNA depletion and muscle wasting in cooperation with the CTSL–BNIP3 axis.

[0323] In summary, increased expression of CTSL in muscle under cachexia conditions induces MHC-I expression, thereby promoting the infiltration of TRM-like CD49a+ CD8+ T cells. These T cells secrete GZMA and, in conjunction with BNIP3, cause mtDNA depletion, mitochondrial dysfunction, and ultimately muscle atrophy. Therefore, CTSL inhibitors can regulate muscle loss by modulating T cell-mediated mitochondrial damage as well as proteasome degradation and autophagy pathways.

[0324]

[0325] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0326]

[0327]

Claims

1. A pharmaceutical composition for treating or preventing immune-related adverse events (irAEs) comprising a cathepsin L inhibitor as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition in which the above immune-related adverse reaction occurs due to the administration of an anticancer drug.

3. In Paragraph 2, The above anticancer agent is a pharmaceutical composition that is an immune checkpoint inhibitor (ICI).

4. In Paragraph 3, A pharmaceutical composition wherein the immune checkpoint inhibitor is one or more selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.

5. In Paragraph 1, A pharmaceutical composition wherein the above immune-related adverse reaction is one or more diseases selected from the group consisting of infectious diseases, anemia, hemorrhagic diseases, liver and kidney diseases, heart diseases, lung diseases, neurological diseases, gastrointestinal diseases, autoimmune diseases, endocrine disorders, and secondary cancers.

6. In Paragraph 1, The above-mentioned cathepsin L inhibitor is a pharmaceutical composition comprising one or more selected from the group consisting of small molecules that bind complementarily to the cathepsin L protein or the transcript sequence of cathepsin L, antisense nucleotides, short interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA, and ribozymes, dsNRA, aptamers, PNA (peptide nucleic acid), ZFN, TALEN, and CRISPR-Cas9.

7. In Paragraph 6, A pharmaceutical composition wherein the above cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine.

8. In Paragraph 7, The above cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Pharmaceutical composition containing N2O5.

9. In Paragraph 8, The above Z-Phe-Tyr-CHO(C 26 H 26 A pharmaceutical composition in which N2O5) is represented by the following chemical formula 1: [Chemical Formula 1] 10. A pharmaceutical composition for the prevention or treatment of cancer comprising a cathepsin L inhibitor as an active ingredient, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredient.

11. In Paragraph 10, A pharmaceutical composition wherein the above cathepsin L inhibitor is one or more selected from the group consisting of CA-074, K11777, Camostat mesylate, Nafamostat mesylate, Protease inhibitor cocktail, CLIK-148, E-64, and Amantadine.

12. In Paragraph 10, The above cathepsin L inhibitor is Z-Phe-Tyr-CHO(C 26 H 26 Pharmaceutical composition containing N2O5.

13. In Paragraph 12, The above Z-Phe-Tyr-CHO(C 26 H 26 A pharmaceutical composition in which N2O5) is represented by the following chemical formula 1: [Chemical Formula 1] 14. In Paragraph 13, A pharmaceutical composition in which the above cancer is gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.

15. A pharmaceutical composition for treating or preventing cachexia comprising a cathepsin L inhibitor as an active ingredient.

16. In Paragraph 15, A pharmaceutical composition in which the above cachexia is caused by the administration of an anticancer drug 17. In Paragraph 16, The above pharmaceutical composition is a pharmaceutical composition that improves and treats one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

18. A pharmaceutical composition for treating or preventing muscle diseases comprising a cathepsin L inhibitor as an active ingredient.

19. In Paragraph 18, A pharmaceutical composition in which the above-mentioned muscle disease was caused by the administration of an anticancer drug 20. In Paragraph 19, A pharmaceutical composition for improving or treating a muscle disease, wherein the muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, muscle loss, and sarcopenia.

21. In Paragraph 20, A pharmaceutical composition for improving or treating a muscle disease, wherein the above muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused or damaged muscle.

22. A pharmaceutical composition for the prevention or treatment of cachexia comprising a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredient.

23. In Paragraph 22, The above pharmaceutical composition is a pharmaceutical composition that improves and treats one or more symptoms of cachexia selected from the group consisting of decreased appetite, weight loss, increased fatigue, muscle weakness, decreased mobility, muscle loss, fat loss, and hematopoietic toxicity.

24. A pharmaceutical composition for improving or treating muscle disease comprising a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredients.

25. In Paragraph 24, A pharmaceutical composition for improving or treating a muscle disease, wherein the muscle disease is selected from the group comprising atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia gravis, muscle loss, and sarcopenia.

26. In Paragraph 25, A pharmaceutical composition for improving or treating a muscle disease, wherein the above muscle disease is caused by aging, decreased muscle function, muscle wasting, muscle degeneration, disused or damaged muscle.

27. A method for treating or preventing immune-related adverse events (irAEs) comprising a cathepsin L inhibitor as an active ingredient.

28. A method for treating or preventing cachexia comprising a cathepsin L inhibitor as an active ingredient.

29. A method for treating or preventing muscle diseases comprising a cathepsin L inhibitor as an active ingredient.

30. A method for the prevention or treatment of cachexia comprising a cathepsin L inhibitor and a pharmaceutically acceptable salt as active ingredients, wherein the subject administers an immunotherapeutic agent before, simultaneously with, or after administering the active ingredients.

Images