Gfral antagonistic antibodies and uses thereof

Abstract

This invention relates to GFRAL antagonistic antibodies and their uses, and more specifically, to anti-GFRAL antibodies comprising heavy chain CDRs and light chain CDRs of specific sequences, and their antigen-binding fragments. Anti-GFRAL antibodies are intended to be useful in alleviating or treating cancer-related anorexia-cachexia syndrome, as well as adverse reactions caused by chemotherapy drugs.

Description

Technical Field

[0001] This invention relates to GFRAL antagonist antibodies and their uses. Background Technology

[0002] Since the advent of the first monoclonal antibody in 1975, which demonstrates strong affinity for antigens, antibody therapeutics were first released in 1994, and therapeutic antibodies have recorded the fastest growth in pharmaceuticals. As of 2007, the global market size for therapeutic antibodies was $27 billion, showing sustained growth from $44.7 billion in 2011 to $57.7 billion in 2016. Based on this growth, antibody drugs accounted for 10% of global drug sales as of 2014, and occupied 6 of the top 10 spots in drug sales in 2016. Compared to existing therapeutics based on chemically synthesized compounds, antibody drugs have been shown to cause relatively fewer side effects while ensuring superior therapeutic efficacy due to their high binding specificity and stability in vivo. Therefore, antibody drug development is attracting attention as a core area of ​​next-generation new drug R&D. Furthermore, due to the rapid development of technologies for the expression, production, purification, and engineering of high molecular weight proteins (which are crucial for antibody drug development) and the high success rate in clinical trials, antibody drugs appear poised to become the next generation of therapeutics.

[0003] Cancer-associated anorexia-cachexia syndrome (CACS) is a catabolic state characterized by persistent anorexia and weight loss. CACS causes increased muscle and fat breakdown, nutritional and metabolic imbalances, and an elevated basal metabolic rate, leading to overall functional deterioration. This type of CACS is one of the leading causes of death in cancer patients and is also the most important independent prognostic factor predicting negative treatment outcomes. Furthermore, the limited nutritional intake associated with treatments (such as chemotherapy, radiation therapy, or surgery), low response rates to treatment, and difficulty in implementing effective treatments are major reasons for reduced patient survival or quality of life. However, CACS is underestimated, and unmet medical needs remain. Currently, common appetite stimulants and muscle synthesis stimulants appear to provide little to no effective treatment results.

[0004] Growth differentiation factor 15 (GDF15) is a cytokine that plays various roles in the body through its involvement in immune responses and metabolism. Under normal conditions, GDF15 is expressed at low concentrations in most tissues, but its levels increase significantly when tissues such as the liver, kidneys, heart, and lungs are damaged. In 2007, it was discovered that GDF15 induces cachexia in prostate cancer patients through anorexia, and there is a significant correlation between weight loss caused by anorexia and GDF15 concentration in cancer patients. The concentration of GDF15 in the blood of prostate cancer patients is 10 to 100 times higher than that in normal individuals (Nature Medicine 2007; 12(10); 1333-40). In 2016, it was disclosed that GDF15 is a major cytokine that induces cachexia in various cancers, and that GDF15 antibody showed cachexia-relieving effects in mouse models of cachexia in various cancers by increasing body weight and reducing the loss of muscle and adipose tissue (Journal of cachexia, sarcopenia and muscle 2016; 7:467-482).

[0005] In 2015, side effects of chemotherapy (such as cisplatin) were reported, including increased GDF15 expression and increased resistance to chemotherapy through anorexia and cachexia (PLoS One 2015; 10(1); e0115372). In 2017, it was found that GDF15 inhibits appetite through the receptor GFRAL, and that GDF15 signal transduction works in a GFRAL-dependent manner after GDF15 treatment alone or cisplatin treatment (Nature 2017; 550(7675); 255-259). GFRAL transmits signals into the cell via RET (a co-receptor) and is specifically expressed in the nucleus tractus solitarius (NTS) region and the area postrema (AP) of the hindbrain, which are accessible to antibody drugs outside the blood-brain barrier.

[0006] Therefore, anti-GFRAL antibodies alleviate anorexia in cancer patients with CACS caused by platinum-based anticancer drugs (such as cisplatin) and reduce resistance to chemotherapy through weight gain and increased skeletal muscle metabolism, thereby helping to improve the welfare and lifespan of cancer patients. Summary of the Invention

[0007] Technical goals

[0008] The purpose of this disclosure is to provide an anti-GFRAL antibody or an antigen-binding fragment thereof.

[0009] Another object of this disclosure is to provide a nucleic acid molecule encoding an anti-GFRAL antibody or an antigen-binding fragment thereof, a recombinant expression vector containing the nucleic acid molecule, and cells transformed with the recombinant expression vector.

[0010] Another object of this disclosure is to provide a composition for the prevention, relief or treatment of cancer-related anorexia-cachexia syndrome (CACS), said composition comprising an anti-GFRAL antibody or an antigen-binding fragment thereof as an active ingredient.

[0011] Another object of the present invention is to provide a composition for the prevention, relief or treatment of anorexia or cachexia caused by anticancer agents, said composition comprising an anti-GFRAL antibody or an antigen-binding fragment thereof as an active ingredient.

[0012] Technical solution

[0013] To achieve the above objectives, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0014] Furthermore, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:7, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:8, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:9; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:10, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:11, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:12.

[0015] Furthermore, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:13, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:14, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:15; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:16, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:17, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:18.

[0016] Furthermore, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:19, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:20, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:21; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:22, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:23, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:24.

[0017] Furthermore, exemplary embodiments of this disclosure provide nucleic acid molecules encoding anti-GFRAL antibodies or antigen-binding fragments thereof.

[0018] Furthermore, exemplary embodiments of this disclosure provide recombinant expression vectors comprising the nucleic acid molecules.

[0019] Furthermore, exemplary embodiments of this disclosure provide cells transformed with the recombinant expression vector.

[0020] Furthermore, exemplary embodiments of this disclosure provide pharmaceutical compositions for the prevention or treatment of cancer-related anorexia-cachexia syndrome (CACS), the pharmaceutical compositions comprising an anti-GFRAL antibody or an antigen-binding fragment thereof as an active ingredient.

[0021] Furthermore, exemplary embodiments of this disclosure provide pharmaceutical compositions for the prevention or treatment of anorexia or cachexia caused by anticancer agents, the pharmaceutical compositions comprising an anti-GFRAL antibody or an antigen-binding fragment thereof as an active ingredient.

[0022] Beneficial effects

[0023] Exemplary embodiments of this disclosure relate to GFRAL antagonistic antibodies and their uses, and more specifically, to anti-GFRAL antibodies or antigen-binding fragments thereof comprising specific sequences of heavy chain CDRs and light chain CDRs. Anti-GFRAL antibodies are intended for the relief or treatment of cancer-related anorexia-cachexia syndrome and the side effects of chemotherapy anticancer drugs. Attached Figure Description

[0024] Figure 1 A phage display method using epoxy resin beads and the antigen GFRAL (mouse and human) is shown.

[0025] Figure 2 The results show the binding ability of phage libraries displayed by each round of phages to GFRAL after elution with acidic buffer by polyphage enzyme-linked immunosorbent assay.

[0026] Figure 3 The results show the results of identifying the binding ability of phage libraries displayed by each round of phages to GFRAL after elution with ligand GDF15 by polyphage enzyme-linked immunosorbent assay.

[0027] Figure 4 The results show the identification of the binding ability of four monoclonal antibodies to GFRAL after a monophagy enzyme-linked reaction adsorption assay.

[0028] Figure 5 The results show the amino acid sequences of the CDR regions of the four monoclonal clones.

[0029] Figure 6 The results show the identification of GFRAL binding ability by enzyme-linked immunosorbent assay after four monoclonal antibodies were expressed and purified into proteins.

[0030] Figure 7 The results of identifying the binding of monoclonal A11 to GFRAL by immunocytochemistry are shown.

[0031] Figure 8 The results of identifying the binding of commercial antibodies to GFRAL by immunocytochemistry are shown.

[0032] Figure 9The results show the identification of the binding ability of monoclonal A11 to GFRAL by surface plasmon resonance.

[0033] Figure 10 The results of reporter assays are shown to identify the reduction in luciferase expression caused by monoclonal A11 in GFRAL / RET / luciferase overexpression cells.

[0034] Figure 11 The results show the identification of pERK expression reduction caused by monoclonal A11 in GFRAL / RET overexpressing cells by Western blotting.

[0035] Figure 12 The numerical representation obtained by density measurement is shown. Figure 11 The result.

[0036] Figure 13 The results show the identification of the mitigation of the weight loss effect of cisplatin by monoclonal A11 in a mouse model.

[0037] Figure 14 The results show the identification of the mitigation of the appetite-depressing effect of cisplatin by monoclonal A11 in a mouse model.

[0038] Figure 15 The results show the identification of GDF15 concentrations altered by cisplatin in a mouse model.

[0039] Figure 16 The results show the identification of the mitigation of the lipid reduction effect of cisplatin by monoclonal A11 in a mouse model.

[0040] Figure 17 The results show the identification of the mitigation of cisplatin-induced muscle loss by monoclonal A11 in a mouse model.

[0041] Figure 18 The results show the results of identifying the inhibitory effect of monoclonal A11 on cisplatin activity in a mouse model by immunohistochemistry.

[0042] Figure 19 The results show the identification of the mitigation of the weight loss effect of cisplatin by monoclonal A11 in an allogeneic mouse tumor model.

[0043] Figure 20 The results show the identification of the mitigation of the appetite-depressing effect of cisplatin by monoclonal A11 in an allogeneic mouse tumor model.

[0044] Figure 21 The results show the identification of cisplatin-modified GDF15 concentrations in an allogeneic mouse tumor model.

[0045] Figure 22 The results show the identification of the mitigation of the fat reduction effect of cisplatin by monoclonal A11 in an allogeneic mouse tumor model.

[0046] Figure 23 The results show the identification of the mitigation of cisplatin-induced muscle loss by monoclonal A11 in an allogeneic mouse tumor model. Detailed Implementation

[0047] Exemplary embodiments of this disclosure provide an anti-GFRAL antibody or an antigen-binding fragment thereof, the anti-GFRAL antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:1, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:2, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:3; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:4, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:5, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:6.

[0048] Furthermore, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:7, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:8, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:9; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:10, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:11, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:12.

[0049] Furthermore, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:13, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:14, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:15; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:16, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:17, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:18.

[0050] Furthermore, exemplary embodiments of this disclosure provide an anti-GFRAL antibody or its antigen-binding fragment thereof, the anti-GFRAL antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO:19, the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO:20, and the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO:21; the light chain variable region comprising light chain CDR1, light chain CDR2 and light chain CDR3, the light chain CDR1 having the amino acid sequence shown in SEQ ID NO:22, the light chain CDR2 having the amino acid sequence shown in SEQ ID NO:23, and the light chain CDR3 having the amino acid sequence shown in SEQ ID NO:24.

[0051] Here, CDRs having the amino acids shown in SEQ ID NO:1 to SEQ ID NO:24 are shown in Table 1.

[0052] Table 1

[0053]

[0054] As used herein, the term "antibody" refers to a protein molecule (including immunoglobulin molecules) that exhibits an immune response to a specific antigen and functions as a receptor that specifically recognizes the antigen. For example, antibodies can encompass all of the following: monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antibody fragments. Furthermore, as used herein, the term "antibody" can also include bivalent or bispecific molecules (such as bispecific antibodies), diabody, trivalent antibody, or tetravalent antibody.

[0055] As used herein, the term "monoclonal antibody" refers to an antibody molecule with a single molecular composition obtained from a group of substantially identical antibodies, and such a monoclonal antibody exhibits a single affinity and affinity for a specific epitope, unlike polyclonal antibodies which can bind to multiple epitopes. The term "full-length antibody" as used herein has a structure with two full-length light chains and two full-length heavy chains, each light chain being linked to the heavy chain by disulfide bonds. The constant regions of the heavy chain have γ, μ, α, δ, and ε types, with subclasses including γ1, γ2, γ3, γ4, α1, and α2. The constant regions of the light chains have κ and λ types. IgG is a subtype, including IgG1, IgG2, IgG3, and IgG4.

[0056] As used herein, the term "heavy chain" may include both the full-length heavy chain and its fragments, comprising a variable region VH and three constant regions, wherein the variable region VH comprises an amino acid sequence having a variable region sequence sufficient to confer specificity against an antigen, and the three constant regions comprise CH1, CH2, and CH3. Furthermore, as used herein, the term "light chain" may include both the full-length light chain and its fragments, comprising a variable region VL and a constant region CL, wherein the variable region VL comprises an amino acid sequence having a variable region sequence sufficient to confer specificity against an antigen.

[0057] In exemplary embodiments of this disclosure, the terms "fragment," "antibody fragment," and "antigen-binding fragment" are used interchangeably to refer to any fragment of an antibody exhibiting antigen-binding functionality in exemplary embodiments of this disclosure. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

[0058] The antibodies or antigen-binding fragments thereof of exemplary embodiments of this disclosure may not only comprise the sequence of the antibodies described herein, but may also comprise their biological equivalents within the range exhibiting specific binding ability to GFRAL. For example, additional modifications may be applied to the amino acid sequence of the antibody to further enhance the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, the deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid variations are based on the relative similarity (e.g., hydrophobicity, hydrophilicity, charge, and size) of the amino acid side chain substituents. Based on the analysis of the size, shape, and type of the amino acid side chain substituents, arginine, lysine, and histidine were found to be positively charged residues; alanine, glycine, and serine had similar sizes; and phenylalanine, tryptophan, and tyrosine had similar shapes. Therefore, based on this, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functionally equivalents.

[0059] Furthermore, exemplary embodiments of this disclosure provide nucleic acid molecules encoding anti-GFRAL antibodies or antigen-binding fragments thereof.

[0060] As used herein, the term "nucleic acid molecule" can broadly include DNA (gDNA and cDNA) and RNA molecules, and nucleotides (the basic structural units of nucleic acid molecules) include natural nucleotides and analogs of modified sugars or bases therein. The nucleic acid molecule sequences encoding the heavy and light chain variable regions of exemplary embodiments of this disclosure can be modified, and said modifications include the addition, deletion, or non-conserved or conserved substitution of nucleotides.

[0061] Furthermore, exemplary embodiments of this disclosure provide recombinant expression vectors comprising the nucleic acid molecules.

[0062] In exemplary embodiments of this disclosure, the term "vector" as used herein refers to a self-replicating DNA molecule used to carry a cloned gene (or another piece of cloned DNA).

[0063] In exemplary embodiments of this disclosure, the term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of an operatively linked coding sequence in a particular host organism. The expression vector may preferably contain one or more selection markers. These markers are nucleic acid sequences having characteristics selectable by conventional chemical methods, including all genes capable of distinguishing transformed cells from untransformed cells. Examples include, but are not limited to, antibiotic resistance genes (e.g., amoxicillin, kanamycin, genimycin (G418), bleomycin, hygromycin, and chloramphenicol), and may be appropriately selected by those skilled in the art.

[0064] To express the DNA sequences of exemplary embodiments of this disclosure, any of a wide variety of expression regulatory sequences may be used in the vector. Examples of useful regulatory sequences may include, for example: early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTR, lac, trp, TAC or TRC systems of retroviruses, T3 and T7 promoters, major operator and promoter regions of λ phage, regulatory regions of fd-encoded proteins, promoters of 3-triphosphate glycerol kinase or other glycolytic enzymes, promoters of phosphatases (e.g., Pho5), promoters of yeast α-cross systems, and other sequences known to regulate the induction and composition of gene expression in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.

[0065] For vectors expressing antibodies according to exemplary embodiments of the present disclosure, it is possible to have a vector system in which the light chain and heavy chain are simultaneously expressed in a single vector, or a system in which the light chain and heavy chain are expressed separately in separate vectors. In the latter case, the two vectors are introduced into host cells via co-conversion and targeted conversion. Co-conversion is a method of selecting cells expressing both the light chain and the heavy chain after simultaneously introducing the vector DNA encoding the light chain and the heavy chain into the host cell. Targeted conversion is a method of selecting cells transformed with a vector containing the light chain (or heavy chain) and re-converting the selected cells expressing the light chain with a vector containing the heavy chain (or light chain) to ultimately select cells expressing both the light chain and the heavy chain.

[0066] Furthermore, exemplary embodiments of this disclosure provide cells transformed with recombinant expression vectors.

[0067] The cells capable of stably and continuously cloning and expressing the vectors of exemplary embodiments of this disclosure can be any host cell known in the art, including, for example, prokaryotic host cells (such as Eschericia coli, Bacillus strains (such as Bacillus subtilis and Bacillus thuringiensis)), Streptomyces, Pseudomonas (such as Pseudomonas putida), Proteus mirabilis, or Staphylococcus (such as Staphylococcus carnosus)), but are not limited thereto.

[0068] In methods for preparing the antibody or its antigen-binding fragment, the transformed cells can be cultured using suitable culture media and conditions known in the art. Such culture processes can be readily adapted for use by those skilled in the art, depending on the selected strain. Cell culture is categorized based on cell growth type as suspension culture and adherent culture, and based on culture type as batch culture, fed-batch culture, and continuous culture. The culture medium used for culture should appropriately meet the requirements of the specific strain.

[0069] Furthermore, exemplary embodiments of this disclosure provide pharmaceutical compositions for the prevention or treatment of cancer-related anorexia-cachexia syndrome (CACS), the pharmaceutical compositions comprising an anti-GFRAL antibody or an antigen-binding fragment thereof as an active ingredient.

[0070] The pharmaceutical compositions of exemplary embodiments of this disclosure may further comprise pharmaceutically acceptable carriers, which are typically used in formulations and include, but are not limited to, lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition to the components described above, the compositions of exemplary embodiments of this disclosure may further comprise lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0071] The pharmaceutical compositions of exemplary embodiments of this disclosure can be administered orally or parenterally. In the case of parenterally administration, they can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, the compositions for oral administration can be formulated by coating the active agent or protecting it from degradation in the stomach due to digestion of proteins or peptides. Furthermore, the compositions of exemplary embodiments of this disclosure can be administered using any device that allows delivery of the active ingredient to target cells.

[0072] The appropriate dosage of the pharmaceutical composition of the exemplary embodiments of this disclosure varies depending on factors such as (e.g., method of formulation, type of administration, patient's age, weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and responsiveness), and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

[0073] The pharmaceutical compositions of exemplary embodiments of this disclosure may be formulated in unit doses using pharmaceutically acceptable carriers and / or excipients, or introduced into multi-dose containers by methods readily practiced by those skilled in the art to which this disclosure pertains. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, suppository, granule, tablet, or capsule, and may additionally contain dispersants or stabilizers.

[0074] Furthermore, exemplary embodiments of this disclosure provide pharmaceutical compositions for the prevention or treatment of anorexia or cachexia caused by anticancer agents, the pharmaceutical compositions comprising an anti-GFRAL antibody or an antigen-binding fragment thereof as an active ingredient.

[0075] More preferably, the anticancer agent may be one or more selected from the group consisting of: cisplatin, oxaliplatin, carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, but is not limited thereto.

[0076] Furthermore, the anti-GFRAL antibody or its antigen-binding fragment of the exemplary embodiments of this disclosure can be used as an anticancer adjuvant. In the exemplary embodiments of this disclosure, an anticancer adjuvant can refer to the relief of side effects caused by administration of an anticancer agent. That is, by administering the anticancer adjuvant of the exemplary embodiments of this disclosure in combination with an anticancer agent, it is possible to prevent the occurrence of various side effects caused by the anticancer agent. The adjuvant of the exemplary embodiments of this disclosure can be administered simultaneously, separately, or sequentially with the anticancer agent. The order of administration of the anticancer adjuvant according to the exemplary embodiments of this disclosure (i.e., which of the anticancer agent and the anticancer adjuvant is administered simultaneously, separately, or sequentially at what time) can be determined by a physician or expert. The order of administration may vary depending on many factors.

[0077] Example

[0078] In the following description, embodiments will be detailed to aid in understanding this disclosure. However, the following embodiments are merely illustrative of the content of this disclosure, and the scope of this disclosure is not limited to the following embodiments. Exemplary implementations of this disclosure are provided to explain this disclosure more fully to those skilled in the art.

[0079] <Example 1> Cell Culture

[0080] HEK-293FT cells (human embryonic kidneys) were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, and Expi-293F cells (human embryonic kidneys) were cultured in Expi-293 expression medium (Gibco).

[0081] <Example 2> Phage Display Method

[0082] Phage display using scFv libraries derived from human patients was performed after attaching recombinant human GFRAL (#9647-GR, R&D systems) or recombinant mouse GFRAL (#9844-GR, R&D systems) to Dynabeads M-270 epoxy beads (#14301, Invitrogen). Alternatingly, each of the human and mouse GFRAL samples was panned twice. After reacting the phages with ordinary beads at room temperature for 1 hour, the unbound supernatant was reacted with GFRAL-bound beads for 2 hours. After washing 3–8 times, the phages bound to the GFRAL were eluted using recombinant human GDF15 ligand (#8146-GD, R&D systems) or acidic buffer (0.1 M glycine-hydrogen chloride, pH 2.2).

[0083] <Example 3> Polyclonal phage enzyme-linked immunosorbent assay

[0084] The 96-well half-region plates (#3690, Corning) were coated with recombinant human GFRAL, recombinant mouse GFRAL, or bovine serum albumin overnight at 4°C, followed by a single wash with PBS. Subsequently, they were blocked with PBS (containing 5% skim milk) at 37°C for 1 hour. After reacting with each round of phage libraries at 37°C for 2 hours, followed by 5 washes with PBS, the plates were reacted with HRP-conjugated antiphage antibody (#11973-MM05T-H, SinoBiological) at 37°C for 1 hour, followed by 5 washes with PBS. Finally, color development was performed using TMB at room temperature for 10 minutes, followed by termination with a stop solution. The absorbance was then measured at 450 nm using a microplate reader.

[0085] <Example 4> Monoclonal phage enzyme-linked immunosorbent assay

[0086] To harvest monoclonal phages, SB medium (containing carbenicillin (#C1389, Sigma) at a concentration of 50 μg / mL) was dispensed into each well of a 96-well deep-well plate (#90060, Bioneer), and colonies containing the phage library were inoculated. The plates were then incubated at 37°C and 250 rpm for 3 hours. Subsequently, M13K07 helper phage was introduced at 10... 10Phages were treated at a concentration of 100 phages / mL and cultured for an additional 2 hours under the same conditions. Subsequently, kanamycin (#K4000, Sigma) was used at a concentration of 70 μg / mL and cultured overnight. During the phage ELISA assay, a portion of the blocking post-treatment medium was used, and the remaining procedures were performed in the same manner as in the polyclonal phage ELISA assay.

[0087] <Example 5> Purification method using high performance liquid chromatography

[0088] The scFv sequence of a single clone capable of binding GFRAL was transferred into an expression vector constructed using the sfiI restriction enzyme (#pfuse hg1fc2, InvivoGen), and then transfected into Expi293F cells. Transfection was performed using the ExpiFectamine 293 transfection kit (#A14524, Gibco). Expi293F cells were transfected at a rate of approximately 2.5 × 10⁻⁶ cells / year. 6 Cells were cultured at a density of 100 cells / mL in 500 mL flasks and treated with a mixture obtained by mixing ExpiFectamine 293 reagent and scFv expression vector in Opti-MEM (#31985-070, Gibco) medium, followed by overnight incubation at 37°C and 125 rpm. The next day, after treatment with ExpiFectamine 293 transfection enhancer 1 and transfection enhancer 2, the cells were cultured for an additional 3 days to generate antibodies in scFv-Fc form. Subsequently, the supernatant was purified using an AKTA Prime Plus (GE Healthcare) purifier and an IgG separation column (#17-0404-01, GE Healthcare). After attaching the antibody to the column with 20 mM sodium phosphate buffer (pH 7.4), elution was performed with 0.1 M glycine-hydrochloride buffer (pH 2.7). The solution was then concentrated using an Amicon Ultra-4-30K filter (#UFC803024, Millipore) and subsequently sterilized using a Spin-X filter (#8160, Corning). Endotoxins were removed using an endotoxin removal column (#88274, Thermo Fisher Scientific) and the concentration was measured using a BCA kit (#23227, Thermo Fisher Scientific).

[0089] <Example 6> Antibody Enzyme-Linked Immunosorbent Assay

[0090] 96-well half-region plates were coated with recombinant human GFRAL, recombinant mouse GFRAL, or bovine serum albumin overnight at 4°C. Enzyme-linked immunosorbent assay (ELISA) was performed using a cloned scFv-Fc antibody as the primary antibody and an HRP-conjugated anti-human IgG antibody (#Ab97225, Abcam) as the secondary antibody.

[0091] <Example 7> Transfection into HEK-293FT cells

[0092] HEK-293FT cells were loaded at approximately 1 × 10⁻⁶ 6 Cells were seeded at a density of [number] cells / well into 6-well plates and treated with a mixture obtained by mixing Lipofectamine 2000 reagent (#11668-027, Thermo Fisher Scientific), GFRAL expression vector (#OHu31183D, GenScript), and RET expression vector (#HG11997-CF, Sino Biological) in Opti-MEM medium at a 1:1 ratio, followed by overnight culture at 37°C. The next day, the supernatant was removed, and the medium was replaced with fresh medium supplemented with 10% fetal bovine serum. After inoculation with a vector capable of expressing luciferase during ERK signal transduction, the plates were then cultured overnight at 37°C, and the supernatant was removed the next day. The process of replacing the medium with fresh medium supplemented with 10% fetal bovine serum was further performed.

[0093] <Example 8> Immunocytochemistry

[0094] The transfected HEK-293FT cells were loaded at approximately 5 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well into 4-well cell culture slides (#154526, Thermo Fisher Scientific) and immobilized by treatment with 4% polyoxymethylene (POM) at room temperature for 10 minutes. Afterward, the slides were blocked for 1 hour with 0.1% PBST (Tween 20) containing 1% BSA and 5% goat serum. Following this, the slides were reacted for 1 hour with a clonal antibody purified with a primary antibody or a commercially available GFRAL antibody (#Ab107719, Abcam). After washing 3–5 times with PBST, the slides were reacted for 1 hour with FITC-conjugated anti-human IgG antibody (#97224, Abcam) or anti-rabbit IgG antibody (#A11008, Invitrogen). After washing 3–5 times with PBST, the slides were treated with PBS containing DAPI for 15 minutes. After mounting, fluorescence was observed under a microscope.

[0095] <Example 9> Surface Plasmon Resonance Analysis

[0096] The binding of clonal antibodies to recombinant human GFRAL was measured using the Biacore SPR system. Recombinant human GFRAL was dissolved in 20 mM sodium acetate as a ligand and then immobilized onto a PEG chip via amine bonds at pH 6.0. Binding was then measured using the clonal antibody dissolved in PBS at pH 7.4 as the analyte, followed by injection at a flow rate of 30 μL / mL. The time intervals for association and dissociation were 4 min and 6 min, respectively. The sensor surface was regenerated by injecting 5 mM–10 mM sodium hydroxide. Association curves and the association constant K were calculated using Scrubber2 (Biologic Software). a dissociation constant K d and equilibrium constant K D The value of .

[0097] As a result of the experiment, the following was obtained: Figure 9 The association curve is shown below. The association constant K is also shown. a The value is 5.556 × 10 -5 / M·s, and the dissociation constant K d The value is 1.083 × 10 -3 / s. Equilibrium constant K D The value is 1.95 nM, using K D =K d / K a The formula is used for calculation.

[0098] <Example 10> Analysis of luciferase expression by antibody

[0099] The transfected HEK-293FT cells were loaded at approximately 7 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well into 96-well plates and kept in serum-deficient conditions for 2 hours, followed by reaction with clonal antibody for 2 hours. Afterward, the cells were treated with recombinant human GDF15 for 5 minutes, and the luminescence intensity was measured using a reagent containing luciferase substrate (#E2610, Promega).

[0100] As such Figure 10 The experimental results shown indicate that the luminescence value increased when treated with GDF15 alone, and decreased in a concentration-dependent manner in the experimental group pretreated with clones A11 and D12.

[0101] <Example 11> Analysis of pERK expression by antibody

[0102] The transfected HEK-293FT cells were loaded at approximately 2 × 10⁻⁶. 5Cells were seeded at a density of 10 cells / well in 24-well plates and incubated for 2 hours under serum-deficient conditions, followed by reaction with clonal antibody for 2 hours. Subsequently, cells were treated with recombinant human GDF15 for 5 minutes, washed with PBS, and harvested for Western blotting. Cytoplasmic or cell membrane proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Cells were then cultured with primary antibody followed by HRP-conjugated secondary antibody. Images were visualized using ECL assay kits (#34095, Thermo Fisher Scientific, #RPN2209, GE Healthcare) and quantified using ECL hyperfilm (AGFA, Morstel). Images on the film were quantified using ImageJ and graphically visualized using an AAT Bioquest IC50 calculator.

[0103] As such Figure 11 The experimental results shown indicate that pERK expression levels increased in the group treated with GDF15 alone, while pERK expression levels decreased in a concentration-dependent manner in the group pretreated with clone A11. The measured IC50 value was 4.9 μg / mL. Images from the film were quantified and shown. Figure 12 middle.

[0104] <Example 12> Analysis of the alleviation of cisplatin side effects by antibodies

[0105] Cisplatin was injected into 8-week-old mice at a concentration of 10 mg / kg, followed by an injection of a 10 mg / kg null control antibody or clone A11 to examine whether cisplatin-induced side effects were alleviated. The drug was administered twice weekly.

[0106] As a result of the experiment, such as Figure 13 As shown, the cisplatin-induced weight loss was recovered by A11. And as... Figure 14 As shown, the appetite-reducing effect was also alleviated. Figure 15 As shown, no difference in GDF15 expression levels was found between the control group and the A11 treatment group. Specific changes in fat and muscle weight were quantified and illustrated. Figure 16 and Figure 17 In the middle. For example Figure 18 As shown, the effect of A11 was found to be produced by inhibiting the action of GFRAL in the mouse brain.

[0107] <Example 13> Immunohistochemistry

[0108] Brain tissue obtained by perfusion fixation of mice used in Example 12 was frozen sectioned to a thickness of 30 μm. The sectioned tissue was blocked for 1 hour with 0.1% PBST (Tween 20) containing 1% BSA and 5% goat serum, followed by reaction for 1 hour with commercial GFRAL antibody (#Ab107719, Abcam) and c-Fos antibody (#sc-166940, Santa Cruz Biotechnology) as primary antibodies. After washing five times with PBST, the tissue was reacted for 1 hour with Alexa fluor 488-conjugated anti-mouse IgG antibody (#A11001, Invitrogen) and Alexa fluor 594-conjugated anti-rabbit IgG antibody (#A11008, Invitrogen). After washing five times with PBST, the tissue was treated with PBS containing DAPI for 15 minutes. After mounting, fluorescence was observed under a microscope.

[0109] <Example 14> Analysis of antibody-mediated cisplatin side effect relief in an allogeneic mouse tumor model

[0110] B16F10-Luc cells (at approximately 1 × 10⁻⁶) 6 (Number of doses) was injected into 8-week-old mice. After the tumors grew to a certain size or larger, cisplatin was injected at a concentration of 10 mg / kg, and a null control antibody or clone A11 was injected at 10 mg / kg to examine whether the side effects induced by cisplatin were alleviated in the chemotherapy model. The drug was administered twice a week.

[0111] As a result of the experiment, it was found that... Figure 19 As shown, the weight loss induced by cisplatin was restored by A11 under conditions involving chemotherapy. Furthermore, as... Figure 20 As shown, the appetite-reducing effect was also alleviated. Figure 21 As shown, no difference in GDF15 expression levels was found between the control group and the A11 treatment group. Specific changes in fat and muscle mass were quantified and illustrated. Figure 22 and Figure 23 middle.

[0112] Although specific parts of this disclosure have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred exemplary embodiments, and the scope of this disclosure is not limited. Therefore, the essential scope of this disclosure will be defined by the appended claims and their equivalents.

Claims

1. An anti-GFRAL antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, the amino acid sequence of the heavy chain CDR1 being set forth in SEQ ID NO: 7, the amino acid sequence of the heavy chain CDR2 being set forth in SEQ ID NO: 8, the amino acid sequence of the heavy chain CDR3 being set forth in SEQ ID NO: 9; the light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3, the amino acid sequence of the light chain CDR1 being set forth in SEQ ID NO: 10, the amino acid sequence of the light chain CDR2 being set forth in SEQ ID NO: 11, the amino acid sequence of the light chain CDR3 being set forth in SEQ ID NO:

12. 2.A nucleic acid molecule encoding the anti-GFRAL antibody or the antigen-binding fragment thereof of claim 1. 3.A recombinant expression vector comprising the nucleic acid molecule of claim 2. 4.A cell transformed with the recombinant expression vector of claim 3. 5.A pharmaceutical composition for preventing or treating cancer-associated cachexia syndrome (CACS), comprising the anti-GFRAL antibody or the antigen-binding fragment thereof of claim 1 as an active ingredient. 6.Use of the anti-GFRAL antibody or the antigen-binding fragment thereof of claim 1 in the manufacture of a medicament for cancer-associated cachexia syndrome. 7.A pharmaceutical composition for preventing or treating anorexia or cachexia caused by an anti-cancer agent, comprising the anti-GFRAL antibody or the antigen-binding fragment thereof of claim 1 as an active ingredient, and further comprising a pharmaceutically acceptable carrier. 8.Use of the anti-GFRAL antibody or the antigen-binding fragment thereof of claim 1 in the manufacture of a medicament for preventing or treating anorexia or cachexia caused by an anti-cancer agent.

9. The use of claim 8, wherein, The anti-cancer agent is one or more selected from the group consisting of cisplatin, oxaliplatin, carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate.

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