Anti-CD24 antibody or antigen-binding fragment thereof, and use thereof

Abstract

The present invention relates to a novel anti-CD24 antibody and use thereof. The CD24-specific antibody according to one aspect increases phagocytosis activity of macrophages in tumor tissues, and thus can be used as a universal anticancer agent for effectively preventing or treating various cancers associated with CD24 without being limited to carcinomas, and has high binding affinity to both humans and mice, and thus can be clinically and preclinically used.

Description

Anti-CD24 antibody or antigen-binding fragment thereof and use thereof

[0001] It relates to a novel anti-CD24 antibody and its use.

[0002] Among the don't eat me signals, the most studied anti-phagocytic protein is CD47, and therapeutic antibodies targeting CD47 have shown promising anti-tumor efficacy in preclinical and clinical trials, but have the problem of causing non-target toxicities such as anemia and thrombocytopenia.

[0003] CD24, a recently discovered novel "don't eat me" signal, has been reported to interact with Siglec-10 on immune cells, enabling immune evasion and protecting tumor cells from immune attack. CD24 is overexpressed in 68% of human cancers, with higher expression compared to other immune checkpoints, but lower expression levels in normal tissues and human red blood cells. Therefore, monoclonal antibodies targeting CD24 could block its interaction with Siglec-10, thereby enhancing phagocytosis and inducing antitumor effects in various cancers, while avoiding the side effects associated with CD47.

[0004] CD24 was first identified as an immune checkpoint in 2019, but was previously developed for tumor targeting. Therefore, previously reported antibodies were limited to targeting CD24 overexpressed on cancer cells, and it was unclear whether they could block the interaction of immune cells with Siglec-10. Furthermore, most antibodies were mouse monoclonal IgG antibodies, making them unsuitable for clinical use.

[0005] These researchers have studied to develop a novel anti-CD24 antibody that can provide a potent antitumor effect not limited to cancer by increasing the phagocytic activity of macrophages, and can be applied to both humans and mice, making it applicable to preclinical and clinical studies.

[0006] One aspect is to provide an antibody or antigen-binding fragment thereof that specifically binds to CD24.

[0007] Another aspect provides a gene encoding the antibody or an antigen-binding fragment thereof; a vector comprising the gene; or a recombinant cell comprising the gene or the vector.

[0008] Another aspect is to provide an antibody-drug conjugate (ADC) comprising the antibody or an antigen-binding fragment thereof.

[0009] Another aspect is to provide a pharmaceutical composition for preventing or treating cancer comprising the antibody or antigen-binding fragment thereof; or the antibody-drug conjugate.

[0010] Another aspect provides a method of preventing or treating cancer comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD24; or an antibody-drug conjugate thereof.

[0011] Another aspect provides the use of the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate, for use in the manufacture of a pharmaceutical preparation for the prevention or treatment of cancer.

[0012] One aspect provides an antibody or antigen-binding fragment thereof that specifically binds to CD24.

[0013] As used herein, “antibody,” “antigen-binding region or site,” or “antigen-binding polypeptide” refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. Since the present invention relates to a CD24 antibody that specifically binds to CD24, “antibody” used herein without any modifiers, unless otherwise specifically designated, may refer to an antibody that specifically binds to CD24.

[0014] The antibody or antigen-binding fragment thereof of the present invention may be a whole antibody; or any antigen-binding fragment or single chain thereof. The term "whole antibody" refers to a structure in which two full-length light chains and two full-length heavy chains are linked by disulfide bonds. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3, and IgG4.

[0015] As used herein, “antigen-binding fragment” refers to a portion of an antibody that contains a portion capable of binding to an antigen, which is a fragment of the entire antibody structure.

[0016] The antibody or antigen-binding fragment thereof of the present invention may comprise any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen among the whole antibody. Examples thereof include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0017] In this specification, “heavy chain (HC or CH)” means a full-length heavy chain and fragments thereof comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region (VR) sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3.

[0018] As used herein, “Light Chain (LC or CL)” means a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0019] As used herein, the term “complementary determining region (CDR)” (i.e., CDR1, CDR2, and CDR3) refers to a region among the variable regions of an antibody that has antigen-binding specificity. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. As used herein, the CDRs included in the heavy chain variable region are referred to as “H-CDRs,” and the CDRs included in the light chain variable region are referred to as “L-CDRs.”

[0020] In one specific example, the antibody or antigen-binding fragment thereof may include at least one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, an antibody variant, a multimeric antibody, a minibody, a diabody, a triabody, a tetrabody, a Bis-scFv, a nanobody, a bispecific antibody, and a multispecific antibody. Preferably, the antibody of the present invention may be an antibody comprising a scFv or scFv-Fc fragment.

[0021] The “Fc” region herein may comprise two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. These two heavy chain fragments may be joined together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0022] As used herein, a “Fab fragment” may be composed of one heavy chain and one light chain, each containing only CH1 and a variable region. The heavy chain of a Fab molecule may not form a disulfide bond with another heavy chain molecule.

[0023] In this specification, “Fab' fragment” includes, in addition to the Fab fragment, a region between the CH1 and CH2 domains of the heavy chain, which can form a disulfide bond between two heavy chains of two molecules of the Fab' fragment to form a F(ab')2 molecule.

[0024] As used herein, the term “F(ab')2 fragment” includes two heavy chains and two light chains, each comprising a variable region, CH1, and a portion of a constant region between the CH1 and CH2 domains, as described above, thereby forming an intrachain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments can bind to each other by the disulfide bond therebetween.

[0025] As used herein, an “Fv fragment” may be an antibody comprising each variable region of a heavy chain and a light chain, but not a constant region. A “single-chain variable fragment,” “single-chain Fv,” or “scFv” antibody fragment may refer to a fusion protein comprising the VH and VL domains of an antibody, wherein these domains are present within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. An scFv-Fc may be an Fc linked to an scFv. A diabody may comprise two molecules of scFv.

[0026] In one specific embodiment, the antibody that specifically binds to CD24 of the present invention comprises a heavy chain complementary determining region (H-CDR) comprising H-CDR1, H-CDR2, and H-CDR3; and a light chain complementary determining region (L-CDR) comprising L-CDR1, L-CDR2, and L-CDR3.

[0027] An H-CDR1 comprising an amino acid sequence of SEQ ID NO: 1 or 2; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith;

[0028] An H-CDR2 comprising the amino acid sequence of SEQ ID NO: 3 or 4; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith;

[0029] An H-CDR3 comprising the amino acid sequence of SEQ ID NO: 5 or 6; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith;

[0030] An L-CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 8; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith;

[0031] An L-CDR2 comprising an amino acid sequence of sequence AAS or sequence EVT; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith; and

[0032] It may comprise an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or 10; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith.

[0033] In one specific example, the heavy chain variable region may include an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 5.

[0034] In one specific example, the heavy chain variable region may include an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 2; an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0035] In one specific example, the light chain variable region may comprise an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; an L-CDR2 comprising the amino acid sequence of sequence AAS; and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0036] In one embodiment, the light chain variable region may comprise an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 8; an L-CDR2 comprising the amino acid sequence of sequence EVT; and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0037] In one specific example, the antibody or antigen-binding fragment thereof may comprise an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 3; an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 5; an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; an L-CDR2 comprising the amino acid sequence of sequence AAS; and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0038] In one specific example, the antibody or antigen-binding fragment thereof may comprise an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 2; an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 8; an L-CDR2 comprising the amino acid sequence of the sequence EVT; and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0039] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11 or 12; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity therewith; and

[0040] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or 14; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity therewith.

[0041] In one specific example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13.

[0042] In one specific example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0043] In one embodiment, the antibody or antigen-binding fragment thereof may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 15 to 18; a portion thereof; or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity therewith.

[0044] In one specific example, the heavy chain variable region and the light chain variable region may be linked directly or by a linker.

[0045] The antibodies or antigen-binding fragments thereof that specifically bind to CD24 of the present invention may include "variants." Such variants may refer to polypeptides in which one or more amino acid residues are inserted, deleted, added, and / or substituted from the polypeptide sequence. Accordingly, as long as the desired biological activity and / or structure of the antibody and / or antigen-binding fragment is maintained, the variants may be linked to other polypeptides to form fusion polypeptides. The variant may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of the antibody or antigen-binding fragment thereof, for example, may have about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity.

[0046] As used herein, “homology” or “identity” may refer to the overall relatedness between nucleic acid molecules and / or between polypeptides. In one embodiment, nucleic acids or polypeptides may be considered “substantially identical” to each other if their sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculating the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes. In certain embodiments, the length of the aligned sequence for comparison purposes can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence.

[0047] The antibody or antigen-binding fragment thereof of the present invention does not occur naturally and may be synthesized chemically or recombinantly. Specifically, it may be synthesized by linking an Fc fragment to an scFv fragment using a linker.

[0048] In one specific example, the antibody or antigen-binding fragment thereof may be a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0049] The antibody of the present invention or an antigen-binding fragment thereof can have high binding affinity to both human CD24 and mouse CD24. That is, the antibody of the present invention can be applied to both clinical and preclinical settings.

[0050] The antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of an anti-CD24 antibody but also a biological equivalent thereof, or may be modified, as long as it can specifically recognize CD24. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. In addition, the antibody or antigen-binding fragment thereof may be modified by conjugation or linkage, glycosylation, tag attachment, or a combination thereof.

[0051]

[0052] Another aspect provides a gene encoding an antibody or antigen-binding fragment thereof that specifically binds to CD24.

[0053] Another aspect provides a vector comprising the above gene.

[0054] The term "coding" herein refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process, either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene in a cell or other biological system produces the protein. Both the coding strand, which is identical to the mRNA sequence and is the nucleotide sequence usually provided in the sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of that gene or cDNA.

[0055] As used herein, the term "vector" refers to any nucleic acid construct capable of delivering or directing the movement of foreign genetic material into a target cell where the polynucleotide can be replicated and / or expressed. The vector comprises the construct to be delivered. The vector may be a linear molecule or a circular molecule. The vector may be integrating or non-integrating.

[0056] In one specific example, the vector may be any one selected from the group consisting of a plasmid, a cosmid, a virus, a phage, a recombinant expression cassette, and a transposon, but is not limited thereto, and it is obvious that any vector that is suitable for the purpose of the present invention among vectors that can be commonly used may be used.

[0057] It is obvious that a gene sequence having a part of the sequence deleted, modified, substituted or added from the gene or vector of the present invention can also be used in the present application, as long as it has the same or corresponding function as the gene of the present invention.

[0058] In one specific example, the gene may comprise a gene sequence of SEQ ID NO: 20 or 21, a portion thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0059]

[0060] Another aspect provides a recombinant cell comprising the gene or the vector.

[0061] The recombinant cell may comprise a single vector containing a single gene encoding the antibody or antigen-binding fragment. The recombinant cell may be introduced with a single vector each containing a gene encoding the light chain and heavy chain of the antibody or antigen-binding fragment; or may be introduced with multiple vectors containing each gene. Each gene or vector may be introduced into the recombinant cell independently, simultaneously, sequentially, or in reverse order.

[0062] In one specific example, the recombinant cell may be an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, a hepatocyte, a plant cell, an animal cell, a mammalian cell, a mouse cell, a non-human primate cell, or a human cell.

[0063]

[0064] Another aspect provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to CD24.

[0065] In this specification, “antibody-drug conjugate (ADC)” may refer to a substance having a structure in which a cytotoxic small molecule drug (payload) is conjugated to an antibody or an antigen-binding fragment thereof that binds to a specific target antigen on the cell surface.

[0066] In one specific example, the antibody drug conjugate may be a therapeutic agent (drug) linked to an antibody or an antigen-binding fragment thereof having target specificity via a linker. That is, the antibody drug conjugate may include an antibody or an antigen-binding fragment thereof that specifically binds to CD24; a drug moiety; and a linker connecting the antibody or antigen-binding fragment thereof and the drug moiety.

[0067] The linker may comprise a cleavable linker or a non-cleavable linker. The cleavable linker, like a peptide linker, can be cleaved by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease. For example, the cleavable linker may be any one selected from the group consisting of, but not limited to, a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, a β-glucuronide-based linker, and a β-galactoside-based linker. The non-cleavable linker can release the drug after the antibody is non-selectively degraded by intracellular hydrolysis.

[0068] In one specific example, the drug or therapeutic agent may be any one selected from the group consisting of a chemotherapeutic compound, a cytotoxic compound, an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, and combinations thereof. For example, the cytotoxic compound may be any one selected from the group consisting of a mitotic inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, and combinations thereof, but is not limited thereto. More specifically, the therapeutic agent may be, but is not limited to, MMAE_Protein A, cotinine-duocarmycin, and Herceptin.

[0069]

[0070] Another aspect provides a pharmaceutical composition for preventing or treating cancer comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD24; or an antibody-drug conjugate thereof.

[0071] Another aspect provides a method of preventing or treating cancer comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD24; or an antibody drug conjugate thereof.

[0072] Another aspect provides the use of the antibody or antigen-binding fragment thereof; or the antibody drug conjugate, for use in the manufacture of a pharmaceutical preparation for the prevention or treatment of cancer.

[0073] As used herein, "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition of the present invention to a subject. For preventive purposes, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0074] As used herein, "treatment" refers to any action that improves the symptoms of a disease or provides benefit by administering the composition of the present invention to a subject. As used herein, the terms "treatment," "palliation," and "improvement" may be used interchangeably. A therapeutic benefit refers to any therapeutically significant improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0075] The antibody or antigen-binding fragment thereof or the antibody-drug conjugate can increase the phagocytic activity of macrophages within tumor tissues. The antibody of the present invention can enhance the ability of macrophages to phagocytose cancer cells within tumor tissues and their ability to penetrate into tumor tissues. In addition, it can increase the expression of TNFα, a representative activity marker of anti-tumor M1 macrophages, and Granzyme B and IFNγ, representative activity markers of cytotoxic T cells (CD8+ T cells).

[0076] In this specification, "cancer" or tumor may collectively refer to a disease caused by cells that have aggressive characteristics, such as dividing and proliferating in defiance of normal growth limits, invasive characteristics, such as infiltrating surrounding tissues, and metastatic characteristics, such as spreading to other parts of the body. The cancer may be a solid tumor or a blood cancer.

[0077] For example, the cancers include pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, It can be heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, triple-negative breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, or thymic cancer.

[0078] Specifically, the cancer may include at least one selected from the group consisting of breast cancer, lung cancer, skin cancer, kidney cancer, colon cancer, head and neck cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, melanoma, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer, and pancreatic cancer.

[0079] The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, "administration" refers to introducing a given substance into a patient by any suitable method.

[0080] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including the type of the patient’s disease, the severity of the disease, the type of active ingredient administered, the type of formulation, the patient’s age, sex, weight, health condition, diet, sensitivity, the time and method of drug administration, the combination of the composition or concurrently used drugs, and other factors well known in the medical field.

[0081] The dosage of the pharmaceutical composition for the prevention or treatment of diseases according to the present invention may range from 0.01 ug / kg to 10 g / kg per day, specifically from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0082] The above dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration may be done in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage per kg as for humans may be used, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (e.g., heart) of the target animal and the human.

[0083] The route of administration of the pharmaceutical composition may be any common route as long as the drug can reach the target tissue. For example, the administration may be oral or parenteral, and may include, but is not limited to, topical ocular administration (e.g., periocular (e.g., subTenon's), subconjunctival, intraocular, intravitreal, intracameral, subretinal, suprachoroidal, and retrobulbar administration), intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In addition, the pharmaceutical composition of the present invention may be administered by any device through which the active ingredient can move to the target cell. It is preferable that the route of administration of the pharmaceutical composition of the present invention be determined depending on the type of disease to which it is applied.

[0084] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., formulated according to conventional methods, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, external preparations, suppositories, sterile injection solutions, and implantable preparations. The pharmaceutical composition may further comprise, in addition to the active ingredient, a pharmaceutically acceptable excipient that can be used in formulation.

[0085] The above excipients include carriers, vehicles, diluents, solvents, for example, monohydric alcohols, for example, ethanol, isopropanol, and polyhydric alcohols, for example, glycerol, and edible oils, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, oily esters, for example, ethyl oleate, isopropyl myristate; It may include at least one selected from the group consisting of binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, glidants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, ion exchange resins, etc., but is not limited thereto.

[0086] The carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0087] The pharmaceutical composition of the present invention being formulated in the form of a parenteral administration dosage form may mean that it is administered by a method such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In this case, in order to formulate the pharmaceutical composition in the parenteral administration dosage form, the active ingredient is mixed with a stabilizer or buffer in water to prepare a solution or suspension, and this solution or suspension can be prepared in a unit dosage form of an ampoule or vial.

[0088] The pharmaceutical composition of the present invention may be formulated in the form of an oral administration dosage form, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc. These oral administration dosage forms may, in addition to the active ingredient according to the typical composition of each dosage form, contain pharmaceutically acceptable carriers, such as diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine, or lubricants such as silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol.

[0089] If the oral dosage form is a tablet, it may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidine, and in some cases, a disintegrating agent such as starch, agar, alginic acid or its sodium salt, an effervescent mixture and / or an absorbent, a coloring agent, a flavoring agent or a sweetening agent.

[0090] In addition, the pharmaceutical composition may be sterilized or may further contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for osmotic pressure control and / or buffers, and may further contain other therapeutically useful substances, and may be formulated according to conventional methods of mixing, granulating or coating.

[0091] The content of the antibody in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the purpose of use of the pharmaceutical composition, the form of the formulation, etc., and may be, for example, 0.001 to 99 wt%, 0.001 to 90 wt%, 0.001 to 50 wt%, 0.01 to 50 wt%, 0.1 to 50 wt%, or 1 to 50 wt% based on the total weight of the pharmaceutical composition.

[0092]

[0093] Another aspect provides a health functional food composition for preventing or improving cancer comprising the anti-CD24 antibody or an antigen-binding fragment thereof.

[0094] The above health functional food composition can be provided for the prevention, improvement or treatment of cancer.

[0095] In this specification, “health functional food” refers to a food manufactured or processed using a specific ingredient as a raw material or a specific ingredient contained in a food raw material by extraction, concentration, purification, mixing, etc. for the purpose of health supplementation, and refers to a food designed and processed so that the above-mentioned ingredient can sufficiently exert bioregulatory functions on the body, such as biodefense, regulation of biological rhythm, and prevention and recovery of diseases.

[0096] There are no specific restrictions on the types of the above foods. Examples of the above foods include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and include all health foods in the conventional sense.

[0097] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0098] The effective dose of the antibody or its antigen-binding fragment contained in the above health functional food composition may be used in accordance with the effective dose of the above pharmaceutical composition, but may be below the above range in the case of long-term intake for the purpose of health and hygiene or health control, and the effective ingredient may be used in an amount above the above range because there is no problem in terms of safety.

[0099]

[0100] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0101] A CD24-specific antibody or antigen-binding fragment thereof according to one aspect can be used as a universal anticancer agent that effectively prevents or treats various cancers related to CD24, not limited to cancer, by increasing the phagocytic activity of macrophages in tumor tissues, and has high binding affinity to both humans and mice, making it suitable for clinical and preclinical use.

[0102] Figure 1 is a schematic diagram of the selection process and development strategy for anti-CD24 antibodies.

[0103] Figures 2 and 3 are graphs showing the results of monoclonal phage ELISA for antibody selection from a 3-round polyclonal pool.

[0104] Figure 4 is a graph analyzing the binding affinity of an anti-CD24 antibody to a breast cancer cell line according to one specific example.

[0105] Figure 5 is a graph analyzing the binding affinity of an anti-CD24 antibody according to one specific example to human and mouse breast cancer cell lines, lung cancer cell lines, liver cancer cell lines, and melanoma cell lines.

[0106] Figure 6 is a schematic diagram of an anti-CD24 antibody and its phagocytic activity according to one specific example.

[0107] Figure 7 is a graph showing flow cytometry analysis of the phagocytic activity of macrophages in a lung cancer cell line after administration of an anti-CD24 antibody according to one specific example.

[0108] Figure 8 is a graph showing flow cytometry analysis of the phagocytic activity of macrophages in a breast cancer cell line after administration of an anti-CD24 antibody according to one specific example.

[0109] Figure 9 is a schematic diagram of the tumor mouse model construction and antibody treatment experiment.

[0110] Figure 10 is a graph showing tumor volume and weight measured after anti-CD24 antibody treatment according to one specific example in a tumor mouse model.

[0111] Figure 11 is a graph showing bioluminescence signals measured after anti-CD24 antibody treatment according to one specific example in a tumor mouse model.

[0112] Figure 12 shows the results of flow cytometry analysis of cancer cells in macrophages after treatment with an anti-CD24 antibody according to one specific example in a tumor mouse model.

[0113] Figure 13 is a graph showing changes in the ratio of M1 and M2 macrophages after treatment with an anti-CD24 antibody according to one specific example in a tumor mouse model.

[0114] Figure 14 is a graph showing changes in the expression of TNFα markers in macrophages after treatment with anti-CD24 antibodies according to one specific example in a tumor mouse model.

[0115] Figure 15 is a graph showing changes in the expression of Granzyme B and IFNγ markers in cytotoxic T cells after treatment with an anti-CD24 antibody according to one specific example in a tumor mouse model.

[0116] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0117]

[0118] Example 1. Biopanning

[0119] Candidate anti-CD24 antibodies were selected through biopanning from a human-binding antibody phage library. Figure 1 is a schematic diagram of the selection process and development strategy for anti-CD24 antibodies.

[0120] Specifically, solution-phase panning was performed using recombinant human CD24 protein (#CD4-H52H3, Acro Biosystems), recombinant mouse CD24 protein (#CD4-M52H7, Acro Biosystems), and Dynabeads M-270 epoxy beads (#14301, Invitrogen; Thermo Fisher Scientific). The beads were bound to human or mouse CD24 protein by incubation overnight at 37°C. Each panning round alternated between bound beads. The protein-bound beads were then exposed to a human combinatorial antibody phage display library for 2 h at 25°C. After incubation, the beads were washed with PBST (0.05% Tween 20 in phosphate-buffered saline (PBS, Cytiva)), and the remaining bound phages were eluted with 0.2 M glycine-HCl containing 0.1% bovine serum albumin (BSA) at pH 2.2. The collected phages were then infected with 100 mg / mL carbenicillin into competent E. coli XL1-Blue cells and cultured at 37°C for 2 h to propagate. The culture was then diluted in Super Broth (SB) medium, and M13KO7 helper phage (#N0315S; New England Biolabs) was added and cultured for an additional 2 h at 37°C. Subsequently, 70 μg / mL kanamycin sulfate was added, and the culture was maintained at 37°C overnight. The following day, the phage particles were concentrated and purified by polyethylene glycol / NaCl precipitation and resuspended in osmotic buffered saline containing 1% BSA.

[0121]

[0122] Example 2. Enzyme-linked immunosorbent assay (ELISA)

[0123] A 96-well half-area plate (#3690, Corning) was prepared by coating with recombinant human CD24 protein, recombinant mouse CD24 protein, and BSA. The plate was incubated overnight at 4°C using carbonate coating buffer. The plate was washed once with phosphate-buffered saline (PBS) and blocked for 1 h at 37°C with PBS containing 5% skim milk. After the blocking step, the phage supernatant was poured into the plate and allowed to interact for 2 h at 37°C. For the phage supernatant, the phage pool obtained from the panning in Example 1 was infected with E. coli, and a single colony grown on a carbenicillin-resistant plate was individually grown in a 96-deep well plate (#90060, Bioneer) containing 1 ml of SB medium at 37°C for 4 h. After that, M13K07 helper phage was added to each well, and the phage supernatant from each well generated overnight was used. After injecting the phage supernatant, each well was washed five times with PBS. After washing, HRP-tagged anti-M13 bacteriophage antibody (#11973-MM05T-H, Sino Biological) was applied and incubated at 37°C for 1 hour. After washing each well five times with PBS, tetramethylbenzidine (TMB) was added for 10 minutes to induce a color reaction, and the reaction was stopped using 2 N sulfuric acid. The absorbance of each well was measured at 450 nm using a microplate reader, and phage with an absorbance of 0.5 or higher for both recombinant human CD24 protein and recombinant mouse CD24 protein were selected (Fig. 2).

[0124] The binding affinity test was repeated for the selected phages, and phages with an absorbance of 0.8 or higher for both recombinant human CD24 protein and recombinant mouse CD24 protein were selected (Fig. 3). Analysis of the DNA of a single well of the selected phages confirmed a common H-CDR3 sequence, as shown in Table 1 below. Table 2 shows the CDR sequences of the two final candidate sequences.

[0125]

[0126] CloneH-CDR3A-12CARDSSSWYGGYYYYYGMDVWB-1CAREYGDYFDYWB-8CARDSSSWYGGYYYYYGMDVWC-1CAREYGDYFDYWC-5CAREYGDYFDYWC-10CAREYGDYFDYWF-10CAREYGDYFDYWG-5CARDSSSWYGGYYYYYGMDVW

[0127]

[0128] CandidateCDR1CDR2CDR31Heavy chainGFTFSSYG(SEQ ID NO: 1)MSGDGLAT(SEQ ID NO: 3)CARDSSSWYGGYYYYYGMDVW(SEQ ID NO: 5)Light chainQGISSW(SEQ ID NO: 7)AASCQQSYSTPLTF(SEQ ID NO: 9)2Heavy chainGFTVSSNY(SEQ ID NO: 2)IYSGGST(SEQ ID NO: 4)CAREYGDYFDYW(SEQ ID NO: 6)Light chainSSDVGGYDH(SEQ ID NO: 8)EVTCSSYAGSNSVLF(SEQ ID NO: 10)

[0129]

[0130]

[0131] Example 3. Antibody purification and production

[0132] An antibody having the CDR sequence confirmed through Example 2 was purified and produced.

[0133] Specifically, the gene of scFv antibody (SEQ ID NO: 20 or SEQ ID NO: 21) was inserted into the pFUSE-Fc expression vector (#pfuse-hg1fc2; InvivoGen) and then transfected into Expi293F cells (#A14524; Gibco; Thermo Fisher Scientific) using the ExpiFectamine 293 Transfection Kit (#A14527; Gibco; Thermo Fisher Scientific). After 3 days, the cell culture supernatant was filtered through a 0.2 μm filter. The filtered supernatant was then purified by AKTA TM Purification was performed using a HiTrap Protein G HP column (#17-0404-01, Cytiva) in a starting protein purification system (Cytiva).

[0134]

[0135] Example 4. Confirmation of CD24 binding affinity

[0136] It was confirmed that an anti-CD24 antibody according to one specific example has high affinity for CD24 expressed in various cancer cell lines.

[0137] First, lung cancer cell lines (A549 and LLC), breast cancer cell lines (4T1 and MCF7), hepatoma cell line (Huh7), and melanoma cell line (B16F10) were maintained in a humidified incubator at 37°C and 5% CO2. LLC, LLC-luc, Huh7, 4T1, and B16F10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Cytiva) supplemented with 10% fetal bovine serum (FBS, Cytiva) and 1% antibiotic-antimycotic (AA, GenDEPOT). A549 and MCF7 cells were cultured in Roswell Park Memorial Institute medium (RPMI1640, Cytiva) supplemented with 10% FBS and 1% AA.

[0138] All cells participating in the study were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at 4°C. After fixation, cells were washed a second time and blocked with PBS-T containing 5% goat serum and 1% BSA. Cells were stained with the indicated primary antibodies for 1 hour at 4°C, followed by incubation with the appropriate secondary antibodies for an additional 1 hour at the same temperature. The primary antibody used was a previously purified anti-CD24 antibody. The secondary antibody used was a FITC-conjugated goat anti-human IgG antibody (#ab97224, Abcam). Sample analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter), and data were processed using FlowJo software.

[0139] As shown in FIGS. 4 and 5, it was confirmed that antibodies #1 and #2 of the present invention have high affinity for CD24 in a breast cancer cell line (MCF-7). In addition, it was confirmed that antibody #1 has high affinity for CD24 in human and mouse breast cancer cell lines (MCF7 and 4T1), lung cancer cell lines (A549 and LLC), liver cancer cell line (Huh7), and melanoma cell line (B16F10).

[0140]

[0141] Example 5. Phagocytosis assay

[0142] The following experiment was conducted to confirm whether anti-CD24 antibodies according to one specific example are involved in the phagocytosis of macrophages.

[0143] Specifically, THP-1 cells (Korean Cell Line Bank) (5 × 10 5(Cells / well) were seeded in 6-well plates in 2 mL RPMI medium (RPMI1640, Cytiva) containing 10% FBS and 1% AA. To optimize differentiation conditions, THP-1 cells were treated with 50 nM phorbol 12-myristate 13-acetate (PMA, Sigma) for 48 h. Differentiated and attached cells were washed twice with PBS and rested in the culture medium for another 24 h to obtain the resting state (M0) of macrophages. Resting macrophages (M0) were stimulated with IL-4 / IL-13 at a concentration of 20 ng / mL for 72 h to differentiate into the M2 phenotype, and these macrophages were used for phagocytosis assays.

[0144] All flow-based in vitro phagocytosis assays were performed by seeding cancer cells and macrophages at a density of 1 × 10 in ultra-low attachment 96-well U-bottom plates (Costar) in serum-free RPMI for 4 h in a humidified 5% CO2 incubator at 37°C. 5 Target cells (cancer cells) vs. 5×10 4 Co-culture was performed at a ratio of 0.5 to 1.5, with macrophages. Cancer cells were labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CSFE, Invitrogen) according to the manufacturer's instructions, cultured at 37°C for 20 minutes under light protection, and then washed twice with culture medium before co-culture. Cancer cells were pretreated with anti-CD24 antibody (#1) according to one specific example, and macrophages were collected from the plates using TrypLE Express (Gibco). After co-culture, phagocytosis was analyzed using flow cytometry using the same method as in Example 4. Macrophages were identified with anti-CD11b-APC (#17-0112-82, 1:400, Invitrogen), and the percentage of phagocytosis was measured as CD11b+ / CSFE+ macrophages using a CytoFLEX flow cytometer (Beckman Coulter) and the data were analyzed using FlowJo software.

[0145] Figure 6 is a schematic diagram of an anti-CD24 antibody and its phagocytic activity according to one specific example.

[0146] As shown in Figures 7 and 8, it was confirmed that the macrophage phagocytic activity of the #1 antibody administration group increased in lung cancer cell lines and breast cancer cell lines.

[0147]

[0148] Example 6. Confirmation of in vivo antitumor efficacy

[0149] The antitumor efficacy of an anti-CD24 antibody according to one specific example was confirmed in a mouse model.

[0150] First, 1 × 10 6 LLC-GFP-luc cells were subcutaneously inoculated. Mice with palpable tumors of approximately 30 mm³ were randomly divided into two groups on the fifth day and then intravenously administered PBS and αCD24 antibody (#1). αCD24 antibody (200 μg / mouse) was injected every three days a week. Tumor growth was measured every two days using a digital caliper, and tumor size was calculated using the ellipsoid formula: V = ½ length × width². Mice were euthanized when signs of health impairment appeared or tumor volume exceeded 2000 mm³ according to humane endpoint criteria. Immediately before euthanasia, in vivo bioluminescence levels were measured using an In Vivo Imaging System (IVIS) after administering D-luciferin (#122799, Perkinelmer). After dissection, the tumor tissues were weighed, and the results are shown in Figs. 10 and 11.

[0151] As shown in Figure 10, it was confirmed that the tumor weight and volume were suppressed in the #1 anti-CD24 antibody administration group compared to the control group.

[0152] As shown in Figure 11, it was confirmed that the bioluminescence signal was suppressed in the #1 anti-CD24 antibody administration group compared to the control group.

[0153] The above results indicate that tumor growth is inhibited by anti-CD24 antibodies according to one specific example.

[0154]

[0155] Example 7. In vivo flow cytometry analysis

[0156] After administration of anti-CD24 antibody according to one specific example to cancer cell lines, increased activity of macrophages was confirmed through flow cytometry analysis.

[0157] First, cells were isolated from tumor tissue for flow cytometry analysis. Mice were prepared using the same method as in Example 6, and cancer tissue was excised from an LLC-bearing mouse model. Each tissue was weighed, placed in a 40 μm cell strainer in a 50 mL conical tube, and the tissue was mashed on the surface using the plunger of a 3 mL syringe. The sample was washed with PBS and resuspended in 5 mL of red blood cell lysis solution (Sigma-Aldrich) for 5 minutes. Furthermore, 25 mL of PBS was added, washed, and centrifuged at 550 × g to obtain cells. The cells were fixed with 4% PFA and used for flow cytometry.

[0158] For in vivo phagocytosis analysis, nonspecific binding to FcR was reduced with 2 μl of TruStain FcX Fc receptor blocking antibody (BioLegend) for 10 min on ice. Intratumoral macrophages were then stained with CD45-PE-Cy7 (#60943S, Cell signaling) and anti-F4 / 80-APC (#86007S, Cell signaling) for analysis. Sample analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter), and data were processed using FlowJo software (Fig. 12).

[0159] For in vivo immunoassays, nonspecific binding to FcR was reduced with 2 μl of TruStain FcX Fc receptor blocking antibody (BioLegend) for 10 min on ice. Afterwards, to analyze intratumoral macrophages, anti-CD45-PE-Cy7 (#60943S, Cell signaling), anti-CD11b-PE (#24956S, Cell signaling), anti-F4 / 80-APC (#86007S, Cell signaling), anti-CD80-eFluor™ 450 (#48-0801-82, Invitrogen), anti-CD206 eFluor™ 450 (#48-2061-82, Invitrogen), and anti-TNFα-eFluor™ 450 (#48-7321-82, Invitrogen) were used to evaluate the frequency and activity of M1-TAM (CD45+CD11b+F4 / 80+CD80+) and M2-TAM (CD45+CD11b+F4 / 80+CD206+) (Fig. 13, Fig. 14). To analyze T cells in tumors, anti-CD45-PE-Cy7 (#60943S, Cell signaling), anti-CD3-APC (#24265S, Cell signaling), anti-CD8α-PE (#56984S, Cell signaling), anti-IFN-γ (#ab9657, Abcam), Granzyme B (#ab4059, Abcam), and Rabbit IgG-Pacific Blue™ (#P-10994, Invitrogen) were used to detect CD8 + T cell activity was assessed. Sample analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter), and data were processed using FlowJo software (Fig. 15).

[0160] As shown in Figure 12, the analysis of cancer cells (GFP) phagocytosed by macrophages (F4 / 80) confirmed that the cancer cell phagocytosis ability in tumor tissue was significantly improved compared to the control group.

[0161] As shown in Figure 13, it was confirmed that the infiltration of macrophages into tumor tissues increased, and the proportion of tumor-suppressive macrophages (M1-TAM) increased compared to tumor-promoting macrophages (M2-TAM).

[0162] As shown in Figure 14, an increase in the expression of TNFα, a representative activation marker of anti-tumor M1 macrophages, was confirmed.

[0163] As shown in Figure 15, an increase in the expression of Granzyme B and IFNγ, which are representative activation markers of cytotoxic T cells (CD8+ T cells) due to macrophage activation in tumor tissues, was confirmed.

[0164] The above results indicate that macrophages within tumor tissues are activated by anti-CD24 antibodies according to one specific example.

[0165]

[0166] In summary, the anti-CD24 antibody of the present invention not only targets cancer cells but also blocks the "don't eat me" signal, thereby enhancing the phagocytosis of macrophages against cancer cells. Therefore, it can be utilized as a universal anticancer agent that effectively prevents or treats a variety of cancers associated with CD24, not just cancer. Increased activity of T cells and NK cells expressing Siglec-10, which interacts with CD24, is also expected, providing a novel immuno-oncology treatment strategy. Furthermore, it exhibits high binding affinity to both humans and mice, making it suitable for clinical and preclinical applications.

[0167]

[0168] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A heavy chain variable region comprising a heavy chain complementary determining region 1 (H-CDR1) comprising an amino acid sequence of SEQ ID NO: 1 or 2; a heavy chain complementary determining region 2 (H-CDR2) comprising an amino acid sequence of SEQ ID NO: 3 or 4; and a heavy chain complementary determining region 3 (H-CDR3) comprising an amino acid sequence of SEQ ID NO: 5 or 6; and An antibody or antigen-binding fragment thereof that specifically binds to CD24, comprising a light chain variable region comprising a light chain complementary determining region 1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 7 or 8; a light chain complementary determining region 2 (L-CDR2) comprising the amino acid sequence of sequence AAS or sequence EVT; and a light chain complementary determining region 3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 9 or 10.

2. In claim 1, the heavy chain variable region is an antibody or antigen-binding fragment thereof comprising a combination of the following (i) or (ii): (i) H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; H-CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and H-CDR3 comprising the amino acid sequence of SEQ ID NO: 5; (ii) H-CDR1 comprising the amino acid sequence of SEQ ID NO: 2; H-CDR2 comprising the amino acid sequence of SEQ ID NO: 4; and H-CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

3. In claim 1, the light chain variable region is an antibody or antigen-binding fragment thereof comprising a combination of the following (i) or (ii): (i) L-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; L-CDR2 comprising the amino acid sequence of sequence AAS; and L-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (ii) L-CDR1 comprising the amino acid sequence of SEQ ID NO: 8; L-CDR2 comprising the amino acid sequence of sequence EVT; and L-CDR3 comprising the amino acid sequence of SEQ ID NO:

10.

4. In claim 1, a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11 or 12; and An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 or 14.

5. An antibody or antigen-binding fragment thereof comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 18 according to claim 1.

6. An antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region are connected directly or by a linker.

7. In claim 1, the antibody or antigen-binding fragment thereof comprises an scFv or scFv-Fc fragment.

8. In claim 1, the antibody or antigen-binding fragment thereof increases the phagocytic activity of macrophages.

9. In claim 1, the antibody or antigen-binding fragment thereof is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

10. A gene encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A vector comprising the gene of claim 10.

12. A recombinant cell comprising the gene of claim 10; or a vector comprising the gene.

13. The antibody of claim 1 or an antigen-binding fragment thereof; drug moiety; and An antibody-drug conjugate (ADC) comprising a linker connecting the antibody or antigen-binding fragment thereof and the drug moiety.

14. A pharmaceutical composition for preventing or treating cancer, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; or an antibody-drug conjugate according to claim 13.

15. A pharmaceutical composition according to claim 14, wherein the cancer comprises at least one selected from the group consisting of breast cancer, lung cancer, skin cancer, kidney cancer, colon cancer, head and neck cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, melanoma, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer, and pancreatic cancer.

16. A method for preventing or treating cancer, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1 to 9; or an antibody drug conjugate of claim 13.

17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for use in the manufacture of a pharmaceutical preparation for the prevention or treatment of cancer; or an antibody-drug conjugate according to claim 13.

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