The present invention relates to uses of an antibody that specifically binds to a specific glycosylation structure of ICAM1 protein, specifically, a hyposialylated n-glycan structure.

Abstract

A composition for detecting ICAM1 protein comprising the anti-ICAM1 antibody of the present invention can selectively detect the protein by recognizing the N-glycan of ICAM1 that is cancer cell-specifically modified. The composition for detecting ICAM1 protein comprising an anti-ICAM1 antibody according to the present invention can selectively detect the corresponding protein by recognizing the N-glycan of ICAM1 that is cancer cell-specifically modified. In particular, the anti-ICAM1 antibody contained in the composition specifically recognizes and binds to a structure in which sialic acid residues at the N-glycan terminal linked to the N130 asparagine residue of ICAM1 are reduced or deficient, that is, a hyposialylated glycosylation pattern. By targeting a glycosylation pattern appearing only in cancer cells, the antibody provides high therapeutic stability due to low non-specific binding to normal cells. Accordingly, the present invention offers an anti-ICAM1 antibody that precisely targets a cancer-specific glycan pattern and a novel immune-oncology therapeutic strategy based thereon, thereby overcoming limitations of existing anticancer agents and providing enhanced cancer treatment effects.

Description

Carbohydrate-specific anticancer antibodies derived from cancer survivors

[0001] The present invention relates to the use of antibodies that specifically bind to specific glycosylation structures of ICAM1 protein, specifically hyposialylated N-glycan structures.

[0002] Cancer is one of the leading causes of death in modern society. It is a disease that occurs when normal cells change due to various causes. It refers to a malignant tumor that does not follow the normal differentiation, proliferation, and growth patterns of cells. Cancer is characterized by "uncontrolled cell growth," and this abnormal cell growth forms a mass of cells called a tumor, which invades surrounding tissues and, in severe cases, metastasizes to other organs in the body.

[0003] Cancer is an incurable, chronic disease that, despite treatments such as surgery, radiation, and drug therapy, often fails to achieve a fundamental cure, causing suffering and ultimately leading to death. In particular, the global cancer incidence rate has been increasing by more than 5% annually, driven by factors such as the growing aging population and environmental deterioration.

[0004] Meanwhile, cancer drug treatments, namely anticancer drugs, typically use cytotoxic compounds to attack and kill cancer cells. However, they cause damage to both cancer cells and normal cells, resulting in significant side effects. Therefore, targeted anticancer drugs have been developed to reduce these side effects. However, while these targeted anticancer drugs have been able to reduce side effects, they also present a limitation: a high probability of developing resistance.

[0005] Accordingly, there has been a recent surge in interest in immunotherapy, which leverages the body's immune system to reduce toxicity and resistance. One example of such immunotherapy is immune checkpoint inhibitors, which bind specifically to PD-L1 on the surface of cancer cells and inhibit T cell binding to PD-1, thereby activating T cells and enabling them to attack cancer cells. However, these immune checkpoint inhibitors also have limitations, as they only respond to a limited range of cancer types.

[0006] However, to date, no studies have reported on antibodies derived from cancer survivors that exhibit anticancer effects. While existing cancer patient-derived antibodies are unlikely to exhibit anticancer effects, those who survive long-term after cancer surgery likely possess antibodies that contribute to their survival.

[0007] Meanwhile, Intercellular Adhesion Molecule 1 (ICAM1) is a protein crucially involved in immune cell adhesion and inflammatory responses, as well as cancer cell invasion and metastasis. It plays a role in inducing distant metastasis through cluster formation of circulating tumor cells (CTCs) and transendothelial migration across vascular endothelial cells. Since ICAM1 is known to be overexpressed in various solid tumors, therapeutic strategies targeting it are theoretically very promising.

[0008] However, there are cases in which previously developed anti-ICAM1 antibodies (such as BI-505) nonspecifically bind to ICAM1 of normal cells as well as cancer cells, causing serious toxicity, especially cardiopulmonary toxicity, and ultimately leading to the discontinuation of clinical development.

[0009] Against this backdrop, the present inventors selected antibodies exhibiting high binding affinity for cancer cells from antibodies isolated from the blood of individuals who survived cancer surgery for more than five years. Among these, they identified an ICAM1 antibody that recognized a hyposialylated N-glycan structure specifically present in cancer cells but did not bind to normal cells. This antibody was confirmed to possess therapeutic efficacy, including cancer specificity and metastasis inhibition, while being non-toxic to normal cells, thereby completing the present invention.

[0010] Therefore, by providing an antibody that selectively binds to the glycosylated ICAM1 structure that appears only in cancer cells, regardless of whether ICAM1 is expressed, and at the same time does not bind to normal cells, this overcomes the toxicity problem caused by non-specific binding of existing anticancer antibodies and provides a new technological means for realizing high-precision immuno-oncology treatment.

[0011] In order to achieve the above object, one aspect of the present invention provides a composition for detecting ICAM1 protein, comprising an anti-ICAM1 antibody, wherein the anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; and wherein the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of SEQ ID NO: 13 is hyposialylated.

[0012] Another aspect of the present invention provides a composition for diagnosing an ICAM1-related disease, comprising a composition for detecting an ICAM1 protein comprising an anti-ICAM1 antibody according to the present invention.

[0013] Another aspect of the present invention provides a kit for detecting ICAM1 protein, comprising a composition for detecting ICAM1 protein comprising an anti-ICAM1 antibody.

[0014] In addition, the present invention provides a diagnostic kit for an ICAM1-related disease comprising a diagnostic composition for an ICAM1-related disease according to the present invention.

[0015] In addition, the present invention provides a method for providing information for diagnosing an ICAM1-related disease, comprising the steps of: (1) contacting a biological sample obtained from an individual with an anti-ICAM1 antibody; (2) detecting an ICAM1 protein bound to the anti-ICAM1 antibody; and (3) evaluating whether the ICAM1 protein is detected in step (2), wherein the anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; The above ICAM1 protein provides a method for providing information for diagnosing an ICAM1-related disease, characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of sequence number 13 is hyposialylated.

[0016] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an anti-ICAM1 antibody, wherein the anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; wherein the anti-ICAM1 antibody recognizes an ICAM1 protein, and the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of SEQ ID NO: 13 is hyposialylated.

[0017] The composition for detecting ICAM1 protein, comprising an anti-ICAM1 antibody according to the present invention, can selectively detect the protein by recognizing the N-glycan of ICAM1 that is specifically modified in cancer cells. Specifically, the anti-ICAM1 antibody included in the present composition specifically recognizes and binds to a structure in which the sialic acid residue at the terminal of the N-glycan linked to the asparagine residue N130 of ICAM1 is reduced or absent, i.e., a hyposialylated glycosylation pattern. This targets a glycosylation pattern that appears only in cancer cells, thereby providing high therapeutic stability due to low non-specific binding to normal cells. Therefore, the present invention provides an anti-ICAM1 antibody that precisely targets a cancer-specific glycosylation pattern and a novel immuno-cancer treatment strategy based thereon, thereby overcoming the limitations of existing anticancer agents and providing improved cancer treatment effects.

[0018] Figure 1 shows the results of performing flow cytometry analysis to select antibodies that exhibit high binding affinity to cancer cells among antibodies derived from the blood of a person who survived for more than 5 years after triple-negative breast cancer surgery.

[0019] Figure 2 shows the results of an in vitro cell proliferation assay performed to confirm the effect of antibodies selected by one aspect on the growth of cancer cells.

[0020] Figure 3 is a photograph taken with an optical microscope after performing an in vitro cell migration assay to confirm the effect of antibodies selected by one aspect on the migration of cancer cells.

[0021] Figure 4 shows the results of measuring the ratio of migrated cells after performing an in vitro cell migration assay to confirm the effect of antibodies selected by one aspect on the migration of cancer cells.

[0022] Figure 5 is a photograph of lung tissue stained with cancer cells taken after administering a selected antibody to a lung metastatic breast cancer animal model to confirm the effect of administration of a selected antibody on metastasis in a lung metastatic breast cancer animal model.

[0023] Figure 6 shows the results of measuring the Metastatic Index value, which quantifies the area of ​​the lung where cancer cells have metastasized, after administering the selected antibody to the lung metastatic breast cancer animal model in order to confirm the effect of administration of the antibody selected by one aspect on metastasis in the lung metastatic breast cancer animal model.

[0024] Figure 7 shows the results of an antibody-dependent cellular cytotoxicity test (ADCC) performed using antibodies selected by one aspect.

[0025] Figure 8 shows the results of confirming whether there is a change in antibody binding by FACS after knockdown of the ICAM1 gene using siRNA to identify the antigen of the antibody selected by one aspect.

[0026] Figure 9 shows the results of Western blot to confirm whether ICAM1 is present in the immunoprecipitation reaction result of the S4-2 antibody selected by one aspect.

[0027] Figure 10 shows the results of modeling the three-dimensional structure of the ICAM1 homodimer and confirming the arrangement of various domains.

[0028] Figure 11 visualizes the results of AlphaFold-multimer analysis confirming the high binding potential of S4-2 antibody and ICAM1.

[0029] Figure 12 shows the results of differential binding patterns in which the S4-2 antibody hardly binds to normal ICAM1 (WT) and forms high binding to mutant ICAM1 (MT) in which N-glycosylation does not occur, depending on the presence or absence of N-glycosylation of ICAM1 N130.

[0030] Figure 13 shows the results showing that BI505, a competitive ICAM1 antibody, forms high binding regardless of whether ICAM1 N130 is mutated (WT or MT), showing binding characteristics different from those of the S4-2 antibody.

[0031] Figure 14 shows the results of different binding patterns in which the binding affinity of the S4-2 antibody to TNBC cancer cells is significantly increased when the cell surface is hyposialylated due to neuraminidase treatment, and the BI-505 antibody maintains high binding affinity regardless of treatment.

[0032] Figure 15 shows that when the cell surface is hyper-oxidized due to ST6GAL1 overexpression, the binding ability of the S4-2 antibody to TNBC cancer cells is inhibited, and the BI-505 antibody maintains a relatively high positive rate.

[0033] Figure 16 shows the results of a Transendothelial Migration Assay (TMA) performed on the triple-negative breast cancer cell line MDA-MB-468 to confirm the in vitro metastasis inhibitory effect of the S4-2 antibody.

[0034] Figure 17 shows the results of administering S4-2 antibody to a lung metastasis mouse model using the triple-negative breast cancer cell line MDA-MB-231 to evaluate the in vivo metastasis inhibitory effect of S4-2 antibody.

[0035] Figure 18 shows the results of a Western blot analysis confirming that ICAM1 protein was expressed in all cell lines (various TNBC cell lines, MCF7, MCF10A) used in the experiment.

[0036] Figure 19 shows the results of flow cytometry analysis (FACS) showing that the S4-2 antibody showed high binding affinity to various TNBC cell lines, but did not bind to MCF10A normal breast epithelial cells expressing ICAM1.

[0037] Figures 20 to 23 show that the S4-2 antibody exhibits binding affinity in cell lines of various cancer types, such as multiple myeloma, non-small cell lung cancer, pancreatic cancer, and ovarian cancer, suggesting its potential as a diagnostic agent for other cancer types with hyposialylation of the N-glycan of ICAM1 and its potential for expanding the range of diseases to be treated.

[0038] Figure 24 shows the cancer specificity of the S4-2 antibody, which effectively binds to the triple-negative breast cancer cell line MDA-MB-436 but not to ICAM1 of the normal breast cell line MCF10A. In contrast, the BI-505 antibody was shown to bind to ICAM1 of both the cancer cell line and the normal cell line MCF10A.

[0039] The present invention provides, in one aspect, a composition for detecting an ICAM1 protein, comprising an anti-ICAM1 antibody, wherein the anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 7; wherein the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of SEQ ID NO: 13 is hyposialylated.

[0040] In the present invention, the anti-ICAM1 antibody according to the present invention may have a heavy chain variable region composed of an amino acid sequence of SEQ ID NO: 4, and a light chain variable region composed of an amino acid sequence of SEQ ID NO: 8.

[0041] As used herein, the term "ICAM1 (intercellular adhesion molecule 1)", also known as CD54, refers to an inflammatory protein involved in the adhesion and migration of inflammatory cells. ICAM1 is a type of intercellular adhesion molecule that is continuously present at low concentrations on the membranes of leukocytes and endothelial cells, and is known to significantly increase in concentration upon cytokine stimulation. In the present invention, the entire sequence of ICAM1 may be comprised of the amino acid sequence of SEQ ID NO: 13.

[0042] The term "antibody" as used herein refers to a substance produced by antigen stimulation within the immune system, and its type is not particularly limited, and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies are very stable not only in vitro but also in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, so they have very high avidity. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of antibodies is divided into the heavy chain constant region and the light chain constant region. The heavy chain constant region has the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses are gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has the kappa (κ) and lambda (λ) types.

[0043] As used herein, the term "heavy chain" is interpreted to mean a full-length heavy chain and fragments thereof, comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2 and CH3 and a hinge. In addition, the term "light chain" is interpreted to mean a full-length light chain and fragments thereof, comprising a variable region domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0044] The term "variable region" or "variable domain" as used herein refers to a portion of an antibody molecule that exhibits many sequence variations while performing the function of specifically binding to an antigen, and the variable region includes complementarity determining regions (CDR1, CDR2, and CDR3). Between the CDRs, there is a framework region (FR) that supports the CDR ring. The "complementarity determining region" is a ring-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

[0045] As used herein, the term "complementarity determining region (CDR)" refers to an amino acid sequence of a hypervariable region of a heavy and light chain of an immunoglobulin. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for binding of an antibody to an antigen or an antigenic determinant.

[0046] As used herein, the terms “specifically bind” or “specifically recognize” have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to produce an immunological response.

[0047] The term "hinge region" as used herein refers to a region contained in the heavy chain of an antibody, which exists between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site within the antibody. For example, the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE or IgG, such as IgG1, IgG2, IgG 3 or IgG4.

[0048] The term “N-glycan” as used herein refers to a sugar chain structure that forms a covalent bond with the amino group of an asparagine (Asn) residue of a protein. The N-glycan located at the 130th amino acid, Asn, of the ICAM1 protein has a composition ratio of HexNAc(7), Hex(8), Fuc(1), and NeuAc(1) as determined by mass spectrometry analysis, and is a complex N-glycan structure formed with various branch structures based on the core structure (GlcNAc₂Man₃) and with sialic acid (NeuAc) attached to the terminal.

[0049] In the present invention, the structure in which “the N-glycan terminal of asparagine (ASN) located at position 130 of SEQ ID NO: 13 is hyposialylated” means a special sugar structure in which the sialic acid at the end of the sugar chain attached to asparagine at position 130 of the ICAM1 protein is reduced or absent, i.e., a hyposialylated structure.

[0050] As used herein, the term “hyposialylation” refers to a state in which the level of sialic acid linkage at the end of a sugar chain is reduced or missing compared to the normal level. In the case of a normal N-glycan structure, sialic acid is attached to the end, but a hyposialylated N-glycosylation structure refers to a sugar structure in which sialic acid is reduced or missing. In one embodiment, the anti-ICAM1 antibody or antigen-binding fragment thereof according to the present invention recognizes a portion of the N-glycan structure formed at Asn present at position 130 of the ICAM1 protein (SEQ ID NO: 13) that is hyposialylated at the end (a state in which fewer sialic acids are attached).

[0051] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, and amino acids referred to herein by abbreviations are described according to the IUPAC-IUB nomenclature as follows: Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S, Threonine: T, Tryptophan: W, Tyrosine: Y, and Valine: V. In one embodiment, “ASN” is a three-letter abbreviation for asparagine, which is represented by the single letter “N.”

[0052] In the present invention, amino acid modification / mutation / substitution refers to a substitution, insertion, and / or deletion, preferably a substitution, of an amino acid in a polypeptide sequence, whereby an amino acid at a specific position in the polypeptide sequence is replaced with another amino acid. In one embodiment, “N130A” refers to a mutation in which asparagine (N) at the 130th amino acid is substituted with alanine (A).

[0053] Meanwhile, in the present invention, the amino acid sequence represented by SEQ ID NO. 12 refers to the amino acid sequence section from positions 116 to 129 of the amino acid sequence of the ICAM1 protein represented by SEQ ID NO. 13, and when the anti-ICAM1 antibody or antigen-binding fragment thereof according to the present invention binds to a hyposialylated N-glycan structure at position 130 of the ICAM1 protein, antibody S4-2 (SEQ ID NO. 11) can recognize and bind together.

[0054] The term “detection composition” as used herein refers to a composition containing biochemical or immunological components for recognizing and detecting a specific protein to be detected, specifically ICAM1, and used to confirm the presence, quantitative change, or structural characteristics of the protein in a sample.

[0055]

[0056] In another aspect, the present invention provides a composition for diagnosing an ICAM1-related disease, comprising a composition for detecting an ICAM1 protein comprising an anti-ICAM1 antibody according to the present invention.

[0057] The term “diagnosis” as used herein means confirming the presence or characteristics of a pathological condition in an individual who has not yet been diagnosed or has been diagnosed. In one embodiment of the present invention, it was confirmed that the S4-2 antibody of the present invention binds to various cancers characterized by ICAM1 hyposialylation, thereby confirming the applicability of the antibody as a diagnostic composition utilizing the same. In this case, the ICAM1-related disease may be cancer.

[0058] The term "cancer" as used herein is a general term for a disease caused by cells that have an aggressive characteristic in which cells divide and proliferate while ignoring normal growth limits, an invasive characteristic in which cells infiltrate surrounding tissues, and a metastatic characteristic in which cells spread to other parts of the body. In the present invention, the cancer may be any one selected from the group consisting of breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, kidney cancer, skin cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Specifically, the breast cancer may be triple-negative breast cancer (TNBC).

[0059] As used herein, the term “triple-negative breast cancer (TNBC)” refers to a type of breast cancer in which all three major receptors (estrogen receptor, progesterone receptor, and HER2 receptor) are negative, accounting for approximately 10 to 15% of all breast cancers, and is a cancer type with a relatively high possibility of recurrence and metastasis and a poor prognosis.

[0060] Additionally, the cancer according to the present invention may be metastatic or relapsed cancer.

[0061]

[0062] In another aspect, the present invention provides a kit for detecting ICAM1 protein, comprising a composition for detecting ICAM1 protein comprising an anti-ICAM1 antibody according to the present invention. In the present invention, the kit can be used to rapidly screen for the presence or absence of ICAM1 protein in a sample prior to detection using an instrument such as HPLC, LC / MS, or GC / MS, or detection using an antibody, but is not limited thereto.

[0063]

[0064] In addition, the present invention provides a diagnostic kit for an ICAM1-related disease comprising a diagnostic composition for an ICAM1-related disease according to the present invention.

[0065] The types of diseases for which the occurrence or possibility of occurrence can be predicted using the diagnostic kit of the present invention may be cancer, specifically, any one selected from the group consisting of breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, kidney cancer, skin cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, but are not limited thereto.

[0066] In the present invention, the diagnostic kit may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0067] The diagnostic kit for the disease of the present invention may further comprise one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the diagnostic kit may further comprise essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit comprises a pair of primers specific for a gene encoding a marker protein. The primers are nucleotides having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may comprise a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0068] Additionally, the diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment thereof are attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control gene or fragment thereof.

[0069] Additionally, the diagnostic kit of the present invention may include essential components necessary for performing an ELISA. The ELISA kit includes an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0070]

[0071] In another aspect, the present invention comprises the steps of: (1) contacting an anti-ICAM1 antibody with a biological sample obtained from an individual; (2) detecting an ICAM1 protein bound to the anti-ICAM1 antibody;

[0072] (3) A method for providing information for diagnosing an ICAM1-related disease, comprising a step of evaluating whether an ICAM1 protein is detected in the step (2), wherein the anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; and wherein the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of SEQ ID NO: 13 is hyposialylated.

[0073] In the information providing method of the present invention, the type of disease for which the occurrence or possibility of occurrence can be predicted may be cancer, specifically, any one selected from the group consisting of breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, kidney cancer, skin cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, but is not limited thereto.

[0074] In addition, the present invention provides a method for diagnosing an ICAM1-related disease, comprising the steps of: (1) contacting a biological sample obtained from an individual with an anti-ICAM1 antibody; (2) detecting an ICAM1 protein bound to the anti-ICAM1 antibody; and (3) evaluating whether the ICAM1 protein is detected in step (2).

[0075] In the present invention, the term "subject" refers to an individual whose onset of the diseases listed above is uncertain, and an individual with a high probability of onset. In addition, the "subject" may be a mammal including a human, and may be selected from the group consisting of, for example, a human, a rat, a mouse, a guinea pig, a hamster, a rabbit, a monkey, a dog, a cat, a cow, a horse, a pig, a sheep, and a goat, and may preferably be a human, but is not limited thereto. In addition, in the present invention, the "biological sample" obtained from the subject may be blood, plasma, or serum of a patient with a high probability of onset.

[0076]

[0077] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an anti-ICAM1 antibody, wherein the anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; wherein the anti-ICAM1 antibody recognizes an ICAM1 protein, and the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of SEQ ID NO: 13 is hyposialylated.

[0078] The above cancer may be any one selected from the group consisting of breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, kidney cancer, skin cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0079] In one embodiment of the present invention, the selected S4-2 antibody was confirmed through in vitro experiments to inhibit the growth and migration of cancer cells and to promote cancer cell death by activating immune cells. Furthermore, in vivo experiments confirmed that when the S4-2 antibody was administered to a mouse model of lung metastatic breast cancer, the amount of metastatic cancer cells was significantly reduced. Therefore, the S4-2 antibody, which specifically binds to ICAM1, was confirmed to have cancer treatment, prevention, and metastasis inhibition effects.

[0080] The pharmaceutical composition of the present invention can be used as a single therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy, and when such combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapeutic agents that can be used together with the composition include cisplatin, procarbazine, carboplatin, mechlorethamine, procarbacyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, Examples of radiation therapy that can be used with the composition of the present invention include X-ray irradiation and γ-ray irradiation.

[0081] In one specific example, the composition of the present invention may further include an immunogenic apoptosis inducer, and the immunogenic apoptosis inducer may be at least one selected from the group consisting of an anthracycline-based anticancer agent, a taxane-based anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide-based anticancer agent, a GADD34 / PP1 inhibitor, LV tSMAC, Measles virus, bleomycin, mitoxantrone, and oxaliplatin, and the anthracycline-based anticancer agent may be at least one selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, It may be sabarubicin or valrubicin, and the taxane family anticancer drug may be paclitaxel or docetaxel.

[0082] As used herein, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention. As used herein, the term "treatment" refers to any action that kills cancer cells or improves or beneficially alters the symptoms of cancer by administering a composition according to the present invention.

[0083] The pharmaceutical composition may contain the antibody or antigen-binding fragment thereof as an active ingredient in an amount of from about 0.1 wt% to about 90 wt%, specifically from about 0.5 wt% to about 75 wt%, and more specifically from about 1 wt% to about 50 wt%, based on the total weight of the composition. The pharmaceutical composition may include conventional, non-toxic, pharmaceutically acceptable additives that are formulated into a formulation according to a conventional method.

[0084] For example, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. Examples of additives used in the pharmaceutical composition may include sweeteners, binders, solvents, solubilizers, wetting agents, emulsifiers, isotonic agents, absorbents, disintegrants, antioxidants, preservatives, lubricants, glidants, fillers, flavoring agents, and the like. For example, the additives may include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, stearin, magnesium stearate, magnesium aluminosilicate, starch, gelatin, gum tragacanth, alginic acid, sodium alginate, methylcellulose, sodium carboxymethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, and the like.

[0085] The pharmaceutical composition may be formulated in various dosage forms for oral administration (e.g., tablets, pills, powders, capsules, syrups, or emulsions) or parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection). Specifically, the pharmaceutical composition may be formulated as a dosage form for oral administration, and the additives used in this case may include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactants, suspending agents, emulsifiers, diluents, and the like. In addition, examples of glidants include colloidal silicon dioxide, magnesium silicate, and the like; Examples of diluents include microcrystalline cellulose, lactose anhydrous, lactose monohydrate, and silicified MCC HD 90; examples of disintegrants include croscarmellose sodium and crospovidone; and examples of lubricants include magnesium stearate, sodium lauryl sulfate, and stearic acid.

[0086] In addition, liquid preparations for oral administration include suspensions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. In addition, preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, etc. Meanwhile, injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0087] The pharmaceutical composition may be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

[0088] As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, the drugs used in combination or simultaneously, and other factors well known in the medical field. The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0089] Specifically, the effective amount of the antibody or antigen-binding fragment thereof in the pharmaceutical composition may vary depending on the patient's age, sex, and weight, and is generally about 0.1 mg to about 1,000 mg per kg of body weight, or about 5 mg to about 200 mg per kg, administered daily or every other day, or divided into one to three times a day. However, the range is not limited thereto, as it may increase or decrease depending on the route of administration, severity of the disease, sex, weight, age, etc. In addition, the pharmaceutical composition may be administered for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, hormone therapy, bone marrow transplantation, stem cell replacement therapy, other biological therapy, surgical intervention, or a combination thereof. For example, it may be used as an adjuvant therapy together with other long-term treatment strategies, or to maintain the condition of a patient after tumor regression or chemoprevention in a severe patient.

[0090]

[0091] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition according to the present invention for preventing or treating cancer.

[0092] The above "subject" may be a mammal including a human, for example, selected from the group consisting of a human, a rat, a mouse, a guinea pig, a hamster, a rabbit, a monkey, a dog, a cat, a cow, a horse, a pig, a sheep and a goat, and preferably, a human, but is not limited thereto.

[0093] The pharmaceutical composition of the present invention can be administered to a subject via various routes. Administration may be, for example, oral, rectal, intravenous, intramuscular, transcutaneous, subcutaneous, intrauterine, or intracerebrovascular injection.

[0094]

[0095]

[0096] The present inventors, through previously registered Korean Patent No. 10-2711074, discovered a cancer-specific antibody from a cancer survivor that effectively inhibits cancer cell proliferation and migration. Subsequently, through follow-up research for the development of anticancer therapeutics, they identified an ICAM1-specific antibody that recognizes hyposialylated N-glycans, which are present only in cancer cells but not in normal cells. They confirmed that this antibody possesses both excellent cancer specificity and therapeutic efficacy without toxicity to normal cells, and based on this, they completed the present invention. The anti-ICAM1 antibody or antigen-binding fragment thereof according to the present invention, and a composition comprising the same, can be usefully used as a composition for the prevention, treatment, or diagnosis of cancer, and can further be applied in combination therapy with existing anticancer agents, thereby contributing to enhancing the effectiveness of cancer treatment.

[0097]

[0098] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are intended only to illustrate the present invention, and the present invention is not limited to the following examples.

[0099]

[0100] Example 1. Production of an antibody library from breast cancer survivors

[0101] An antibody library from survivors of triple-negative breast cancer who survived for more than 5 years was constructed using the following method. cDNA was synthesized from RNA extracted from lymph node (LN) and peripheral blood mononuclear cell (PBMC) samples from triple-negative breast cancer survivors who survived for more than 5 years, and the λ (Vλ) and κ (Vκ) chain genes of the heavy (VH) and light (LH) chains of the variable regions were amplified using polymerase chain reaction (PCR). Assembly PCR was performed to combine the heavy and light chains into scFv form, and the combined scFv genes were then amplified by extension PCR.

[0102] Next, to construct the scFv library, the amplified scFv gene was inserted into the phagemid vector pDR-D1. The amplified scFv gene and the pDR-D1 vector were digested with SfiI restriction enzyme, and then the library was cloned through ligation. The constructed scFv library plasmid was transformed into E. coli ER2738 using electroporation, and finally about 2 Х 10 8 Antibody libraries with cfu sizes were secured.

[0103]

[0104] Example 2. Identification of antibodies that specifically bind to cancer cells.

[0105] In order to identify antibodies that specifically bind to cancer cells from the antibody library produced in Example 1, biopanning was performed using the following method. MDA-MB-468 cells (ATCC, USA; HTB-132™), a breast cancer cell line, were used for biopanning, and MCF-10A cells (ATCC, USA; CRL 10317™), a breast epithelial cell line, were used as a negative control cell line. The biopanning process was performed four times in total, and in each panning process, the library phage was first treated with MCF-10A cells, a negative control cell line, to remove phage binding to MCF-10A cells as much as possible. Then, only the supernatant containing phage that did not bind to MCF-10A cells was separated and treated with MDA-MB-468, the target cells. Afterwards, the phages that did not bind to MDA-MB-468 cells were washed away, and only the phages bound to MDA-MB-468 were eluted and re-infected into E. coli. This process was repeated to amplify the phages that bind to MDA-MB-468. After the third and fourth panning, the sequences of the amplified phages were analyzed, and seven antibodies (S3-2, S3-3, S3-4, S3-5, S4-2, S4-4, S4-9) that specifically bind to MDA-MB-468 were identified.

[0106] Afterwards, to produce and purify experimental antibodies, the variable regions of the seven identified antibodies were each cloned into a whole IgG expression vector, and these were transfected into human embryonic kidney cell line HEK293E cells (ATCC, USA; CRL-1573™) using lipofectamine. After culturing the transfected cells for 3 days, the expressed and produced IgG in the supernatant was separated and purified using a protein G affinity column. The purified protein was analyzed using SDS-PAGE and showed a purity of over 95%.

[0107]

[0108] Example 3. Selection of antibodies exhibiting high binding affinity to cancer cells

[0109] In order to select antibodies exhibiting high binding affinity to cancer cells among the antibodies selected in Example 2 above, flow cytometry analysis was performed using the following method.

[0110] Patient-derived cells (PDC) obtained from survivors of triple-negative breast cancer who had survived for more than 5 years after surgery were trypsinized and cultured at a density of 1 x 10 in FACS buffer (3% FBS in PBS). 6 Cells / ㎖ were resuspended at a final concentration of 100 μg. 2 μg of each of the seven antibodies selected in Example 2 were incubated with PDC for 2 hours, and then combined with Fluorescein (FITC)-conjugated AffiniPure Goat-Anti Human IgG (109-095-098, Jackson ImmunoResearch) for 1 hour. After binding, the cells were filtered using a 100 μm strainer. The filtered cells were evaluated using a BD Accuri™ C6 flow cytometer and a BD Accuri™ C6 Plus flow cytometer, and analyzed using BD Accuri C6 software and BD Accuri C6 Plus software.

[0111] As a result, it was confirmed that the S4-2 antibody exhibited excellent binding affinity to cancer cells (Fig. 1), and this was selected.

[0112]

[0113] Example 4. Confirmation of anticancer efficacy of selected antibodies

[0114] 4.1 In vitro cell proliferation assay

[0115] To determine the effect of the S4-2 antibody selected in Example 3 on the growth of cancer cells, the following experiment was performed. Cancer cells (PDC) obtained from survivors of triple-negative breast cancer surgery who had survived for more than 5 years were seeded in 96-well plates at a density of 3,000 to 10,000 cells / well in 50 μL of culture medium. After seeding, the cancer cells were treated for 24 hours with the five antibodies (S3-2, S3-5, S4-2, S4-4, S4-9) identified in Example 2 at concentrations of 0.2 μg / mL, 1 μg / mL, and 5 μg / mL, respectively.

[0116] To monitor cell proliferation, 1 / 10 volume of Quanti-Max™ reagent was added directly to untreated control and antibody-treated cells 24 and 48 hours after antibody treatment. After incubating the cancer cells for an additional 3 hours, cell viability was measured using fluorescence measurements using a microplate fluorescence spectrophotometer.

[0117] As a result, it was confirmed that the S4-2 antibody inhibited the growth of cancer cells in a concentration-dependent manner compared to other antibodies (Fig. 2).

[0118]

[0119] 4.2 In vitro cell migration assay

[0120] To confirm the effect of the S4-2 antibody selected in Example 3 on the migration of cancer cells, the following experiment was performed. The migration ability of cells was analyzed using transwell chambers with 6.5 mm diameter polycarbonate filters (8.0 μm pore size). MDA-MB-468 cells, a breast cancer cell line, were trypsinized and seeded at 2 to 4 x 10 in serum-free medium. 4 Resuspended to a final concentration of 10 cells / ㎖.

[0121] A cell suspension was spread into each upper chamber, and 5 μg of each of the five antibodies (S3-2, S3-5, S4-2, S4-4, S4-9) identified in Example 2 were treated. 10% FBS was used in the lower chamber, and the chambers were cultured at 37°C for 24 hours. After the culture was completed, the cells were fixed and stained with hematoxylin and eosin. Non-migratory cells in the upper chamber of the filter were removed with a cotton swab, and the migration ability was measured by photographing and counting cells that had migrated to the lower part of the filter using an optical microscope.

[0122] As a result, it was confirmed that the S4-2 antibody inhibited the migration of cancer cells compared to other antibodies (Figs. 3 and 4).

[0123]

[0124] 4.3 In vivo lung metastasis animal model experiment

[0125] To confirm the effect of the S4-2 antibody selected in Example 3 on cancer cell metastasis in a lung metastatic breast cancer animal model, the following experiment was performed. Immunodeficient NSG mice (JA Bio, Korea) injected with MDA-MB-468 cells, a human breast cancer cell line, were used as a lung metastatic breast cancer animal model. Specifically, 5 x 10 5 MDA-MB-468 cells were suspended in 150 μl of PBS and injected into the tail vein of NSG mice. Three days after the cancer cell injection, IgG (2.5 mg / kg) was administered intravenously twice a week for three weeks as a control group and S4-2 (2.5 mg / kg) as an experimental group.

[0126] After 3 weeks, the mice were euthanized, and their lungs were removed, fixed in formalin, and paraffin-embedded to obtain tissue slide sections. The tissue slides were stained with H&E and observed. As a result, it was confirmed that the lung metastasis rate was significantly reduced in the lung metastatic breast cancer mouse model administered with the S4-2 antibody compared to the control group (Fig. 5). To quantify this, the area of ​​the area where cancer cells had metastasized was calculated using Image J and divided by the total area to obtain the Metastatic Index. As shown in Fig. 6, it was confirmed that the Metastatic Index was significantly reduced when the S4-2 antibody was administered compared to the control group.

[0127]

[0128] 4.4 Antibody-dependent cell cytotoxicity (ADCC)

[0129] MDA-MB-468 cells, a breast cancer cell line, and cancer cells (patient-derived cells, PDC) obtained from survivors of triple-negative breast cancer surgery who had survived for more than 5 years were seeded in a 96-well plate at a density of 1 x 10 4 / 100 ㎕ / well density and cultured at 37°C for 24 to 48 hours to prepare cancer cells as target cells.

[0130] Eight milliliters of peripheral blood were collected from breast cancer patients and normal controls, and whole blood was diluted with citrate-dextrose solution (ACD solution, Sigma; C3821) or 1:1 with phosphate-buffered saline (PBS). 3 milliliters of Ficoll-Paque (Miltenyl Biotec) was added to a 15-ml conical tube, and 10 milliliters of the diluted blood was carefully added thereto. The tube was then centrifuged at 400 g (or 2,000 to 2,500 rpm) for 30 to 40 minutes (or 20 to 30 minutes) at room temperature in a swinging bucket rotor without brakes. The supernatant was removed using a pipette, leaving the buffy coat (containing PBMCs) at the interface. After carefully transferring the PBMC layer to a new 15-mL conical tube filled with PBS, the tube was centrifuged at 200 g (or 1,500 to 1,800 rpm) for 10 to 15 minutes (or 5 minutes). The supernatant was discarded, the pellet was resuspended in PBS, and centrifuged again at 1,500 rpm for 5 minutes. The pellet was resuspended in PBS, and the cell number was counted to prepare PBMC.

[0131] Plated breast cancer cells were stained by adding CFSE (Carboxyfluorescein succinimidyl ester, Invitrogen; final concentration 5 μM, 100 μl / well) solution at 37°C for 20 min. After removing CFSE not bound to cancer cells, the stained cancer cells were washed twice for 5 min each with DPBS containing 10% FBS.

[0132] S4-2 antibody-stained cancer cells were added to the wells at a concentration of 1 μg / 100 μl / well and incubated at 37°C for 1 hour. Fc gamma blocking antibody as a control was added at the same concentration 1 hour before adding S4-2 antibody. Next, PBMC cell suspension was cultured at 2.5 x 105 / 100 ㎕ / well were added to the wells (cancer cells:PBMC ratio 1:25) and incubated overnight (16 hours) at 37℃. After incubation, floating and attached cells (by adding trypsin / EDTA) were harvested, and the cell suspension was transferred to a new E. tube. Then, centrifugation was performed at 3,000 rpm for 5 minutes at 4℃, and the cell pellet was resuspended and stained with propidium bromide (BD, #. 51-66211E). Single-cell suspensions were analyzed for CSFE and PI double-positive cells using a flow cytometer (Accuri C6, BD).

[0133] As a result, it was confirmed that the S4-2 antibody promotes cancer cell death through activation of immune cells (Figure 7).

[0134]

[0135] Example 5. Identification of antigen for S4-2 antibody

[0136] To identify the antigen binding to the S4-2 antibody selected in Example 3, immunoprecipitation-mass spectrometry was performed as follows. First, the S4-2 antibody was mixed with the lysate of MDA MB-468 breast cancer cell line and captured with Protein G agarose conjugated beads (immunoprecipitation). Through this, proteins binding to the antibody were secured and mass spectrometry was performed using LC-MS / MS equipment to confirm their identities. Among the antigen candidate genes detected through mass spectrometry, to identify the genes essential for binding to the S4-2 antibody, siRNA was used to knockdown specific genes and FACS was used to test for changes in antibody binding. As a positive control, Cetuximab, a well-known antibody against EGFR, was used and it was confirmed that the binding of Cetuximab was reduced when siEGFR was treated. Similarly, when ICAM1 gene expression was inhibited by treatment with ICAM1 siRNA, binding of the S4-2 antibody was reduced (Fig. 8). This confirmed that the antigen of the S4-2 antibody is ICAM1.

[0137] In addition, the presence of ICAM1 in the immunoprecipitation reaction result of the S4-2 antibody was confirmed through western blot, thereby verifying that the antigen of the S4-2 antibody is ICAM1 (Fig. 9).

[0138]

[0139] Example 6. Binding analysis of ICAM1 and antibodies

[0140] We structurally analyzed the interaction between ICAM1 protein and antibody. By modeling the three-dimensional structure of the ICAM1 homodimer, we confirmed the arrangement of various domains, such as Ib, IIb, IIIb, IVa, IVb, Va, and Vb (Fig. 10). In addition, we distinguished lower and higher exposure regions within the ICAM1 Ex3 monomer structure, and mapped the three-dimensional binding interface between ICAM1 and antibody using hydrogen / deuterium exchange (HDX) mass spectrometry and cell penetrating peptide (CPP).

[0141] As a result, AlphaFold-multimer analysis predicted that the S4-2 antibody would have a high binding potential to the antibody-binding site. Furthermore, the S4-2 antibody was confirmed to bind to the hyposialylated 130th asparagine (Asn) residue of ICAM1 at a close distance of 6.0 Å (Fig. 11).

[0142]

[0143] Example 7. Confirmation of ICAM1 N130 hyposialylation specificity of S4-2

[0144] In this example, we confirmed that S4-2 specifically recognizes a specific glycosylation modification in the ICAM1 protein, particularly hyposialylation in the N-glycan of asparagine (Asn, N) residue at position 130 of the ICAM1 protein.

[0145]

[0146] 7.1. Effect of N-glycosylation of ICAM1 on antibody binding

[0147] To determine whether the binding properties of the S4-2 antibody differ depending on the presence or absence of N-glycosylation at asparagine (N) residue 130 of ICAM1, a cell model was established in HEK293 normal cells expressing the ICAM1 gene. Specifically, HEK293 cells expressing normal ICAM1 (WT) with N-glycosylation at residue N130 and HEK293 cells expressing mutant ICAM1 (MT, N130A) in which residue N130 is mutated to alanine (A) and thus N-glycosylation does not occur were prepared. After treating each cell with the S4-2 antibody and the comparative antibody BI505, the degree of antibody binding to the cell surface was quantitatively analyzed.

[0148] As a result, the S4-2 antibody showed a specific binding pattern in which it barely bound to the wild type ICAM1 (WT) but strongly bound to the mutant ICAM1 (MT) lacking N-glycosylation. This shows that the S4-2 antibody selectively binds to the state in which N-glycosylation is removed at the N130 residue of ICAM1. In contrast, the comparative antibody BI505 bound to both ICAM1 (WT) and ICAM1 (MT) cells at similar levels, regardless of whether the N130 residue of ICAM1 was modified (Fig. 12a and Fig. 12b).

[0149] These results clearly demonstrate that the S4-2 antibody selectively binds to ICAM1 depending on whether or not N-glycosylation is present at the N130 residue. The S4-2 antibody strongly binds only to ICAM1 lacking N-glycosylation, demonstrating a mechanism of action that allows it to specifically bind to cancer cells while maintaining non-toxicity toward normal cells. In contrast, the comparator antibody BI505 exhibited high binding to wild-type ICAM1 (WT) regardless of mutations at the N130 residue.

[0150]

[0151] 7.2. Effect of Sialylation on Cancer Cell-Specific S4-2 Antibody Binding

[0152] We analyzed the relationship between cancer cell-specific binding of the S4-2 antibody and sialic acid modifications of cell surface N-glycans, specifically hyposialylation or hypersialylation. Using triple-negative breast cancer (TNBC) cell lines, we evaluated the effect of cell surface sialic acid modifications on the binding of the S4-2 and BI505 antibodies.

[0153] First, a hyposialylated state was artificially induced by removing sialic acid from the surface of TNBC cells using neuraminidase enzyme treatment. The neuraminidase-treated and untreated control groups were treated with S4-2, BI-505, and Human IgG antibodies, respectively, and the cell binding affinity of each antibody was quantitatively measured.

[0154] As a result, as shown in Figure 13, the S4-2 antibody showed a significantly increased binding affinity to TNBC cancer cells hyposialylated by neuraminidase treatment. In contrast, the BI-505 antibody maintained high binding affinity regardless of sialic acid removal, demonstrating binding characteristics unaffected by sialylation status.

[0155]

[0156] Additionally, the binding affinity of S4-2, BI505, and IgG antibodies was measured in TNBC cells in which the ST6GAL1 gene was overexpressed (OE) to increase sialic acid on the cell surface.

[0157] As a result, as shown in Figure 14, the binding affinity of S4-2 was significantly reduced in cells overexpressing ST6GAL1, and a lower 'Positive Rate' was observed compared to the control IgG and BI505 antibodies. In contrast, BI505 maintained a relatively high 'Positive Rate' even under conditions with increased sialylation.

[0158] These results clearly demonstrate that the S4-2 antibody is sensitive to the sialylation status of cell surfaces, particularly hyposialylation, and has the property of selectively binding to hyposialylated cancer cells. This reflects the mechanism by which the S4-2 antibody recognizes and binds to N-glycosylation abnormalities, particularly hyposialylation, present in cancer cells as cancer-specific targets. In contrast, BI505 bound regardless of changes in sialic acid content, confirming a difference from the hyposialylation-based cancer-specific mechanism of S4-2. This suggests that the S4-2 antibody has the potential to perform cancer-specific targeting by targeting abnormal glycosylation patterns, particularly hyposialylation, in cancer cells. Therefore, the S4-2 antibody may provide a cancer-specific targeting strategy differentiated from existing ICAM1 antibodies in cancer therapy.

[0159]

[0160] Example 8. Evaluation of the in vitro and in vivo metastasis inhibition effects of S4-2 antibody

[0161] 8.1. In vitro metastasis inhibition effect of S4-2 antibody

[0162] In this example, a Transendothelial Migration Assay (TMA) was performed to analyze the effect of the S4-2 antibody according to the present invention on cell migration and metastasis of a triple-negative breast cancer cell line (MDA-MB-468).

[0163] MDA-MB-468 cells are a representative triple-negative breast cancer cell line with high EGFR expression and strong invasiveness. In this study, the amount of cell migration between the S4-2-treated group and the control group (human IgG1 isotype control antibody) was compared using Transwell inserts coated with endothelial cells.

[0164] As a result, as shown in Figure 15, the number of cells that passed through the endothelial cell layer was significantly reduced in the group treated with S4-2 antibody compared to the control group, suggesting that the S4-2 antibody effectively inhibits the mobility of cancer cells by targeting ICAM1.

[0165]

[0166] 8.2. In vivo metastasis inhibition effect of S4-2 antibody

[0167] To confirm the metastasis inhibitory effect of the S4-2 antibody, an in vivo test was performed using a mouse lung metastasis model of breast cancer.

[0168] A lung metastasis model was established by intravenously injecting MDA-MB-231 cells, a triple-negative breast cancer cell line, into immunodeficient mice. S4-2 antibody (10 mg / kg) was then administered repeatedly intravenously at regular intervals. The control group was administered an equivalent amount of IgG1 antibody.

[0169] As a result, as shown in Fig. 16, the number of metastatic nodules in lung tissue was significantly reduced in the S4-2 antibody administration group, and histopathological analysis confirmed that the metastatic area and degree of cell infiltration were markedly reduced. This result proves that the antibody according to the present invention has significant metastasis-inhibiting activity.

[0170]

[0171] Example 9. Confirmation of broad TNBC cell line-specific binding and non-toxicity to normal cells of the S4-2 antibody.

[0172] In this example, it was confirmed that the S4-2 antibody has the property of broadly and specifically binding to various TNBC cell lines, while not binding to normal breast cells expressing the ICAM1 protein.

[0173] Various TNBC cell lines (MDA-MB-468, MDA-MB-436, MDA-MB-231, HS578T, 057239, 050786), a hormone receptor-positive breast cancer cell line (MCF7), and a normal breast epithelial cell line (MCF10A) were prepared. After protein extraction from each cell line, Western blotting was performed to confirm the expression and amount of ICAM1 protein, and β-actin was used as a standard for comparison of protein loading.

[0174] As a result, as shown in Figure 17, it was confirmed that ICAM1 protein was expressed in all cell lines used in the experiment. In particular, although ICAM1 was expressed in the normal cell line MCF10A, the binding affinity of the S4-2 antibody was almost nonexistent in the subsequent antibody binding experiment.

[0175]

[0176] After treatment with the S4-2 antibody for each cell line, flow cytometry (FACS) analysis was performed using a fluorescently labeled secondary antibody, and the cell surface binding rate of the S4-2 antibody was quantitatively measured. Based on the FACS data, the antibody binding rate to each cell line was calculated and compared.

[0177] Western blot analysis results, as shown in Figure 18, confirmed that ICAM1 protein was expressed in all cell lines used in the experiment (MDA-MB-468, MDA-MB-436, MDA-MB-231, HS578T, 057239, 050786, MCF7, and MCF10A). Notably, MCF10A normal breast epithelial cells were also found to express ICAM1. Furthermore, as shown in Figure 19, flow cytometry (FACS) results showed that the S4-2 antibody exhibited high binding affinity to various TNBC cell lines, as follows:

[0178] ㆍ MDA-MB-468 (TNBC): 98.85% binding

[0179] ㆍ MDA-MB-436 (TNBC): 99.11% binding

[0180] ㆍ MDA-MB-231 (TNBC): 68.23% binding

[0181] ㆍ 057239 (TNBC): 34.40% and 87.68% combined

[0182] ㆍ 050786 (TNBC): 87.61% combined

[0183] ㆍ HS578T (TNBC): 'x'

[0184] In contrast, the S4-2 antibody showed little binding to MCF10A normal breast epithelial cells expressing ICAM1, indicating that it does not bind to normal breast cells. Furthermore, it showed low binding affinity of 6.61% to MCF7 (ER+ breast cancer) cells.

[0185] The above experimental results demonstrated that the S4-2 antibody potently and specifically binds to a wide range of TNBC cancer cell lines. Notably, despite expressing ICAM1 protein, the S4-2 antibody exhibited minimal binding to normal breast epithelial cells, such as MCF10A, confirming the high non-toxicity of S4-2 to normal cells. These results strongly suggest that S4-2 does not simply bind based on the presence of ICAM1 protein, but rather selectively binds to cancer-specific targets within the ICAM1 protein. Therefore, the S4-2 antibody has the potential to be an effective and safe targeted antibody therapeutic for the treatment of TNBC.

[0186]

[0187] Example 10. Confirmation of the applicability of the S4-2 antibody to multiple myeloma, non-small cell lung cancer, pancreatic cancer, and ovarian cancer.

[0188] The therapeutic or diagnostic potential of S4-2 and BI-505 was confirmed for various cancers characterized by ICAM1 hyposialylation (multiple myeloma, non-small cell lung cancer, pancreatic cancer, and ovarian cancer). Prior to clinical application of the S4-2 antibody, circulating tumor cells (CTCs) FACS (circulating tumor cell flow cytometry) can be used for diagnostic purposes to determine whether ICAM1 hyposialylation is present in patients' circulating tumor cells (CTCs). This can be used to select patients eligible for the S4-2 antibody.

[0189] The binding affinity of the S4-2 antibody was determined for multiple myeloma, non-small cell lung cancer, pancreatic cancer, and ovarian cancer cell lines, and compared with that of the competitive drug BI-505. RPMI8226, H929, and U266 cell lines were used as multiple myeloma cell lines; H460, H1299, PC9, and A549 cell lines were used as non-small cell lung cancer cell lines; AsPC-1 and BxPC3 cell lines were used as pancreatic cancer cell lines; and JHOM-1 and JHOM-2B cell lines were used as ovarian cancer cell lines.

[0190] As a result, as shown in FIGS. 20 to 23, the S4-2 antibody also showed binding affinity to cell lines of multiple myeloma, non-small cell lung cancer, pancreatic cancer, and ovarian cancer, suggesting that it can be applied to various types of cancer (multiple myeloma, non-small cell lung cancer, pancreatic cancer, and ovarian cancer) with ICAM1 hyposialylation.

[0191]

[0192] Comparative Example 1. Confirmation of cancer-specific binding of S4-2 antibody to TNBC cells and its superiority over competing antibodies.

[0193] In this example, the cancer specificity of the S4-2 antibody was verified by confirming its specific binding to triple-negative breast cancer (TNBC) cell lines and simultaneously evaluating its non-binding characteristics toward normal cells expressing ICAM1. In addition, the safety and differentiation of S4-2 were evaluated by analyzing the differences in binding characteristics with BI-505, a comparative antibody whose development was discontinued in Phase 2 clinical trials due to cardiopulmonary toxicity.

[0194] Triple-negative breast cancer cell line MDA-MB-436 and human breast normal cell line MCF10A were cultured in appropriate media and prepared at the cell density required for the experiment. Each cell line was treated with S4-2 and BI-505 antibodies at a given concentration. Cell surface binding of the antibodies was analyzed by flow cytometry using fluorescently labeled secondary antibodies.

[0195] As a result, as shown in Figure 24, the S4-2 antibody showed a high binding rate of 95.8% to MDA-MB-436 cancer cells, indicating strong binding to the cancer cell surface. On the other hand, the same antibody showed a very low binding rate of 1.8% to MCF10A normal cells. In contrast, the BI-505 antibody showed a binding rate of 97.3% to MDA-MB-436 cancer cells, but also a high binding rate of 90.2% to MCF10A normal cells.

[0196] These results clearly demonstrate that the S4-2 antibody possesses excellent cancer specificity, specifically binding to cancer cells but not to normal cells. Specifically, while BI-505 nonspecifically binds to normal cells, S4-2 does not bind to ICAM1 on normal cells, offering significant superiority in selectivity and safety as a therapeutic target. This strongly suggests that S4-2 has high potential as a cancer therapeutic antibody that can overcome the toxicity issues of existing ICAM1-targeting antibodies.

[0197] The sequence numbers (SEQ ID NO: 1 to SEQ ID NO: 13) of the present invention are described in the Sequence Listing submitted together with the present application and constitute a part of the present invention.

Claims

A composition for detecting ICAM1 protein comprising an anti-ICAM1 antibody, - The anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; - A composition for detecting ICAM1 protein, characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of sequence number 13 of the ICAM1 protein is hyposialylated. In the first paragraph, A composition for detecting ICAM1 protein, wherein the heavy chain variable region is composed of an amino acid sequence of SEQ ID NO: 4, and the light chain variable region is composed of an amino acid sequence of SEQ ID NO:

8. A composition for diagnosing an anti-ICAM1 related disease comprising the composition of claim 1. In the third paragraph, A diagnostic composition for an anti-ICAM1-related disease, characterized in that the anti-ICAM1-related disease is cancer. In paragraph 4, A composition for diagnosing an anti-ICAM1 related disease, wherein the cancer is any one selected from the group consisting of breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, kidney cancer, skin cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. In paragraph 5, A composition for diagnosing an anti-ICAM1 related disease, characterized in that the breast cancer is triple negative breast cancer. In paragraph 5 or 6, A composition for diagnosing an anti-ICAM1 related disease, characterized in that the cancer is metastatic or relapsed cancer. A kit for detecting ICAM1 protein comprising the composition of claim 1. A diagnostic kit for anti-ICAM1 related diseases comprising the composition of claim 3. (1) A step of contacting an anti-ICAM1 antibody with a biological sample obtained from an individual; (2) A step of detecting ICAM1 protein bound to anti-ICAM1 antibody; (3) A method for providing information for diagnosing an anti-ICAM1 related disease, comprising a step of evaluating whether ICAM1 protein is detected in the above step (2), The anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; A method for providing information for diagnosing an anti-ICAM1 related disease, wherein the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of sequence number 13 is hyposialylated. A pharmaceutical composition for preventing or treating cancer comprising an anti-ICAM1 antibody, - The anti-ICAM1 antibody comprises a heavy chain variable region comprising a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 7; - A pharmaceutical composition for preventing or treating cancer, wherein the anti-ICAM1 antibody recognizes the ICAM1 protein, and the ICAM1 protein is characterized in that the N-glycan terminal of asparagine (ASN) located at position 130 of sequence number 13 is hyposialylated.

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