Integrin β2-specific antibody

Abstract

The present invention relates to the use of an antibody or an immunologically active fragment thereof, which specifically binds to integrin β2, as an immuno-oncology agent. The chimeric and humanized antibodies of the present invention specifically bind to integrin β2, which is a tumor-specific antigen and is expressed on the cell membrane of M2 tumor-associated macrophages, and thus can be used as an antibody therapeutic agent for cancer treatment using a cancer cell death mechanism by immune cell activation, an antibody therapeutic agent for cancer treatment, or a cell therapeutic agent for cancer treatment, and can also be used for cancer diagnosis.

Description

Integrin β2 specific antibody

[0001] The present invention relates to the use of an antibody that specifically binds to integrin β2 or a fragment having the same’s immunological activity as an immunotherapeutic agent.

[0002] Conventional anticancer treatments have been researched in the direction of directly attacking cancer cells or enhancing the activity of the body's immune cells that attack cancer cells. However, these anticancer drugs also attack normal cells other than cancer cells, leading to numerous side effects such as hair loss, nausea, and vomiting, as well as causing adverse reactions due to the excessive proliferation of immune cells. Anticancer immunotherapy, which can minimize side effects compared to conventional chemotherapy or radiation therapy, is a method of treating cancer by utilizing the body's immune system. Among these anticancer immunotherapy techniques, active research is being conducted on cell therapy methods, which involve activating therapeutic immune cells such as T cells (including CAR-T), dendritic cells, and natural killer cells outside the body and injecting them directly into the body, and anticancer vaccine methods, which enhance anticancer efficacy by directly activating existing immune cells through the injection of cancer antigens and immune-activating substances. However, these cell therapies and cancer vaccines are primarily used for blood cancers and generally have the disadvantage of having very low therapeutic efficacy in solid tumors. One of the reasons for this is attributed to microenvironmental factors that suppress immune function around solid tumors. In fact, cells that impair immune cell function (MDSC: myeoloid-derived stromal cells, Treg: regulatory T cell, TAM: tumor-associated macrophages, CAF: cancer-associated fibroblast), as well as immunosuppressive cytokines and metabolites, act actively in the tumor microenvironment, thereby drastically reducing the activity of immune-activating substances and therapeutic immune cells. Therefore, it is becoming important to develop therapeutic agents that possess anticancer effects by regulating only the microenvironment surrounding tumor cells without directly affecting tumor cells or immune cells, thereby blocking the supply of nutrients to tumor cells and angiogenesis around tumor cells.The tumor microenvironment is considered a major therapeutic target as it contributes to the proliferation and survival of malignant cells, angiogenesis, metastasis, abnormal adaptive immunity, and reduced response to hormones and chemotherapy agents. Recently, various antibody therapies that inhibit the action of immune checkpoint proteins such as CTLA-4 (Cytotoxic T-Lymphocyte-Associated Protein 4) and PD-1 (Programmed Cell Death Protein 1) have been developed, leading to an explosive increase in clinical demand. Consequently, the immune checkpoint inhibitor market is projected to grow continuously at an average annual rate of 21.8% from 2020 to 2027.

[0003] The tumor microenvironment (TME) consists of endothelial cells, inflammatory cells, and fibroblasts, and it was discovered in the 1970s that tumor-associated macrophages (TAMs) play a crucial role in tumor growth. TAMs play a vital role in the overall tumor microenvironment, including cancer growth and metastasis, and are classified into two phenotypes: tumor-suppressing M1 macrophages or tumor-supporting M2 macrophages. M1 macrophages possess a potent ability to present antigens and are generally activated by interferon-γ, lipoglycans (LPS), and tumor necrosis factor (TNF)-α, performing pro-inflammatory and bactericidal functions. M2 macrophages are known to promote immunosuppression, tumorigenesis, and angiogenesis by releasing various extracellular matrix components, angiogenic factors, and chemotactic factors. Generally, they are induced by IL-4 and IL-13 and are distinguished from M1 tumor-associated macrophages by expressing markers such as arginase-1, mannose (MMR, CD206), scavenger receptors (SR-A, CD204), CD163, and IL-10. Tumor-associated macrophages present around tumors are closely associated with tumor cell growth and metastasis, and it has been reported that the presence of a large number of M2 tumor-associated macrophages around tumors in cancer patients leads to poor prognosis and survival rates. M2 macrophages produce cytokines such as IL-10, TGFβ, and CCL18 that promote cancer growth, and receptors such as PDL1 and B7-1 / 2 present on the surface of M2 tumor-associated macrophages are reported to inhibit the anti-tumor activity of T cells and NK cells. Since tumor growth, differentiation, and metastasis occur actively in a microenvironment where M2 tumor-associated macrophages are abundant, the development of targeted therapies targeting M2 tumor-associated macrophages is necessary.

[0004] The object of the present invention is to provide an antibody that specifically binds to integrin β2 or a fragment having the same’s immunological activity.

[0005] In addition, the object of the present invention is to provide an antibody-drug conjugate comprising the antibody or a fragment having the immunological activity thereof, and a drug.

[0006] In addition, the object of the present invention is to provide a chimeric antigen receptor comprising a nucleic acid encoding the antibody or a fragment having immunological activity thereof.

[0007] In addition, the object of the present invention is to provide a chimeric antigen receptor-expressing cell transformed with a recombinant vector comprising a gene encoding the chimeric antigen receptor.

[0008] In addition, the object of the present invention is to provide a T cell agent comprising the antibody or a fragment having the immunological activity thereof.

[0009] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer.

[0010] In addition, the objective of the present invention is to provide a composition for diagnosing cancer.

[0011] In addition, the objective of the present invention is to provide a pharmaceutical composition for removing tumor-associated macrophages in the tumor microenvironment.

[0012] In addition, the objective of the present invention is to provide a method for preventing or treating cancer.

[0013] To solve the above problem, the present invention provides an antibody that specifically binds to integrin β2 or a fragment having the immunological activity thereof.

[0014] In addition, the present invention provides an antibody-drug conjugate comprising the antibody or a fragment having immunological activity thereof, and a drug.

[0015] In addition, the present invention provides a chimeric antigen receptor comprising a nucleic acid encoding the antibody or a fragment having immunological activity thereof.

[0016] In addition, the present invention provides a chimeric antigen receptor-expressing cell transformed with a recombinant vector comprising a gene encoding the chimeric antigen receptor.

[0017] In addition, the present invention provides a T cell agent comprising the antibody or a fragment having immunological activity thereof.

[0018] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer.

[0019] In addition, the present invention provides a composition for diagnosing cancer.

[0020] In addition, the present invention provides a pharmaceutical composition for removing tumor-associated macrophages in a tumor microenvironment.

[0021] In addition, the present invention provides a method for preventing or treating cancer.

[0022] Since the chimeric and humanized antibodies of the present invention are tumor-specific antigens and specifically bind to integrin β2 expressed on the cell membrane of M2 tumor-associated macrophages, they have the effect of being usable as antibody therapeutics for cancer treatment or cell therapeutics for cancer treatment that utilize the mechanism of cancer cell death by immune cell activation, and also have the effect of being usable for the diagnosis of cancer.

[0023] Figures 1 and 2 show the amino acid and nucleotide sequences of mouse (ms) anti-CD18 / CR3 antibodies (clone KIM127) VL and VH against integrin β2:

[0024] Red mark: CDR.

[0025] Figure 3 shows the human antibody sequences selected as most similar based on the nucleotide sequence of the KIM127 mouse antibody sequence:

[0026] Underlined: CDR.

[0027] Figure 4 shows the amino acid sequence of the chimeric antibody chKIM127, in which the CDR sequence in a selected human antibody sequence was substituted with the KIM127 mouse antibody sequence:

[0028] Underlined: CDR.

[0029] Figure 5 shows the VH and VL amino acid sequences of two humanized antibodies designed by humanizing chKIM127:

[0030] Underlined: CDR.

[0031] Figure 6 is a figure showing the purity of the chimeric antibody and two types of humanized antibodies designed in the present invention, synthesized and purified into IgG form.

[0032] Figure 7 is a figure analyzing the binding affinity of the culture supernatant of cells transiently expressing chKIM127 to target cells (THP-1):

[0033] KIM127 chIgG culture supernatant: Culture supernatant of cells transiently expressing chKIM127.

[0034] Figure 8 is a figure analyzing the binding affinity of three purified antibodies of the present invention to a target cell (THP-1):

[0035] chKIM127: refined chKIM127;

[0036] hzKIM127.11: refined hzKIM127.1; and

[0037] KIM127.22: Refined hzKIM127.2.

[0038] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0039] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0040] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0041] Throughout this specification, not only are conventional one- and three-character codes for naturally occurring amino acids used, but generally accepted three-character codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc., are also used. Additionally, amino acids referred to by abbreviations in this invention are described according to the IUPAC-IUB nomenclature as follows:

[0042] 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.

[0043]

[0044] In one aspect, the present invention relates to an antibody that specifically binds to integrin β2 or a fragment having the same’s immunological activity.

[0045] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof can specifically bind to an active conformation in which integrin β2 is extended.

[0046] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may include a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 having the amino acid sequence of SEQ ID NO. 1, CDRH2 having the amino acid sequence of SEQ ID NO. 2, and CDRH3 having the amino acid sequence of SEQ ID NO. 3.

[0047] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may include a VL domain comprising a CDRL (Complementarity determining regions Light chain) 1 having the amino acid sequence of SEQ ID NO. 4, a CDRL 2 having the amino acid sequence of SEQ ID NO. 5, and a CDRL 3 having the amino acid sequence of SEQ ID NO. 6.

[0048] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may comprise a VH domain comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 7 to 9.

[0049] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may comprise a VL domain comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 10 to 12.

[0050] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may comprise a heavy chain comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 13 to 15.

[0051] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may comprise a light chain comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 16 to 18.

[0052] In one embodiment, the antibody chKIM127 of the present invention may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 13 and a light chain comprising the amino acid sequence of SEQ ID NO. 16.

[0053] In one embodiment, the antibody hzKIM127.1 of the present invention may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 14 and a light chain comprising the amino acid sequence of SEQ ID NO. 17.

[0054] In one embodiment, the antibody hzKIM127.2 of the present invention may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 15 and a light chain comprising the amino acid sequence of SEQ ID NO. 18.

[0055] In one embodiment, the antibody of the present invention or the fragment having immunological activity thereof may be an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, a single-chain antibody, an Fv dimer, a complementarity determining region fragment, a single-domain antibody, a diabody, a multispecific antibody, a dual-specific antibody, or a bispecific antibody, and is more preferably a chimeric antibody or a humanized antibody.

[0056] In one embodiment, the antibody of the present invention may be IgG1, IgG2, or IgG4 or a fragment thereof.

[0057] The above antibody is in the form of a whole antibody as well as includes functional fragments of the antibody molecule. The whole antibody has a structure comprising two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. A functional fragment of the antibody molecule refers to a fragment possessing antigen-binding function, and examples of antibody fragments include: (i) a Fab fragment consisting of the variable region (VL) of the light chain and the variable region (VH) of the heavy chain, and the constant region (CL) of the light chain and the first constant region (CH1) of the heavy chain; (ii) an Fd fragment consisting of the VH and CH1 domains; and (iii) an Fv fragment consisting of the VL and VH domains of a monoclonal antibody. (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) a separated CDR region; (vi) a divalent fragment F(ab')2 fragment containing two linked Fab fragments; (vii) a single-stranded Fv molecule (scFv) linked by a peptide linker that links the VH domain and VL domain to form an antigen-binding site; (viii) a bispecific single-stranded Fv dimer (PCT / US92 / 09965); and (ix) a diabody WO94 / 13804, a multivalent or multispecific fragment produced by gene fusion.

[0058] The antibody of the present invention or the fragment having immunological activity thereof may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments having immunological activity thereof. The antibody may be produced recombinantly or synthetically.

[0059] Animal-derived antibodies, produced by immunizing animals with a desired antigen, can generally cause immune rejection when administered to humans for therapeutic purposes; chimeric antibodies were developed to suppress this rejection. Chimeric antibodies are created by using genetic engineering methods to replace the constant region of animal-derived antibodies—which causes anti-isotype reactions—with the constant region of human antibodies. While chimeric antibodies show significant improvement in anti-isotype reactions compared to animal-derived antibodies, they still carry potential side effects related to anti-idiotypic reactions because animal-derived amino acids remain in the variable region. Humanized antibodies were developed to address these side effects. They are produced by transplanting the Complementarity Determining Regions (CDRs), which play a crucial role in antigen binding within the variable region of chimeric antibodies, into a human antibody framework.

[0060] In CDR grafting technology for producing humanized antibodies, the most critical aspect is selecting an optimized human antibody capable of best accepting the CDR site of an animal-derived antibody; to achieve this, techniques such as antibody databases, crystal structure analysis, and molecular modeling are utilized. However, even when the CDR site of an animal-derived antibody is grafted onto an optimized human antibody scaffold, there are a significant number of cases where antigenic binding affinity is not preserved due to the presence of amino acids on the animal antibody scaffold that affect antigen binding. Therefore, the application of additional antibody engineering techniques to restore antigenic binding affinity is essential.

[0061] The above antibody or the fragment having immunological activity thereof may be isolated from a living organism (not present in the living organism) or non-naturally occurring, for example, may be produced synthetically or recombinantly.

[0062] In the present invention, the term "antibody" refers to a substance produced within the immune system by stimulation of an antigen; its type is not particularly limited and can be obtained naturally or unnaturally (e.g., synthetically or recombinantly). Antibodies are advantageous for mass expression and production because they are very stable and have a long half-life, both in vitro and in vivo. Furthermore, antibodies have a very high avidity because they inherently possess a dimer structure. A complete antibody has a structure comprising two full-length light chains and two full-length heavy chains, with each light chain connected to a heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0063] In the present invention, the term "heavy chain" is interpreted to mean a full-length heavy chain and a fragment thereof, comprising a variable region domain VH having an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a hinge with three constant region domains CH1, CH2, and CH3. Additionally, the term "light chain" is interpreted to mean a full-length light chain and a fragment thereof, comprising a variable region domain VL having an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL.

[0064] In the present invention, the term "variable region" or "variable domain" refers to a part of an antibody molecule that performs the function of specifically binding to an antigen and exhibits many variations in sequence, and the variable region contains complementary determining regions CDR1, CDR2, and CDR3. Between the CDRs, there exists a framework region (FR) that serves to support the CDR loop. The "complementary determining region" is a loop-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

[0065] In the present invention, the term "scFv (single chain fragment variable)" refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to a single-chain antibody in which the VH region and the VL region of the antibody are connected by a short peptide chain. Unless otherwise specified or understood from the context, the term "scFv" is intended to include scFv fragments, including antigen-binding fragments. This is obvious to a person skilled in the art.

[0066] In the present invention, the term "complementarity determining region (CDR)" refers to the amino acid sequence of a hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy chain and light chain may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide major contact residues for the antibody to bind to an antigen or an epitope.

[0067] In the present invention, the terms "specifically binding" or "specifically recognizing" have the same meaning as commonly known to those skilled in the art, and refer to an antigen and an antibody specifically interacting to produce an immunological reaction.

[0068] In the present invention, the term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that includes a portion of a polypeptide capable of binding to an antigen. For example, it may be scFv, (scFv)2, scFv-Fc, Fab, Fab', or F(ab')2, but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Recombinant technology for generating the Fv fragment from a minimal antibody fragment having only a heavy chain variable region and a light chain variable region is widely known in the art. In a two-chain Fv, the variable region of the heavy chain and the variable region of the light chain are connected by non-covalent bonds, and in a single-chain Fv, the variable region of the heavy chain and the variable region of the single chain are generally connected by covalent bonds through a peptide linker or directly at the C-terminus, so they can form a dimer-like structure similar to that of a two-chain Fv. The linker may be a peptide linker composed of 1 to 100 or 2 to 50 any amino acids, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, a Fab fragment can be obtained by restriction cleaving a whole antibody with papain, and an F(ab')2 fragment can be obtained by cleaving it with pepsin), and can be produced through genetic recombination technology.

[0069] In the present invention, the term "hinge region" refers to a region included in the heavy chain of an antibody, existing between the CH1 and CH2 regions, and functioning to provide flexibility to the antigen binding site within the antibody. For example, the hinge may be derived from a human antibody, specifically, from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

[0070] In the present invention, the term "amino acid modification / mutation" means the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence, preferably substitution. As used in the present invention, the term "amino acid substitution" or "substitution" means that an amino acid at a specific position in the polypeptide sequence of an antibody is replaced with another amino acid.

[0071] The antibody of the present invention or the fragment having immunological activity thereof may be prepared by any method known in the art. After encoding the polypeptide sequence of the antibody of the present invention or the fragment having immunological activity thereof, it is used to form nucleic acids that are cloned into host cells, expressed, and tested, if desired. Various methods for this are described in the literature (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).

[0072] The nucleic acid encoding the antibody of the present invention or the fragment having immunological activity thereof may be inserted into an expression vector for protein expression. The expression vector typically comprises a protein that is functionally linked, i.e., functionally related, to a regulatory or control sequence, a selection marker, any fusion partner, and / or additional elements. Under appropriate conditions, the antibody according to the present invention or the fragment having immunological activity thereof may be produced by a method of inducing protein expression by culturing a host cell transformed with nucleic acid, preferably a nucleic acid-containing expression vector encoding the antibody of the present invention or the fragment having immunological activity thereof. Various suitable host cells, including mammalian cells, bacteria, insect cells, and yeast, may be used, but are not limited thereto. Methods for introducing exogenous nucleic acid into host cells are known in the art and will vary depending on the host cell used. Preferably, the antibody according to the present invention or the fragment having immunological activity thereof is produced using Escherichia coli, which has high industrial value due to its low production cost, as the host cell.

[0073] Accordingly, the scope of the present invention includes a method for producing an antibody or a fragment having immunological activity, comprising the steps of: introducing a nucleic acid encoding the antibody of the present invention or a fragment having immunological activity thereof into a host cell under conditions suitable for protein expression; and purifying or isolating the antibody or the fragment having immunological activity thereof expressed from the host cell.

[0074] Antibodies may be separated or purified by various methods known in the art. Standard purification methods include chromatography, electrophoresis, immunoassay, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As known in the art, various natural proteins, such as bacterial proteins A, G, and L, bind to antibodies, and said proteins may be used for purification. Often, purification by specific fusion partners may be possible.

[0075] In one aspect, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody of the present invention or a fragment having immunological activity thereof, and a drug.

[0076] In one embodiment, the drug may be an immunogenic apoptosis inducer, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, or a radionuclide, and the immunogenic apoptosis inducer may be one or more selected from the group consisting of anthracycline anticancer agents, taxane anticancer agents, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosphamide anticancer agents, GADD34 / PP1 inhibitors, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin.

[0077] In one embodiment, the drug is SN-38 (7-ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, Triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine,Capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin It may be selected from the group consisting of F(auristatin F), monomethyl auristatin E (MMAE), monomethyl auristatin F (monomethyl auristatin F), and derivatives thereof.

[0078] In one embodiment, the antibody-drug conjugate may further comprise an ADC linker, and the ADC linker may be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyl oxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyl oxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

[0079] In one embodiment, the antibody-drug conjugate may form a complex with a drug through an ADC linker, wherein the antibody of the present invention or a fragment having immunological activity thereof may form a complex with the drug.

[0080] In one aspect, the present invention relates to a bispecific or multispecific antibody comprising the antibody of the present invention or a fragment having immunological activity thereof, and a portion that binds to a target antigen other than integrin β2.

[0081] In one embodiment, the portion binding to the target antigen may include an antibody or a fragment having the same immunological activity.

[0082]

[0083] In one embodiment, the target antigen is 17-1A antigen, GD3 ganglioside R24, EGFRvⅢ PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, atypically glycosylated MUC1, fibronectin isoform containing an ED-B domain, HER2 / neu, carcinoma-embryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, antigen specific to A3 and A33 antibodies, Ba 733, BrE3-antigen, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, Colon-specific antigen-p (CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, Folate receptor, HLA-DR, Human chorionic gonadotropin (HCG) and its subunits, Hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, Insulin-growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, Macrophage inhibitor factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigens specific to PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, SLOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,ED-B may be one or more selected from the group consisting of fibronectin, angiogenesis markers, oncogene markers, or oncogene products.

[0084] In one embodiment, the apoptosis-related gene is ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, PAK1, It may be PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ, or YWHAZ;상기 전사인자 유전자는 AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1,PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A, ZIC2, ZNF217 또는 ZNF268일 수 있고;The above metastasis-related genes may be AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1, PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3, or WT1; and the above angiogenesis-related genes may be BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3, FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB, or SPARC; The above tyrosine-kinase gene may be ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC, or YES1.

[0085] 일 구현예에서, 상기 종양유전자는 SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, ENTPD5, EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A,FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ, GNAS, GOLPH3, GOPC, GPAT4, GPM6A, GPM6B, GPR132, GREM1, GRM1, GSK3A, GSM1, H19, HAS1, HAX1, HDGFRP2, HMGN5, HNRNPA1, HOTAIR, HOTTIP, HOXA-AS2, HRAS, HSPA1A, HSPA4, HSPB1, HULC, IDH1, IFNG, IGF2BP1, IKBKE, IL7R, INPPL1, INTS1, INTS2, INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28A, LIN28B, LMO1, LMO2, LMO3, LMO4, LSM1, LUADT1, MACC1, MACROD1, MAGEA11, MALAT1, MAML2, MAP3K8, MAPRE1, MAS1, MCC, MCF2, MCF2L, MCTS1, MEFV, MFHAS1, MFNG, MIEN1, MINA, MKL2, MLANA, MLLT1, MLLT11, MLLT3, MLLT4, MMP12, MMS22L, MN1, MNAT1, MOS, MPL, MPST, MRAS, MRE11A, MSI1, MTCP1, MTDH, MTOR, MUC1, MUC4, MUM1, MYD88, NAAA, NANOGP8, NBPF12, NCOA4, NEAT1, NECTIN4, NEDD4, NEDD9, NET1, NINL, NME1, NOTCH1, NOTCH4, NOV, NSD1, NUAK2, NUP214, NUP98, NUTM1, OLR1,PA2G4, PADI2, PAK7, PARK7, PARM1, PBK, PCAT1, PCAT5, PDGFA, PDZK1IP1, PELP1, PFN1P3, PIGU, PIK3CA, PIK3R1, PIM1, PIM2, PIM3, PIRD,PLKPLM1,PICWIL1, PPP1R10, PPP1R14A, PPP2R1A, PRAME, PRDM12, PRMT5, PSIP1, PSMD10, PTCH2, PTMA, PTP4A1, PTP4A2, PTP4A3, PTTG1, PTTG1IP, PTTG2, R21, RABAB, PVTA1, RAB8A, RALGDS, RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF14, S100T, S1X1T S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLCNO3A2, SCG9, SMR1,B, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZTA171CSTD, SSW TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEMPR140,2, TMPRSSPD2, TMPOP2 TRE17, TREH, TRIB1, TRIB2,It may be any one or more selected from the group consisting of TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, XIAP, YAP1, YEATS4, YY1AP1, ZEB1-AS1, ZFAND4, ZFAS1, ZMYM2, ZNF703, and ZNHIT6.

[0086] In one embodiment, the target antigen may be a cell surface antigen or an autoantigen.

[0087] In one embodiment, the cell surface antigen may be one or more selected from the group consisting of CEA, ED-B fibronectin, CD20, CD22, CD19, EGFR, IGF1R, VEFGR1 / Flt-1, VEGFR2 / KDR, VEGRF3 / Flt-4, HER2 / neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, adenovirus fibrous nop, PfMSP-1, HN / NDV, EpCAM / 17-1A, hTR, IL-2R / Tac, CAl9-9, MUCl, HLA class II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, M13 envelope protein, and GPIIb / IIIa.

[0088] In one aspect, the present invention relates to an antibody of the present invention or a fragment having immunological activity thereof, or an isolated nucleic acid molecule encoding a bispecific or multispecific antibody of the present invention, a vector comprising the same, and a host cell transformed therefrom.

[0089] The nucleic acid molecules of the present invention may be isolated or recombinant and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. In the case of nucleic acids isolated from natural sources, isolated nucleic acids are nucleic acids separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acids produced from these procedures may be understood as isolated nucleic acid molecules. Isolated nucleic acid molecules represent nucleic acid molecules in the form of separate fragments or as components of larger nucleic acid constructs. Nucleic acids are operably linked when arranged in a functional relationship with other nucleic acid sequences. For example, the DNA of a presequence or secretion leader is operably linked to the polypeptide DNA when the polypeptide is expressed as a preprotein, which is the form prior to secretion; a promoter or enhancer is operably linked to the coding sequence when it influences the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to the coding sequence when positioned to facilitate translation. Generally, operably linked means that the DNA sequences to be linked are located adjacently, and in the case of a secretion leader, this means they exist adjacently within the same reading frame. However, enhancers do not need to be located adjacently. Linkage is achieved by ligation at a convenient restriction enzyme site. If such a site is not present, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0090] The isolated nucleic acid molecule encoding the antibody of the present invention, the fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention may undergo various modifications to the coding region within a range that does not alter the amino acid sequence of the antibody expressed from the coding region, due to codon degeneracy or considering the codons preferred by the organism to express said antibody; and may also undergo various modifications or alterations to parts excluding the coding region within a range that does not affect gene expression, and such modified genes are also included within the scope of the present invention, as will be well understood by those skilled in the art. That is, as long as the nucleic acid molecule of the present invention codes for a protein having equivalent activity, one or more nucleic acid bases may be modified by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention. The sequence of such nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0091] An isolated nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention, according to the present invention, may be inserted into an expression vector for protein expression. The expression vector typically comprises a protein operably linked, i.e., functionally related, to a regulatory or control sequence, a selection marker, any fusion partner, and / or additional elements. Under appropriate conditions, the antibody of the present invention or a fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention, may be produced by a method of inducing protein expression by culturing a host cell transformed with nucleic acid, preferably, an expression vector containing the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof, or the bispecific or multispecific antibody of the present invention. Various suitable host cells, including mammalian cells, bacteria, insect cells, and yeast, may be used, but are not limited thereto. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, Escherichia coli, which has low production costs and high industrial utility value, can be produced as a host cell.

[0092] The vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable vectors may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequences may include, but are not limited to, PhoA signal sequences, OmpA signal sequences, etc. when the host is Escherichia sp., α-amylase signal sequences, subtilisin signal sequences, etc. when the host is Bacillus sp., MFα signal sequences, SUC2 signal sequences, etc. when the host is yeast, and insulin signal sequences, α-interferon signal sequences, antibody molecule signal sequences, etc. when the host is an animal cell. Additionally, the vector may include a selection marker for selecting a host cell containing the vector, and if it is a replicable expression vector, it includes a replication origin.

[0093] In the present invention, the term “vector” refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art may construct vectors using standard recombinant techniques (Maniatis, et al., *Molecular Cloning*, *A Laboratory Manual*, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., *Current Protocols in Molecular Biology*, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0094] In one embodiment, when producing the vector, expression control sequences such as a promoter, terminator, and enhancer, sequences for membrane targeting or secretion, etc., can be appropriately selected and combined in various ways according to the purpose, depending on the type of host cell to be used to produce the antibody.

[0095] In the present invention, the term “expression vector” means a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. The expression vector may include various regulatory sequences. Along with regulatory sequences that regulate transcription and translation, the vector and the expression vector may also include nucleic acid sequences that provide other functions.

[0096] In the present invention, the term "host cell" refers to any transgenic organism, including eukaryotes and prokaryotes, capable of replicating said vector or expressing a gene encoded by said vector. The host cell may be transfected or transformed by said vector, which refers to the process in which an exogenous nucleic acid molecule is delivered or introduced into the host cell.

[0097] In one embodiment, the host cell may be a bacterium or an animal cell, the animal cell line may be a CHO cell, an HEK cell, or an NSO cell, and the bacterium may be E. coli.

[0098] In one aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising a nucleic acid encoding an antibody of the present invention or a fragment having immunological activity thereof.

[0099] In one embodiment, the chimeric antigen receptor may include an integrin β2 binding domain, a transmembrane domain, and an intracellular signaling domain, and the integrin β2 binding domain may include CDRH (Complementarity determining regions Heavy chain) 1 including the amino acid sequence of SEQ ID NO. 1, CDRH2 including the amino acid sequence of SEQ ID NO. 2, CDRH3 including the amino acid sequence of SEQ ID NO. 3, CDRL (Complementarity determining regions Light chain) 1 including the amino acid sequence of SEQ ID NO. 4, CDRL2 including the amino acid sequence of SEQ ID NO. 5, and CDRL3 including the amino acid sequence of SEQ ID NO. 6.

[0100] In one aspect, the present invention relates to a recombinant vector comprising a gene encoding the chimeric antigen receptor.

[0101] In one aspect, the present invention relates to a chimeric antigen receptor-expressing cell transformed with the recombinant vector.

[0102] In one embodiment, the chimeric antigen receptor-expressing cell may be a chimeric antigen receptor-expressing macrophage (CAR-macrophage), a chimeric antigen receptor-expressing T (CAR-T) cell, a chimeric antigen receptor-expressing gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

[0103] In the present invention, the term "CAR (chimeric antigen receptor)" refers to a receptor that does not naturally exist and can confer specificity for a specific antigen to immune effector cells. Typically, the CAR refers to a receptor used to transfer the specificity of a monoclonal antibody to T cells. A CAR is generally composed of an extracellular domain (Ectodomain), a transmembrane domain, and an intracellular domain (Ectodomain).

[0104] The above extracellular domain includes an antigen recognition region, and the transmembrane domain of the CAR is connected to the extracellular domain and may be derived from natural or synthetic sources. If derived from a naturally occurring source, it may be derived from a membrane-bound or membrane-permeable protein, and may be a portion derived from the membrane-permeable region of various proteins such as the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or CD8. The sequence of such a transmembrane domain may be obtained from literature known in the ordinary art that discloses the membrane-permeable region portion of a membrane-permeable protein, but is not limited thereto.

[0105] In addition, when the above-mentioned transmembrane domain is synthesized, it may mainly contain hydrophobic amino acid residues such as leucine and valine, and for example, triplets of phenylalanine, tryptophan, and valine may be present in the synthesized transmembrane domain, but are not limited thereto. Sequence information for such transmembrane domains may be obtained from literature known in the ordinary art regarding synthesized transmembrane domains, but is not limited thereto.

[0106] In the CAR of the present invention, the intracellular domain is a part of the CAR domain present within the cell and is connected to a transmembrane domain. The intracellular domain of the present invention may include an intracellular signaling domain characterized by bringing about T cell activation, preferably T cell proliferation, when an antigen binds to the antigen binding site of the CAR. The intracellular signaling domain is not particularly limited in type as long as it is a part that transmits a signal capable of bringing about T cell activation when an antibody binds to an antigen binding site present outside the cell, and various types of intracellular signaling domains may be used. Examples include an immune receptor tyrosine-based activation motif or ITAM, and the ITAM includes, but is not limited to, those derived from CD3 zeta (ζ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ.

[0107] A CAR contains a short-chain fragment variable (scFv) of an antibody specific to a tumor-associated antigen (TAA) that is coupled to the cytoplasmic domain of a T-cell signaling molecule via a hinge and a transmembrane region. Most conventional lymphocyte-activating moiety contains T-cell co-stimulating (e.g., CD28, CD137, OX40, ICOS, and CD27) domains in a tandem with a T-cell-triggering (e.g., CD3ζ) moiety. CAR-mediated adoptive immunotherapy enables CAR-transplanted cells to directly recognize TAAs on target tumor cells in a non-HLA-restricted manner.

[0108] In the present invention, the term "chimeric antigen receptor expressing T (CAR-T) cell" refers to a T cell that expresses CAR. The chimeric antigen receptor expressing T (CAR-T) cell has the advantage of being able to treat cancers that evade the action of anticancer drugs by reducing HLA expression on the cell surface, as it recognizes cancer antigens in a manner independent of HLA (human leukocyte antigen); the advantage of being able to treat cancers that evade the action of anticancer drugs by reducing HLA expression on the cell surface; the advantage of being able to use it for treatment regardless of the patient's HLA type, as it is independent of HLA type; and the advantage of being able to produce a large amount of cancer-specific T cells in a short period of time, as it can exhibit an excellent anticancer effect.

[0109] The above T cells include CD4+ T cells (helper T cells, TH cells), CD8+ T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc.

[0110] In one embodiment, the present invention relates to a T-cell engager comprising an antibody or a fragment having immunological activity thereof.

[0111] In one embodiment, the T cell engager may be, for example, a bispecific T-cell engager (BiTE). The BiTE is a class of artificial bispecific monoclonal antibodies and is a fusion protein consisting of amino acid sequences from four different genes or two short-chain variable fragments (scFv) of different antibodies on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to the T cell via a CD3 receptor, and the other binds to the tumor cell via a tumor-specific molecule. Similar to other bispecific antibodies, and unlike ordinary monoclonal antibodies, the BiTE forms a link between the T cell and the tumor cell. This enables the T cell to exert cytotoxic activity on the tumor cell independently of the presence of MHC I or co-stimulating molecules by producing proteins such as perforin and granzyme. These proteins enter the tumor cell and initiate apoptosis.

[0112] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, an antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor-expressing cell or a T cell participant.

[0113] In one embodiment, cancer is a brain tumor, melanoma, multiple myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, proanal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, It may be any one selected from the group consisting of brainstem gliomas and pituitary adenomas, and may be a cancer refractory to immune checkpoint inhibitors.

[0114] In one embodiment, the composition of the present invention may further comprise an immunogenic apoptosis inducer, and the immunogenic apoptosis inducer may be one or more selected from the group consisting of anthracycline anticancer agents, taxane anticancer agents, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosphamide anticancer agents, GADD34 / PP1 inhibitors, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, and the anthracycline anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, picantrone, sabarubicin, or It may be valrubicin, and the taxane-based anticancer agent may be paclitaxel or docetaxel.

[0115] The pharmaceutical composition for the prevention or treatment of cancer according to the present invention can increase the cancer treatment effect of conventional anticancer agents through the effect of cancer cell death by administering it together with a chemical anticancer drug (anticancer agent), etc. Co-administration may be carried out simultaneously or sequentially with the said anticancer agent. Examples of said anticancer agents include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; Anticancer antibiotics include dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids include vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan, but are not limited thereto.

[0116] In the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention.

[0117] The term "treatment" as used in the present invention refers to any act of causing the death of cancer cells or improving or beneficially altering the symptoms of cancer through the administration of the composition of the present invention. A person skilled in the art to which the present invention pertains would be able to determine the precise criteria for diseases for which the composition of the present invention is effective, and judge the degree of improvement, enhancement, and treatment by referring to materials provided by the Korean Medical Association, etc.

[0118] In the present invention, the term "therapeutically effective amount" used in combination with the active ingredient refers to a pharmaceutically acceptable amount of salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0119] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used in the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of cancer, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention 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 as a single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0120] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0121] The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more thereof that are conventionally used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary dosage forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0122] The composition of the present invention may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally depending on the intended method, and the dosage varies depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or divided into multiple doses per day.

[0123] Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc.

[0124] In one aspect, the present invention relates to an anticancer adjuvant comprising the antibody of the present invention or a fragment having immunological activity thereof, the chimeric antigen receptor protein of claim 11, or the immune cell of claim 12.

[0125] In one embodiment, the adjuvant can enhance the anticancer effect of immune checkpoint inhibitors and may be administered concurrently, separately, or sequentially with the immune checkpoint inhibitors.

[0126] In one embodiment, the immune checkpoint inhibitor may be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a variant thereof.

[0127] In one aspect, the present invention relates to a composition for diagnosing cancer comprising, as an active ingredient, an antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor-expressing cell or a T cell participant.

[0128] In one embodiment, the label may be a chromogenic enzyme, a radioisotope, a chromopore, a luminescent material, a fluorescent material, a probe, or a tag, and the fluorescent material may be a cyanine (Cy) series, rhodamine series, Alexa series, BODIPY series, or ROX series fluorescent material, and may be Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine, or Alexa.

[0129] In one aspect, the present invention relates to a cancer diagnostic kit comprising a cancer diagnostic composition of the present invention.

[0130] In one embodiment, the kit may further include tools and / or reagents for collecting biological samples from a subject or patient, as well as tools and / or reagents for preparing genomic DNA, cDNA, RNA, or protein from the sample.

[0131] In the present invention, the term "cancer diagnostic kit" refers to a kit containing the cancer diagnostic composition of the present invention. Accordingly, the expression "cancer diagnostic kit" may be used interchangeably or in combination with "cancer diagnostic composition." In this specification, the term "diagnosis" includes determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object afflicted with a specific disease or condition (e.g., identification of a pre-metastatic or metastatic cancer state, determination of the stage of cancer, or determination of the responsiveness of cancer to treatment), or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy).

[0132] As used in the present invention, the term “sample” means a biological sample obtained from a subject or patient. Sources of the biological sample may be fresh, frozen and / or preserved organ or tissue samples or solid tissue from a biopsy or aspirate; blood or any blood globulic components; or cells at any point in the subject’s pregnancy or development.

[0133] In one aspect, the present invention relates to a pharmaceutical composition for removing tumor-associated macrophages (TAMs) in a tumor microenvironment, comprising an antibody of the present invention or a fragment having immunological activity thereof as an active ingredient.

[0134] In one embodiment, the tumor-associated macrophage may be an M2 tumor-associated macrophage expressing integrin β2.

[0135] In one embodiment, the composition can remove only M2 macrophages without removing M1 macrophages.

[0136] In one aspect, the present invention relates to a method for preventing or treating cancer comprising the step of administering a pharmaceutical composition of the present invention to an individual.

[0137] The term "individual" as used in this invention refers to a subject requiring a method for the prevention, control, or treatment of a disease, and may be used without limitation and includes humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0138] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0139]

[0140] Example 1. Sequence analysis of integrin β2-activating antibodies and selection of human antibody sequences

[0141] RNA was extracted from the KIM127 Hybridoma (CRL-2838) cell line (Organism: Mus musculus) to synthesize cDNA, and the genes for the variable region of the light chain (Kappa) and heavy chain were PCR amplified. The PCR-amplified genes for the variable region of the light chain and heavy chain were cloned into a T-vector, and sequencing was performed to confirm the amino acid and nucleotide sequences. The CDRs were then indicated in red using the Kabat numbering method (Figs. 1 and 2). Subsequently, to determine the human antibody sequences required for humanization, a search of the antibody sequence database was conducted, and the most similar human antibody sequences calculated based on the nucleotide sequences of the identified KIM127 mouse antibody sequences were found to be heavy chain IGHV2-5*08 (78.01%) and IGHJ6*03 (74.19%), and light chain IGKV2-18*01 (82.99%) and IGKJ1*01 (86.49%) (Fig. 3).

[0142]

[0143] Example 2. Design of Humanized Antibodies

[0144] hzKIM127 was designed by replacing the CDR region in the human antibody sequence selected in Example 1 with the msKIM127 sequence, and two heavy chain types hzKIM127.1 VH (Sequence No. 8) and hzKIM127.2 VH (Sequence No. 9), and two light chain types hzKIM127.1 VL (Sequence No. 11) and hzKIM127.2 VL (Sequence No. 12) were designed by back-mutating the sequence of the framework portion in the designed humanized antibody sequence to the mouse antibody sequence (Fig. 5). At this time, hzKIM127.2 is closer to the human antibody sequence than hzKIM127.1. In addition, a chimeric antibody chKIM127 (VH: SEQ ID NO. 7, VL: SEQ ID NO. 10, heavy chain: SEQ ID NO. 13 and light chain: SEQ ID NO. 16) was designed (Fig. 4).

[0145]

[0146] Example 3. IgG Synthesis and Purification

[0147] Gene synthesis was performed by concatenating the variable region sequences of one chimeric antibody (chKIM127) and two humanized antibodies (hzKIM127.1 and hzKIM127.2) designed in Example 2 above with the human IgG1 / kappa constant region sequences to obtain an IgG form in which the heavy chain is IgG1 and the light chain is a kappa-type constant region (GenScript). The heavy and light chain genes of each synthesized antibody were cloned into the pcDNA3.4(+) expression vector (GenScript), transformed into HEK293F cells for expression, and each IgG was purified from the cell culture medium using Protein-A chromatography. The purity of the antibodies was confirmed by performing SDS-PAGE on the purified antibodies under reducing (R) and non-reducing (NR) conditions and staining with CBB (Coomassie Brilliant Blue) (Fig. 6). As a result, it was confirmed that all purified antibodies were purified to a high purity.

[0148]

[0149] Example 4. Analysis of antibody binding affinity to target cells

[0150] The binding affinity of the IgG antibodies (chKIM127, hzKIM127.1, and KIM127.2) synthesized and purified in Example 3 above to the target cell, the THP-1 cell line (a leukemia cell line derived from human monocytes, possessing the ability to differentiate into macrophages) (Twinpig Biolab), and the non-target cell, the Raji cell line (a lymphoma cell line derived from human B cells) (Twinpig Biolab), was analyzed by FACS. Specifically, 1.0 X 10⁻⁶ 5 Each of the purified antibodies (chKIM127, hzKIM127.1 and KIM127.2) or the culture supernatant (60 nM) of cells transiently expressing chKIM127 was treated as the primary antibody in THP-1 and Raji cells of cells / sample, respectively, and incubated at 4°C for 60 minutes. Afterward, anti-hFc-FITC (1:200 dilution) was treated as the secondary antibody, incubated at 4°C for 60 minutes, and analyzed by FACS.

[0151] hFITCchKIM127hzKIM127.11hzKIM127.22THP-113.1766729740Raji3.5611.111.88.02

[0152] As a result, both the chimeric antibody (chKIM127) and the humanized antibody (hzKIM127.1 and KIM127.2) of KIM127 bound to the target cell THP-1 but did not bind to Raji cells (Figs. 7 and 8), and the binding affinity (Mean Fluorescence Intensity, MFI) of these antibodies to the target cell was found to be no different between the antibodies (Table 1).

Claims

1. An antibody that specifically binds to integrin β2 or a fragment having the same's immunological activity.

2. The antibody or a fragment having the same’s immunological activity, wherein the antibody specifically binds to an active conformation in which integrin β2 is extended.

3. The antibody or the fragment having immunological activity thereof, comprising a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 having the amino acid sequence of SEQ ID NO. 1, CDRH2 having the amino acid sequence of SEQ ID NO. 2, and CDRH3 having the amino acid sequence of SEQ ID NO.

3.

4. The antibody or the fragment having immunological activity thereof, comprising a VL domain comprising a CDRL (Complementarity determining regions Light chain) 1 having the amino acid sequence of SEQ ID NO. 4, a CDRL 2 having the amino acid sequence of SEQ ID NO. 5, and a CDRL 3 having the amino acid sequence of SEQ ID NO.

6.

5. The antibody or the fragment having the same’s immunological activity, comprising a VH domain selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs 7 to 9, in claim 1.

6. The antibody or the fragment having the same’s immunological activity, comprising a VL domain selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs 10 to 12.

7. The antibody or the fragment having the same’s immunological activity, comprising a heavy chain including any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 13 to 15.

8. The antibody or the fragment having the same’s immunological activity, comprising a light chain comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs 16 to 18.

9. In claim 1, the antibody or the fragment having immunological activity thereof is an antibody or the fragment having immunological activity thereof, wherein the antibody is an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, a single-chain antibody, an Fv dimer, a complementarity determining domain fragment, a single-domain antibody, a diabody, a multispecific antibody, a bispecific antibody, or a bispecific antibody.

10. In paragraph 1, the antibody is an antibody or a fragment having immunological activity thereof, which is IgG1, IgG2, or IgG4 or a fragment thereof.

11. An antibody-drug conjugate (ADC) comprising the antibody of claim 1 or a fragment having immunological activity thereof, and a drug.

12. In paragraph 11, the drug is an antibody-drug conjugate that is an immunogenic apoptosis inducer, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, or a radionuclide.

13. A chimeric antigen receptor (CAR) comprising the antibody of claim 1 or a nucleic acid encoding a fragment having immunological activity thereof.

14. A chimeric antigen receptor-expressing cell transformed with a recombinant vector containing a gene encoding the chimeric antigen receptor of claim 13.

15. In paragraph 14, the chimeric antigen receptor-expressing cells are chimeric antigen receptor-expressing cells, which are chimeric antigen receptor-expressing T (CAR-T) cells or natural killer (CAR-NK) cells.

16. A T-cell engager comprising the antibody of claim 1 or a fragment having the immunological activity thereof.

17. A pharmaceutical composition for the prevention or treatment of cancer, comprising the antibody of claim 1 or a fragment having immunological activity thereof, the chimeric antigen receptor of claim 13, the cell of claim 14, or the T cell agent of claim 16.

18. In paragraph 13, cancer is any one selected from the group consisting of brain tumor, melanoma, multiple myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, procanal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma A pharmaceutical composition for the treatment or prevention of cancer.

19. A composition for diagnosing cancer comprising the antibody of claim 1 or a fragment having immunological activity thereof, the chimeric antigen receptor of claim 13, the cell of claim 14, or the T cell participant of claim 16.

20. A composition for diagnosing cancer, wherein, in accordance with claim 19, a label is additionally included.

21. A pharmaceutical composition for removing tumor-associated macrophages (TAMs) in a tumor microenvironment, comprising as an active ingredient the antibody of claim 1 or a fragment having immunological activity thereof.

22. A method for the prevention or treatment of cancer comprising the step of administering the pharmaceutical composition of claim 17 to an individual.

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