Antxr1-binding antibody and use thereof

Abstract

The present invention relates to an antibody specifically binding to anthrax toxin receptor 1 (ANTXR1), a nucleic acid molecule encoding the antibody, a vector comprising the nucleic acid molecule, a host cell comprising the vector, a pharmaceutical composition for preventing or treating cancer, such as solid cancer, the composition comprising the antibody, or a conjugate of the antibody and a drug, a radioligand therapeutic agent comprising the antibody, a bispecific antibody, and the like.

Description

ANTXR1 binding antibody and its uses

[0001] The present invention relates to an antibody that specifically binds to ANTXR1 (anthrax toxin receptor 1), a nucleic acid molecule encoding said antibody, a vector comprising said nucleic acid molecule, a host cell comprising said vector, a pharmaceutical composition for the prevention or treatment of ANTXR1-overexpressing cancer, particularly solid tumors, comprising said antibody, or a conjugate of said antibody and a drug, a radioactive ligand therapeutic agent comprising said antibody, a bispecific antibody, etc.

[0002] Anthrax toxin receptor 1 (ANTXR1) was originally discovered as a receptor for anthrax toxin, and its role in enabling the bacterium to deliver the toxin into the cell has been elucidated. Anthrax toxin is secreted by the Gram-positive bacterium Bacillus anthracis and consists of three toxin proteins: protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa) (Morton, N. (2001) New Engl. J. Med. 345:1621-1626).

[0003] Subsequent studies have confirmed that ANTXR1 is upregulated in tumor blood vessels of various tumor types in both mice and humans (Carson-Walter et al., 2001; Fernando and Fletcher, 2009; Nanda et al., 2004), and is also expressed in the tumor cells themselves in some tumors (Carson-Walter et al., 2001; Jinnin et al., 2008; Yang et al., 2011b). In particular, overexpression of ANTXR1 can be observed in various solid tumors, including gastrointestinal cancer, colorectal cancer, breast cancer, and pancreatic cancer, and this is also associated with a poor prognosis in cancer patients.

[0004] ANTXR1, also known as TEM8 (tumor endothelial marker 8) (Bradley et al., 2001), is a highly conserved single-pass cell surface glycoprotein identified based on its overexpression in endothelial cells (ECs) lining tumor blood vessels in human colorectal cancer, and has a molecular weight of 80–85 kDa (St Croix et al., 2000). Because ANTXR1 acts as a receptor for extracellular ligands, it can serve as a target for the treatment of angiogenesis (Cancer Cell. 2012 February 14; 21(2): 212-226. doi:10.1016 / j.ccr.2012.01.004.).

[0005] Various forms of response to cancer treatment are mixed, and conventional methods of treating cancer, including chemotherapy and radiation therapy, have limited utility due to toxic side effects. Immunotherapy using therapeutic antibodies offers limited success, partly due to poor pharmacokinetic profiles, rapid clearance of antibodies by serum proteases, filtration in the glomeruli, limited permeation into the tumor site, and the expression levels of target antigens on tumor cells.

[0006] Based on the characteristic that ANTXR1 is hardly expressed in normal tissues and is specifically expressed only in cancer tissues, the inventors intend to provide a novel ANTXR1-binding antibody for the treatment of solid tumors and its use, thereby providing an effective treatment method for solid tumors.

[0007] [Prior Art Literature]

[0008] [Patent Literature]

[0009] Korean Patent Application Publication No. 10-2019-0013612

[0010] Republic of Korea Registered Patent No. 10-2156822

[0011] [Non-patent literature]

[0012] Morton, N. (2001) New Engl. J. Med. 345:1621-1626

[0013] Cancer Cell. February 14, 2012; 21(2): 212-226. doi:10.1016 / j.ccr.2012.01.004

[0014] One objective of the present invention is to provide an antibody or a fragment thereof having a specifically high binding affinity to ANTXR1, an effective pharmaceutical composition for cancer treatment or diagnosis comprising said antibody, etc., and an antibody-drug conjugate (conjugate) having high antitumor activity.

[0015] One aspect of the present invention is an antibody that binds to ANTXR1 or an antigen-binding fragment thereof, wherein

[0016] A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 1 or SEQ ID NO. 2,

[0017] A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 3 or SEQ ID NO. 4, and

[0018] A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 5 or SEQ ID NO. 6; and

[0019] A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 7 or SEQ ID NO. 8,

[0020] A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 9 or SEQ ID NO. 10, and

[0021] The invention relates to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO. 11 or SEQ ID NO. 12.

[0022] Another aspect of the present invention relates to an antibody or an antigen-binding fragment thereof that binds to an epitope of ANTXR1 represented by SEQ ID NO. 13.

[0023] Another aspect of the present invention relates to a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described herein.

[0024] Another aspect of the present invention relates to a vector comprising the nucleic acid molecule.

[0025] Another aspect of the present invention relates to a host cell comprising the vector.

[0026] Another aspect of the present invention relates to a method for generating the antibody or its antigen-binding fragment described herein by culturing the host cell.

[0027] Another aspect of the present invention relates to a pharmaceutical composition for treating cancer comprising the antibody or antigen-binding fragment thereof described herein.

[0028] Another aspect of the present invention relates to an antibody-drug conjugate in which a drug is bound to the antibody or its antigen-binding fragment described herein.

[0029] The antibody or its antigen-binding fragment according to the present invention can specifically bind to ANTXR1 and effectively treat cancer characterized by the overexpression of ANTXR1, such as solid tumors (e.g., colorectal cancer or pancreatic cancer). In addition, it can be used as an antibody-drug conjugate by binding to cells in which ANTXR1 is overexpressed.

[0030] FIG. 1 illustrates the amino acid sequence of an antibody (1T1C12) of one example of the present invention and the nucleic acid sequence encoding it.

[0031] FIG. 2 illustrates the amino acid sequence of an antibody (1T1H12B1) of an example of the present invention and the nucleic acid sequence encoding it.

[0032] Figure 3 illustrates data showing the process of deriving clone candidates through ELISA screening.

[0033] FIG. 4 shows K according to SPR of an antibody (1T1C12: POR-CAF-001-01) of an example of the present invention. D This is a graph showing the measurement results.

[0034] FIG. 5 shows K according to SPR of an antibody (1T1H12B1: POR-CAF-001-02) of an example of the present invention. D This is a graph showing the measurement results.

[0035] Figure 6 illustrates the binding characteristics of antibodies (1T1C12: : POR-CAF-001-01) and 1T1H12B1: : POR-CAF-001-02) of an example of the present invention to ANTXR1 overexpressing cells.

[0036]

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0038] As a result of efforts to develop an anti-ANTXR1 antibody for the treatment of cancer, specifically solid tumors, the inventors identified a specific epitope of ANTXR1 and provide an anti-ANTXR1 IgG antibody having a high binding affinity to said epitope.

[0039] The antibody or antigen-binding fragment thereof that binds to the above ANTXR1 is,

[0040] A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 1 or SEQ ID NO. 2,

[0041] A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 3 or SEQ ID NO. 4, and

[0042] A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 5 or SEQ ID NO. 6; and

[0043] A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 7 or SEQ ID NO. 8,

[0044] A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 9 or SEQ ID NO. 10, and

[0045] It includes a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO. 11 or SEQ ID NO. 12.

[0046] The above antibody or its antigen-binding fragment is,

[0047] A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 1,

[0048] A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 3, and

[0049] A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 5; and

[0050] A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 7,

[0051] A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 9, and

[0052] It comprises a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO. 11, or

[0053] A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 2,

[0054] A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 4, and

[0055] A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 6; and

[0056] A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 8,

[0057] A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 10, and

[0058] It may include a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO. 12.

[0059] The above antibody or its antigen-binding fragment may comprise a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 14 or 16; and / or a variable light chain comprising the amino acid sequence of SEQ ID NO. 15 or 17.

[0060] The above antibody or its antigen-binding fragment comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 14; and / or a variable light chain comprising the amino acid sequence of SEQ ID NO. 15, or

[0061] It may include a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 16; and / or a variable light chain comprising the amino acid sequence of SEQ ID NO. 17.

[0062] The above antibody or its antigen-binding fragment may include the linker of SEQ ID NO. 18 or SEQ ID NO. 19.

[0063] The above antibody or its antigen-binding fragment may include the heavy chain constant region of SEQ ID NO. 20 or SEQ ID NO. 21.

[0064] In the present invention, the CDR of each variable region of the light chain and the CDR of each variable region of the heavy chain may be freely combined. For example, it may include a light chain variable region having a light chain CDR1 of SEQ ID NO. 1, a light chain CDR2 region of SEQ ID NO. 4, and a light chain CDR3 region of SEQ ID NO. 5; and a heavy chain variable region having a heavy chain CDR1 region of SEQ ID NO. 8, a heavy chain CDR2 region of SEQ ID NO. 9, and a heavy chain CDR3 region of SEQ ID NO. 12.

[0065] The antibody or its antigen-binding fragment may include a "variant." The variant may refer to a polypeptide in which one or more amino acid residues from the amino acid sequence are inserted, deleted, added, and / or substituted. Accordingly, the antibody and / or antigen-binding fragment may include a fusion polypeptide by linking to another polypeptide, provided that the desired biological activity and / or structure is maintained. The above variant may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the sequence of the antibody or its antigen-binding fragment, for example, approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0066] According to one embodiment, a light chain variable region comprising a CDR1 region represented by the amino acid sequence of SEQ ID NO. 1 or 2, a CDR2 region represented by the amino acid sequence of SEQ ID NO. 3 or 4, and a CDR3 region represented by the amino acid sequence of SEQ ID NO. 5 or 6; The sequence of an antibody or fragment thereof that specifically binds to ANTXR1, comprising a CDR1 region represented by the amino acid sequence of SEQ ID NO. 7 or 8, a CDR2 region represented by the amino acid sequence of SEQ ID NO. 9 or 10, and a CDR3 region represented by the amino acid sequence of SEQ ID NO. 11 to 12, may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and for example, may have sequence identity of about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0067] According to one embodiment, the sequence of an antibody or fragment thereof that specifically binds to ANTXR1, comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO. 15 or 17; and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO. 14 or 16, may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and for example, may have sequence identity of about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0068] In this specification, the term “identity” may refer to the overall relationship between nucleic acid molecules and / or polypeptides. In one embodiment, nucleic acids or polypeptides may be considered “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The calculation of the percentage identity of two nucleic acid or polypeptide sequences may be performed, for example, by aligning the two sequences for the purpose of optimal comparison. In a specific embodiment, for comparison purposes, the length of the aligned sequence may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence.

[0069] ANTXR1 is an extracellular matrix protein, also known as TEM8 (tumor endothelial marker 8). ANTXR1 is a type 1 transmembrane protein expressed in the endothelium of various tumors. It has already been identified as a gene whose expression increases during tumor angiogenesis (St. Croix et al., (2000) Sciences 289:1197), and it is known to be involved in extracellular matrix protein-mediated cell adhesion and migration in tumor vascular endothelial cells (Bradely et al., (2001) Nature 414:228-229; Nanda et al., (2004) Curr Opin Oncol 16:44-49). Its specific nucleic acid sequence is available on NCBI (NCBI Reference Sequence: NM_032208.2).

[0070] As described above, since ANTXR1 acts as a receptor for extracellular ligands, it can be a target for the treatment of angiogenesis and tumors (Carson-Walter, EB et al., (2001) Cancer Res 61:6649).

[0071] Other studies have confirmed that ANTXR1 shows high expression in cancer tissues, including cancer-associated fibroblasts. In particular, overexpression of the TEM8 protein was observed in cancer types including gastrointestinal cancer, colorectal cancer, breast cancer, and pancreatic cancer. Furthermore, this has been confirmed to be associated with a poor prognosis in cancer patients.

[0072] Accordingly, antibodies with high binding affinity to ANTXR1 can be used to treat or diagnose cancer, specifically solid tumors such as gastrointestinal cancer, colorectal cancer, breast cancer, and pancreatic cancer, by inhibiting the angiogenesis process of tumors or inhibiting cancer-associated fibroblasts.

[0073] In one aspect, the present invention provides an isolated antibody that binds to ANTXR1. The binding activity of the anti-ANTXR1 antibody is K D It can be expressed as a (Dissociation constant) value. The antibody of the present invention has 0.1 nM to 10 nM of KD, or 0.2 nM to 8 nM, or 1.0 nM to 5.0 nM of K D It can exhibit binding activity having a value.

[0074] The antibody or its antigen-binding fragment described herein can bind to the epitope of ANTXR1 represented by SEQ ID NO. 13.

[0075] Another aspect of the present invention relates to an antibody that binds to the epitope of SEQ ID NO. 13 or an antigen-binding fragment thereof.

[0076] In this specification, "antibody" means a polypeptide that specifically binds to and recognizes the epitope of ANTXR1. The present invention includes not only a complete antibody form that binds to the epitope of ANTXR1, but also an antigen-binding fragment of said antibody molecule.

[0077] In this specification, an “antigen-binding fragment” is an antibody molecule, or a part or site thereof, or a derivative thereof, which possesses all or a significant portion of the antigen binding of the corresponding full-length antibody. The antigen-binding fragment may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. The variable regions of the heavy and light chains (VH and VL) comprise three highly variable regions referred to as “complementarity-determining regions (CDRs).” The CDRs bind to epitopes of the antigen, and the CDRs of the light and heavy chains typically have three regions (CDR1, CDR2, and CDR3).

[0078] In this specification, “antibody or antigen-binding fragment thereof” includes not only full-length or original polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, fusion proteins comprising one or more antibody portions, artificial spaced antibodies, chimeric antibodies, minibodies, diabodies, tribodies, tetrabodies, linear antibodies, monochain antibodies, multispecific antibodies (e.g., bispecific antibodies), glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies as other modified arrangement forms of immunoglobulin molecules comprising antigen recognition sites of required specificity. In one example, a bispecific antibody comprising an anti-ANTXR1 antibody or an antigen-binding fragment thereof is provided. In this specification, “bispecific antibody” may be a hybrid antigen-binding protein or antibody having two different antigen-binding sites. These bispecific antibodies are a type of multispecific antigen-binding protein or multispecific antibody, which can be generated by various known methods, such as the linkage of Fab' fragments or the fusion of hybridomas. For example, see Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553, etc. The two different epitopes to which the two antigen-binding sites of a bispecific antigen-binding protein or antibody bind may be located on the same or different protein targets.

[0079] The above "antigen-binding fragments" include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, (scFv)2 fragments, and scFv-Fc fragments.

[0080] In this specification, "full-length antibody" means an antibody composed of two "full-length antibody heavy chains" and two "full-length antibody light chains." Typical full-length antibody classes are six types: IgA, IgD, IgE, IgG, IgM, and IgY. Some of these may be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0081] In this specification, “monoclonal antibody” refers to an antibody having a monovalent affinity (binding ability), meaning that each antibody molecule in a sample of monoclonal antibodies binds to the same epitope of the antigen, and can be produced using the same immune cell that is a clone of a specific parent cell (e.g., a hybridoma cell). For reference, “polyclonal antibody” refers to a set of antibodies that react to a specific antigen, within which they may be different antibody molecules, e.g., antibody molecules that react to different epitopes of the antigen.

[0082] In this specification, "epitope" comprises any polypeptide determinant capable of specifically binding to an antibody, and the epitope is a region of the antigen to which the antibody binds. The epitope determinant may comprise groups of chemically active surfaces of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and may also have specific three-dimensional structural features and / or specific charge features.

[0083] In this specification, "human antibody" means an antibody having variable and constant regions substantially corresponding to an antibody obtained from or originating from a human.

[0084] One aspect of the present invention relates to a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof described herein. The nucleic acid molecule may include, for example, the sequence of SEQ ID NO. 22 or 23. As used herein, the terms “nucleic acid” or “polynucleotide” may include single-stranded and double-stranded nucleotide polymers. The nucleotides constituting the polynucleotide may be ribonucleotides or deoxyribonucleotides or modified forms of both types of nucleotides. Such modifications may include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladate, and phosphoramidate.

[0085] Another aspect of the present invention relates to a vector comprising the nucleic acid molecule. The nucleic acid sequence encoding the antibody and / or its antigen-binding fragment may be cloned into a number of types of vectors. For example, the nucleic acid may be cloned into a plasmid, phagemid, phage derivative, animal virus, and cosmid. Other vectors may include expression vectors, replication vectors, probe-generating vectors, sequencing vectors, and viral vectors. In other examples, the vector may be a spore-forming viral (FV) vector, which is a type of retroviral vector prepared from a spumavirus. Viral vector design and techniques are widely known in the art as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2001) and other virology and molecular biology manuals. The vector may refer to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. The vector may include a nucleic acid sequence that allows replication in the host cell, such as a replication origin. The vector may also include one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. The vector may transduce, transform, or infect cells to cause the cells to express nucleic acids and / or proteins other than those native to the cells. The vector may optionally include materials that aid in achieving the entry of nucleic acids into the cell, such as viral particles, liposomes, protein coatings, etc.

[0086] Another aspect of the present invention relates to a host cell that is transformed with the vector and expresses the nucleic acid. In this specification, the term "host cell" may mean a cell that has been transformed with a nucleic acid sequence or can be transformed to express the gene of interest. The host cell may include offspring of the parent cell, regardless of whether the offspring have the same morphology or genetic composition as the original parent cell, as long as the gene of interest is present.

[0087] Another aspect of the present invention relates to an antibody-drug conjugate (ADC) comprising an anti-ANTXR1 antibody or an antigen-binding fragment thereof.

[0088] Specifically, the antibody-drug conjugate may comprise the anti-ANTXR1 antibody or its antigen-binding fragment described herein; a drug moiety; and a linker connecting the drug moiety to the antibody or its antigen-binding fragment.

[0089] In this specification, the term “antibody-drug conjugate (ADC)” may refer to a substance having a structure in which a cytotoxic small molecule drug (payload) is conjugated to an antibody that binds to a specific target antigen on the surface of a cell or to an antigen-binding fragment thereof.

[0090] In one embodiment, the antibody-drug conjugate may be formed by attaching a therapeutic agent (drug) to an antibody having target specificity or an antigen-binding fragment thereof through a linker.

[0091] The above linker may include a cleavable linker or a non-cleavable linker. The cleavable linker may be cleaved by intracellular peptidase or protease enzymes, such as lysosome or endosome proteases, as with peptide linkers. For example, the cleavable linker may be any one selected from the group consisting of protease cleavable linkers, acid-cleavable linkers, disulfide linkers, β-glucuronide-based linkers, and β-galactoside-based linkers, but is not limited thereto. The non-cleavable linker may release a drug after the antibody is non-selectively degraded by intracellular hydrolysis.

[0092] According to one embodiment, the therapeutic agent may be any one selected from the group consisting of chemotherapeutic compounds, cytotoxic compounds, immunomodulatory compounds, anticancer agents, antiviral agents, antimicrobial agents, antifungal agents, antiparasitic agents, and combinations thereof. For example, the cytotoxic compound may be any one selected from the group consisting of mitotic inhibitors, DNA alkyalting agents, topoisomerase inhibitors, and combinations thereof, but is not limited thereto. More specifically, the therapeutic agent may be MMAE_Protein A, cotinine-duocarmycin, or Herceptin, but is not limited thereto.

[0093] In another aspect of the present invention, the invention relates to a pharmaceutical composition for treating cancer, particularly solid tumors, comprising the antibody, antigen-binding fragment, or antibody-drug conjugate.

[0094] The term "solid tumor" in the present invention refers to a tumor composed of blood vessels or connective tissue that has a certain hardness and shape, and is not limited thereto, but includes, for example, pancreatic cancer, stomach cancer, colorectal cancer, lung cancer, breast cancer, germ cell carcinoma, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, etc.

[0095] The above pharmaceutical composition may contain the antibody or its antigen-binding fragment as an active ingredient in an amount of about 0.1% to about 90% by weight, specifically about 0.5% to about 75% by weight, more specifically about 1% to about 50% by weight, based on the total weight of the composition.

[0096] The above pharmaceutical composition may include conventional, non-toxic, and pharmaceutically acceptable additives that are formulated into a formulation according to conventional methods. For example, the above pharmaceutical composition may further include pharmaceutically acceptable carriers, diluents, or excipients.

[0097] Examples of additives used in the above pharmaceutical composition may include sweeteners, binders, solvents, solubilizing aids, wetting agents, emulsifiers, isotonic agents, absorbents, disintegrants, antioxidants, preservatives, lubricants, lubricants, fillers, flavoring agents, etc. For example, the additives may include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, stearin, magnesium stearate, magnesium aluminosilicate, starch, gelatin, tragacanth gum, alginic acid, sodium alginate, methylcellulose, sodium carboxymethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, etc.

[0098] The above pharmaceutical composition may be formulated into various formulations for oral administration (e.g., tablets, pills, powders, capsules, syrups, or emulsions) or parenteral administration (e.g., intramuscular, intravenous, or subcutaneous injection).

[0099] Specifically, the above pharmaceutical composition may be formulated as an oral administration preparation, and the additives used therein may include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactant, suspending agent, emulsifier, diluent, etc.

[0100] In addition, formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

[0101] The above pharmaceutical composition may be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

[0102] The terms "therapeutically effective amount" or "pharmaceuticalally effective amount" refer to an amount of a compound or composition effective for preventing or treating a target disease, which is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause adverse effects. The level of the said effective amount may be determined based on factors including the patient's health status, type and severity of the disease, 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.

[0103] The above 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 as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by a person skilled in the art.

[0104] Specifically, the effective amount of the antibody or its antigen-binding fragment in the above pharmaceutical composition may vary depending on the patient's age, gender, and body weight, and generally, about 0.1 mg to about 1,000 mg or about 5 mg to about 200 mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 doses per day. However, the range is not limited thereto, as it may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc.

[0105] Additionally, the above pharmaceutical composition may be administered for tumor therapy in combination with chemotherapy, radiation therapy, immunotherapy, hormone therapy, bone marrow transplantation, stem cell replacement therapy, other biological therapies, surgical intervention, or combinations thereof. For example, it may be used as an adjuvant therapy in conjunction with other long-term treatment strategies, or to promote tumor regression in severe patients or to maintain the patient's condition after chemoprophylaxis.

[0106] In this specification, "phage display" is a concept first introduced by G. Smith in 1985 and a technology first applied to the manufacture of antibodies by the UK MRC in 1990.

[0107] "Bacteriophage" is a type of virus that parasitizes Escherichia coli, and M13, a filamentous bacteriophage, is mainly used.

[0108] "Antibody phage display" refers to a technology that enables the production of an antibody library of various combinations of heavy and light chain variable regions displayed on the surface of phages by amplifying the heavy and light chain variable regions of numerous antibodies present in the human body by PCR, cloning them in a form fused to phage surface proteins contained within a phagemid vector, expressing them in E. coli, and then infecting with a helper phage. The antibody forms used in this process may be scFv or Fab. Human monoclonal antibodies that bind to specific antigens can be isolated and produced from the above library through a panning process.

[0109] In this specification, "panning" refers to a process of amplifying the number of phages by binding phages to a target antigen, removing unbound phages, recovering the bound phages, and infecting E. coli, and repeating this process 2 to 4 times.

[0110] Using the above technology, human monoclonal antibodies can be isolated in just a few weeks.

[0111] In this specification, "nucleic acids" or "nucleic acid molecules" have a meaning that comprehensively includes DNA (gDNA and cDNA) or RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified.

[0112] In this specification, "cloning" refers to a genetic engineering technique for mass-producing identical genes, meaning a technique for replicating genetically identical clones through cell division by introducing a gene or DNA fragment into a plasmid or virus that acts as a vector for mass production.

[0113] In this specification, "clone" means an identical copy, referring to an identical copy of the same cell population or a specific gene segment.

[0114] In this specification, "transformation" refers to the introduction of a foreign gene into a host cell that changes the genetic properties of the host cell, and if the host is a eukaryotic cell, the term "transfection" may be used.

[0115] The term "transformation" as used in the present invention may include any method of introducing a foreign gene into an organism, cell, tissue, or organ, and preferably refers to introducing a foreign gene into Escherichia coli. Methods of transformation include electroporation, liposome-based methods, and Ca 2+ Methods commonly used in the industry, such as DNA transport methods using [the device], can be used.

[0116]

[0117] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples.

[0118]

[0119] 1. Antibody screening

[0120] A phage display method was used to select antibodies with selectivity and specificity for specific antigens by constructing a single-strand variable fragment (scFv) phage library in which variable regions (VH and VL) of various antibodies were expressed on the surface of M13 bacteriophages. A panning process to select antibodies binding to ANTXR1 from a diverse antibody pool was performed three times under the following experimental methods and conditions.

[0121]

[0122] Biopanning

[0123] The target antigen ANTXR1 was immobilized on the surface of a solid. A phage library was prepared, and display phages generated therefrom were cultured together with the immobilized antigen. Unbound phages were washed and removed using PBS-T buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.3–7.4, 0.1% Tween20). Phages with high affinity were eluted from the immobilized antigen. The eluted phages were cultured with E. coli host cells to amplify a phage subpopulation displaying antibodies with affinity for ANTXR1 through infection and proliferation. The above process was repeated two more times to perform a total of three phage displays.

[0124] The conditions for the above biopanning are as follows:

[0125] Phage library, human immobilized immunotube, targeted ANTXR1 extract-coating solution, sodium bicarbonate buffer (pH 9.6), blocking solution, 5% skim milk-PBST

[0126] 1st 2nd 3rd Input Output Input Output Input Output 1X10 13 <8.5X10 5 1x10 12 <7.5X10 5 1x10 12 <1.0X10 5

[0127] (Unit: cfu / ml)

[0128]

[0129] Screening

[0130] A subpopulation of the above biopanning process was cultured on a plate using colonies of infected bacteria obtained from the amplification step. Monoclonal scFvs were produced from each colony. Monoclonal complexes were identified through ELISA (Enzyme Linked ImmunoSorbent Assay) screening under the following conditions.

[0131] Immobilization Corning (Cat. No. 3690) Target ANTXR1(1) Negative control - Coating solution Sodium bicarbonate buffer (pH 9.6) Blocking solution 3% M-PBST Washing solution PBST secondary Ab-HRP1:2000 TMB Substrate incubation time 5 min Measurement filter 450 nm Plate reader Epoch, BIOTEK

[0132] The results of the above ELISA are shown in Figure 3. In the ELISA results, a total of three clone candidates (i.e., 1T1C12, 1T1G10, and 1T1H12) were identified as positive clones without background values, and antibody production was carried out for 1T1C12 and 1T1H12 among them.

[0133]

[0134] production of IgG

[0135] VH (variable heavy chain) and VL (variable light chain) fragments binding to the target antigen were amplified via PCR. The VH fragment was inserted into an IgG1 subtype VH expression vector. The VH gene was designed to be positioned at the 5' top of the constant region gene (CH1-hinge-CH2-CH3). The VL fragment was inserted into a VL expression vector. The VL gene was designed to be positioned at the 5' top of the constant region gene (CL). After mixing the plasmid DNA of the VH and VL vectors in appropriate proportions, they were transfected into 293F cells. The cell culture supernatant was collected after an appropriate incubation period. Monoclonal antibodies were purified using affinity chromatography, such as protein A resin.

[0136] The above production and purification conditions are as shown in the following Tables 4 and 5:

[0137] Expression source of anti-ANTXR1 IgG antibody 293f Cell culture volume each 100 ml (1T1C12, 1T1H12B1) 9 Form recombinant whole human IgG composition IgG1 subtype

[0138] Affinity Chromatography (Resin) Protein A Resin (Cytiva) Equipment FPLC (AKTA prime plus, Cytiva) Purity >90% when measured by SDS-PAGE and CBB staining

[0139] Amino acid and gene sequence information for two antibodies purified by the above method are shown in Figures 1 and 2, respectively.

[0140]

[0141] Identification of the antibody recognition site (epitope) on the antigen

[0142] The antibody recognition site and sequence information on the ANTXR1 antigen were investigated using Uniprot (a database that provides basic information such as protein sequences, gene names, descriptions of sequences, phylogenetic classification, and citation information, as well as clear annotation information based on biological existence, biological classification, cross-references, experimental data, and computer data).

[0143] The epitope sequence information of the corresponding antigen is the same as SEQ ID NO. 13, and recombinant human TEM8 protein expressed in HEK293 cells was used.

[0144]

[0145] Measurement of antigen-antibody binding strength

[0146] Hit substances were selected to evaluate whether they bind well to the corresponding target. ELISA, SPR (Surface Plasmon Resonance), and FACS (Fluorescence Activated Cell Sorting) methods were primarily used.

[0147] ELISA is a method that measures the presence and extent of an antigen-antibody reaction using an enzyme as a marker. Through this method, antibodies favorable for new drug development were screened among various antibodies.

[0148] SPR analysis is a technique that observes intermolecular binding, interactions, and dissociation rates in real time. Through this, binding rate constants and dissociation rate constants can be determined, making it useful for studying protein interactions.

[0149] FACS is an experimental technique that can classify cell mixtures into groups of cells with various characteristics and traits through fluorescent staining of the cell surface and interior.

[0150]

[0151] ELISA

[0152] Binding affinity by ELISA was measured under the following conditions:

[0153] Coated Protein ANTXR1 (1 µg / ml) Primary Antibody Anti-ANTXR1 IgG Antibody (2 µg / ml) Secondary Antibody-HRP1:20,000 (Anti-Human IgG Antibody, HRP-conjugated)

[0154] The measurement results are as shown in the following Table 7:

[0155] Anti-ANTXR1 Antibody Antigen ANTXR1 Background Control IgG1T1C122.0522.0120.0600.0561T1H12B12.7552.7470.0550.053

[0156] As a result of the above, it can be confirmed that the antibodies produced recognize and bind to the target antigen.

[0157]

[0158] SPR

[0159] Data was analyzed using an SPR spectrometer (SR7500DC, Reichert Analytical Instrument, Depew, NY) and Scrubber 2.0 (BioLogic Software, Australia) software. The sensor chip was a product (13206061, Reichert, Depew, NY) consisting of a self-assembled monolayer mixed with 90% monothiol alkanes PEG3KOH and 10% monothiol alkanes PEG6K-COOH, which was mounted in the instrument using immersion oil.

[0160] As a surface activation step, a mixture of 0.1M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.05M N-hydroxysuccinimide was injected at a flow rate of 10 µl / min to modify the free carboxyl groups on the surface, thereby creating an activated succinimide ester surface.

[0161] Subsequently, immobilization and blocking steps were performed. Specifically, a ligand was prepared at 20 μg / ml in 10M sodium acetate buffer at pH 4.5 and bound to the sensor via free amine coupling to the immobilized succinimide. The remaining active succinimide was quenched in 1M ethanolamine at pH 8.5.

[0162] As a test step for experimental conditions, the binding time was determined by flowing a few μM levels of analyte into each ligand-immobilized channel at a flow rate of 30 μL / min. The dissociation time was determined by flowing a running buffer at the same flow rate. After the binding / dissociation steps, 10 mM–40 mM NaOH was injected as a regeneration solution to find ideal conditions. The regeneration conditions were selected based on the time, flow rate, and concentration that could remove the analyte quickly and restore the baseline. In this test, 20 mM NaOH was used as the regeneration solution.

[0163] Stock solutions of each analyte were prepared in PBS running buffer (0.01 M phosphate buffer, 2.7 mM KCl, 0.137 M NaCl, pH 7.4), and then serially diluted twofold to prepare five sample concentrations. The binding and dissociation times were each set to 3 minutes.

[0164] SPR sensorgrams were acquired in real time from each sample channel and reference channel, and the difference was calculated by subtracting the value of the reference channel from the value of the sample channel. Data was then derived by double-referencing with a blank value of concentration 0. The equilibrium dissociation constant was calculated using the kd / ka ratio.

[0165] The above results are shown in Figures 4 and 5, respectively.

[0166] K of 1.15 nM and 4.1 nM for the target antigen in the two antibodies, respectively D It represented.

[0167]

[0168] FACS

[0169] NCI-H460, a cell line overexpressing ANTXR1, and NCl-H1437, a cell line underexpressing ANTXR1, were obtained from the Korean Cell Line Bank. Subsequently, to measure antibody function, each cell was collected and incubated with different concentrations of test antibodies at 4°C for 16 to 18 hours. After washing the cells twice, a secondary antibody at a dilution ratio of 1:1000 was added and incubated at room temperature for 1 hour. The cells were washed twice more. Afterward, samples were read using a flow cytometer.

[0170] The above experimental results are shown in Figure 6.

[0171] It was confirmed that both of the two antibodies discovered from the above results bind highly only to cells overexpressing ANTXR1.

Claims

1. As an antibody that binds to ANTXR1 or an antigen-binding fragment thereof, A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 1 or SEQ ID NO. 2, A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 3 or SEQ ID NO. 4, and A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 5 or SEQ ID NO. 6; and A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 7 or SEQ ID NO. 8, A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 9 or SEQ ID NO. 10, and An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO. 11 or SEQ ID NO.

12.

2. In paragraph 1, the above antibody, A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 1, A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 3, and A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 5; and A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 7, A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 9, and An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO.

11.

3. In paragraph 1, the above antibody, A light chain CDR1 region having the amino acid sequence described in SEQ ID NO. 2, A light chain CDR2 region having the amino acid sequence described in SEQ ID NO. 4, and A light chain variable region having a light chain CDR3 region having the amino acid sequence described in SEQ ID NO. 6; and A heavy chain CDR1 region having the amino acid sequence described in SEQ ID NO. 8, A heavy chain CDR2 region having the amino acid sequence described in SEQ ID NO. 10, and An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having a heavy chain CDR3 region having the amino acid sequence described in SEQ ID NO.

12.

4. In any one of claims 1 to 3, the antibody is an antibody or an antigen-binding fragment thereof that binds to an epitope of ANTXR1 represented by SEQ ID NO.

13.

5. An antibody or an antigen-binding fragment thereof, comprising, in claim 1 or 2, a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 14; and / or a variable light chain comprising the amino acid sequence of SEQ ID NO.

15.

6. An antibody or an antigen-binding fragment thereof, comprising, in claim 1 or 3, a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 16; and / or a variable light chain comprising the amino acid sequence of SEQ ID NO.

17.

7. An antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO. 13 of ANTXR1.

8. An antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a single-strand variable fragment (scFv), a (scFv)2 fragment, or a scFv-Fc fragment.

9. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof of any one of claims 1 to 3.

10. A vector containing the nucleic acid molecule of claim 9.

11. A host cell containing the vector of claim 10.

12. A method of producing any one of the antibodies of claims 1 to 3 or an antigen-binding fragment thereof by culturing the host cells of claim 11.

13. A pharmaceutical composition for treating cancer comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

14. A pharmaceutical composition according to claim 13, wherein the cancer is a cancer selected from the group consisting of pancreatic cancer, gastric cancer, colorectal cancer, lung cancer, breast cancer, germ cell carcinoma, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.

15. An antibody-drug conjugate in which a drug is bound to an antibody or an antigen-binding fragment thereof described in any one of paragraphs 1 to 3.

16. An antibody-drug conjugate according to paragraph 15, wherein the above drug is an antitumor drug.

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