Anti-BCAM antibody or antigen-binding fragment thereof

Abstract

The present invention relates to an anti-BCAM antibody or an antigen-binding fragment thereof, and uses thereof. In addition, the anti-BCAM antibody or an antigen-binding fragment thereof according to the present invention can be used to prevent or treat pathologies associated with cells expressing BCAM, such as cancer.

Description

[Technical Field]

[0001] The present invention relates to anti-BCAM antibodies or antigen-binding fragments thereof, and uses thereof. Furthermore, the anti-BCAM antibodies or antigen-binding fragments thereof of the present invention can be used to prevent or treat pathologies associated with cells expressing BCAM, such as cancer. [Background technology]

[0002] Basal cell adhesion molecule (BCAM) is a transmembrane protein of the Ig superfamily, also known as Lutheran blood group glycoprotein (Lu). Lu was initially studied as a Lutheran blood group antigen, while BCAM was identified as an antigen upregulated in ovarian cancer. Lu and BCAM share the same extracellular domain but differ in their cytoplasmic tails. Specifically, BCAM lacks the COOH-terminal 40 amino acids present in the Lu cytoplasmic tail. Furthermore, the Lu-specific cytoplasmic region contains an SH3-binding motif, a dileucine motif, and potential phosphorylation sites. The shared region of the cytoplasmic tail of Lu and BCAM contains a spectrin-binding motif. Because the structures of BCAM and Lu overlap, it is difficult to distinguish Lu from BCAM in actual tissues; therefore, Lu and BCAM are referred to interchangeably, sometimes as Lu / BCAM or CD239.

[0003] The extracellular domain of BCAM contains one V-set, one C1-set, and three I-set (V-C1-III) domains. BCAM specifically binds to laminin α5, a major component of basement membranes, and is thought to be involved in cell adhesion to basement membranes. Laminin α5 binds to the β and γ chains to form a heterotrimer, which is found in many basement membranes of normal and diseased tissues. Additionally, BCAM promotes lung cancer cell migration with laminin-511 (LM-511), which is composed of the α5, β1, and γ1 chains. Furthermore, tumor cell migration with LM-511 is inhibited in the presence of a function-blocking antibody against BCAM. Overexpression of BCAM has been observed not only in ovarian carcinomas but also in skin cancers and hepatocellular carcinomas. Therefore, BCAM has been proposed as a useful antigen for diagnostics and the development of antibody drugs. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yamato Kikkawa et al., Scientific Reports, 2018 Apr 26;8(1):6612 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an anti-BCAM antibody or an antigen-binding fragment thereof that specifically binds to the BCAM protein.

[0006] Furthermore, the invention provides the use of the antibody or antigen-binding fragment to prevent or treat a condition associated with cells expressing BCAM, such as cancer. [Means for solving the problem]

[0007] The present invention provides anti-BCAM antibodies or antigen-binding fragments thereof.

[0008] The present invention provides pharmaceutical compositions for preventing or treating cancer, comprising an anti-BCAM antibody or an antigen-binding fragment thereof. Additionally, the present invention provides antibodies or antigen-binding fragments in the form of antibody-drug conjugates (ADCs) that can selectively deliver inhibitors to target BCAM-expressing cells.

[0009] In one embodiment, the present invention provides an anti-BCAM antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, and 11; a heavy chain CDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17; a heavy chain CDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, and 23; a light chain CDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, and 12; a light chain CDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, and 18; and a light chain CDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, and 24.

[0010] In one embodiment, the present invention provides an anti-BCAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 1, 3, and 5, and a light chain variable region selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

[0011] In one embodiment, the antigen-binding fragment of the present invention may be a Fab, Fab', Fab'-SH, Fv, single-chain antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody (scFV(CH3)2), IgG deltaCH2, scFv-Fc, (scFv)2-Fc, fynomer, dual affinity retargeting (DART), or TRIDENT comprising the CDR sequences of the present invention. The antibody of the present invention may be a chimeric antibody, a humanized antibody, a human antibody, or a multispecific antibody.

[0012] In another embodiment, the invention provides nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, and recombinant expression vectors comprising the nucleic acid molecules.

[0013] The present invention also provides a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof as an active ingredient. The pharmaceutical composition can be used in combination with an additional anti-cancer agent, such as another anti-cancer agent or a chemotherapeutic agent, or can be used in combination with radiation therapy.

[0014] The present invention also provides a composition for analyzing or detecting a BCAM protein, the composition comprising an antibody or antigen-binding fragment thereof. [Effects of the Invention]

[0015] The anti-BCAM antibodies or antigen-binding fragments thereof of the present invention bind to human BCAM protein with high affinity, and as a result, can be effectively used in the prevention or treatment of cancer. [Brief explanation of the drawings]

[0016] [Figure 1a] 1 shows the amino acid sequences of the heavy chain variable region (1a) of selected antibodies, with the bold and underlined sequences representing the CDR sequences of each region. [Figure 1b] 1 shows the amino acid sequences of the light chain variable region (1b) of selected antibodies, with the bold and underlined sequences representing the CDR sequences of each region. [Figure 2] FIG. 1 shows the binding ability of the antibody of the present invention to BCAM by ELISA analysis. [Figure 3] FIG. 1 shows the binding ability of the antibodies of the present invention to BCAM by FACS analysis (values ​​for PBS and human IgG overlap). [Figure 4a] FIG. 1 shows the binding ability of the antibodies of the present invention to BCAM by Octet analysis. [Figure 4b]FIG. 1 shows the binding ability of the antibodies of the present invention to BCAM by Octet analysis. [Figure 4c] FIG. 1 shows the binding ability of the antibodies of the present invention to BCAM by Octet analysis. [Figure 5] FIG. 1 shows the degree of cellular internalization of the antibody of the present invention as observed with a confocal laser scanning microscope. DETAILED DESCRIPTION OF THE INVENTION

[0017] Anti-BCAM antibody or antigen-binding fragment thereof The present invention provides anti-BCAM antibodies or antigen-binding fragments thereof that specifically bind to a BCAM protein.

[0018] In one embodiment, the anti-BCAM antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain CDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, and 11; a heavy chain CDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17; a heavy chain CDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, and 23; a light chain CDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, and 12; a light chain CDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, and 18; and A light chain CDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, and 24.

[0019] In specific embodiments, the anti-BCAM antibody or antigen-binding fragment thereof of the invention comprises: (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 21; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; or (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and A light chain CDR3 comprising the amino acid sequence of SEQ ID NO:24.

[0020] As used herein, each chain or its variable region comprises a particular amino acid sequence means that the chain or variable region comprises, has, or consists of the entire amino acid sequence.

[0021] In one example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:7, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:13, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:19, a light chain CDR1 having the amino acid sequence of SEQ ID NO:8, a light chain CDR2 having the amino acid sequence of SEQ ID NO:14, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:20.

[0022] In another example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 consisting essentially of the amino acid sequence of SEQ ID NO:7, a heavy chain CDR2 consisting essentially of the amino acid sequence of SEQ ID NO:13, a heavy chain CDR3 consisting essentially of the amino acid sequence of SEQ ID NO:19, a light chain CDR1 consisting essentially of the amino acid sequence of SEQ ID NO:8, a light chain CDR2 consisting essentially of the amino acid sequence of SEQ ID NO:14, and a light chain CDR3 consisting essentially of the amino acid sequence of SEQ ID NO:20.

[0023] In another example, the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 19, a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 20.

[0024] As used herein, the terms "comprising," "comprise," or variations thereof refer to an open meaning. By way of example, an antibody or antigen-binding fragment thereof comprising a recited amino acid sequence can include additional amino acid sequences that are not recited, whether required or not.

[0025] As used herein, the term "consisting essentially of" or variations thereof includes any of the recited elements and permits the presence of elements that do not substantially affect the basic and novel properties or functional characteristics of the embodiment. As an example, an antibody or antigen-binding fragment thereof consisting essentially of a recited amino acid sequence can include substitutions of one or more amino acid residues that do not substantially affect the properties of the antibody or fragment thereof.

[0026] As used herein, the term "consisting of" or variations thereof refers to each component described herein without permitting any element not described or listed in that description of the embodiment.

[0027] Each chain or variant region of another antibody or antigen-binding fragment thereof defined herein may also comprise, have or consist of an amino acid sequence such as the examples above.

[0028] The term "complementarity determining region" (CDR; CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable region that are necessary for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0029] In another embodiment, the anti-BCAM antibody or antigen-binding fragment thereof of the invention comprises: any one heavy chain variable region selected from the group consisting of SEQ ID NOs: 1, 3, and 5; and Any one light chain variable region selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

[0030] In specific embodiments, the anti-BCAM antibody or antigen-binding fragment thereof of the invention comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6.

[0031] The antibody variable region refers to the light chain and heavy chain portions of an antibody molecule comprising the amino acid sequences of the CDRs and framework regions (FRs).

[0032] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, or protein, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The term "antibody" is used herein in the broadest sense and thus broadly encompasses not only intact polyclonal or monoclonal antibodies, but also dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antigen-binding fragments thereof, antibody fragments thereof, fusion proteins including any other modified configuration of an immunoglobulin molecule containing an antigen recognition site (e.g., a variable region), synthetic antibodies (e.g., "antibody mimetics"), "FynomAbs," and the like.

[0033] There are five classes of antibodies: immunoglobulin (Ig) M, IgD, IgG, IgA, and IgE, each of which contains a heavy chain made from the heavy chain constant region genes μ, δ, γ, α, and ε. The light and heavy chains of antibodies are divided into variable regions, which have different amino acid sequences for each antibody, and constant regions, which have the same amino acid sequence. The heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each domain consists of two β sheets connected by an intramolecular disulfide bond.

[0034] In this specification, an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 is referred to as "111a," an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 is referred to as "111b," and an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 is referred to as "111c."

[0035] The antibodies of the present invention may be chimeric, humanized, or human antibodies.

[0036] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985), the entire contents of which are incorporated herein by reference.

[0037] As used herein, the term "humanized antibody" refers to an antibody in which one or more CDR sequences derived from the germline of another mammalian species, e.g., a non-human species such as mouse or chicken, have been grafted onto framework sequences of a human immunoglobulin molecule. The framework sequences may be further modified, for example, by mutation. Human Ig sequences may be disclosed by reference, for example, in the NCBI Database (Entez Gene). Appropriate sequences may be used to reduce the immunogenicity of the antibody or to reduce, enhance, or modify its binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or other suitable properties.

[0038] As used herein, the term "human antibody" refers to an antibody comprising variable regions in which both the framework and CDR regions are derived from human immunoglobulin sequences, and the antibody constant regions are also derived from human immunoglobulin sequences.

[0039] As used herein, the term "antigen-binding fragment" or "antibody fragment" generally refers to at least a portion of the antigen-binding domain or variable region (e.g., one or more CDRs) of a parent antibody. An antibody fragment retains at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments that can be used herein include, but are not limited to, Fab, Fab', Fab'-SH, Fv, single-chain antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody ((scFV-CH3)2), IgG deltaCH2, scFv-Fc, (scFv)2-Fc, fynomer, dual affinity retargeting (DART), anticalin, FN3 monobody, DARPin, affibody, affilin, affimer, affitin, alphabody, avimer, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, nanobody, binobody, or di-sdFv.

[0040] Specifically, a Fab fragment refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains.

[0041] Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the CH1 domain including at least one cysteine ​​from the antibody hinge region.

[0042] Fab'-SH refers to Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group.

[0043] F(ab')2 antibody fragments are produced as pairs of Fab' fragments separated by hinge cysteines.

[0044] Fv is the minimum antibody fragment that completely contains the antigen-recognition and antigen-binding sites. This fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region tightly bound by noncovalent bonds. The folding of these two regions results in six hypervariable loops (three loops from the heavy chain and three loops from the light chain), which provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable region can recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0045] A single-chain antibody scFv is an antibody fragment comprising a VH antibody domain and a VL antibody domain linked in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Herein, scFv polypeptides are also referred to as scFv antibody fragments, antigen-binding fragment scFvs, scFv antibodies, antibody scFvs, or simply scFvs.

[0046] Diabodies refer to small antibody fragments prepared by constructing scFv fragments using a short linker (approximately 5-10 residues) between the VH and VL domains, allowing interchain but not intrachain pairing of the V domains, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers consisting of two "crossover" scFv fragments in which the VH and VL domains of two antibodies are present on different polypeptide chains. Similarly, triabodies and tetrabodies contain three and four polypeptide chains, respectively, forming three and four antigen-binding sites, which may be identical or different.

[0047] Fynomers refer to non-immunoglobulin-derived binding polypeptides derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are known in the art and are disclosed, for example, in Grabulovski et al. (2007) JBC 282, 3196-3204 and WO 2008 / 022759. Fynomers can be genetically fused to another molecule (e.g., an antibody) to generate a "FynomAb," i.e., a form that can be engineered to have bispecificity.

[0048] Dual affinity retargeting (DART) and TRIDENT refer to those designed to simultaneously bind to two or more targets. DART refers to a covalently linked bispecific diabody, for example, a diabody linked via a C-terminal disulfide bridge, and its structure and definition are disclosed in J. Mol. Biol. (2010) 399, 436-449, etc.

[0049] The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to a BCAM protein, such as a human BCAM protein.

[0050] In a specific embodiment, the present invention provides an anti-BCAM antibody or antigen-binding fragment thereof that specifically binds to human BCAM and binds to the same epitope as antibody 111a, 111b, or 111c. The CDR1, CDR2, and CDR3 of each of the heavy and light chains of the antibody or antigen-binding fragment thereof are at least 80%, preferably at least 90%, and particularly preferably 100% identical to the CDR1, CDR2, and CDR3 of each of the heavy and light chains of antibody 111a, 111b, or 111c. Similarly, the heavy and light chains of the antibody or antigen-binding fragment thereof are at least 80%, preferably at least 90%, and particularly preferably 100% identical to the heavy and light chains of antibody 111a, 111b, or 111c.

[0051] As used herein, the term "BCAM" refers to basal cell adhesion molecule, a surface glycoprotein that acts as a receptor for the extracellular matrix protein laminin. "BCAM" is also known as Lutheran blood group glycoprotein (Lu) or CD239, and as used herein, "BCAM," "Lu," "Lu / BCAM," and "CD239" refer to the same protein and can be used interchangeably.

[0052] As used herein, the terms "specifically bind" or "specific" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a particular target (e.g., epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets.

[0053] As used herein, the term "specifically binds to human BCAM protein" refers to a specific antibody that binds to 1×10 -7 M or less, or preferably 5 x 10 -8 The bond dissociation equilibrium constant (K D ) can refer to an antibody that binds to human BCAM protein. Therefore, the anti-BCAM antibody or antigen-binding fragment thereof of the present invention can be used in a concentration of 1 × 10 -7 The bond dissociation equilibrium constant (K D ), preferably 5 × 10 -8 K below M D It can bind to the human BCAM protein at

[0054] As used herein, "K D The term "" refers to the binding-dissociation equilibrium constant of a particular antibody-antigen interaction and is given by the formula K D =K d / K a (In the formula, K a is the association rate constant, and Kd is the dissociation rate constant), and the constant K D The unit of is M. K of antibody DThe K value can be determined using methods well established in the art. D A preferred method for measuring the K value is to use surface plasmon resonance (SRP), preferably a biosensor system such as the Biacore® system, or biolayer interferometry (BLI) such as the Octet® system. D may be a value obtained by BLI using the Octet system.

[0055] In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention can be in a drug-conjugated form, i.e., antibody-drug conjugates (ADCs). As used herein, the term "antibody-drug conjugate" or "ADC" refers to a compound of the formula M-[LD] n where M represents an antibody molecule, i.e., an anti-BCAM antibody of the present invention or an antigen-binding fragment thereof, L is an optional linker or linker unit, D is a suitable drug or prodrug, and n is an integer of about 1 to about 20. The drug contained in the ADC can be appropriately selected depending on the therapeutic or diagnostic application, as long as it does not interfere with the specific binding of the antibody of the present invention. In one embodiment, the drug includes, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent), a prodrug-converting enzyme, a radioactive isotope or compound, or a toxin. The drug and linker that can be contained in the ADC, as well as the method for preparing them, can be determined according to methods known in the art. Thus, the present invention provides an antibody-drug conjugate comprising an anti-BCAM antibody or an antigen-binding fragment thereof.

[0056] In another embodiment, the anti-BCAM antibody or antigen-binding fragment thereof of the present invention binds to BCAM protein with an EC50 of 150 nM or less, e.g., 130 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, or 10 nM or less, as determined by ELISA assay.

[0057] As used herein, the term "EC50" refers to an in vitro or in vivo assay using an antibody and refers to the concentration of antibody that induces 50% of the maximal response, i.e., a response halfway between the maximal response and the baseline.

[0058] Nucleic Acid Molecules and Vectors Another aspect of the invention pertains to nucleic acid molecules encoding the anti-BCAM antibodies, or antigen-binding fragments thereof, of the invention.

[0059] Nucleic acids can be present in whole cells, in a cell lysate, or in a specifically purified or substantially pure form. Nucleic acids are "isolated" or "made substantially pure" when they have been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art.

[0060] Nucleic acids of the invention may be, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0061] In one embodiment, a nucleic acid molecule of the present invention encodes the light chain region, the heavy chain region, or both the light and heavy chain regions of an anti-BCAM antibody or antigen-binding fragment thereof of the present invention, and preferably encodes the light chain variable region, the heavy chain variable region, or both the light and heavy chain variable regions.

[0062] Once DNA fragments encoding the VL and / or VH regions are obtained, such DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0063] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, CH2, and CH3). The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region.

[0064] For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0065] To prepare an scFv gene, a DNA fragment encoding VL and VH is operably linked to another fragment encoding a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3, so that the VL and VH regions are joined by the flexible linker and the VL and VH sequences can be expressed as a continuous single-chain protein.

[0066] The nucleic acid sequences of the invention, such as RNA or DNA, can be isolated, genetically engineered, amplified, and / or recombinantly expressed from a variety of sources. Any recombinant expression system can be used, including, in addition to bacteria, yeast, insect, or mammalian systems. Nucleic acid manipulations, such as subcloning into expression vectors, probe labeling, sequencing, and hybridization, can be performed as known in the art.

[0067] Accordingly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0068] As used herein, the term "vector" refers to a DNA molecule capable of autonomous replication in prokaryotic and / or eukaryotic cells, and is used interchangeably with recombinant vectors, cloning vectors, or expression vectors, which are commonly used as carriers for delivering genes or DNA fragments into cells, etc. Vectors generally include, but are not limited to, an origin of replication that allows replication in prokaryotic and / or eukaryotic cells, a selectable marker gene that can confer resistance to certain conditions / substances such as antibiotic-degrading enzymes, a promoter that allows transcription of genes in eukaryotic or prokaryotic cells, and a translatable sequence.

[0069] One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).

[0070] Preparation of antibodies or antigen-binding fragments thereof The antibody or antigen-binding fragment thereof of the present invention can be prepared according to conventional methods known in the art.

[0071] In one embodiment, the antibodies or antigen-binding fragments thereof of the present invention can be prepared using antibody display techniques, such as phage display techniques.

[0072] An antibody phage library is prepared by cloning the heavy and light chain variable region genes of human antibodies into a phagemid vector in the form of fusion with the phage surface protein (pIII), expressing the phage in Escherichia coli (E. coli), and then infecting the cells with M13 helper phage to prepare an antibody library in which antibody fragments (scFv or Fab) with various combinations of heavy and light chain variable region sequences are displayed on the phage surface. From this library, antibody fragments that bind to specific antigens are isolated by panning, characterized, converted to whole IgG format, and mass-expressed in animal cells to produce specific human monoclonal antibodies.

[0073] The library can be a naive antibody library that uses antibody genes already present in the human body, or a synthetic antibody library that has increased diversity by adding random synthetic sequences to the CDRs of antibodies.

[0074] As used herein, the term "phagemid vector" refers to a plasmid DNA that has a phage origin of replication and usually has an antibiotic resistance gene as a selectable marker.

[0075] The phagemid vector used for phage display contains the gill gene of M13 phage or a part thereof, and the ScFv gene is ligated to the 5' end of the gill gene and expressed through transformants.

[0076] A "helper phage" is a phage that provides the genetic information necessary for a phagemid to be packaged into a phage particle. Because the phagemid contains the gIII gene or a portion thereof, the helper phage infects host cells transformed with the phagemid (transformant), providing the remaining phage genes. Helper phages include M13K07 or VCSM13, and most contain antibiotic resistance genes, such as a kanamycin resistance gene, to allow selection of transformants infected with the helper phage. Additionally, because the packaging signal is missing, the phagemid genes, rather than the helper phage genes, are preferentially packaged into phage particles.

[0077] In another embodiment, antibodies of the present invention, e.g., in the form of monoclonal antibodies, can be prepared by injecting a test subject (e.g., a mouse) with a BCAM antigen according to methods known in the art, and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics.

[0078] DNA encoding a monoclonal antibody is readily isolated and sequenced by conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed into an expression vector and transfected into host cells that do not produce immunoglobulin proteins, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, resulting in the synthesis of the monoclonal antibody in the recombinant host cells.

[0079] Use and Method The antibodies or antigen-binding fragments thereof of the present invention specifically bind to BCAM and prevent or treat cancer.

[0080] Thus, in one embodiment, the invention provides a method for preventing, ameliorating, or treating cancer, comprising administering to a subject an effective amount of an anti-BCAM antibody or antigen-binding fragment thereof.

[0081] In another embodiment, the invention provides the use of an anti-BCAM antibody or antigen-binding fragment thereof to prevent, ameliorate, or treat cancer.

[0082] In another embodiment, the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating cancer, comprising an anti-BCAM antibody or antigen-binding fragment thereof. The anti-BCAM antibody or antigen-binding fragment thereof can be contained in an effective amount in the pharmaceutical composition.

[0083] As used herein, the term "subject" is meant to include both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, including non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with non-human primates, sheep, dogs, cats, cows, and horses being preferred. A preferred subject is a human in need of cancer prevention or treatment.

[0084] Preferably, the anti-BCAM antibodies or antigen-binding fragments thereof of the present invention specifically bind to the BCAM protein, thereby effectively binding to BCAM-expressing cancer cells and inhibiting cancer cell proliferation in vivo, and can be effectively used for the prevention, amelioration, or treatment of cancer. BCAM is associated with tumor migration, and its overexpression is known to be particularly observed in skin cancer, ovarian cancer, pancreatic cancer, breast cancer, and the like (see, e.g., FRM Latini et al., Blood Cells, Molecules and Diseases 50 (2013) 161-165). Therefore, the anti-BCAM antibodies of the present invention can be used for all cancers in which tumor migration is observed, such as metastatic cancer, and can also be used for specific carcinomas in which its overexpression is observed.

[0085] Examples of cancers whose growth may be inhibited using the antibodies of the invention include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colorectal cancer, rectal cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer), bone cancer, pancreatic cancer, liver cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, These include, but are not limited to, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, urethral carcinoma, penile carcinoma, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder carcinoma, ureteral carcinoma, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and environmentally induced cancers including those induced by asbestos.

[0086] In one embodiment, the antibodies or antigen-binding fragments thereof of the present invention can be used to prevent or treat cancer by specifically binding to the BCAM protein to form an antibody-drug conjugate (ADC). Thus, in one embodiment, the present invention provides an anti-BCAM antibody or antigen-binding fragment conjugated to a drug. In one embodiment, the drug includes, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent), a prodrug-converting enzyme, a radioisotope or compound, or a toxin. The drugs and linkers that can be included in the ADC, as well as methods for preparing them, can be prepared according to methods known in the art.

[0087] The antibodies or antigen-binding fragments of the invention can be used alone or in combination with other anti-cancer therapies, which can include, for example, standard cancer treatments (e.g., chemotherapy, radiation therapy, or surgery) or other anti-cancer agents such as cytotoxic, cytostatic, anti-hormonal, anti-angiogenic or antimetabolite agents, targeted agents, immunostimulatory or immunomodulatory agents, immune checkpoint inhibitors, or antibodies conjugated to cytotoxic, cytostatic, and other toxic agents.

[0088] Preferably, the antibodies or antigen-binding fragments of the present invention can be used in combination with other anti-cancer agents, such as immune checkpoint inhibitors, chemotherapeutic agents, and radiotherapeutic agents.

[0089] Immune checkpoint inhibitors may be, for example, anti-CTLA-4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab), or anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab). Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antineoplastic antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterones, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Specific examples of chemotherapeutic agents of the present invention include gemcitabine, vinorelbine, etoposide (VP-16), platinum analogs such as cisplatin or carboplatin, and taxoids such as paclitaxel, albumin-bound paclitaxel, and doxetaxel.

[0090] When the antibody or antigen-binding fragment of the present invention is used in combination with other anticancer drugs, they may be administered separately or in the form of a combined pharmaceutical in which multiple active ingredients are present in a single pharmaceutical formulation. When administered as separate formulations, the two formulations may be administered sequentially or simultaneously. When administered simultaneously, they are given to a patient together. When administered sequentially, they can be administered at a short time interval, for example, the formulations can be administered to a patient within a period of 12 hours or 6 hours.

[0091] In one embodiment, the present invention provides a method for preventing, ameliorating, or treating cancer, comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof in combination with an additional anti-cancer agent. This embodiment includes not only simultaneous administration of a single composition containing an anti-BCAM antibody or antigen-binding fragment together with the additional anti-cancer agent to a patient in need thereof, but also simultaneous or sequential administration of separate compositions containing the anti-BCAM antibody or antigen-binding fragment and the additional anti-cancer agent to a patient in need thereof.

[0092] In another embodiment, the invention provides the use of an anti-BCAM antibody or antigen-binding fragment thereof in combination with an additional anti-cancer agent to prevent, ameliorate, or treat cancer.

[0093] In another embodiment, the present invention provides a pharmaceutical composition or combination for preventing, ameliorating, or treating cancer, the pharmaceutical composition or combination comprising an anti-BCAM antibody or antigen-binding fragment thereof and an additional anti-cancer agent. As used herein, a pharmaceutical composition or combination comprising an anti-BCAM antibody or antigen-binding fragment thereof and an additional anti-cancer agent includes not only cases in which the two components are physically present together in the form of a single formulation, but also cases in which the two components are administered simultaneously or sequentially in separate formulations. In this case, the two drugs may be provided separately or together in a single kit. Accordingly, the present invention provides a kit for preventing, ameliorating, or treating cancer, the kit comprising an anti-BCAM antibody or antigen-binding fragment thereof and an additional anti-cancer agent.

[0094] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising an anti-BCAM antibody or an antigen-binding fragment thereof. The compositions may contain inactive ingredients, i.e., pharmaceutically acceptable excipients (see, e.g., Handbook of Pharmaceutical Excipients). Therapeutic and diagnostic compositions can be prepared, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension, by mixing the formulation with a physiologically acceptable carrier, excipient, or stabilizer.

[0095] Suitable routes of administration include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration. Administration of antibodies used in the pharmaceutical compositions or used to practice the methods of the invention can be carried out by a variety of conventional methods, such as topical application or intradermal, subcutaneous, intraperitoneal, parenteral, intraarterial, or intravenous injection. In one embodiment, the antibodies of the invention are administered intravenously or subcutaneously. Further embodiments are as follows. [Embodiment 1] A heavy chain CDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, and 11; a heavy chain CDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17; A heavy chain CDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, and 23; a light chain CDR1 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, and 12; a light chain CDR2 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, and 18; a light chain CDR3 comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, and 24; An anti-BCAM antibody or antigen-binding fragment thereof comprising: [Embodiment 2] (a) the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7; the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13; the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8; the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 21; the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; or (c) the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; the heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; the heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12; the light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 24 2. The anti-BCAM antibody or antigen-binding fragment thereof of embodiment 1, comprising: [Embodiment 3] any one heavy chain variable region selected from the group consisting of SEQ ID NOs: 1, 3, and 5; any one light chain variable region selected from the group consisting of SEQ ID NOs: 2, 4, and 6; 2. The anti-BCAM antibody or antigen-binding fragment thereof of embodiment 1, comprising: [Embodiment 4] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 2. The anti-BCAM antibody or antigen-binding fragment thereof of embodiment 1, comprising: [Embodiment 5] The anti-BCAM antibody or antigen-binding fragment thereof of embodiment 1, wherein the anti-BCAM antibody is a multispecific antibody. [Embodiment 6] The anti-BCAM antibody or antigen-binding fragment thereof of embodiment 1, wherein the antigen-binding fragment is a Fab, Fab', Fab'-SH, Fv, single-chain antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody (scFV(CH3)2), IgG deltaCH2, scFv-Fc, (scFv)2-Fc, Fynomer, dual affinity retargeting (DART), or TRIDENT. [Embodiment 7] 1×10 -7 The bond dissociation equilibrium constant (K D 2. An anti-BCAM antibody or antigen-binding fragment thereof according to embodiment 1, which binds to human BCAM protein at [Embodiment 8] The anti-BCAM antibody or antigen-binding fragment thereof of embodiment 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody. [Embodiment 9] An antibody-drug conjugate comprising the anti-BCAM antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8. [Embodiment 10] A nucleic acid molecule encoding the anti-BCAM antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 8. [Embodiment 11] 11. A recombinant expression vector comprising the nucleic acid molecule of embodiment 10. [Embodiment 12] A pharmaceutical composition for preventing or treating cancer, comprising the anti-BCAM antibody or antigen-binding fragment thereof described in any one of embodiments 1 to 8. [Embodiment 13] 13. The pharmaceutical composition of embodiment 12, further comprising an additional anticancer agent. [Embodiment 14] The pharmaceutical composition of embodiment 13, wherein the anti-BCAM antibody or antigen-binding fragment thereof and the additional anti-cancer agent are administered simultaneously in a single formulation, or simultaneously or sequentially in separate formulations. [Embodiment 15] An anti-BCAM antibody or antigen-binding fragment thereof that specifically binds to human BCAM and binds to the same epitope as antibody 111a, 111b, or 111c. [Example]

[0096] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely intended to explain the present invention in more detail, and the scope of the present invention, according to the subject matter of the present invention, is not limited by these examples.

[0097] Example 1: Preparation of BCAM-specific antibodies by phage display 1.1 Preparation and selection of scFv antibody libraries by phage display The antibodies of the present invention were prepared by phage display methods.

[0098] The mRNAs of the heavy and light chain variable regions of antibodies obtained from human blood or bone marrow were amplified by PCR, and cDNA was synthesized. The cDNAs were cloned into a phagemid vector using restriction enzymes and expressed in E. coli by electroporation. Subsequently, the cells were infected with helper phage to prepare a human library in the form of scFv.

[0099] The library was screened by biopanning for antibodies that bind to the target antigen BCAM with high affinity. Positive clones that bind to BCAM were selected, and unique scFv sequences against BCAM were selected by sequencing.

[0100] 1.2 Preparation of anti-BCAM IgG4 antibodies 111a to 111c To produce antibodies in the form of human IgG4, the light and heavy chain regions of the scFv selected in Example 1.1 were cloned into an expression vector. The DNA was transiently transfected and cultured to express the antibodies. The culture medium was then bound to a κ region capture body, and the antibodies were eluted to prepare three types of anti-BCAM human IgG4 antibodies (111a, 111b, and 111c).

[0101] The amino acid sequences of the light-chain and heavy-chain variable regions of each IgG4 antibody, as well as the amino acid sequences of the CDRs of each variable region, are shown in Figures 1a and 1b and Tables 1 and 2, respectively (the bold and underlined sequences in Figures 1a and 1b and Table 1 represent each CDR).

[0102] [Table 1]

[0103] [Table 2]

[0104] Example 2: Confirmation of binding activity of anti-BCAM IgG4 antibody 2.1 Confirmation of binding ability to antigen protein by ELISA To confirm the binding ability of each antibody prepared in Example 1 to the antigen protein (BCAM protein), an ELISA (Enzyme-Linked Immunosorbent Assay) test was performed. The antigen protein was diluted with PBS buffer to a concentration of 15 nM. 50 μL of the antigen protein was coated onto each well of a 96-well half plate (Costar, catalog number 3690) and incubated overnight at 4°C. The next day, all the solution in the plate was removed, and blocking buffer (3% BSA in PBS) was added to each well and incubated at 37°C for 1 hour. Three types of antibody samples (111a, 111b, and 111c) were serially diluted 10-fold in blocking buffer from 1 μM to 7 points (1 pM). After the blocking step, the buffer was removed from the wells, and 50 μL of the diluted antibody was added to each well and incubated at 37°C for 2 hours. The incubated wells were washed with 0.1% PBST (0.1% Tween 20 in PBS) and treated with HRP-conjugated anti-human IgG Fc antibody (ThermoFisher Scientific, Cat. No. 31423), a secondary antibody for antibody detection, at 37°C for 1 hour, and then washed again with 0.1% PBST. 50 μL of ABTS solution (ThermoFisher Scientific, Cat. No. 00-2024) was added to each well for coloring, and the reaction was allowed to proceed at room temperature for 10 minutes. The optical density at 405 nm (OD ) was measured. 405 ) was measured. The OD 405 The values ​​are shown in Figure 2 and Table 3. To compare the degree of binding activity of each antibody to the antigen, the EC50 concentration of each antibody was calculated, and the results are shown in Table 4.

[0105] As a result, it was confirmed that antibodies 111a, 111b, and 111c tested by ELISA experiments have excellent binding ability to BCAM.

[0106] [Table 3]

[0107] [Table 4]

[0108] 2.2 Confirmation of antigen protein binding ability by Fluorescence Activated Cell Sorting (FACS, flow cytometry) The three types of antibodies prepared in Example 1 were subjected to FACS testing.

[0109] Human BCAM antigen was expressed in HEK293FT cells by transfection (using the 293FT / BCAM1 plasmid). Transfected HEK293FT cells (1x10 5 The cells were suspended in PBS, and 50 μL of the suspension was seeded onto the plate.

[0110] Separately, the anti-BCAM antibody and isotype control (human IgG) antibody of Example 1 were initially diluted to 45 μg / mL using PBS, and then diluted 12 times in 3-fold dilutions. The plates were incubated for an appropriate time at an appropriate temperature. PBS buffer was used as a negative control.

[0111] After washing, AlexaFluor® 647 AffiniPure Goat Anti-Human IgG (H+L) (min × Bov, Hrs, Ms, Sr, Prot) (Jackson) (1 μg / mL) was added, the plate was incubated again, washed again with PBS, and the antigen-antibody binding was analyzed using a flow cytometer.

[0112] The results are shown in Table 5 and FIG.

[0113] [Table 5]

[0114] 2.3 Confirmation of binding ability to antigen protein by biolayer interferometry (BLI) The three types of antibodies prepared in Example 1 were subjected to a binding ability confirmation test by BLI using Octet (Octet QK, ForteBio).

[0115] Specifically, a Reactive 2nd Generation (AR2G) biosensor (catalog no. 18-5094, Sartorius) was mounted and activated using the AE2G reagent kit (catalog no. 18-5095) to bind human BCAM (antigen) using amine coupling. The biosensor was then placed in the antigen solution for 10 minutes to allow binding of human BCAM, and then placed in an ethanolamine solution for 5 minutes to terminate the reaction. The human BCAM-bound biosensor was placed in a buffer solution containing the three antibodies 111a, 111b, and 111c. The binding reaction was monitored for 5 minutes, followed by a dissociation reaction for 15 minutes. The antibody solution was serially diluted to examine the association and dissociation rates at different concentrations. The association rate constant (Ka) and dissociation rate constant (Kd) were determined by fitting the data to a 1:1 binding model using curve fitting software. The binding / dissociation equilibrium constant (KD) was calculated as KD = Kd / Ka. The binding dynamics data are shown in Table 6 below, and the binding sensorgrams are shown in Figures 4a, 4b, and 4c.

[0116] [Table 6]

[0117] This confirmed that the antibody of the present invention binds to human BCAM protein with high affinity.

[0118] Example 3: Confirmation of cellular internalization of antibodies using confocal laser scanning microscopy Using the three types of antibodies prepared in Example 1, an experiment was carried out to confirm the cellular internalization of the antibodies.

[0119] 5×10 4MKN-1 (human gastric cancer cell line, KCLB) cells were seeded onto cell culture slides (SPL, Korea). The next day, cells were confirmed to have adhered to the plate. After removing the existing cell culture medium, antibodies 111a, 111b, and 111c prepared in Example 1 were diluted with PBS to prepare 10 μg / mL or 40 μg / mL. Each well was treated with 500 μL of antibody. After incubation at 4°C for 1 hour, the remaining antibody was removed, and the cell slides were washed three times with PBS. The cell slides were divided into two groups: one group served as a negative control for cellular internalization, and the other group served as a cellular internalization induction group. The cell slide used as the negative control was fixed with 4% paraformaldehyde (PFA, Sigma, USA) for 10 minutes at room temperature. The cell slide used as the cellular internalization induction group was transferred to a 37°C incubator and incubated for 1 hour, after which it was fixed with 4% PFA, as in the negative control group. Each fixed cell slide was washed with PBS to completely remove PFA, then treated with 0.1% Triton X100 for 15 minutes at room temperature to prepare for secondary antibody penetration. After washing with PBS and blocking (3% BSA for 20 minutes at room temperature), the cell slides were treated with a fluorescently conjugated secondary antibody (10 μg / mL Alexa 488-conjugated goat anti-human IgG, Invitrogen, USA) for 1 hour at 4°C. For nuclear staining, each cell slide was treated with Hoechst 33342 (Invitrogen, USA) for 3 minutes at room temperature, washed with PBS, treated with mounting solution (Agilent Technologies, USA), and covered with a coverslip to complete the slide sample. The extent of antibody internalization in cells was observed at 400x magnification using an LSM710 confocal laser microscope (Carl Zeiss, Germany).

[0120] The results are shown in Figure 5. In the negative control group (antibody treatment at 4°C for 1 hour, which did not induce cellular internalization; top panel of Figure 5), all three antibodies bound to the cancer cell surface. However, in the antibody treatment at 37°C for 1 hour, which induces cellular internalization (bottom panel of Figure 5), each antibody was internalized into the cells, and the fluorescent signal on the cell surface decreased, while the fluorescent signal inside the cells increased (fluorescent signals in the nuclear stained area are indicated by asterisks, and fluorescent signals in the antibody-stained area are indicated by triangles). These results indicate that the antibodies of the present invention have the potential to deliver cytotoxic drugs into cells through cellular internalization; i.e., the antibodies may be used in the form of drug-conjugated ADCs in the future.

Claims

1. (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 21; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; or (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 17; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and Light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 24 An anti-BCAM antibody or antigen-binding fragment thereof comprising:

2. (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 The anti-BCAM antibody or antigen-binding fragment thereof of claim 1 , comprising:

3. The anti-BCAM antibody or antigen-binding fragment thereof of claim 1 , wherein the anti-BCAM antibody is a multispecific antibody.

4. 2. The anti-BCAM antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, single-chain antibody scFv, F(ab')2 fragment, diabody, triabody, tetrabody, minibody (scFv(CH3)2), IgG deltaCH2, scFv-Fc, (scFv)2-Fc, or TRIDENT™.

5. 1 x 10 －７ The bond dissociation equilibrium constant (K Ｄ 2. The anti-BCAM antibody or antigen-binding fragment thereof of claim 1, which binds to human BCAM protein at the nucleotide sequence (SEQ ID NO: 1).

6. The anti-BCAM antibody or antigen-binding fragment thereof of claim 1 , wherein the anti-BCAM antibody is a chimeric antibody or a human antibody.

7. An antibody-drug conjugate comprising the anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A nucleic acid molecule encoding the anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

9. A recombinant expression vector comprising the nucleic acid molecule of claim 8.

10. A pharmaceutical composition for preventing or treating cancer, comprising the anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

11. 11. The pharmaceutical composition of claim 10, further comprising an additional anti-cancer agent.

12. The pharmaceutical composition of claim 11 , wherein the anti-BCAM antibody or antigen-binding fragment thereof and the additional anti-cancer agent are administered simultaneously in a single formulation, or simultaneously or sequentially in separate formulations.

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