Anti-BCAM antibodies and antibody-drug conjugates

Abstract

The present invention provides an anti-BCAM antibody or its antigen-binding fragment that binds to basal cell adhesion molecule (BCAM) proteins, an antibody-drug conjugate containing the antibody or its antigen-binding fragment, a nucleic acid encoding the antibody or its antigen-binding fragment, and a method for obtaining the antibody or its antigen-binding fragment and the antibody-drug conjugate. Furthermore, the present invention relates to the use of the antibody or its antigen-binding fragment and the antibody-drug conjugate for the prevention, improvement, or treatment of diseases (e.g., cancer) related to the function or expression of BCAM.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority based on Korean Patent Application No. 10-2023-0044305, titled "Anti-BCAM Antibody and Antibody-Drug Conjugate," filed on April 4, 2023, and all disclosures in the specification and drawings of said application are incorporated herein by reference.

[0002] Technical field to which the invention belongs The present invention relates to an anti-BCAM antibody or its antigen-binding fragment that binds to a basal cell adhesion molecule (BCAM) protein, an antibody-drug conjugate containing the antibody or its antigen-binding fragment, a pharmaceutical composition containing the antibody or its antigen-binding fragment or the antibody-drug conjugate, a method for producing the antibody or its antigen-binding fragment or the antibody-drug conjugate and its use, a polynucleotide encoding the antibody or its antigen-binding fragment, a vector containing the polynucleotide, and isolated host cells for recombinant production of the antibody or its antigen-binding fragment. The antibody, its antigen-binding fragment or antibody-drug conjugate of the present invention can be used for the prevention, improvement, or treatment of diseases related to the function or expression of BCAM, such as cancer. [Background technology]

[0003] BCAM is a transmembrane protein belonging to the immunoglobulin superfamily, also known as Lu (Lutheran blood group glycoprotein) or CD239. Lu and BCAM are interchangeable and are sometimes referred to as Lu / BCAM. Lu was initially studied as an antigen of the Lutheran blood group system, while BCAM was identified as an upregulated antigen in ovarian cancer. Lu and BCAM share the same extracellular domain but differ in their cytoplasmic tails. Specifically, BCAM lacks the 40 C-terminal amino acids present in the Lu cytoplasmic tail. Furthermore, the Lu-specific cytoplasmic region contains an SH3-binding motif, a dileucine motif, and a potential phosphorylation site. However, the common region of the Lu and BCAM cytoplasmic tails contains a spectrin-binding motif. Due to this structural overlap between BCAM and Lu, it is known to be difficult to distinguish between Lu and BCAM in actual tissues. The extracellular domain of BCAM contains one V set, one C1 set, and three I set domains (V-C1-III). BCAM specifically binds to laminin α5, a major component of the basement membrane. Laminin α5 associates with β and γ chains to form a heterotrimer, which is found in many basement membranes of normal and diseased tissues. BCAM also promotes the migration of lung cancer cells on laminin 511 (LM-511), which consists of α5, β1, and γ1 chains, and the migration of tumor cells on LM-511 is inhibited in the presence of functional inhibitory antibodies against BCAM. In addition to ovarian cancer, BCAM overexpression has also been observed in breast cancer, prostate cancer, skin cancer, hepatocellular carcinoma, and KRAS-mediated colorectal cancer (CRC), leading to the proposal of BCAM as a useful antigen for use in antibody drugs and cancer diagnostics. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yamato Kikkawa et al., Scientific Report, 2018 Apr 26;8(1):6612 [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide an anti-BCAM antibody or its antigen-binding fragment that binds to the BCAM protein with high affinity, or an antibody-drug conjugate containing the anti-BCAM antibody, and a method for producing the same. Another object of the present invention is to provide the use of an anti-BCAM antibody or its antigen-binding fragment or an antibody-drug conjugate containing an anti-BCAM antibody for the prevention, improvement, treatment, or diagnosis of diseases (e.g., cancer) related to the function or expression of BCAM. [Means for solving the problem]

[0006] [1] The present invention provides an anti-BCAM antibody or antigen-binding fragment thereof that binds to a basal cell adhesion molecule (BCAM) protein, comprising a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 5, a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 6, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 7, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 8. [2] The present invention provides an anti-BCAM antibody or antigen-binding fragment thereof as described in [1] above, comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 2. [3] The antibody is an IgG isotype, and is an anti-BCAM antibody or antigen-binding fragment thereof as described in [1] or [2] above. [4] The antibody is a multispecific antibody, an anti-BCAM antibody or its antigen-binding fragment as described in any one of items [1] to [3]. [5] An anti-BCAM antibody or antigen-binding fragment thereof, which is a humanized antibody, a chimeric antibody, a CDR-transplanted antibody, or a recombinant human antibody, as described in any one of items [1] to [4]. [6]An anti-BCAM antibody or its antigen-binding fragment as described in any one of [1] to [5], which is Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 fragment, VL, VH, bispecific antibody, tripspecific antibody, quadruplespecific antibody, minibody, IgG delta CH2, scFv-Fc, (scFv)2-Fc, Fynomer, DART (dual-affinity re-targeting) protein, antikalin, FN3 monobody, DARPin, afibody, affin, affimer, afitin, alphabody, avimer, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, nanobody, bi-nanobody, or di-sdFv. [7] 1 × 10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1 × 10 -11 It is an anti-BCAM antibody or its antigen-binding fragment described in any one of [1] to [6] that binds to the human BCAM protein with a binding-dissociation equilibrium constant (KD) of M or less.

[0007] [8] The present invention provides an antibody-drug conjugate comprising an anti-BCAM antibody or an antigen-binding fragment thereof as described in any one of [1] to [7]. [9] The present invention provides a pharmaceutical composition for the prevention, improvement or treatment of a disease related to the function or expression of BCAM, comprising an anti-BCAM antibody or its antigen-binding fragment as described in any one of [1] to [7]; or an antibody-drug conjugate as described in [8]; and a pharmaceutically acceptable carrier.

[10] The pharmaceutical composition according to [9], wherein the disease related to the function or expression of BCAM is cancer.

[11] The pharmaceutical composition may be the one described in [9] or

[10] , which is used in combination with a further anticancer agent.

[12] Further anticancer agents are the pharmaceutical compositions according to

[11] , which are administered simultaneously as a single formulation with an antibody or an antigen-binding fragment thereof or an antibody-drug conjugate, or administered simultaneously or sequentially with separate formulations.

[13] The present invention provides a nucleic acid (nucleotide) encoding an antibody or an antigen-binding fragment thereof according to any one of [1] to [7].

[14] The present invention provides a recombinant expression vector containing the nucleic acid according to

[13] .

[15] The present invention provides an isolated host cell for recombinantly producing an anti-BCAM antibody or an antigen-binding fragment thereof according to any one of [1] to [7].

[16] The present invention provides a method for producing an antibody or an antigen-binding fragment thereof, comprising: culturing the host cell according to

[15] under conditions capable of producing the antibody or an antigen-binding fragment thereof, and isolating the antibody or an antigen-binding fragment thereof from the host cell or the culture medium.

[17] The present invention provides an anti-BCAM antibody or an antigen-binding fragment thereof according to any one of [1] to [7] for use in the prevention, improvement or treatment of a disease related to the function or expression of BCAM.

Advantages of the Invention

[0008] The anti-BCAM antibody, antigen-binding fragment thereof, or antibody-drug conjugate containing the antibody or antigen-binding fragment thereof of the present invention has an action of binding to human BCAM protein with high affinity, internalizing and accumulating in cancer cells expressing BCAM protein. Therefore, the anti-BCAM antibody, antigen-binding fragment thereof, or antibody-drug conjugate of the present invention can be usefully used for the prevention, improvement, or treatment of diseases related to the function or expression of BCAM.

Brief Description of the Drawings

[0009] [Figure 1] It is a diagram showing the amino acid sequences of the heavy chain variable region and the light chain variable region of the Ab-I antibody, which is an example of the anti-BCAM antibody of the present invention. The underlined sequences indicate the CDR sequences of each region. [Figure 2]This is a diagram showing the results of analyzing the binding ability of the Ab-I antibody, which is an example of the anti-BCAM antibody of the present invention, to BCAM by the ELISA method. [Figure 3] This is a diagram showing the results of analyzing the binding ability of the Ab-I antibody, which is an example of the anti-BCAM antibody of the present invention, to BCAM by FACS, and the values of PBS and human IgG are shown superimposed. [Figure 4] This is a diagram showing the results of analyzing the binding ability of the Ab-I antibody, which is an example of the anti-BCAM antibody of the present invention, to BCAM by Octet analysis. [Figure 5] This is an image obtained by observing the degree of internalization of the Ab-I antibody, which is an example of the anti-BCAM antibody of the present invention, in the human cancer cell line SK-BR3 using a confocal laser microscope. [Figure 6] This is a diagram showing the results of analyzing the binding ability of Ab-II, which is an example of the anti-BCAM antibody-drug conjugate of the present invention, to BCAM by the ELISA method. [Figure 7] This is a diagram showing the results of analyzing the in vitro anti-cancer effect of Ab-II, which is an example of the anti-BCAM antibody-drug conjugate of the present invention, against the human cancer cell lines SK-BR3, OVCAR3, and DU145.

Modes for Carrying out the Invention

[0010] [Definition] As used herein, the term "BCAM" refers to the basal cell adhesion molecule, a surface glycoprotein that acts as a receptor for laminin, an extracellular matrix protein. "BCAM" is also known as the Lutheran blood group glycoprotein (Lu) or CD239, and the terms "BCAM", "Lu", "Lu / BCAM", and "CD239" as used herein refer to the same protein and may be used interchangeably.

[0011] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, polypeptides, and proteins via at least one antigen-recognizing site located within the variable region of the immunoglobulin molecule. As used herein, the term "antigen" is used in its broadest sense and broadly includes complete polyclonal or monoclonal antibodies, as well as dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), their antigen-binding fragments, antibody fragments, and fusion proteins, synthetic antibodies (e.g., "antibody mimes"), "FynomAbs," and the like, including any other modified configuration of an immunoglobulin molecule (including an antigen-recognizing site (e.g., variable region)). There are five types of antibodies: immunoglobulin (Ig) M, IgD, IgG, IgA, and IgE, each containing a heavy chain produced from heavy chain constant region genes μ, δ, γ, α, and ε, respectively. The light and heavy chains of antibodies are divided into a variable region, where the amino acid sequence differs between antibodies, and a constant region, where the amino acid sequence is the same. The heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each domain consists of two β-sheets, linked by intramolecular disulfide bonds. As used herein, the term "antibody variable region" refers to the light chain and heavy chain portions of an antibody molecule, including the amino acid sequences of the CDR and framework region (FR). As used herein, the term "complementarity-determining region (CDR)" refers to the amino acid residues in the antibody variable region necessary for antigen binding. Each variable region has three CDRs, usually identified as CDR1, CDR2, and CDR3.

[0012] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity to at least two different sites. As used herein, the terms "humanized antibody" and "CDR-transplanted antibody" refer to an antibody in which one or more CDR sequences derived from non-human species, such as other mammalian species, are inserted into a framework sequence derived from a human immunoglobulin molecule. The framework sequence may be further modified, for example, by mutation. Human Ig sequences can be referenced, for example, from the NCBI database (Entrez Gene). The immunogenicity of an antibody can be reduced using an appropriate sequence, or its binding properties, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other appropriate characteristics can be reduced, enhanced, or altered. As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody. Methods for producing chimeric antibodies are well known in the art. For example, one can refer to Morrison, Science 229:1202 (1985), which is incorporated herein by reference in its entirety. As used herein, the term "human antibody" refers to an antibody that contains a variable region in which both the framework region and the CDR region are derived from a human immunoglobulin sequence. The constant region of the antibody is also derived from a human immunoglobulin sequence. As used herein, the term "anti-BCAM antibody" includes both monovalent antibodies with single specificity and multispecific antibodies comprising at least two arms (e.g., a first arm bound to BCAM and a second arm bound to a second (target) antigen).

[0013] As used herein, the terms "antigen-binding fragment" or "antibody fragment" usually refer to a fragment that includes at least a portion of the antigen-binding domain (e.g., one or more CDRs) or variable region of a parent antibody. The antibody fragment retains at least a portion of the binding specificity of the parent antibody. Examples of antigen-binding fragments that may be used herein include, but are not limited to, Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 fragment, VL, VH, bispecific antibodies, tripspecific antibodies, quadruplespecific antibodies, minibodies, IgG delta CH2, scFv-Fc, (scFv)2-Fc, finomers, DART (dual-affinity re-targeting) proteins, antikalin, FN3 monobodies, DARPin, afibodies, afirin, afimers, afitin, alphabodies, avimers, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, nanobodies, bi-nanobody, or di-sdFv. The variable light chain region (VL) refers to a variable light chain, and is also called the VL domain. VH refers to the variable heavy chain, also known as the VH domain. Fab refers to a monovalent fragment consisting of VL, VH, CL, and CH1 domains. Fab' differs from the Fab fragment in that one or more cysteine ​​residues from the antibody hinge region are attached to the carboxyl terminus of the CH1 domain. Fab'-SH refers to a Fab' molecule that has a free thiol group at the cysteine ​​residue in its constant domain. Fv is the smallest antibody fragment containing a complete antigen recognition site and antigen binding site, consisting of a dimer in which one heavy chain variable region and one light chain variable region are tightly and non-covalently associated. These two regions fold to form six hypervariable loops (three from the heavy chain and three 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 has the function of recognizing and binding to the antigen, but with lower affinity than the entire binding site.

[0014] A single-chain antibody scFv is an antibody fragment comprising VH and VL antibody domains linked by a single-chain polypeptide, preferably the scFv polypeptide further comprising a polypeptide linker between the VH and VL domains such that the scFv can form a desired structure for antigen binding. In this specification, the terms scFv antibody fragment, antigen-binding fragment scFv, scFv antibody, antibody scFv, or simply scFv may also be used. The F(ab')2 fragment is generated as a pair of Fab' fragments via hinge cysteine ​​between Fab' fragments. A bispecific antibody is a small antibody fragment prepared by constructing an scFv fragment using a short linker (approximately 5-10 residues) between the VH domain and the VL domain, so that it forms a bivalent fragment, i.e., a fragment with two antigen-binding sites, through pairing within the V domain chain rather than through inter-chain pairing. A bispecific antibody is a heterodimer consisting of two "cross-linked" scFv fragments, in which the VH and VL domains of the two antibodies are located on different polypeptide chains. The triplicate and quadruplicate antibodies each contain three and four polypeptide chains, which may be identical or different, and each forms three and four antigen-binding sites. The minibody, also known as (scFv-CH3)2, is a dimeric molecule consisting of two polypeptide chains, each containing an scFv molecule fused to the CH3 domain or its portion via a linked peptide (for example, a modified scFv molecule containing the altered VH domain described above). scFv-Fc refers to a polypeptide formed by the ligation of scFv and Fc. (scFv)2-Fc refers to a polypeptide formed by the linking of two types of scFv and Fc.

[0015] Fynomers refer to non-immunoglobulin-derived conjugated polypeptides derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well-known in the industry and are described, for example, in D Grabulovski et al. (2007), Journal of Biological Chemistry, Volume 282, Issue 5, 2007, pp. 3196-3204, and International Publication No. 2008 / 022759. Fynomers can be genetically fused with other molecules (e.g., antibodies) to produce "FynomAbs," which are forms that can be manipulated to have bispecificity. Biaffinity retargeting (DART) proteins and TRIDENTs are engineered molecules designed to bind to two or more targets simultaneously. DARTs are covalently linked bispecific antibodies, such as bispecific antibodies linked via a C-terminal disulfide crosslink. Their specific structure and definition are described, for example, in S. Johnson et al., Journal of Molecular Biology, Volume 399, Issue 3, 2010, pp. 436-449. Antikarin is a type of antibody mimetic and belongs to a family of molecules related to human lipocalin. For characteristics and types of antikarin, see A. Skerra, Current Opinion in Biotechnology, Volume 18, Issue 4, 2007, pp. 295-304. FN3 monobodies are synthetic binding proteins constructed using fibronectin type III (FN3) domains as a molecular scaffold. Monobodies were first introduced in 1998 by the Koide group as an alternative to antibodies against target-binding proteins (Koide et al., Journal of Molecular Biology, Volume 284, Issue 4, 1998, pp. 1141-1151). DARPin (designed ankyrin repeat protein) refers to an ankyrin repeat domain (166 residues) designed to provide a robust interface typically resulting from three repeating β-turns. DARPin generally has three repeats corresponding to an artificial consensus sequence, with six randomized positions in each repeat.

[0016] Affibody molecules are a family of antibody mimetic molecules derived from the Z domain of Staphylococcus protein A. Affibody molecules are based on a 3-helix bundle domain, which can structurally be found in fusion proteins. Affibody molecules themselves have a molecular weight of approximately 6 kDa and are stable under high temperature and acidic or alkaline conditions. Target specificity is obtained by randomizing 13 amino acids located in the two α-helices involved in the binding activity of the parent protein domain (Feldwisch, J., Tolmachev, V. (2012). Engineering of Affibody Molecules for Therapy and Diagnostics. In: Voynov, V., Caravella, J. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 899. Humana Press, Totowa, NJ). Affilins are antibody mimics developed by modifying amino acids on the surface of a protein using γ-B crystallin or ubiquitin as a scaffold through random mutagenesis. The selection of affilins with desired target specificity is performed, for example, by phage display or ribosome display technology. Affimers represent the evolution of peptide aptamers. Affimers are small, highly stable proteins engineered to present peptide loops that provide a high-affinity binding surface to specific target proteins or antigens. Affimers may offer the same advantages as antibody specificity, but with the advantages of being smaller, chemically synthesized or modified, and free from cell culture contaminants. Affimers are typically low-molecular-weight proteins, usually 12–14 kDa, derived from the cystatin family of cysteine ​​protease inhibitors. The affimer backbone is a stable protein based on the folding of cystatin proteins. It exhibits two peptide loops and an N-terminal sequence that can be randomized to bind to different target proteins with high affinity and specificity. Afitin, also known as nanophytin, is an antibody-mimetic protein derived from the DNA-binding protein Sac7d of Sulfolobus acidocaldarius. Afitin generally has a molecular weight of approximately 7 kDa and is designed to bind specifically to target molecules by randomizing the amino acids on its binding surface (B Mouratou et al. Biomolecules. 2015 Jan 30;5(1), pp. 60-75). Alpha bodies are small 10 kDa proteins engineered to bind to various antigens. Alpha bodies are developed as scaffolds with a set of amino acid residues that can be modified to bind to protein targets while maintaining correct folding and thermal stability. The alpha body backbone is computer-designed based on a double-coil structure, but there are no known native counterparts. Initially, the scaffold was constructed from three peptides that non-covalently associate to form a parallel double-coiled trimer (US Patent Application Publication 20100305304), but was later redesigned into a single polypeptide chain containing three α-helices linked by a linker region (J Desmet et al. Nature Communications volume 5, Article number: 5237 (2014)).

[0017] Abimers are a class of antibody mimetics consisting of two or more peptide sequences, each containing 30-35 amino acids, derived from the A domain of various membrane receptors and linked by a linker peptide. Binding of the target molecule occurs via the A domain, and the domain with the desired binding specificity can be selected, for example, using phage display technology. The binding specificity of the different A domains contained in the avimer may or may not be identical (UH Weidle et al. Cancer Genomics Proteomics. 2013 Jul-Aug; 10(4), pp.155-68). Im7 refers to immunoprotein 7 that binds to ColE7 colisin, a 60 kDa toxin produced by E. coli. VLR is an abbreviation for variable lymphocyte receptor, a molecule that governs adaptive immune responses in Agna (jawless fish) such as hagfish and lampreys. Unlike conventional antibodies, it is a receptor belonging to the single-chain leucine-rich repeat family and has specific binding ability to certain antigens (Boehm et al., Annual Review of Immunology Vol. 30, pp.203-220). Recombinant proteins produced by mutating VLRs to increase their affinity for specific proteins, known as lipobodies (Lee et al., Molecular Therapy, 2014 22(7), pp. 1254-1265), can also be considered to belong to the VLR family. VNAR is an important component of the shark's adaptive immune system and is the smallest IgG-like protein in the animal kingdom. Trimab refers to a triple-specific antibody. CrossMab is a chimeric antibody composed of halves of two full-length antibodies, prepared by combining (i) a knob-into-hole technique to promote correct pairing between the two heavy chains, and (ii) an exchange technique between the heavy and light chains of one of the two Fabs to introduce asymmetry to avoid light chain pairing errors. Nanobodies, also known as VHH antibodies or single-domain antibody fragments (sdAbs), are antibody fragments consisting of a single monomeric variable antibody domain. Binanobodies are tetramers created by chemically bonding two types of scFv. di-sdFv is an sdFv dimer in which the heavy and light chains of sdFv are linked by disulfide bonds.

[0018] The term "vector" as used in this invention is used interchangeably with recombinant vectors, cloning vectors, expression vectors, etc., and refers to a DNA molecule capable of self-replication in prokaryotic and / or eukaryotic cells, and is typically used as an intermediate carrier for transferring genes or DNA fragments into cells. A vector typically includes, but is not limited to, a replication origin that can replicate in prokaryotic and / or eukaryotic cells, a selectable marker gene that can confer resistance to specific conditions / substances such as antibiotic-degrading enzymes, and a promoter that can transcribe genes in eukaryotic or prokaryotic cells. One type of vector is a plasmid, which is a circular double-stranded DNA loop to which further DNA fragments can be ligated. Other vectors are viral vectors, to which further DNA fragments can be ligated into a viral genome. Certain types of vectors can replicate autonomously within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors).

[0019] As used herein, the terms "specifically binds to" or "specific to" mean determining the presence of a target in a heterogeneous population of molecules, including biological molecules, and refer to measurable and reproducible interactions, such as binding between the target and the antibody. For example, an antibody that specifically binds to a particular target (e.g., an epitope) is an antibody that binds to the target more readily, with higher affinity, higher avidity, and / or for a longer duration than an antibody that binds to other targets. As used herein, the term "anti-BCAM antibody or its antigen-binding fragment that specifically binds to human BCAM protein" refers to 1 × 10⁻¹⁴ -6 This refers to antibodies or their antigen-binding fragments that bind to human BCAM proteins with a binding-dissociation equilibrium constant (KD) of M or less. As used herein, the term "KD" refers to the binding equilibrium dissociation constant for a specific antibody-antigen interaction, calculated using the formula KD = Kd / Ka (wherein Ka is the association rate constant and Kd is the dissociation rate constant), with the unit of the constant KD being M. The KD value of an antibody can be measured using methods widely established in the industry. Preferred methods for measuring the KD value of an antibody include surface plasmon resonance (SPR) using a biosensor system such as the Biacore® system, or Bio-layer interferometry (BLI) using, for example, the Octet® system. In one specific example, the KD referred to herein may be a value obtained by Bio-Layer Interferometry using the Octet® system. As used herein, the term "EC50" is a term related to in vitro or in vitro assays using antibodies, and refers to 50% of the maximum response, i.e., the antibody concentration that induces an intermediate response between the maximum response and the baseline. As used herein, the term "operably linked" means that two DNA fragments are linked together such that the amino acid sequence encoded by those DNA fragments remains in frame.

[0020] As used herein, the term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, reptiles, and other mammals, but non-human primates, sheep, dogs, cats, cattle, horses, and other mammals are preferred. The preferred subject is a human being in need of cancer prevention or treatment. The terms “comprising,” “comprises,” and variations thereof as used herein are non-limiting. For example, an antibody or antigen-binding fragment thereof containing an enumerated amino acid sequence may contain further amino acid sequences not enumerated, whether essential or not. In this specification, “each chain or its variable region contains a particular amino acid sequence” means all cases in which it contains the entire amino acid sequence, has an entire amino acid sequence, or consists of an amino acid sequence. As used herein, the term "consisting of" or variations thereof means that no element not mentioned or enumerated in the description of each component of an embodiment is permitted.

[0021] Anti-BCAM antibodies or their antigen-binding fragments, or antibody-drug conjugates containing them. In one embodiment, the present invention provides an anti-BCAM antibody or an antigen-binding fragment thereof that binds to the BCAM protein, or an antibody-drug conjugate containing the same. In one embodiment of the present invention, the anti-BCAM antibody or its antigen-binding fragment may be an anti-BCAM antibody or its antigen-binding fragment that binds to a basal cell adhesion molecule (BCAM) protein, comprising a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 5, a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 6, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 7, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 8. In other embodiments of the present invention, the anti-BCAM antibody or antigen-binding fragment thereof may be an anti-BCAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region containing an amino acid sequence having at least 95% sequence identity (preferably at least 96%, 97%, 98%, or 99% sequence identity) with the amino acid sequence represented by SEQ ID NO: 1, and a light chain variable region containing an amino acid sequence having at least 95% sequence identity (preferably at least 96%, 97%, 98%, or 99% sequence identity) with the amino acid sequence represented by SEQ ID NO: 2. In yet another embodiment of the present invention, the anti-BCAM antibody or the antigen-binding fragment thereof may be an anti-BCAM antibody or the antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 2. In one embodiment of the present invention, an antibody comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 2 may be referred to as "Ab-I".

[0022] In one embodiment of the present invention, the anti-BCAM antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), a heteroconjugated antibody, a humanized antibody, or any other modified form of an immunoglobulin molecule containing an antigen recognition site with the required specificity (e.g., a glycosylated variant of the antibody, an amino acid sequence variant of the antibody, and a covalently modified antibody). Furthermore, the antigen-binding fragment of the anti-BCAM antibody may be Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 fragment, VL, VH, bispecific antibody, tripspecific antibody, quadruplespecific antibody, minibody, IgG delta CH2, scFv-Fc, (scFv)2-Fc, finomer, DART (biaffinity retargeting) protein, antikalin, FN3 monobody, DARPin, afibody, affin, affimer, afitin, alphabody, avimer, Im7, VLR, VNAR, trimab, CrossMab, TRIDENT, nanobody, bi-nanobody, or di-sdFv. In one embodiment of the present invention, an anti-BCAM antibody or its antigen-binding fragment may specifically bind to a BCAM protein, such as human BCAM protein. In other embodiments of the present invention, an anti-BCAM antibody or its antigen-binding fragment can bind to the BCAM protein with an EC50 of 150 nM or less, for example, 130 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, or 10 nM or less, as measured by an ELISA assay. Preferably, the BCAM protein is human BCAM protein. In yet another aspect of the present invention, the anti-BCAM antibody or its antigen-binding fragment can bind to human BCAM protein with a binding dissociation equilibrium constant (KD) of 1×10 -6 M or less. Preferably, the anti-BCAM antibody or its antigen-binding fragment can bind to human BCAM protein with a binding dissociation equilibrium constant (KD) of 1×10 -6 M to 1×10 -11 M, 10 -6 M to 1×10 -8 M, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, or 1×10 -11 M or less.

[0023] In other embodiments of the present invention, the antibody or its antigen-binding fragment of the present invention can be in a conjugated form with a drug, i.e., can form an antibody-drug conjugate (ADC). As used herein, the term "antibody-drug conjugate" or "ADC" can be represented by the formula M-[L-D] n (where M represents an antibody molecule, i.e., the anti-BCAM antibody or its antigen-binding fragment of the present invention, L is a linker or a linker unit in some cases where the linker can be cleaved or not, D is a suitable drug or prodrug, and n is from 1 to 20). The n can be 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less.

[0024] The drug contained in the ADC can be appropriately selected according to therapeutic or diagnostic uses, etc., as long as it does not interfere with the specific binding of the antibody of the present invention. In one embodiment, the drug can include, but is not limited to, a cytotoxic agent (e.g., a chemotherapeutic agent), a prodrug-converting enzyme, a radioisotope or a compound or a toxin. The drugs and linkers that can be contained in the ADC and their production methods can follow methods known in the art. Furthermore, in one embodiment of the present invention, the present invention provides the antibody-drug conjugate containing the anti-BCAM antibody or its antigen-binding fragment.

[0025] Nucleic acids and vectors encoding anti-BCAM antibodies or their antigen-binding fragments. One aspect of the present invention relates to nucleic acids encoding the anti-BCAM antibody or its antigen-binding fragment. Nucleic acids may be present in whole cells or cell lysates, or in a specifically purified or substantially pure form. Nucleic acids are “isolated” or “substantially pure isolated” nucleic acids, purified from other cellular components or contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques such as alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods known in the art. In one embodiment of the present invention, the nucleic acid 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. In other embodiments of the present invention, the nucleic acid encodes the light chain region, heavy chain region, or both the light and heavy chain regions of an anti-BCAM antibody or its antigen-binding fragment, and preferably encodes the light chain variable region, heavy chain variable region, or both the light and heavy chain variable regions. After obtaining a DNA fragment encoding the VL and / or VH regions, the DNA fragment can be further recombinant, for example, using standard recombinant DNA techniques, to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In this process, the DNA fragment encoding VL or VH is operably linked to another protein, for example, another DNA fragment encoding an antibody constant region or a flexible linker.

[0026] In one embodiment of the present invention, isolated DNA encoding a VH region can be converted into a full-length heavy chain gene by operably ligating the VH-encoding DNA to other DNA molecules encoding heavy chain constant regions (CH1, CH2, and CH3). The heavy chain constant regions may be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions. In another embodiment of the present invention, in a Fab fragment heavy chain gene, the DNA encoding VH may be operably ligated to another DNA molecule encoding only the heavy chain CH1 constant region. In yet another embodiment of the present invention, to construct the scFv gene, DNA fragments encoding VL and VH may be operably linked to another fragment encoding a mobile linker, such as the amino acid sequence (Gly4-Ser)3, so that the VL and VH sequences can be expressed as a continuous single-strand protein with VL and VH regions linked by the mobile linker. In another aspect of the present invention, the present invention provides a recombinant expression vector encoding an anti-BCAM antibody or an antigen-binding fragment thereof, or a recombinant expression vector comprising the nucleic acid.

[0027] Preparation of anti-BCAM antibodies or their antigen-binding fragments In one aspect of the present invention, the anti-BCAM antibody or its antigen-binding fragment may be produced by any suitable method known in the art to which the present invention belongs. In one embodiment of the present invention, the anti-BCAM antibody may be produced using techniques well established in the industry, such as hybridoma, recombination, phage display, transfection, synthesis, or a combination thereof, or other techniques readily known in the industry (e.g., Jayasena, SD, Clin. Chem., 45: 1628-50 (1999) and Fellouse, FA, et al., J. Mol. Biol., 373(4):924-40 (2007)). In other embodiments of the present invention, anti-BCAM antibodies can be produced using phage display technology. Specifically, anti-BCAM antibodies can be produced by cloning genes encoding the heavy and light chain variable regions of human antibodies into phagemide vectors, fusing them to phage surface proteins (pIII), and expressing them in E. coli. Subsequently, an antibody library can be generated in which scFv or Fab antibody fragments containing various heavy and light chain variable region sequences are displayed on the surface of phages by infection with M13 helper phages. Then, antibody fragments that specifically bind to target antigens can be isolated using panning technology with the library. After characterizing the isolated antibody fragments, they can be converted to whole IgG form and expressed in mammalian cells for large-scale production of specific human monoclonal antibodies. As the phage display library, a naive antibody library using antibody genes already present in the human body can be used. Alternatively, a synthetic antibody library with increased diversity by inserting randomly synthesized sequences into the CDR region of the antibody may be used. The phagemide vector is plasmid DNA containing a phage replication origin, and typically includes an antibiotic resistance gene as a selection marker. Phagemide vectors used for phage display typically contain gene III (gIII) or a portion thereof derived from the M13 phage, with the scFv gene ligated to the 5' end of gIII and expressed in transformed host cells. Helper phages provide the genetic information necessary for phagemide assembly into phage particles. Since phagemide vectors contain only phage gene III or a portion thereof, phagemide-transformed host cells should be infected with helper phages to supply the remaining phage genes. Various phagemides exist, such as M13K07 or VCSM13, and usually contain kanamycin resistance genes to select helper phage-infected transformants. Furthermore, because phagemides have a defect in their packaging signal, the phagemide genome preferentially assembles into phage particles over the helper phage genome. In another embodiment of the present invention, anti-BCAM antibodies may be produced using hybridoma technology. Specifically, after injecting a test subject (e.g., a mouse) with the BCAM antigen, hybridomas expressing antibodies with desired sequences or functional properties may be isolated, and antibodies may be produced, for example, in the form of monoclonal antibodies.

[0028] In one embodiment of the present invention, the anti-BCAM antibody may be produced in the form of a monoclonal antibody, and the nucleotide sequence encoding the monoclonal antibody may be immediately isolated and sequenced using a known method (for example, using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody), and hybridoma cells may be used as a preferred source of the nucleotide sequence. At this point, once the nucleotide sequence is isolated, it can be placed in an expression vector and transfected into host cells, such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, to obtain the synthesis of the monoclonal antibody in recombinant host cells.

[0029] In another aspect of the present invention, the present invention provides isolated host cells for recombinantly producing an anti-BCAM antibody or an antigen-binding fragment thereof. In one embodiment of the present invention, an anti-BCAM antibody or its antigen-binding fragment can be expressed by introducing a nucleic acid into any recombinant expression system, including bacteria, yeast, insects, or mammalian lines. The recombinant expression system may include isolated host cells. In other embodiments of the present invention, the expression system may be isolated mammalian host cells, for example, but not limited to, CHO cells, DG44 or DUXB11 cells, NS0 myeloma cells, monkey kidney cell lines (e.g., CV1 cells, COS cells, etc.), SP2 cells, human fetal kidney (HEK) cells, Chinese hamster fibroblasts, human cervical cancer cells (e.g., HELA), mouse fibroblasts (e.g., BALBC / 3T3), mouse myeloma cells (P3x63-Ag3.653;NS0;SP2 / O), hamster kidney cells (e.g., HAK), mouse L cells (e.g., L-929), human lymphocytes (e.g., RAJI), human kidney cells (e.g., 293 and 293T).

[0030] In another aspect of the present invention, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing host cells under conditions capable of producing an antibody or an antigen-binding fragment thereof, and isolating an antibody or an antigen-binding fragment thereof from host cells or a culture medium. In one embodiment of the present invention, after introducing a recombinant expression vector into a recombinant expression system, host cells are cultured under conditions sufficient for the antibody to be expressed intracellularly or secreted into the culture medium, and the antibody or its antigen-binding fragment is recovered from the host cells or culture medium using a standard protein purification method. In other embodiments of the present invention, the recombinant expression vector may be transfected into host cells by standard techniques, at which point known techniques used for intracellular introduction of exogenous nucleic acids, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection, may be used.

[0031] Uses of anti-BCAM antibodies or their antigen-binding fragments, and anti-BCAM antibody-drug conjugates The antibodies and antibody-drug conjugates of the present invention can be usefully applied to a variety of purposes, including but not limited to therapeutic treatment methods and diagnostic methods. In one aspect of the present invention, an antibody or its antigen-binding fragment or antibody-drug conjugate specifically binds to the BCAM protein and can be used to prevent, improve, or treat diseases related to the function or expression of BCAM. In other embodiments of the present invention, the disease associated with the function or expression of BCAM may be cancer. The anti-BCAM antibody, its antigen-binding fragment, or antibody-drug conjugate of the present invention specifically binds to the BCAM protein, effectively binds to cancer cells expressing BCAM, inhibits the proliferation of cancer cells in vivo, and can be usefully used for the prevention, improvement, or treatment of cancer. BCAM is known to be involved in tumor migration, and its overexpression is known to occur particularly in skin cancer, ovarian cancer, pancreatic cancer, breast cancer, etc. (see, for example, FRM Latini et al., Blood Cells, Molecules and Diseases 50 (2013), pp. 161-165). Therefore, the anti-BCAM antibody of the present invention may be used in any cancer in which tumor migration is observed, such as metastatic cancer, and may also be used in other specific cancers in which BCAM overexpression is observed.

[0032] Cancers whose proliferation can be inhibited using the anti-BCAM antibody, its antigen-binding fragment, or drug conjugate of the present invention include, for example, gallbladder cancer, melanoma (e.g., metastatic malignant melanoma, malignant melanoma of the skin and eye), kidney cancer (e.g., clear cell carcinoma, renal cell carcinoma), prostate cancer (e.g., hormone-resistant prostate adenocarcinoma), breast cancer (e.g., invasive breast cancer, non-invasive breast cancer), colorectal cancer, rectal cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer), osteosarcoma, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), skin cancer, head or neck cancer, uterine cancer, ovarian cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, This includes, but is not limited to, pharyngeal cancer, laryngeal cancer, esophageal cancer, oral cancer, tongue cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bone marrow cancer, chronic and acute leukemia (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), pediatric solid tumors, lymphocytic lymphoma, bladder cancer, ureteral cancer, renal pelvis cancer, neoplasia of the central nervous system (CNS), primary CNS lymphoma, tumor neovascularization, spinal axial tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (e.g., asbestos-induced cancer), brain cancer, glioma, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, and urothelial carcinoma.

[0033] In one embodiment of the present invention, the present invention provides a method for preventing, improving, or treating cancer, comprising administering an effective amount of an anti-BCAM antibody or its antigen-binding fragment or an anti-BCAM antibody-drug conjugate to a subject. In other embodiments of the present invention, the present invention provides the use of anti-BCAM antibodies or their antigen-binding fragments or anti-BCAM antibody-drug conjugates for the prevention, improvement, or treatment of cancer. In another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention, improvement, or treatment of cancer, comprising an anti-BCAM antibody or its antigen-binding fragment or an anti-BCAM antibody-drug conjugate. The anti-BCAM antibody or its antigen-binding fragment or the anti-BCAM antibody-drug conjugate may be included in an effective amount in the pharmaceutical composition. The anti-BCAM antibody of the present invention, its antigen-binding fragment, or antibody-drug conjugate may be used alone or in combination with other anticancer therapies. Other anticancer therapies may include, for example, standard cancer therapies (e.g., chemotherapy, radiotherapy, or surgery); or other anticancer agents, such as cytotoxic agents, cell proliferation inhibitors, antihormone agents, anti-angiogenic agents or antimetabolites, targeted anticancer agents, immunostimulants or immunomodulators, immune checkpoint inhibitors, or antibodies conjugated with cytotoxic agents, cell proliferation inhibitors, and other toxic agents. Preferably, the anti-BCAM antibody or its antigen-binding fragment or the anti-BCAM antibody-drug conjugate of the present invention may be used in combination with other anticancer agents, such as immune checkpoint modulators, chemotherapeutic agents, or radiotherapy. Immune checkpoint modulators may be, for example, anti-CT LA-4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab), or anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab). Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle toxin plant alkaloids, cytotoxic / antitemocyte antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogestins, 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 (e.g., cisplatin or carboplatin), and taxoids (e.g., paclitaxel, albumin-conjugated paclitaxel, or docetaxel).

[0034] When the anti-BCAM antibody or its antigen-binding fragment, or antibody-drug conjugate of the present invention is used in combination with other anticancer agents, they may be administered separately or as a combination product 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. In the case of simultaneous administration, they are both provided to the subject. In the case of sequential administration, they may be provided with a time interval that is not too long (for example, they may be administered to the subject within 12 or 6 hours), and may be administered to the subject at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks or more.

[0035] Pharmaceutical compositions may contain inactive components, i.e., pharmaceutically acceptable excipients (see, for example, Handbook of Pharmaceutical Excipients). Pharmaceutical compositions may be prepared, for example, by mixing with physiologically acceptable carriers, excipients, or stabilizers, in the form of lyophilized powders, slurries, aqueous solutions, or suspensions. Suitable routes of administration for the pharmaceutical composition include parenteral administration, such as intramuscular, intravenous, or subcutaneous administration. The antibody for use in the pharmaceutical composition of the present invention, or for carrying out the method of the present invention, may be administered by topical application or by various conventional methods such as skin, subcutaneous, intraperitoneal, parenteral, intra-arterial, or intravenous injection. In one embodiment, the antibody of the present invention is administered intravenously or subcutaneously. The pharmaceutical composition may further contain an anticancer agent. In one embodiment, the pharmaceutical composition may be used in combination with further anticancer agents. The anticancer agent may be administered simultaneously with an antibody or its antigen-binding fragment or antibody-drug conjugate as a single formulation, or simultaneously or sequentially as separate formulations, and the type of anticancer agent and the method of administration are as described above.

[0036] In one embodiment, the present invention provides a method for preventing, improving, or treating cancer, comprising administering an effective amount of an anti-BCAM antibody or its antigen-binding fragment or antibody-drug conjugate to a subject in combination with other anticancer agents. This embodiment includes administering the anti-BCAM antibody or antigen-binding fragment together with further anticancer agents in a single composition for co-administration, and administering separately contained compositions simultaneously or sequentially to a subject in need. In this case, the route of administration includes parenteral administration, such as intramuscular, intravenous, or subcutaneous administration. In another aspect of the present invention, the present invention provides the use of an anti-BCAM antibody or its antigen-binding fragment or an anti-BCAM antibody-drug conjugate for combination therapy with other anticancer agents for the prevention, improvement, or treatment of cancer.

[0037] In one aspect of the present invention, the present invention provides a pharmaceutical composition or combination (combination) for the prevention, improvement, or treatment of cancer, comprising an anti-BCAM antibody or its antigen-binding fragment or an anti-BCAM antibody-drug conjugate and another anticancer agent. In this specification, a pharmaceutical composition or combination comprising an anti-BCAM antibody or its antigen-binding fragment and a further anticancer agent includes cases where the two components are physically present together in a single formulation and cases where they are administered simultaneously or sequentially as separate formulations, at which point the two drugs may be provided individually or together in a single kit. Accordingly, the present invention provides a kit for the prevention, improvement, or treatment of cancer, comprising an anti-BCAM antibody or its antigen-binding fragment or an anti-BCAM antibody-drug conjugate and another anticancer agent. In another aspect of the present invention, the present invention provides the use of an anti-BCAM antibody or its antigen-binding fragment or an anti-BCAM antibody-drug conjugate for the manufacture of a pharmaceutical product for the treatment of a disease related to the function or expression of BCAM.

[0038] [Examples] The present invention will be described in more detail below through the examples. These examples are provided to illustrate the present invention more concretely, and it will be clear to those skilled in the art that the scope of the present invention is not limited by these examples, as is evident from the gist of the invention. [Examples]

[0039] Production of BCAM-specific antibodies by phage display 1.1. Construction and selection of scFv antibody libraries by phage display The antibody of the present invention was produced by phage display. The mRNA of the heavy and light chain variable regions of antibodies obtained from human blood or bone marrow was amplified by PCR to synthesize cDNA. The cDNA was cloned into a phagemide vector using restriction enzymes and then expressed in E. coli by electroporation. Subsequently, the vectors were infected with helper phages to create a human library in scFv format. The library was screened via biopanning for antibodies that bind to the target antigen BCAM with high affinity. Positive clones that bind to the human BCAM protein were selected, and BCAM-specific scFv sequences were selected by sequencing.

[0040] 1.2. Production of Ab-I antibody The amino acid sequences of the light and heavy chain regions of the scFv selected in Example 1.1 were cloned into DNA sequences and synthesized, and then cloned into pcDNA3.4 expression vectors to produce human IgG4 format antibodies with the S228P mutation. Using these vectors, transient transfection was performed in the ExpiCHO-S cell line, followed by incubation for 8 days to express the antibodies. The resulting culture supernatant was purified using affinity chromatography with protein A, eluted with a low pH buffer, and then formulated to produce anti-BCAM human IgG4 antibody, Ab-I, with a purity of 97% (by SEC-HPLC). The heavy and light chain variable regions of the prepared Ab-I antibody are shown in Figure 1 and Table 1, and the amino acid sequences of the heavy and light chain CDRs of the Ab-I antibody are shown in Table 2. The CDRs were defined based on the IMGT CDR and Martin CDR standards (in Table 1 and Figure 1, regions marked in bold or underlined indicate each CDR).

[0041] [Table 1] [Examples]

[0042] Confirmation of the binding affinity of Ab-I antibody to BCAM. 2.1 Confirmation of BCAM binding ability by ELISA An ELISA (Enzyme-Linked Immunosorbent Assay) test was performed to confirm the binding ability of the Ab-I antibody prepared in Example 1 to human BCAM protein. The antigen protein (human BCAM protein, 10238-H08H, SinoBiological, China) was diluted to a concentration of 25 nM in PBS buffer, and 50 μL was coated into each well of a 96-well half plate (Costar, USA, product number 3690), and then incubated overnight at 4°C. The next day, all solution was removed from the plate, blocking buffer (3% BSA in PBS) was added to each well, and the plate was incubated at 37°C for 1 hour. The Ab-I antibody sample was sequentially diluted in blocking buffer from 1 μM to 6 pM in a 3-fold dilution series over 12 points. After the blocking process was complete, the buffer was removed from the wells, and 50 μL of the prepared diluted antibody was added to each well and incubated at 37°C for 2 hours. After washing with 0.1% PBST (0.1% Tween 20 in PBS), the HRP-conjugated anti-human IgG Fc antibody (ThermoFisher Scientific, US product number 31423), a secondary antibody for detection, was added and treated at 37°C for 1 hour, followed by further washing with 0.1% PBST. For color development, 50 μL of TMB solution (ThermoFisher Scientific, US product number 34028) was added to each well and reacted at room temperature for 10 minutes. Subsequently, the absorbance at 450 nm (OD450) was measured, and the OD450 values ​​for antibody concentration are shown in Figure 2 and Table 3. To represent the degree of binding affinity of the Ab-I antibody to the antigen protein, the EC50 concentration was calculated, and the results are shown in Table 4. As a result, ELISA testing confirmed that the tested Ab-I antibody had excellent binding ability to the human BCAM protein.

[0043] [Table 2] 2.2. Confirmation of binding ability to BCAM using FACS A FACS (Fluorescence Activated Cell Sorting, Flow Cytometry) test was performed to confirm the binding ability of the Ab-I antibody prepared in Example 1 to the antigen protein (human BCAM protein) expressed on the cell surface. Human BCAM antigen protein was expressed in HEK293FT cells via transfection using a 293FT / BCAM plasmid. 1 × 10 5 HEK293FT cells transfected with PBS were suspended, and 50 μL of the suspension was seeded onto a plate. Separately, the Ab-I antibody and isotype control (human IgG4) antibody from Example 1 were sequentially diluted 12-fold in PBS, starting at a concentration of 111 nM, via 3-fold dilutions, added to plates, and incubated for an appropriate time and temperature. PBS buffer was used as a negative control. The plates were then washed, and goat anti-human IgG(H+L) cross-adsorbed secondary antibody, Alexa Fluor® 647 (ThermoFisher Scientific, U.S. product number A21445) (1:400), was added. The plates were then incubated again. After washing with PBS, the degree of antigen-antibody binding was analyzed using a flow cytometer. The results are shown in Table 5 and Figure 3. Therefore, FACS testing confirmed that the Ab-I antibody has excellent binding ability to human BCAM expressed on the cell surface.

[0044] [Table 3] 2.3 Confirmation of binding ability to antigen proteins by biolayer interferometry (BLI) The Ab-I antibody prepared in Example 1 was subjected to a binding affinity test using BLI with Octet (Octet® R8, Sartorius). Specifically, to immobilize human BCAM protein (antigen) using amine coupling, a reactive second-generation (AR2G) biosensor (product number 18-5094, Sartorius) was mounted and activated using an AR2G reagent kit (product number 18-5095). The human BMP protein was then immobilized by immersion in the antigen solution for 10 minutes, and the reaction was stopped by immersion in ethanolamine solution for 5 minutes. The biosensor with the immobilized human BCAM protein was placed in a buffer containing the prepared antibody, and the association reaction was monitored for 5 minutes, followed by the dissociation reaction for 5 minutes. During this time, the antibody solution was serially diluted to confirm the association and dissociation rates at different concentrations. After setting the dissociation phase to 100 seconds for optimal fitting, the data was fitted to a 1:1 binding model using curve fitting software to determine the association (Ka) and dissociation (Kd) rate constants. The binding-dissociation equilibrium constant (KD) was calculated as KD = Kd / Ka. The binding kinetic data is shown in Table 6 below, and the binding sensor gram is shown in Figure 4.

[0045] [Table 4] This confirms that the antibody of the present invention binds to human BCAM protein with high affinity. [Examples]

[0046] Confirmation of antibody intracellular relocation into cancer cells by confocal laser microscopy observation. Using the Ab-I antibody prepared in Example 1, we conducted an experiment to confirm the transfer of the antibody into cells. 2.5 × 10 4SK-BR-3 cells (human breast cancer cell line, KCLB) were plated onto cell culture chamber slides (SPL Korea). The following day, after confirming cell adhesion to the plate, the existing cell culture medium was removed. The Ab-I antibody prepared in Example 1 was diluted with PBS to prepare a 10 μg / mL solution, and 500 μl was added to each well. After reacting 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 was used as a negative control for intracellular integration, and the other group was used as an internalization induction group. The cell slides used as the negative control group were fixed with 4% PFA (paraformaldehyde; Sigma USA) (fixation, treatment at room temperature for 10 minutes), and the cell slides used as the internalization induction group were transferred to a 37°C incubator and incubated for 1 hour, and then fixed with 4% PFA in the same way as the negative control group. After fixation was complete, the PFA was washed off with PBS to completely remove it from each cell slide. Subsequently, 0.1% Triton X-100 was applied at room temperature for 15 minutes to prepare for cell membrane permeabilization with the secondary antibody. After washing with PBS and blocking (treatment with 3% BSA at room temperature for 20 minutes), fluorescently labeled secondary antibody, goat anti-human IgG (H+L) cross-adsorbed secondary antibody, and Alexa Fluor® 488 (ThermoFisher Scientific, US product number A11013) were applied at 10 μg / mL at 4°C for 1 hour. The cell slides with completed secondary antibody treatment were stained nuclearly with DAPI staining solution (Abcam, product number ab228549) at room temperature for 3 minutes. After washing with PBS, mounting solution (Agilent Technologies, US product number S3023) was applied, and the slides were covered with coverslips to complete the slide specimens. The degree of antibody internalization was observed under a confocal laser scanning microscope (LSM 700, Carl Zeiss, Germany) at 400× magnification. The results are shown in Figure 5. In the negative control group (4°C, 1-hour antibody treatment condition that does not induce intracellular internalization, upper photograph in Figure 5), the Ab-I antibody bound to the surface of cancer cells. However, under intracellular internalization-inducing conditions of 37°C and 1-hour antibody treatment (lower photograph in Figure 5), each antibody was internalized into the cell, resulting in a decrease in the fluorescence signal on the cell surface and an increase in the fluorescence signal inside the cell. These results indicate that the antibodies of the present invention have the function of delivering cytotoxic drugs into cells via intracellular internalization, and that they can be used in the form of antibody-drug conjugates (ADCs). [Examples]

[0047] Preparation of antibody-drug conjugates (Ab-II) using Ab-I antibody 4.1. Preparation of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)pyrroridine-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (Compound 1)

[0048] [ka] 151 mg (0.205 mmol) of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl(4-nitrophenyl)carbonate (compound 1b) was dissolved in 10 mL of N,N-dimethylformamide, and 22 mg (0.16 mmol) of 1-hydroxybenzotriazole was added and the mixture was stirred at room temperature for 30 minutes. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (compound 1a) 100 mg (0.137 mmol), pyridine 3.78 mL (46.8 mmol), and diisopropylethylamine 148 mg (1.15 mmol) were added to the reaction mixture and the mixture was stirred at room temperature for 20 hours. After the reaction was complete, the reaction mixture was distilled under reduced pressure, and the resulting residue was purified using a high-performance liquid chromatography apparatus. The resulting residue was lyophilized to obtain 40 mg of the title compound (yield 22%).

[0049] HRMS(ESI+):m / z=1330.7665[M+H]+.

[0050] 4.2. Preparation of Ab-II antibody-drug conjugates The antibody-drug conjugate Ab-II was produced by conjugating compound 1, prepared in Example 4.1, with the antibody Ab-I from Example 1. In this invention, a method was applied to partially reduce the disulfide bonds of the antibody using a conjugation technique that utilizes the maleimide functional group present in the linker and the cysteine ​​residue side chain of the antibody, thereby activating the necessary side chains. Specifically, the reducing agent DTT was added at a concentration of 2 mM under pH 8.0 conditions, and the antibody was partially reduced by reacting it at 37°C for 1 hour. Subsequently, the reducing agent was removed using Amicon ultra (Millipore), and the buffer was replaced with 100 mM Phosphate (pH 6.5). Then, 15 equivalents of compound 1 dissolved in DMSO were added to allow the conjugation reaction to proceed. At this time, the antibody concentration during the reaction was diluted to 5 mg / mL, and the reaction was carried out in a chamber at 25°C for 1 hour. The reaction material was purified using size exclusion chromatography (SEC). The final Ab-II was formulated in PBS. The purity of Ab-II was analyzed by SEC-HPLC. Specifically, 20 μg of Ab-II was loaded onto an SEC-HPLC column, and the analysis was performed by flowing 50 mM sodium phosphate buffer (pH 6.8) containing 150 mM sodium chloride at a flow rate of 1 mL / min, and detecting the sample at 214 nm using a UV detector. The analysis results confirmed that the purity of the generated Ab-II was 92%. UV spectrophotometry was applied to measure the concentration of the product using absorbance at 280 nm, and the drug-to-antibody ratio (DAR) was also calculated using the absorbance ratio at 248 nm. The final analysis yielded a calculated DAR of 5.3 for Ab-II. [Examples]

[0051] Confirmation of Ab-II antibody-drug conjugate binding affinity to BCAM using ELISA. An ELISA (Enzyme-Linked Immunosorbent Assay) test was performed to confirm the binding ability of the Ab-II antibody-drug conjugate prepared in Example 4 to the human BCAM protein. The antigen protein (human BCAM protein, 10238-H08H, SinoBiological China) was diluted to a concentration of 7 nM in PBS buffer, and 50 μL was coated into each well of a 96-well plate (9018, Corning, USA), and incubated at room temperature for 2 hours. All solution was removed from the plate, blocking buffer (3% BSA in PBS) was added to each well, and the plate was incubated at 37°C for 1 hour. The Ab-II antibody-drug conjugate sample was serially diluted in blocking buffer from 100 nM to 1.7 pM in a 3-fold dilution series across 11 points. After the blocking process was complete, the buffer was removed from the wells, and 50 μL of the prepared diluted antibody was added to each well and incubated at 37°C for 1 hour. After washing with 0.1% PBST (0.1% Tween 20 in PBS), a secondary antibody for antibody-drug conjugate detection (HRP-complex anti-human IgG Fc antibody (109-035-098, Jackson ImmunoResearch Inc., USA)) was added and treated at 37°C for 1 hour, followed by further washing with 0.1% PBST. 50 μL of TMB solution (ThermoFisher Scientific, US product number 34028) was added to each well and reacted at room temperature for 10 minutes to develop color. Subsequently, the absorbance at 450 nm (OD450) was measured, and the OD450 values ​​for antibody concentration are shown in Figure 6. To represent the degree of binding affinity of the Ab-II antibody-drug conjugate to the antigen protein, the EC50 concentration was calculated, and the results are shown in Table 7. From this, ELISA testing confirmed that the tested Ab-II antibody-drug conjugate has excellent binding ability to human BCAM protein.

[0052] [Table 5] [Examples]

[0053] Confirmation of the in vitro anticancer effect of the anti-BCAM antibody-drug conjugate Ab-II In vitro studies were conducted to confirm the anticancer effect of the Ab-II antibody-drug conjugate BCAM produced in Example 4 against human cell lines that highly express the latter. SK-BR-3 (human breast cancer cell line, KCLB), OVCAR3 (human ovarian cancer cell line, KCLB), DU145 (human prostate cancer cell line, KCLB) 5 × 10 6 Individual cancer cells were inoculated into a 96-well plate (3595, Corning, Corning USA). The following day, after confirming cell adhesion to the plate, the existing cell culture was removed. Ab-II prepared in Example 4 was serially diluted in cell culture medium in a 5-fold dilution series over eight points (from 1 μM to 12.8 pM), and 200 μL was added to each well. On the drug treatment day, the luminescence of the negative control (0% control, not treated with the drug) was measured. After removing the medium, 100 μL of CellTiter-Glo® (Promega, USA) solution was added to each well, and after reacting at room temperature for 10 minutes, the luminescence was measured using a microplate reader (Synergy H1, Biotek, USA). The drug-treated experimental group and the positive control group (100% control, untreated with the drug) were reacted in a 37°C CO2 incubator for 72 hours. Subsequently, the culture medium was removed, and 100 μL of CellTiter-Glo® solution was added to each well. After reacting at room temperature for 10 minutes, luminescence was measured using a microplate reader. The % cell viability of Ab-II was calculated by substituting the values ​​into the formula: [(Ab-II treatment group) - (0% control group)] / [(100% control group) - (0% control group)]. The GI50 (maximum half-mass growth inhibition concentration) value of Ab-II for each cell line was calculated based on the % cell viability using GraphPad Prism (GraphPad software Inc., USA). The results are shown in Figure 7 and Table 8. This confirms that the Ab-II antibody-drug conjugate of the present invention has excellent inhibitory activity against human cancer cell lines that highly express BCAM.

[0054] [Table 6]

Claims

1. An anti-BCAM antibody or its antigen-binding fragment that binds to a basal cell adhesion molecule (BCAM) protein, comprising a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 5, a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 6, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 7, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO:

8.

2. The anti-BCAM antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:

2.

3. The anti-BCAM antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is an IgG isotype.

4. An anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a multispecific antibody.

5. An anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a humanized antibody, a chimeric antibody, a CDR-transplanted antibody, or a recombinant human antibody.

6. Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 fragment, VL, VH, bispecific antibody, triplicate antibody, quadruplicate antibody, minibody, IgG delta CH ２ , scFv-Fc, (scFv) ２ An anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is -Fc, Fynomer, DART (dual-affinity retargeting) protein, antikalin, FN3 monobody, DARPin, afibody, affin, afimer, afitin, alphabody, avimer, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, nanobody, bi-nanobody, or di-sdFv.

7. 1 x 10 －６ An anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which binds to human BCAM proteins with a binding-dissociation equilibrium constant (KD) of M or less.

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

9. A pharmaceutical composition for the prevention, improvement, or treatment of diseases related to the function or expression of BCAM. (i) The anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; or (ii) The antibody-drug conjugate according to claim 8; and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, wherein the disease related to the function or expression of BCAM is cancer.

11. The pharmaceutical composition according to claim 9 or 10, wherein the pharmaceutical composition is used in combination with a further anticancer agent.

12. The pharmaceutical composition according to claim 11 for the prevention, improvement, or treatment of diseases related to the function or expression of BCAM, wherein the further anticancer agent is administered simultaneously with an antibody or its antigen-binding fragment or antibody-drug conjugate as a single formulation, or simultaneously or sequentially as a separate formulation.

13. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

14. A recombinant expression vector comprising the nucleic acid described in claim 13.

15. Isolated host cells for recombinant production of an anti-BCAM antibody or its antigen-binding fragment according to any one of claims 1 to 7.

16. A method for producing an antibody or its antigen-binding fragment, the following: A method comprising culturing the host cells described in claim 15 under conditions capable of producing an antibody or an antigen-binding fragment thereof, and isolating the antibody or the antigen-binding fragment thereof from the host cells or culture medium.

17. An anti-BCAM antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, for use in the prevention, improvement, or treatment of diseases related to the function or expression of BCAM.

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