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WO2025239669A1 - Anticorps anti-bcam et son conjugué - Google Patents

Anticorps anti-bcam et son conjugué

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Publication number
WO2025239669A1
WO2025239669A1 PCT/KR2025/006521 KR2025006521W WO2025239669A1 WO 2025239669 A1 WO2025239669 A1 WO 2025239669A1 KR 2025006521 W KR2025006521 W KR 2025006521W WO 2025239669 A1 WO2025239669 A1 WO 2025239669A1
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WO
WIPO (PCT)
Prior art keywords
antibody
cancer
drug conjugate
ala
bcam
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2025/006521
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English (en)
Inventor
Mi Young Cha
Hyun Uk Kim
Hyun Kyung Yu
Young Eun Ha
Mi Ra Kim
Ki Tae Park
Seung Min Byun
Bu Nam Jeon
Soo Jung Moon
Gyeong Jin CHEON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genome&company Inc
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Genome&company Inc
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Publication date
Priority claimed from KR1020240063246A external-priority patent/KR20250164033A/ko
Application filed by Genome&company Inc filed Critical Genome&company Inc
Publication of WO2025239669A1 publication Critical patent/WO2025239669A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Definitions

  • the present invention relates to an anti-BCAM antibody or an antigen-binding fragment thereof that specifically binds to BCAM protein, a conjugate comprising the antibody or the antigen-binding fragment thereof (e.g., an antibody-drug conjugate), a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof or the conjugate, a method for producing the antibody or the antigen-binding fragment thereof or the conjugate, a use thereof.
  • a conjugate comprising the antibody or the antigen-binding fragment thereof (e.g., an antibody-drug conjugate)
  • a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof or the conjugate
  • a method for producing the antibody or the antigen-binding fragment thereof or the conjugate a use thereof.
  • the antibody or the antigen-binding fragment thereof or the conjugate of the present invention may be used for the prevention, amelioration, or treatment of diseases associated with the function or expression of BCAM protein, such as cancer.
  • ADC Antibody-Drug Conjugate
  • ADC binds to an antigen specifically expressed on the surface of cancer cells to bind a target cancer cell, subsequently undergoing internalization into the cancer cell. Following internalization, proteolytic enzymes present within cellular lysosomes degrade the linking between the antibody and the drug, thereby releasing the drug, which exerts its cytotoxic effect and leads to the destruction of the cancer cell. Consequently, ADCs are expected to provide greater anticancer effects than standalone antibody therapies. Since the FDA approved Mylotarg® (Gemcitabine ozogamicin) in 2000 as the first ADC, various ADCs targeting different antigens have been developed. In addition to anticancer drugs, targeted therapies for inflammatory diseases and other conditions are also actively being explored.
  • basal cell adhesion molecule is a transmembrane protein belonging to the Ig superfamily and is also known as Lutheran blood group glycoprotein (Lu). Since BCAM specifically binds to laminin ⁇ 5, a major component of basement membrane, it is considered to be involved in cell adhesion through its interaction with laminin. Laminin ⁇ 5 forms a heterotrimer by associating with ⁇ and ⁇ chains, and are found in the basement membranes of both normal and diseased tissues. Furthermore, BCAM promotes the migration of lung cancer cells on laminin-511 (LM-511), which consists of ⁇ 5, ⁇ 1, and ⁇ 1 chains. The migration of tumor cells on LM-511 is inhibited by antibodies that suppress the function of BCAM.
  • LM-511 laminin-511
  • BCAM a useful antigen for the diagnosis and development of antibody-based therapeutics.
  • BCAM is suitable as a target antigen for ADCs, as well as antibodies that specifically bind to BCAM with high internalization efficiency and ADCs derived therefrom, have not yet been reported.
  • the inventors of the present invention have sought to develop an antibody that specifically binds to BCAM protein, particularly human BCAM protein, and exhibits tumor cell internalization capability, thereby effectively delivering a payload with cytotoxicity or a therapeutic moiety into the cells.
  • BCAM protein particularly human BCAM protein
  • the inventors have successfully developed an anti-BCAM antibody with high binding affinity for human BCAM protein and strong tumor cell internalization capability.
  • an antibody-drug conjugate prepared by conjugating a cytotoxic therapeutic moiety and a linker to the anti-BCAM antibody can release the therapeutic moiety within cancer cells in vivo, thereby completing the present invention.
  • the present invention relates to an antibody-drug conjugate (ADC) represented by Formula I:
  • M is an anti-BCAM antibody or an antigen-binding fragment thereof that binds to BCAM protein; L is a linker; D is a therapeutic moiety; and n is the average number of L-D structures conjugated per antibody, ranging from 1 to 20.
  • an anti-BCAM antibody or an antigen-binding fragment thereof includes: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7; a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the antibody-drug conjugate of [1] or [2], wherein an antibody or an antigen-binding fragment thereof includes: a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2.
  • amino acid residue is selected from a group consisting of alanine, cysteine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, and arginine.
  • amino acid unit is selected from a group consisting of: Valine-Citrulline (Val-Cit), Alanine-Alanine (Ala-Ala), Alanine-Citrulline (Ala-Cit), Citrulline-Asparagine (Cit-Asn), Valine-Glutamic Acid (Val-Glu), Citrulline-Aspartic Acid (Cit-Asp), Alanine-Valine (Ala-Val), Valine-Alanine (Val-Ala), Phenylalanine-Lysine (Phe-Lys), Valine-Lysine (Val-Lys), Alanine-Lysine (Ala-Lys), Phenylalanine-Citrulline (Phe-Cit), Leucine-Citrulline (Leu-Cit), Isoleucine-Citrulline (Ile-Cit), Phenylalanine-Arginine (Phe-Arg), Alan
  • maleimide moiety comprises Maleimidocaproyl (MC), Maleimidopropanoyl (MP), or Maleimidobutanoyl (MB).
  • a is an integer from 0 to 24,
  • b is an integer of 0 or 1
  • X is selected from a group consisting of alkyl, alkyl-cycloalkyl, and cycloalkyl, wherein the alkyl, alkyl-cycloalkyl, and cycloalkyl are substituted or unsubstituted with C 1-30 alkyl or C 3-31 cycloalkyl, and
  • Mal is a moiety comprising a maleic acid derivative.
  • PAB p-aminobenzyl
  • PABC p-aminobenzyl oxycarbonyl
  • SPP N-succinimidyl 4-(2-pyridyldithio)pentanoate
  • SCC N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-
  • the linker comprises at least one selected from the group consisting of: a combination of maleimidocaproyl, valine-citrulline (Val-Cit), and PABC; a combination of maleimidopropanoyl, polyethylene glycol (PEG), and valine-alanine (Val-Ala); a combination of maleimidopropanoyl, PEG, Val-Ala, and PABC; a combination of maleimidopropanamide, PEG, carbonyl, and Val-Ala; a combination of maleimidopropanamide, PEG, carbonyl, Val-Ala, and PABC; a combination of maleimidopropanamide, PEG, amide, and Val-Ala; a combination of maleimidopropanamide, PEG, amide, and Val-Ala; a combination of maleimidopropanamide, PEG, amide, Val-Ala, and PABC; a combination of maleimidopropanamide, alkyl-carbonyl, and
  • tubulin polymerase inhibitor is Monomethyl Auristatin F (MMAF) or Monomethyl Auristatin E (MMAE).
  • camptothecin analogue may be selected from a group consisting of Irinotecan, Rubitecan, Topotecan, Gimatecan, Pegamotecan, Lutotecan, Karenitecan, Apellatecan, Homocamptothecin, Diflomotecan, Belotecan, 9-Aminocamptothecin, Exatecan, SN-38, Dxd derivatives, or Exatecan analogues.
  • DNA alkylating agent is selected from a group consisting of pyrrolobenzodiazepine (PBD) dimer, pyrrolobenzodiazepine (PBD) dimer analogue, calicheamicin, calicheamicin analogue, duocarmycin, duocarmycin analogue, cyclophosphamide, ifosfamide, bendamustine, cisplatin, melphalan, or carboplatin.
  • PBD pyrrolobenzodiazepine
  • PBD pyrrolobenzodiazepine
  • Another aspect of the present invention relates to an anti-BCAM antibody or an antigen-binding fragment thereof, which binds to BCAM protein and comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2.
  • the anti-BCAM antibody or antigen-binding fragment thereof according to [44], wherein the tubulin polymerase inhibitor is Monomethyl Auristatin F (MMAF) or Monomethyl Auristatin E (MMAE).
  • MMAF Monomethyl Auristatin F
  • MMAE Monomethyl Auristatin E
  • the topoisomerase inhibitor is a camptothecin analogue
  • the camptothecin analogue may be selected from a group consisting of Irinotecan, Rubitecan, Topotecan, Gimatecan, Pegamotecan, Lutotecan, Karenitecan, Apellatecan, Homocamptothecin, Diflomotecan, Belotecan, 9-Aminocamptothecin, Exatecan, SN-38, Dxd derivatives, or Exatecan analogues.
  • DNA alkylating agent selected from a group consisting of pyrrolobenzodiazepine (PBD) dimer, pyrrolobenzodiazepine (PBD) dimer analogue, calicheamicin, calicheamicin analogue, duocarmycin, duocarmycin analogue, cyclophosphamide, ifosfamide, bendamustine, cisplatin, melphalan, or carboplatin.
  • PBD pyrrolobenzodiazepine
  • PBD pyrrolobenzodiazepine
  • One aspect of the present invention relates to a pharmaceutical composition for treating a disease associated with the function or expression of BCAM protein, comprising any one of the antibody-drug conjugates according to [1] to [38], or any one of the anti-BCAM antibodies or antigen-binding fragments thereof according to [39] to [51], and a pharmaceutically acceptable carrier.
  • composition according to [52], wherein the disease associated with the function or expression of BCAM protein is selected from a group consisting of leukemia, lymphoma, multiple myeloma, bone and connective tissue sarcoma, brain cancer, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, eye cancer, vaginal cancer, vulvar cancer, uterine cancer, ovarian cancer, esophageal cancer, stomach cancer, colorectal cancer, liver cancer, gallbladder cancer, bile duct cancer, lung cancer, testicular cancer, prostate cancer, penile cancer, oral cancer, salivary gland cancer, skin cancer, kidney cancer, bladder cancer, head and neck cancer, melanoma, bone cancer, anal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, neoplasia of the central nervous system (CNS), primary CNS lymphoma, tumor neovascularization, spinal axis
  • CNS
  • Another aspect of the present invention relates to a method for treating a subject having a disease associated with the function or expression of BCAM protein, or suspected of having such a disease, comprising the step of administering a therapeutically effective amount of any one of the antibody-drug conjugates according to [1] to [38], any one of the antibodies or antigen-binding fragments thereof according to [39] to [51], or any one of the pharmaceutical compositions according to [52] to [54], to the subject.
  • Another aspect of the present invention relates to the use of a therapeutically effective amount of any one of the antibody-drug conjugates according to [1] to [38], any one of the antibodies or antigen-binding fragments thereof according to [39] to [51], or any one of the pharmaceutical compositions according to [52] to [54], for the manufacture of a medicament for treating a disease associated with the function or expression of BCAM protein.
  • the anti-BCAM antibody or an antigen-binding fragment thereof, or the antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof according to the present invention binds to human BCAM protein with high affinity and exhibits excellent tumor cell internalization activity and cytotoxic activity.
  • anti-BCAM antibody or an antigen-binding fragment thereof, or the antibody-drug conjugate according to the present invention can be usefully employed for the prevention, amelioration, or treatment of diseases associated with the function or expression of human BCAM protein, such as cancer.
  • Figure 1 shows the SEC-HPLC analysis results of the Ab1 antibody as an anti-BCAM antibody.
  • Figure 2 shows the SEC-HPLC analysis results of the Ab2 antibody as an anti-BCAM antibody.
  • FIG. 3 shows the SDS-PAGE analysis results of Ab1 and Ab2 antibodies as anti-BCAM antibodies.
  • Figure 4 shows the ELISA analysis results of the binding affinity of the Ab1 antibody, a type of anti-BCAM antibody, to BCAM protein antigen, depicting changes in OD450 at 450 nm as the antibody concentration increases.
  • FIG. 5 shows the FACS analysis results of the binding affinity of the Ab1 antibody to BCAM protein antigen, depicting Mean Fluorescence Intensity (MFI) changes according to the concentration of the antibody.
  • MFI Mean Fluorescence Intensity
  • Figure 6 shows the biolayer interferometry analysis results of the dynamic binding affinity of the Ab1 antibody to BCAM protein antigen, depicting a binding sensor-gram showing sensor response changes over time.
  • Figure 7 shows the fluorescence signal changes over an incubation time when VCaP cells expressing human BCAM antigen protein were treated with Ab1 antibody, a type of anti-BCAM antibody, or a negative control human IgG4 antibody (hIgG4) at varying concentrations (0.5, 1, 2, or 4 ⁇ g/ml).
  • Figure 8 shows the SEC-HPLC chromatogram of ADC1-1, an anti-BCAM antibody-drug conjugate.
  • Figure 9 shows the SEC-HPLC chromatogram of ADC1-2, an anti-BCAM antibody-drug conjugate.
  • Figure 10 shows the SEC-HPLC chromatogram of ADC2-1, an anti-BCAM antibody-drug conjugate.
  • Figure 11 shows the HIC-HPLC chromatogram of ADC1-1, an anti-BCAM antibody-drug conjugate.
  • Figure 12 shows the HIC-HPLC chromatogram of ADC1-2, an anti-BCAM antibody-drug conjugate.
  • Figure 13 shows the HIC-HPLC chromatogram of ADC2-1, an anti-BCAM antibody-drug conjugate.
  • Figure 14 shows the ELISA analysis results of the binding affinity of ADC1-1, an anti-BCAM antibody-drug conjugate, to BCAM protein antigen, depicting changes in OD 450 as the antibody-drug conjugate concentration increases.
  • Figure 15 shows the FACS analysis results of the binding affinity of ADC1-1, an anti-BCAM antibody-drug conjugate, to BCAM protein antigen, depicting MFI changes according to antibody-drug conjugate concentration.
  • Figure 16 shows the biolayer interferometry analysis results of the dynamic binding affinity of ADC1-1, an anti-BCAM antibody-drug conjugate, to BCAM protein antigen, depicting a binding sensor-gram showing sensor response changes over time.
  • Figure 17 shows the fluorescence signal changes over an incubation time when ADC1-1, an anti-BCAM antibody-drug conjugate, and a negative control human IgG4-vcMMAF (hIgG4-vcMMAF) were treated at varying concentrations (0.5, 1, 2, or 4 ⁇ g/ml).
  • Figure 18 shows the cell viability (%) of ADC1-1 and ADC1-2, anti-BCAM antibody-drug conjugates, and their respective negative controls (human IgG4-vcMMAF (hIgG4-vcMMAF) and human IgG4-vcMMAE (hIgG4-vcMMAE)) in BCAM-expressing cell lines DU145, KURAMOCHI, SNU-119, OVCAR3, T47D, VCaP, and EFM-192A.
  • Figure 19a shows the tumor growth inhibition effect in a DU145 human prostate cancer xenograft model, comparing hIgG4-vcMMAF (negative control) and ADC1-1 (anti-BCAM antibody-drug conjugate, experimental group) administered at 10 mg/kg.
  • Figure 19b shows the tumor growth inhibition effect in an OVCAR3 human ovarian cancer xenograft model, comparing PBS (negative control) and ADC1-1 (anti-BCAM antibody-drug conjugate, experimental group) administered at 10 mg/kg.
  • Figure 19c shows the tumor growth inhibition effect in a VCaP human prostate cancer xenograft model, comparing PBS (negative control) and ADC1-1 (anti-BCAM antibody-drug conjugate, experimental group) administered at 10 mg/kg.
  • Figure 19d shows the tumor growth inhibition effect in an RERF-GC-1b human gastric cancer xenograft model, comparing PBS (negative control) and ADC1-1 (anti-BCAM antibody-drug conjugate, experimental group) administered at 10 mg/kg.
  • Figure 20 shows the tumor growth profiles following ADC1-1 treatment in a xCAROV3 xenograft model.
  • the graph displays the tumor growth curves, represented by the mean tumor volume ( ⁇ SD), and the table shows tumor growth inhibition (TGI, %, vs. PBS group) and tumor regression (TR, %).
  • TGI tumor growth inhibition
  • % %
  • vs. PBS group tumor regression
  • Figure 21 shows the body weight change (%) following ADC1-1 treatment in a xCAROV3 xenograft model.
  • the graph shows the percentage of body weight change during ADC1-1.
  • the table represents the mean ( ⁇ SD) body weight change on Day 3, highlighting the maximum body weight loss observed on that day.
  • ADC1-1 did not induce significant body weight loss, with changes remaining within a 2 % range.
  • Figure 22 shows the experimental schedule of the study regarding the in vivo efficacy of ADC1-1 in a xCAOV3 xenograft model (Example 9)
  • BCAM 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. Lu was initially studied as an antigen of the Lutheran blood group system, and BCAM has been identified as an upregulated antigen in various cancers, including ovarian cancer, breast cancer, and prostate cancer. Lu and BCAM share the same extracellular domain, but their cytoplasmic tails differ. Specifically, BCAM lacks the C-terminal 40 amino acids that are present in the cytoplasmic tail of Lu.
  • the Lu-specific cytoplasmic region contains an SH3-binding motif, a dileucine motif, and potential phosphorylation sites.
  • the common region of the Lu and BCAM cytoplasmic tails includes a spectrin-binding motif. Due to the structural overlap between BCAM and Lu, it is difficult to distinguish between them in actual tissue samples. Therefore, in the present application, the terms "BCAM”, “Lu”, “Lu/BCAM”, and “CD239” refer to the same protein and may be used interchangeably.
  • the extracellular domain of BCAM comprises one V-set domain, one C1-set domain, and three I-set domains (V-C1-I-I-I).
  • antibody refers to an immunoglobulin molecule that specifically binds to a target ⁇ such as a carbohydrate, polynucleotide, lipid, polypeptide, or protein via at least one antigen-recognition site located within its variable region.
  • antibody is used in its broadest sense and thus encompasses, polyclonal antibodies, monoclonal antibodies, as well as dimers, multimers, and multispecific antibodies (e.g., bispecific antibodies), antigen-binding fragments thereof, antibody fragments, fusion proteins containing any modified arrangements of immunoglobulin molecules comprising antigen-recognition regions (e.g., variable regions), synthetic antibodies (e.g., "antibody mimetics”), "FynomAb” and related formats.
  • Immunoglobulin (Ig) M Immunoglobulin (Ig) M, IgD, IgG, IgA, and IgE, each containing a heavy chain derived from the constant region genes ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Each light chain and heavy chain of an antibody comprises a variable region, which has a unique amino acid sequence among different antibodies, and a constant region, which has a conserved amino acid sequence.
  • antibody variable region refers to the light chain portion and heavy chain portion of an antibody molecule that includes complementarity-determining regions (CDRs), and framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • CDR refers to amino acid residues in the variable region of an antibody that are essential for antigen binding.
  • Each variable region typically contains three CDRs, designated as CDR1, CDR2, and CDR3.
  • CDRs contain most of the residues responsible for the specific interaction between an antibody (or its antigen-binding fragment) and its target antigen. Thus, CDRs contribute significantly to the functional activity of the antibody, and they serve as the primary determinants of antigen specificity.
  • multispecific antibody refers to an antibody that has binding specificity for at least two different sites.
  • humanized antibody and CDR-grafted antibody refer to an antibody in which one or more CDR sequences derived from a non-human species, such as another mammalian species, are inserted into a framework sequence from a human immunoglobulin molecule.
  • the framework sequence may be further modified, for example, through mutation methods.
  • Human Ig sequences can be referenced from sources such as the NCBI Database (Entrez Gene).
  • the immunogenicity of the antibody may be reduced, or characteristics such as binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable feature may be reduced, enhanced, or altered.
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.
  • Methods for producing chimeric antibodies are well known in the art, and references such as Morrison, Science 229:1202 (1985) can be consulted, which is hereby incorporated in its entirety by reference.
  • human antibody refers to an antibody that includes a variable region in which both the framework and CDR regions are derived from human immunoglobulin sequences.
  • the constant region of the antibody is also derived from human immunoglobulin sequences.
  • Fab fragment refers to a monovalent fragment consisting of VL, VH, CL, and CH1 domains.
  • Fab' fragment differs from the Fab fragment in that it includes one or more cysteines from the antibody hinge region, with a few additional residues at the carboxyl terminus of the CH1 domain.
  • Fab'-SH refers to a Fab' fragment in which the cysteine residues in the constant domain retain a free thiol group.
  • F(ab') 2 antibody fragment refers to a dimer of two Fab' fragments linked through hinge cysteines.
  • Fv refers to the smallest antibody fragment containing a complete antigen-recognition and antigen-binding site, consisting of a tightly associated non-covalent dimer of a single heavy chain variable region and a single light chain variable region. These two regions fold together to form 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.
  • six hypervariable loops three loops from the heavy chain and three loops from the light chain
  • single-chain antibody refers to an antibody fragment comprising VH and VL antibody domains that are linked into a single polypeptide chain.
  • an scFv polypeptide further includes a polypeptide linker between the VH and VL domains to allow the scFv to form the desired structure for antigen binding.
  • scFv antibody fragments may also be referred to as "scFv antigen-binding fragments", “scFv antibodies”, “antibody scFv”, or simply "scFv”.
  • diabody refers to a small antibody fragment produced by constructing an scFv fragment using a short linker (approximately 5-10 residues) between the VH and VL domains, thereby allowing intrachain pairing of V domains instead of interchain pairing of V domains, resulting in a bivalent fragment with two antigen-binding sites.
  • a bispecific diabody is a heterodimer consisting of two "cross-linked" scFv fragments, where the VH and VL domains of two different antibodies are present on separate polypeptide chains.
  • a triabody and a tetrabody include three and four polypeptide chains, respectively, forming either identical or different antigen-binding sites in sets of three and four, respectively.
  • Fynomer refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain.
  • Fyn SH3-derived polypeptides are well known in the art and are described, for example, in Grabulovski et al. (2007) JBC, 282, p. 3196-3204, as well as in WO 2008/022759.
  • a Fynomer may be genetically fused with other molecules (e.g., antibodies) to generate "FynomAb", which can be engineered for bispecificity.
  • DART dual-affinity re-targeting
  • TRIDENT TRIDENT
  • prevention refers to any action that suppresses or delays the onset of a disease associated with the function or expression of BCAM.
  • treatment refers to any action that improves or beneficially alters the symptoms of a disease associated with the function or expression of BCAM.
  • Non-human animals include all vertebrates, such as mammals and non-mammals, for example, non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles.
  • the subject may be a mammal such as a non-human primate, sheep, dog, cat, cattle, or horse.
  • the subject may be a human.
  • alkyl refers to a fully saturated aliphatic hydrocarbon group that may be either branched or unbranched.
  • the alkyl group may have 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, or 1 to 6 carbon atoms.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, eicosyl, and tetracosyl, and the alkyl group may be substituted with C 1-30 linear alkyl or C 3-31 cycloalkyl.
  • alkenyl refers to an unsaturated branched or straight-chain hydrocarbon group containing at least one carbon-carbon double bond.
  • alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, and decenyl, but are not limited thereto.
  • alkynyl refers to an unsaturated branched or straight-chain hydrocarbon group containing at least one carbon-carbon triple bond.
  • alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl, but are not limited thereto.
  • cycloalkyl refers to a fully saturated cyclic hydrocarbon ring with no double or triple bonds. If composed of two or more rings, the rings may be fused, bridged, or spiro-connected.
  • the cycloalkyl group may have 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, or 3 to 7 carbon atoms.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and the cycloalkyl group may be substituted with C 1-30 linear alkyl or C 3-31 cycloalkyl.
  • aryl refers to a multi-unsaturated aromatic hydrocarbon group that may be a single ring or multiple rings, either fused or covalently linked. Examples of aryl groups include phenyl, naphthyl, and tetrahydronaphthyl, but are not limited thereto.
  • heterocyclyl refers to a ring structure containing at least one heteroatom (e.g., N, O, P, or S) and may be single-ring or multi-ring, which may include one or more double or triple bonds within the ring. If composed of two or more rings, the rings may be fused, bridged, or spiro-connected.
  • the heterocyclyl group may include heteroaryl, heterocycloalkenyl, or heterocycloalkynyl or fused-ring compounds containing these groups, but is not limited thereto.
  • esters refers to a group in which an organic radical replaces the hydrogen atom of an acid.
  • prodrug refers to a compound that can be converted into a pharmacologically active compound through enzymatic action, pH conditions, etc..
  • the anti-BCAM antibody or an antigen-binding fragment thereof may comprise: a heavy chain (VH) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a heavy chain (VH) CDR2 comprising the amino acid sequence of SEQ ID NO: 7; a heavy chain (VH) CDR3 comprising the amino acid sequence of SEQ ID NO: 8; a light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; a light chain (VL) CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and a light chain (VL) CDR3 comprising the amino acid sequence of SEQ ID NO: 11:
  • VH-CDR2 YTSSSGSTIYYADSVKG (SEQ ID NO: 7)
  • VL-CDR1 TGTSSDVGGYNYVS (SEQ ID NO: 9)
  • VL-CDR2 EVNKRPS (SEQ ID NO: 10)
  • the CDRs are designated according to the Kabat numbering system.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may comprise a heavy chain variable region including the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 1, and a light chain variable region including the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 95% sequence identity (e.g., at least 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 2.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may comprise a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 2, as follows:
  • VH Sequence (SEQ ID NO: 1): EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYTSSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDITAFDIWGQGTMVTVSS
  • VL Sequence (SEQ ID NO: 2): LPVLTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCCSYAGSNNYVFGTGTKVTVL
  • the anti-BCAM antibody may be a monoclonal antibody, polyclonal antibody, chimeric antibody, multispecific antibody (e.g., bispecific antibody), heterodimeric antibody, humanized antibody, or any other modified form of an immunoglobulin molecule containing an antigen recognition site with the required specificity, including glycosylation variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies.
  • the anti-BCAM antibody or an antigen-binding fragment thereof is a multispecific antibody molecule, such as a bispecific or trispecific antibody molecule.
  • a multispecific antibody molecule includes multiple variable regions, each of which has binding specificity for a different epitope.
  • the first variable region of a bispecific antibody molecule has first binding specificity for a first epitope, such as BCAM protein, while the second variable region has second binding specificity for a second epitope, such as a target protein other than BCAM protein.
  • a multispecific antibody molecule may bind to a protein that selectively binds to an antigen expressed specifically in cancer cells compared to normal cells, such as an antibody that binds to a tumor-specific antigen.
  • the tumor-specific antigen refers to an antigen that is known to be overexpressed specifically in cancer cells, including but not limited to ADAM9, ALCAM, ALPP, ANGPT2, AXL, CA9, CD22, CD269, CD27, CD274, CD276, CD30, CD33, CD44, CD46, CD70, CD79b, CDH3, CDH6, CEACAM5, CLDN18, CLDN6, CLDN9, CRIPTO, DLL3, DPEP3, EDNRB, EEF1A2, EGFR, EPCAM, EPHA2, EPHA4, EPHA5, ERBB2, ERBB3, F3, FAP, FGFR2, FGFR3, FN1, FOLH1, FOLR1, FUT3, GPC1, GPNMB, GPR20, GUCY1A2, HAVCR
  • any combination of the above molecules may be used to construct a multispecific antibody molecule, such as a trispecific antibody containing first binding specificity for BCAM and second and third binding specificities for two or more tumor-specific antigens.
  • the multispecific antibody molecules of the present invention may be produced using standard molecular biology techniques known to those skilled in the art, such as recombinant DNA and protein expression technologies.
  • the antigen-binding fragment of the anti-BCAM antibody may be Fab, Fab', Fab'-SH, Fv, single-chain antibody (scFv), F(ab') 2 fragment, VL, VH, diabody, triabody, tetrabody, minibody, IgG-deltaCH 2 , scFv-Fc, (scFv) 2 -Fc, Fynomer, dual-affinity re-targeting protein, anticalin, FN3-based monobody, DARPin, Affibody, Affilin, Affimer, Affitin, Alphabody, Avimer, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, nanobody, binanobody, or di-(di)-sdFv.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may specifically bind to BCAM protein, such as human BCAM protein.
  • BCAM protein such as human BCAM protein.
  • the human BCAM protein may be represented by the amino acid sequences of K7ENU8, K7ERB7, or A0A087WXM8 as provided in Uniprot (https:/www.uniprot.org/uniprotkb/P50895/entry#sequences), but is not limited thereto and includes any protein classified as BCAM that exhibits known BCAM functions.
  • the ability of the anti-BCAM antibody or an antigen-binding fragment thereof to bind to BCAM protein may be confirmed using Flow Cytometry, ELISA Assay, and/or Bio-Layer Interferometry (BLI).
  • the anti-BCAM antibody or an antigen-binding fragment thereof may bind to human BCAM protein with a binding dissociation equilibrium constant (K D ) of 1 ⁇ 10 -3 M, 1 ⁇ 10 -4 M, 1 ⁇ 10 -5 M, 1 ⁇ 10 -6 M, 1 ⁇ 10 -7 M, 1 ⁇ 10 -8 M, 1 ⁇ 10 -9 M, 1 ⁇ 10 -10 M, 9 ⁇ 10 -10 M, or 1 ⁇ 10 -11 M or lower.
  • K D binding dissociation equilibrium constant
  • K D value for an antibody can be measured using widely established industry methods. Representative methods for measuring K D values of antibodies include surface plasmon resonance (SPR), such as using a biosensor system like Biacore®, or Bio-Layer Interferometry (BLI), such as using the Octet® system. In a certain example, the K D values mentioned in this application may be obtained using SPR.
  • SPR surface plasmon resonance
  • BLI Bio-Layer Interferometry
  • the anti-BCAM antibody or an antigen-binding fragment thereof may bind to BCAM protein with an EC 50 of 150 nM or lower, for example, 130 nM or lower, 100 nM or lower, 50 nM or lower, 20 nM or lower, 10 nM, or 2 nM or lower, as measured by ELISA assay or Flow Cytometry.
  • EC 50 refers to the concentration of an antibody that induces 50% of the maximum response, meaning the midpoint response between the maximum reaction and the baseline, in an in vitro or in vivo assay using an antibody.
  • the anti-BCAM antibody exhibits tumor cell internalization activity.
  • the anti-BCAM antibody of the present invention may be internalized into BCAM-expressing cancer cells, thereby delivering a payload, cytotoxic agent, or therapeutic moiety.
  • nucleotides encoding the anti-BCAM antibody or an antigen-binding fragment thereof.
  • the nucleotides may be present in whole cells or cell lysates, or they may exist in a specifically purified or substantially pure form.
  • the nucleotides are "isolated” or “substantially purified and isolated” through purification methods that remove other cellular components or contaminants, such as other cellular nucleic acids or proteins, using standard techniques including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other well-known methods in the art.
  • the nucleotide may be DNA or RNA and may or may not contain intron sequences.
  • the nucleotide is a cDNA molecule.
  • the nucleotide encodes the light chain region, heavy chain region, or both the light and heavy chain regions of the anti-BCAM antibody or an antigen-binding fragment thereof.
  • the nucleotide may encode the light chain variable region, the heavy chain variable region, or both the light and heavy chain variable regions of the anti-BCAM antibody or an antigen-binding fragment thereof.
  • the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as the antibody constant region or a flexible linker.
  • operably linked means that two DNA fragments are joined such that the amino acid sequences encoded by both DNA fragments remain in-frame.
  • an isolated DNA encoding the VH region may be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the constant regions of the heavy chain (CH1, CH2, and CH3).
  • the heavy chain constant region may be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region.
  • the heavy chain constant region may be IgG4, specifically hIgG4(S228P) or hIgG1(LALA).
  • the VH-encoding DNA in the Fab fragment heavy chain gene, may be operably linked only to another DNA molecule encoding the CH1 constant region of the heavy chain.
  • VL- and VH-encoding DNA fragments may be operably linked to another fragment encoding a flexible linker, for example, an amino acid sequence (Gly4-Ser)3, so that the VL and VH sequences can be expressed as a continuous single-chain protein with the VL and VH regions which are connected by a flexible linker.
  • a flexible linker for example, an amino acid sequence (Gly4-Ser)3, so that the VL and VH sequences can be expressed as a continuous single-chain protein with the VL and VH regions which are connected by a flexible linker.
  • nucleic acid sequences of the present invention may be isolated from various sources, genetically engineered, amplified, and/or expressed through recombinant methods. Any recombinant expression system, including bacteria, insect cells, or mammalian systems, in addition to yeast, may be used.
  • nucleic acid manipulations such as subcloning into an expression vector, probe labeling, sequencing, and hybridization, may be performed using methods known in the art.
  • the invention provides a recombinant expression vector encoding the anti-BCAM antibody or an antigen-binding fragment thereof, or a recombinant expression vector containing the nucleic acid.
  • vector refers to a recombinant vector, cloning vector, or expression vector, which is a self-replicating DNA molecule in prokaryotic and/or eukaryotic cells. It is generally used as an intermediate carrier for introducing genes or DNA fragments into cells.
  • a vector typically includes a replication origin capable of replication in prokaryotic and/or eukaryotic cells, a selectable marker gene that can confer resistance to specific conditions/substances such as antibiotic-degrading enzymes (an antibiotic resistance gene), a promoter that allows transcription of the gene in eukaryotic or protist cells, and a translatable sequences, but is not limited thereto.
  • vector refers to a circular double-stranded DNA loop that allows the insertion of additional DNA fragments.
  • viral vector refers to a viral genome.
  • Certain vectors can replicate autonomously within the host cell into which they are introduced, such as bacterial vectors containing bacterial origins of replication or episomal mammalian vectors.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may be produced by any suitable method known in the art.
  • the anti-BCAM antibody may be produced using well-established techniques, such as hybridoma technology, recombinant techniques, phage display, transfection, synthetic methods, combinations thereof, or other methods readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45:1628-50 (1999) and Fellouse, F.A., et al., J. Mol. Biol., 373(4):924-40 (2007)).
  • the anti-BCAM antibody may be produced using phage display technology. Specifically, genes encoding the variable regions of human antibody heavy and light chains may be cloned into a phagemid vector, where they are fused to a phage surface protein (pIII) and expressed in Escherichia coli. Infection with an M13 helper phage then allows the generation of an antibody library, in which scFv or Fab antibody fragments with various heavy and light chain variable region sequences are displayed on the surface of the phage.
  • pIII phage surface protein
  • Panning techniques with the library may then be used to isolate antibody fragments that specifically bind to a target antigen, and after characterizing the isolated antibody fragments, they may be converted into whole IgG form and expressed in mammalian cells for large-scale production of specific human monoclonal antibodies.
  • a naive antibody library which utilizes antibody genes already present in the human body, may be used as the phage display library.
  • a synthetic antibody library in which randomly synthesized sequences are inserted into antibody CDR regions to enhance diversity, may be used.
  • a phagemid vector is a plasmid DNA containing a phage origin of replication, typically including an antibiotic resistance gene as a selection marker.
  • Phagemid vectors used in phage display generally include gene III (gIII) or a portion thereof from the M13 phage, and scFv genes are ligated to the 5' terminus of gIII to allow expression in transformed host cells.
  • a helper phage provides the necessary genetic information for phagemid assembly into phage particles. Since the phagemid vector contains the phage gene III or only a part thereof, the transformed host cells must be infected with a helper phage to supply the remaining phage genes.
  • Various phagemids, such as M13K07 or VCSM13 exist and typically include kanamycin resistance genes to allow selection of helper phage-infected transformants. Furthermore, since phagemids have defective packaging signals, the phagemid genome is preferentially assembled into phage particles over the helper phage genome.
  • the anti-BCAM antibody may be produced using hybridoma technology. Specifically, after injecting a test subject (e.g., a mouse) with the BCAM antigen, hybridomas expressing antibodies with the desired sequence or functional properties may be isolated, thereby producing the antibody in the form of a monoclonal antibody.
  • a test subject e.g., a mouse
  • hybridomas expressing antibodies with the desired sequence or functional properties may be isolated, thereby producing the antibody in the form of a monoclonal antibody.
  • 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 conventional methods, for example, using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of monoclonal antibodies.
  • Hybridoma cells may serve as a primary source of these nucleotide sequences. Once the nucleotide sequence is isolated, it may be inserted into an expression vector, followed by transfection into a host cell such as Escherichia coli (E.
  • coli simian COS cells
  • Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulin proteins, thereby enabling the recombinant host cell to synthesize the monoclonal antibody.
  • the invention provides an isolated host cell capable of recombinantly producing the anti-BCAM antibody or an antigen-binding fragment thereof.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may be expressed by introducing the nucleic acid into any recombinant expression system, including bacterial, yeast, insect, or mammalian systems.
  • the recombinant expression system may include the isolated host cell.
  • Bacterial cells may be Gram-positive or Gram-negative bacteria, for example, species of Escherichia, such as cells derived from E. coli or Pseudomonas.
  • yeast cells may be used, and yeast expression may be carried out using yeast strains such as Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha.
  • insect cells for example, Drosophila and Sf9-derived cells, may be used as host cells.
  • the expression system may include an isolated mammalian host cell, such as CHO cells, DG44 or DUXB11 cells, NS0 myeloma cells, monkey kidney cell lines (e.g., CV1 cells, COS cells), SP2 cells, human embryonic kidney (HEK) cells, Chinese hamster fibroblasts, human cervical carcinoma cells (e.g., HeLa), BHK cells, NS0 cells, Bowes melanoma cells, murine fibroblasts (e.g., BALB/c 3T3), murine myeloma cells (e.g., P3X63-Ag3.653, NS0, SP2/O), hamster kidney cells (e.g., HAK), murine L cells (e.g., L-929), human lymphocytes (e.g., RAJI), and human kidney cells (e.g., 293 and 293T), but is not limited thereto.
  • CHO cells such as CHO cells, DG44 or DUXB11
  • the host cell may be a human cell, such as HeLa, 911, AT1080, A549, 293, and HEK293 cells, and in a specific example, Expi293F cells.
  • the invention provides a method for producing the antibody or an antigen-binding fragment thereof, comprising culturing the host cell under conditions that allow production of the antibody or antigen-binding fragment thereof and isolating the antibody or antigen-binding fragment thereof from the host cell or the culture medium.
  • the host cells when the recombinant expression vector is introduced into a recombinant expression system, the host cells are cultured under conditions that allow sufficient expression of the antibody within the host cell or secretion of the antibody or antigen-binding fragment into the culture medium in which the host cells grow.
  • the antibody or antigen-binding fragment thereof may then be recovered using standard protein purification methods.
  • the recombinant expression vector may be transfected into the host cell using standard techniques, and known methods for introducing exogenous nucleic acids into cells, such as electroporation, calcium-phosphate precipitation, and DEAE-dextran transfection, may be used.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may be conjugated to a therapeutic moiety.
  • the aforementioned anti-BCAM antibody or an antigen-binding fragment thereof may be linked to a therapeutic moiety via a linker, forming an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the therapeutic moiety, linker, and conjugation type and method may be the same as those used in the antibody-drug conjugate represented by Formula I, but are not limited thereto.
  • the invention provides an antibody-drug conjugate (ADC) represented by Formula I:
  • M is an anti-BCAM antibody or an antigen-binding fragment thereof that binds to BCAM protein
  • L is a linker
  • D is a therapeutic moiety
  • n is the average number of L-D structures conjugated per antibody, ranging from 1 to 20.
  • the anti-BCAM antibody or an antigen-binding fragment thereof included in the antibody-drug conjugate of the present invention is an anti-BCAM antibody or an antigen-binding fragment thereof that specifically binds to BCAM protein.
  • it may be an antibody or an antigen-binding fragment thereof comprising: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 6; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 7; VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 9; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may be an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 1; and a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 2.
  • VH heavy chain variable region
  • VL light chain variable region
  • a reducing agent may be used for this purpose, and tris(2-carboxyethyl)phosphine (TCEP) may be used as the reducing agent.
  • TCEP tris(2-carboxyethyl)phosphine
  • the anti-BCAM antibody or an antigen-binding fragment thereof may comprise an engineered amino acid residue at one or more selected positions to facilitate or inhibit the binding of the linker.
  • the anti-BCAM antibody or an antigen-binding fragment thereof may comprise an engineered amino acid residue at the carboxyl terminus of the light chain or heavy chain to facilitate or inhibit the binding of the linker.
  • the engineered amino acid residue may be selected from a group consisting of alanine, cysteine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, and arginine.
  • the linker (L) is a substance that can connect an antibody to a therapeutic moiety using any known linker or linker technology.
  • the linker (L) may be a cleavable linker, a non-cleavable linker, or a combination thereof.
  • the linker (L) may be a cleavable linker comprising a cleavable peptide moiety.
  • the cleavable peptide moiety may be cleaved by enzymes.
  • the cleavable peptide moiety may be recognized and cleaved by lysosomal proteolytic (internalized) enzymes, such as Cathepsin B or beta-glucuronidase.
  • the linker (L) may comprise natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- ⁇ -amino acids. Additionally, the linker (L) may include amino acid units.
  • the amino acid unit may include alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, their derivatives, or combinations thereof.
  • the amino acid unit may be selected from a group consisting of valine-citrulline (Val-Cit), alanine-alanine (Ala-Ala), alanine-citrulline (Ala-Cit), citrulline-asparagine (Cit-Asn), valine-glutamic acid (Val-Glu), citrulline-aspartic acid (Cit-Asp), alanine-valine (Ala-Val), valine-alanine (Val-Ala), phenylalanine-lysine (Phe-Lys), valine-lysine (Val-Lys), alanine-lysine (Ala-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), phenylalanine-arginine (Phe-Arg), alanine-alanine-aspart
  • the linker (L) may be a moiety comprising a maleic acid derivative.
  • the maleic acid derivative may be maleic acid, maleimide, maleamic acid, or maleamic ester, which has an ⁇ , ⁇ -unsaturated carbonyl group in its molecular structure.
  • the moiety comprising a maleic acid derivative may include a maleimide moiety, a maleamic acid moiety, or a maleamic ester moiety.
  • the maleimide moiety may be maleimidocaproyl (MC), maleimidopropanoyl (MP), or maleimidobutanoyl (MB).
  • the linker may include one or more alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl groups, and any carbon in the alkyl, alkenyl, alkynyl, or cycloalkyl group may be substituted with one or more of carbonyl, amide, oxygen, sulfur, -S(O) 2 -, -NH-, or nitrogen.
  • the linker may include a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • the linker (L) may include a structure represented by -(CH 2 CH 2 O)m-, where m is an integer from 1 to 24.
  • the linker (L) may include a moiety represented by Formula II:
  • a is an integer from 0 to 24, b is an integer of 0 or 1
  • X is selected from a group consisting of alkyl, alkyl-cycloalkyl, and cycloalkyl, wherein the alkyl, alkyl-cycloalkyl, and cycloalkyl groups may be substituted or unsubstituted with C 1-30 alkyl or C 3-31 cycloalkyl
  • Mal is a moiety comprising a maleic acid derivative.
  • X may be selected from a group consisting of C 1-6 alkyl, C 1-6 alkyl-C 3-7 cycloalkyl, and C 3-7 cycloalkyl, wherein these groups may be further substituted with C 1-30 linear alkyl or C 3-31 cycloalkyl.
  • the linker may include a self-immolative moiety.
  • the self-immolative moiety may be selected from a group consisting of p-aminobenzyl (PAB), p-aminobenzyl oxycarbonyl (PABC), aminomethylene, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).
  • PAB p-aminobenzyl
  • PABC p-aminobenzyl oxycarbonyl
  • SPP N-succinimidyl 4-(2-pyridyldithio)pentanoate
  • SCC N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • SIAB N-
  • the self-immolative moiety may be PABC or aminomethylene.
  • the linker may include a sugar or sugar acid, wherein the sugar or sugar acid is linked via a bond susceptible to enzymatic cleavage in vivo.
  • the moiety comprising a maleic acid derivative may be attached to the anti-BCAM antibody or an antigen-binding fragment thereof via a cysteine residue on the antibody or antigen-binding fragment thereof.
  • the linker may include one or more combinations selected from the following group: a combination of maleimidocaproyl, valine-citrulline (Val-Cit), and PABC; a combination of maleimidopropanoyl, polyethylene glycol (PEG), and valine-alanine (Val-Ala); a combination of maleimidopropanoyl, PEG, Val-Ala, and PABC; a combination of maleimidopropanamide, PEG, carbonyl, and Val-Ala; a combination of maleimidopropanamide, PEG, carbonyl, Val-Ala, and PABC; a combination of maleimidopropanamide, PEG, amide, and Val-Ala; a combination of maleimidopropanamide, PEG, amide, and Val-Ala; a combination of maleimidopropanamide, PEG, amide, Val-Ala, and PABC; a combination of maleimidopropanamide, alkyl-carbonyl, and
  • the therapeutic moiety is a substituent or structure that can bind to the linker (L), and as long as it does not interfere with the BCAM protein-specific binding of the anti-BCAM antibody, it may be appropriately selected based on therapeutic or diagnostic applications without limitation.
  • the therapeutic moiety included in the antibody-drug conjugate (ADC) of the present invention may function as a substance that increases the in vivo half-life of the antibody, enhances the in vivo function or effect of the antibody, enables the detection of the antibody, or serves as a therapeutic agent.
  • the therapeutic moiety may include cytotoxic agents (e.g., chemotherapeutic agents), therapeutic proteins, biocompatible polymers, oligonucleotides, immunomodulators, imaging agents, radionuclides, anti-tumor agents, agents for blood disorders, agents for autoimmune diseases, anti-inflammatory agents, antibacterial agents, antifungal agents, antiparasitic agents, antiviral agents, or anesthetic agents. Additionally, the therapeutic moiety may be released into its original form upon cleavage from the linker (L), thereby exerting its intended effect (e.g., anti-cancer effects, anti-inflammatory effects, etc.).
  • the therapeutic moiety may be a cytotoxic agent.
  • the cytotoxic agent may be selected from the group consisting of anthracyclines, camptothecin or camptothecin analogs (e.g., irinotecan, rubitecan, topotecan, gimatecan, pegamotecan, lutotecan, karenitecan, apellatecan, homocamptothecin, diflomotecan, belotecan, 9-aminocamptothecin, exatecan, SN-38, Dxd derivatives), combretastatin, dolastatin, enediynes, geldanamycin, indolino-benzodiazepine dimers, maytansine, puromycin, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterlin, spliceostatin, pladienolides, calicheamic
  • the therapeutic moiety may be a prodrug of a cytotoxic agent.
  • the cytotoxic prodrug may be converted into a pharmacologically active cytotoxic agent under physiological conditions in vivo through enzymatic action or pH changes.
  • the cytotoxic agent may be a DNA-damaging agent or a tubulin inhibitor that inhibits tubulin polymerization, but is not limited thereto.
  • the DNA-damaging agent includes topoisomerase inhibitors and DNA alkylating agents, while the tubulin inhibitor includes auristatins or maytansinoids.
  • the DNA alkylating agent refers to a drug that disrupts DNA cross-linking and may include pyrrolobenzodiazepine (PBD) dimers, pyrrolobenzodiazepine (PBD) dimer analogs, calicheamicins, calicheamicin analogs, duocarmycins or duocarmycin analogs, cyclophosphamide, ifosfamide, bendamustine, cisplatin, melphalan, and carboplatin, etc.
  • PBD pyrrolobenzodiazepine
  • PBD pyrrolobenzodiazepine
  • the tubulin inhibitor may be an auristatin or a maytansinoid.
  • the therapeutic moiety may be a tubulin polymerase inhibitor.
  • the tubulin polymerase inhibitor may be MMAF or MMAE.
  • the cytotoxic agent may be a DNA-damaging agent, and the DNA-damaging agent may be a topoisomerase inhibitor.
  • the therapeutic moiety may be a topoisomerase inhibitor.
  • the topoisomerase inhibitor may be a camptothecin (CPT) analogue.
  • the camptothecin analogue may include irinotecan, rubitecan, topotecan, gimatecan, pegamotecan, lutotecan, karenitecan, apellatecan, homocamptothecin, diflomotecan, belotecan, 9-aminocamptothecin, exatecan, SN-38, Dxd derivatives, and exatecan analogues.
  • the camptothecin analogue may be exatecan, an exatecan analogue, SN-38, or a Dxd derivative.
  • the therapeutic moiety may be a therapeutic protein, such as but not limited to toxins, hormones, enzymes, and growth factors.
  • the toxin protein (or polypeptide) may include, but is not limited to, diphtheria toxin (e.g., diphtheria A chain), Pseudomonas exotoxin and endotoxin, lysin (e.g., lysin A chain), abrin (e.g., abrin A chain), modecin (e.g., modecin A chain), alpha-sarcin, Aleurites fordii protein, dianthin protein, ribonuclease (RNase), DNase I, staphylococcus enterotoxin-A, Phytolacca americana antiviral protein, gelonin, diphtherin toxin, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitors, curcin
  • the therapeutic moiety may be a biocompatible polymer that increases and/or prolongs the serum half-life and bioactivity of the antibody-drug conjugate.
  • the biocompatible polymer may be selected from a group consisting of water-soluble polymers or derivatives thereof and zwitterionic biocompatible polymers, but is not limited thereto.
  • An example of a water-soluble polymer is polyethylene glycol (PEG), while an example of a zwitterionic biocompatible polymer is a phosphorylcholine-containing polymer.
  • the therapeutic moiety may be an immunomodulator.
  • the immunomodulator may be used to regulate immune cell activity in the vicinity of the antibody-drug conjugate (ADC).
  • the immunomodulator may include, but is not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factors (TNF), hematopoietic factors, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage,
  • the therapeutic moiety may additionally include an imaging agent.
  • the imaging agent may be a substance that enables the visualization of the antibody-drug conjugate (ADC) in a subject or a biological sample of a subject.
  • the imaging agent may be a fluorescent substance.
  • the imaging agent may include fluorescein, rhodamine, lanthanide phosphate and its derivatives, or a radioactive isotope bound to a chelator.
  • the fluorescent substance may be selected from a group consisting of fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosin, Alexa FluorTM dyes (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101), but is not limited thereto.
  • FITC fluorescein isothiocyanate
  • FAM fluorescein amidite
  • eosin carboxyfluorescein,
  • the chelator may be selected from a group consisting of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), but is not limited thereto.
  • DOTA 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid
  • NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • the therapeutic moiety may be a radionuclide or include a radionuclide, which may be selected from a group consisting of 225 Ac, 212 Bi, 213 Bi, 131 I, 186 Re, 227 Th, 222 Rn, 223 Ra, 224 Ra, and 90 Y, but is not limited thereto.
  • the antibody-drug conjugate may be labeled with a detectable or functional marker.
  • the detectable label may include, but is not limited to, a radioactive label (e.g., isotopes such as 2H, 3H, 14C, etc.), a fluorescent label, a label commonly used in MRI-CT imaging technologies, or a chemical label such as biotin.
  • the functional label may include a substance designed to target tumor sites and induce tumor tissue destruction. Examples of functional labels may include cytotoxic drugs (e.g., 5-fluorouracil, ricin) or enzymes (e.g., bacterial carboxypeptidase or nitroreductase), but are not limited thereto.
  • n is synonymous with DAR (Drug-to-Antibody Ratio), which represents the number of therapeutic moieties (e.g., drugs) conjugated per antibody.
  • DAR Drug-to-Antibody Ratio
  • n is a key factor affecting the efficacy and safety of an ADC. While the manufacturing process of ADCs aims to ensure a specific binding number of therapeutic moieties conjugated per antibody by controlling reaction conditions such as reagent amounts, ADC production differs from small-molecule chemical reactions and typically results in a mixture of ADCs with different drug conjugation levels. The binding number of therapeutic moieties conjugated per antibody is specified and reported as an average value. Unless otherwise stated in the present invention, n represents the average DAR of ADCs in a ADCs mixture with varying numbers of drug conjugations.
  • the DAR (n) of the ADC may be between 1 and 20, 1 and 10, 2 and 8, or may be 8 or lower, 7 or lower, 6 or lower, or 5 or lower.
  • the DAR (n) of the ADC may be 3 or higher or 4 or higher. Furthermore, in another embodiment, the DAR (n) of the ADC may be between 3 and 8.
  • the binding ability of the antibody-drug conjugate (ADC) to BCAM protein may be confirmed using Flow Cytometry, ELISA Assay, and/or Bio-Layer Interferometry (BLI).
  • the antibody-drug conjugate may exhibit an EC 50 or equilibrium dissociation constant (K D ) similar to that of the unconjugated anti-BCAM antibody.
  • the antibody-drug conjugate when measured by ELISA assay or Flow Cytometry, may bind to BCAM protein with an EC 50 of 150 nM or lower, for example, 130 nM or lower, 100 nM or lower, 50 nM or lower, 20 nM or lower, 10 nM, or 1 nM or lower.
  • the antibody-drug conjugate may bind to human BCAM protein with an equilibrium dissociation constant (K D ) of 1 ⁇ 10 -3 M, 1 ⁇ 10 -4 M, 1 ⁇ 10 -5 M, 1 ⁇ 10 -6 M, 1 ⁇ 10 -7 M, 1 ⁇ 10 -8 M, 1 ⁇ 10 -9 M, 1 ⁇ 10 -10 M, or 1 ⁇ 10 -11 M or lower.
  • K D equilibrium dissociation constant
  • the antibody-drug conjugate exhibits tumor cell internalization activity.
  • the antibody-drug conjugate of the present invention may exert cytotoxic or cytostatic effects on BCAM-expressing cancer cells.
  • the antibody-drug conjugate is internalized and accumulated within BCAM-expressing cells and may exert therapeutic effects such as cytotoxicity, cytostasis, or immunosuppression in BCAM-expressing cells.
  • the antibody or antigen-binding fragment used to produce the antibody-drug conjugate of the present invention is not particularly limited as long as it is capable of specifically binding to BCAM protein.
  • the therapeutic moiety, linker, and antibody-drug conjugate may be produced using known techniques.
  • the therapeutic moiety may be conjugated or linked to the anti-BCAM antibody or an antigen-binding fragment thereof to produce a conjugate.
  • the antibody-drug conjugate (ADC) may be prepared through the following steps:
  • the reducing agent tris(2-carboxyethyl)phosphine (TCEP) is added to the anti-BCAM antibody.
  • the reaction is performed at 25°C for 2 hours, allowing partial reduction of the antibody’s disulfide bonds.
  • the compound obtained in Step 1 is dissolved in DMSO.
  • the antibody of which disulfide bond is partially reduced is mixed with 15 equivalents of the compound from Step 1 dissolved in DMSO.
  • the antibody concentration is maintained at 10.0 mg/mL, and the reaction proceeds in a 25°C chamber for 3 hours.
  • the mixture is passed through a desalting column, followed by buffer exchange with 50 mM sodium phosphate buffer (pH 6.5) containing 400 mM sodium chloride to remove unreacted Compound 1 or Compound 2.
  • the desalting column eluate is concentrated using a 50 kDa cut-off PES membrane centrifugal filter unit, which further removes residual unreacted Compound 1 or Compound 2.
  • the purification of the antibody-drug conjugate may be performed using known methods to achieve an optimized Drug-to-Antibody Ratio (DAR) with a specific number. These methods include Batch purification, Cyclic processing, Flow-through processing, Separation using hydrophobic resins, etc.
  • the antibody and/or antibody-drug conjugate (ADC) of the present invention can be usefully applied to various purposes, including but not limited to therapeutic and/or diagnostic methods.
  • the antibody or an antigen-binding fragment thereof and/or the antibody-drug conjugate may specifically bind to BCAM protein, thereby being used for the prevention, amelioration, and/or treatment of diseases associated with the function or expression of BCAM.
  • the disease associated with the function or expression of BCAM may be cancer.
  • the anti-BCAM antibody or an antigen-binding fragment thereof, or the antibody-drug conjugate can effectively bind to BCAM-expressing cancer cells, thereby inhibiting tumor growth in vivo. Consequently, it can be usefully applied in the prevention, amelioration, or treatment of cancer.
  • BCAM is known to be involved in tumor cell migration, and its overexpression has been reported particularly in skin cancer, ovarian cancer, pancreatic cancer, and breast cancer (F.R.M. Latini et al., Blood Cells, Molecules and Diseases 50 (2013) 161-165).
  • the anti-BCAM antibody of the present invention may be used for any cancer exhibiting tumor migration, such as metastatic cancer, and it may also be applied to specific cancer types where BCAM overexpression is observed.
  • the anti-BCAM antibody, antigen-binding fragment thereof, or antibody-drug conjugate (ADC) of the present invention may be used to inhibit the growth of various cancers, including but not limited to: (1) Leukemia (e.g., acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) such as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, myelodysplastic syndrome (MDS), preleukemia, and chronic myelomonocytic leukemia (CMML)); (2) Lymphoma (e.g., T-cell lymphoma, lymphoblastic lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma); (3) Multiple myeloma; (4) Bone and connective tissue sarcomas (e.g., osteosarcom
  • the ADC of the present invention may be applied to other cancers not limited to those listed above, including environmentally induced cancers (e.g., asbestos-induced cancer) (see Fishman et al., 1985, Medicine, 2nd Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).
  • the tumors may be metastatic, unresectable, or locally advanced.
  • the disease associated with the function or expression of BCAM protein may be a solid tumor.
  • the disease associated with the function or expression of BCAM protein may be a cancer that is unresponsive or resistant to conventional cancer therapies, such as immune checkpoint inhibitors, chemotherapy, or radiation therapy.
  • the invention provides a method for preventing, ameliorating, or treating diseases associated with the function or expression of BCAM, such as cancer, comprising administering an effective amount of an anti-BCAM antibody, an antigen-binding fragment thereof, and/or an anti-BCAM antibody-drug conjugate to a subject.
  • the invention provides the use of an anti-BCAM antibody, an antigen-binding fragment thereof, and/or an anti-BCAM antibody-drug conjugate for preventing, ameliorating, or treating diseases associated with the function or expression of BCAM, such as cancer.
  • the invention provides a pharmaceutical composition for preventing, ameliorating, or treating diseases associated with the function or expression of BCAM, such as cancer, comprising an anti-BCAM antibody, an antigen-binding fragment thereof, and/or an anti-BCAM antibody-drug conjugate.
  • BCAM antibody, antigen-binding fragment thereof, and/or antibody-drug conjugate may be included in the pharmaceutical composition in a therapeutically effective amount.
  • the pharmaceutical composition may include inactive ingredients, i.e., pharmaceutically acceptable excipients (see, e.g., Handbook of Pharmaceutical Excipients).
  • the pharmaceutical composition may be prepared as a lyophilized powder, slurry, aqueous solution, or suspension, by mixing with a physiologically acceptable carrier, excipient, or stabilizer.
  • the pharmaceutical composition of the present invention may be administered via parenteral routes, for example, intramuscular, intravenous, or subcutaneous injection.
  • parenteral routes for example, intramuscular, intravenous, or subcutaneous injection.
  • the administration of the pharmaceutical composition or the method of the present invention may be carried out using various conventional methods, including topical application, skin administration, subcutaneous administration, intraperitoneal administration, parenteral administration, intra-arterial injection, or intravenous injection.
  • the anti-BCAM antibody, antigen-binding fragment thereof, and/or antibody-drug conjugate (ADC) of the present invention may be administered intravenously or subcutaneously.
  • the anti-BCAM antibody, antigen-binding fragment thereof, or ADC of the present invention may be used alone or in combination with other therapeutic agents, such as other cancer treatments.
  • Other cancer treatments may include, for example, standard cancer therapies (e.g., chemotherapy, radiation therapy, or surgery) or other anticancer agents, such as cytotoxic agents, cytostatic agents, antihormonal agents, anti-angiogenesis agents or antimetabolites, targeted cancer therapies, immune stimulators or immunomodulators, immune checkpoint inhibitors, antibody-drug conjugates (ADCs) conjugated with cytotoxic agents, cytostatic agents, or other toxic compounds
  • the anti-BCAM antibody, antigen-binding fragment thereof, and/or ADC of the present invention may be used in combination therapy with other anticancer agents, such as immune checkpoint regulators, chemotherapeutic agents, or radiation therapy.
  • the chemotherapeutic agents may include, but are not limited to, alkylating agents, antimetabolites, kinase inhibitors, spindle toxin plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs), anti-progestins, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone (LHRH) agonists, anti-androgens, aromatase inhibitors, epidermal growth factor receptor (EGFR) inhibitors, vascular endothelial growth factor (VEGF) inhibitors, antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or
  • chemotherapeutic agents include Gemcitabine, Vinorelbine, Etoposide (VP-16), Platinum-based agents, such as cisplatin or carboplatin, Taxoids, such as paclitaxel, albumin-bound paclitaxel, or docetaxel.
  • the anti-BCAM antibody, an antigen-binding fragment thereof, and/or an antibody-drug conjugate (ADC) of the present invention may be used for patients who do not respond to other immune checkpoint inhibitor treatments.
  • ADC antibody-drug conjugate
  • the anti-BCAM antibody, an antigen-binding fragment thereof, or an ADC When used in combination with other anticancer agents, they may be administered separately or as a combination product in which multiple active ingredients are contained in a single pharmaceutical formulation. If administered as separate formulations, the two formulations may be administered sequentially or simultaneously. In simultaneous administration, both are delivered together to the subject, whereas in sequential administration, they may be administered with a time interval, such as within 12 hours or 6 hours, or 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.
  • the pharmaceutical composition described above may additionally include an anticancer agent.
  • the anticancer agent may be administered together with the antibody, antigen-binding fragment, or ADC in a single formulation, or may be administered separately, either simultaneously or sequentially, with the type and administration method of the anticancer agent being as described above.
  • the present invention provides a method for preventing, ameliorating, or treating cancer, comprising administering an effective amount of an anti-BCAM antibody, an antigen-binding fragment thereof, or an ADC in combination with an additional anticancer agent to a subject.
  • This embodiment includes administering tThe anti-BCAM antibody or antigen-binding fragment together with an additional anticancer agent in a single composition for simultaneous administration, or administering each component separately in different compositions, which are administered either simultaneously or sequentially to a subject in need thereof.
  • the route of administration may be parenteral, including intramuscular, intravenous, or subcutaneous administration.
  • the additional anticancer agent may be an immune checkpoint inhibitor, such as anti-CTLA-4 antibody (e.g., ipilimumab), anti-PD-1 antibody (e.g., pembrolizumab, nivolumab), anti-PD-L1 antibody (e.g., atezolizumab, avelumab, durvalumab), other additional anticancer agents may include chemotherapeutic agents, such as Gemcitabine, Vinorelbine, Etoposide (VP-16), Platinum-based agents (e.g., cisplatin or carboplatin), Taxoids (e.g., paclitaxel, albumin-bound paclitaxel, or docetaxel).
  • anti-CTLA-4 antibody e.g., ipilimumab
  • anti-PD-1 antibody e.g., pembrolizumab, nivolumab
  • anti-PD-L1 antibody e.g., atezolizumab,
  • the invention provides the use of an anti-BCAM antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate (ADC) in combination with an additional anticancer agent for the prevention, amelioration, or treatment of cancer.
  • ADC antibody-drug conjugate
  • the invention provides a pharmaceutical composition or combination for preventing, ameliorating, or treating cancer, comprising an anti-BCAM antibody, an antigen-binding fragment thereof, or an ADC, and an additional anticancer agent.
  • the pharmaceutical composition or combination described in this invention includes cases where the two components are physically combined into a single formulation, or the two components are separately formulated and administered simultaneously or sequentially.
  • the two drugs may be provided separately or as part of a single kit.
  • the present invention provides a kit for preventing, ameliorating, or treating cancer, comprising: an anti-BCAM antibody, an antigen-binding fragment thereof, or an ADC and an additional anticancer agent.
  • the invention provides the use of an anti-BCAM antibody, an antigen-binding fragment thereof, or an ADC in the manufacture of a medicament for the treatment of diseases associated with the function or expression of BCAM.
  • the antibody of the present invention was produced using the phage display method.
  • the mRNA of the variable regions of the heavy and light chains of antibodies from human blood or bone marrow was amplified via PCR, followed by cDNA synthesis.
  • the resulting cDNA was cloned into a phagemid vector using restriction enzymes and expressed in E. coli via electroporation.
  • the expressed cDNA was then infected with a helper phage, generating a human scFv antibody library.
  • Biopanning was performed on the library to screen antibodies that bind with high affinity to the target antigen, BCAM. Positive clones binding to BCAM protein were selected, and sequencing was performed to identify BCAM-specific scFv sequences.
  • Two anti-BCAM antibodies (Ab1 and Ab2) were produced, each comprising a heavy chain variable region corresponding to SEQ ID NO: 1, and a light chain variable region corresponding to SEQ ID NO: 2.
  • the constant region of the heavy chain of Ab1 is derived from IgG4 with an S228P mutation (SEQ ID NO: 3), while the constant region of the heavy chain of Ab2 is derived from human IgG1 with an LALA mutation (SEQ ID NO: 4).
  • the constant region of the light chain of both Ab1 and Ab2 corresponds to SEQ ID NO: 5.
  • amino acid sequences of SEQ ID NO: 1 to 5 are listed in Table 1, and the CDR amino acid sequences of the variable regions of Ab1 and Ab2 are provided in Table 2.
  • Region Sequence SEQ ID NO: 1 Heavy Chain Variable Region (VH) EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYTSSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDITAFDIWGQGTMVTVSS SEQ ID NO: 2 Light Chain Variable Region (VL) LPVLTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVNKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCCSYAGSNNYVFGTGTKVTVL SEQ ID NO: 3 Human Mutant Heavy Chain Constant Region (CH-hIgG4(S228P) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR
  • the heavy chain variable region protein corresponding to SEQ ID NO: 1 was fused with the human mutant IgG4 heavy chain constant region protein corresponding to SEQ ID NO: 3.
  • the light chain variable region protein corresponding to SEQ ID NO: 2 was fused with the human light chain constant region protein corresponding to SEQ ID NO: 5.
  • the DNA sequences encoding the fused amino acid sequences were codon-optimized, and the optimized codon sequences were synthesized and cloned into the pcDNA3.4 expression vector. Using this vector, transient transfection was performed in ExpiCHO-S cells, followed by antibody expression for 8 days.
  • the expressed antibody underwent two-step purification, first purification: Affinity chromatography using Protein A, and second purification: Ion exchange chromatography using a cation exchange resin. Specifically, the antibody was bound to an affinity chromatography resin equilibrated with a 20 mM Tris-acetate buffer (pH 7.4) containing 150 mM sodium chloride, followed by washing with a 20 mM Tris-acetate buffer (pH 7.4) containing 500 mM sodium chloride and a 25 mM sodium acetate buffer (pH 5.0). The target protein was then eluted using a 25 mM sodium acetate buffer (pH 3.6) at low pH.
  • affinity chromatography resin equilibrated with a 20 mM Tris-acetate buffer (pH 7.4) containing 150 mM sodium chloride, followed by washing with a 20 mM Tris-acetate buffer (pH 7.4) containing 500 mM sodium chloride and a 25 mM sodium acetate buffer (
  • the eluted protein was titrated to pH 5.0 using 1 M sodium acetate and subjected to a second cation exchange chromatography. Specifically, the affinity chromatography eluate titrated to pH 5.0 was bound to a cation exchange chromatography resin equilibrated with a 50 mM sodium acetate buffer (pH 5.0), washed with a 50 mM sodium acetate buffer (pH 5.0), and then eluted using a 50 mM sodium acetate buffer (pH 5.0) containing 1 M sodium chloride with a 30% linear gradient. The final Ab1 antibody was formulated in phosphate-buffered saline (PBS, pH 7.4) to complete its preparation.
  • PBS phosphate-buffered saline
  • the heavy chain variable region protein corresponding to SEQ ID NO: 1 was fused with the human mutant IgG1 heavy chain constant region protein corresponding to SEQ ID NO: 4.
  • the light chain variable region protein corresponding to SEQ ID NO: 2 was fused with the human light chain constant region protein corresponding to SEQ ID NO: 5.
  • the DNA sequences encoding the fused amino acid sequences were codon-optimized, and the optimized codon sequences were synthesized and cloned into the pcDNA3.4 expression vector. Using this vector, transient transfection was performed in ExpiCHO-S cells, followed by antibody expression for 8 days.
  • the expressed antibody was purified using Protein A affinity chromatography. Specifically, the antibody was bound to an affinity chromatography resin equilibrated with a 20 mM Tris-acetate buffer (pH 7.4) containing 150 mM sodium chloride. The resin was then washed with a 20 mM sodium acetate buffer (pH 5.0) containing 1 M sodium chloride and a 25 mM sodium acetate buffer (pH 5.0), followed by elution of the target protein using a 25 mM sodium acetate buffer (pH 3.6) at low pH. The eluted protein was then diluted twofold with a 75 mM sodium acetate buffer (pH 5.5) containing 300 mM sodium chloride for formulation, completing the preparation of the final Ab2 antibody.
  • a 20 mM Tris-acetate buffer pH 7.4
  • the resin was then washed with a 20 mM sodium acetate buffer (pH 5.0) containing 1 M sodium chloride and a 25 mM
  • the absorbance of the Ab1 and Ab2 antibodies produced in Preparation Example 1.2 was measured using Nanodrop, and the measured absorbance values were used to determine the concentration using the A280/Extinction coefficient formula. Further, the purity of the purified proteins was analyzed using SDS-PAGE and SEC-HPLC.
  • each of the Ab1 and Ab2 antibodies were loaded onto an SEC-HPLC column, and the analysis was conducted under conditions where a 50 mM sodium phosphate buffer (pH 6.8) containing 150 mM sodium chloride was applied at a flow rate of 1 mL/min. The antibody purity was assessed using a UV detector at 214 nm.
  • the SEC-HPLC analysis results for the Ab1 and Ab2 antibodies are shown in Figure 1 and Figure 2, respectively, while the SDS-PAGE analysis results for the Ab1 and Ab2 antibodies are presented in Figure 3. Additionally, the measured purity of the Ab1 and Ab2 antibodies is provided in Table 3.
  • the binding ability of the Ab1 antibody produced in Preparation Example 1 to human BCAM protein was analyzed using the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method.
  • a solution of anti-His tag antibody (Biolegend, USA, Cat. No. 652505) was prepared at a concentration of 4 ⁇ g/mL by diluting it in PBS buffer.
  • a 96-well plate (Thermo, USA, Cat. No. 439454) was coated with 100 ⁇ L of the antibody solution per well and incubated overnight at 4°C. The next day, all solutions were removed from the plate, and each well was washed three times with 300 ⁇ L of washing buffer (0.1% Tween-20 in PBS). After washing, blocking buffer (3% skim milk in PBS) was added to each well (300 ⁇ L per well) and incubated at room temperature for 1 hour.
  • the Ab1 antibody exhibited excellent binding affinity for human BCAM protein, demonstrating its high specificity and effectiveness.
  • the binding ability of the Ab1 antibody produced in Preparation Example 1 to human BCAM protein expressed on the cell surface was analyzed using FACS (Fluorescence-Activated Cell Sorting, Flow Cytometry) (FACS CantoTM II, BD, USA).
  • FACS Fluorescence-Activated Cell Sorting, Flow Cytometry
  • HEK293 cells were purchased from ThermoFisher Scientific (MA, USA). The HEK293 cells were transfected using a human BCAM plasmid vector, and the HEK293 cells expressing the human BCAM antigen protein were designated as HEK293/BCAM cells.
  • the HEK293/BCAM cells were suspended in FACS buffer and seeded into a 96-well plate at a density of 1 ⁇ 10 5 cells per well.
  • the Ab1 antibody from Example 1 was serially diluted in FACS buffer from an initial concentration of 333 nM in a 3-fold dilution series down to 1.88 pM across 12 points.
  • the diluted antibody solution was added to the seeded cells in the plate, followed by incubation at 4°C for 30 minutes. After washing the wells with FACS buffer, Goat Anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 (ThermoFisher Scientific, USA, Cat. No. A21445) (1:400) was added, and the plate was further incubated. The wells were then washed again with FACS buffer. The binding levels of antigen-antibody interactions at different antibody concentrations were measured using flow cytometry, and the Mean Fluorescence Intensity (MFI) values were obtained. Based on these values, the EC 50 concentration was calculated.
  • MFI Mean Fluorescence Intensity
  • the Ab1 antibody exhibited excellent binding ability to human BCAM protein expressed on the cell surface, demonstrating its high specificity and strong binding affinity.
  • the binding ability of the Ab1 antibody produced in Preparation Example 1 was evaluated using Bio-Layer Interferometry (BLI) with the Octet® R8 system (Sartorius).
  • a Reactive 2nd Generation (AR2G) biosensor (Cat. No. 18-5094, Sartorius) was mounted onto the Octet instrument, and the AR2G biosensor was activated using the AR2G Reagent Kit (Cat. No. 18-5095).
  • the activated biosensor was then immersed in an antigen solution containing human BCAM protein for 10 minutes, allowing the human BCAM protein to be immobilized onto the biosensor via amine coupling.
  • the biosensor was immersed in an ethanolamine solution for 5 minutes.
  • the biosensor with immobilized human BCAM protein was then placed into a buffer solution containing the Ab1 antibody from Example 1, and the binding reaction was monitored for 5 minutes, followed by a dissociation reaction for another 5 minutes.
  • the antibody solution was serially diluted.
  • the dissociation phase was set to 100 seconds, and the binding (Ka) and dissociation (Kd) rate constants were determined by fitting the data to a 1:1 binding model using curve-fitting software.
  • the binding kinetics data are presented in Table 6, and the binding sensor-gram is shown in Figure 6.
  • the Ab1 antibody demonstrated excellent dynamic binding ability to human BCAM protein, confirming its high affinity and specificity.
  • the VcaP prostate cancer cell line was purchased from ATCC (American Type Culture Collection, USA). A total of 2.5 ⁇ 104 VcaP cells were seeded into each well of a 96-well plate (Corning, USA, Cat. No. 3595) at a volume of 50 ⁇ L per well. The next day, after confirming cell attachment to the plate, the existing culture medium was removed.
  • the Ab1 antibody prepared in Example 1 and the isotype control human IgG4 (hIgG4) antibody were each diluted in cell culture medium (DMEM, 90%; heat-inactivated fetal bovine serum (FBS), 10%) to final concentrations of 0.5, 1, 2, and 4 ⁇ g/mL, with a two-fold dilution series.
  • DMEM cell culture medium
  • FBS heat-inactivated fetal bovine serum
  • the diluted antibodies were mixed with IncuCyte® FabFluor-pH Red Antibody labeling reagent (Sartorius, Cat. No. 4722) and incubated at 37°C for 15 minutes. Then, 50 ⁇ L of the mixture was added to each well, and the plate was placed into the IncuCyte® S3 Live-Cell Analysis System. Images were captured every hour for 24 hours. The results measured over 24 hours are shown in Figure 7.
  • the antibody of the present invention can facilitate intracellular delivery of cytotoxic drugs or therapeutic moieties via internalization into target cells.
  • the antibody of the present invention can be used in the form of an antibody-drug conjugate (ADC).
  • Compound 1 was synthesized to be used as the L-D moiety (Linker-Therapeutic Moiety) in the antibody-drug conjugate (ADC) represented by Formula I in the present invention.
  • Compound 1a (Monomethylauristatin F, MMAF) ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine
  • Compound 2 which serves as the L-D moiety (Linker-Therapeutic Moiety) in the antibody-drug conjugate (ADC) represented by Formula I, was purchased from BLDpharm (#BD317613, vcMMAE).
  • ADC1-1, ADC1-2, and ADC2-1 Three different ADCs (ADC1-1, ADC1-2, and ADC2-1) were prepared using the method described below.
  • payload therapeutic moiety
  • linker the same method can be applied or slight modifications can be made to synthesize ADCs.
  • the antibody, payload (therapeutic moiety), and linker are as described previously.
  • the antibody used was Ab1 prepared in Example 1, and the L-D of the antibody-drug conjugate (ADC) represented by General Formula I of the present invention was compound 1 prepared in Example 2.1.
  • the resulting ADC was designated as ADC1-1.
  • ADC prepared using Ab1, which was produced in Example 1, as the antibody, and compound 2 (#BD317613, vcMMAE) purchased from BLDpharm as the L-D of the antibody-drug conjugate (ADC) represented by Formula I of the present invention, was designated as ADC1-2.
  • the reducing agent TCEP was added to Ab1 antibody at a concentration of 1.09 mM.
  • TCEP was added to Ab2 antibody at a concentration of 0.28 mM under the same conditions. The reaction was carried out at 25°C for 2 hours to partially reduce the disulfide bonds of the antibodies.
  • Compound 1 or Compound 2 was dissolved in DMSO.
  • the partially reduced Ab1 and Ab2 antibodies were each combined with Compound 1 or Compound 2 dissolved in DMSO at a molar ratio of 15 equivalents.
  • the reaction was adjusted to maintain an antibody concentration of 10.0 mg/mL and was carried out for 3 hours at 25°C in a controlled chamber.
  • the reaction mixture was passed through a desalting column, and buffer exchange was performed with 50 mM phosphate-buffered saline (PBS, pH 6.5) containing 400 mM sodium chloride to remove unreacted Compound 1 and Compound 2.
  • PBS phosphate-buffered saline
  • the eluate from the desalting column was further concentrated and residual unreacted compounds were removed using a 50 kDa cut-off PES membrane centrifugal filter unit.
  • the prepared ADC1-1, ADC1-2, and ADC2-1 were each loaded onto an SEC-HPLC column at a concentration of 20 ⁇ g.
  • SEC-HPLC analysis was performed under conditions using 50 mM phosphate-buffered saline (pH 6.8) containing 150 mM sodium chloride at a flow rate of 1 mL/min.
  • the purity of the antibody-drug conjugates was confirmed using a UV detector at 280 nm.
  • the SEC-HPLC chromatograms of ADC1-1, ADC1-2, and ADC2-1 are shown in Figures 8, 9, and 10, respectively.
  • ADC1-1, ADC1-2, and ADC2-1 were each loaded onto an HIC-HPLC column at a concentration of 40 ⁇ g.
  • the drug-to-antibody ratio (DAR) analysis was conducted under a gradient elution condition from 100% of 20 mM sodium phosphate buffer (pH 6.8) containing 1 M ammonium sulfate to 70% of 20 mM sodium phosphate buffer (pH 6.8) and 30% of 20% isopropanol over 25 minutes, at 30°C, with a flow rate of 0.6 mL/min.
  • the DAR of the prepared antibody-drug conjugates was determined using a UV detector at 280 nm.
  • the HIC-HPLC chromatograms of ADC1-1, ADC1-2, and ADC2-1 are shown in Figures 11, 12, and 13, respectively.
  • ADC1-1 prepared by the Preparation Example 2 To evaluate the binding ability of ADC1-1 prepared by the Preparation Example 2 to human BCAM protein, a sandwich ELISA (Enzyme-Linked Immunosorbent Assay) test was performed.
  • the anti-His tag antibody (Biolegend, USA, Cat. No. 652505) was diluted in PBS buffer to a final concentration of 4 ⁇ g/mL and coated onto a 96-well plate (Thermo, USA, Cat. No. 439454) by adding 100 ⁇ L per well, followed by incubation at 4°C overnight. The next day, all solutions were removed from the plate, and each well was washed three times with washing buffer (0.1% Tween-20 in PBS, 300 ⁇ L per well). After washing, blocking buffer (3% skim milk in PBS, 300 ⁇ L per well) was added and incubated at room temperature for 1 hour.
  • human BCAM protein (Biointron, China) was prepared by diluting to 50 nM in blocking buffer and added to each well (100 ⁇ L per well), then incubated at room temperature for 1 hour.
  • ADC1-1 samples were serially diluted in blocking buffer from 111 nM in a 3-fold dilution series across 10 points, down to 6 pM.
  • the prepared ADC1-1 dilutions were added (100 ⁇ L per well) and incubated at room temperature for 2 hours.
  • a secondary antibody HRP-conjugated anti-human IgG Fc antibody, Jackson ImmunoResearch, USA, Cat. No.
  • the sandwich ELISA assay demonstrated that ADC1-1 exhibits excellent binding affinity to human BCAM protein. This indicates that even when Ab1 antibody is formulated as an antibody-drug conjugate (ADC), its binding ability to BCAM protein remains unaffected.
  • ADC antibody-drug conjugate
  • ADC1-1 produced in Preparation Example 2 was analyzed using FACS (Fluorescence-Activated Cell Sorting, Flow Cytometry) (FACS CantoTM II, BD, USA).
  • HEK293 cells were purchased from ThermoFisher Scientific (MA, USA). The HEK293 cells were transfected using a human BCAM plasmid vector, and the HEK293 cells expressing the human BCAM antigen protein were designated as HEK293/BCAM cells. The HEK293/BCAM cells, suspended in FACS buffer, were seeded into a 96-well plate at 1 ⁇ 10 5 cells per well.
  • the antibody-drug conjugate (ADC1-1) of Preparation Example 2 was serially diluted in FACS buffer from 333 nM at a 3-fold dilution ratio down to 1.88 pM, covering 12 different concentrations.
  • the diluted ADC was then added to the seeded cells in the plate and incubated at 4°C for 30 minutes. After incubation, the wells were washed with FACS buffer, followed by the addition of Goat Anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 (ThermoFisher Scientific, USA, Cat. No. A21445) at a dilution of 1:400.
  • the plate was incubated again, then washed once more with FACS buffer. Using flow cytometry, the Mean Fluorescence Intensity (MFI) values were measured to assess the binding of ADC1-1 to the antigen at various antibody concentrations. The EC 50 value was calculated based on the obtained data.
  • MFI Mean Fluorescence Intensity
  • the antibody-drug conjugate (ADC1-1) exhibited excellent binding affinity to human BCAM protein expressed on the cell surface. Therefore, it can be interpreted that the BCAM-binding ability of the Ab1 antibody remains unaffected even after being formulated as an antibody-drug conjugate.
  • Binding affinity verification test for ADC1-1 prepared in Example 2 was conducted using Bio-layer Interferometry (BLI) with the Octet (Octet® R8, Sartorius).
  • a Reactive 2 nd Generation (AR2G) biosensor (Cat. No. 18-5094, Sartorius) was mounted on the Octet instrument, and the AR2G biosensor was activated using the AR2G Reagent Kit (Cat. No. 18-5095).
  • the activated biosensor was then immersed in an antigen solution containing human BCAM protein for 10 minutes, thereby conjugating human BCAM protein to the biosensor via amine coupling.
  • the biosensor was immersed in an ethanolamine solution for 5 minutes to terminate the coupling reaction.
  • the biosensor conjugated with human BCAM protein was placed in a buffer solution containing the antibody-drug conjugate ADC1-1 prepared in Preparation Example 2, and the binding reaction was monitored for 5 minutes, followed by observation of the dissociation reaction for 5 minutes.
  • the binding kinetic data are presented in Table 6, and the binding sensor-gram is shown in Figure 16.
  • the prostate cancer cell line VCaP was purchased from ATCC (American Type Culture Collection, USA). A total of 2.5 ⁇ 104 VCaP cells were seeded into each well of a 96-well plate (Corning, USA, Cat. No. 3595) with 50 ⁇ L per well. The next day, after confirming that the cells had attached to the plate, the existing culture medium was removed.
  • the antibody-drug conjugate ADC1-1 prepared in Preparation Example 2 and the isotype control human IgG4-vc.MMAF (hIgG4-vc.MMAF) were each diluted in cell culture medium (DMEM, 90%; heat-inactivated fetal bovine serum (FBS), 10%) at final concentrations of 0.5, 1, 2, and 4 ⁇ g/mL.
  • DMEM cell culture medium
  • FBS heat-inactivated fetal bovine serum
  • the diluted antibody-drug conjugate was mixed with IncuCyte® FabFluor-pH Red Antibody labeling reagent (Sartorius, Cat. No. 4722) and incubated at 37°C for 15 minutes. Then, 50 ⁇ L of the mixture was added to each well, and images were acquired every hour for 24 hours using the IncuCyte® S3 Live-Cell Analysis System. The results obtained over 24 hours are shown in Figure 17.
  • the antibody-drug conjugate ADC1-1 was internalized into cancer cells over time and as the concentration increased, leading to an increase in the cellular fluorescence signal.
  • no change in the fluorescence signal was observed when cells were treated with the hIgG4-vcMMAF antibody-drug conjugate. Therefore, similar to the cellular internalization properties of the Ab1 antibody confirmed in Example 2, it was demonstrated that the antibody-drug conjugate of the present invention can deliver a cytotoxic drug into cells through internalization.
  • the human prostate cancer cell line DU145 (KCLB), the human ovarian cancer cell lines SNU-119 (KCLB) and OVCAR3 (KCLB), and the human breast cancer cell line T47D were purchased from KCLB (Korean Cell Line Bank, South Korea).
  • the human ovarian cancer cell line KURAMOCHI was purchased from the JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank, Japan), the human prostate cancer cell line VCaP was purchased from ATCC (American Type Culture Collection, USA), and the human breast cancer cell line EFM-192A was purchased from DSMZ (Leibniz-Institut DSMZ, Germany).
  • DU145, KURAMOCHI, SNU-119, OVCAR3, T47D, VCaP, and EFM-192A cells were seeded at a density of 5 ⁇ 10 3 cells per well in a 96-well plate (Corning, USA, Cat. No. 3595). The next day, after confirming that the cells had adhered to the plate, the existing culture medium was removed.
  • ADC1-1 and ADC1-2 the antibody-drug conjugates prepared in Preparation Example 2 were sequentially diluted in the culture medium from an initial concentration of 1 ⁇ M, using a five-fold dilution series, across eight concentration points down to 12.8 pM. The diluted antibody-drug conjugates were added to each well at 200 ⁇ L per well.
  • the negative control for ADC1-1 and ADC1-2 was an antibody-drug conjugate in which the Ab1 antibody from Preparation Example 2 was replaced with a human IgG4 antibody.
  • the experimental groups treated with ADC1-1 and ADC1-2, along with the 100% control group were incubated in a 37°C CO 2 incubator for 72 hours. After incubation, the culture medium was removed, and 100 ⁇ L each of CellTiter-Glo® solution and culture medium were added to each well, followed by a 10-minute incubation at room temperature. The luminescence of the experimental groups and the 100% control group was then measured using a microplate reader.
  • the cell viability (%) in the experimental groups treated with ADC1-1 and ADC1-2 was calculated using the following formula:
  • the GI 50 (half-maximal growth inhibition concentration) values of ADC1-1 and ADC1-2 in DU145, KURAMOCHI, SNU-119, OVCAR3, T47D, VCaP, and EFM-192A cells were determined using GraphPad Prism (GraphPad Software Inc., USA) based on cell viability data.
  • the cell viability (%) of ADC1-1 and ADC1-2 in DU145, KURAMOCHI, SNU-119, OVCAR3, T47D, VCaP, and EFM-192A cells is shown in Figure 18, and the GI50 values are presented in Table 11.
  • the antibody-drug conjugates ADC1-1 and ADC1-2 of the present invention inhibited the cell growth of DU145, KURAMOCHI, SNU-119, OVCAR3, T47D, VCaP, and EFM-192A in a concentration-dependent manner. Therefore, it was confirmed that the antibody-drug conjugates ADC1-1 and ADC1-2 of the present invention exhibit excellent cell growth inhibitory activity against human cancer cell lines expressing BCAM.
  • the antibody-drug conjugate ADC1-1 prepared in Preparation Example 2, was evaluated for its ability to inhibit the growth of human cancer cells under in vivo conditions using an immunodeficient mouse xenograft model.
  • the human prostate cancer cell line DU145 and the human ovarian cancer cell line OVCAR3 were purchased from the Korean Cell Line Bank (KCLB, South Korea).
  • the human prostate cancer cell line VCaP was purchased from the American Type Culture Collection (ATCC, USA), and the human gastric cancer cell line RERF-GC-1b was purchased from the JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank, Japan).
  • a total of 1 ⁇ 10 6 DU145 human prostate cancer cells per 100 ⁇ L were mixed with 100 ⁇ L of Matrigel at a 1:1 ratio, and a total volume of 200 ⁇ L was subcutaneously implanted into six-week-old male NOD/SCID mice to establish a prostate cancer xenograft mouse model. Twenty-two days after DU145 subcutaneous implantation, when the tumor volume had grown to approximately 80-150 mm 3 , the mice were divided into two groups (nine mice per group) so that the average tumor volume in each group was as similar as possible (approximately 118 mm 3 per group). The mice were then assigned to the negative control group and the experimental group.
  • an isotype control antibody-drug conjugate (hereinafter referred to as hIgG4-vcMMAF) was administered at a dose of 10 mg/kg and a volume of 10 mL/kg.
  • the antibody-drug conjugate ADC1-1 prepared in Preparation Example 2 was administered at a dose of 10 mg/kg and a volume of 10 mL/kg. Both groups received intravenous injections through the tail vein once per week for a total of two doses (on Day 0 and Day 7).
  • the tumor volume of both the negative control group and the experimental group was measured three times per week.
  • the tumor growth inhibition (TGI) and tumor regression (TR) efficacy of the ADC1-1 antibody were calculated using the following formulas, setting the tumor growth rate of the negative control group to 100% (Relative tumor growth, 100%).
  • ADC1-1 demonstrated a statistically significant tumor growth inhibition effect in the DU145 xenograft model compared to the negative control group (p-value ⁇ 0.0001) ( Figure 19a).
  • the tumor growth inhibition efficacy and tumor regression efficacy observed on the final tumor measurement day (D16) were 72.39% and 32.04%, respectively (Table 12).
  • a total of 2 ⁇ 10 6 OVCAR3 human ovarian cancer cells per 100 ⁇ L were mixed with 100 ⁇ L of Matrigel in a 1:1 ratio, and a total volume of 200 ⁇ L was subcutaneously implanted into 7-week-old female NOD/SCID mice, establishing an ovarian cancer xenograft mouse model. Fifty days after subcutaneous implantation, when the tumor volume reached approximately 50-210 mm3, the mice were divided into two groups (9 mice per group) so that the average tumor volume was as similar as possible between groups (approximately 132 mm3 per group).
  • the negative control group was administered phosphate-buffered saline (PBS) at a dose volume of 10 mL/kg, while the experimental group was administered ADC1-1, the antibody-drug conjugate (ADC) prepared in Preparation Example 2, at a dose of 10 mg/kg with a volume of 10 mL/kg via tail vein injection, administered once per week for a total of two doses (D0 and D7).
  • PBS phosphate-buffered saline
  • ADC1-1 the antibody-drug conjugate
  • TGI tumor growth inhibition efficacy
  • TR tumor regression efficacy
  • ADC1-1 demonstrated statistically significant tumor growth inhibition efficacy against OVCAR3 tumors compared to the negative control group (p-value ⁇ 0.0001) ( Figure 19b).
  • TGI tumor growth inhibition
  • TR tumor regression
  • a VCaP human prostate cancer cell line (2 ⁇ 10 6 cells/100 ⁇ L) was mixed with 100 ⁇ L of Matrigel at a 1:1 ratio, and a total of 200 ⁇ L was subcutaneously implanted into 9-week-old male NOD/SCID mice to establish a VCaP xenograft prostate cancer mouse model. After 34 days, when the tumor volume reached approximately 50 to 210 mm 3 , the mice were divided into two groups (9 mice per group), ensuring the average tumor volume was as similar as possible between groups (approximately 138 - 140 mm 3 per group).
  • the negative control group received phosphate-buffered saline (PBS) at 10 mL/kg, while the experimental group received ADC1-1, the antibody-drug conjugate (ADC) manufactured in Example 2, at a dose of 10 mg/kg and a volume of 10 mL/kg, administered via tail vein injection once per week for a total of two doses (D0 and D7).
  • PBS phosphate-buffered saline
  • ADC1-1 the antibody-drug conjugate manufactured in Example 2
  • the tumor volumes of the negative control group and the experimental group were measured three times per week.
  • the tumor growth inhibition (TGI) and tumor regression (TR) of ADC1-1 were calculated using the following formulas, with the tumor growth rate of the negative control group set at 100% (Relative Tumor Growth, 100%):
  • ADC1-1 demonstrated statistically significant tumor growth inhibition efficacy against VCaP tumors compared to the negative control group (p-value ⁇ 0.0001) ( Figure 19c).
  • TGI tumor growth inhibition
  • TR tumor regression
  • a RERF-GC-1b human gastric cancer cell line (5 ⁇ 10 cells/100 ⁇ L) was mixed with 100 ⁇ L of Matrigel at a 1:1 ratio, and a total of 200 ⁇ L was subcutaneously implanted into 8-week-old female NOD/SCID mice to establish a RERF-GC-1b xenograft gastric cancer mouse model. After 47 days, when the tumor volume reached approximately 70 to 140 mm 3 , the mice were divided into two groups (negative control group: 6 mice, experimental group: 4 mice), ensuring the average tumor volume was as similar as possible between groups (approximately 93 - 96 mm 3 per group).
  • the negative control group received phosphate-buffered saline (PBS) at 10 mL/kg, while the experimental group received ADC1-1, the antibody-drug conjugate (ADC) manufactured in Preparation Example 2, at a dose of 10 mg/kg and a volume of 10 mL/kg, administered via tail vein injection once per week for a total of two doses (D0 and D7).
  • PBS phosphate-buffered saline
  • ADC1-1 the antibody-drug conjugate manufactured in Preparation Example 2
  • the tumor volumes of the negative control group and the experimental group were measured three times per week.
  • the tumor growth inhibition (TGI) and tumor regression (TR) of ADC1-1 were calculated using the following formulas, with the tumor growth rate of the negative control group set at 100% (Relative Tumor Growth, 100%):
  • ADC1-1 demonstrated statistically significant tumor growth inhibition efficacy against RERF-GC-1b tumors compared to the negative control group (p-value ⁇ 0.01) ( Figure 19d).
  • TGI tumor growth inhibition
  • TR tumor regression
  • Example 7 Cell growth inhibition assay of ADC1-1 in human normal cells
  • ADC1-1 is composed of humanized monoclonal antibody Ab1, which target CD239, and conjugated with MMAF through mc-VC-PABC (Compound 1; maleimidocaproyl-L-valine-L-citrulline-para-aminobenzyl carbamate).
  • test and control materials are described in Tables 16 and 17 below, respectively.
  • cell information is described in Table 18 below.
  • the cell viability (%) was calculated after subtracting the luminescence (RLU, relative light unit) values of negative control (0% viability control) as 0% cell growth, and by setting the RLU values of positive control (100% viability control) as 100% cell growth.
  • the RLU values were transformed to cell viability (%) using the following formula.
  • Cell viability (%) ⁇ (RLU of test group - RLU of 0% viability control) / (RLU of 100% viability control - RLU of 0% viability control) ⁇ * 100
  • the graphs were displayed as means ⁇ S.D. (standard deviation) from triplicate, and the half-maximal growth inhibition (GI 50 ) values were determined by nonlinear regression with normalized response using GraphPad Prism program (GraphPad Software, Inc., San Diego, CA).
  • Hs27 cells or HUVECs were seeded into a 96-well plate at 5.0X10 3 cells/100 ⁇ L and incubated at 37°C, 5% CO 2 incubator overnight.
  • test or control articles were prepared by serial dilution in culture media to 2X of final concentration (12.8 pM ⁇ 1 ⁇ M, 5-fold, 8 points).
  • test or control article treatment Days 0
  • media were removed from the negative control (0% cell growth; no treatment of test or control articles) plate.
  • 100 ⁇ L of fresh culture media and 100 ⁇ L of CellTiter-Glo ® reagent were sequentially added to the plate and then incubated for 10 min at room temperature with gentle mixing on an orbital shaker.
  • Luminescence was measured with a microplate reader with 1 sec integration time per well.
  • test group cells treated with test or control articles
  • media were removed from the test group (cells treated with test or control articles) plate.
  • 100 ⁇ L of fresh culture media and 100 ⁇ L CellTiter-Glo ® reagent were sequentially added to the plate and then incubated for 10 min at room temperature with gentle mixing on an orbital shaker.
  • Luminescence was measured with a microplate reader with 1 sec integration time per well.
  • the GI 50 value of ADC1-1 was > 1,000 nM.
  • the GI 50 value of ADC1-1 was 40 nM.
  • MMAF showed no inhibition of the cell growth in both Hs27 cells and HUVECs, with GI 50 values more than 1,000 nM (Table 20).
  • the cytotoxic activity of ADC1-1 was higher than those in Hs27 cells. Moreover, they showed higher cytotoxic activities than their corresponding isotype control ADC.
  • Example 8 Cell growth inhibition assay of ADC1-1 in CD239 negative human cancer cell lines
  • the aim of this study is to assess the cell growth inhibitory effect of ADC1-1, an ADC (antibody-drug conjugate) comprising Ab1 conjugated with MMAF through mc-VC-PABC (Compound 1; maleimidocaproyl-L-valine-L-citrulline-para-aminobenzyl carbamate) linker-payload.
  • ADC1-1 an ADC (antibody-drug conjugate) comprising Ab1 conjugated with MMAF through mc-VC-PABC (Compound 1; maleimidocaproyl-L-valine-L-citrulline-para-aminobenzyl carbamate) linker-payload.
  • mc-VC-PABC Compound 1; maleimidocaproyl-L-valine-L-citrulline-para-aminobenzyl carbamate linker-payload.
  • This evaluation is conducted on CD239 negative human cancer cell lines, BT-549 (Breast cancer) and MK
  • test and control materials are described in Tables 21 and 22 below, respectively.
  • cell information is described in Table 23 below.
  • the cell viability (%) was calculated after subtracting the luminescence (RLU, relative light unit) values of negative control (0% viability control) as 0% cell growth, and by setting the RLU values of positive control (100% viability control) as 100% cell growth.
  • the RLU values were transformed to cell viability (%) using the following formula.
  • Cell viability (%) ⁇ (RLU of test group - RLU of 0% viability control) / (RLU of 100% viability control - RLU of 0% viability control) ⁇ * 100
  • the graphs were displayed as means ⁇ S.D. (standard deviation) from triplicate, and the half-maximal growth inhibition (GI 50 ) values were determined by nonlinear regression with normalized response using GraphPad Prism program (GraphPad Software, Inc., San Diego, CA).
  • test or control articles were prepared by serial dilution in culture media to 2X of final concentration (0.0128 nM ⁇ 1 ⁇ M, 5-fold, 8 points).
  • test or control article treatment Days 0
  • media were removed from the negative control (0% cell growth; no treatment of test or control articles) plate.
  • 100 ⁇ L of fresh culture media and 100 ⁇ L of CellTiter-Glo ® reagent were sequentially added to the plate and then incubated for 10 min at room temperature with gentle mixing on an orbital shaker.
  • Luminescence was measured with a microplate reader with 1 sec integration time per well.
  • test group cells treated with test or control articles
  • media were removed from the test group (cells treated with test or control articles) plate.
  • 100 ⁇ L of fresh culture media and 100 ⁇ L CellTiter-Glo ® reagent were sequentially added to the plate and then incubated for 10 min at room temperature with gentle mixing on an orbital shaker.
  • Luminescence was measured with a microplate reader with 1 sec integration time per well.
  • the GI 50 values of ADC1-1 against BT-549 and MKN-45 cells are given in Table 25 below.
  • the GI 50 value for ADC1-1 was 321 nM (Table 25).
  • ⁇ hIgG4-VC.MMAF showed no inhibition of growth in both BT-549 and MKN-45 cells, with GI 50 values > 1,000 nM (Table 25).
  • Test and control article DAR GI 50 (nM) BT-549 MKN-45 hIgG4-VC.MMAF 3.8 > 1,000 > 1,000 ADC1-1 4.7 321 > 1,000
  • ADC1-1 exhibits a low potential for inhibiting the growth of CD239 negative human cancer cells.
  • Example 9 In vivo efficacy of ADC1-1 in a xCAOV3 xenograft model
  • the aim of this study is to evaluate efficacy of ADC1-1 in a xCAOV3 xenograft model.
  • Control article 3 hIgG4-VC.MMAF
  • xCAOV3 Human ovarian cancer, In vivo recycled CAOV3 cells referred to as xCAOV3 from Wuxi; Note: xCAOV3 cell was derived from CAOV3 by isolation from tumor, to optimize the taking ratio and volume variation. The cell line identity was confirmed by STR)
  • the tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA. The cells growing in an exponential growth phase was harvested and counted for tumor inoculation.
  • Each mouse was inoculated subcutaneously at the right flank with xCAOV3 tumor cells (5 ⁇ 10 6 ) in 0.2 mL DPBS mixed with matrigel for tumor development.
  • the mean tumor volumes reached averages of 192 mm 3 on Day 24 after xCAOV3 cell implantation, which was one day before group assignment.
  • the formulation preparation is given in Table 29 below.
  • the Relative tumor growth rate (RTG, %), Tumor growth inhibition (TGI, %), Tumor regression (TR, %) were determined as follows:
  • TGI (%) [1 - (RTV of the treated group)/(Mean RTV of the vehicle control)] ⁇ 100 (%)
  • TR (%) [1 - (final tumor volume / initial tumor volume)] ⁇ 100 (%)
  • ADC1-1 treatment (5 and 10 mg/kg) once weekly for four weeks significantly inhibited the xCAOV3 tumor growth in a dose-dependent manner.
  • the maximum tumor growth inhibition (TGI, vs. PBS group) was observed on Day 13 or 20 after initiating treatment, while the maximum tumor regression (TR) was specifically observed in the ADC1-1 10 mg/kg treatment group on Day 20 ( Figure 20).
  • ADC1-1 exhibited significant anti-tumor effects that were dose-dependent.
  • the maximum body weight losses were limited to 2 % across all groups; however, one mouse each from the 5 mg/kg and 10 mg/kg ADC1-1 group was found dead on Day 24 and Day 25, respectively, and abdominal swelling was observed in 10 mg/kg ADC1-1 group on Day 24. Further studies are needed to elucidate the underlying reasons for this observed toxic profiles.
  • xCAOV3 cell line The sensitivity of xCAOV3 cell line to platinum-based drugs (cisplatin and carboplatin) was validated by in vitro cell viability assay at Wuxi, and xCAOV3 cell line showed resistance to both drugs.
  • the sequence listing electronic file is attached.

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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un anticorps ou son fragment liant l'antigène qui se lie à la protéine BCAM, un conjugué anticorps-médicament comprenant l'anticorps, et des procédés de préparation de celui-ci. De plus, la présente invention concerne l'utilisation de l'anticorps ou de son fragment liant l'antigène, ou du conjugué anticorps-médicament, pour la prévention, le soulagement ou le traitement de maladies associées à la fonction ou à l'expression de BCAM (par exemple, le cancer). Figure représentative : Figure 18
PCT/KR2025/006521 2024-05-14 2025-05-14 Anticorps anti-bcam et son conjugué Pending WO2025239669A1 (fr)

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