WO2023008858A1 - Novel antibody for prevention or treatment of fibrotic disease - Google Patents

Novel antibody for prevention or treatment of fibrotic disease Download PDF

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WO2023008858A1
WO2023008858A1 PCT/KR2022/010881 KR2022010881W WO2023008858A1 WO 2023008858 A1 WO2023008858 A1 WO 2023008858A1 KR 2022010881 W KR2022010881 W KR 2022010881W WO 2023008858 A1 WO2023008858 A1 WO 2023008858A1
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antibody
tsp
antigen
present
binding fragment
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PCT/KR2022/010881
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French (fr)
Korean (ko)
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윤호근
박수연
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • the present invention predicts the occurrence of fibrotic disease using an antibody that specifically binds to thrombospondin (TSP-2) protein involved in fibrosis, or prevents or prevents fibrotic disease by inhibiting the activity of the protein. It's about how to treat.
  • TSP-2 thrombospondin
  • Idiopathic pulmonary fibrosis is a serious chronic interstitial lung disease, a fatal and progressive disease that affects millions of patients worldwide. The disease scars the patient's lungs and hardens them, resulting in serious symptoms such as shortness of breath. Because the symptoms usually progress slowly over several years, it is not until symptoms become severe enough to interfere with daily life. It is a more dangerous disease because it is often treated by a doctor.
  • idiopathic pulmonary fibrosis is known as a disease that is very difficult to treat and is almost impossible to cure.
  • Fibrosis is a reparative or reactive process characterized by the formation and deposition of excessive fibrous connective tissue, and serves as a major etiology of several fibrotic diseases and chronic diseases.
  • pulmonary fibrosis is known to occur due to excessive deposition of extracellular matrix (ECM) and damage to abnormal alveolar epithelial cells, although the exact mechanism is not known.
  • ECM extracellular matrix
  • fibroblasts play an important role in wound healing and connective tissue generation, but excessive immune cells and ECM proteins accumulate in foci due to uncontrolled expression of excessive fibroblasts, resulting in pathological conditions. will develop into
  • predictors and pathogenesis of pulmonary fibrosis are still ambiguous, so identifying the exact molecular and biological mechanisms for them is a very important factor in diagnosing or treating pulmonary fibrosis.
  • ECM is a non-structural matrix protein, and abnormal deposition is observed in fibrosis.
  • ECM is a non-structural matrix protein, and abnormal deposition is observed in fibrosis.
  • About 30 kinds of extracellular matrix-adhesion proteins that bind to such ECM are currently known, and Thrombospondin (TSP) is one of them.
  • TSP Thrombospondin
  • These extracellular matrix-adhesion proteins serve to connect cells and extracellular matrix and play an important role as regulators of cell-matrix interactions.
  • the TSP-2 protein belonging to the TSP family is secreted by fibroblasts and is involved in matrix remodeling.
  • the present inventors focused on the relationship between TSP-2 and pulmonary fibrosis among the TSP family, and tried to prove that TSP-2 contributes to the development of pulmonary fibrosis, and through this study, the relationship between TSP-2 protein and fibrosis At the same time, we tried to develop a monoclonal antibody specific to TSP-2 using it as a treatment target for fibrosis, and demonstrate the anti-fibrotic effect of the antibody.
  • TSP-2 fibroblast-mediated fibrosis
  • an object of the present invention is to provide an antibody or antigen-binding fragment thereof against the TSP-2 protein and a nucleic acid molecule encoding the same.
  • Another object of the present invention is to provide a composition for preventing or treating fibrotic disease.
  • Another object of the present invention is a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention; It is to provide a recombinant vector containing the nucleic acid molecule and a host cell containing the recombinant vector.
  • Another object of the present invention is to provide a method for producing the antibody or antigen-binding fragment thereof of the present invention.
  • Another object of the present invention is to provide a method for confirming the presence or absence of TSP-2 protein in a sample using the antibody or antigen-binding fragment thereof of the present invention.
  • Another object of the present invention is to provide a diagnostic composition comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.
  • the present invention is a heavy chain having a heavy chain CDR (complementarity determining region) amino acid sequence of HCDR1 consisting of the sequence of SEQ ID NO: 1, HCDR2 consisting of the sequence of SEQ ID NO: 2, and HCDR3 consisting of the sequence of SEQ ID NO: 3
  • An antibody or antigen-binding fragment thereof against a Thrombospondin-2 (TSP-2) protein comprising a variable region is provided.
  • TSP-2 fibroblast-mediated fibrosis
  • antibody refers to an antibody against TSP-2, which specifically recognizes and binds to a specific epitope thereof, and includes antigen-binding fragments (antibody fragments) of antibody molecules as well as complete antibody forms. .
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bonds.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types, and subclasses include gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain has kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • the term “antigen-binding fragment of an antibody” refers to a fragment that retains an antigen-antibody binding function within the entire antibody molecule, and includes Fab, F(ab'), F(ab')2, and Fv. do.
  • Fab has a structure having light chain and heavy chain variable regions, light chain constant region, and heavy chain first constant region (C H1 ) and has one antigen binding site.
  • Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C H1 domain.
  • An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'.
  • Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombinant technology for generating Fv fragments is described in PCT International Publication Patent Applications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344.
  • the heavy chain variable region and the light chain variable region are connected by a non-covalent bond
  • the heavy chain variable region and the short chain variable region are generally shared through a peptide linker. They are linked by bonds or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv.
  • Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.
  • heavy chain refers to a full-length variable region domain V H comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen and three constant region domains C H1 , C H2 and C H3 . Both heavy chains and fragments thereof are meant.
  • CDR complementarity determining region
  • the scope of the antibody or antibody fragment of the present invention includes variants with conservative amino acid substitutions in the CDR regions.
  • the antibody or antibody fragment of the present invention may include a variant of the amino acid sequence described in the accompanying sequence listing within the scope of specifically recognizing phosphorylated PLC ⁇ 2.
  • additional changes may be made to the amino acid sequence of the antibody to further improve its binding affinity and/or other biological properties.
  • Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody.
  • Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
  • each amino acid is given a hydrophobicity index according to its hydrophobicity and charge: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is a known fact that amino acids having similar hydrophobicity indexes should be substituted to retain similar biological activities. When a mutation is introduced with reference to the hydrophobicity index, substitution is made between amino acids exhibiting a hydrophobicity index difference within ⁇ 2 in one specific example, ⁇ 1 in another specific example, and ⁇ 0.5 in another specific example.
  • Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979).
  • the most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
  • the antibodies of the present invention or the nucleic acid molecules encoding them are construed to include sequences showing substantial identity with the sequences listed in the Sequence Listing.
  • the above substantial identity is at least 61% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art.
  • Homology refers to a sequence exhibiting 70% homology, according to another specific example, 80% homology, and according to another specific example, 90% homology.
  • Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math .
  • J. Mol. Biol . (1990) 215:403-10 is accessible from NBCI and the like, and sequences such as blastp, blasm, blastx, tblastn and tblastx are found on the Internet. It can be used in conjunction with an analysis program.
  • the BLAST is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparison methods using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 11.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the light chain CDR amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO: 4, LCDR2 consisting of the sequence of SEQ ID NO: 5, and LCDR3 consisting of the sequence of SEQ ID NO: 6 It further comprises a light chain variable region having.
  • light chain herein refers to both a full-length light chain and fragments thereof comprising a variable region domain V L and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen.
  • the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 16.
  • Antibodies of the present invention include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idio type (anti-Id) antibodies, and epitope-binding fragments of the antibodies, and the like, but are not limited thereto.
  • the antibody of the present invention is a monoclonal antibody.
  • the term "monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
  • the antibody of the present invention is a neutralizing antibody.
  • neutralizing antibody refers to an antibody that has a form specific to a structure that exhibits a biological function among surface structures of proteins, antigens, or infectious particles, and is capable of specifically binding to the corresponding structure. It refers to an antibody that plays a role in inhibiting the biological function of an infectious particle.
  • the antibody of the present invention is a chimeric antibody, a humanized antibody or a human antibody.
  • the antibody or antigen-binding fragment of the present invention is a Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment, more specifically a single chain Fvs(scFV) antibody. .
  • the antibody of the present invention can be prepared in various forms of the antibody.
  • the antibody of the present invention can be prepared as a sFv or Fab antibody, and also recombination with a human-derived constant region using the light and heavy chain variable regions obtained from the sFv or Fab antibody By doing so, it is also possible to provide a whole antibody.
  • the present invention provides a composition for preventing or treating fibrotic disease comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.
  • prevention refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed with the disease or disease, but is likely to suffer from the disease or disease.
  • the term “treatment” refers to (a) inhibition of the development of a disease, condition or condition; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition.
  • the composition of the present invention When the composition of the present invention is administered to a subject, the activity of the TSP-2 protein that causes fibrosis is inhibited, preventing excessive fibrosis, thereby inhibiting the development of symptoms due to fibrotic disease, removing or alleviating it . Therefore, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients to be applied as a treatment adjuvant for the above diseases. Accordingly, the term “treatment” or “therapeutic agent” in the present specification includes the meaning of "therapeutic aid” or "therapeutic aid”.
  • fibrotic disease refers to all pathological conditions that accompany the progress of fibrosis of pathological tissues and have direct or indirect causes of such fibrosis.
  • fibrosis also refers to the excessive accumulation of fibrous connective tissue (components of the extracellular matrix, such as collagen and fibronectin) in and around inflamed or damaged tissue, resulting in permanent scarring, organ dysfunction, and eventual means a disease that can cause death.
  • the fibrotic disease of the present invention is pulmonary fibrosis (Pulmonary Fibrosis).
  • pulmonary fibrosis refers to a disease in which the lungs become fibrotic due to repeated lung or lung tissue damage and wounds, and may include dyspnea; dry cough; fatigue; weight loss; and symptoms of nail clubbing.
  • the pulmonary fibrosis of the present invention is at least one disease selected from the group consisting of interstitial lung disease (ILD) and idiopathic pulmonary fibrosis (IPF). .
  • ILD interstitial lung disease
  • IPF idiopathic pulmonary fibrosis
  • ILD interstitial lung disease
  • idiopathic pulmonary fibrosis refers to a progressive respiratory disease characterized by thickening and rigidity of lung tissue and the formation of scar tissue, characterized by a gradual and irreversible decrease in lung function.
  • 'idiopathic' As a type of chronic scarring lung disease, it is referred to as 'idiopathic' because no clear cause is known.
  • the present invention provides a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention.
  • nucleic acid molecule has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also analogs with modified sugar or base sites. (analogue) is also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584).
  • sequences of nucleic acid molecules encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
  • nucleic acid molecules of the present invention are also construed to include nucleotide sequences exhibiting substantial identity to the nucleotide sequences described above.
  • the above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a nucleotide sequence showing homology, at least 90% homology in one specific example, and at least 95% homology in another specific example.
  • the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 27.
  • the present invention provides a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention.
  • the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 32.
  • the present invention provides a nucleic acid molecule encoding the above-described heavy chain variable region, a nucleic acid molecule encoding a light chain variable region, or a recombinant vector containing both of the above nucleic acid molecules.
  • vector refers to a plasmid vector as a means for expressing a target gene in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and the like.
  • a nucleic acid molecule encoding a light chain variable region and a nucleic acid molecule encoding a heavy chain variable region are operatively linked to a promoter.
  • the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence will control the transcription and/or translation of said other nucleic acid sequence.
  • the recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) .
  • Vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence.
  • a strong promoter capable of promoting transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter , rac5 promoter, amp promoter, recA promoter, SP6 promote
  • E. coli eg, HB101, BL21, DH5 ⁇ , etc.
  • promoter and operator regions of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. J. Bacteriol . 158: 1018-1024 (1984))
  • the leftward promoter of phage ⁇ (pL ⁇ promoter, Herskowitz, I. and Hagen, D. Ann. Rev. Genet . 14:399-445 (1980)
  • phage ⁇ pL ⁇ promoter, Herskowitz, I. and Hagen, D. Ann. Rev. Genet . 14:399-445 (1980)
  • the promoter of the toxin protein gene of Bacillus thuringiensis ( Appl. Environ. Microbiol . 64: 3932-3938 (1998); Mol. Gen. Genet . 250: 734-741 (1996) ) or any promoter that can be expressed in Bacillus strains can be used as a regulatory site.
  • the recombinant vector of the present invention is a plasmid often used in the art (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, It can be produced by manipulating pGEX series, pET series, and pUC19, etc.), phages (eg, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, and M13, etc.) or viruses (eg, SV40, etc.).
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter, ⁇ -actin promoter, human hemoglobin promoter, and human muscle
  • a promoter derived from a mammalian virus e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter
  • the LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Lowe's Sarcoma Virus (RSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.
  • the recombinant vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom.
  • Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA); maltose binding protein (NEB, USA); FLAG (IBI, USA); tag sequences such as 6x His (hexahistidine; Quiagen, USA), Pre-S1, c-Myc; There are leader sequences such as OmpA and PelB.
  • the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.
  • the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
  • an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
  • the vector expressing the antibody of the present invention may be either a vector system in which the light chain and the heavy chain are simultaneously expressed in one vector or a system in which the light chain and the heavy chain are expressed in separate vectors. In the latter case, the two vectors are introduced into the host cell through co-transformation and targeted transformation.
  • Co-transformation is a method of simultaneously introducing each vector DNA encoding the light chain and the heavy chain into a host cell, and then selecting cells expressing both the light chain and the heavy chain.
  • Targeted transformation selects cells transformed with a vector containing a light chain (or heavy chain) and transforms the selected cells expressing the light chain with a vector containing a heavy chain (or light chain) to express both light and heavy chains. It is a method for final selection of cells.
  • the present invention provides a host cell comprising the recombinant vector of the present invention.
  • the host cell is a cell transformed with the recombinant vector of the present invention.
  • Any host cell that can stably and continuously clone and express the vector of the present invention can be used as any host cell known in the art, for example, Escherichia coli , Bacillus subtilis and Bacillus thuringen. strains of the genus Bacillus, such as Cis, Streptomyces , Pseudomonas (e.g. Pseudomonas putida ), Proteus mirabilis or Staphylococcus (e.g. For example, Staphylococcus carnosus ( Staphylocus carnosus )), but includes prokaryotic host cells such as, but are not limited thereto.
  • Suitable eukaryotic host cells of the vector are fungi such as Aspergillus species , Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces ) and Neurospora crassa , other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.
  • transformation refers to introducing a desired gene into a host cell using the recombinant vector of the present invention, and is used in the same sense as “transfection”.
  • transformation and/or “transfection” into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue, or organ, and as is known in the art, using standard techniques suitable for the host cell. You can choose to do it.
  • These methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation using silicon carbide fibers, agrobacterium mediated transformation, PEG, dextran sulfate, lipo pectamine and desiccation/inhibition mediated transformation methods; and the like.
  • the present invention provides a method for detecting TSP-2 (Thrombospondin-2) protein in a sample comprising the step of contacting the antibody or antigen-binding fragment thereof of the present invention to the sample.
  • TSP-2 Thrombospondin-2
  • the antibody of the present invention can be applied to a biological sample to detect TSP-2 (Thrombospondin-2) protein.
  • biological sample means tissue, cell, whole blood, serum, plasma, saliva, urine, lymph fluid, spinal fluid, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic and bacterial expression systems, etc., but are not limited thereto.
  • the sample is tissue, cell, whole blood, serum, plasma, saliva, urine, lymph or spinal fluid.
  • Detection of TSP-2 (Thrombospondin-2) protein in biological samples includes colorimetric method, electrochemical method, fluorescence method, luminometry, particle counting method, It can be performed through detection of antigen-antibody complex formation using visual assessment or scintillation counting method. “Detection” in the present specification is for detecting an antigen-antibody complex and can be performed using various markers. Specific examples of the label include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioactive isotopes.
  • Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, ⁇ -D-galactosidase, horseradish peroxidase and ⁇ -latamase, etc. Fluorescein Phosphorus, Eu 3+ , Eu 3+ chelate or cryptate, etc. are included, ligands include biotin derivatives, etc., luminous substances include acridinium esters and isoluminol derivatives, etc., and microparticles include colloids. It includes gold and colored latex, etc., and radioactive isotopes include 57 Co, 3 H, 125 I and 125 I-Bolton Hunter reagent and the like.
  • antigen-antibody complexes can be detected using enzyme immunosorbent assay (ELISA).
  • Enzyme immunosorbent methods include direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and solid support Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support It includes a variety of ELISA methods, such as indirect sandwich ELISA using labeled secondary antibodies.
  • the antibody of the present invention may have a detection label, and when it does not have a detection label, the antibody of the present invention can be captured and another antibody having a detection label can be treated and identified.
  • the present invention provides a composition for diagnosis of fibrotic disease comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.
  • the TSP-2 protein of the present invention can be detected according to an immunoassay method using an antigen-antibody reaction and used to analyze whether or not a fibrotic disease occurs.
  • an immunoassay can be performed according to various immunoassay or immunostaining protocols previously developed.
  • antibodies labeled with radioactive isotopes may be used.
  • the antibody specifically recognizing the TSP-2 protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
  • Antibodies of the present invention can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,567 ) or phage antibody library methods (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). .
  • fusion methods Kellow and Milstein, European Journal of Immunology , 6:511-519 (1976)
  • diagnosis includes determination of a subject's susceptibility to a particular disease, determination of whether a subject currently has a particular disease, and determination of the prognosis of a subject suffering from a particular disease. do.
  • the term “diagnostic composition” refers to a conventional mixture or equipment including a means for measuring the expression level of a TSP-2 protein or a gene encoding the same in order to determine whether a subject has a fibrotic disease or to predict the possibility of occurrence of a fibrotic disease ( device), which can also be expressed as a “diagnostic kit”.
  • the present invention provides a composition for diagnosis of fibrotic disease comprising, as an active ingredient, an agent for measuring the expression level of TSP-2 (Thrombospondin-2) protein or a gene encoding it do.
  • TSP-2 Thrombospondin-2
  • the agent for measuring the expression level of the gene encoding TSP-2 is a primer or probe that specifically binds to the nucleic acid molecule of the gene.
  • primer refers to conditions in which synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of nucleotides and a polymerizer such as DNA polymerase, synthesis under conditions of suitable temperature and pH. refers to an oligonucleotide that serves as the starting point of Specifically, the primer is a single chain deoxyribonucleotide.
  • Primers used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides, and may also include ribonucleotides.
  • the primer of the present invention may be an extension primer that anneals to a target nucleic acid to form a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which is extended to a position where the immobilized probe is annealed, so that the probe becomes occupies the annealed area.
  • the extension primer used in the present invention includes a hybrid nucleotide sequence complementary to a specific nucleotide sequence of a gene encoding a target nucleic acid, for example, the TSP-2 protein.
  • the term "complementary" means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, substantially complementary and perfectly complementary. ), and specifically means completely complementary cases.
  • substantially complementary sequence is intended to include not only completely identical sequences, but also sequences that are partially inconsistent with the sequence to be compared, within the range of annealing to a specific sequence and acting as a primer.
  • the primer must be long enough to prime the synthesis of the extension product in the presence of the polymerization agent.
  • the suitable length of a primer depends on a number of factors, such as temperature, pH and the source of the primer, but is typically 15-30 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.
  • the design of such primers can be easily performed by those skilled in the art by referring to the target nucleotide sequence, and can be performed using, for example, a primer design program (eg, PRIMER 3 program).
  • the term “probe” refers to a natural or modified monomer including deoxyribonucleotide and ribonucleotide capable of hybridizing to a specific nucleotide sequence, or a linear oligomer having a linkage. Specifically, the probe is single-stranded for maximum efficiency in hybridization, more specifically a deoxyribonucleotide.
  • a sequence perfectly complementary to a specific nucleotide sequence of the gene encoding the TSP-2 protein may be used, but substantially within a range that does not interfere with specific hybridization. ) complementary sequences may be used. In general, since the stability of a duplex formed by hybridization tends to be determined by the matching of the terminal sequence, it is preferable to use a probe complementary to the 3'-end or 5'-end of the target sequence. do.
  • an aptamer that specifically binds to the TSP-2 protein may be used instead of an antibody to measure the activity or expression level of the protein.
  • the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity.
  • RNA or DNA nucleic acid
  • peptide molecule that binds to a specific target substance with high affinity and specificity.
  • the present invention has a light chain CDR (complementarity determining region) amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO: 4, LCDR2 consisting of the sequence of SEQ ID NO: 5, and LCDR3 consisting of the sequence of SEQ ID NO: 6
  • LCDR1 consisting of the sequence of SEQ ID NO: 4
  • LCDR2 consisting of the sequence of SEQ ID NO: 5
  • LCDR3 consisting of the sequence of SEQ ID NO: 6
  • TSP-2 Thrombospondin-2
  • LCDRs light chain CDRs
  • the present invention provides a composition for preventing or treating fibrotic disease comprising an inhibitor for TSP-2 (Thrombospondin-2) as an active ingredient.
  • TSP-2 Thrombospondin-2
  • composition for preventing or treating fibrotic diseases using the antibody used in the present invention and the meaning of the prevention or treatment have already been described above, the description thereof will be omitted to avoid excessive redundancy.
  • the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of a target gene, whereby the activity or expression of the target gene becomes undetectable or present at an insignificant level, as well as the It refers to a substance that reduces activity or expression to the extent that biological function can be significantly reduced.
  • Inhibitors of target genes are, for example, shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) antisense oligonucleotides or targets that inhibit the expression of the gene at the gene level, the sequence of which is already known in the art All known in the art including, but not limited to, CRISPR systems containing guide RNAs recognizing genes, antibodies or aptamers that inhibit at the protein level, as well as compounds, peptides and natural products that inhibit their activity Means of inhibition at the gene and protein level can be used.
  • Antibodies and aptamers that specifically bind to and inhibit the activity or expression of the TSP-2 protein have already been described in detail, so description thereof is omitted to avoid excessive redundancy.
  • small hairpin RNA is a single strand consisting of 50-70 nucleotides forming a stem-loop structure in vivo , which is used to suppress the expression of a target gene through RNA interference. It refers to the RNA sequence that creates a tight hairpin structure.
  • long RNAs of 19-29 nucleotides complementary to both sides of the loop region of 5-10 nucleotides form a double-stranded stem, which is introduced into the cell through a vector containing a U6 promoter so that it is always expressed. It is transduced and is usually passed on to daughter cells, allowing inheritance of suppression of the target gene.
  • RNA refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, the blunt end or cohesive All ends are possible. As for the sticky end structure, both a structure with 3 ends protruding and a structure with 5 ends protruding are possible.
  • miRNA refers to a single-stranded RNA molecule that inhibits target gene expression through complementary binding with mRNA of a target gene while having a short stem-loop structure as an oligonucleotide that is not expressed in cells. do.
  • ribozyme is a type of RNA and refers to an RNA molecule having a function such as an enzyme that recognizes a specific RNA base sequence and cuts it itself.
  • a ribozyme is composed of a region that binds with specificity to a complementary nucleotide sequence of a target mRNA strand and a region that cleaves a target RNA.
  • PNA peptide nucleic acid
  • antisense oligonucleotide refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and performs translation into a protein, translocation into the cytoplasm, maturation, or all other functions.
  • Antisense oligonucleotides can be modified at one or more bases, sugars or backbone positions to enhance potency (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). .
  • the oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomic, heterocyclic sugarsulfones, and the like.
  • gRNA guideRNA
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the present invention provides a screening method for a composition for preventing or treating fibrotic disease comprising the following steps:
  • TSP-2 Thrombospondin-2
  • the test substance is determined as a composition for preventing or treating fibrotic disease.
  • TSP-2 protein used in the present invention and diseases that can be prevented or treated through regulation of its expression have already been described in detail, description thereof will be omitted to avoid excessive redundancy.
  • biological sample is any sample containing cells expressing the above-described proteins obtained from mammals, including humans, and includes, but is not limited to, tissues, organs, cells, or cell cultures. More specifically, the biological sample may be a lung-derived tissue, cell, or culture thereof.
  • test substance used while referring to the screening method of the present invention is added to a sample containing cells expressing the protein or gene of the present invention to examine whether or not it affects the activity or expression level of these proteins or genes.
  • the test substance includes, but is not limited to, compounds, nucleotides, peptides and natural extracts.
  • the step of measuring the expression level or activity of the protein or gene in the biological sample treated with the test substance may be performed by various methods for measuring the expression level and activity known in the art.
  • the present invention provides a method for diagnosing fibrotic disease comprising administering to a subject a composition comprising the antibody or antigen-binding fragment thereof as an active ingredient.
  • the present invention is a fibrosis comprising the step of administering to a subject a composition comprising, as an active ingredient, an agent for measuring the expression level of TSP-2 (Thrombospondin-2) protein or a gene encoding it.
  • TSP-2 Thrombospondin-2
  • a method for diagnosing fibrotic disease is provided.
  • the present invention provides a method for preventing or treating fibrotic disease (fibrotic disease) comprising a composition containing an inhibitor for TSP-2 (Thrombospondin-2) as an active ingredient.
  • fibrotic disease fibrotic disease
  • TSP-2 Thrombospondin-2
  • the present invention uses an antibody that specifically binds to thrombospondin-2 (TSP-2) protein involved in fibrosis to predict the occurrence of fibrotic disease or suppresses the activity of the protein to prevent fibrotic disease.
  • TSP-2 thrombospondin-2
  • the present invention newly presents TSP-2 as a novel diagnosis and treatment target for idiopathic pulmonary fibrosis, for which no effective treatment method has been available due to unknown pathogenesis, and effective diagnosis, prevention and treatment of idiopathic pulmonary fibrosis. Provides effective antibodies that can be used for
  • the present invention can be usefully used to efficiently block fibrosis in various tissues by ultimately providing a method for fundamentally suppressing pathological fibrosis mechanisms.
  • FIG. 1B is a diagram showing the results of performing Robust Multi-array Average (RMA) background correction and data normalization of TSP-2 using GSE110147, a publicly available database.
  • RMA Robust Multi-array Average
  • Figure 2 is a picture showing the results of the serum level of TSP-2 in a healthy control group and interstitial lung disease (ILD) patients through a quantitative sandwich enzyme immunoassay (Quantitative Sandwich Enzyme Immunoassay).
  • Figure 3a is a diagram showing an experimental plan for a mouse lung fibrosis model through bleomycin.
  • Figure 3b is a picture showing the result of confirming the increased TSP-2 due to bleomycin in concentrated bronchoalveolar lavage fluid (BALF) by Western blot.
  • Figure 3c is a picture showing the increase in inflammatory cells in the mouse lung fibrosis model as a result of BAL fluid total cell counting.
  • Figure 3d is a picture showing the results of qRT-PCR for TSP-2 in mouse lung tissue.
  • Figure 3e is a diagram illustrating the doxycycline dosing regimen for generating epithelial Ccsp-TGF- ⁇ 1-TG mice.
  • FIG. 3f is a picture showing the result of immunoblot confirmation of increased TSP-2 due to doxycycline in concentrated bronchoalveolar lavage fluid (BALF).
  • FIG. 3g is a diagram showing an increase in inflammatory cells due to doxycycline as a result of total cell counting in BAL fluid.
  • Figure 3h is a picture showing that TSP-2 expression increased in mouse lung tissue by doxycycline administration.
  • Figure 4a is a graph showing the decrease in mRNA representing TSP-2 expression induced by TGF- ⁇ as a result of knockdown of Smad4 in MLg and C22 cell lines.
  • Figure 4b is a picture showing the result of immunoblot confirming that the amount of TSP-2 protein induced by TGF- ⁇ was reduced as a result of Smad4 knockdown in MLg and C22 cell lines.
  • FIG. 5A is a graph showing that a selective inhibitor of TGF- ⁇ 1 inhibits TSP-2 expression induced by TGF- ⁇ 1 in a dose dependent manner.
  • Figure 5b is a picture showing the result of Western blot confirming that a selective inhibitor of TGF- ⁇ 1 inhibits TSP-2 expression induced by TGF- ⁇ 1 in a dose dependent manner.
  • Figure 6a is a schematic diagram showing Smad-binding elements within the 4 kb TSP-2 gene promoter.
  • Figure 6b is a diagram showing the results of examining the activity of the promoter according to the deletion of the promoter portion by luciferase reporter assay.
  • Figure 6c is a diagram showing the results of examining the activity of the promoter according to the promoter partial deletion in the absence of TGF- ⁇ 1 by luciferase reporter assay.
  • Figure 6d is a picture showing the results of performing a luciferase reporter assay using #6 Smad-binding site deletion in the presence of TGF- ⁇ 1.
  • FIG. 7A is a schematic diagram illustrating an autocrine and paracrine experiment.
  • Figure 7b is a picture showing the results of qRT-PCR analysis on the expression of the TSP-2 gene in lung cells, which was performed to determine the knockdown efficiency of TSP-2.
  • Figure 7c is a picture showing the results of Western blotting on the expression of the TSP-2 gene in lung cells, which was performed to determine the knockdown efficiency of TSP-2.
  • Figure 7d is a picture showing the results of measuring fibroblast counts to determine TGF- ⁇ 1-induced fibroblast (autocrine) activation.
  • Figure 7e is a picture showing the results of qRT-PCR to determine whether fibrosis markers (target genes) were upregulated by TGF- ⁇ 1 administration and downregulated by TSP-2 knockdown.
  • FIG. 8A is a diagram showing the results of qRT-PCR analysis for the expression of the TSP-2 gene, which was performed to examine the knockdown efficiency of TSP-2 in lung epithelial cells.
  • FIG. 8B is a picture showing the results of Western blotting on the expression of the TSP-2 gene, which was performed to examine the knockdown efficiency of TSP-2 in lung epithelial cells.
  • 8c is a diagram showing the results of measuring fibroblast counts in order to determine TGF- ⁇ 1-induced fibroblast (paracrine) activation.
  • FIG. 8D is a diagram showing qRT-PCR results for determining whether fibrosis markers (target genes) in lung epithelial cells were up-regulated by TGF- ⁇ 1 administration and down-regulated by TSP-2 knockdown.
  • FIG. 9a is a graph showing that target inhibition of TSP-2 reduces TGF- ⁇ 1-induced fibroblast activation in a dose-dependent manner, in terms of fibroblast cell number.
  • FIG. 9B is a graph showing the results of qRT-PCR showing that the expression of TGF- ⁇ 1 -induced fibrosis markers (target genes) is reduced by target inhibition of TSP-2 by a single neutralizing antibody specific for TSP-2.
  • 10a is a diagram showing that target inhibition of TSP-2 in epithelial cells (C22) reduces TGF- ⁇ 1 -induced fibroblast activation in a dose-dependent manner, in terms of the number of fibroblast cells.
  • Figure 10b shows that the expression of fibrosis markers (target genes) induced by TGF- ⁇ 1 is reduced by the targeted inhibition of TSP-2 by a single neutralizing antibody specific for TSP-2 in epithelial cells (C22). This is a PCR result graph.
  • 10c is a diagram showing that target inhibition of TSP-2 in epithelial cells (RLE-6TN) reduces TGF- ⁇ 1 -induced fibroblast activation in a dose-dependent manner, in terms of the number of fibroblast cells.
  • TGF- ⁇ 1 -induced fibrosis markers targets genes
  • RLE-6TN epithelial cells
  • FIG. 11a is a diagram showing the results of dot-blot for 7 clones performed to produce a monoclonal antibody against TSP-2.
  • Figure 11b is a picture showing the results of Western blotting in lung fibroblasts performed to determine the antibody affinity of 7 clones.
  • Figure 11c is a graph comparing the fibroblast proliferation inhibitory ability of #2 and #4 clone antibodies.
  • 11d is a graph comparing the ability of the final antibody and commercially available antibody to inhibit fibroblast proliferation after TGF- ⁇ treatment.
  • Serum was separated using serum separation tubes, and serum levels of TSP-2 (excluding patients with late-stage ILD) were measured using the Human Thrombospondin-2 quantikine ELISA kit (R&D Systems Inc., USA) according to the manufacturer's instructions.
  • mice were from the C57BL/6 genetic background and housed in a 12h-day/12h-night cycle.
  • mice were anesthetized with an intraperitoneal injection of Zoletil (30 mg/kg) and Rompun (10 mg/kg). Mice were injected intratracheally with PBS (vehicle control) or 4 mg/kg bleomycin (Santa Cruz Biotechnology, Dallas, TX, USA). An average of 8 mice were used in each group, and the mice were sacrificed on day 14 after administration of bleomycin and bronchial lavage (BAL), and lung tissues were collected.
  • TGF- ⁇ 1-TG mice were provided by Professor Myeong-Hyeon Sohn's laboratory at Yonsei University. To induce TGF- ⁇ 1 expression, adult transgenic mice (8-12 weeks old) were housed for 4 weeks with drinking water containing 2% sucrose and 0.5 mg/mL doxycycline. Water containing doxycycline was changed three times a week.
  • mice were anesthetized with zoletyl/lumpoon, and an incision was made in the abdomen and thorax to expose the heart and lungs.
  • Lungs were perfused via the pulmonary artery using 10 mL PBS.
  • the left lobe of the mouse lung was removed, fixed in formaldehyde (4% w/v) for 48 hours at room temperature, and embedded in paraffin. Sectionalization of paraffin block sections was performed at the Histology Core, Yonsei University College of Medicine, Seoul.
  • Sections were deparaffinized and hydrated with water to confirm collagen production by Mason's tricolor staining. Sections were stained in Bouin's solution (#HT10132, Merck, Darmstadt, Germany) for 1 minute using a microwave and then left at room temperature for 15 minutes. After washing with running tap water, the sections were immersed in hematoxylin for 10 minutes, rinsed again with running tap water, and stained with Biebrich scarlet for 5 minutes. Then, the sections were stained with phosphotungstic/phosphomolybdic for 15 min, immediately transferred to aniline blue for 5 min, dehydrated and covered with a coverslip.
  • Bouin's solution #HT10132, Merck, Darmstadt, Germany
  • the mouse fibroblast cell line MLg and the mouse alveolar type 2 cell line RLE-6TN were purchased from Korea Cell Line Bank (Seoul, Korea). MLg and RLE-6TN were cultured in Dulbecco's modified Eagles' medium supplemented with 10% (v/v) fetal bovine serum and 1% antibiotic/antimycotic solution (Corning, Manassas, VA, USA) at 37°C; Incubated in 5% CO 2 .
  • the amplified mixture was treated with DpnI (Aglient Technologies, Santa Clara, CA, USA) at 37°C for 1 hour and 30 minutes, and the PCR product was used to transform water-soluble E.coli (Real Biotech Corporation, Banqiao, Taiwan). . All products were confirmed through DNA sequencing.
  • a specific region (-1700 to +300 nucleotides) of the TSP-2 gene was prepared by amplifying genomic DNA obtained from wild-type C57BL/6 mouse tissue (QIAGEN, DNA mini kit). The amplified product was purified, digested with Nhe-IHF and XhoI restriction enzymes, and cloned into the pGL3-Control vector (Promega). This TSP-2 reporter gene was used as a template for a deletion construct. To perform the luciferase reporter assay, cells were transiently transfected with 1 ⁇ g of the TSP-2 reporter gene per well in 24-well plates using Lipofectamine 3000 (Invitrogen, Carlsbad, Calif., USA).
  • Luminescence was measured with a MicroLumatPlus LB96V Microplate Luminometer (Berthold).
  • the lentiviral vector (shTSP-2) used in this study to generate cell lines for stable knockdown of TSP-2 was purchased from Sigma-Aldrich, USA.
  • Auxiliary plasmid liposomes (pxPAX.2 and pMD2.G) and lentiviral vectors were transfected into 293FT cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) to produce lentiviruses. 48 hours after transfection, the supernatant was collected and centrifuged to remove cell debris. The centrifuged supernatant was filtered using a 0.45 ⁇ m polyvinylidene difluoride filter.
  • C22, RLE-6TN and MLg cells were infected with virus suspension mixed with 10 ⁇ g/ml polybrene. 48 hours after infection, puromycin at 2 ⁇ g/ml was added to screen for stably transduced positive cell lines.
  • lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100, 1.5% MgCl2, 1 mM EDTA, 1 mM Na2VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail, pH 7.5).
  • PMSF phenylmethylsulfonyl fluoride
  • the lysate was briefly vortexed and sonicated, followed by centrifugation at 12,000 x g for 20 min at 4°C to remove by-products. The supernatant was collected and transferred to a new tube. Protein concentration was determined with 660 nm protein assay reagent (Thermo Scientific, Waltham, MA, USA).
  • Equal amounts of protein extracts were electrophoresed on SDS-polyacrylamide gels and then transferred to nitrocellulose transfer membranes (Whatman, Dassel, Germany). Membranes were formulated with 0.1% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA), 5% (w/v) nonfat DifcoTM skim milk (BD Biosciences, Sparks, MD, USA) and 3% BSA. (Affymetrix, Santa clara OH, USA), and primary antibodies were added.
  • anti-TSP-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) purchased from Cell Signaling (Danvers, MA, USA), anti-Smad3, anti-p- Smad3 and anti-Smad4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti- ⁇ -actin (Sigma-Aldrich, St. Louis, MO, USA).
  • the membrane was then washed with 1x PBST and incubated for 1 hour with an appropriate secondary anti-rabbit or anti-mouse horseradish peroxidase conjugated antibody (Thermo Scientific, Rockford IL, USA), followed by a LAS-3000 system (Fujifilm , Stamford, CT, USA) and visualized through enhanced chemiluminescence detection reagent (Thermo Scientific).
  • RNA isolation and quantitative real-time chain reaction qRT-PCR
  • RNA concentration was measured with Nanodrop1000 (Thermo Scientific, Waltham, MA, USA). After RNA isolation, 1 ⁇ g of total RNA was used and cDNA was synthesized using CellScript (CellSafe, Seoul, Korea) according to the manufacturer's protocol.
  • qRT-PCR analysis was performed using an ABI PRISM 7000 Sequence Detection System instrument and software (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's protocol with some modifications. Briefly, an appropriate amount of the reverse transcription reaction mixture was amplified with specific primers using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). PCR primer sequences for qRT-PCR are listed in Table 1.
  • Expression levels of genes were determined by generating a 5-point continuous standard curve. The concentration of cDNA was normalized through ⁇ -Tubulin. All reactions were performed in triplicate, and relative expression levels and SD values were calculated using a comparative method.
  • mice Thbs2 forward primer GACCAAACCTACGCTGGTGGA (SEQ ID NO: 33)
  • mice Thbs2 reverse primer AGCACATTGCTGGGAGCTGGA (SEQ ID NO: 34)
  • mouse ⁇ -SMA forward primer GTGACTCACAACGTGCCTATC (SEQ ID NO: 35)
  • mouse ⁇ -SMA reverse primer CTCGGCAGTAGTCACGAAGG (SEQ ID NO: 36)
  • mouse FN forward primer AAGACCATACCTGCCGAATG (SEQ ID NO: 37)
  • mouse FN reverse primer GAACATGACCGATTTGGACC (SEQ ID NO: 38)
  • mouse col1a1 forward primer ATGGATTCCCGTTCGAGTACG (SEQ ID NO: 39)
  • mouse col1a1 reverse primer TCAGCTGGATAGCGACATCG (SEQ ID NO: 40)
  • mouse col1a2 forward primer TGCAGTAACTTCGTGCCTAGC (SEQ ID NO: 41)
  • mouse col1a2 reverse primer ACGTGGTCCTTCTGTCTCCA (SEQ ID NO: 42)
  • mouse col3a1 forward primer CTAAAATTCTGCCACCCCGAA (SEQ ID NO: 43)
  • mouse col3a1 reverse primer AGGATCAACCCAGTATTCTCCACTC (SEQ ID NO: 44)
  • mouse Snail forward primer TCTGAAGATGCACATCCGAAGCCA (SEQ ID NO: 45)
  • mouse Snail reverse primer AGGAGAATGGCTTCTCACCAGTGT (SEQ ID NO: 46)
  • mouse Smad4 forward primer CAGCCATAGTGAAGGACTGTTGC (SEQ ID NO: 47)
  • mouse Smad4 reverse primer CCTACTTCCAGTCCAGGTGGTA (SEQ ID NO: 48)
  • rat Thbs2 forward primer CAAGGACATGCAGTTTGGGC (SEQ ID NO: 49)
  • rat Thbs2 reverse primer GAAGACCAGGGTGACCAGTG (SEQ ID NO: 50)
  • rat ⁇ -SMA forward primer TTCATTGGAATGGAGTCGGCG (SEQ ID NO: 51)
  • rat ⁇ -SMA reverse primer CTGTCAGCAATGCCTGGGTA (SEQ ID NO: 52)
  • the medium of C22, RLE-6TN or MLg cells cultured in a 60-mm cell culture dish was harvested and passed through a 0.45 ⁇ m filter (Sartorius, Stonehouse, UK) and used as a conditioned medium. Additionally, after the culture medium was applied to a VIVASPIN6 column (Sartorius) and centrifuged at 10,000 x g for 2 hours, enriched proteins were calculated using a protein assay and then immunoblotted using antibodies. In the case of paracrine, a conditioned medium of C22 or RLE-6TN secreting Matricellular proteins was transferred to MLg.
  • Results were visualized and analyzed with Prism software version 5 (GraphPad Software, San Diego, CA, USA). When comparing two or more sample groups, one-way ANOVA was used for statistical significance. Data values are expressed as mean ⁇ standard deviation (SD). Student's t-test was used to determine the statistical significance of the two groups, and values of P ⁇ 0.05 were considered statistically significant.
  • An antibody was prepared using a peptide including a.a 928-930 (RGD domain) among 1,172 amino acids of TSP-2.
  • a commercially available Santacruz antibody sc-136238, mouse mono
  • recognizing a.a 173-295 was used.
  • TSP-2 amino acid sequence (NCBI Reference Sequence: NP_003238.2, thrombospondin-2 precursor [Homo sapiens]):
  • TSP-2 TSP-2-induced pulmonary fibrosis
  • Fibrosis-inducing secreted factors function as predictive biomarkers of fibrosis and regulators of ECM synthesis, and are thus promising potential therapeutic targets. Therefore, among the TSPs of fibrosis-inducing secreted factors, TSP-2 and the pathological relevance of lung diseases are investigated. To do this, the expression of TSP-2 was measured in lung tissue of IPF patients, serum of interstitial lung disease patients, and mouse models of pulmonary fibrosis.
  • TSP-1 and TSP-2 were found to be significantly elevated in samples from IPF patients compared to controls (FIGS. 1a and 1b) .
  • TSP-2 levels in healthy subjects and ILD patients were investigated, and the serum level of human TSP-2 in ILD patients was evaluated using a TSP-2 ELISA kit. As a result, it was confirmed that the level of serum TSP-2 was up-regulated in ILD patients.
  • a bleomycin-induced pulmonary fibrosis model and Ccsp-TGF 1-TG mice were used to investigate TSP-2 expression in a pulmonary fibrosis mouse model.
  • Bleomycin (BLM) is widely used for acute induction of pulmonary fibrosis in mice.
  • Bleomycin increases the level of TGF- ⁇ 1, a potent fibrotic cytokine, which plays an important role in IPF38.
  • TGF- ⁇ is a major inducer of epithelial-mesenchymal transition (EMT) and a major mediator of fibrosis in many tissues, including the lung.
  • EMT epithelial-mesenchymal transition
  • TSP-2 protein As shown in Figure 3a, the experiment was conducted in a bleomycin-induced pulmonary fibrosis model, and collagen accumulation could be observed.
  • BALF concentrated bronchoalveolar lavage fluid
  • FIG. 3b The upregulated expression of TSP-2 was confirmed by western blot analysis (FIG. 3b), and an increase in the total number of cells in BAL fluid was confirmed in the mouse lung fibrosis model by counting total cells in BAL fluid (FIG. 3c).
  • the expression of TSP-2 in the mouse lung fibrosis model was also elevated in mouse lung tissue (FIG. 3d). Accordingly, it was confirmed that the level of TSP-2 significantly increased in the lungs of mice after bleomycin injection (FIGS. 3a to 3d).
  • TSP-2 expression was elevated in IPF patients and mouse models of pulmonary fibrosis.
  • the transcription of the TSP-2 gene is activated through TGF - ⁇ -mediated SMAD-dependent signaling.
  • TSP-2 Since TSP-2 has been shown to activate TGF- ⁇ 28, the mRNA level of TSP-2 was evaluated in MLg (mouse fibroblasts) and C22 (mouse club cells) after TGF- ⁇ 1 treatment. According to the results of qRT-PCR and Western blot analysis, it was observed that the level of TSP-2 was increased by TGF- ⁇ 1 treatment.
  • TGF- ⁇ -induced TSP-2 expression is mediated by canonical TGF- ⁇ signaling
  • Smad4 knockdown was examined. Knockdown of Smad4 in MLg and C22 cell lines abolished mRNA levels in TGF- ⁇ -induced TSP-2 expression (FIG. 4A). In addition, the protein level of TGF- ⁇ -induced TSP-2 expression was also decreased (Fig. 4b). These results suggest that Smad-dependent canonical TGF- ⁇ signaling is required for transcriptional activation of the TSP-2 gene.
  • LY2157299 a selective TGF- ⁇ 1 inhibitor.
  • TSP-2 In order to analyze the transcription factor of the promoter region of TSP-2, it was searched using PROMO 3.0.2 tool. In particular, to further investigate whether Smad directly regulates TSP-2 transcription in response to TGF- ⁇ 1 treatment, the Smad-binding motif CAGAC or GTCTG in the TSP-2 gene promoter was searched. 6A schematically depicts several Smad binding elements of the TSP-2 promoter. Promoter activity was significantly reduced in the TSP-2 gene construct (-30 ⁇ 300 bp) promoter compared to the 2 kb (-1700 ⁇ 300 bp) promoter (Fig. 6b).
  • TSP-2 Knockdown of TSP-2 inhibits TGF- ⁇ 1-induced fibroblast activation.
  • TGF- ⁇ is a major mediator of fibrosis in many tissues and TSP-2 is regulated by TGF- ⁇ signaling
  • TGF- ⁇ increases the secretion of TSP-2 in both epithelial cells and fibroblasts, followed by autologous It was hypothesized to promote ECM deposition in fibroblasts via a secretory or paracrine mode. Therefore, TGF- ⁇ -induced fibroblast proliferation and target gene expression in fibroblasts were evaluated after blocking TSP-2 expression in lung epithelial cells (C22 and RLE-6TN) or lung fibroblasts (MLg).
  • qRT-PCR Fig. 7b, 8a
  • Western blot analysis Fig. 7c, 8b
  • Fig. 7a effects of TSP-2 knockdown on fibroblast activation were investigated.
  • paracrine signals the conditioned medium after knockdown of TSP-2 in epithelial cells was transferred to fibroblasts, and cell proliferation of fibroblasts was measured (FIG. 7a).
  • TSP-2 is required for TGF- ⁇ 1-induced fibroblast proliferation.
  • TGF- ⁇ 1 treatment significantly increased fibrosis target genes such as Collagen1a1 (Col1a1), Collagen1a2 (Col1a2), Collagen3a1 (Col3a1), Fibronectin (FN), and ⁇ -Smooth Muscle Actin ( ⁇ -SMA), whereas TSP Knockdown of -2 substantially inhibited TGF- ⁇ 1-induced upregulation of target genes (FIG. 7e).
  • TSP Knockdown of -2 in epithelial cells abolished the induction of fibrosis target genes in fibroblasts (Fig. 8d).
  • TSP-2 When TSP-2 is blocked with a TSP-2 monoclonal neutralizing antibody, fibroblast activation is inhibited.
  • TSP-2 Since inhibition of TSP-2 expression inhibited fibroblast activation, we investigated the effectiveness of a monoclonal antibody (mAb) against TSP-2 that can effectively inhibit TSP-2 function. Autocrine and paracrine effects of TSP-2 blockade on fibroblast activation were examined. First, to confirm the blocking action of the TSP-2 monoclonal antibody, the inhibitory effect of TGF- ⁇ 1-induced fibroblast activation on fibroblast proliferation was evaluated. Then, whether treatment with TSP-2 monoclonal antibody suppresses target genes of fibrosis markers similarly to knockdown results was investigated. Next, it was investigated whether treatment with TSP-2 monoclonal antibody suppresses target genes of fibrosis markers similarly to knockdown results.
  • mAb monoclonal antibody
  • Fibroblasts were treated with TGF- ⁇ 1 (20 ng/ml) and TSP-2 monoclonal antibody according to the concentration, and then TGF- ⁇ 1-induced fibroblast proliferation was analyzed by cell counting.
  • CTGF monoclonal antibody treatment was used as a positive control for antibody blocking experiments, and TGF- ⁇ 1-induced fibroblast proliferation in conditioned medium with anti-TSP-2 monoclonal antibody was significantly reduced in a dose-dependent manner (Fig. 9a).
  • TGF- ⁇ 1-induced fibrosis target gene expression could be decreased in fibroblasts in a dose-dependent manner through targeted inhibition of TSP-2 through a TSP-2 monoclonal neutralizing antibody (FIG. 9B).
  • epithelial cells were treated with TSP-2 monoclonal antibody and TGF- ⁇ 1 (20 ng/ml), and the conditioned medium was transferred to fibroblasts, and cell proliferation of the fibroblasts was measured.
  • TGF- ⁇ 1-induced fibroblast proliferation in conditioned medium with anti-TSP-2 monoclonal antibody was reduced in a dose dependent manner (FIG. 10A).
  • target inhibition of TSP-2 by the anti-TSP-2 monoclonal antibody efficiently suppressed TGF- ⁇ 1-induced fibrosis target gene expression in fibroblasts in a dose-dependent manner (FIG. 10B). Therefore, these results demonstrated that anti-TSP-2 mAb inhibits TGF- ⁇ 1-induced fibroblast activation by binding to secreted TSP-2 protein and neutralizing TSP-2 in an autocrine and paracrine manner. .
  • HCDR1 GYSFTGYY (SEQ ID NO: 1)
  • HCDR2 VNPNNGGI (SEQ ID NO: 2)
  • HCDR3 ARDGAY (SEQ ID NO: 3)
  • LCDR1 QSLLDSDGKTY (SEQ ID NO: 4)
  • LCDR2 LVS (SEQ ID NO: 5)
  • LCDR3 WQGTHFPFT (SEQ ID NO: 6)
  • HFR1 EVQLQQSGPDLVKPGASVKISCKAS (SEQ ID NO: 7)
  • HFR2 MHWVKQSHEKSLEWIGR (SEQ ID NO: 8)
  • HFR3 SYNQKFRGKAILTVDRSSNTAYMELRSLTSEDSAVYYC (SEQ ID NO: 9)
  • HFR4 WGQGTLVTVSA (SEQ ID NO: 10)
  • LFR1 DVVMTQTPLTLSVTIGQPASISCKSS (SEQ ID NO: 12)
  • LFR2 LNWLLQRPGQSPKRLIY (SEQ ID NO: 13)
  • LFR3 KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYYC (SEQ ID NO: 14)
  • LFR4 FGSGTKLEIK (SEQ ID NO: 15)
  • HCDR1 GGTTACTCATTCACTGGCTACTAC (SEQ ID NO: 17)
  • HCDR2 GTTAATCCTAACAATGGTGGTATA (SEQ ID NO: 18)
  • HCDR3 GCAAGAGATGGTGCTAC (SEQ ID NO: 19)
  • LCDR1 CAGAGCCTCTTAGATAGTGATGGAAAGACATAT (SEQ ID NO: 20)
  • LCDR2 CTGGTGTCT (SEQ ID NO: 21)
  • LCDR3 TGGCAAGGTACACATTTTCCATTCACG (SEQ ID NO: 22)
  • HFR2 ATGCACTGGGTGAAACAGAGCCATGAAAAGAGCCTTGAGTGGATTGGACGT (SEQ ID NO: 24)
  • HFR4 TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 26)
  • LFR2 TTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGGCTAATCTAT (SEQ ID NO: 29)
  • LFR4 TTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 31)

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Abstract

The present invention relates to a method for prevention or treatment of fibrotic disease, wherein an antibody binding specifically to thrombospondin-2 (TSP-2) protein involved in fibrosis is used for predicting the onset of fibrotic disease or for inhibiting the activity of the corresponding protein. The present invention presents TSP-2 as a new diagnostic and therapeutic target for idiopathic pulmonary fibrosis, for which there have been no effective treatment methods because a clear pathogenesis is not known, and provides an effective antibody that can be used for effective diagnosis, prevention, or treatment of idiopathic pulmonary fibrosis. In addition, the present invention can be advantageously used to effectively block fibrosis in various tissues by ultimately providing a fundamental inhibition method for pathological fibrosis mechanisms.

Description

섬유화 질환의 예방 또는 치료용 신규 항체 Novel antibodies for prevention or treatment of fibrotic diseases
본 발명은 섬유화에 관여하는 트롬보스폰딘-2(Thrombospondin, TSP-2) 단백질에 특이적으로 결합하는 항체를 이용하여 섬유화 질환 발생 여부를 예측하거나, 해당 단백질의 활성을 억제함으로써 섬유화 질환을 예방 또는 치료하는 방법에 관한 것이다.The present invention predicts the occurrence of fibrotic disease using an antibody that specifically binds to thrombospondin (TSP-2) protein involved in fibrosis, or prevents or prevents fibrotic disease by inhibiting the activity of the protein. It's about how to treat.
특발성 폐섬유증(IPF)은 심각한 만성 간질성 폐질환으로, 전 세계에 수백만 명의 환자에게 영향을 미치는 치명적이고 진행성인 질환이다. 해당 질환은 환자의 폐에 흉터를 남기면서 폐를 딱딱하게 만들어 호흡 곤란 등의 중대한 증상을 초래하며, 증상이 대개 몇 년간에 걸쳐 천천히 진행하기 때문에, 일상 생활에 불편이 생길 정도로 증상이 심해지고 나서야 의사의 진료를 받는 경우가 많아서 더욱 위험한 질환이다. 또한, 종래에 특발성 폐섬유증은 치료가 매우 어려워 완치가 거의 불가능한 질환으로 알려져 있으며, 현재 해당 질병에 특이적으로 효과적인 약물이 전무하며, 말기 환자들의 경우에 제한적으로 폐 이식만이 이용되고 있는 실정이다. 이러한 이유로 본 질환의 치료제에 대한 업계의 요구가 높으나, 병인이 정확하게 알려져 있지 않아서 치료제 개발에 어려움이 많은 상황이다.Idiopathic pulmonary fibrosis (IPF) is a serious chronic interstitial lung disease, a fatal and progressive disease that affects millions of patients worldwide. The disease scars the patient's lungs and hardens them, resulting in serious symptoms such as shortness of breath. Because the symptoms usually progress slowly over several years, it is not until symptoms become severe enough to interfere with daily life. It is a more dangerous disease because it is often treated by a doctor. In addition, conventionally, idiopathic pulmonary fibrosis is known as a disease that is very difficult to treat and is almost impossible to cure. Currently, there is no drug specifically effective for the disease, and only lung transplantation is used limitedly for terminally ill patients. . For this reason, there is a high demand in the industry for a therapeutic agent for this disease, but it is difficult to develop a therapeutic agent because the etiology is not accurately known.
섬유증(Fibrosis)은 과도한 섬유질 결합 조직의 형성 및 침착을 주 특징으로 하는 회복 또는 반응성 과정으로, 여러 섬유화 질환(Fibrotic Disease) 및 만성 질환의 주요 병인으로 작용한다. 특히, 폐 섬유증은 정확한 기작은 알려져 있지 않으나, 세포 외 기질(Extracellular Matrix, ECM)의 과도한 침착 및 비정상적인 폐포 상피 세포의 손상으로 발생한다고 알려져 있다. 정상적인 상태에서 섬유아세포(fibroblast)는 상처 회복 및 결합 조직 생성에 중요한 역할을 수행하나, 조절되지 않은 지나친 섬유아세포의 발현으로 인하여 병소(foci)에 과도한 면역세포 및 ECM 단백질이 축적되는 경우 병리학적인 상태로 발전하게 되는 것이다. 이러한 개략적인 기작 외에, 폐섬유증의 예측인자 및 발병기전은 여전히 모호하기 때문에, 이에 대한 정확한 분자 및 생물학적 기작을 밝히는 것은 폐섬유증의 진단 또는 치료에 매우 중요한 요소라고 할 수 있다.Fibrosis is a reparative or reactive process characterized by the formation and deposition of excessive fibrous connective tissue, and serves as a major etiology of several fibrotic diseases and chronic diseases. In particular, pulmonary fibrosis is known to occur due to excessive deposition of extracellular matrix (ECM) and damage to abnormal alveolar epithelial cells, although the exact mechanism is not known. In a normal state, fibroblasts play an important role in wound healing and connective tissue generation, but excessive immune cells and ECM proteins accumulate in foci due to uncontrolled expression of excessive fibroblasts, resulting in pathological conditions. will develop into In addition to these schematic mechanisms, predictors and pathogenesis of pulmonary fibrosis are still ambiguous, so identifying the exact molecular and biological mechanisms for them is a very important factor in diagnosing or treating pulmonary fibrosis.
한편, ECM은 비-구조적(non-structural) 기질 단백질로, 섬유증에서 비정상적인 침착이 관찰된다. 이러한 ECM과 결합하는 30여 종의 세포외기질-부착 단백질이 현재 알려져 있는데, 트롬보스폰딘(Thrombospondin, TSP)도 그 중 하나이다. 이러한 세포외기질-부착 단백질은 세포와 세포외기질을 연결하는 역할을 하여 세포-기질 상호작용의 조절자로서 중요한 역할을 수행한다. 특히 TSP 패밀리에 속하는 TSP-2 단백질의 경우, 섬유아세포에 의하여 분비되며 기질(matrix)의 리모델링에 관여한다.On the other hand, ECM is a non-structural matrix protein, and abnormal deposition is observed in fibrosis. About 30 kinds of extracellular matrix-adhesion proteins that bind to such ECM are currently known, and Thrombospondin (TSP) is one of them. These extracellular matrix-adhesion proteins serve to connect cells and extracellular matrix and play an important role as regulators of cell-matrix interactions. In particular, the TSP-2 protein belonging to the TSP family is secreted by fibroblasts and is involved in matrix remodeling.
이에, 본 발명자들은 TSP 패밀리 중 특히 TSP-2와 폐 섬유증과의 연관성에 집중하여, TSP-2가 폐 섬유증의 발생에 기여함을 입증하고자 하였으며, 해당 연구를 통하여 TSP-2 단백질과 섬유화의 관계성을 입증하는 동시에 이를 섬유증의 치료 타겟으로 하여 TSP-2에 특이적인 단일클론 항체를 개발하고, 해당 항체의 항-섬유화 효과를 입증하고자 하였다.Therefore, the present inventors focused on the relationship between TSP-2 and pulmonary fibrosis among the TSP family, and tried to prove that TSP-2 contributes to the development of pulmonary fibrosis, and through this study, the relationship between TSP-2 protein and fibrosis At the same time, we tried to develop a monoclonal antibody specific to TSP-2 using it as a treatment target for fibrosis, and demonstrate the anti-fibrotic effect of the antibody.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.A number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.
본 발명자들은 전 세계적으로 높은 유병률을 가지면서 폐 이식 등을 제외하고는 유효한 치료 방법이 전무한 난치성 질환인 특발성 폐섬유증(Idiopathic Pulmonary Fibrosis, IPF)의 발병 기전을 밝히는 동시에, 이에 대한 효율적인 치료 방법을 발굴하고자 예의 연구 노력하였다. 그 결과, 폐 섬유화의 발병 기전에서 TSP-2가 섬유아세포-매개 섬유화(섬유증)를 일으킨다는 사실을 최초로 규명하고 TSP-2가 치료 타겟이 될 수 있음을 확인하였을 뿐 아니라, 동시에 TSP-2를 특이적으로 인식 및 억제하는 신규한 단일클론 중화항체를 개발함으로써, 본 발명을 완성하게 되었다.The present inventors uncovered the pathogenesis of idiopathic pulmonary fibrosis (IPF), an incurable disease for which there is no effective treatment method except for lung transplantation, which has a high prevalence worldwide, and at the same time discovered an efficient treatment method for it. I tried diligently to do research. As a result, the fact that TSP-2 causes fibroblast-mediated fibrosis (fibrosis) in the pathogenesis of pulmonary fibrosis was identified for the first time, and TSP-2 could be a therapeutic target. At the same time, TSP-2 The present invention has been completed by developing a novel monoclonal neutralizing antibody that specifically recognizes and inhibits.
따라서 본 발명의 목적은 TSP-2 단백질에 대한 항체 또는 그의 항원 결합 단편 및 이를 코딩하는 핵산분자를 제공하는 데 있다.Accordingly, an object of the present invention is to provide an antibody or antigen-binding fragment thereof against the TSP-2 protein and a nucleic acid molecule encoding the same.
본 발명의 다른 목적은 섬유화 질환(fibrotic disease)의 예방 또는 치료용 조성물을 제공하는 데 있다. Another object of the present invention is to provide a composition for preventing or treating fibrotic disease.
본 발명의 또 다른 목적은 본 발명의 항체 또는 그의 항원 결합 단편을 코딩하는 핵산분자; 상기 핵산분자를 포함하는 재조합 벡터 및 상기 재조합 벡터를 포함하는 숙주세포를 제공하는 데 있다.Another object of the present invention is a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention; It is to provide a recombinant vector containing the nucleic acid molecule and a host cell containing the recombinant vector.
본 발명의 또 다른 목적은 본 발명의 항체 또는 그의 항원 결합 단편의 제조방법을 제공하는 데 있다.Another object of the present invention is to provide a method for producing the antibody or antigen-binding fragment thereof of the present invention.
본 발명의 또 다른 목적은 본 발명의 항체 또는 그의 항원 결합 단편을 이용한 시료 내 TSP-2 단백질의 존재여부를 확인하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for confirming the presence or absence of TSP-2 protein in a sample using the antibody or antigen-binding fragment thereof of the present invention.
본 발명의 또 다른 목적은 본 발명의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 진단용 조성물을 제공하는데 있다.Another object of the present invention is to provide a diagnostic composition comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 서열번호 1의 서열로 이루어진 HCDR1, 서열번호 2의 서열로 이루어진 HCDR2 및 서열번호 3의 서열로 이루어진 HCDR3의 중쇄 CDR(complementarity determining region) 아미노산 서열을 갖는 중쇄 가변영역을 포함하는, TSP-2 (Thrombospondin-2) 단백질에 대한 항체 또는 그의 항원 결합 단편을 제공한다.According to one aspect of the present invention, the present invention is a heavy chain having a heavy chain CDR (complementarity determining region) amino acid sequence of HCDR1 consisting of the sequence of SEQ ID NO: 1, HCDR2 consisting of the sequence of SEQ ID NO: 2, and HCDR3 consisting of the sequence of SEQ ID NO: 3 An antibody or antigen-binding fragment thereof against a Thrombospondin-2 (TSP-2) protein comprising a variable region is provided.
본 발명자들은 전 세계적으로 높은 유병률을 가지면서 폐 이식 등을 제외하고는 유효한 치료 방법이 전무한 난치성 질환인 특발성 폐섬유증(Idiopathic Pulmonary Fibrosis, IPF)의 발병 기전을 밝히는 동시에, 이에 대한 효율적인 치료 방법을 발굴하고자 예의 연구 노력하였다. 그 결과, 폐 섬유화의 발병 기전에서 TSP-2가 섬유아세포-매개 섬유화(섬유증)를 일으킨다는 사실을 최초로 규명하고 TSP-2가 치료 타겟이 될 수 있음을 확인하였을 뿐 아니라, 동시에 TSP-2를 특이적으로 인식 및 억제하는 신규한 단일클론 중화항체를 개발함으로써, 본 발명을 완성하게 되었다.The present inventors uncovered the pathogenesis of idiopathic pulmonary fibrosis (IPF), an incurable disease for which there is no effective treatment method except for lung transplantation, which has a high prevalence worldwide, and at the same time discovered an efficient treatment method for it. I tried diligently to do research. As a result, the fact that TSP-2 causes fibroblast-mediated fibrosis (fibrosis) in the pathogenesis of pulmonary fibrosis was identified for the first time, and TSP-2 could be a therapeutic target. At the same time, TSP-2 The present invention has been completed by developing a novel monoclonal neutralizing antibody that specifically recognizes and inhibits.
본 명세서에서 용어“항체(antibody)”는 TSP-2에 대한 항체로서, 이의 특정 에피토프를 특이적으로 인식하여 이에 결합하며, 완전한 항체 형태뿐만 아니라 항체 분자의 항원 결합 단편(항체 단편)을 포함한다.As used herein, the term “antibody” refers to an antibody against TSP-2, which specifically recognizes and binds to a specific epitope thereof, and includes antigen-binding fragments (antibody fragments) of antibody molecules as well as complete antibody forms. .
완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다.A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bonds. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.
본 명세서에서 용어“항체의 항원 결합 단편”은 전체 항체 분자 내에서 항원-항체 결합 기능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등을 포함한다. 항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다.As used herein, the term “antigen-binding fragment of an antibody” refers to a fragment that retains an antigen-antibody binding function within the entire antibody molecule, and includes Fab, F(ab'), F(ab')2, and Fv. do. Among antibody fragments, Fab has a structure having light chain and heavy chain variable regions, light chain constant region, and heavy chain first constant region (C H1 ) and has one antigen binding site.
Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변영역 및 경쇄 가변영역만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 PCT 국제 공개특허출원 WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 및 WO 88/09344에 개시되어 있다. 이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변영역과 경쇄 가변영역이 연결되어 있고 단쇄 Fv(single-chain Fv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 단쇄의 가변영역이 공유결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 유전자 재조합 기술을 통하여 제작할 수도 있다.Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C H1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombinant technology for generating Fv fragments is described in PCT International Publication Patent Applications WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv, the heavy chain variable region and the short chain variable region are generally shared through a peptide linker. They are linked by bonds or directly linked at the C-terminus, so that they can form a dimer-like structure like double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.
본 명세서에서 용어“중쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3 개의 불변영역 도메인 CH1, CH2 및 CH3을 포함하는 전체길이 중쇄 및 이의 단편을 모두 의미한다.As used herein, the term "heavy chain" refers to a full-length variable region domain V H comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen and three constant region domains C H1 , C H2 and C H3 . Both heavy chains and fragments thereof are meant.
본 명세서에서, 용어“CDR(complementarity determining region)”은 면역글로블린 중쇄 및 경쇄의 고가변 영역(hypervariable region)의 아미노산 서열을 의미한다(Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). 중쇄(HCDR1, HCDR2 및 HCDR3) 및 경쇄(LCDR1, LCDR2 및 LCDR3)에는 각각 3개의 CDR이 포함되어 있으며, 이들 CDR은 항체가 항원 또는 에피토프에 결합하는 데 있어서 주요한 접촉 잔기를 제공한다.As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of immunoglobulin heavy and light chains (Kabat et al . Sequences of Proteins of Immunological Interest , 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.
본 발명의 항체 또는 항체 단편의 범위에는 CDR 영역에 보존적 아미노산 치환을 갖는 변이체가 포함된다. 또한, 본 발명의 항체 또는 항체 단편은, 인산화된 PLCγ2를 특이적으로 인식할 수 있는 범위 내에서 첨부한 서열목록에 기재된 아미노산 서열의 변이체를 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은, 예를 들어 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 갖으며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.The scope of the antibody or antibody fragment of the present invention includes variants with conservative amino acid substitutions in the CDR regions. In addition, the antibody or antibody fragment of the present invention may include a variant of the amino acid sequence described in the accompanying sequence listing within the scope of specifically recognizing phosphorylated PLCγ2. For example, additional changes may be made to the amino acid sequence of the antibody to further improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Accordingly, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
변이를 도입하는 데 있어서, 아미노산의 소수성 인덱스(hydropathic index)가 고려될 수 있다. 각각의 아미노산은 소수성과 전하에 따라 소수성 인덱스가 부여되어 있다: 아이소루이신(+4.5); 발린(+4.2); 루이신(+3.8); 페닐알라닌(+2.8); 시스테인/시스타인(+2.5); 메티오닌(+1.9); 알라닌(+1.8); 글라이신(-0.4); 쓰레오닌(-0.7); 세린(-0.8); 트립토판(-0.9); 타이로신(-1.3); 프롤린(-1.6); 히스티딘 (-3.2); 글루타메이트(-3.5); 글루타민(-3.5); 아스파르테이트(-3.5); 아스파라긴 (-3.5); 라이신(-3.9); 및 아르기닌(-4.5).In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is given a hydrophobicity index according to its hydrophobicity and charge: Isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
단백질의 상호적인 생물학적 기능(interactive biological function)을 부여하는 데 있어서 소수성 아미노산 인덱스는 매우 중요하다. 유사한 소수성 인덱스를 가지는 아미노산으로 치환하여야 유사한 생물학적 활성을 보유할 수 있다는 것은 공지된 사실이다. 소수성 인덱스를 참조하여 변이를 도입시키는 경우, 일특정예에서는 ± 2 이내, 다른 특정예에서는 ± 1 이내, 또 다른 특정예에서는 ± 0.5 이내의 소수성 인덱스 차이를 나타내는 아미노산 사이에 치환을 한다.The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is a known fact that amino acids having similar hydrophobicity indexes should be substituted to retain similar biological activities. When a mutation is introduced with reference to the hydrophobicity index, substitution is made between amino acids exhibiting a hydrophobicity index difference within ± 2 in one specific example, ± 1 in another specific example, and ± 0.5 in another specific example.
한편, 유사한 친수성 값(hydrophilicity value)을 가지는 아미노산 사이의 치환이 균등한 생물학적 활성을 갖는 단백질을 초래한다는 것도 잘 알려져 있다. 미국 특허 제4,554,101호에 개시된 바와 같이, 다음의 친수성 값이 각각의 아미노산 잔기에 부여되어 있다: 아르기닌(+3.0); 라이신(+3.0); 아스팔테이트(+3.0± 1); 글루타메이트(+3.0±1); 세린(+0.3); 아스파라긴(+0.2); 글루타민(+0.2); 글라이신(0); 쓰레오닌(-0.4); 프롤린(-0.5±1); 알라닌(-0.5); 히스티딘(-0.5); 시스테인(-1.0); 메티오닌(-1.3); 발린(-1.5); 루이신(-1.8); 아이소루이신 (-1.8); 타이로신(-2.3); 페닐알라닌(-2.5); 트립토판(-3.4). 친수성 값을 참조하여 변이를 도입시키는 경우, 일특정예에서는 ± 2 이내, 다른 특정예에서는 ± 1 이내, 또 다른 특정예에서는 ± 0.5 이내의 친수성 값 차이를 나타내는 아미노산 사이에 치환을 한다.On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in proteins having equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); Asphaltate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); Alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4). When a mutation is introduced by referring to the hydrophilicity value, substitution is made between amino acids showing a difference in hydrophilicity value within ± 2 in one specific example, ± 1 in another specific example, and ± 0.5 in another specific example.
분자의 활성을 전체적으로 변경시키지 않는 단백질에서의 아미노산 교환은 당해 분야에 공지되어 있다(H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu 및 Asp/Gly 간의 교환이다.Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 항체 또는 이를 코딩하는 핵산 분자는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인 하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인 된 서열을 분석한 경우에, 최소 61%의 상동성, 일특정예에 따르면 70%의 상동성, 다른 특정예에 따르면 80%의 상동성, 또 다른 특정예에 따르면 90%의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Smith and Waterman, Adv. Appl. Math. (1981) 2:482 Needleman and Wunsch, J. Mol. Bio. (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol. (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al. Nuc. Acids Res. (1988) 16:10881-90; Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 및 Pearson et al. Meth. Mol. Biol. (1994) 24:307-31에 개시되어 있다. NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al. J. Mol. Biol. (1990) 215:403-10)은 NBCI 등에서 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn 및 tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLSAT는 www.ncbi.nlm.nih.gov/BLAST/에서 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 www.ncbi.nlm.nih.gov/BLAST/blast_help.html에서 확인할 수 있다.Considering the mutations having the above-described biologically equivalent activity, the antibodies of the present invention or the nucleic acid molecules encoding them are construed to include sequences showing substantial identity with the sequences listed in the Sequence Listing. The above substantial identity is at least 61% when the sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Homology, according to one specific example, refers to a sequence exhibiting 70% homology, according to another specific example, 80% homology, and according to another specific example, 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math . (1981) 2:482 Needleman and Wunsch, J. Mol. Bio . (1970) 48:443; Pearson and Lipman, Methods in Mol. Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73:237-44; Higgins and Sharp, CABIOS (1989) 5:151-3; Corpet et al . Nuc. Acids Res . (1988) 16:10881-90; Huang et al . Comp. Appl. BioSci . (1992) 8:155-65 and Pearson et al . Meth. Mol. Biol . (1994) 24:307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al . J. Mol. Biol . (1990) 215:403-10) is accessible from NBCI and the like, and sequences such as blastp, blasm, blastx, tblastn and tblastx are found on the Internet. It can be used in conjunction with an analysis program. The BLAST is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparison methods using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체 또는 그의 항원 결합 단편의 중쇄 가변영역은 서열번호 11의 아미노산 서열을 갖는다.According to a specific embodiment of the present invention, the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 11.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체 또는 그의 항원 결합 단편은 서열번호 4의 서열로 이루어진 LCDR1, 서열번호 5의 서열로 이루어진 LCDR2 및 서열번호 6의 서열로 이루어진 LCDR3의 경쇄 CDR 아미노산 서열을 갖는 경쇄 가변영역을 추가적으로 포함한다.According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof of the present invention comprises the light chain CDR amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO: 4, LCDR2 consisting of the sequence of SEQ ID NO: 5, and LCDR3 consisting of the sequence of SEQ ID NO: 6 It further comprises a light chain variable region having.
본 명세서 용어“경쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 의미한다.The term "light chain" herein refers to both a full-length light chain and fragments thereof comprising a variable region domain V L and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체 또는 그의 항원 결합 단편의 경쇄 가변영역은 서열번호 16의 아미노산 서열을 갖는다.According to a specific embodiment of the present invention, the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 16.
본 발명의 항체는 단일클론 항체, 인간 항체, 인간화 항체, 키메라 항체, 단쇄 Fvs(scFV), 단쇄항체, Fab 단편, F(ab')단편, 다이설파이드-결합 Fvs(sdFV) 및 항-이디오타입(항-Id) 항체, 그리고 상기 항체들의 에피토프-결합 단편 등을 포함하나, 이에 한정되는 것은 아니다.Antibodies of the present invention include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idio type (anti-Id) antibodies, and epitope-binding fragments of the antibodies, and the like, but are not limited thereto.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체는 단일클론 항체이다.According to a specific embodiment of the present invention, the antibody of the present invention is a monoclonal antibody.
본 명세서에서 용어“단일클론 항체”는, 실질적으로 동일한 항체 집단에서 수득한 단일 분자 조성의 항체분자를 의미하며, 단일클론 항체는 특정 에피토프에 대해 단일 결합 특이성 및 친화도를 나타낸다.As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체는 중화 항체(Neutralizing Antibody)이다.According to a specific embodiment of the present invention, the antibody of the present invention is a neutralizing antibody.
본 발명에서 용어 “중화 항체”는 단백질, 항원 또는 감염성 입자의 표면 구조 중 특히 생물학적 기능을 나타내는 구조에 특화된 형태를 가져 해당 구조에 특이적으로 결합이 가능한 항체로써, 결합을 통하여 타겟 단백질, 항원 또는 감염성 입자의 생물학적 기능을 억제하는 역할을 수행하는 항체를 의미한다.In the present invention, the term “neutralizing antibody” refers to an antibody that has a form specific to a structure that exhibits a biological function among surface structures of proteins, antigens, or infectious particles, and is capable of specifically binding to the corresponding structure. It refers to an antibody that plays a role in inhibiting the biological function of an infectious particle.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체는 키메라 항체, 인간화 항체 또는 인간 항체이다.According to a specific embodiment of the present invention, the antibody of the present invention is a chimeric antibody, a humanized antibody or a human antibody.
보다 구체적으로는 본 발명의 본 발명의 항체 또는 항원 결합 단편은 Fab 단편, F(ab') 단편, F(ab')2 단편 또는 Fv 단편이며, 보다 더 구체적으로는 단쇄 Fvs(scFV) 항체이다.More specifically, the antibody or antigen-binding fragment of the present invention is a Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment, more specifically a single chain Fvs(scFV) antibody. .
본 발명에 따르면, 본 발명의 항체는 다양한 형태의 항체로 제조될 수 있다. 예를 들어, 하기의 실시예에 기재된 바와 같이, 본 발명의 항체는 sFv 또는 Fab 항체로 제조될 수 있고, 또한 sFv 또는 Fab 항체에 얻은 경쇄 및 중쇄 가변영역을 이용하여 사람 유래의 불변영역과 재조합함으로써 완전한(whole) 형태의 항체를 제공할 수도 있다.According to the present invention, the antibody of the present invention can be prepared in various forms of the antibody. For example, as described in the Examples below, the antibody of the present invention can be prepared as a sFv or Fab antibody, and also recombination with a human-derived constant region using the light and heavy chain variable regions obtained from the sFv or Fab antibody By doing so, it is also possible to provide a whole antibody.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for preventing or treating fibrotic disease comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.
본 명세서에서 용어“예방”은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병에 걸릴 가능성이 있는 대상체에서 질환 또는 질병의 발생을 억제하는 것을 의미한다.As used herein, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed with the disease or disease, but is likely to suffer from the disease or disease.
본 명세서에서 용어“치료”는 (a) 질환, 질병 또는 증상의 발전의 억제; (b) 질환, 질병 또는 증상의 경감; 또는 (c) 질환, 질병 또는 증상을 제거하는 것을 의미한다. 본 발명의 조성물을 대상체에 투여하면 섬유화를 일으키는 TSP-2 단백질의 활성이 억제되면서 과도한 섬유화를 방지하여 섬유화 질환(fibrotic disease)로 인한 증상의 발전을 억제하거나, 이를 제거하거나 또는 경감시키는 역할을 한다. 따라서, 본 발명의 조성물은 그 자체로 이들 질환 치료의 조성물이 될 수도 있고, 혹은 다른 약리성분과 함께 투여되어 상기 질환에 대한 치료 보조제로 적용될 수도 있다. 이에, 본 명세서에서 용어“치료”또는“치료제”는“치료 보조”또는“치료 보조제”의 의미를 포함한다.As used herein, the term “treatment” refers to (a) inhibition of the development of a disease, condition or condition; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition. When the composition of the present invention is administered to a subject, the activity of the TSP-2 protein that causes fibrosis is inhibited, preventing excessive fibrosis, thereby inhibiting the development of symptoms due to fibrotic disease, removing or alleviating it . Therefore, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients to be applied as a treatment adjuvant for the above diseases. Accordingly, the term "treatment" or "therapeutic agent" in the present specification includes the meaning of "therapeutic aid" or "therapeutic aid".
본 발명에서 용어“섬유화 질환(fibrotic disease)”는 병리학적인 조직의 섬유화(fibrosis) 진행을 수반하고, 이러한 섬유화를 직접 또는 간접적인 원인으로 하는 모든 병적 상태를 통칭한다. 또한 용어“섬유화 또는 섬유증(fibrosis)”는 염증이 있거나 손상된 조직 내부 및 주변 부위에 섬유질 결합조직(콜라겐 및 피브로넥틴과 같은 세포외기질의 구성요소)이 과도하게 축적되어 영구적인 흉터, 장기 기능 장애 및 궁극적으로는 사망을 초래할 수 있는 질환을 의미한다.In the present invention, the term "fibrotic disease" refers to all pathological conditions that accompany the progress of fibrosis of pathological tissues and have direct or indirect causes of such fibrosis. The term “fibrosis” also refers to the excessive accumulation of fibrous connective tissue (components of the extracellular matrix, such as collagen and fibronectin) in and around inflamed or damaged tissue, resulting in permanent scarring, organ dysfunction, and eventual means a disease that can cause death.
본 발명의 구체적인 구현예에 따르면, 본 발명의 섬유화 질환은 폐섬유증(Pulmonary Fibrosis)이다.According to a specific embodiment of the present invention, the fibrotic disease of the present invention is pulmonary fibrosis (Pulmonary Fibrosis).
본 발명에서 용어 “폐섬유증(pulmonary fibrosis)”은, 반복된 폐 또는 폐 조직의 손상 및 상처로 인하여 폐가 섬유화되는 질환을 의미하며, 호흡곤란; 마른기침; 피로감; 체중저하; 및 곤봉손발톱(nail clubbing)의 증상을 동반한다.In the present invention, the term "pulmonary fibrosis" refers to a disease in which the lungs become fibrotic due to repeated lung or lung tissue damage and wounds, and may include dyspnea; dry cough; fatigue; weight loss; and symptoms of nail clubbing.
본 발명의 구체적인 구현예에 따르면, 본 발명의 폐섬유증(Pulmonary Fibrosis)은 간질성폐질환(Interstitial Lung Disease, ILD) 및 특발성 폐섬유증(Idiopathic Pulmonary Fibrosis, IPF)으로 구성된 군으로부터 선택되는 하나 이상의 질환이다.According to a specific embodiment of the present invention, the pulmonary fibrosis of the present invention is at least one disease selected from the group consisting of interstitial lung disease (ILD) and idiopathic pulmonary fibrosis (IPF). .
본 발명에서 용어 “간질성폐질환(interstitial lung disease, ILD)”는 폐의 간질(interstitium of lung)에 주로 침범하는 비종양성, 비감염성 호흡기 질환들의 총칭으로써, 폐섬유증의 원인이 되기도 한다.In the present invention, the term “interstitial lung disease (ILD)” is a general term for non-neoplastic, non-infectious respiratory diseases that mainly invade the interstitium of the lung, and may also cause pulmonary fibrosis.
본 발명에서 용어“특발성 폐 섬유증(idiopathic pulmonary fibrosis)”는 폐 조직이 두꺼워지고 경직되어 흉터 조직이 형성되는 것을 특징으로 하는 진행성 호흡기 질환을 의미하며, 폐 기능의 점진적이고 비가역적인 감소를 특징으로 하는 만성 흉터성 폐 질환의 일종으로써, 명확한 원인이 알려져 있지 않아서‘특발성’이라고 지칭된다.In the present invention, the term “idiopathic pulmonary fibrosis” refers to a progressive respiratory disease characterized by thickening and rigidity of lung tissue and the formation of scar tissue, characterized by a gradual and irreversible decrease in lung function. As a type of chronic scarring lung disease, it is referred to as 'idiopathic' because no clear cause is known.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 그의 항원 결합 단편의 중쇄 가변영역을 코딩하는 핵산 분자를 제공한다.According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention.
본 명세서에서 용어“핵산 분자”는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다(Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman 및 Peyman, Chemical Reviews, (1990) 90:543-584). 본 발명의 중쇄 및 경쇄 가변영역을 코딩하는 핵산 분자의 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다.As used herein, the term “nucleic acid molecule” has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also analogs with modified sugar or base sites. (analogue) is also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequences of nucleic acid molecules encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
본 발명의 핵산 분자는 상기한 뉴클레오타이드 서열에 대하여 실질적인 동일성을 나타내는 뉴클레오타이드 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 뉴클레오타이드 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인 된 서열을 분석한 경우에, 최소 80%의 상동성, 일 특정예에서는 최소 90%의 상동성, 다른 특정예에서는 최소 95%의 상동성을 나타내는 뉴클레오타이드 서열을 의미한다.The nucleic acid molecules of the present invention are also construed to include nucleotide sequences exhibiting substantial identity to the nucleotide sequences described above. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence described above are aligned so as to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a nucleotide sequence showing homology, at least 90% homology in one specific example, and at least 95% homology in another specific example.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체 또는 그의 항원 결합 단편의 중쇄 가변영역을 코딩하는 핵산분자는 서열번호 27의 아미노산 서열을 갖는다.According to a specific embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 27.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 그의 항원 결합 단편의 경쇄 가변영역을 코딩하는 핵산분자를 제공한다.According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항체 또는 그의 항원 결합 단편의 중쇄 가변영역을 코딩하는 핵산분자는 서열번호 32의 아미노산 서열을 갖는다.According to a specific embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention has the amino acid sequence of SEQ ID NO: 32.
본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 중쇄 가변영역을 코딩하는 핵산 분자, 경쇄 가변영역을 코딩하는 핵산 분자 또는 상기 핵산분자 모두를 포함하는 재조합 벡터를 제공한다.According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the above-described heavy chain variable region, a nucleic acid molecule encoding a light chain variable region, or a recombinant vector containing both of the above nucleic acid molecules.
본 명세서에서 용어 “벡터”는 숙주세포에서 목적 유전자를 발현시키기 위한 수단으로 플라스미드 벡터; 코즈미드 벡터; 그리고 박테리오파아지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스 벡터 같은 바이러스 벡터 등을 포함한다.As used herein, the term "vector" refers to a plasmid vector as a means for expressing a target gene in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and the like.
본 발명의 일구현예에 따르면, 본 발명의 벡터에서 경쇄 가변영역을 코딩하는 핵산 분자 및 중쇄 가변영역을 코딩하는 핵산 분자는 프로모터와 작동적으로 결합(operatively linked)되어 있다. 본 명세서에서 용어“작동적으로 결합된”은 핵산 발현조절서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다.According to one embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule encoding a light chain variable region and a nucleic acid molecule encoding a heavy chain variable region are operatively linked to a promoter. As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence will control the transcription and/or translation of said other nucleic acid sequence.
본 발명의 재조합 벡터 시스템은 당업계에 공지된 다양한 방법을 통해 구축될 수 있으며, 이에 대한 구체적인 방법은 Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press(2001)에 개시되어 있다.The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) .
본 발명의 벡터는 전형적으로 클로닝을 위한 벡터 또는 발현을 위한 벡터로서 구축될 수 있다. 또한, 본 발명의 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다. 예를 들어, 본 발명의 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예컨대, tac 프로모터, lac 프로모터, lacUV5 프로모터, lpp 프로모터, pLλ프로모터, pRλ프로모터, rac5 프로모터, amp 프로모터, recA 프로모터, SP6 프로모터, trp 프로모터 및 T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 숙주세포로서 E. coli(예컨대, HB101, BL21, DH5α 등)가 이용되는 경우, E. coli 트립토판 생합성 경로의 프로모터 및 오퍼레이터 부위(Yanofsky, C. J. Bacteriol. 158:1018-1024(1984)) 그리고 파아지 λ의 좌향 프로모터(pLλ프로모터, Herskowitz, I. and Hagen, D. Ann. Rev. Genet. 14:399-445(1980))가 조절부위로서 이용될 수 있다. 숙주세포로서 바실러스 균이 이용되는 경우, 바실러스 츄린겐시스의 독소단백질 유전자의 프로모터(Appl. Environ. Microbiol. 64:3932-3938(1998); Mol. Gen. Genet. 250:734-741(1996)) 또는 바실러스균에서 발현 가능한 어떠한 프로모터라도 조절부위로 이용될 수 있다.Vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as a host cell, promoter and operator regions of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. J. Bacteriol . 158: 1018-1024 (1984)) And the leftward promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D. Ann. Rev. Genet . 14:399-445 (1980)) can be used as a control region. When a Bacillus strain is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis ( Appl. Environ. Microbiol . 64: 3932-3938 (1998); Mol. Gen. Genet . 250: 734-741 (1996) ) or any promoter that can be expressed in Bacillus strains can be used as a regulatory site.
한편, 본 발명의 재조합 벡터는 당업계에서 종종 사용되는 플라스미드(예: pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈 및 pUC19 등), 파지(예: λgt4·λB, λ-Charon, λΔz1 및 M13 등) 또는 바이러스(예: SV40 등)를 조작하여 제작될 수 있다.On the other hand, the recombinant vector of the present invention is a plasmid often used in the art (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, It can be produced by manipulating pGEX series, pET series, and pUC19, etc.), phages (eg, λgt4·λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.).
한편, 본 발명의 벡터가 발현 벡터이고, 진핵 세포를 숙주로 하는 경우에는, 포유동물 세포의 지놈으로부터 유래된 프로모터(예: 메탈로티오닌 프로모터, β-액틴 프로모터, 사람 헤로글로빈 프로모터 및 사람 근육 크레아틴 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예: 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, 사이토메갈로바이러스(CMV) 프로모터, HSV의 tk 프로모터, 마우스 유방 종양 바이러스(MMTV) 프로모터, HIV의 LTR 프로모터, 몰로니 바이러스의 프로모터 엡스타인바 바이러스(EBV)의 프로모터 및 로우스 사코마 바이러스(RSV)의 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle) creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter) , the LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Lowe's Sarcoma Virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.
본 발명의 재조합 벡터는 그로부터 발현되는 항체의 정제를 용이하게 하기 위하여 다른 서열과 융합될 수도 있다. 융합되는 서열은, 예컨대 글루타티온 S-트랜스퍼라제(Pharmacia, USA); 말토스 결합 단백질(NEB, USA); FLAG(IBI, USA); 6x His(hexahistidine; Quiagen, USA), Pre-S1, c-Myc와 같은 태그 서열; OmpA, PelB와 같은 선도서열 등이 있다. 또한, 본 발명의 벡터에 의해 발현되는 단백질이 항체이기 때문에 정제를 위한 추가적인 서열 없이도, 발현된 항체는 단백질 A 컬럼 등을 통하여 용이하게 정제할 수 있다.The recombinant vector of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA); maltose binding protein (NEB, USA); FLAG (IBI, USA); tag sequences such as 6x His (hexahistidine; Quiagen, USA), Pre-S1, c-Myc; There are leader sequences such as OmpA and PelB. In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.
한편, 본 발명의 재조합 벡터는 선택표지로서 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.On the other hand, the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a gene for resistance to tetracycline.
본 발명의 항체를 발현하는 벡터는, 경쇄와 중쇄가 하나의 벡터에서 동시에 발현되는 벡터 시스템이거나 또는 경쇄와 중쇄를 각각 별도의 벡터에서 발현시키는 시스템 모두 가능하다. 후자의 경우, 두 벡터는 동시 형질전환(co-transfomation) 및 표적 형질전환(targeted transformation)을 통하여 숙주세포로 도입된다. 동시 형질전환은 경쇄 및 중쇄를 코딩하는 각각의 벡터 DNA를 동시에 숙주세포로 도입한 뒤 경쇄와 중쇄를 모두 발현하는 세포를 선별하는 방법이다. 표적 형질전환은 경쇄(또는 중쇄)를 포함하는 벡터로 형질전환 된 세포를 선별하고 경쇄를 발현하는 선별된 세포를 중쇄(또는 경쇄)를 포함하는 벡터로 다시 형질전환 하여 경쇄 및 중쇄 모두를 발현하는 세포를 최종적으로 선별하는 방법이다.The vector expressing the antibody of the present invention may be either a vector system in which the light chain and the heavy chain are simultaneously expressed in one vector or a system in which the light chain and the heavy chain are expressed in separate vectors. In the latter case, the two vectors are introduced into the host cell through co-transformation and targeted transformation. Co-transformation is a method of simultaneously introducing each vector DNA encoding the light chain and the heavy chain into a host cell, and then selecting cells expressing both the light chain and the heavy chain. Targeted transformation selects cells transformed with a vector containing a light chain (or heavy chain) and transforms the selected cells expressing the light chain with a vector containing a heavy chain (or light chain) to express both light and heavy chains. It is a method for final selection of cells.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 재조합 벡터를 포함하는 숙주세포를 제공한다. 일 구체예에서, 상기 숙주세포는 본 발명의 재조합 벡터로 형질전환된 세포이다. 본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주 세포는 당업계에 공지되어 어떠한 숙주세포도 이용할 수 있으며, 예컨대, 에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스 및 바실러스 츄린겐시스와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주세포를 포함하나, 이로 제한되는 것은 아니다.According to another aspect of the present invention, the present invention provides a host cell comprising the recombinant vector of the present invention. In one embodiment, the host cell is a cell transformed with the recombinant vector of the present invention. Any host cell that can stably and continuously clone and express the vector of the present invention can be used as any host cell known in the art, for example, Escherichia coli , Bacillus subtilis and Bacillus thuringen. strains of the genus Bacillus, such as Cis, Streptomyces , Pseudomonas (e.g. Pseudomonas putida ), Proteus mirabilis or Staphylococcus (e.g. For example, Staphylococcus carnosus ( Staphylocus carnosus )), but includes prokaryotic host cells such as, but are not limited thereto.
상기 벡터의 적합한 진핵세포 숙주세포는 아스페르길러스 속(Aspergillus species)과 같은 진균, 피치아 파스토리스(Pichia pastoris), 사카로마이세스 세르비시아(Saccharomyces cerevisiae), 쉬조사카로마세스(Schizosaccharomyces) 및 뉴로스포라 크라사(Neurospora crassa)와 같은 효모, 그 밖의 하등 진핵세포, 곤충-유래 세포와 같은 고등 진핵생물의 세포, 그리고 식물 또는 포유동물로부터 유래한 세포를 이용할 수 있다.Suitable eukaryotic host cells of the vector are fungi such as Aspergillus species , Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces ) and Neurospora crassa , other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.
본 명세서에서 용어“형질전환”은 본 발명의 재조합 벡터를 이용하여 숙주 세포에 목적하는 유전자를 도입하는 것을 말하며, “형질감염”과 동일한 의미로 사용된다. 따라서, 숙주세포로의“형질전환”및/또는“형질감염”은 핵산을 유기체, 세포, 조직 또는 기관에 도입하는 어떤 방법도 포함되며 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 전기충격유전자전달법(electroporation), 원형질 융합, 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 실리콘 카바이드 섬유 이용한 교반, 아그로 박테리아 매개된 형질전환, PEG, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 포함되나, 이로 제한되지 않는다.In this specification, the term "transformation" refers to introducing a desired gene into a host cell using the recombinant vector of the present invention, and is used in the same sense as "transfection". Thus, “transformation” and/or “transfection” into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue, or organ, and as is known in the art, using standard techniques suitable for the host cell. You can choose to do it. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation using silicon carbide fibers, agrobacterium mediated transformation, PEG, dextran sulfate, lipo pectamine and desiccation/inhibition mediated transformation methods; and the like.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 그의 항원 결합 단편을 시료에 접촉시키는 단계를 포함하는 시료 내 TSP-2(Thrombospondin-2) 단백질을 검출하는 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for detecting TSP-2 (Thrombospondin-2) protein in a sample comprising the step of contacting the antibody or antigen-binding fragment thereof of the present invention to the sample.
본 발명에서 사용되는 항체 또는 그의 항원결합 단편은 이미 상술하였으므로, 과도한 중복을 피하기 위하여 그 기재를 생략한다.Since the antibody or antigen-binding fragment thereof used in the present invention has already been described above, its description is omitted to avoid excessive redundancy.
본 발명의 항체는 생물학적 시료에 적용되어 TSP-2 (Thrombospondin-2) 단백질을 검출할 수 있다. 본 명세서에서 사용된 용어, “생물학적 시료”란 조직, 세포, 전혈, 혈청, 혈장, 타액, 뇨, 림프액, 척수액, 조직 부검 시료(뇌, 피부, 림프절, 척수 등), 세포 배양 상등액, 파열된 진핵세포 및 세균 발현계 등을 들 수 있지만, 이에 제한되지는 않는다. 구체적으로는, 상기 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 뇨, 림프액 또는 척수액이다.The antibody of the present invention can be applied to a biological sample to detect TSP-2 (Thrombospondin-2) protein. As used herein, the term “biological sample” means tissue, cell, whole blood, serum, plasma, saliva, urine, lymph fluid, spinal fluid, tissue autopsy sample (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic and bacterial expression systems, etc., but are not limited thereto. Specifically, the sample is tissue, cell, whole blood, serum, plasma, saliva, urine, lymph or spinal fluid.
생물학적 시료에서 TSP-2 (Thrombospondin-2) 단백질의 검출은, 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 또는 섬광계수법(scintillation counting method)을 이용한 항원-항체 복합체 형성의 검출을 통하여 수행할 수 있다. 본 명세서상의“검출”은 항원-항체 복합체를 검출하기 위한 것으로 여러 가지 표지체를 사용하여 실시할 수 있다. 표지체의 구체적인 예로는 효소, 형광물, 리간드, 발광물, 미소입자 또는 방사성 동위원소를 포함한다.Detection of TSP-2 (Thrombospondin-2) protein in biological samples includes colorimetric method, electrochemical method, fluorescence method, luminometry, particle counting method, It can be performed through detection of antigen-antibody complex formation using visual assessment or scintillation counting method. “Detection” in the present specification is for detecting an antigen-antibody complex and can be performed using various markers. Specific examples of the label include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, or radioactive isotopes.
검출 표지체로서 사용되는 효소로는 아세틸콜린에스테라제, 알칼라인 포스파타제, β-D-갈락토시다제, 호스래디쉬 퍼옥시다제 및 β-라타마제 등을 포함하며, 형광물로는 플루오레세인, Eu3+, Eu3+ 킬레이트 또는 크립테이트 등을 포함하며, 리간드로는 바이오틴 유도체 등을 포함하며, 발광물로는 아크리디늄 에스테르 및 이소루미놀 유도체 등을 포함하며, 미소입자로는 콜로이드 금 및 착색된 라텍스 등을 포함하며, 방사성 동위원소로는 57Co, 3H, 125I 및 125I-볼톤(Bonton) 헌터(Hunter) 시약 등을 포함한다.Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase and β-latamase, etc. Fluorescein Phosphorus, Eu 3+ , Eu 3+ chelate or cryptate, etc. are included, ligands include biotin derivatives, etc., luminous substances include acridinium esters and isoluminol derivatives, etc., and microparticles include colloids. It includes gold and colored latex, etc., and radioactive isotopes include 57 Co, 3 H, 125 I and 125 I-Bolton Hunter reagent and the like.
본 발명의 일구현예에 따르면, 항원-항체 복합체를 효소면역흡착법(ELISA) 을 이용하여 검출할 수 있다. 효소면역흡착법에는 고체 지지체에 부착된 항원을 인지하는 표지된 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 이차항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 2차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다. 본 발명의 항체는 검출 표지를 가질 수 있으며, 검출표지를 가지지 않을 경우는 본 발명의 항체를 포획할 수 있고 검출 표지를 가지는 또 다른 항체를 처리하여 확인할 수 있다.According to one embodiment of the present invention, antigen-antibody complexes can be detected using enzyme immunosorbent assay (ELISA). Enzyme immunosorbent methods include direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and solid support Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support It includes a variety of ELISA methods, such as indirect sandwich ELISA using labeled secondary antibodies. The antibody of the present invention may have a detection label, and when it does not have a detection label, the antibody of the present invention can be captured and another antibody having a detection label can be treated and identified.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 진단용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for diagnosis of fibrotic disease comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.
본 발명에 따르면, 본 발명의 TSP-2 단백질을 항원-항체 반응을 이용한 면역분석(immunoassay) 방법에 따라 검출하여 섬유화 질환 발생 여부를 분석하는 데 이용될 수 있다. 이러한 면역분석은 종래에 개발된 다양한 면역분석 또는 면역염색 프로토콜에 따라 실시될 수 있다.According to the present invention, the TSP-2 protein of the present invention can be detected according to an immunoassay method using an antigen-antibody reaction and used to analyze whether or not a fibrotic disease occurs. Such an immunoassay can be performed according to various immunoassay or immunostaining protocols previously developed.
예를 들어, 본 발명의 방법이 방사능면역분석 방법에 따라 실시되는 경우, 방사능동위원소(예컨대, C14, I125, P32 및 S35)로 표지된 항체가 이용될 수 있다. 본 발명에서 TSP-2 단백질을 특이적으로 인식하는 항체는 폴리클로날 또는 모노클로날 항체이며, 바람직하게는 모노클로날 항체이다.For example, when the method of the present invention is performed according to the radioimmunoassay method, antibodies labeled with radioactive isotopes (eg, C 14 , I 125 , P 32 and S 35 ) may be used. In the present invention, the antibody specifically recognizing the TSP-2 protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.
본 발명의 항체는 당업계에서 통상적으로 실시되는 방법들, 예를 들어, 융합 방법(Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), 재조합 DNA 방법(미국 특허 제4,816,567호) 또는 파아지 항체 라이브러리 방법(Clackson et al, Nature, 352:624-628(1991) 및 Marks et al, J. Mol. Biol., 222:58, 1-597(1991))에 의해 제조될 수 있다. 항체 제조에 대한 일반적인 과정은 Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York (1999)에 상세하게 기재되어 있다. Antibodies of the present invention can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,567 ) or phage antibody library methods (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). . General procedures for antibody preparation are described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York (1999).
상술한 면역분석 과정에 의한 최종적인 시그널의 강도를 분석함으로써, 종양의 전이 여부 또는 전이 가능성을 예측할 수 있다. 즉, 개체의 시료에서 TSP-2 단백질에 대한 시그널이 정상 시료 보다 강하게 나오는 경우에는 개체의 종야의 전이가 진행되었거나 향후 진행될 가능성이 높은 것으로 판단된다.By analyzing the strength of the final signal by the above-described immunoassay process, it is possible to predict whether or not the tumor has metastasized or whether it is possible to metastasize. That is, if the signal for the TSP-2 protein in the sample of the subject is stronger than that of the normal sample, the subject's The metastasis has progressed or is likely to progress in the future It is judged to be
본 명세서에서 용어“진단”은 특정 질환에 대한 개체의 감수성(susceptibility)의 판정, 특정 질환을 현재 개체가 가지고 있는 지 여부의 판정, 및 특정 질환에 걸린 한 객체의 예후(prognosis)의 판정을 포함한다.As used herein, the term "diagnosis" includes determination of a subject's susceptibility to a particular disease, determination of whether a subject currently has a particular disease, and determination of the prognosis of a subject suffering from a particular disease. do.
본 명세서에서 용어“진단용 조성물”은 대상체의 섬유화 질환 발생 여부를 판단하거나, 발생 가능성을 예측하기 위해 TSP-2 단백질 또는 이를 인코딩하는 유전자의 발현량 측정수단을 포함하는 통상적인(mixture) 또는 장비(device)를 의미하며, 이에“진단용 키트”로 표현될 수도 있다.As used herein, the term “diagnostic composition” refers to a conventional mixture or equipment including a means for measuring the expression level of a TSP-2 protein or a gene encoding the same in order to determine whether a subject has a fibrotic disease or to predict the possibility of occurrence of a fibrotic disease ( device), which can also be expressed as a “diagnostic kit”.
본 발명의 또 다른 양태에 따르면, 본 발명은 TSP-2 (Thrombospondin-2) 단백질 또는 이를 코딩하는 유전자의 발현량을 측정하는 제제를 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 진단용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for diagnosis of fibrotic disease comprising, as an active ingredient, an agent for measuring the expression level of TSP-2 (Thrombospondin-2) protein or a gene encoding it do.
본 발명의 구체적인 구현예에 따르면, 상기 TSP-2를 인코딩하는 유전자의 발현량을 측정하는 제제는 상기 유전자의 핵산 분자에 특이적으로 결합하는 프라이머 또는 프로브이다.According to a specific embodiment of the present invention, the agent for measuring the expression level of the gene encoding TSP-2 is a primer or probe that specifically binds to the nucleic acid molecule of the gene.
본 명세서에서 사용되는 용어“프라이머”는 핵산쇄(주형)에 상보적인 프라이머 연장 산물의 합성이 유도되는 조건, 즉, 뉴클레오타이드와 DNA 중합효소와 같은 중합제의 존재, 적합한 온도와 pH의 조건에서 합성의 개시점으로 작용하는 올리고뉴클레오타이드를 의미한다. 구체적으로는, 프라이머는 디옥시리보뉴클레오타이드 단일쇄이다. 본 발명에서 이용되는 프라이머는 자연(naturally occurring) dNMP(즉, dAMP, dGMP, dCMP 및 dTMP), 변형 뉴클레오타이드 또는 비-자연 뉴클레오타이드를 포함할 수 있으며, 리보뉴클레오타이드도 포함할 수 있다.As used herein, the term “primer” refers to conditions in which synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of nucleotides and a polymerizer such as DNA polymerase, synthesis under conditions of suitable temperature and pH. refers to an oligonucleotide that serves as the starting point of Specifically, the primer is a single chain deoxyribonucleotide. Primers used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides, and may also include ribonucleotides.
본 발명의 프라이머는 타겟 핵산에 어닐링 되어 주형-의존성 핵산 중합효소에 의해 타겟 핵산에 상보적인 서열을 형성하는 연장 프라이머(extension primer)일 수 있으며, 이는 고정화 프로브가 어닐링 되어 있는 위치까지 연장되어 프로브가 어닐링 되어 있는 부위를 차지한다.The primer of the present invention may be an extension primer that anneals to a target nucleic acid to form a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which is extended to a position where the immobilized probe is annealed, so that the probe becomes occupies the annealed area.
본 발명에서 이용되는 연장 프라이머는 타겟 핵산, 예를 들어 TSP-2 단백질을 코딩하는 유전자의 특정 염기서열에 상보적인 혼성화 뉴클레오타이드 서열을 포함한다. 용어“상보적”은 소정의 어닐링 또는 혼성화 조건하에서 프라이머 또는 프로브가 타겟 핵산 서열에 선택적으로 혼성화할 정도로 충분히 상보적인 것을 의미하며, 실질적으로 상보적(substantially complementary)인 경우 및 완전히 상보적(perfectly complementary)인 경우를 모두 포괄하는 의미이며, 구체적으로는 완전히 상보적인 경우를 의미한다. 본 명세서에서 용어“실질적으로 상보적인 서열”은 완전히 일치되는 서열뿐만 아니라, 특정 서열에 어닐링하여 프라이머 역할을 할 수 있는 범위 내에서, 비교 대상의 서열과 부분적으로 불일치되는 서열도 포함되는 의미이다.The extension primer used in the present invention includes a hybrid nucleotide sequence complementary to a specific nucleotide sequence of a gene encoding a target nucleic acid, for example, the TSP-2 protein. The term "complementary" means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, substantially complementary and perfectly complementary. ), and specifically means completely complementary cases. As used herein, the term "substantially complementary sequence" is intended to include not only completely identical sequences, but also sequences that are partially inconsistent with the sequence to be compared, within the range of annealing to a specific sequence and acting as a primer.
프라이머는, 중합제의 존재 하에서 연장 산물의 합성을 프라이밍시킬 수 있을 정도로 충분히 길어야 한다. 프라이머의 적합한 길이는 다수의 요소, 예컨대, 온도, pH 및 프라이머의 소스(source)에 따라 결정되지만 전형적으로 15-30 뉴클레오타이드이다. 짧은 프라이머 분자는 주형과 충분히 안정된 혼성 복합체를 형성하기 위하여 일반적으로 보다 낮은 온도를 요구한다. 이러한 프라이머의 설계는 타겟 뉴클레오티드 서열을 참조하여 당업자가 용이하게 실시할 수 있으며, 예컨대, 프라이머 디자인용 프로그램(예: PRIMER 3 프로그램)을 이용하여 할 수 있다.The primer must be long enough to prime the synthesis of the extension product in the presence of the polymerization agent. The suitable length of a primer depends on a number of factors, such as temperature, pH and the source of the primer, but is typically 15-30 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The design of such primers can be easily performed by those skilled in the art by referring to the target nucleotide sequence, and can be performed using, for example, a primer design program (eg, PRIMER 3 program).
본 명세서에서 용어“프로브”는 특정 뉴클레오타이드 서열에 혼성화될 수 있는 디옥시리보뉴클레오타이드 및 리보뉴클레오타이드를 포함하는 자연 또는 변형되는 모노머 또는 결합을 갖는 선형의 올리고머를 의미한다. 구체적으로, 프로브는 혼성화에서의 최대 효율을 위하여 단일가닥이며, 더욱 구체적으로는 디옥시리보뉴클레오타이드이다. 본 발명에 이용되는 프로브로서, 상기 TSP-2 단백질을 코딩하는 유전자의 특정 염기서열에 완전하게(perfectly) 상보적인 서열이 이용될 수 있으나, 특이적 혼성화를 방해하지 않는 범위 내에서 실질적으로(substantially) 상보적인 서열이 이용될 수도 있다. 일반적으로, 혼성화에 의해 형성되는 듀플렉스(duplex)의 안정성은 말단의 서열의 일치에 의해 결정되는 경향이 있기 때문에, 타겟 서열의 3’-말단 또는 5’-말단에 상보적인 프로브를 사용하는 것이 바람직하다.As used herein, the term “probe” refers to a natural or modified monomer including deoxyribonucleotide and ribonucleotide capable of hybridizing to a specific nucleotide sequence, or a linear oligomer having a linkage. Specifically, the probe is single-stranded for maximum efficiency in hybridization, more specifically a deoxyribonucleotide. As the probe used in the present invention, a sequence perfectly complementary to a specific nucleotide sequence of the gene encoding the TSP-2 protein may be used, but substantially within a range that does not interfere with specific hybridization. ) complementary sequences may be used. In general, since the stability of a duplex formed by hybridization tends to be determined by the matching of the terminal sequence, it is preferable to use a probe complementary to the 3'-end or 5'-end of the target sequence. do.
혼성화에 적합한 조건은 Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.(2001) 및 Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985)에 개시된 사항을 참조하여 결정할 수 있다.Conditions suitable for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington , can be determined by referring to the matters disclosed in DC (1985).
본 발명은 단백질의 활성 또는 발현량을 측정하기 위하여 항체 대신 TSP-2 단백질에 특이적으로 결합하는 앱타머를 이용할 수도 있다. 본 명세서에서 용어“앱타머”는 특정 표적물질에 높은 친화력과 특이성으로 결합하는 단일 줄기의(single-stranded) 핵산(RNA 또는 DNA) 분자 또는 펩타이드 분자를 의미한다. 앱타머의 일반적인 내용은 Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R . "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7(1998)에 상세하게 개시되어 있다.In the present invention, an aptamer that specifically binds to the TSP-2 protein may be used instead of an antibody to measure the activity or expression level of the protein. As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. For a general discussion of aptamers see Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).
본 발명의 또 다른 양태에 따르면, 본 발명은 서열번호 4의 서열로 이루어진 LCDR1, 서열번호 5의 서열로 이루어진 LCDR2 및 서열번호 6의 서열로 이루어진 LCDR3의 경쇄 CDR(complementarity determining region) 아미노산 서열을 갖는 경쇄 가변영역을 포함하는 TSP-2(Thrombospondin-2) 단백질에 대한 항체 또는 그의 항원 결합 단편을 제공한다.According to another aspect of the present invention, the present invention has a light chain CDR (complementarity determining region) amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO: 4, LCDR2 consisting of the sequence of SEQ ID NO: 5, and LCDR3 consisting of the sequence of SEQ ID NO: 6 An antibody or antigen-binding fragment thereof against the TSP-2 (Thrombospondin-2) protein containing a light chain variable region is provided.
본 발명에서 사용되는 경쇄 CDR(LCDR); 경쇄 가변영역; 및 이를 포함하는 항체 또는 그의 항원결합 단편에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위하여 그 기재를 생략한다.light chain CDRs (LCDRs) used in the present invention; light chain variable region; And since the antibodies or antigen-binding fragments thereof containing them have already been described above, their descriptions are omitted to avoid excessive redundancy.
본 발명의 또 다른 양태에 따르면, 본 발명은 TSP-2 (Thrombospondin-2)에 대한 억제제를 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 예방 또는 치료용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for preventing or treating fibrotic disease comprising an inhibitor for TSP-2 (Thrombospondin-2) as an active ingredient.
본 발명에서 사용되는 항체를 이용한 섬유화질환의 예방 또는 치료용 조성물 및 예방 또는 치료의 의미에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위하여 그 기재를 생략한다.Since the composition for preventing or treating fibrotic diseases using the antibody used in the present invention and the meaning of the prevention or treatment have already been described above, the description thereof will be omitted to avoid excessive redundancy.
본 명세서에서 용어“억제제”는 타겟 유전자의 활성 또는 발현의 저하를 야기시키는 물질을 의미하며, 이에 의해 타겟 유전자의 활성 또는 발현이 탐지 불가능해지거나 무의미한 수준으로 존재하게 되는 경우 뿐 아니라, 타겟 유전자의 생물학적 기능이 유의하게 저하될 수 있을 정도로 활성 또는 발현을 저하시키는 물질을 의미한다.As used herein, the term "inhibitor" refers to a substance that causes a decrease in the activity or expression of a target gene, whereby the activity or expression of the target gene becomes undetectable or present at an insignificant level, as well as the It refers to a substance that reduces activity or expression to the extent that biological function can be significantly reduced.
타겟 유전자의 억제제는 예를 들어 당업계에 이미 그 서열이 공지된 상기 유전자의 발현을 유전자 수준에서 억제하는 shRNA, siRNA, miRNA, 리보자임(ribozyme), PNA(peptide nucleic acids) 안티센스 올리고뉴클레오타이드 또는 타겟 유전자를 인식하는 가이드 RNA를 포함하는 CRISPR 시스템과, 단백질 수준에서 억제하는 항체 또는 앱타머 뿐 아니라, 이들의 활성을 억제하는 화합물, 펩타이드 및 천연물을 포함하나, 이에 제한되지 않고 당업계에 공지된 모든 유전자 및 단백질 수준의 억제수단이 사용될 수 있다. Inhibitors of target genes are, for example, shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids) antisense oligonucleotides or targets that inhibit the expression of the gene at the gene level, the sequence of which is already known in the art All known in the art including, but not limited to, CRISPR systems containing guide RNAs recognizing genes, antibodies or aptamers that inhibit at the protein level, as well as compounds, peptides and natural products that inhibit their activity Means of inhibition at the gene and protein level can be used.
TSP-2 단백질에 특이적으로 결합하여 이의 활성 또는 발현을 억제하는 항체 및 앱타머에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위해 그 기재를 생략한다.Antibodies and aptamers that specifically bind to and inhibit the activity or expression of the TSP-2 protein have already been described in detail, so description thereof is omitted to avoid excessive redundancy.
본 명세서에서 용어“shRNA(small hairpin RNA)”는 인 비보 상에서 스템-루프(stem-loop) 구조를 이루는 단일 가닥으로 50-70개로 구성된 뉴클레오타이드로서, RNA 간섭을 통해 타겟 유전자의 발현을 억제하기 위한 타이트한 헤어핀 구조를 만드는 RNA 서열을 의미한다. 통상적으로 5-10개의 뉴클레오타이드의 루프 부위 양쪽으로 상보적으로 19-29개의 뉴클레오타이드의 긴 RNA가 염기쌍을 이루어 이중가닥의 스템을 형성하며, 언제나 발현되도록 하기 위하여 U6 프로모터를 포함하는 벡터를 통해 세포 내로 형질도입되며 대개 딸세포로 전달되어 타겟 유전자의 발현억제가 유전되도록 한다.In the present specification, the term "small hairpin RNA (shRNA)" is a single strand consisting of 50-70 nucleotides forming a stem-loop structure in vivo , which is used to suppress the expression of a target gene through RNA interference. It refers to the RNA sequence that creates a tight hairpin structure. Usually, long RNAs of 19-29 nucleotides complementary to both sides of the loop region of 5-10 nucleotides form a double-stranded stem, which is introduced into the cell through a vector containing a U6 promoter so that it is always expressed. It is transduced and is usually passed on to daughter cells, allowing inheritance of suppression of the target gene.
본 명세서에서 용어“siRNA”는 특정 mRNA의 절단(cleavage)을 통하여 RNAi(RNA interference) 현상을 유도할 수 있는 짧은 이중사슬 RNA를 의미한다. 타겟 유전자의 mRNA와 상동인 서열을 가지는 센스 RNA 가닥과 이와 상보적인 서열을 가지는 안티센스 RNA 가닥으로 구성된다. 전체 길이는 10 내지 100 염기, 바람직하게는 15 내지 80 염기, 가장 바람직하게는 20 내지 70 염기이고, 타겟 유전자의 발현을 RNAi 효과에 의하여 억제할 수 있는 것이면 평활(blunt)말단 혹은 점착(cohesive) 말단 모두 가능하다. 점착 말단 구조는 3 말단 돌출한 구조와 5 말단 쪽이 돌출한 구조 모두 가능하다.As used herein, the term "siRNA" refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) through cleavage of a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, the blunt end or cohesive All ends are possible. As for the sticky end structure, both a structure with 3 ends protruding and a structure with 5 ends protruding are possible.
본 명세서에서 용어“miRNA(microRNA)”는 세포내에서 발현되지 않는 올리고뉴클레오타이드로서 짧은 스템-루프 구조를 가지면서 타겟 유전자의 mRNA와 상보적인 결합을 통하여 타겟 유전자 발현을 억제하는 단일 가닥 RNA분자를 의미한다.As used herein, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that inhibits target gene expression through complementary binding with mRNA of a target gene while having a short stem-loop structure as an oligonucleotide that is not expressed in cells. do.
본 명세서에서 용어“리보자임(ribozyme)”은 RNA의 일종으로 특정한 RNA의 염기 서열을 인식하여 자체적으로 이를 절단하는 효소와 같은 기능을 가진 RNA 분자를 의미한다. 리보자임은 타겟 mRNA 가닥의 상보적인 염기서열로 특이성을 가지고 결합하는 영역과 타겟 RNA를 절단하는 영역으로 구성된다.In the present specification, the term “ribozyme” is a type of RNA and refers to an RNA molecule having a function such as an enzyme that recognizes a specific RNA base sequence and cuts it itself. A ribozyme is composed of a region that binds with specificity to a complementary nucleotide sequence of a target mRNA strand and a region that cleaves a target RNA.
본 명세서에서 용어“PNA(Peptide nucleic acid)”는 핵산과 단백질의 성질을 모두 가지면서 DNA 또는 RNA와 상보적으로 결합이 가능한 분자를 의미한다. PNA는 자연계에서는 발견되지 않고 인공적으로 화학적인 방법으로 합성되며, 상보적인 염기 서열의 천연 핵산과 혼성화(hybridization)를 통해 이중가닥을 형성하여 타겟 유전자의 발현을 조절한다.In the present specification, the term “peptide nucleic acid (PNA)” refers to a molecule that has properties of both nucleic acid and protein and can complementarily bind to DNA or RNA. PNA is not found in nature, but is artificially synthesized by chemical methods, and forms double strands through hybridization with natural nucleic acids having complementary nucleotide sequences to regulate the expression of target genes.
본 명세서에서 용어 “안티센스 올리고뉴클레오타이드”는 특정 mRNA의 서열에 상보적인 뉴클레오타이드 서열로서 타겟 mRNA 내의 상보적 서열에 결합하여 이의 단백질로의 번역, 세포질내로의 전위(translocation), 성숙(maturation) 또는 다른 모든 전체적인 생물학적 기능에 대한 필수적인 활성을 저해하는 핵산 분자를 의미한다. 안티센스 올리고뉴클레오타이드는 효능을 증진시키기 위하여 하나 이상의 염기, 당 또는 골격(backbone)의 위치에서 변형될 수 있다(De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). 올리고뉴클레오타이드 골격은 포스포로티오에이트, 포스포트리에스테르, 메틸 포스포네이트, 단쇄 알킬, 시클로알킬, 단쇄 헤테로아토믹, 헤테로시클릭 당숄포네이 등으로 변형될 수 있다.As used herein, the term “antisense oligonucleotide” refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and performs translation into a protein, translocation into the cytoplasm, maturation, or all other functions. A nucleic acid molecule that inhibits an essential activity for overall biological function. Antisense oligonucleotides can be modified at one or more bases, sugars or backbone positions to enhance potency (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). . The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomic, heterocyclic sugarsulfones, and the like.
본 명세서에서 용어“gRNA(guideRNA)”는 타겟 유전자를 인식하여 핵산분해효소(nuclease)를 유도함으로써 인식된 분위를 특이적으로 절단하는 유전자 편집 시스템에 사용되는 RNA 분자를 의미한다. 이러한 유전자 편집 시스템에는 대표적으로 CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) 시스템이 있다.As used herein, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cut the recognized locus. A typical example of such a gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.
본 발명의 또 다른 양태에 따르면, 본 발명은 다음의 단계를 포함하는 섬유화 질환의 예방 또는 치료용 조성물의 스크리닝 방법을 제공한다:According to another aspect of the present invention, the present invention provides a screening method for a composition for preventing or treating fibrotic disease comprising the following steps:
(a) TSP-2 (Thrombospondin-2) 단백질 또는 이를 발현하는 세포를 포함하는 생물학적 시료에 시험물질을 접촉시키는 단계; 및(a) contacting a test substance with a biological sample containing TSP-2 (Thrombospondin-2) protein or cells expressing the same; and
(b) 상기 생물학적 시료 내 TSP-2 단백질 또는 이를 인코딩하는 유전자의 발현량을 측정하는 단계,(b) measuring the expression level of the TSP-2 protein or a gene encoding the same in the biological sample;
상기 생물학적 시료 내 상기 단백질 또는 이를 인코딩하는 유전자의 발현량이 감소하는 경우, 상기 시험물질은 섬유화 질환의 예방 또는 치료용 조성물로 판정한다.When the expression level of the protein or the gene encoding the protein in the biological sample decreases, the test substance is determined as a composition for preventing or treating fibrotic disease.
본 발명에서 사용되는 TSP-2 단백질 및 이의 발현 조절을 통해 예방 또는 치료할 수 있는 질환에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위하여 그 기재를 생략한다.Since the TSP-2 protein used in the present invention and diseases that can be prevented or treated through regulation of its expression have already been described in detail, description thereof will be omitted to avoid excessive redundancy.
본 발명에서 용어“생물학적 시료”는 인간을 포함한 포유동물로부터 얻어지는, 상술한 단백질을 발현하는 세포를 포함하고 있는 모든 시료로서, 조직, 기관, 세포 또는 세포 배양액을 포함하나, 이에 제한되지 않는다. 보다 구체적으로는, 상기 생물학적 시료는 폐 유래의 조직, 세포 또는 이의 배양액일 수 있다.In the present invention, the term "biological sample" is any sample containing cells expressing the above-described proteins obtained from mammals, including humans, and includes, but is not limited to, tissues, organs, cells, or cell cultures. More specifically, the biological sample may be a lung-derived tissue, cell, or culture thereof.
본 발명의 스크리닝 방법을 언급하면서 사용되는 용어 “시험물질”은 본 발명의 단백질 또는 유전자를 발현하는 세포를 포함하는 시료에 첨가되어 이들 단백질 또는 유전자의 활성 또는 발현량에 영향을 미치는지 여부를 검사하기 위하여 스크리닝에서 이용되는 미지의 물질을 의미한다. 상기 시험물질은 화합물, 뉴클레오타이드, 펩타이드 및 천연 추출물을 포함하나, 이에 제한되는 것은 아니다. 시험물질을 처리한 생물학적 시료에서 상기 단백질 또는 유전자의 발현량 또는 활성을 측정하는 단계는 당업계에 공지된 다양한 발현량 및 활성 측정방법에 의해 수행될 수 있다.The term "test substance" used while referring to the screening method of the present invention is added to a sample containing cells expressing the protein or gene of the present invention to examine whether or not it affects the activity or expression level of these proteins or genes. means an unknown substance used in screening for The test substance includes, but is not limited to, compounds, nucleotides, peptides and natural extracts. The step of measuring the expression level or activity of the protein or gene in the biological sample treated with the test substance may be performed by various methods for measuring the expression level and activity known in the art.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 조성물을 대상체에 투여하는 단계를 포함하는 섬유화 질환(fibrotic disease)의 진단 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for diagnosing fibrotic disease comprising administering to a subject a composition comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명의 또 다른 양태에 따르면, 본 발명은 TSP-2 (Thrombospondin-2) 단백질 또는 이를 코딩하는 유전자의 발현량을 측정하는 제제를 유효성분으로 포함하는 조성물을 대상체에 투여하는 단계를 포함하는 섬유화 질환(fibrotic disease)의 진단 방법을 제공한다.According to another aspect of the present invention, the present invention is a fibrosis comprising the step of administering to a subject a composition comprising, as an active ingredient, an agent for measuring the expression level of TSP-2 (Thrombospondin-2) protein or a gene encoding it. A method for diagnosing fibrotic disease is provided.
본 발명의 또 다른 양태에 따르면, 본 발명은 TSP-2 (Thrombospondin-2)에 대한 억제제를 유효성분으로 포함하는 조성물을 포함하는 섬유화 질환(fibrotic disease)의 예방 또는 치료 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for preventing or treating fibrotic disease (fibrotic disease) comprising a composition containing an inhibitor for TSP-2 (Thrombospondin-2) as an active ingredient.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 섬유화에 관여하는 트롬보스폰딘-2(Thrombospondin, TSP-2) 단백질에 특이적으로 결합하는 항체를 이용하여 섬유화 질환 발생 여부를 예측하거나, 해당 단백질의 활성을 억제함으로써 섬유화 질환을 예방 또는 치료하는 방법을 제공한다.(a) The present invention uses an antibody that specifically binds to thrombospondin-2 (TSP-2) protein involved in fibrosis to predict the occurrence of fibrotic disease or suppresses the activity of the protein to prevent fibrotic disease. Provides a method for preventing or treating
(b) 본 발명은 명확한 발병 기전이 알려져 있지 않아 효율적인 치료 방법이 부재했던 특발성 폐섬유증에 대한 신규한 진단 및 치료 타겟으로서 TSP-2를 새롭게 제시하고, 특발성 폐섬유증에 대한 유효한 진단, 예방 또한 치료에 사용될 수 있는 효과적인 항체를 제공한다.(b) The present invention newly presents TSP-2 as a novel diagnosis and treatment target for idiopathic pulmonary fibrosis, for which no effective treatment method has been available due to unknown pathogenesis, and effective diagnosis, prevention and treatment of idiopathic pulmonary fibrosis. Provides effective antibodies that can be used for
(c) 아울러, 본 발명은 궁극적으로 병리학적 섬유화 기전에 대한 근원적인 억제 방법을 제공함으로써 다양한 조직에서의 섬유화를 효율적으로 차단하는 데에 유용하게 이용될 수 있다.(c) In addition, the present invention can be usefully used to efficiently block fibrosis in various tissues by ultimately providing a method for fundamentally suppressing pathological fibrosis mechanisms.
도 1a는 건강한 지원자(n=35) 및 IPF 환자(n=49)의 총 폐 조직에서 TSP-2의 FPKM(백만당 전사체 염기의 킬로 단위) 매핑 판독을 공개적으로 사용 가능한 데이터베이스인 GSE124685를 사용하여 비교한 결과를 도시한 그림이다. 도 1b는 공개적으로 이용 가능한 데이터베이스인 GSE110147를 사용하여 TSP-2의 RMA(Robust Multi-array Average) 배경 보정(background correction) 및 데이터 정규화를 수행한 결과를 도시한 그림이다.1A shows FPKM (kilo transcript bases per million) mapping reads of TSP-2 in total lung tissue of healthy volunteers (n=35) and IPF patients (n=49) using the publicly available database GSE124685. The figure shows the result of comparison. FIG. 1B is a diagram showing the results of performing Robust Multi-array Average (RMA) background correction and data normalization of TSP-2 using GSE110147, a publicly available database.
도 2는 정량적 샌드위치 효소 면역분석(Quantitative Sandwich Enzyme Immunoassay)를 통하여 건강한 대조군과 간질성 폐질환(ILD)환자에서 TSP-2의 혈청 수준을 대조군 결과를 도시한 그림이다.Figure 2 is a picture showing the results of the serum level of TSP-2 in a healthy control group and interstitial lung disease (ILD) patients through a quantitative sandwich enzyme immunoassay (Quantitative Sandwich Enzyme Immunoassay).
도 3a는 블레오마이신을 통한 마우스 폐 섬유증 모델에 대한 실험 계획을 도시한 그림이다. 도 3b는 농축된 기관지 폐포 세척액(BALF)에서 블레오마이신으로 인하여 증가된 TSP-2를 웨스턴 블롯으로 확인한 결과를 도시한 그림이다. 도 3c는 BAL 액 총 세포 계수 결과, 마우스 폐 섬유증 모델에서 염증세포가 증가된 것을 도시한 그림이다. 도 3d는 마우스 폐 조직에서 TSP-2에 대한 qRT-PCR의 결과를 도시한 그림이다. 도 3e는 상피 Ccsp-TGF-β1-TG 마우스를 생성하기 위한 독시사이클린 투여 계획을 도시한 그림이다. 도 3f는 농축된 기관지 폐포 세척액(BALF)에서 독시사이클린으로 인하여 증가된 TSP-2을 면역블롯으로 확인한 결과를 도시한 그림이다. 도 3g는 BAL 액 총 세포 계수 결과, 독시사이클린으로 인하여 염증세포가 증가한 것을 도시한 그림이다. 도 3h는 독시사이클린 투여로 TSP-2 발현이 마우스 폐 조직에서 증가한 것을 도시한 그림이다.Figure 3a is a diagram showing an experimental plan for a mouse lung fibrosis model through bleomycin. Figure 3b is a picture showing the result of confirming the increased TSP-2 due to bleomycin in concentrated bronchoalveolar lavage fluid (BALF) by Western blot. Figure 3c is a picture showing the increase in inflammatory cells in the mouse lung fibrosis model as a result of BAL fluid total cell counting. Figure 3d is a picture showing the results of qRT-PCR for TSP-2 in mouse lung tissue. Figure 3e is a diagram illustrating the doxycycline dosing regimen for generating epithelial Ccsp-TGF-β1-TG mice. Figure 3f is a picture showing the result of immunoblot confirmation of increased TSP-2 due to doxycycline in concentrated bronchoalveolar lavage fluid (BALF). FIG. 3g is a diagram showing an increase in inflammatory cells due to doxycycline as a result of total cell counting in BAL fluid. Figure 3h is a picture showing that TSP-2 expression increased in mouse lung tissue by doxycycline administration.
도 4a는 MLg 및 C22 세포주에서 Smad4의 넉다운(Knockdown)결과, TGF-β에 의해 유도되는 TSP-2 발현을 나타내는 mRNA가 감소된 것을 도시한 그래프이다. 도 4b는 MLg 및 C22 세포주에서 Smad4의 넉다운(Knockdown)결과, TGF-β에 의해 유도되는 TSP-2의 단백질 양이 감소된 것을 면역 블롯으로 확인한 결과를 도시한 그림이다.Figure 4a is a graph showing the decrease in mRNA representing TSP-2 expression induced by TGF-β as a result of knockdown of Smad4 in MLg and C22 cell lines. Figure 4b is a picture showing the result of immunoblot confirming that the amount of TSP-2 protein induced by TGF-β was reduced as a result of Smad4 knockdown in MLg and C22 cell lines.
도 5a는 TGF-β1의 선택적 억제제가 TGF-β1에 의해 유도되는 TSP-2 발현을 양-의존적(dose dependent manner)로 억제함을 도시한 그래프이다. 도 5b는 TGF-β1의 선택적 억제제가 TGF-β1에 의해 유도되는 TSP-2 발현을 용량-의존적 방식(dose dependent manner)으로 억제함을 웨스턴 블롯으로 확인한 결과를 도시한 그림이다.5A is a graph showing that a selective inhibitor of TGF-β 1 inhibits TSP-2 expression induced by TGF-β 1 in a dose dependent manner. Figure 5b is a picture showing the result of Western blot confirming that a selective inhibitor of TGF-β 1 inhibits TSP-2 expression induced by TGF-β 1 in a dose dependent manner.
도 6a는 4kb 크기의 TSP-2 유전자 프로모터 내의 Smad-결합 요소를 표시한 개략도이다. 도 6b는 프로모터 부분 결실에 따른 프로모터의 활성을 루시퍼레이즈 리포터 분석으로 조사한 결과를 나타낸 그림이다. 도 6c는 TGF-β1이 없는 경우, 프로모터 부분 결실에 따른 프로모터의 활성을 루시퍼레이즈 리포터 분석으로 조사한 결과를 나타낸 그림이다. 도 6d는 TGF-β1이 있는 경우 #6 Smad-결합 부위 결실을 사용하여 루시퍼레이즈 리포터 분석을 수행한 결과를 도시한 그림이다.Figure 6a is a schematic diagram showing Smad-binding elements within the 4 kb TSP-2 gene promoter. Figure 6b is a diagram showing the results of examining the activity of the promoter according to the deletion of the promoter portion by luciferase reporter assay. Figure 6c is a diagram showing the results of examining the activity of the promoter according to the promoter partial deletion in the absence of TGF-β 1 by luciferase reporter assay. Figure 6d is a picture showing the results of performing a luciferase reporter assay using #6 Smad-binding site deletion in the presence of TGF-β1.
도 7a는 자가분비(Autocrine) 및 측분비(Paracrine) 실험을 도시한 개략도이다. 도 7b는 TSP-2의 녹다운 효율을 알아보기 위하여 수행한, 폐 세포에서 TSP-2 유전자의 발현에 대한 qRT-PCR 분석 결과를 도시한 그림이다. 도 7c는 TSP-2의 녹다운 효율을 알아보기 위하여 수행한, 폐 세포에서 TSP-2 유전자의 발현에 대한 웨스턴 블롯 결과를 도시한 그림이다. 도 7d는 TGF-β1-유도 섬유아세포(자가분비) 활성화를 알아보기 위하여 섬유아세포 계수를 측정한 결과를 도시한 그림이다. 도 7e는 섬유화 마커(표적 유전자)들의 TGF-β1 투여에 의한 상향조절 여부 및 TSP-2 녹다운에 의한 하향조절 여부를 알아보기 위한 qRT-PCR 결과를 도시한 그림이다.7A is a schematic diagram illustrating an autocrine and paracrine experiment. Figure 7b is a picture showing the results of qRT-PCR analysis on the expression of the TSP-2 gene in lung cells, which was performed to determine the knockdown efficiency of TSP-2. Figure 7c is a picture showing the results of Western blotting on the expression of the TSP-2 gene in lung cells, which was performed to determine the knockdown efficiency of TSP-2. Figure 7d is a picture showing the results of measuring fibroblast counts to determine TGF-β1-induced fibroblast (autocrine) activation. Figure 7e is a picture showing the results of qRT-PCR to determine whether fibrosis markers (target genes) were upregulated by TGF-β1 administration and downregulated by TSP-2 knockdown.
도 8a는 폐 상피 세포(Lung Epithelial Cell)에서 TSP-2의 녹다운 효율을 알아보기 위하여 수행한, TSP-2 유전자의 발현에 대한 qRT-PCR 분석 결과를 도시한 그림이다. 도 8b는 폐 상피 세포(Lung Epithelial Cell)에서 TSP-2의 녹다운 효율을 알아보기 위하여 수행한, TSP-2 유전자의 발현에 대한 웨스턴 블롯 결과를 도시한 그림이다. 도 8c는 TGF-β1-유도 섬유아세포(측분비) 활성화를 알아보기 위하여 섬유아세포 계수를 측정한 결과를 도시한 그림이다. 도 8d는 폐 상피 세포에서 섬유화 마커(표적 유전자)들의 TGF-β1 투여에 의한 상향조절 여부 및 TSP-2 녹다운에 의한 하향조절 여부를 알아보기 위한 qRT-PCR 결과를 도시한 그림이다.FIG. 8A is a diagram showing the results of qRT-PCR analysis for the expression of the TSP-2 gene, which was performed to examine the knockdown efficiency of TSP-2 in lung epithelial cells. FIG. 8B is a picture showing the results of Western blotting on the expression of the TSP-2 gene, which was performed to examine the knockdown efficiency of TSP-2 in lung epithelial cells. 8c is a diagram showing the results of measuring fibroblast counts in order to determine TGF-β1-induced fibroblast (paracrine) activation. FIG. 8D is a diagram showing qRT-PCR results for determining whether fibrosis markers (target genes) in lung epithelial cells were up-regulated by TGF-β 1 administration and down-regulated by TSP-2 knockdown.
도 9a는 TSP-2의 표적 억제는 용량 의존적 방식으로 TGF-β1 유도 섬유아세포 활성화를 감소시킴을 섬유아세포의 세포수로 나타낸 그래프이다. 도 9b는 TSP-2에 특이적인 단일 중화 항체에 의한 TSP-2의 표적 억제에 의하여 TGF-β1으로 유도된 섬유화 마커(표적 유전자)들의 발현이 감소됨을 도시한 qRT-PCR 결과 그래프이다.9a is a graph showing that target inhibition of TSP-2 reduces TGF-β1-induced fibroblast activation in a dose-dependent manner, in terms of fibroblast cell number. FIG. 9B is a graph showing the results of qRT-PCR showing that the expression of TGF-β 1 -induced fibrosis markers (target genes) is reduced by target inhibition of TSP-2 by a single neutralizing antibody specific for TSP-2.
도 10a는 상피 세포(C22)에서 TSP-2의 표적 억제는 용량 의존적 방식으로 TGF-β1 유도 섬유아세포 활성화를 감소시킴을 섬유아세포의 세포수로 나타낸 그림이다. 도 10b는 상피 세포(C22)에서 TSP-2에 특이적인 단일 중화 항체에 의한 TSP-2의 표적 억제에 의하여 TGF-β1으로 유도된 섬유화 마커(표적 유전자)들의 발현이 감소됨을 도시한 qRT-PCR 결과 그래프이다. 도 10c는 상피 세포(RLE-6TN)에서 TSP-2의 표적 억제는 용량 의존적 방식으로 TGF-β1 유도 섬유아세포 활성화를 감소시킴을 섬유아세포의 세포수로 나타낸 그림이다. 도 10d는 상피 세포(RLE-6TN)에서 TSP-2에 특이적인 단일 중화 항체에 의한 TSP-2의 표적 억제에 의하여 TGF-β1으로 유도된 섬유화 마커(표적 유전자)들의 발현이 감소됨을 도시한 qRT-PCR 결과 그래프이다.10a is a diagram showing that target inhibition of TSP-2 in epithelial cells (C22) reduces TGF-β 1 -induced fibroblast activation in a dose-dependent manner, in terms of the number of fibroblast cells. Figure 10b shows that the expression of fibrosis markers (target genes) induced by TGF-β 1 is reduced by the targeted inhibition of TSP-2 by a single neutralizing antibody specific for TSP-2 in epithelial cells (C22). This is a PCR result graph. 10c is a diagram showing that target inhibition of TSP-2 in epithelial cells (RLE-6TN) reduces TGF-β 1 -induced fibroblast activation in a dose-dependent manner, in terms of the number of fibroblast cells. 10d shows that the expression of TGF-β 1 -induced fibrosis markers (target genes) is reduced by target inhibition of TSP-2 by a single neutralizing antibody specific for TSP-2 in epithelial cells (RLE-6TN). This is a graph of qRT-PCR results.
도 11a는 TSP-2에 대한 단일클론항체를 제작하기 위하여 시행한 7개 클론에 대한 닷-블롯(Dot-Blot) 결과를 도시한 그림이다. 도 11b는 7개 클론의 항체 친화도(Affinity)를 알아보기 위하여 시행한 폐섬유아세포에서의 웨스턴 블롯 결과를 도시한 그림이다. 도 11c는 #2 및 #4 클론 항체의 섬유아세포 증식 저해능을 비교한 그래프이다. 도 11d는 최종 제작 항체와 시판항체의 TGF-β처리 후 섬유아세포 증식 억제능을 비교한 그래프이다.11a is a diagram showing the results of dot-blot for 7 clones performed to produce a monoclonal antibody against TSP-2. Figure 11b is a picture showing the results of Western blotting in lung fibroblasts performed to determine the antibody affinity of 7 clones. Figure 11c is a graph comparing the fibroblast proliferation inhibitory ability of #2 and #4 clone antibodies. 11d is a graph comparing the ability of the final antibody and commercially available antibody to inhibit fibroblast proliferation after TGF-β treatment.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
실험방법 및 분석방법Experiment method and analysis method
환자 샘플patient sample
인간 혈청 샘플은 세브란스 병원(서울, 한국)에서 얻었다. 총 8개의 ILD 환자 샘플과 정상 조직을 가진 총 4개의 대조군 샘플을 연구에 포함시켰다. 환자는 미국 흉부 학회 및 유럽 호흡기 학회의 기준을 충족했으며, ILD 진단은 병력, 신체 검사, 폐 기능 연구, 흉부 고해상도 컴퓨터 단층 촬영으로 입증하였고, 비디오-보조 흉강경 폐 생검 또는 이식편 외식(transplant explants)으로 확증하였다. 본 연구는 세브란스병원 기관심사위원회의 승인받아 수행하였다.Human serum samples were obtained from Severance Hospital (Seoul, Korea). A total of 8 ILD patient samples and a total of 4 control samples with normal tissue were included in the study. The patient met the criteria of the American Thoracic Society and the European Respiratory Society, and the diagnosis of ILD was substantiated by history, physical examination, pulmonary function study, high-resolution computed tomography of the chest, and video-assisted thoracoscopic lung biopsy or transplant explants. Confirmed. This study was conducted with the approval of the Institutional Review Board of Severance Hospital.
효소-연결 면역흡착측정법 (ELISA)Enzyme-Linked Immunosorbent Assay (ELISA)
혈청 분리 튜브를 사용하여 혈청을 분리하고 제조업체의 지침에 따라 Human Thrombospondin-2 quantikine ELISA 키트(R&D Systems Inc., USA)를 사용하여 TSP-2의 혈청 수준(말기 ILD 환자 제외)을 측정하였다.Serum was separated using serum separation tubes, and serum levels of TSP-2 (excluding patients with late-stage ILD) were measured using the Human Thrombospondin-2 quantikine ELISA kit (R&D Systems Inc., USA) according to the manufacturer's instructions.
동물 연구animal research
모든 동물 실험은 연세대학교 의과대학 동물연구소 동물관리위원회(인증번호 IACUC-2018-0087)의 승인을 받았고, 프로토콜은 연세대학교 의과대학 동물실험윤리위원회의 승인을 받았다. 모든 마우스는 C57BL/6 유전적 배경의 마우스를 사용하였고, 12h-낮/12h-밤 주기로 수용하였다.All animal experiments were approved by the Animal Care Committee of the Institute of Animal Research, Yonsei University College of Medicine (accreditation number IACUC-2018-0087), and the protocol was approved by the Animal Experimentation Ethics Committee of Yonsei University College of Medicine. All mice were from the C57BL/6 genetic background and housed in a 12h-day/12h-night cycle.
마우스들을 Zoletil(30 mg/kg) 및 Rompun(10 mg/kg)을 복강내 주사하여 마취하였다. 마우스에 PBS(비히클 대조군) 또는 4mg/kg 블레오마이신(Santa Cruz Biotechnology, Dallas, TX, USA)을 기관내 주사하였다. 각 그룹에 평균 8마리의 마우스를 사용했으며, 마우스는 블레오마이신 및 기관지 세척액(Bronchial Lavage, BAL)을 투여받은 후 14일째에 희생시키고 폐조직을 채취하였다. TGF-β1-TG 마우스는 연세대학교 손명현 교수의 연구실에서 제공받았다. TGF-β1 발현을 유도하기 위해 성체 형질전환 마우스(8-12주령)를 4주 동안 2% 자당(Sucrose)과 0.5 mg/mL 독시사이클린(Doxycycline)을 함유한 식수와 함께 4주 동안 수용하였다. 독시사이클린을 함유하는 물은 일주일에 세 번 교체하였다.Mice were anesthetized with an intraperitoneal injection of Zoletil (30 mg/kg) and Rompun (10 mg/kg). Mice were injected intratracheally with PBS (vehicle control) or 4 mg/kg bleomycin (Santa Cruz Biotechnology, Dallas, TX, USA). An average of 8 mice were used in each group, and the mice were sacrificed on day 14 after administration of bleomycin and bronchial lavage (BAL), and lung tissues were collected. TGF-β1-TG mice were provided by Professor Myeong-Hyeon Sohn's laboratory at Yonsei University. To induce TGF-β1 expression, adult transgenic mice (8-12 weeks old) were housed for 4 weeks with drinking water containing 2% sucrose and 0.5 mg/mL doxycycline. Water containing doxycycline was changed three times a week.
폐의 채취 및 고정Lung harvest and fixation
마우스는 졸레틸/룸푼으로 마취하였으며, 복부와 흉곽을 절개하여 심장과 폐를 노출시켰다. 10mL PBS를 사용하여 폐동맥을 통해 폐를 관류하였다. 마우스 폐의 좌엽을 제거하고 실온에서 48시간 동안 포름알데히드(4% w/v)에 고정하고 파라핀에 포매하였다. 파라핀 블록 절편의 구획화는 서울의 연세대 의과대학 조직학 코어에서 수행하였다.Mice were anesthetized with zoletyl/lumpoon, and an incision was made in the abdomen and thorax to expose the heart and lungs. Lungs were perfused via the pulmonary artery using 10 mL PBS. The left lobe of the mouse lung was removed, fixed in formaldehyde (4% w/v) for 48 hours at room temperature, and embedded in paraffin. Sectionalization of paraffin block sections was performed at the Histology Core, Yonsei University College of Medicine, Seoul.
메이슨 삼색 염색법(Masson’s Trichrome Staining)Masson's Trichrome Staining
메이슨 삼색 염색법으로 콜라겐 생성을 확인하기 위해 절편을 탈파라핀화하고 물로 수화하였다. 절편을 전자레인지를 사용하여 Bouin’s 용액(#HT10132, Merck, Darmstadt, Germany)에 1분 동안 염색한 다음, 실온에서 15분 동안 방치하였다. 흐르는 수돗물로 씻은 후 절편을 헤마톡실린에 10분간 담그고, 다시 흐르는 수돗물로 헹구고 비브리히 스칼렛(Biebrich scarlet)으로 5분간 염색하였다. 그 후, 절편을 15분 동안 인텅스텐(phosphotungstic) / 인몰리브덴산(phosphomolybdic)으로 염색하고 아닐린 블루로 5분 동안 바로 옮긴 후, 탈수하고 커버슬립을 덮었다.Sections were deparaffinized and hydrated with water to confirm collagen production by Mason's tricolor staining. Sections were stained in Bouin's solution (#HT10132, Merck, Darmstadt, Germany) for 1 minute using a microwave and then left at room temperature for 15 minutes. After washing with running tap water, the sections were immersed in hematoxylin for 10 minutes, rinsed again with running tap water, and stained with Biebrich scarlet for 5 minutes. Then, the sections were stained with phosphotungstic/phosphomolybdic for 15 min, immediately transferred to aniline blue for 5 min, dehydrated and covered with a coverslip.
세포 배양 및 시약Cell culture and reagents
마우스 섬유아세포주 MLg 및 쥐 폐포 2형 세포주 RLE-6TN은 한국세포주은행(서울, 한국)에서 구입하였다. MLg와 RLE-6TN은 10%(v/v) 태아 소 혈청과 1% 항생제/항진균 용액(Corning, Manassas, VA, USA)이 첨가된 둘베코수정이글배지(Dulbecco’s modified Eagles’s medium)에서 37℃, 5% CO2에서 배양하였다. 불멸화 마우스 폐 클럽 세포주(Immortalized Mouse Cell Line), C22(Sigma-Aldrich)는 2% 소 태아 혈청, 페니실린[100U/ml], 스트렙토마이신[100 μg/ml], 2mM 글루타민, 엔도텔린-1[0.25㎍/ml], 인터페론-γ[0.01㎍/ml], 인슐린[10㎍/ml], 트랜스페린[5㎍/ml], 내피세포 성장 보충 [7.5 μg/ml], 표피 성장 인자 [0.025 μg/ml], 히드로코르티손 [0.36 μg/ml] 및 T3 [0.02 μg/ml] 이 보충된 허용 조건의 둘베코수정이글배지(Corning, Manassas, VA, USA)에서 5% CO2에서 33℃로 실험 전까지 유지하였고, 그런 다음 T 대항원(Large T-antigen)을 비활성화하기 위해 37 ℃에서 비허용 조건(인터페론-γ 없음)으로 옮겼다. 블레오마이신은 Santa Cruz Biotechnology (Dallas, TX, USA)에서 구입하였고, TGF-β1은 ProSpec (East Brunswick, NJ, USA)에서 구입하였다. 일시적 형질감염(Transient transfection)은 Lipofectamine 2000, Lipofectamine 3000 및 Lipofectamine RNAiMAX(Invitrogen, Carlsbad, CA, USA)를 사용하여 수행하였다.The mouse fibroblast cell line MLg and the mouse alveolar type 2 cell line RLE-6TN were purchased from Korea Cell Line Bank (Seoul, Korea). MLg and RLE-6TN were cultured in Dulbecco's modified Eagles' medium supplemented with 10% (v/v) fetal bovine serum and 1% antibiotic/antimycotic solution (Corning, Manassas, VA, USA) at 37°C; Incubated in 5% CO 2 . Immortalized Mouse Lung Club Cell Line, C22 (Sigma-Aldrich), 2% fetal bovine serum, penicillin [100 U/ml], streptomycin [100 μg/ml], 2 mM glutamine, endothelin-1 [0.25 μg/ml], interferon-γ [0.01 μg/ml], insulin [10 μg/ml], transferrin [5 μg/ml], endothelial cell growth supplement [7.5 μg/ml], epidermal growth factor [0.025 μg/ml] ], hydrocortisone [0.36 μg/ml] and T3 [0.02 μg/ml] were maintained at 33°C in 5% CO 2 in permissive Dulbecco's Modified Eagle's Medium (Corning, Manassas, VA, USA) until the experiment. and then transferred to non-permissive conditions (no interferon-γ) at 37°C to inactivate the Large T-antigen. Bleomycin was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and TGF-β1 was purchased from ProSpec (East Brunswick, NJ, USA). Transient transfection was performed using Lipofectamine 2000, Lipofectamine 3000 and Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA).
국부 돌연변이 실험(Site-directed Mutagenesis)Site-directed Mutagenesis
High-Fidelity DNA Polymerases & Master Mixes(Thermo science, Waltham, Massachusetts, USA)를 사용하여 다양한 결실 구조가 생성하였고, PCR 반응을 위해 10pM 프라이머를 혼합하였다. 국부 돌연변이 실험에서 사용된 PCT 사이클링 조건은 다음과 같다: 98℃에서 2분간 초기 변성, 98℃에서 30초 변성, 65℃에서 30초 동안 어닐링 및 72℃에서 45초 동안 연장 반응을 수행하였다. 그리고 최종 extension은 72℃에서 5분간 진행하였다. 증폭된 혼합물을 DpnⅠ(Aglient Technologies, Santa clara, CA, USA)로 37℃에서 1시간 30분 동안 처리하였고, PCR 산물을 사용하여 수용성 E.coli(Real Biotech Corporation, Banqiao, Taiwan)를 형질전환하였다. 모든 생성물들은 DNA 시퀀싱을 통해 확인하였다.Various deletion structures were generated using High-Fidelity DNA Polymerases & Master Mixes (Thermo science, Waltham, Massachusetts, USA), and 10 pM primers were mixed for the PCR reaction. The PCT cycling conditions used in the local mutagenesis experiments were as follows: initial denaturation at 98°C for 2 minutes, denaturation at 98°C for 30 seconds, annealing at 65°C for 30 seconds, and extension reaction at 72°C for 45 seconds. And the final extension was carried out at 72℃ for 5 minutes. The amplified mixture was treated with DpnI (Aglient Technologies, Santa Clara, CA, USA) at 37°C for 1 hour and 30 minutes, and the PCR product was used to transform water-soluble E.coli (Real Biotech Corporation, Banqiao, Taiwan). . All products were confirmed through DNA sequencing.
루시퍼레이즈 리포터 분석 (Luciferase reporter assay)Luciferase reporter assay
마우스 TSP-2 유전자 프로모터 구조체을 만들기 위해 TSP-2 유전자의 특정 영역(-1700 ~ +300 뉴클레오티드)을 야생형 C57BL/6 마우스 조직(QIAGEN, DNA mini kit)에서 얻은 유전체DNA를 증폭시킴으로써 준비하였다. 증폭된 생성물을 정제하고 Nhe-IHF 및 XhoI 제한 효소로 절단하고 pGL3-Control 벡터(Promega)에 클로닝하였다. 이 TSP-2 리포터 유전자를 결실 구조체(deletion construct)의 주형으로 사용하였다. 루시퍼라제 리포터 분석을 수행하기 위해 리포펙타민 3000(Invitrogen, Carlsbad, CA, USA)을 사용하여 24웰 플레이트에서 웰당 1㎍의 TSP-2 리포터 유전자로 세포를 일시적 형질감염시켰다. 24시간 동안 인큐베이션한 후, 세포를 추가 24시간 동안 무혈청 배지에서 20ng/mL의 TGF-β1으로 처리하였다. 세포를 수확하고 용해하여 루시퍼라제 분석 시스템(Promega)을 사용하여 루시퍼라제 활성을 분석하였다. 발광성은 MicroLumatPlus LB96V Microplate Luminometer(Berthold)로 측정하였다.To create a mouse TSP-2 gene promoter construct, a specific region (-1700 to +300 nucleotides) of the TSP-2 gene was prepared by amplifying genomic DNA obtained from wild-type C57BL/6 mouse tissue (QIAGEN, DNA mini kit). The amplified product was purified, digested with Nhe-IHF and XhoI restriction enzymes, and cloned into the pGL3-Control vector (Promega). This TSP-2 reporter gene was used as a template for a deletion construct. To perform the luciferase reporter assay, cells were transiently transfected with 1 μg of the TSP-2 reporter gene per well in 24-well plates using Lipofectamine 3000 (Invitrogen, Carlsbad, Calif., USA). After incubation for 24 hours, cells were treated with 20 ng/mL of TGF-β1 in serum-free medium for an additional 24 hours. Cells were harvested, lysed and assayed for luciferase activity using the Luciferase Assay System (Promega). Luminescence was measured with a MicroLumatPlus LB96V Microplate Luminometer (Berthold).
안정적인 형질감염 세포주의 구축Construction of stable transfected cell lines
TSP-2의 안정적인 녹다운을 위한 세포주를 생성하기 위해 이 연구에 사용된 렌티바이러스 벡터(shTSP-2)를 Sigma-Aldrich, U.S.A.에서 구입하였다. 보조 플라스미드 리포솜(pxPAX.2 및 pMD2.G) 및 렌티바이러스 벡터는 Lipofectamine 2000이라는 형질감염 시약(Invitrogen, Carlsbad, CA, USA)을 이용하여 293FT cell에 형질감염하여 렌티바이러스를 생산하였다. 형질감염 48시간 후, 상층액을 수집하고 원심분리하여 세포 파편(cell debris)를 제거하였다. 원심분리된 상층액은 0.45 μm 폴리비닐리덴 디플루오라이드 필터를 사용하여 여과하였다. C22, RLE-6TN 및 MLg 세포를 10㎍/ml 폴리브렌과 혼합된 바이러스 현탁액으로 감염시켰다. 감염 48시간 후, 안정적으로 형질도입된 양성 세포주를 스크리닝하기 위해 2㎍/ml의 퓨로마이신을 첨가하였다.The lentiviral vector (shTSP-2) used in this study to generate cell lines for stable knockdown of TSP-2 was purchased from Sigma-Aldrich, USA. Auxiliary plasmid liposomes (pxPAX.2 and pMD2.G) and lentiviral vectors were transfected into 293FT cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) to produce lentiviruses. 48 hours after transfection, the supernatant was collected and centrifuged to remove cell debris. The centrifuged supernatant was filtered using a 0.45 μm polyvinylidene difluoride filter. C22, RLE-6TN and MLg cells were infected with virus suspension mixed with 10 μg/ml polybrene. 48 hours after infection, puromycin at 2 μg/ml was added to screen for stably transduced positive cell lines.
웨스턴 블롯 분석Western blot analysis
세포는 용해 완충액(20mM Tris-Cl, 150mM NaCl, 1% Triton X-100, 1.5% MgCl2, 1mM EDTA, 1mM Na2VO4, 1mM 페닐메틸설포닐 플루오라이드(PMSF) 및 프로테아제 억제제 칵테일, pH 7.5)에서 용해하였다. 용해물을 간단히 볼텍싱하고 초음파 처리한 후, 4℃에서 20분 동안 12,000 x g에서 원심분리하여 부산물을 제거하였다. 상층액을 수집하고 새로운 튜브로 옮겼다. 단백질 농도는 660 nm 단백질 분석 시약(Thermo Scientific, Waltham, MA, USA)으로 결정하였다. 동일한 양의 단백질 추출물을 SDS-폴리아크릴아미드 겔에서 전기영동한 다음 니트로셀룰로오스 전달막(Whatman, Dassel, Germany)으로 옮겼다. 멤브레인은 0.1%(v/v) Tween 20(Sigma-Aldrich, St. Louis, MO, USA), 5%(w/v) 무지방 DifcoTM 탈지유(BD Biosciences, Sparks, MD, USA) 및 3% BSA(Affymetrix, Santa clara OH, USA)를 포함하는 PBS를 통해 차단되었고, 1차 항체를 첨가하였다. 실험에는 다음과 같은 항체를 사용하였다: Cell Signaling(Danvers, MA, USA)에서 구입한 항-TSP-2(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 항-Smad3, 항-p-Smad3 및 항- Smad4(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 항-β-액틴(Sigma-Aldrich, St. Louis, MO, USA). 그런 다음 멤브레인을 1x PBST로 세척하고 적절한 이차 항-토끼 또는 항-마우스 양고추냉이 퍼옥시다제 결합 항체(Thermo Scientific, Rockford IL, USA)와 함께 1시간 동안 인큐베이션한 다음, LAS-3000 시스템 (Fujifilm, Stamford, CT, USA)과 함께 enhanced chemiluminescence detection reagent (Thermo Scientific)을 통해 시각화하였다.Cells were lysed in lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100, 1.5% MgCl2, 1 mM EDTA, 1 mM Na2VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail, pH 7.5). did The lysate was briefly vortexed and sonicated, followed by centrifugation at 12,000 x g for 20 min at 4°C to remove by-products. The supernatant was collected and transferred to a new tube. Protein concentration was determined with 660 nm protein assay reagent (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein extracts were electrophoresed on SDS-polyacrylamide gels and then transferred to nitrocellulose transfer membranes (Whatman, Dassel, Germany). Membranes were formulated with 0.1% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA), 5% (w/v) nonfat DifcoTM skim milk (BD Biosciences, Sparks, MD, USA) and 3% BSA. (Affymetrix, Santa clara OH, USA), and primary antibodies were added. The following antibodies were used in the experiment: anti-TSP-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) purchased from Cell Signaling (Danvers, MA, USA), anti-Smad3, anti-p- Smad3 and anti-Smad4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). The membrane was then washed with 1x PBST and incubated for 1 hour with an appropriate secondary anti-rabbit or anti-mouse horseradish peroxidase conjugated antibody (Thermo Scientific, Rockford IL, USA), followed by a LAS-3000 system (Fujifilm , Stamford, CT, USA) and visualized through enhanced chemiluminescence detection reagent (Thermo Scientific).
RNA 분리 및 정량적 실시간 연쇄 반응(qRT-PCR)RNA isolation and quantitative real-time chain reaction (qRT-PCR)
제조사의 프로토콜(Takara, Kyoto, Japan)에 따라 TRIzol 시약을 사용하여 총 RNA를 추출하였다. 500㎕의 Trizol을 세포 배양 접시(6웰)에 첨가하고 세포를 튜브에 수집하였다. 100 μl의 클로로포름을 샘플에 첨가하고 샘플을 볼텍싱하였다. 샘플을 실온에서 3분 동안 인큐베이션하고 4℃에서 15분 동안 12,000 x g에서 원심분리하였다. 250 ul의 상청액을 수집하고 새로운 튜브로 옮겼다. 250㎕의 이소프로판올을 샘플에 첨가하고 샘플을 4℃에서 10분 동안 12,000 xg에서 원심분리하였다. 상층액을 제거하고 500㎕의 75% 에탄올을 샘플에 첨가하였다. 그 후 샘플을 4℃에서 5분 동안 13,000 x g에서 원심분리하였다. 상층액을 제거하고 펠렛을 실온에서 건조하였다. DEPC 30㎕를 건조된 펠렛에 첨가하였다. RNA의 농도는 Nanodrop1000(Thermo Scientific, Waltham, MA, USA)으로 측정하였다. RNA 분리 후 1 μg의 total RNA를 사용하고 제조사의 프로토콜에 따라 CellScript(CellSafe, Seoul, Korea)를 사용하여 cDNA를 합성하였다. qRT-PCR 분석은 ABI PRISM 7000 Sequence Detection System 기기 및 소프트웨어(Applied Biosystems, Carlsbad, CA, USA)를 사용하여 일부 수정된 제조업체의 프로토콜에 따라 수행하였다. 간단히 요약하면, 적절한 양의 역전사 반응 혼합물을 SYBR 그린 PCR 마스터 믹스(Applied Biosystems, Carlsbad, CA, USA)를 사용하여 특정 프라이머로 증폭하였다. qRT-PCR에 대한 PCR 프라이머 서열은 표 1에 나열하였다.Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol (Takara, Kyoto, Japan). 500 μl of Trizol was added to the cell culture dish (6 well) and the cells were collected in a tube. 100 μl of chloroform was added to the sample and the sample was vortexed. Samples were incubated at room temperature for 3 minutes and centrifuged at 12,000 x g for 15 minutes at 4°C. 250 ul of supernatant was collected and transferred to a new tube. 250 μl of isopropanol was added to the sample and the sample was centrifuged at 12,000 xg for 10 minutes at 4°C. The supernatant was removed and 500 μl of 75% ethanol was added to the sample. The samples were then centrifuged at 13,000 x g for 5 minutes at 4°C. The supernatant was removed and the pellet was dried at room temperature. 30 μl of DEPC was added to the dried pellet. RNA concentration was measured with Nanodrop1000 (Thermo Scientific, Waltham, MA, USA). After RNA isolation, 1 μg of total RNA was used and cDNA was synthesized using CellScript (CellSafe, Seoul, Korea) according to the manufacturer's protocol. qRT-PCR analysis was performed using an ABI PRISM 7000 Sequence Detection System instrument and software (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's protocol with some modifications. Briefly, an appropriate amount of the reverse transcription reaction mixture was amplified with specific primers using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). PCR primer sequences for qRT-PCR are listed in Table 1.
유전자의 발현 수준은 5점 연속 표준 곡선을 생성하여 결정하였다. cDNA의 농도는 α-Tubulin를 통하여 정규화하였다. 모든 반응은 삼중으로 수행하였으며, 상대 발현 수준 및 SD 값은 비교 방법(comparative method)을 사용하여 계산하였다. Expression levels of genes were determined by generating a 5-point continuous standard curve. The concentration of cDNA was normalized through α-Tubulin. All reactions were performed in triplicate, and relative expression levels and SD values were calculated using a comparative method.
Figure PCTKR2022010881-appb-img-000001
Figure PCTKR2022010881-appb-img-000001
mouse Thbs2 forward primer : GACCAAACCTACGCTGGTGGA (서열번호 33)mouse Thbs2 forward primer: GACCAAACCTACGCTGGTGGA (SEQ ID NO: 33)
mouse Thbs2 reverse primer : AGCACATTGCTGGAGCTGGA (서열번호 34)mouse Thbs2 reverse primer: AGCACATTGCTGGGAGCTGGA (SEQ ID NO: 34)
mouse α-SMA forward primer : GTGACTCACAACGTGCCTATC (서열번호 35)mouse α-SMA forward primer: GTGACTCACAACGTGCCTATC (SEQ ID NO: 35)
mouse α-SMA reverse primer : CTCGGCAGTAGTCACGAAGG (서열번호 36)mouse α-SMA reverse primer: CTCGGCAGTAGTCACGAAGG (SEQ ID NO: 36)
mouse FN forward primer : AAGACCATACCTGCCGAATG (서열번호 37)mouse FN forward primer: AAGACCATACCTGCCGAATG (SEQ ID NO: 37)
mouse FN reverse primer : GAACATGACCGATTTGGACC (서열번호 38)mouse FN reverse primer: GAACATGACCGATTTGGACC (SEQ ID NO: 38)
mouse col1a1 forward primer : ATGGATTCCCGTTCGAGTACG (서열번호 39)mouse col1a1 forward primer: ATGGATTCCCGTTCGAGTACG (SEQ ID NO: 39)
mouse col1a1 reverse primer : TCAGCTGGATAGCGACATCG (서열번호 40)mouse col1a1 reverse primer: TCAGCTGGATAGCGACATCG (SEQ ID NO: 40)
mouse col1a2 forward primer : TGCAGTAACTTCGTGCCTAGC (서열번호 41)mouse col1a2 forward primer: TGCAGTAACTTCGTGCCTAGC (SEQ ID NO: 41)
mouse col1a2 reverse primer : ACGTGGTCCTCTGTCTCCA (서열번호 42)mouse col1a2 reverse primer: ACGTGGTCCTTCTGTCTCCA (SEQ ID NO: 42)
mouse col3a1 forward primer : CTAAAATTCTGCCACCCCGAA (서열번호 43)mouse col3a1 forward primer: CTAAAATTCTGCCACCCCGAA (SEQ ID NO: 43)
mouse col3a1 reverse primer : AGGATCAACCCAGTATTCTCCACTC (서열번호 44)mouse col3a1 reverse primer: AGGATCAACCCAGTATTCTCCACTC (SEQ ID NO: 44)
mouse Snail forward primer : TCTGAAGATGCACATCCGAAGCCA (서열번호 45)mouse Snail forward primer: TCTGAAGATGCACATCCGAAGCCA (SEQ ID NO: 45)
mouse Snail reverse primer : AGGAGAATGGCTTCTCACCAGTGT (서열번호 46) mouse Snail reverse primer: AGGAGAATGGCTTCTCACCAGTGT (SEQ ID NO: 46)
mouse Smad4 forward primer : CAGCCATAGTGAAGGACTGTTGC (서열번호 47) mouse Smad4 forward primer: CAGCCATAGTGAAGGACTGTTGC (SEQ ID NO: 47)
mouse Smad4 reverse primer : CCTACTTCCAGTCCAGGTGGTA (서열번호 48)mouse Smad4 reverse primer: CCTACTTCCAGTCCAGGTGGTA (SEQ ID NO: 48)
rat Thbs2 forward primer : CAAGGACATGCAGTTTGGGC (서열번호 49)rat Thbs2 forward primer: CAAGGACATGCAGTTTGGGC (SEQ ID NO: 49)
rat Thbs2 reverse primer : GAAGACCAGGGTGACCAGTG (서열번호 50)rat Thbs2 reverse primer: GAAGACCAGGGTGACCAGTG (SEQ ID NO: 50)
rat α-SMA forward primer : TTCATTGGAATGGAGTCGGCG (서열번호 51)rat α-SMA forward primer: TTCATTGGAATGGAGTCGGCG (SEQ ID NO: 51)
rat α-SMA reverse primer : CTGTCAGCAATGCCTGGGTA (서열번호 52)rat α-SMA reverse primer: CTGTCAGCAATGCCTGGGTA (SEQ ID NO: 52)
배지 농도medium concentration
60-mm 세포 배양 접시에서 배양된 C22, RLE-6TN 또는 MLg 세포의 배지를 수확하고 0.45 μm 필터(Sartorius, Stonehouse, UK)를 통과시킨 후, 조절 배지(conditioned medium)로 사용하였다. 추가적으로, 배양 배지를 VIVASPIN6 컬럼(Sartorius)에 적용하고 10,000 x g에서 2시간 동안 원심분리한 후, 단백질 분석을 사용하여 농축된 단백질을 계산한 다음 항체를 사용하여 면역블롯팅하였다. 주변분비(paracrine)의 경우, 섬유화 유발 분비인자(Matricellular proteins)를 분비하는 C22 또는 RLE-6TN의 조건 배지를 MLg로 옮겼다.The medium of C22, RLE-6TN or MLg cells cultured in a 60-mm cell culture dish was harvested and passed through a 0.45 μm filter (Sartorius, Stonehouse, UK) and used as a conditioned medium. Additionally, after the culture medium was applied to a VIVASPIN6 column (Sartorius) and centrifuged at 10,000 x g for 2 hours, enriched proteins were calculated using a protein assay and then immunoblotted using antibodies. In the case of paracrine, a conditioned medium of C22 or RLE-6TN secreting Matricellular proteins was transferred to MLg.
통계 분석statistical analysis
결과는 Prism 소프트웨어 버전 5(GraphPad Software, San Diego, CA, USA)로 시각화 및 분석하였다. 2개 이상의 샘플 그룹을 비교할 때, 통계적 유의성을 위해 일원 분산 분석(one-way ANOVA)을 이용하였다. 데이터 값은 평균±표준편차(SD)로 나타내었다. 두 그룹의 통계적 유의성을 결정하는 데 스튜던트 t-검정를 사용하였으며, P < 0.05의 값은 통계적으로 유의한 것으로 간주하였다.Results were visualized and analyzed with Prism software version 5 (GraphPad Software, San Diego, CA, USA). When comparing two or more sample groups, one-way ANOVA was used for statistical significance. Data values are expressed as mean ± standard deviation (SD). Student's t-test was used to determine the statistical significance of the two groups, and values of P < 0.05 were considered statistically significant.
항체 제작antibody production
TSP-2의 1,172개의 아미노산 중, a.a 928-930 (RGD 도메인)을 포함하는 펩타이드를 이용하여 항체를 제작하였다. 대조군으로는 a.a 173-295를 인식하는 산타크루즈(Santacruz) 시판 항체(sc-136238, mouse mono)를 사용하였다.An antibody was prepared using a peptide including a.a 928-930 (RGD domain) among 1,172 amino acids of TSP-2. As a control, a commercially available Santacruz antibody (sc-136238, mouse mono) recognizing a.a 173-295 was used.
TSP-2 아미노산 서열 (NCBI Reference Sequence: NP_003238.2 , thrombospondin-2 precursor [Homo sapiens]) :TSP-2 amino acid sequence (NCBI Reference Sequence: NP_003238.2, thrombospondin-2 precursor [Homo sapiens]):
MVWRLVLLALWVWPSTQAGHQDKDTTFDLFSISNINRKTIGAKQFRGPDPGVPAYRFVRFDYIPPVNADDMVWRLVLLALWVWPSTQAGHQDKDTTFDLFSISNINRKTIGAKQFRGPDPGVPAYRFVRFDYIPPVNADD
LSKITKIMRQKEGFFLTAQLKQDGKSRGTLLALEGPGLSQRQFEIVSNGPADTLDLTYWIDGTRHVVSLELSKITKIMRQKEGFFLTAQLKQDGKSRGTLLALEGPGLSQRQFEIVSNGPADTLDLTYWIDGTRHVVSLE
DVGLADSQWKNVTVQVAGETYSLHVGCDLIDSFALDEPFYEHLQAEKSRMYVAKGSARESHFRGLLQNVHDVGLADSQWKNVTVQVAGETYSLHVGCDLIDSFALDEPFYEHLQAEKSRMYVAKGSARESHFRGLLQNVH
LVFENSVEDILSKKGCQQGQGAEINAISENTETLRLGPHVTTEYVGPSSERRPEVCERSCEELGNMVQELLVFENSVEDILSKKGCQQGQGAEINAISENTETLRLGPHVTTEYVGPSSERRPEVCERSCEELGNMVQEL
SGLHVLVNQLSENLKRVSNDNQFLWELIGGPPKTRNMSACWQDGRFFAENETWVVDSCTTCTCKKFKTICSGLHVLVNQLSENLKRVSNDNQFLWELIGGPPKTRNMSACWQDGRFFAENETWVVDSCTTCTCKKFKTIC
HQITCPPATCASPSFVEGECCPSCLHSVDGEEGWSPWAEWTQCSVTCGSGTQQRGRSCDVTSNTCLGPSIHQITCPPATCASPSFVEGECCPSCLHSVDGEEGWSPWAEWTQCSVTCGSGTQQRGRSCDVTSNTCLGPSI
QTRACSLSKCDTRIRQDGGWSHWSPWSSCSVTCGVGNITRIRLCNSPVPQMGGKNCKGSGRETKACQGAPQTRACSLSKCDTRIRQDGGWSHWSPWSSCSVTCGVGNITRIRLCNSPVPQMGGKNCKGSGRETKACQGAP
CPIDGRWSPWSPWSACTVTCAGGIRERTRVCNSPEPQYGGKACVGDVQERQMCNKRSCPVDGCLSNPCFPCPIDGRWSPWSPWSACTVTCAGGIRERTRVCNSPEPQYGGKACVGDVQERQMCNKRSCPVDGCLSNPCFP
GAQCSSFPDGSWSCGSCPVGFLGNGTHCEDLDECALVPDICFSTSKVPRCVNTQPGFHCLPCPPRYRGNQGAQCSSFPDGSWSCGSCPVGFLGNGTHCEDLDECALVPDICFSTSKVPRCVNTQPGFHCLPCPPRYRGNQ
PVGVGLEAAKTEKQVCEPENPCKDKTHNCHKHAECIYLGHFSDPMYKCECQTGYAGDGLICGEDSDLDGWPVGVGLEAAKTEKQVCEPENPCKDKTHNCHKHAECIYLGHFSDPMYKCECQTGYAGDGLICGEDSDLDGW
PNLNLVCATNATYHCIKDNCPHLPNSGQEDFDKDGIGDACDDDDDNDGVTDEKDNCQLLFNPRQADYDKDPNLNLVCATNATYHCIKDNCPHLPNSGQEDFDKDGIGDACDDDDDNDGVTDEKDNCQLLFNPRQADYDKD
EVGDRCDNCPYVHNPAQIDTDNNGEGDACSVDIDGDDVFNERDNCPYVYNTDQRDTDGDGVGDHCDNCPLEVGDRCDNCPYVHNPAQIDTDNNGEGDACSVDIDGDDVFNERDNCPYVYNTDQRDTDGDGVGDHCDNCPL
VHNPDQTDVDNDLVGDQCDNNEDIDDDGHQNNQDNCPYISNANQADHDRDGQGDACDPDDDNDGVPDDRDVHNPDQTDVDNDLVGDQCDNNEDIDDDGHQNNQDNCPYISNANQADHDRDGQGDACDPDDDNDGVPDDRD
NCRLVFNPDQEDLDGDGRGDICKDDFDNDNIPDIDDVCPENNAISETDFRNFQMVPLDPKGTTQIDPNWVNCRLVFNPDQEDLDGDGRGDICKDDFDNDNIPDIDDVCPENNAISETDFRNFQMVPLDPKGTTQIDPNWV
IRHQGKELVQTANSDPGIAVGFDEFGSVDFSGTFYVNTDRDDDYAGFVFGYQSSSRFYVVMWKQVTQTYWIRHQGKELVQTANSDPGIAVGFDEFGSVDFSGTFYVNTDRDDDYAGFVFGYQSSSRFYVVMWKQVTQTYW
EDQPTRAYGYSGVSLKVVNSTTGTGEHLRNALWHTGNTPGQVRTLWHDPRNIGWKDYTAYRWHLTHRPKTEDQPTRAYGYSGVSLKVVNSTTGTGEHLRNALWHTGNTPGQVRTLWHDPRNIGWKDYTAYRWHLTHRPKT
GYIRVLVHEGKQVMADSGPIYDQTYAGGRLGLFVFSQEMVYFSDLKYECRDI (서열번호 53)GYIRVLVHEGKQVMADSGPIYDQTYAGGRLGLFVFSQEMVYFSDLKYECRDI (SEQ ID NO: 53)
닷 블롯(Dot-blot)을 통하여 TSP-2에 대한 제작 항체의 후보군으로 7개의 클론을 확보하였고, 해당 클론들로 폐섬유아세포에서 웨스턴 블롯 테스트을 수행하여, 항체 친화도를 기반으로 2개의 클론으로 후보군을 좁혔다. 그 후, 해당 2개의 항체의 섬유아세포 증식 저해능을 비교하여, 최종적으로 1개의 항체를 선별하였다. 그 후, 제작된 항체와 시판항체의 TGF-β처리 후 섬유아세포 증식 억제능력을 세포 수 계수(Cell Counting)방법을 통하여 비교하였다.Through dot-blot, seven clones were secured as candidates for the antibody produced against TSP-2, and Western blotting was performed on lung fibroblasts with the corresponding clones, and two clones were divided based on antibody affinity. narrowed down the pool of candidates. Thereafter, the ability of the two antibodies to inhibit fibroblast proliferation was compared, and one antibody was finally selected. Thereafter, the ability of the manufactured antibody and the commercially available antibody to inhibit fibroblast proliferation after TGF-β treatment was compared through a cell counting method.
실험결과Experiment result
TSP-2의 발현이 IPF 환자의 폐 및 폐 섬유증 마우스 모델에서 증가되었다.Expression of TSP-2 was increased in the lungs of patients with IPF and in a mouse model of pulmonary fibrosis.
섬유화 유발 분비인자(Matricellular proteins)는 섬유증의 예측 바이오마커 및 ECM 합성의 조절자로 기능하며, 따라서 유망한 잠재적 치료 타겟이므로, 섬유화 유발 분비인자의 TSP들 중 TSP-2와 폐 질환의 병리학적 관련성을 조사하기 위하여 IPF 환자의 폐 조직, 간질성 폐 질환 환자의 혈청 및 폐 섬유증의 마우스 모델에서 TSP-2의 발현을 측정하였다.Fibrosis-inducing secreted factors (Matricellular proteins) function as predictive biomarkers of fibrosis and regulators of ECM synthesis, and are thus promising potential therapeutic targets. Therefore, among the TSPs of fibrosis-inducing secreted factors, TSP-2 and the pathological relevance of lung diseases are investigated. To do this, the expression of TSP-2 was measured in lung tissue of IPF patients, serum of interstitial lung disease patients, and mouse models of pulmonary fibrosis.
NCBI(National Center for Biotechnology Information)의 유전자 발현 옴니버스(GEO)에 따르면, TSP-1 및 TSP-3이 아닌 TSP-2가 대조군에 비해 IPF 환자의 샘플에서 유의하게 상승한 것으로 나타났다(도 1a 및 1b). 또한 건강한 피험자와 ILD 환자의 TSP-2 수치를 조사하였고, ILD 환자에서 인간 TSP-2의 혈청 수준은 TSP-2 ELISA 키트를 통해 평가하였다. 그 결과, 혈청 TSP-2의 수준은 ILD 환자에서 상향 조절됨을 확인하였다.According to the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI), TSP-1 and TSP-2, but not TSP-3, were found to be significantly elevated in samples from IPF patients compared to controls (FIGS. 1a and 1b) . In addition, TSP-2 levels in healthy subjects and ILD patients were investigated, and the serum level of human TSP-2 in ILD patients was evaluated using a TSP-2 ELISA kit. As a result, it was confirmed that the level of serum TSP-2 was up-regulated in ILD patients.
다음으로, 폐 섬유증 마우스 모델에서 TSP-2 발현을 조사하기 위해 블레오마이신 유도 폐 섬유증 모델 및 Ccsp-TGF 1-TG 마우스를 사용하였다. Bleomycin(BLM)은 마우스에서 폐 섬유증의 급성 유도에 널리 사용되며, Bleomycin에 의하여 강력한 섬유화 사이토카인인 TGF-β1의 수준이 증가하고 이는 IPF38에서 중요한 역할을 수행한다. TGF-β는 상피-중간엽 전이(EMT)의 주요 유도제이며 폐를 포함한 많은 조직에서 섬유증의 주요 매개체이다.Next, a bleomycin-induced pulmonary fibrosis model and Ccsp-TGF 1-TG mice were used to investigate TSP-2 expression in a pulmonary fibrosis mouse model. Bleomycin (BLM) is widely used for acute induction of pulmonary fibrosis in mice. Bleomycin increases the level of TGF-β1, a potent fibrotic cytokine, which plays an important role in IPF38. TGF-β is a major inducer of epithelial-mesenchymal transition (EMT) and a major mediator of fibrosis in many tissues, including the lung.
도 3a에 나타낸 바와 같이, 블레오마이신 유도 폐 섬유증 모델에서 실험을 진행하였고 콜라겐 축적을 관찰할 수 있었다. 분비된 TSP-2 단백질의 발현을 조사하기 위해 마우스 폐 섬유증 모델에서 농축된 기관지 폐포 세척액(BALF)을 조사하였다. 상향 조절된 TSP-2 발현은 웨스턴 블롯 분석을 통하여 확인할 수 있었고(도 3b), BAL 액체 총 세포 계산를 통하여 마우스 폐 섬유증 모델에서 BAL 액 총 세포 수의 증가를 확인할 수 있었다(도 3c). 또한, 마우스 폐 조직에서 마우스 폐 섬유증 모델의 TSP-2 발현도 상승하였다(도 3d). 따라서, TSP-2 수준은 블레오마이신 주사 후 마우스의 폐에서 유의하게 증가함을 확인하였다(도 3a 내지 3d).As shown in Figure 3a, the experiment was conducted in a bleomycin-induced pulmonary fibrosis model, and collagen accumulation could be observed. To investigate the expression of secreted TSP-2 protein, concentrated bronchoalveolar lavage fluid (BALF) was examined in a mouse lung fibrosis model. The upregulated expression of TSP-2 was confirmed by western blot analysis (FIG. 3b), and an increase in the total number of cells in BAL fluid was confirmed in the mouse lung fibrosis model by counting total cells in BAL fluid (FIG. 3c). In addition, the expression of TSP-2 in the mouse lung fibrosis model was also elevated in mouse lung tissue (FIG. 3d). Accordingly, it was confirmed that the level of TSP-2 significantly increased in the lungs of mice after bleomycin injection (FIGS. 3a to 3d).
TGF-β 구동 폐 섬유증에서 TSP-2 발현의 유도를 추가로 확인하기 위해, TGF-β1(Ccsp-TGF 1-TG 마우스)의 유도성 과발현이 있는 형질전환 마우스 모델에서 TSP-2 발현의 변화를 평가하였다. 독시사이클린(Dox)의 투여는 폐 섬유증을 유도하는데(도 3e), Dox 투여 30일 후, TSP-2는 야생형 마우스의 폐와 비교하여 Ccsp-TGF 1-TG 마우스의 폐에서 유의하게 증가하였다. BALF를 농축하고 면역블롯을 사용하여 TSP-2의 발현 증가를 관찰할 수 있었다(도 3f). 또한, 많은 염증 세포와 콜라겐 축적으로 인해 BAL 액의 총 세포 수가 증가하였고(도 3g), 마우스 폐 조직에서 TSP-2 발현도 상향 조절되었다(도 3h). 상기 결과를 통하여, TSP-2 발현이 IPF 환자 및 폐 섬유증의 마우스 모델에서 상승한다는 것을 입증할 수 있었다.To further confirm the induction of TSP-2 expression in TGF-β driven pulmonary fibrosis, changes in TSP-2 expression were examined in a transgenic mouse model with inducible overexpression of TGF-β1 (Ccsp-TGF 1-TG mice). evaluated. Administration of doxycycline (Dox) induced pulmonary fibrosis (Fig. 3e), and 30 days after administration of Dox, TSP-2 was significantly increased in the lungs of Ccsp-TGF 1-TG mice compared to the lungs of wild-type mice. BALF was enriched and increased expression of TSP-2 was observed using immunoblot (FIG. 3f). In addition, the total number of cells in BAL fluid increased due to the accumulation of many inflammatory cells and collagen (Fig. 3g), and the expression of TSP-2 was also upregulated in mouse lung tissue (Fig. 3h). Through the above results, it could be demonstrated that TSP-2 expression is elevated in IPF patients and mouse models of pulmonary fibrosis.
TGF-β 매개 SMAD 의존적 신호전달을 통하여 TSP-2 유전자의 전사가 활성화된다. The transcription of the TSP-2 gene is activated through TGF -β -mediated SMAD-dependent signaling.
TSP-2는 TGF-β28을 활성화시키는 것으로 나타났으므로, TGF-β1 처리 후 MLg(마우스 섬유아세포), C22(마우스 클럽 세포)에서 TSP-2의 mRNA 수준을 평가하였다. qRT-PCR 및 Western blot 분석 결과에 의하여 TGF-β1 처리에 의해 TSP-2의 수준이 증가됨을 관찰할 수 있었다.Since TSP-2 has been shown to activate TGF-β28, the mRNA level of TSP-2 was evaluated in MLg (mouse fibroblasts) and C22 (mouse club cells) after TGF-β1 treatment. According to the results of qRT-PCR and Western blot analysis, it was observed that the level of TSP-2 was increased by TGF-β1 treatment.
TGF-β-유도된 TSP-2 발현이 표준 TGF-β 신호전달에 의해 매개되는지 여부를 조사하기 위해, TSP-2 발현에 대한 Smad4 녹다운의 효과를 조사하였다. MLg 및 C22 세포주에서 Smad4의 녹다운은 TGF-β-유도된 TSP-2 발현에서의 mRNA 수준을 없앴다(도 4a). 또한, TGF-β-유도된 TSP-2 발현의 단백질 수준도 감소하였다(도 4b). 이러한 결과를 통하여 TSP-2 유전자의 전사 활성화에 Smad 의존적 표준 TGF-β 신호전달이 필요함을 알 수 있었다. 다음으로, 선택적 TGF-β1 억제제인 LY2157299를 이용하여 TGF-β 억제가 TSP-2의 발현에 미치는 영향을 시험하였다. 그 결과, LY2157299는 용량 의존적 방식으로 TGF-β1 유도 TSP-2 발현을 억제함을 알 수 있었다(도 5a, 5b). 상기 결과를 종합하여, TSP-2 발현이 TGF-β 신호전달에 의해 조절된다는 것을 입증할 수 있었다.To investigate whether TGF-β-induced TSP-2 expression is mediated by canonical TGF-β signaling, the effect of Smad4 knockdown on TSP-2 expression was examined. Knockdown of Smad4 in MLg and C22 cell lines abolished mRNA levels in TGF-β-induced TSP-2 expression (FIG. 4A). In addition, the protein level of TGF-β-induced TSP-2 expression was also decreased (Fig. 4b). These results suggest that Smad-dependent canonical TGF-β signaling is required for transcriptional activation of the TSP-2 gene. Next, the effect of TGF-β inhibition on TSP-2 expression was tested using LY2157299, a selective TGF-β1 inhibitor. As a result, it was found that LY2157299 inhibited TGF-β1-induced TSP-2 expression in a dose-dependent manner (FIGS. 5a and 5b). Taken together, it can be demonstrated that TSP-2 expression is regulated by TGF-β signaling.
TSP-2의 프로모터 영역의 전사 인자를 분석하기 위해 PROMO 3.0.2 도구를 사용하여 검색하였다. 특히, Smad가 TGF-β1 처리에 대한 반응으로 TSP-2의 전사를 직접 조절하는지 여부를 추가로 조사하기 위해 TSP-2 유전자 프로모터의 Smad 결합 모티프 CAGAC 또는 GTCTG를 검색하였다. 도 6a는 TSP-2 프로모터의 여러 Smad 결합 요소를 개략적으로 도시한 것이다. 프로모터 활성은 2kb (-1700 ~ 300 bp) 프로모터와 비교하여 TSP-2 유전자의 구조체(-30 ~ 300 bp) 프로모터에서 유의하게 감소하였다(도 6b). 전사 시작 부위의 업스트림에 초점을 맞추기 위해 TSP-2 유전자의 1.6kb, 400 및 350 염기쌍 크기의 프로모터를 사용하여 루시퍼라제 리포터 유전자 분석을 추가로 수행하였다. 400 및 350 염기쌍 크기의 프로모터에서 유사하게 루시페라제 활성이 증가했기 때문에(도 6c) #6 Smad 결합 요소가 중요할 것으로 예상하였다. #6(-43에서 -39) Smad 결합 요소가 결실된 TSP-2 유전자 프로모터에서 대조군과 비교하여 루시페라제 활성의 상당한 감소를 관찰할 수 있었다. 또한, #6이 결실된 돌연변이 프로모터의 루시페라제 활성은 TGF-β1 처리에 의해 영향을 받지 않았다(도 6d). 이러한 결과를 통하여 #6 Smad-결합 요소가 TSP-2 유전자의 전사 조절에 필수적임을 입증할 수 있었다. 따라서, Smad가 TSP-2 프로모터에 결합하고 TGF-β 신호전달에 반응하여 TSP-2 유전자 전사를 활성화한다는 것을 입증할 수 있었다.In order to analyze the transcription factor of the promoter region of TSP-2, it was searched using PROMO 3.0.2 tool. In particular, to further investigate whether Smad directly regulates TSP-2 transcription in response to TGF-β1 treatment, the Smad-binding motif CAGAC or GTCTG in the TSP-2 gene promoter was searched. 6A schematically depicts several Smad binding elements of the TSP-2 promoter. Promoter activity was significantly reduced in the TSP-2 gene construct (-30 ~ 300 bp) promoter compared to the 2 kb (-1700 ~ 300 bp) promoter (Fig. 6b). Further luciferase reporter gene analysis was performed using promoters of 1.6 kb, 400 and 350 base pairs in size of the TSP-2 gene to focus upstream of the transcriptional start site. Since luciferase activity was similarly increased in promoters of 400 and 350 base pair size (Fig. 6c), we expected the #6 Smad binding element to be important. #6 (-43 to -39) A significant decrease in luciferase activity compared to the control could be observed in the TSP-2 gene promoter in which the Smad binding element was deleted. In addition, the luciferase activity of the mutant promoter in which #6 was deleted was not affected by TGF-β1 treatment (Fig. 6d). These results demonstrate that #6 Smad-binding element is essential for the transcriptional regulation of the TSP-2 gene. Thus, it could be demonstrated that Smad binds to the TSP-2 promoter and activates TSP-2 gene transcription in response to TGF-β signaling.
TSP-2의 녹다운은 TGF-β1 유도 섬유아세포 활성화를 억제한다. Knockdown of TSP-2 inhibits TGF-β1-induced fibroblast activation.
TGF-β는 여러 조직에서 섬유증의 주요 매개체이고 TSP-2는 TGF-β 신호전달에 의해 조절되기 때문에, TGF-β가 상피 세포 및 섬유아세포 모두에서 TSP-2의 분비를 증가시키고, 그 후 자가분비 또는 측분비 방식을 통해 섬유아세포에서 ECM의 침착을 촉진시키는 것으로 가정하였다. 따라서, 폐 상피 세포(C22 및 RLE-6TN) 또는 폐 섬유아세포(MLg)에서 TSP-2 발현을 차단한 후 TGF-β에 의해 유도된 섬유아세포 증식 및 섬유아세포에서 표적 유전자 발현을 평가하였다.Since TGF-β is a major mediator of fibrosis in many tissues and TSP-2 is regulated by TGF-β signaling, TGF-β increases the secretion of TSP-2 in both epithelial cells and fibroblasts, followed by autologous It was hypothesized to promote ECM deposition in fibroblasts via a secretory or paracrine mode. Therefore, TGF-β-induced fibroblast proliferation and target gene expression in fibroblasts were evaluated after blocking TSP-2 expression in lung epithelial cells (C22 and RLE-6TN) or lung fibroblasts (MLg).
TSP-2의 녹다운 효율을 확인하기 위해 MLg, C22, RLE-6TN(쥐 AT2 세포)에서 qRT-PCR(도 7b, 8a) 및 웨스턴 블롯 분석(도 7c, 8b)을 수행하였다. 섬유아세포 활성화에 대한 TSP-2 녹다운의 자가분비 및 측분비(도 7a) 효과를 조사하였다. 측분비 신호의 경우, 상피 세포에서 TSP-2의 녹다운 후의 조절 배지를 섬유아세포로 이동시켰고, 섬유아세포의 세포 증식을 측정하였다(도 7a). TGF-β1 처리와 함께 배양된 조건 배지는 섬유아세포 증식을 증가시켰으나, TSP-2의 녹다운은 자가분비 및 측분비 방식 모두에서 TGF-β1 유도 섬유아세포 활성화를 감소시켰다(도 7d, 8c). 따라서 TGF-β1에 의한 섬유아세포 증식에는 TSP-2가 필요함을 알 수 있었다.To confirm the knockdown efficiency of TSP-2, qRT-PCR (Fig. 7b, 8a) and Western blot analysis (Fig. 7c, 8b) were performed on MLg, C22, and RLE-6TN (rat AT2 cells). The autocrine and paracrine (Fig. 7a) effects of TSP-2 knockdown on fibroblast activation were investigated. In the case of paracrine signals, the conditioned medium after knockdown of TSP-2 in epithelial cells was transferred to fibroblasts, and cell proliferation of fibroblasts was measured (FIG. 7a). Conditioned media cultured with TGF-β1 treatment increased fibroblast proliferation, but knockdown of TSP-2 reduced TGF-β1-induced fibroblast activation in both autocrine and paracrine modes (FIG. 7d, 8c). Therefore, it was found that TSP-2 is required for TGF-β1-induced fibroblast proliferation.
다음으로, 섬유아세포에서 섬유화 표적 유전자 발현에 대한 TSP-2의 넉다운 효과를 조사하였다. 예상대로 TGF-β1 처리는 Collagen1a1(Col1a1), Collagen1a2(Col1a2), Collagen3a1(Col3a1), Fibronectin(FN), α-Smooth Muscle Actin(α-SMA)과 같은 섬유화 표적 유전자를 유의하게 증가시켰고, 반면 TSP-2의 녹다운은 표적 유전자의 TGF-β1-유도된 상향조절을 실질적으로 억제하였다(도 7e). 또한, 상피 세포에서 TSP-2의 녹다운은 섬유아세포에서 섬유화 표적 유전자의 유도를 없앴다(도 8d). 이러한 결과를 통해 TSP-2의 녹다운에 의하여 자가분비 또는 측분비 방식을 통해 TGF-β 유도 섬유아세포 활성화를 억제되며, 따라서 TSP-2는 TGF-β로 유도된 섬유아세포 활성화를 조절함을 알 수 있었다.Next, the knockdown effect of TSP-2 on the expression of fibrosis target genes in fibroblasts was investigated. As expected, TGF-β1 treatment significantly increased fibrosis target genes such as Collagen1a1 (Col1a1), Collagen1a2 (Col1a2), Collagen3a1 (Col3a1), Fibronectin (FN), and α-Smooth Muscle Actin (α-SMA), whereas TSP Knockdown of -2 substantially inhibited TGF-β1-induced upregulation of target genes (FIG. 7e). In addition, knockdown of TSP-2 in epithelial cells abolished the induction of fibrosis target genes in fibroblasts (Fig. 8d). These results suggest that knockdown of TSP-2 inhibits TGF-β-induced fibroblast activation through autocrine or paracrine methods, and thus TSP-2 regulates TGF-β-induced fibroblast activation. there was.
TSP-2 단클론 중화항체로 TSP-2를 차단하는 경우 섬유아세포 활성화가 억제된다.When TSP-2 is blocked with a TSP-2 monoclonal neutralizing antibody, fibroblast activation is inhibited.
TSP-2의 발현을 억제하면 섬유아세포 활성화가 억제되는 것으로 관찰되어 TSP-2의 기능을 효과적으로 억제할 수 있는 TSP-2에 대한 단클론항체(mAb)의 유효성을 조사하였다. 섬유아세포 활성화에 대한 TSP-2 차단의 자가분비 및 측분비 효과를 실험하였다. 먼저, TSP-2 단일클론항체의 차단 작용을 확인하기 위해 TGF-β1 유도 섬유아세포 활성화가 섬유아세포 증식에 미치는 억제 효과를 평가하였다. 그 다음 TSP-2 단일클론항체의 처리가 넉다운 결과와 유사하게 섬유화 마커의 표적 유전자를 억제하는지 여부를 조사하였다. 다음으로, TSP-2 단일클론항체의 처리가 넉다운 결과와 유사하게 섬유화 마커의 표적 유전자를 억제하는지 여부를 조사하였다.Since inhibition of TSP-2 expression inhibited fibroblast activation, we investigated the effectiveness of a monoclonal antibody (mAb) against TSP-2 that can effectively inhibit TSP-2 function. Autocrine and paracrine effects of TSP-2 blockade on fibroblast activation were examined. First, to confirm the blocking action of the TSP-2 monoclonal antibody, the inhibitory effect of TGF-β1-induced fibroblast activation on fibroblast proliferation was evaluated. Then, whether treatment with TSP-2 monoclonal antibody suppresses target genes of fibrosis markers similarly to knockdown results was investigated. Next, it was investigated whether treatment with TSP-2 monoclonal antibody suppresses target genes of fibrosis markers similarly to knockdown results.
섬유아세포에 TGF-β1(20ng/ml)과 농도에 따라 TSP-2 단일클론항체를 처리한 후, TGF-β1에 의해 유도된 섬유아세포 증식을 세포 계수로 분석하였다. CTGF 모노클로날 항체 처리는 항체 차단 실험의 양성 대조군을 위하여 사용되었고, 항-TSP-2 단일클론 항체가 있는 조건 배지의 TGF-β1-유도 섬유아세포 증식은 용량 의존적 방식으로 유의하게 감소되었다(도 9a). 또한, TSP-2 모노클로날 중화 항체를 통하여 TSP-2의 표적화 억제를 통하여 용량 의존적 방식으로 섬유아세포에서 TGF-β1 유도 섬유화 표적 유전자 발현을 감소실 수 있음을 확인하였다(도 9b).Fibroblasts were treated with TGF-β1 (20 ng/ml) and TSP-2 monoclonal antibody according to the concentration, and then TGF-β1-induced fibroblast proliferation was analyzed by cell counting. CTGF monoclonal antibody treatment was used as a positive control for antibody blocking experiments, and TGF-β1-induced fibroblast proliferation in conditioned medium with anti-TSP-2 monoclonal antibody was significantly reduced in a dose-dependent manner (Fig. 9a). In addition, it was confirmed that TGF-β1-induced fibrosis target gene expression could be decreased in fibroblasts in a dose-dependent manner through targeted inhibition of TSP-2 through a TSP-2 monoclonal neutralizing antibody (FIG. 9B).
측분비 신호전달에 대해서는, 상피세포에 TSP-2 단일클론항체와 TGF-β1(20 ng/ml) 처리하고, 조건배지를 섬유아세포로 이동시킨 후 섬유아세포의 세포 증식을 측정하였다. 항-TSP-2 모노클로날 항체를 갖는 조절 배지의 TGF-β1-유도된 섬유아세포 증식은 용량 의존적 방식으로 감소하였다(도 10a). 또한, 항-TSP-2 단일클론 항체에 의한 TSP-2의 표적 억제는 용량 의존적 방식으로 섬유아세포에서 TGF-β1-유도 섬유화 표적 유전자 발현을 효율적으로 억제하였다(도 10b). 따라서, 이러한 결과를 통하여 항-TSP-2 mAb가 분비된 TSP-2 단백질에 결합하고 자가분비 및 측분비 방식으로 TSP-2를 중화함으로써 TGF-β1 유도 섬유아세포 활성화를 억제함을 입증할 수 있었다.Regarding paracrine signaling, epithelial cells were treated with TSP-2 monoclonal antibody and TGF-β1 (20 ng/ml), and the conditioned medium was transferred to fibroblasts, and cell proliferation of the fibroblasts was measured. TGF-β1-induced fibroblast proliferation in conditioned medium with anti-TSP-2 monoclonal antibody was reduced in a dose dependent manner (FIG. 10A). In addition, target inhibition of TSP-2 by the anti-TSP-2 monoclonal antibody efficiently suppressed TGF-β1-induced fibrosis target gene expression in fibroblasts in a dose-dependent manner (FIG. 10B). Therefore, these results demonstrated that anti-TSP-2 mAb inhibits TGF-β1-induced fibroblast activation by binding to secreted TSP-2 protein and neutralizing TSP-2 in an autocrine and paracrine manner. .
최종 제작 항체의 CDR 서열을 확보하였다.The CDR sequence of the final antibody was secured.
상기 항체 제작 및 선별과정을 통하여, 기존 시판항체(sc-136238)보다 섬유아세포 저해능이 우월한 제작항체를 선별할 수 있었다. 7개의 클론(#1 내지 #7)을 확보하기 위하여 닷 블롯을 사용하였고(도 11a), 폐섬유아세포에서 웨스턴 블롯을 기반으로 2개의 후보를 선정하였으며(도 11b), 최종적으로 2개의 후보(#2, #4)의 섬유아세포증식 저해능을 비교하여 최종 제작 항체(#4)를 선별하였다(도 11c).Through the above antibody preparation and selection process, it was possible to select a manufactured antibody superior in fibroblast inhibitory ability to the existing commercially available antibody (sc-136238). Dot blot was used to secure 7 clones (#1 to #7) (FIG. 11a), two candidates were selected based on Western blotting in lung fibroblasts (FIG. 11b), and finally two candidates ( The final antibody (#4) was selected by comparing the fibroblast proliferation inhibitory ability of #2 and #4) (FIG. 11c).
또한, 해당 제작항체의 CDR 서열을 분석하였으며, 분석결과는 다음과 같다.In addition, the CDR sequence of the produced antibody was analyzed, and the analysis results are as follows.
아미노산 서열amino acid sequence
HCDR1 : GYSFTGYY (서열번호 1)HCDR1: GYSFTGYY (SEQ ID NO: 1)
HCDR2 : VNPNNGGI (서열번호 2)HCDR2: VNPNNGGI (SEQ ID NO: 2)
HCDR3 : ARDGAY (서열번호 3)HCDR3: ARDGAY (SEQ ID NO: 3)
LCDR1 : QSLLDSDGKTY (서열번호 4)LCDR1: QSLLDSDGKTY (SEQ ID NO: 4)
LCDR2 : LVS (서열번호 5)LCDR2: LVS (SEQ ID NO: 5)
LCDR3 : WQGTHFPFT (서열번호 6)LCDR3: WQGTHFPFT (SEQ ID NO: 6)
HFR1 : EVQLQQSGPDLVKPGASVKISCKAS (서열번호 7)HFR1: EVQLQQSGPDLVKPGASVKISCKAS (SEQ ID NO: 7)
HFR2 : MHWVKQSHEKSLEWIGR (서열번호 8)HFR2: MHWVKQSHEKSLEWIGR (SEQ ID NO: 8)
HFR3 : SYNQKFRGKAILTVDRSSNTAYMELRSLTSEDSAVYYC (서열번호 9)HFR3: SYNQKFRGKAILTVDRSSNTAYMELRSLTSEDSAVYYC (SEQ ID NO: 9)
HFR4 : WGQGTLVTVSA (서열번호 10)HFR4: WGQGTLVTVSA (SEQ ID NO: 10)
중쇄 가변영역 : Heavy chain variable region:
EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHEKSLEWIGRVNPNNGGISYNQKFRGKAILTVDRSSNTAYMELRSLTSEDSAVYYCARDGAYWGQGTLVTVSA (서열번호 11)EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHEKSLEWIGRVNPNNGGISYNQKFRGKAILTVDRSSNTAYMELRSLTSEDSAVYYCARDGAYWGQGTLVTVSA (SEQ ID NO: 11)
LFR1 : DVVMTQTPLTLSVTIGQPASISCKSS (서열번호 12)LFR1: DVVMTQTPLTLSVTIGQPASISCKSS (SEQ ID NO: 12)
LFR2 : LNWLLQRPGQSPKRLIY (서열번호 13)LFR2: LNWLLQRPGQSPKRLIY (SEQ ID NO: 13)
LFR3 : KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYYC (서열번호 14)LFR3: KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYYC (SEQ ID NO: 14)
LFR4 : FGSGTKLEIK (서열번호 15)LFR4: FGSGTKLEIK (SEQ ID NO: 15)
경쇄 가변영역 : Light chain variable region:
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYYCWQGTHFPFTFGSGTKLEIK (서열번호 16)DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYYCWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 16)
핵산 서열nucleic acid sequence
HCDR1 : GGTTACTCATTCACTGGCTACTAC (서열번호 17)HCDR1: GGTTACTCATTCACTGGCTACTAC (SEQ ID NO: 17)
HCDR2 : GTTAATCCTAACAATGGTGGTATA (서열번호 18)HCDR2: GTTAATCCTAACAATGGTGGTATA (SEQ ID NO: 18)
HCDR3 : GCAAGAGATGGTGCTTAC (서열번호 19)HCDR3: GCAAGAGATGGTGCTAC (SEQ ID NO: 19)
LCDR1 : CAGAGCCTCTTAGATAGTGATGGAAAGACATAT (서열번호 20)LCDR1: CAGAGCCTCTTAGATAGTGATGGAAAGACATAT (SEQ ID NO: 20)
LCDR2 : CTGGTGTCT (서열번호 21)LCDR2: CTGGTGTCT (SEQ ID NO: 21)
LCDR3 : TGGCAAGGTACACATTTTCCATTCACG (서열번호 22)LCDR3: TGGCAAGGTACACATTTTCCATTCACG (SEQ ID NO: 22)
HFR1 : HFR1:
GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCT (서열번호 23)GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCT (SEQ ID NO: 23)
HFR2 : ATGCACTGGGTGAAACAGAGCCATGAAAAGAGCCTTGAGTGGATTGGACGT (서열번호 24)HFR2: ATGCACTGGGTGAAACAGAGCCATGAAAAGAGCCTTGAGTGGATTGGACGT (SEQ ID NO: 24)
HFR3 : HFR3:
AGCTACAACCAGAAGTTCAGGGGCAAGGCCATATTAACTGTAGACAGGTCATCCAATACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCGGTCTATTACTGT (서열번호 25)AGCTACAACCAGAAGTTCAGGGGCAAGGCCATATTAACTGTAGACAGGTCATCCAATACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCGGTCTATTACTGT (SEQ ID NO: 25)
HFR4 : TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (서열번호 26)HFR4: TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 26)
중쇄 가변영역 : Heavy chain variable region:
GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTGAAACAGAGCCATGAAAAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAACAATGGTGGTATAAGCTACAACCAGAAGTTCAGGGGCAAGGCCATATTAACTGTAGACAGGTCATCCAATACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGATGGTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (서열번호 27)GAGGTCCAGCTGCAGCAGTCTGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTGAAACAGAGCCATGAAAAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAACAATGGTGGTATAAGCTACAACCAGAAGTTCAGGGGCAAGGCCATATTAACTGTAGACAGGTCATCCAATACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGATGGTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (서열번호 27)
LFR1 : LFR1:
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGT (서열번호 28)GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGT (SEQ ID NO: 28)
LFR2 : TTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGGCTAATCTAT (서열번호 29)LFR2: TTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGGCTAATCTAT (SEQ ID NO: 29)
LFR3 : LFR3:
AAATTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTATTGC (서열번호 30)AAATTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTATTGC (SEQ ID NO: 30)
LFR4 : TTCGGCTCGGGGACAAAGTTGGAAATAAAA (서열번호 31)LFR4: TTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 31)
경쇄 가변영역 : Light chain variable region:
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGGCTAATCTATCTGGTGTCTAAATTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTATTGCTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (서열번호 32)GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGGCTAATCTATCTGGTGTCTAAATTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAATTTATTATTATTGCTGGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (서열번호 32)
제작된 항체와 기존 시판항체의 TGF-β처리 후 섬유아세포 증식 억제능을 비교한 결과, 제작항체의 억제능이 시판항체보다 뛰어남을 확인할 수 있었다(도 11d).As a result of comparing the inhibitory ability of the manufactured antibody and the commercially available antibody after TGF-β treatment, it was confirmed that the inhibitory ability of the manufactured antibody was superior to that of the commercially available antibody (FIG. 11d).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (17)

  1. 서열번호 1의 서열로 이루어진 HCDR1, 서열번호 2의 서열로 이루어진 HCDR2 및 서열번호 3의 서열로 이루어진 HCDR3의 중쇄 CDR(complementarity determining region) 아미노산 서열을 갖는 중쇄 가변영역을 포함하는, TSP-2 (Thrombospondin-2) 단백질에 대한 항체 또는 그의 항원 결합 단편.TSP-2 (Thrombospondin -2) Antibodies to proteins or antigen-binding fragments thereof.
  2. 제 1 항에 있어서, 상기 중쇄 가변영역은 서열번호 11의 아미노산 서열을 포함하는 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The antibody or antigen-binding fragment of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11.
  3. 제 1 항에 있어서, 상기 항체 또는 그의 항원 결합 단편은 서열번호 4의 서열로 이루어진 LCDR1, 서열번호 5의 서열로 이루어진 LCDR2 및 서열번호 6의 서열로 이루어진 LCDR3의 경쇄 CDR 아미노산 서열을 갖는 경쇄 가변영역을 추가적으로 포함하는 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The light chain variable region of claim 1, wherein the antibody or antigen-binding fragment thereof has a light chain CDR amino acid sequence of LCDR1 consisting of the sequence of SEQ ID NO: 4, LCDR2 consisting of the sequence of SEQ ID NO: 5, and LCDR3 consisting of the sequence of SEQ ID NO: 6 An antibody or antigen-binding fragment thereof, characterized in that it further comprises.
  4. 제 3 항에 있어서, 상기 경쇄 가변영역은 서열번호 16의 아미노산 서열을 포함하는 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 3, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16.
  5. 제 1 항에 있어서, 상기 항원 결합 단편은 Fab 단편, F(ab') 단편, F(ab')2 단편 또는 Fv 단편인 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is a Fab fragment, F(ab') fragment, F(ab')2 fragment or Fv fragment.
  6. 제 1 항에 있어서, 상기 항체는 단일클론 항체인 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.
  7. 제 1 항에 있어서, 상기 항체는 중화 항체(Neutralizing Antibody)인 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a neutralizing antibody.
  8. 제 1 항에 있어서, 상기 항체는 키메라 항체, 인간화 항체 또는 인간 항체인 것을 특징으로 하는 항체 또는 그의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.
  9. 제 1 항 내지 제 8 항 중 어느 한 항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 예방 또는 치료용 조성물.A composition for preventing or treating fibrotic disease comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8 as an active ingredient.
  10. 제 9 항에 있어서, 상기 섬유화 질환은 폐섬유증(Pulmonary Fibrosis)인 것을 특징으로 하는 조성물.The composition according to claim 9, wherein the fibrotic disease is pulmonary fibrosis.
  11. 제 10 항에 있어서, 상기 폐섬유증(Pulmonary Fibrosis)은 간질성폐질환(Interstitial Lung Disease, ILD) 및 특발성 폐섬유증(Idiopathic Pulmonary Fibrosis, IPF)으로 구성된 군으로부터 선택되는 하나 이상의 질환인 것을 특징으로 하는 조성물.The composition according to claim 10, wherein the pulmonary fibrosis is one or more diseases selected from the group consisting of interstitial lung disease (ILD) and idiopathic pulmonary fibrosis (IPF). .
  12. 제 1 항 내지 제 4 항 중 어느 한 항의 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자.A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.
  13. 제 1 항 내지 제 8 항 중 어느 한 항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 진단용 조성물.A composition for diagnosis of fibrotic disease comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8 as an active ingredient.
  14. TSP-2 (Thrombospondin-2) 단백질 또는 이를 코딩하는 유전자의 발현량을 측정하는 제제를 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 진단용 조성물. A composition for diagnosis of fibrotic disease comprising, as an active ingredient, an agent for measuring the expression level of TSP-2 (Thrombospondin-2) protein or a gene encoding the same.
  15. 서열번호 4의 서열로 이루어진 LCDR1, 서열번호 5의 서열로 이루어진 LCDR2 및 서열번호 6의 서열로 이루어진 LCDR3의 경쇄 CDR(complementarity determining region) 아미노산 서열을 갖는 경쇄 가변영역을 포함하는 TSP-2(Thrombospondin-2) 단백질에 대한 항체 또는 그의 항원 결합 단편.TSP-2 (Thrombospondin- 2) Antibodies to proteins or antigen-binding fragments thereof.
  16. TSP-2 (Thrombospondin-2)에 대한 억제제를 유효성분으로 포함하는 섬유화 질환(fibrotic disease)의 예방 또는 치료용 조성물.A composition for preventing or treating fibrotic disease comprising an inhibitor for TSP-2 (Thrombospondin-2) as an active ingredient.
  17. 다음의 단계를 포함하는 섬유화 질환의 예방 또는 치료용 조성물의 스크리닝 방법:A screening method for a composition for preventing or treating fibrotic disease comprising the following steps:
    (a) TSP-2 (Thrombospondin-2) 단백질 또는 이를 발현하는 세포를 포함하는 생물학적 시료에 시험물질을 접촉시키는 단계; 및(a) contacting a test substance with a biological sample containing TSP-2 (Thrombospondin-2) protein or a cell expressing the same; and
    (b) 상기 생물학적 시료 내 TSP-2 단백질 또는 이들을 인코딩하는 유전자의 발현량을 측정하는 단계,(b) measuring the expression level of TSP-2 protein or genes encoding them in the biological sample;
    상기 생물학적 시료 내 상기 단백질 또는 상기 유전자의 발현량이 감소하는 경우, 상기 시험물질은 섬유화 질환의 예방 또는 치료용 조성물로 판정한다.When the expression level of the protein or gene in the biological sample decreases, the test substance is determined as a composition for preventing or treating fibrotic disease.
PCT/KR2022/010881 2021-07-26 2022-07-25 Novel antibody for prevention or treatment of fibrotic disease WO2023008858A1 (en)

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