WO2017116212A1 - 마이코박테리아 유래 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편 - Google Patents
마이코박테리아 유래 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Definitions
- the present invention relates to an antigen-binding fragment that specifically binds to CFP-10 or Ag85B, an antigen derived from mycobacteria, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a host cell comprising the recombinant vector.
- tuberculosis is a disease caused by the infection of Mycobacterium tuberculosis , it is known to be the number 1 incidence among statutory infectious diseases in Korea. Therefore, the early diagnosis and treatment of obtaining reliable information in a short time from patients suspected of having tuberculosis is important.
- the tuberculin skin test (TST) method is a common method for diagnosing tuberculosis.
- TST tuberculin skin test
- the cultivation method is the most reliable test among tuberculosis diagnosis methods, and it is a test for confirming tuberculosis by multiplying tuberculosis bacteria, and because it isolates and cultures bacteria, the risk of infection of the experimenter can not be excluded. At least four to six weeks are required for early diagnosis, which is difficult.
- BACTEC system Mycobacteria Growth Indicator Tube
- MGIT Mycobacteria Growth Indicator Tube
- the method shortens the incubation period by using a liquid medium, but can confirm the growth of Mycobacterium tuberculosis in about two weeks.
- an additional confirmation test is required to distinguish it from atypical Mycobacterium tuberculosis.
- problems concerns about the infection of Mycobacterium tuberculosis, and expensive equipment are required.
- One aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to antigen CFP-10 derived from mycobacteria, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a host cell comprising the recombinant vector.
- Another aspect of the present invention provides an antibody or antigen-binding fragment that specifically binds to antigen Ag85B from mycobacteria, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a host cell comprising the recombinant vector.
- One aspect of the present invention is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; And an antibody or antigen-binding fragment thereof that specifically binds to a mycobacterial-derived CFP-10 antigen comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6.
- heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; And an antibody or antigen-binding fragment thereof that specifically binds to a mycobacterial-derived CFP-10 antigen comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
- Another aspect of the invention is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9; And an antibody or antigen-binding fragment thereof that specifically binds to a mycobacterial-derived Ag85B antigen comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
- heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and an antibody or antigen-binding fragment thereof that specifically binds to a mycobacterial-derived Ag85B antigen comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.
- heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; And an antibody or antigen-binding fragment thereof that specifically binds to a mycobacterial-derived Ag85B antigen comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.
- Mycobacterium tuberculosis Mycobacterium bovis , Mycobacterium leprae , Mycobacterium kansasii , Mycobacterium kansasii It may be derived from Mycobacterium intracellularlare , Mycobacterium abscessus or Mycobacterium avium .
- antibody is a specific antibody against Ag85B or CFP-10 antigen derived from Mycobacterium, which specifically binds to Ag85B or CFP-10 antigen, and is an antibody as well as a complete antibody form. Antigen binding fragments of molecules.
- a complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds.
- the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types and subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), Gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
- the constant regions of the light chains have kappa ( ⁇ ) and lambda ( ⁇ ) types.
- variable chain refers to a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and an entirety comprising three constant region domains CH1, CH2 and CH3. It means both length heavy chains and fragments thereof.
- light chain is also used herein to refer to the full-length light chain and its variable length domain VL and constant region domain CL, which comprises an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. It means all fragments.
- the term "antigen binding fragment” refers to a portion of a polypeptide that includes a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin.
- it may be, but is not limited to, F (ab ') 2, Fab', Fab, Fv, or scFv.
- Fab in the antigen-binding fragment has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain.
- F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy chain variable regions and light chain variable regions are well known in the art.
- Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single-chain Fv is generally shared by the variable region of the heavy chain and the short chain variable region through a peptide linker. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv.
- the antigen binding fragments can be obtained using proteolytic enzymes (e.g., restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') 2 fragments), It can be produced through genetic recombination technology.
- proteolytic enzymes e.g., restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') 2 fragments
- the term “specifically binding” or “specifically recognizing” has the same meaning as is commonly known to those skilled in the art, wherein the antigen and the antibody (or antigen-binding fragment) are specific. Interact with each other to produce an immunological response.
- the antigen-binding fragment that specifically binds to the mycobacteria-derived CFP-10 antigen or Ag85B antigen is a compound of the amino acid sequence described in the above-mentioned sequence number within a range capable of specifically recognizing the CFP-10 antigen or Ag85B antigen.
- Variants may be included.
- changes can be made to the amino acid sequence of the antibody to improve the binding affinity and / or other biological properties of the antibody.
- modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues 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.
- arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes.
- arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be referred to as biologically functional equivalents.
- amino acid substitutions in proteins that do not alter the activity of the molecule as a whole are known in the art.
- the most commonly occurring substitutions are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.
- the antigen-binding fragment that specifically binds to the CFP-10 antigen or Ag85B antigen may be interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO. Can be. Such substantial identity is obtained by aligning the amino acid sequence of the above SEQ ID NO. To any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art. In some cases, at least 60% homology, at least 70% homology, at least 80% homology, or at least 90% homology. Alignment methods for the comparison of sequences are known in the art. For example, through the NCBI Basic Local Alignment Search Tool (BLAST), sequence analysis programs such as blastp, blastx, tblastn and tblastx can be used on the Internet.
- BLAST NCBI Basic Local Alignment Search Tool
- Another aspect of the invention provides a polynucleotide encoding the heavy chain variable region of an antibody that specifically binds to a mycobacterial-derived CFP-10 antigen comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7 and SEQ ID NO: 6 or SEQ ID NO:
- a polynucleotide polynucleotide encoding a light chain variable region of an antibody that specifically binds to a mycobacterial-derived CFP-10 antigen comprising the amino acid sequence of is provided.
- polynucleotide encoding the heavy chain variable region of the antibody specifically binding to the mycobacterial-derived Ag85B antigen comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 and SEQ ID NO: 10, SEQ ID NO: 12, or sequence
- a polynucleotide polynucleotide encoding a light chain variable region of an antibody that specifically binds to a mycobacterial derived Ag85B antigen comprising the amino acid sequence of amino acid sequence of No. 14 is provided.
- polynucleotide is a polymer of deoxyribonucleotides or ribonucleotides present in single- or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom and includes analogs of natural polynucleotides unless specifically stated otherwise.
- the polynucleotide includes not only the nucleotide sequence encoding the amino acid sequence of an antigen binding fragment that specifically binds to the CFP-10 antigen or Ag85B antigen, but also a sequence complementary to the sequence.
- Such complementary sequences include not only perfectly complementary sequences, but also substantially complementary sequences, which are specific for stringent conditions known in the art, for example, for the CFP-10 antigen or Ag85B antigen.
- nucleotide sequence encoding the amino acid sequence of the antigen-binding fragment that specifically binds to the CFP-10 antigen or Ag85B antigen can be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides.
- Polynucleotides encoding the amino acid sequence of the antigen-binding fragment that specifically binds to the CFP-10 antigen or Ag85B antigen are to be interpreted to include a nucleotide sequence showing substantial identity to the nucleotide sequence. The substantial identity is at least 80% when the nucleotide sequence is aligned with any other sequence as closely as possible, and the aligned sequence is analyzed using algorithms commonly used in the art. A sequence exhibiting homology, at least 90% homology, or at least 95% homology.
- the polynucleotide encoding the heavy chain variable region of the antibody specifically binding to the CFP-10 antigen may have a nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 16.
- the polynucleotide encoding the light chain variable region of the antibody that specifically binds to the CFP-10 antigen may have a nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18.
- the polynucleotide encoding the heavy chain variable region of the antibody specifically binding to the Ag85B antigen may be any one of a polynucleotide having a nucleotide sequence of SEQ ID NO: 19 to SEQ ID NO: 21.
- the polynucleotide encoding the light chain variable region of the antibody that specifically binds to the Ag85B antigen may be any one of the colinucleotide having a nucleotide sequence of SEQ ID NO: 22 to SEQ ID NO: 24.
- Another aspect of the invention provides a recombinant vector comprising said polynucleotide.
- the term "vector” refers to a means for expressing a gene of interest in a host cell.
- it may include, but is not limited to, plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.
- Vectors that can be used as the recombinant vector are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14).
- phages eg, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1 and M13, etc.
- viruses eg, CMV, SV40, etc.
- the preparation vector may include a base sequence of the polynucleotide and a promoter operatively linked to the base sequence.
- operably linked refers to a functional bond between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence, whereby the control sequence is responsible for the transcription and And / or control detoxification.
- a nucleotide expression control sequence eg, a promoter sequence
- another nucleotide sequence whereby the control sequence is responsible for the transcription and And / or control detoxification.
- the recombinant vector can typically be constructed as a vector for cloning or a vector for expression.
- the expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms.
- the recombinant vector may be constructed through various methods known in the art.
- the recombinant vector can be constructed using a prokaryotic or eukaryotic cell as a host.
- a prokaryotic or eukaryotic cell for example, when the vector used is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pL promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) capable of promoting transcription It is common to include ribosome binding sites and transcription / detox termination sequences for initiation of translation.
- a strong promoter for example, a pL promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.
- replication origins that operate in eukaryotic cells included in the vector include f1 origin, SV40 origin, pMB1 origin, adeno origin, AAV origin, CMV origin, and BBV origin. Including but not limited to.
- promoters derived from the genome of mammalian cells eg, metallothionine promoters
- promoters derived from mammalian viruses eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus (CMV) promoter and tk promoter of HSV
- CMV Cytomegalovirus
- recombinant vectors that can be used in the present invention include plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (eg, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, M13, etc.) or viruses (eg SV40, etc.) can be engineered.
- plasmids eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.
- phage eg, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, M13, etc.
- viruses eg SV40, etc.
- the recombinant vector of the present invention may be fused with other sequences to facilitate purification of the antigen binding fragment expressed therefrom.
- Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Qiagen, USA), and most preferably Is 6 ⁇ His. Because of the additional sequence for the purification, the protein expressed in the host can be purified quickly and easily through affinity chromatography.
- the expression vector of the present invention may include antibiotic resistance genes commonly used in the art as selection markers, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneeti Resistance genes for shin, neomycin, hygromycin and tetracycline.
- antibiotic resistance genes commonly used in the art as selection markers, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneeti Resistance genes for shin, neomycin, hygromycin and tetracycline.
- Another aspect of the invention provides a host cell comprising said recombinant vector, ie, a cell transformed with said recombinant vector.
- genomic DNA sequences and their transcripts can be used.
- the method for preparing a transcript may be carried out according to a method known in the art, and when preparing a transcript using a recombinant vector, it is preferable to first prepare a transcript by linearizing the vector.
- a host cell capable of continuously cloning or expressing the recombinant vector can be any host cell known in the art.
- prokaryotic cells for example, E. coli JM109, E. coli BL21, E Bacillus sp. strains such as coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E.
- coli W3110 Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marsonsons And enterobacteria and strains such as various Pseudomonas species, and when transforming to eukaryotic cells, as host cells, yeast (Saccharomyce cerevisiae), insect cells, plant cells and animal cells, such as SP2 / 0, CHO ( Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines and the like can be used.
- yeast Sacharomyce cerevisiae
- insect cells such as various Pseudomonas species
- plant cells and animal cells such as SP2 / 0, CHO ( Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3,
- the delivery of the polynucleotide or the recombinant vector comprising the same into the host cell may employ a delivery method well known in the art.
- a delivery method well known in the art.
- the host cell is a prokaryotic cell
- a CaCl2 method or an electroporation method may be used.
- the host cell is a eukaryotic cell
- a micro-injection method, calcium phosphate precipitation method, electroporation method, liposomes, or the like may be used.
- -Mediated transfection and gene bombardment may be used, but is not limited thereto.
- productivity is higher than that of animal cells, but is not suitable for the production of intact Ig-type antibodies due to glycosylation problems.
- antigen-binding fragments such as Fab and Fv Can be used for production.
- the method of selecting the transformed host cell can be easily carried out according to methods well known in the art using a phenotype expressed by a selection label.
- the selection marker is a specific antibiotic resistance gene
- the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.
- CFP-10 or Ag85B which is a mycobacteria-derived antigen
- CFP-10 or Ag85B which is a mycobacteria-derived antigen
- Figure 2 confirms the binding of Ag85B-specific monophages and Ag85B antigen isolated from the human antibody-phage library.
- Figure 3 shows the binding site for Ag85B-specific monophages to Ag85B.
- Figure 5 confirms the binding capacity of CFP-10 specific monophages and CFP-10 antigen.
- Figure 6 shows a schematic of the human IgG heavy or light chain expression vector for Ag85B.
- FIG. 7 is a graph of surface plasmon resonance results showing the antigen binding capacity of C12 and 8B3 antibodies.
- 8 is a graph of surface plasmon resonance results showing antigen binding capacity of 50B14 and 8B3 antibodies.
- Figure 9 shows a schematic diagram of the human IgG heavy chain or light chain expression vector for CFP-10.
- Figure 10 shows surface plasmon resonance results showing the antigen binding capacity of Group 2 and Group 3 antibodies.
- Example 1 Mycobacterium tuberculosis antigen CFP-10 and Ag85B Recombinant Protein Production and Purification
- CFP-10 antibody and Ag85B (p85) antibody In order to prepare CFP-10 antibody and Ag85B (p85) antibody, recombinant antigens CFP-10 and Ag85B were first constructed. Using the genomic DNA of M. tuberculosis as a template, the CFP-10-F primer (SEQ ID NO: 1), the CFP-10-R primer (SEQ ID NO: 2), Ag85B-F primer (SEQ ID NO: 3), and Ag85B-R primer ( Each gene was amplified by PCR using SEQ ID NO: 4 (Shin et al., Clin. Vaccine Immunol., 15: 1788, 2008) (Table 1).
- the amplified PCR products were digested with restriction enzymes BamHI and EcoRI, respectively, and inserted into the BamHI / EcoRI positions of pET28a (Novagen, USA).
- Recombinant plasmids were transformed into Escherichia coli BL21 (DE3), and then overexpressed by inducing IPTG to a final concentration of 1 mM at OD 600 values of 0.4 to 0.5 at 37 ° C.
- Cultured E. coli were collected and suspended with 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride (PMSF). Thereafter, cells were disrupted by ultrasound and recombinant proteins were purified through a Ni-NTA agarose column (Invitrogen, USA).
- Antigen binding fragments that specifically bind to Ag85B antigen were identified using human antibody-phage libraries.
- Example 2-1 To Ag85B (p85) Screening for Specific Binding Antigen Binding Fragments
- Biopanning which coats 2 ml of Ag85B antigen (10 ⁇ g / ml) obtained from Example 1 on an immunotube, and then reacts the human antibody-phage library with the coated antigen to select initial binding phage. ) was performed. At this time, the total number of phages in the library was about 2 ⁇ 10 13 CFU. Subsequently, the remaining phage after 10 washing procedures for removal of non-specific phage was infected with E. coli XL-1 Blue to amplify the phage, and then a primary sublibrary was obtained, and the antigen-phage using the sub-library. The reaction and selection process were carried out three times in total. The gripping number in each panning process is shown in Table 2.
- the selected phage was reacted with a plate coated with Ag85B antigen, and a secondary antibody (anti-M13-HRP) was bound to the bound phage. .
- a secondary antibody anti-M13-HRP
- the signal at 490nm wavelength was compared with the analytical values of myc-ELISA.
- the monoclonal phage antibody was isolated using the phage library obtained in the fourth panning. Specifically, about 40 colonies were taken from the fourth phage library (XL-1 blue), and phages were harvested from the culture by individually expressing phages. Then, monophage ELISA analysis was performed on Ag85B and c-myc in individually produced phage.
- phagemids of the monoclonals were extracted and DNA fingerprinting was performed through Bstl restriction enzyme treatment to analyze the characteristics of the monophages.
- three scFv antibodies (8B3, 50B14, Each amino acid sequence was analyzed (heavy chain variable region of 8B3: light chain variable region of SEQ ID NO: 9, 8B3: heavy chain variable region of SEQ ID NO: 10, 50B14: light chain variable region of SEQ ID NO: 11, 50B14: sequence Heavy chain variable region of No. 12, C12: SEQ ID NO: 13, light chain variable region of C12: SEQ ID NO: 14).
- Example 3-1 Screening for Antigen-Binding Fragments Specific for CFP-10
- the phages were selected using the same method as Example 2 except for the difference of three panning processes, and confirmed whether the proteins displayed on the phages recognized CFP-10 (10 kDa culture filtrate antigen) antigens. It was.
- the phage library was separated from the phage library obtained in the third panning by the same method as Example 2 above.
- the whole IgG antibody was prepared using the sequence of the scFV antibody obtained in Example 2 above.
- Example 4-1 On Ag85B For human Ig G Heavy chain or Light chain Construction of Expression Vector
- variable regions of each heavy and light chain were amplified using primers respectively, and the amplified variable and heavy chain variable regions were cleaved using SfiI / NheI or SfiI / BglII restriction enzymes. Subsequently, fragments were inserted by ligation into pNATABH and pNATABL vectors, which are human IgG heavy or light chain expression vectors digested using the same restriction enzyme (FIG. 6). After transforming the vector into XL-1 blue and culturing, colony-PCR was performed to confirm that the antibody sequence was properly incorporated into the vector (FIG. 6).
- Heavy- and light-chain expression vectors were co-transfected into HEK293 cells, and cell culture fluid was recovered every 3 days and concentrated about 500 ml 10-fold to obtain expression-secreted antibodies from HEK293 cells, followed by Protein-A bead (sepharose). Pharmacia) was used to purify the antibody.
- Antigen binding analysis of the obtained Ag85B monoclonal antibody was measured by surface plasmon resonance (SPR) method.
- Gold-bound protein (GBP) bound C12 was reacted at 25, 50, 100 and 200 ⁇ g / ml concentrations on the chip coated with gold, and then nonspecific with 500 ⁇ g / ml BSA. The reaction was removed. Thereafter, 10 ⁇ g / ml of the Ag85B antigen prepared in Example 1 was bound, the nonspecific reaction was further removed using 100 ⁇ g / ml of BSA, and 8B3 was reacted at a concentration of 50 ⁇ g / ml.
- Example 5 specifically binds to CFP-10 IgG Antibody Construction
- Example 5-1 Human to CFP-10 IgG Heavy chain or Light chain Construction of Expression Vector
- Example 4-1 Using the same method as Example 4-1, the sequence of the CFP-10 antibody was inserted into the vector and transduced into XL-1 blue (FIG. 9).
- Light and heavy chain expression vectors for Group2 or Group3 were co-transfected into HEK293 cells and cell cultures were recovered every 3 days to obtain expression-secreted antibodies from transduced HEK293 cells.
- About 500 ml of the recovered cell culture solution was ultra-filtration (millipore) and concentrated to about 50 ml, the antibody was bound using Protein-A bead (sepharose; Pharmacia), and purified using glycine (glycin).
- Purified CFP-10 antibody (about 10ml) was stored at -20 after dialysis, and the obtained antibody was confirmed using SDS-PAGE and silver stain.
- Antigen binding of the obtained CFP-10 monoclonal antibody was measured by surface plasmon resonance (SPR) method. After reacting Group3 to which gold-binding protein is bound on the gold-coated chips at concentrations of 100, 50, 10 and 1 ⁇ g / ml, respectively, and then removing the nonspecific reaction using 100 ⁇ g / ml of BSA, 100 ⁇ g / ml of CFP-10 antigen prepared in 1 was bound, and the non-specific reaction was removed at a concentration of 100 ⁇ g / ml of BSA, and then 100 ⁇ g / of Group 2 having a silica binding protein (SBP) bound thereto. The reaction was carried out at a concentration of ml. As a result, as shown in Figure 10, it was confirmed that Group2 and Group3 binds to the CFP-10 antigen with high specificity (Fig. 10).
- SBP silica binding protein
- Mycobacteria ELISA OD value for Anti-Ag85B complex (Group2) Anti-CFP-10 (8B3) M. tuberculosis CF 0.3403 0.2359 M. avium CF 0.0607 0.0557 M. kansasii CF 0.0667 0.0427 M. bovis BCG CF 0.0858 0.0369 M. intracellularlare CF 0.0789 0.0583 M. abscessus CF 0.0631 0.038
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Abstract
Description
번호 | 프라이머 이름 | 염기서열 |
서열번호 1 | CFP-10-F | GGCCGGGGATCCATGGCAGAGATGAAGACCG |
서열번호 2 | CFP-10-R | GGCCGGGAATTCGAAGCCCATTTGCGAGGAC |
서열번호 3 | Ag85B-F | GGCCGGGGATCCAGCGAAGAGCCGGACGATG |
서열번호 4 | Ag85B-R | GGCCGGGAATTCGAGCATGAGACTCGATCAG |
인풋 타이터(cfu/㎖) | 아웃풋 타이터(cfu/㎖) | ||
Ag85B(20㎍) | Ag85B(Neg) | ||
1차 | sublibrary titer not evaluated | ||
2차 | 1.9x1013 | 3.0x107 | 1.3x105 |
3차 | 4.4x1013 | 8.64x107 | 8.0x105 |
4차 | 1.97x1013 | 1.8x1013 | 1.05x106 |
총 클론(clone) 개수 62개 | ||
그룹 1 | IGHV1-69*01/IGKV2D-40*01 | 32개 |
그룹 2 | IGHV3-9*01/IGLV1-47*02 | 20개 |
그룹 3 | IGHV3-33*01/IGLV1-47*01 | 5개 |
그룹 4 | IGHV1-69*06 | 1개 |
그룹 5 | IGHV3-30*18 | 1개 |
그룹 6 | IGHV3-72*01/IGKV1-17*02 | 2개 |
No signal | 1개 |
Mycobacteria | ELISA OD value for | |
Anti-Ag85B complex(Group2) | Anti-CFP-10 (8B3) | |
M. tuberculosis CF | 0.3403 | 0.2359 |
M. avium CF | 0.0607 | 0.0557 |
M. kansasii CF | 0.0667 | 0.0427 |
M. bovis BCG CF | 0.0858 | 0.0369 |
M. intracelullare CF | 0.0789 | 0.0583 |
M. abscessus CF | 0.0631 | 0.038 |
Claims (15)
- 서열번호 5의 아미노산 서열을 포함하는 중쇄 가변 영역; 및서열번호 6의 아미노산 서열을 포함하는 경쇄 가변 영역을 포함하는 마이코박테리아 유래 CFP-10 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편.
- 서열번호 7의 아미노산 서열을 포함하는 중쇄 가변 영역; 및서열번호 8의 아미노산 서열을 포함하는 경쇄 가변 영역을 포함하는 마이코박테리아 유래 CFP-10 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편.
- 제1항 또는 제2항에 있어서, 상기 마이코박테리아는 마이코박테리움 튜버큘로시스(Mycobacterium tuberculosis), 마이코박테리움 보비스(Mycobacterium bovis), 마이코박테리움 레프레(Mycobacterium leprae), 마이코박테리움 칸사시(Mycobacterium kansasii), 마이코박테리움 인트라셀룰라레(Mycobacterium intracelullare), 마이코박테리움 앱세서스(Mycobacterium abscessus) 및 마이코박테리움 아비움(Mycobacterium avium)으로 이루어진 군으로부터 선택되는 것인 항체 또는 그의 항원 결합 단편.
- 서열번호 5 또는 서열번호 7의 아미노산 서열을 포함하는 마이코박테리아 유래 CFP-10 항원에 특이적으로 결합하는 항체의 중쇄 가변 영역을 코딩하는 폴리뉴클레오티드.
- 서열번호 6 또는 서열번호 8의 아미노산 서열을 포함하는 마이코박테리아 유래 CFP-10 항원에 특이적으로 결합하는 항체의 경쇄 가변 영역을 코딩하는 폴리뉴클레오티드.
- 제4항 또는 제5항의 폴리뉴클레오티드를 포함하는 재조합 벡터.
- 제6항의 재조합 벡터를 포함하는 숙주세포.
- 서열번호 9의 아미노산 서열을 포함하는 중쇄 가변 영역; 및서열번호 10의 아미노산 서열을 포함하는 경쇄 가변 영역을 포함하는 마이코박테리아 유래 Ag85B 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편.
- 서열번호 11의 아미노산 서열을 포함하는 중쇄 가변 영역; 및서열번호 12의 아미노산 서열을 포함하는 경쇄 가변 영역을 포함하는 마이코박테리아 유래 Ag85B 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편.
- 서열번호 13의 아미노산 서열을 포함하는 중쇄 가변 영역; 및서열번호 14의 아미노산 서열을 포함하는 경쇄 가변 영역을 포함하는 마이코박테리아 유래 Ag85B 항원에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편.
- 제8항 내지 제10항 중 어느 한 항에 있어서, 상기 마이코박테리아는 마이코박테리움 튜버큘로시스(Mycobacterium tuberculosis), 마이코박테리움 보비스(Mycobacterium bovis), 마이코박테리움 레프레(Mycobacterium leprae), 마이코박테리움 칸사시(Mycobacterium kansasii), 마이코박테리움 인트라셀룰라레(Mycobacterium intracelullare), 마이코박테리움 앱세서스(Mycobacterium abscessus) 및 마이코박테리움 아비움(Mycobacterium avium)으로 이루어진 군으로부터 선택되는 것인 항체 또는 그의 항원 결합 단편.
- 서열번호 9, 서열번호 11 또는 서열번호 13의 아미노산 서열을 포함하는 마이코박테리아 유래 Ag85B 항원에 특이적으로 결합하는 항체의 중쇄 가변 영역을 코딩하는 폴리뉴클레오티드.
- 서열번호 10, 서열번호 12 또는 서열번호 14의 아미노산 서열을 포함하는 마이코박테리아 유래 Ag85B 항원에 특이적으로 결합하는 항체의 경쇄 가변 영역을 코딩하는 폴리뉴클레오티드.
- 제12항 또는 제13항의 폴리뉴클레오티드를 포함하는 재조합 벡터.
- 제14항의 재조합 벡터를 포함하는 숙주세포.
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WO2021118997A1 (en) * | 2019-12-09 | 2021-06-17 | Versiti Blood Research Institute Foundation Inc. | Bcg car constructs and methods of their manufacture and use |
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KR101631054B1 (ko) * | 2015-12-31 | 2016-06-16 | 중앙대학교 산학협력단 | 마이코박테리아 유래 CFP-10 또는 Ag85B에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편 |
WO2019117585A2 (ko) * | 2017-12-11 | 2019-06-20 | 주식회사 딕스젠 | 결핵진단용 조성물 및 광학적 특성 변화에 기반한 결핵 진단방법 |
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