KR101631054B1 - Antibody or Antigen Binding Fragment Binding to CFP-10 or Ag85B from Mycobacteria - Google Patents

Abstract

The present invention relates to an antigen binding fragment specifically binding to CFP-10 or Ag85B which are antibodies derived from mycobacteria; polynucleotide which codes the same; a recombinant vector comprising the polynucleotide; and a host cell comprising the recombinant vector. The CFP-10 or Ag85B which are antibodies derived from mycobacteria can be detected with high sensitivity by using the antigen binding fragment of the present invention. Thus, the antigen binding fragment can be developed into a kit which can rapidly and accurately detect mycobacteria. An antibody or an antigen binding fragment thereof comprises: a heavy chain variable region including an amino acid sequence represented by SEQ ID NO: 5; and a light chain variable including an amino acid sequence represented by SEQ ID NO: 6.

Description

(Antibody or Antigen Binding Fragment Binding to CFP-10 or Ag85B from Mycobacteria) which specifically binds to Mycobacterium-derived CFP-10 or Ag85B,

The present invention relates to an antigen-binding fragment which specifically binds to an antigen derived from mycobacteria, CFP-10 or Ag85B, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a host cell comprising the recombinant vector .

In general, tuberculosis is a disease caused by infection with Mycobacterium tuberculosis, and it is known that it is the highest disease incidence among the legal infectious diseases in Korea. Therefore, early diagnosis and treatment of obtaining reliable information from a patient suspected of being infected with tuberculosis within a short period of time is of paramount importance.

Tuberculin skin test (TST) is a common method for diagnosing tuberculosis. However, because of its low specificity, it is not highly reliable in terms of tuberculosis infection but it is still widely used because of its high sensitivity and low cost. . In addition, the culture method is the most reliable method among the various methods of diagnosis of tuberculosis. Since the test for confirming tuberculosis by proliferation of the Mycobacterium tuberculosis or culturing the bacteria separately is not possible, the risk of infection of the experimenter can not be excluded. It takes a minimum of 4 to 6 weeks to take, which makes it difficult to diagnose early.

Recently, the BACTEC system or the Mycobacteria Growth Indicator Tube (MGIT) method using a liquid medium has been implemented. Although this method can confirm the proliferation of Mycobacterium tuberculosis in about 2 weeks by shortening the incubation period by using the liquid medium, there is a disadvantage that the additional confirmatory test for distinguishing it from atypical Mycobacterium tuberculosis is required, and the waste treatment There is a problem that it is a problem, infection concern of Mycobacterium tuberculosis, expensive equipment is required.

Therefore, there is a continuing need to develop techniques for rapidly and accurately detecting the Mycobacterium tuberculosis. For this purpose, it is essential to develop a method capable of specifically detecting the secreted antigen upon proliferation of Mycobacterium tuberculosis.

An aspect of the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to antigen CFP-10 derived from mycobacterium, a polynucleotide encoding the antigen, 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 the antigen Ag85B derived from mycobacterium, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a host cell comprising the recombinant vector will be.

One aspect of the invention provides a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 5; And an antibody or antigen-binding fragment thereof that specifically binds to a CFP-10 antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6.

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; And an antigen binding fragment thereof, which specifically binds to a CFP-10 antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

Another aspect of the present invention is a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 9; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10, or an antigen-binding fragment thereof, which specifically binds to the Ag85B antigen derived from mycobacteria.

A 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 an Ag85B antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; And an antigen binding fragment thereof, which specifically binds to a Maichoobacterium-derived Ag85B antigen comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

As used herein, the term "mycobacteria" refers to a group of bacteria classified as aerobic, acid-fast gram-positive bacteria with thick cell walls containing a waxy, hydrophobic, rich mycolic acid . Mycobacteria can be classified as non-pathogenic mycobacteria that grow rapidly during culture, and pathogenic mycobacteria that grow slowly when cultured, and the CFP-10 or Ag85B antigens are pathogenic mycobacteria, For example, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium may be derived from Mycobacterium intracelulla, Mycobacterium abscessus or Mycobacterium avium.

As used herein, the term " antibody " refers to a specific antibody against Ag85B or CFP-10 antigen from Mycobacterium, which specifically binds to Ag85B or CFP-10 antigen, Binding fragment of the molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has kappa (kappa) and lambda (lambda) types.

As used herein, the term " heavy chain " refers to a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, and a variable region domain VH comprising three constant region domains CH1, CH2 and CH3 Quot; means both the heavy chain and the fragment thereof. The term " light chain " as used herein also refers to a full-length light chain comprising the variable region domain VL and the constant region domain CL comprising an amino acid sequence with sufficient variable region sequence to confer specificity to the antigen, It means all fragments.

As used herein, the term "antigen binding fragment" refers to a fragment of the immunoglobulin whole structure, which portion of the polypeptide includes a portion to which the antigen can bind. For example, it may be, but is not limited to, F (ab ') 2, Fab', Fab, Fv or scFv. Fab among the antigen-binding fragments has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only a heavy chain variable region and a light chain variable region are well known in the art. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. The antigen-binding fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is restricted to papain, a Fab can be obtained and when cut with pepsin, an F (ab ') 2 fragment can be obtained) Can be produced through recombinant DNA technology.

As used herein, the terms "specifically binding" or " specifically recognizing "are the same as commonly known to those of ordinary skill in the art and refer to an antigen and an antibody (or antigen binding fragment) This means that they interact with each other to perform an immunological reaction.

The antigen-binding fragment specifically binding to the CFP-10 antigen or the Ag85B antigen derived from the mycobacteria may be an antigen-binding fragment specifically binding to the CFP-10 antigen or the Ag85B antigen within the range capable of specifically recognizing the antigenic sequence Or a variant thereof. For example, the amino acid sequence of an antibody may be altered to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of the amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. For example, arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine are similar in size, phenylalanine, tryptophan and tyrosine are similar. Thus, based on the above considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

On the other hand, amino acid substitutions in proteins that do not globally alter the activity of the molecule 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 / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. Considering the mutation having the biological equivalent activity, the antigen-binding fragment specifically binding to the CFP-10 antigen or the Ag85B antigen is interpreted to include a sequence showing substantial identity with the sequence set forth in SEQ ID NO: . The above-mentioned substantial identity can be obtained by aligning the amino acid sequence of the above sequence number with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art Sequence may be at least 60% homologous, at least 70% homologous, at least 80% homologous or at least 90% homologous. Alignment methods for comparing sequences are well known in the art. For example, a sequence analysis program such as blastp, blastx, tblastn and tblastx can be used on the Internet via the NCBI Basic Local Alignment Search Tool (BLAST).

Another aspect of the present invention is a polynucleotide encoding a 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 a polynucleotide encoding a heavy chain variable region of SEQ ID NO: A polynucleotide polynucleotide encoding a light chain variable region of an antibody that specifically binds to a CFP-10 antigen derived from mycobacteria comprising an amino acid sequence of SEQ ID NO:

A polynucleotide encoding a heavy chain variable region of an antibody that specifically binds to an Ag85B antigen derived from mycobacteria comprising an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; and a polynucleotide encoding a heavy chain variable region of SEQ ID NO: There is provided a polynucleotide polynucleotide encoding a light chain variable region of an antibody that specifically binds to an Ag85B antigen derived from mycobacteria comprising an amino acid sequence of the amino acid sequence of SEQ ID NO: 14.

As used herein, the term "polynucleotide" is a polymer of a deoxyribonucleotide or a ribonucleotide present in single-stranded or double-stranded form. RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and include analogs of natural polynucleotides unless otherwise specified.

The polynucleotide includes a nucleotide sequence encoding an amino acid sequence of an antigen-binding fragment that specifically binds to the CFP-10 antigen or the Ag85B antigen as well as a complementary sequence to the sequence. The complementary sequence includes a perfectly complementary sequence as well as a substantially complementary sequence that is specific for the CFP-10 or Ag85B antigen under stringent conditions known in the art Quot; means a sequence that can hybridize with a nucleotide sequence of a nucleotide sequence that codes for an amino acid sequence of an antigen-binding fragment that binds to an antigen.

In addition, the nucleotide sequence encoding the amino acid sequence of the antigen-binding fragment that specifically binds to the CFP-10 antigen or the Ag85B antigen may be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence of the antigen-binding fragment that specifically binds to the CFP-10 antigen or the Ag85B antigen is interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above substantial identity is obtained by aligning the nucleotide sequence with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art to obtain at least 80% Homology, at least 90% homology, or at least 95% homology.

According to one embodiment, the polynucleotide encoding the heavy chain variable region of the antibody that specifically binds to the CFP-10 antigen may be one having the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

According to one embodiment, the polynucleotide encoding the light chain variable region of the antibody that specifically binds to the CFP-10 antigen may have the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

According to one embodiment, the polynucleotide encoding the heavy chain variable region of the antibody that specifically binds to the Ag85B antigen may be any of the polynucleotides having the nucleotide sequence of SEQ ID NO: 19 to SEQ ID NO: 21.

According to one embodiment, the polynucleotide encoding the light chain variable region of the antibody that specifically binds to the Ag85B antigen may be any of the colony nucleotides having the nucleotide sequence of SEQ ID NO: 22 to SEQ ID NO: 24.

Yet another aspect of the present invention provides a recombinant vector comprising said polynucleotide.

As used herein, the term "vector" means means for expressing a gene of interest in a host cell. But are not limited to, viral vectors such as, for example, plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors. The vector that can be used as the recombinant vector may be a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series and pUC19), phage (e.g.,? gt4? B,? -charon,?? z1 and M13) or viruses (e.g. CMV, SV40 and the like).

The first combination vector may comprise a base sequence of the polynucleotide and a promoter operatively linked to the base sequence.

As used herein, the term "operably linked" refers to a functional linkage between a nucleotide expression control regulatory sequence (e. G., A promoter sequence) and another nucleotide sequence, whereby the regulatory sequence is a transcription of the other nucleotide sequence / ≪ / RTI >

The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be any conventional vector used in the art to express an exogenous protein in plants, animals or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.

In addition, the recombinant vector can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, a pL promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) , A ribosome binding site for initiation of translation, and a transcription / translation termination sequence. When the eukaryotic cell is used as a host, the origin of replication that functions in the eukaryotic cells contained in the vector is f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, CMV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus (CMV) promoter and the tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The recombinant vectors that can be used in the present invention include plasmids such as pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET etc. which are frequently used in the art, phages such as λgt4λB, ?? z1 and M13) or a virus (e.g., SV40 and the like).

The recombinant vector of the present invention may be fused with other sequences to facilitate purification of the antigen-binding fragment expressed therefrom. Fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexagenidine; Qiagen, USA) Is 6x His. Because of the additional sequence for such purification, the protein expressed in the host can be rapidly and easily purified through affinity chromatography.

On the other hand, the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, and examples thereof include ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, Neomycin, hygromycin, and tetracycline. ≪ / RTI >

Another aspect of the present invention provides a host cell comprising the recombinant vector, i. E., A cell transformed with the recombinant vector.

To prepare the transformed cells of the present invention, a genomic DNA sequence and its transcripts can be used. The method for producing the transcript can be carried out according to a method known in the art. When a transcript is produced by using a recombinant vector, it is preferable to first linearize the vector to prepare a transcript.

Any host cell known in the art may be used as a host cell capable of continuously cloning or expressing the recombinant vector stably. Examples of the prokaryotic cell include E. coli JM109, E. coli BL21, E Bacillus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Bacillus turginensis, and Salmonella typhimurium and Serratia marcesensis Yeast (insect cells, plant cells and animal cells, for example, SP2 / 0, CHO (CH3), etc.) as yeast cells when transforming eukaryotic cells Chinese hamster ovary K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines.

The delivery of the polynucleotide or a recombinant vector containing the polynucleotide into a host cell can be carried out by a method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl2 method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome -Mediated transfection, and gene bombardment, but are not limited thereto.

When E. coli and other microorganisms are used, the productivity is higher than that of animal cells, but it is not suitable for the production of intact Ig-type antibody due to glycosylation problem. However, since the antigen binding fragments such as Fab and Fv It can be used for production.

The method of selecting the transformed host cells can be easily carried out according to a method widely known in the art by using a phenotype expressed by a selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

When the antigen-binding fragment of the present invention is used, CFP-10 or Ag85B, an antigen derived from mycobacteria, can be detected with high sensitivity, so that it can be developed as a kit capable of detecting mycobacteria rapidly and accurately.

Figure 1 identifies an Ag85B specific human antibody-phage library according to a bio-panning cycle.
FIG. 2 shows binding of Ag85B-specific monophage and Ag85B antigen isolated from a human antibody-phage library.
FIG. 3 shows binding sites for Ag85B of Ag85B-specific monophages.
Figure 4 identifies a CFP-10 specific human antibody-phage library according to a bio-panning cycle.
FIG. 5 shows the binding force between the CFP-10 specific monophage and the CFP-10 antigen.
Fig. 6 is a schematic diagram of a human IgG heavy chain or light chain expression vector for Ag85B.
Fig. 7 is a graph of surface plasmon resonance results showing the antigen binding ability of C12 and 8B3. Fig.
Figure 8 is a graph of surface plasmon resonance results showing antigen binding capacities of 50B14 and 8B3.
9 is a schematic diagram of a human IgG heavy chain or light chain expression vector for CFP-10.
Fig. 10 shows the results of surface plasmon resonance showing antigen binding forces of Group 2 and Group 3. Fig.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example 1 Production and Purification of Recombinant Proteins of Mycobacterial Antigens CFP-10 and Ag85B

To prepare the CFP-10 antibody and the Ag85B (p85) antibody, recombinant antigens CFP-10 and Ag85B were prepared. To the genomic DNA of M. tuberculosis as the template, CFP-10-F primer (SEQ ID NO: 1), CFP-10-R primer (SEQ ID NO: 2), Ag85B-F primer (SEQ ID NO: 3) and Ag85B-R primer ( (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 sites of pET28a (Novagen, USA). The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), and then IPTG was added at 37 ° C to a final concentration of 1 mM at an OD ₆₀₀ value of 0.4-0.5. Overexpression was induced by centrifugation The cultured E. coli was collected and suspended in 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 20 mM imidazole and 1 mM phenylmethylsulfonyl fluoride (PMSF). After that, the cells were disrupted by ultrasonication and the recombinant protein was purified through a Ni-NTA agarose column (Invitrogen, USA).

number Name of the primer Base sequence SEQ ID NO: 1 CFP-10-F GGCCGG GGATCC ATGGCAGAGATGAAGACCG SEQ ID NO: 2 CFP-10-R GGCCGG GAATTC GAAGCCCATTTGCGAGGAC SEQ ID NO: 3 Ag85B-F GGCCGG GGATCC AGCGAAGAGCCGGACGATG SEQ ID NO: 4 Ag85B-R GGCCGG GAATTC GAGCATGAGACTCGATCAG

(The underlined part represents the restriction enzyme site.)

Example  2: Mycobacterium tuberculosis antigen Ag85B on  Screening and purification of specifically binding antigen-binding fragments

Antigen binding fragments that specifically bind to the Ag85B antigen were identified using a human antibody-phage library.

Example  2-1: On Ag85B (p85)  Screening of specifically binding antigen-binding fragments

2 ml of the Ag85B antigen (10 μg / ml) obtained from Example 1 was coated on an Immunotube, and then the human antibody-phage library was reacted with the coated antigen to obtain Biopanning ) Were performed. At this time, the total number of phages in the library was about 2 × 10 ¹³ CFU. Then, to remove nonspecific phage, the phage remaining after 10 washing steps were infected with E. coli XL-1 Blue to amplify the phage, and then a primary sub-library was obtained. The antigen-phage using the sub- The reaction and screening procedures were performed three times in total. The wave index for each panning process is shown in Table 2.

Input Titer (cfu / ml) Output Titer (cfu / ml) Ag85B (20 [mu] g) Ag85B (Neg) Primary sublibrary titer not evaluated Secondary 1.9x10 ¹³ 3.0x10 ⁷ 1.3x10 ⁵ Third 4.4 x ^{10 13} 8.64x10 ⁷ 8.0x10 ⁵ Fourth 1.97x10 ¹³ 1.8x10 ¹³ 1.05x10 ⁶

To confirm the recognition of the Ag85B antigen in the phage displayed on the plate coated with the Ag85B antigen, the selected phage was reacted and the secondary antibody (anti-M13-HRP) was bound to the bound phage . After that, OPD / H ₂ O ₂ After addition of the substrate, the signal was analyzed at a wavelength of 490 nm. In order to compare the number of phages specifically bound to Ag85B and the total number of phages, we compared the values of myc-ELISA with those of recombinant C-myc tags expressing all phages.

As a result, the number of phage obtained from the library obtained at each panning was similar, and it was confirmed that the phage showing a specific binding to Ag85B increased with the lapse of the panning cycle (FIG. 1).

Example 2-2: Purification of an antigen-binding fragment that specifically binds to Ag85B

Since the binding capacity of the phage library to Ag85B did not increase significantly after the fourth panning, the monoclonal phage antibody was isolated using the phage library obtained in the 4th panning. Specifically, about 40 colonies were taken from the quaternary phage library (XL-1 blue), and the phage were separately expressed to harvest the phage in the culture medium. Monophage ELISA analysis was then performed on Ag85B and c-myc in individually produced phages.

As a result, it was confirmed that more than 60% of the total phages had binding ability to Ag85B (FIG. 2). To confirm which part of Ag85B the resulting monophagy binds to, the two subunits p32 and p33 As a result, it was confirmed that they were selectively bound to p32 and not to p33 (FIG. 3).

Then, phagemid of each monoclone was extracted and DNA fingerprinting was performed by Bstl restriction enzyme treatment. As a result, three kinds of scFv antibodies (8B3, 50B14, (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 12, heavy chain variable region of C12: SEQ ID NO: 13, light chain variable region of C12: SEQ ID NO: 14).

Example 3: Screening and purification of antigen-binding fragments specifically binding to Mycobacterium tuberculosis antigen CFP-10

Example 3-1: Selection of antigen-binding fragments specifically binding to CFP-10

The purging was selected using the same method as in Example 2 except for the difference that the panning procedure was performed three times, and it was confirmed whether the protein displayed in the phage recognized the CFP-10 antigen.

As a result, it was confirmed that c-myc was similar for each panning, but the phage exhibiting specific binding to CFP-10 increased with the lapse of the panning cycle (Fig. 4).

Example 3-2: Purification of an antigen-binding fragment that specifically binds to CFP-10

Since the binding strength of the phage library to CFP-10 in the tertiary panning was greatly increased, the monophage was separated from the phage library obtained in the third panning in the same manner as in Example 2 above.

96 clones were selected from the separated monophages, and the binding ability to CFP-10 was analyzed by ELISA. As a result, antibodies capable of binding to CFP-10 were confirmed at a high ratio (FIG. 5). The sequences of 62 monophages with strong signal were analyzed, and the sequences of the antigen recognition sites were compared. As a result, six kinds of antibodies were identified (Table 3).

Total number of clones 62 Group 1 IGHV1-69 * 01 / IGKV2D-40 * 01 32 Group 2 IGHV3-9 * 01 / IGLV1-47 * 02 20 Group 3 IGHV3-33 * 01 / IGLV1-47 * 01 5 Group 4 IGHV1-69 * 06 One Group 5 IGHV3-30 * 18 One Group 6 IGHV3-72 * 01 / IGKV1-17 * 02 2 No signal One

In order to confirm the binding ability of the monoclonal antibodies in each group, the concentration of the antigen CFP-10 was diluted step by step at 1 / / ml, coated on a plate, and analyzed by ELISA. As a result, quantitative detection was obtained even when the antigen was diluted to 1/3125 times (about 350 pg / ml). Groups 4 and 6 showed the best binding force, and two kinds of scFv antibodies (Group 2 and Group 3) (Heavy chain variable region of Group 2: light chain variable region of Group 2: SEQ ID NO: 6, heavy chain variable region of Group 3: SEQ ID NO: 7, light chain variable region of Group 3: SEQ ID NO: 8).

Example  4: Ag85B on  Specifically binding IgG  Antibody production

The whole IgG antibody was prepared using the sequence of the scFV antibody obtained in Example 2 above.

Example  4-1: Ag85B on  Human Ig G Heavy chain  or Light chain  Production of expression vector

The variable region of each heavy chain and the light chain was amplified using a primer in the scFV antibody sequence of Ag85B, and the amplified heavy and light chain variable regions were cleaved using SfiI / NheI or SfiI / BglII restriction enzyme. Thereafter, fragments were inserted into pNATABH and pNATABL vectors, which are human IgG heavy chain or light chain expression vectors, which had been cleaved using the same restriction enzymes (Fig. 6). The vector was transformed into XL-1 blue and cultured, and then colony-PCR was performed to confirm whether the antibody sequence was properly inserted into the vector (FIG. 6).

Example  4-2: Expression and purification of Ag85B monoclonal antibody

The heavy and light chain expression vectors were co-transfected into HEK293 cells. To obtain antibodies expressed from HEK293 cells, the cell culture medium was collected every 3 days, and about 500 ml was concentrated 10 times. Protein-A beads (sepharose ; Pharmacia).

The antigen binding affinity of the obtained Ag85B monoclonal antibody was measured by a surface plasmon resonance (SPR) method. Gold-binding protein-bound C12 was reacted at 25, 50, 100, and 200 μg / ml, respectively, on gold coated chip, followed by non-specific reaction with 500 μg / ml BSA Respectively. Subsequently, 10 μg / ml of the Ag85B antigen prepared in Example 1 was ligated, followed by removal of nonspecific reaction using 100 μg / ml of BSA, and then 8B3 was reacted at a concentration of 50 μg / ml. As a result, as shown in FIG. 7, it was confirmed that C12 and 8B3 bind to Ag85B antigen with high specificity (FIG. 7). In addition, 50B14 to which gold-binding proteins were bound was reacted at a concentration of 25, 50, 100 and 200 μg / ml on a chip coated with gold, followed by removal of nonspecific reaction using 500 μg / ml of BSA, 10 μg / ml of the Ag85B antigen prepared in Example 1 was ligated, followed by removing the nonspecific reaction at a concentration of 100 μg / ml of BSA. Then, 8B3 was reacted at a concentration of 50 μg / ml. As a result, as shown in FIG. 8, it was confirmed that 50B14 and 8B3 bind to Ag85B antigen with high specificity (FIG. 8).

Example 5: Production of IgG antibody specifically binding to CFP-10

Example 5-1: Preparation of human IgG heavy chain or light chain expression vector in CFP-10

The sequence of the CFP-10 antibody was inserted into the vector and transfected into XL-1 blue using the same method as in Example 4-1 (FIG. 9).

Example 5-2: Expression and purification of CFP-10 monoclonal antibody

Light and heavy chain expression vectors for Group 2 or Group 3 were co-transfected into HEK293 cells and cell culture medium was collected every 3 days to obtain expression-secreted antibodies from transduced HEK293 cells. Approximately 500 ml of the recovered cell culture was concentrated to about 50 ml by ultra-filtration (millipore), and the antibody was bound using Protein-A bead (sepharose; Pharmacia) and purified using glycine. The purified CFP-10 antibody (about 10 ml) was dialyzed and stored at -20 ° C. The obtained antibody was confirmed by SDS-PAGE and silver stain.

The antigen-binding property of the obtained CFP-10 monoclonal antibody was measured by a surface plasmon resonance (SPR) method. Group 3, in which gold-binding proteins were bound, was reacted at a concentration of 100, 50, 10, and 1 μg / ml on a chip coated with gold, followed by removal of nonspecific reaction using 100 μg / ml of BSA, 100 μg / ml of the CFP-10 antigen prepared in Example 1 was combined with 100 μg / ml of BSA. Then, Group 2 was reacted at a concentration of 100 μg / ml. As a result, as shown in FIG. 10, it was confirmed that Group 2 and Group 3 bound with CFP-10 antigen with high specificity (FIG. 10).

Example 6: Analysis of specificity of Ag85B antibody and CFP-10 antibody

To determine the specificity of the antibodies produced in Examples 4 and 5, the antigens present in the culture medium of other mycobacteria were detected by ELISA.

As a result, it was confirmed that CFP-10 antigen and Ag85B antigen were specifically detected only in the culture medium of Mycobacterium tuberculosis (Table 4).

Mycobacteria ELISA OD value for Anti-Ag85B complex
(Group 2) 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

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Antibody or Antigen Binding Fragment Binding to CFP-10 or Ag85          from Mycobacteria <130> PN150170 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CFP-10-F primer <400> 1 ggccggggat ccatggcaga gatgaagacc g 31 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CFP-10-R primer <400> 2 ggccgggaat tcgaagccca tttgcgagga c 31 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Ag85-F primer <400> 3 ggccggggat ccagcgaaga gccggacgat g 31 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Ag85-R primer <400> 4 ggccgggaat tcgagcatga gactcgatca g 31 <210> 5 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> heavy chain of Group 2 <400> 5 Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg Ser Leu Arg   1 5 10 15 Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp Phe Ala Met His              20 25 30 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile          35 40 45 Thr Trp Asn Ser Gly Thr Ile Ala Tyr Ala Asp Ser Val Lys Gly Arg      50 55 60 Phe Thr Leu Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu Glu Met  65 70 75 80 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly                  85 90 95 His Tyr Gly Leu Asp Val Trp Gly His Gly Thr Lys Val Thr Val Ser             100 105 110 <210> 6 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> variable light chain of Group 2 <400> 6 Asp Val His Ser Gln Phe Val Leu Thr Gln Pro Pro Ser Val Ser Gly   1 5 10 15 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Tyr Ser Asn              20 25 30 Ile Gly Thr Asn Tyr Val Tyr Trp Tyr His Gln Leu Pro Gly Thr Ala          35 40 45 Pro Lys Leu Val Ile Gln Lys Asn Thr Gln Arg Pro Ser Gly Val Ser      50 55 60 Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile  65 70 75 80 Ser Gly Leu Arg Ser Glu Asp Glu Gly Asn Tyr Phe Cys Ser Ala Trp                  85 90 95 Asp Asp Ser Leu Ser Ala Val Leu Phe Gly Gly Gly Thr Lys Leu Thr             100 105 110 Val Leu         <210> 7 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain of Group 3 <400> 7 Asp Val His Ser Gln Val Gln Leu Val Lys Ser Gly Gly Gly Leu Val   1 5 10 15 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr              20 25 30 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly          35 40 45 Leu Glu Trp Val Ser Ala Ile Thr Gly Gly Gly Gly Ser Thr Tyr Tyr      50 55 60 Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys  65 70 75 80 Asn Thr Leu Tyr Val Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala                  85 90 95 Val Tyr Tyr Cys Ala Lys Pro Lys Tyr Ser Ser Gly Trp Tyr Asp Ala             100 105 110 Phe Asp Ile Trp Asp Gln Gly Thr Met Val Thr Val Ser         115 120 125 <210> 8 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> variable light chain of Group3 <400> 8 Val Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser   1 5 10 15 Tyr Ser Asn Ile Gly Thr Asn Tyr Val Tyr Trp Tyr His Gln Leu Pro              20 25 30 Gly Thr Ala Pro Lys Leu Val Ile Gln Lys Asn Thr Gln Arg Pro Ser          35 40 45 Gly Val Ser Asp Arg Phe Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser      50 55 60 Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Gly Asn Tyr Phe Cys  65 70 75 80 Ser Ala Trp Asp Asp Ser Leu Ser Ala Val Leu Phe Gly Gly Gly Thr                  85 90 95 Lys Val Thr Val             100 <210> 9 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain of 8B3 <400> 9 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Ser              20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Arg Ile Arg Ser Ser Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala      50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr  65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Ala Tyr Tyr Asp Phe Trp Ser Gly Tyr Tyr Arg Leu             100 105 110 Val Lys Val Ala Arg Pro Pro Thr Tyr Phe Asp Tyr Trp Gly Gln Gly         115 120 125 Thr Leu Val Thr Val Ser     130 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> variable light chain of 8B3 <400> 10 Arg Leu Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Ser Pro Gly   1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ala Lys Thr Asn              20 25 30 Val Ala Trp Tyr Gln Gln Lys Phe Gly Gln Ala Pro Arg Val Leu Ile          35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Arg Leu Glu Pro  65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Trp                  85 90 95 Thr Phe Gly Gln Gly Thr Gln Val Glu Ile Lys             100 105 <210> 11 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain of 50B14 <400> 11 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln   1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn              20 25 30 Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Ser Ser Gly Leu Glu          35 40 45 Trp Leu Gly Arg Thr Tyr Phe Arg Ser Lys Trp Tyr Asp Asp Tyr Ala      50 55 60 Val Ser Val Lys Ser Arg Leu Thr Ile Asn Pro Asp Thr Ser Lys Asn  65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val                  85 90 95 Tyr Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala             100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 12 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> variable light chain of 50B14 <400> 12 Arg Ile Val Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asn Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile          35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Thr Asn Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr His Cys Gln Gln Tyr Asn Ser Phe Pro Pro                  85 90 95 Lys Thr Phe Gly Gln Gly Thr Gln Val Glu Ile Lys Arg Leu Glu             100 105 110 <210> 13 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain of C12 <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Thr Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met          35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu      50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Ala Phe Gly Ala Ala Gly Arg Tyr Gly Met Asp Val Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser         115 120 <210> 14 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> variable light chain of C12 <400> 14 Asp Ile Val Met Thr Gln Ser Ser Thr Leu Ser Ala Ser Thr Gly   1 5 10 15 Asp Arg Val Asn Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Gly Trp              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile          35 40 45 Tyr Lys Val Ser Ser Leu Gln Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Val Pro Val                  85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys Arg Leu Glu             100 105 110 <210> 15 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable heavy chain of Group 2 <400> 15 ctggtggagt ccgggggaga cttggtacag cctggcaggt ccctgagact ctcctgtgta 60 gcctctggat tcacctttga tgattttgcc atgcactggg tccggcaagc tccggggaag 120 ggcctggagt gggtctcagg tattacttgg aatagtggta ccatcgccta tgcggactct 180 gtgaagggcc gcttcaccct ctccagagac aacgccaaga attccctgtc tctggaaatg 240 aacagtctga gagccgagga cacggccgtg tattactgtg cgagaggcca ctacggtttg 300 gacgtctggg gccatgggac caaggtcacc gtctcc 336 <210> 16 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable heavy chain of Group 3 <400> 16 gatgtccact cgcaggtgca gctggtaaag tctgggggag gcttggtaca gcctgggggg 60 tccctgagac tctcctgtgc agcctctgga ttcaccttta gcagctatgc catgagctgg 120 gtccgccagg ctccagggga ggggctggag tgggtctctg ctattactgg tggtggcggt 180 agcacatact acgcagactc cgtgaggggc cggttcacca tctcccgaga caattccaag 240 aacacgctgt atgtgcaaat gaacagtctg agagccgagg acacggccgt gtattactgt 300 gcgaaaccga agtatagcag tggctggtat gatgcttttg atatctggga ccaagggaca 360 atggtcaccg tctcc 375 <210> 17 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding variable light chain of Group 2 <400> 17 gatgtccact cgcagttcgt gctgactcag ccgccctcag tctctgggac ccccgggcag 60 agggtcacca tctcttgttc tggaagttac tccaacatcg gaactaatta tgtctactgg 120 taccaccaac tcccgggaac ggcccccaaa cttgtcatcc aaaagaatac tcagcggccc 180 tcaggtgtca gtgaccgatt ctcgggctct aggtctggca cctcagcctc cctggccatc 240 agtgggctcc ggtccgagga tgagggtaat tatttttgtt ccgcatggga tgacagcctg 300 agtgccgtac ttttcggcgg agggaccaag ctgaccgtcc ta 342 <210> 18 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable light chain of Group 3 <400> 18 gtctctggga cccccgggca gagggtcacc atctcttgtt ctggaagtta ctccaacatc 60 ggaactaatt atgtctactg gtaccaccaa ctcccgggaa cggcacccaa acttgtcatc 120 caaaagaata ctcagcggcc ctcaggtgtc agtgaccgat tctcgggctc taggtctggc 180 acctcagcct ccctggccat cagtgggctc cggtccgagg atgagggtaa ttatttttgt 240 tccgcatggg atgacagcct gagtgccgta cttttcggcg gagggaccaa ggtcaccgtc 300                                                                          300 <210> 19 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable heavy chain of 8B3 <400> 19 caggtgcagc tggtgcagtc tgggggaggc ttggtccagc ctggggggtc cctgaaactc 60 tcctgtgcag cctctgggtt caccctcagt ggctctgcta tgcactgggt ccgccaggct 120 tccgggaaag ggctggagtg ggttggccgt attagaagca aatctaacaa ttacgcgaca 180 gcatatgctg cgccggtgaa aggcaggttc accatctcca gagatgattc aaagaacacg 240 gcgtatcttc aaatgaacag cctgaaaacc gaggacacgg ccgtctatta ctgtgcaaga 300 gcctattacg atttttggag tggttattat cggttagtga aagtggctcg acctccgaca 360 tactttgact actggggcca aggaaccctg gtcaccgtct cc 402 <210> 20 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding variable heavy chain of 50B14 <400> 20 caggtgcagc tggtgcagtc tggtccagga ctggtgaagc cctcacagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg 120 cagtccccat cgagaggcct tgagtggctg ggaaggacat acttcaggtc caagtggtat 180 gatgattatg cagtatctgt gaaaagtcga ctaaccatca acccagacac atccaagaac 240 cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggccgtcta ttactgtgct 300 ccgtttggtt actacgtgtc tgactatgct atggcctact ggggccaagg gaccctggtc 360 accgtctcc 369 <210> 21 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable heavy chain of C12 <400> 21 caggtgcagc tggtgcagtc tggagctgag gtgaggacgc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat 180 gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag cacagcctac 240 atggagctga ggagcctgag atctgacgac acggccgtct attactgtgc aagagcattc 300 ggggaagcag ctggtaggta cggtatggac gtctggggcc aaggaaccct ggtcaccgtc 360 tcc 363 <210> 22 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding variable light chain of 8B3 <400> 22 cgactcgtgt tgacgcagtc tccaggcaca ctgtcagtgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgctaaa accaacgtag cctggtacca acagaaattt 120 ggccaggctc ccagggtcct catctatggt gcatccacca gggccactga tatcccagcc 180 aggttcagtg gcagtgggtc tgggacagat ttcagtctca ccatcagcag actggagcct 240 gaagattttg cagtgtatta ctgtcagcag tatggtagct caccgtggac gttcggccaa 300 gggacacagg tggagatcaa a 321 <210> 23 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable light chain of 50B14 <400> 23 cgaattgtga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaaa cagagtcacc 60 atcacttgcc gggcgagtca gggcattagc aattatttag cctggtatca gcagaaacca 120 gggaaagttc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180 cggttcagcg gcagtggatc tgggacagag ttcagtctca ccatcaccaa tctgcaacct 240 gaagattttg caacttacca ctgtcaacag tataacagtt tccctccgaa gacgttcggc 300 caagggacac aggtggaaat taaacgtctc gag 333 <210> 24 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding variable light chain of C12 <400> 24 gatattgtga tgacccagtc tccttccacc ctgtctgcat ctactggaga cagggtcaac 60 atcacttgcc gggccagtca gagtattagt ggctggttgg cctggtatca gcagaagcca 120 gggaaagccc ctaacctcct aatctataag gtgtctagtt tacaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agtgacagtg tcccggtcac cttcggccaa 300 gggaccagac tggatattaa acgtctcgag 330

Claims (15)

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 CFP-10 antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6.
A 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 CFP-10 antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
The method according to claim 1 or 2, wherein the mycobacteria are Mycobacterium tuberculosis , Mycobacterium bovis , Mycobacterium leprae , Mycobacterium kansasii , Mycobacterium intracelullare , Mycobacterium abscessus and Mycobacterium &lt; RTI ID = 0.0 &gt; Mycobacterium avium , or an antigen-binding fragment thereof.
A polynucleotide encoding a heavy chain variable region of an antibody that specifically binds to a CFP-10 antigen derived from mycobacteria comprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.
A polynucleotide encoding a light chain variable region of an antibody that specifically binds to a CFP-10 antigen derived from mycobacteria comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.
A recombinant vector comprising the polynucleotide of claim 4 or 5.
A host cell comprising the recombinant vector of claim 6.
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9; And
An antigen or antigen-binding fragment thereof that specifically binds to an Ag85B antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11; And
An antibody or an antigen-binding fragment thereof that specifically binds to an Ag85B antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; And
An antigen or an antigen-binding fragment thereof which specifically binds to an Ag85B antigen derived from mycobacteria comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.
11. The method according to any one of claims 8 to 10, wherein the mycobacteria are Mycobacterium tuberculosis , Mycobacterium bovis , Mycobacterium leprae , , Mycobacterium kansasii , Mycobacterium intracelullare , Mycobacterium abscessus , and Mycobacterium avium . The present invention also relates to a method for the treatment and / or prophylaxis of a disease or condition selected from the group consisting of Mycobacterium spp ., Mycobacterium kansasii , Mycobacterium intracelullare , Mycobacterium abscessus and Mycobacterium avium &Lt; / RTI &gt; or an antigen-binding fragment thereof.
A polynucleotide encoding a heavy chain variable region of an antibody that specifically binds to an Ag85B antigen derived from mycobacteria comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.
A polynucleotide encoding a light chain variable region of an antibody that specifically binds to an Ag85B antigen derived from mycobacteria comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.
12. A recombinant vector comprising the polynucleotide of claim 12 or 13.
15. A host cell comprising the recombinant vector of claim 14.

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