RU2802944C1 - Thioredoxin-1 epitope binding specifically to monoclonal antibody - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is proposed: the use of the human thioredoxin-1 (Trx1) antigen epitope consisting of the amino acid sequence of the following SEQ ID NO: 174, for specific binding to a monoclonal antibody or antigen-binding fragment thereof, which contains a light chain variable region containing a CDR1 of the following SEQ ID NO: 1, CDR2 with the following SEQ ID NO: 2, CDR3 with the following SEQ ID NO: 3 and a heavy chain variable region containing CDR1 of the following SEQ ID NO: 4, CDR2 with the following SEQ ID NO: 5, CDR3 with the following SEQ ID NO: 6.

EFFECT: epitope can be used to develop an antibody with improved Trx1 binding affinity, high level of sensitivity and specificity, which is useful for diagnosing breast cancer.

3 cl, 22 dwg, 25 tbl, 17 ex

Description

This work was supported by the Promotion of Innovative Businesses for Regulation-Free Special Zones funded by the Ministry of SMEs and Startups (MSS, Korea).

FIELD OF THE INVENTION

The present invention relates to an epitope of the thioredoxin-1 (Trx1) antigen and a monoclonal antibody specifically binding thereto, and more particularly to an epitope, a monoclonal antibody specifically binding thereto, an antigen binding fragment thereof, a nucleic acid molecule encoding a heavy chain and/or a light chain of said antibody or an antigen-binding fragment thereof, a recombinant vector containing said nucleic acid molecule, a host cell containing said recombinant vector, a method for producing said antibody or an antigen-binding fragment thereof, a kit for diagnosing breast cancer and a method of providing information necessary for diagnosis breast cancer.

BACKGROUND ART

Thioredoxin (Trx) is a small redox protein of approximately 12 kDa that is present in a reduced state due to NADPH-dependent reduction by thioredoxin reductase and in mammals includes thioredoxin-1 (Trx1) and thioredoxin-2 (Trx2). Thioredoxin acts as a growth factor, removes hydrogen peroxide, which is toxic to cells, stimulates the binding of critical factors associated with the role of ribonucleotide reductase and transcription in bacteria to DNA, and influences the activity of transcription factors such as nuclear transcription factor kB (NF-kB) in eukaryotic cells. Thus, thioredoxin influences cell and tumor death and therefore plays a key role in regulating the growth of cancer cells, and also cleaves the disulfide bond in another oxidized protein, helping to maintain activity in a reduced state. Thioredoxin-1- and thioredoxin-2-reductases remove nitric oxide from cysteines in mammalian cells, which influences cell death, and have potential relevance in various diseases, including inflammatory disease, heart attack and cancer. In addition, immunohistochemical analysis using anti-thioredoxin antibody demonstrates the expression of thioredoxin in human cancer tissues, including liver, colon, pancreas and cervix, and such expression indicates a possible role for thioredoxin in tumor development.

In these circumstances, the inventors have studied a marker for the diagnosis of breast cancer, which allows the diagnosis of breast cancer or its early prognosis; the expression of thioredoxin-1 was low in normal breast tissue but very high in breast cancer tissue, demonstrating that thioredoxin-1 is useful as a marker for breast cancer diagnosis (Korea Patent No. 10-1058230).

To develop enzyme-linked immunosorbent assay (ELISA)-based in vitro diagnostics (IVD) with high accuracy and high precision, a pair of antibodies to the same antigenic protein having different sites with different affinities is required. Moreover, a low-cost system for producing antibodies with a certain affinity is needed. In the present invention, to detect thioredoxin-1 (Trx1) present in human serum, two types of highly potent recombinant monoclonal antibodies were developed that bind to thioredoxin-1 with very high specificity and thus can be useful for screening to detect patients with breast cancer. Moreover, by identifying the site of the human Trx1 antigen to which these two types of antibodies bind, the present invention has been completed.

DESCRIPTION OF THE INVENTION

Technical problem

The present invention is proposed to solve the above problems and is aimed at providing a monoclonal antibody or an antigen-binding fragment thereof that allows diagnosing breast cancer with high sensitivity and specificity.

The present invention is also directed to providing a nucleic acid molecule encoding the heavy chain and/or light chain of said monoclonal antibody or antigen binding fragment thereof.

The present invention is also directed to providing a recombinant vector containing the specified nucleic acid molecule.

The present invention is also directed to providing a host cell containing said recombinant vector.

The present invention is also directed to providing an epitope of a human Trx1 antigen to which a monoclonal antibody or binding fragment thereof binds, a nucleic acid molecule encoding said epitope, a recombinant vector containing said nucleic acid molecule, and a host cell containing said recombinant vector.

The present invention is also directed to providing a method for producing a monoclonal antibody that specifically binds to Trx1, or an antigen-binding fragment thereof, comprising culturing said host cell.

The present invention is also directed to providing a kit for the diagnosis of breast cancer containing the above-described monoclonal antibody or an antigen binding fragment thereof.

The present invention is also directed to providing a method of providing information necessary for the diagnosis of breast cancer using the above-described monoclonal antibody or an antigen binding fragment thereof.

Technical solution

To solve the problems described above, the present invention provides a monoclonal antibody that specifically binds Trx1, or an antigen binding fragment thereof, comprising a light chain variable region comprising a light chain CDR1 (complementarity determining region 1) consisting of the amino acid sequence SEQ ID NO: 1, CDR2 a light chain consisting of the amino acid sequence SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 4, CDR2 heavy chain consisting of the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6.

According to one exemplary embodiment of the present invention, an antibody may comprise a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 14.

According to another exemplary embodiment of the present invention, the antibody may comprise a light chain consisting of the amino acid sequence of SEQ ID NO: 17 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18.

The present invention also provides a monoclonal antibody that specifically binds Trx1, or an antigen binding fragment thereof, comprising a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: : 8, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 9, and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 10, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: : 11, and a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 12.

According to one exemplary embodiment of the present invention, an antibody may comprise a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16.

According to another exemplary embodiment of the present invention, the antibody may comprise a light chain consisting of the amino acid sequence of SEQ ID NO: 19 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 20.

According to yet another exemplary embodiment of the present invention, the antibody may comprise a light chain consisting of the amino acid sequence of SEQ ID NO: 25 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 26.

According to yet another exemplary embodiment of the present invention, the antibody may comprise an IgG1 heavy chain and a kappa (κ) light chain.

According to yet another exemplary embodiment of the present invention, the antigen binding fragment may be a Fab, F(ab'), F(ab') ₂ , Fv, or a single chain antibody molecule.

According to yet another exemplary embodiment of the present invention, the antibody may be a chimeric antibody, a humanized antibody, or a human antibody.

The present invention also provides a nucleic acid molecule encoding the heavy chain and/or light chain of the above-described antibody or antigen binding fragment thereof.

According to one exemplary embodiment of the present invention, the nucleic acid molecule encoding the light chain may consist of the nucleotide sequence of SEQ ID NO: 21, the nucleotide sequence of SEQ ID NO: 23, or the nucleotide sequence of SEQ ID NO: 27.

According to one exemplary embodiment of the present invention, the nucleic acid molecule encoding the heavy chain may consist of the nucleotide sequence of SEQ ID NO: 22, the nucleotide sequence of SEQ ID NO: 24, or the nucleotide sequence of SEQ ID NO: 28.

The present invention also provides a recombinant vector containing a nucleic acid molecule encoding a heavy chain, a nucleic acid encoding a light chain, or both of nucleic acid molecules encoding a heavy chain and a light chain, and a host cell containing said recombinant vector.

The present invention also provides an epitope of a human Trx1 antigen consisting of any amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34 and 172-176, and a nucleic acid molecule encoding said epitope.

According to one exemplary embodiment of the present invention, the nucleic acid molecule may consist of any nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-37 and 177-181.

The present invention also provides a recombinant vector containing said nucleic acid molecule and a host cell containing said recombinant vector.

The present invention also provides a method for producing a monoclonal antibody that specifically binds to Trx1, or an antigen-binding fragment thereof, comprising culturing a host cell containing a recombinant vector containing a nucleic acid molecule encoding the heavy chain of the antibody described above, a nucleic acid encoding its light chain, or both of the nucleic acid molecules encoding its heavy chain and light chain.

The present invention also provides a breast cancer diagnostic kit containing the above-described antibody or an antigen-binding fragment thereof.

According to one typical embodiment of the present invention, the kit may be an enzyme-linked immunosorbent assay (ELISA) kit.

According to another exemplary embodiment of the present invention, the ELISA may be any selected from the group consisting of direct ELISA, indirect ELISA, direct sandwich ELISA and indirect sandwich ELISA.

The present invention also provides a method of providing information necessary for the diagnosis of breast cancer, comprising: (a) contacting the above-described monoclonal antibody or an antigen binding fragment thereof with a biological sample isolated from a subject suspected of having breast cancer; (b) measuring the level of expression of the Trx1 protein that binds to the monoclonal antibody or an antigen-binding fragment thereof in the specified biological sample through the formation of an antigen-antibody complex; and (c) comparing the Trx1 protein expression level measured in step (b) with a control and, if the protein expression level is greater than the control, designating the subject as a breast cancer patient.

In addition, according to the present invention, there is provided a method of providing information necessary for the diagnosis of breast cancer, comprising: (a) coating a solid support with a monoclonal antibody or an antigen binding fragment thereof containing light chain CDR1-CDR3 and heavy chain CDR1-CDR3 of a B266 or B266- antibody. 1, a monoclonal antibody or an antigen-binding fragment thereof containing a light chain variable region and a heavy chain variable region of an antibody B266 or B266-1, or an antibody B266 or B266-1 or an antigen-binding fragment thereof; (b) applying a biological sample isolated from a subject suspected of having breast cancer onto a coated solid support; (c) removing the unbound sample; or its antigen-binding fragment onto a solid support; (e) removing the unbound monoclonal antibody or antigen-binding fragment thereof; (f) measuring the expression level of Trx1 protein; and (g) comparing the Trx1 protein expression level measured in step (e) with a control and, if the protein expression level is higher than the control, designating the subject as a breast cancer patient.

According to one exemplary embodiment of the present invention, the level of Trx1 protein expression can be measured by any method selected from the group consisting of Western blot, ELISA, sandwich ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, immunoprecipitation assay, complement fixation assay, immunochromatographic analysis, FACS (fluorescence activated cell sorting) and protein chip analysis.

According to another exemplary embodiment of the present invention, the isolated biological sample may be any one or more selected from the group consisting of whole blood, serum, plasma, breast tissue and breast cells.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one skilled in the art to which this invention relates. In general, the nomenclature used here is well known and widely used in the field.

The following definitions of key terms are used in this application.

The term "antigen" refers to a molecule that can be bound by an antibody and can be used in an animal to produce an antibody capable of binding to an epitope of an antigen or part of the molecule thereof. An antigen may have one or more than one epitope.

The term "antibody" or "Ab" is an immunoglobulin molecule that can recognize a specific target or antigen, such as a carbohydrate, polynucleotide, lipid or polypeptide, through one or more antigen recognition sites located in the variable region of the immunoglobulin molecule and bind to her. The term "antibody" as used herein can refer to any type of antibody, including, without limitation: monoclonal antibody; polyclonal antibody; an "antigen-binding fragment" of an antibody having the ability to specifically bind to a particular antigen (eg, Trx1), for example, Fab, Fab', F(ab') ₂ , Fd, Fv, Fc, and so on; selected complementarity determining region (CDR); bispecific antibody; heteroconjugated antibody or mutant thereof; an antibody or fusion protein having an antigen binding fragment (eg, a single domain antibody); single chain variable fragment (ScFv) and single domain antibody (eg, shark and camelid antibodies); maxibody, minibody, intrabody, diabody, tribody, tetrabody, v-NAR and bis-scFv; humanized antibody; chimeric antibody; and all other modified configurations of the immunoglobulin molecule containing an antigen recognition site with the required specificity (including glycosylated variants of the antibody, variants of the amino acid sequence of the antibody and covalently modified antibody). The antibody may be of mouse, rat, human, or any other source (including a chimeric or humanized antibody).

An antibody or polypeptide that "specifically binds" to a particular target or antigen (eg, Trx1 protein) is a term commonly understood in the art to which this invention relates, and methods for determining such specific binding are also well known in the art. A certain molecule is considered to have "specific binding" when it interacts or binds to a particular cell or substance more often, faster, more consistently, and/or with higher affinity than to another type of cell or substance. A particular antibody "specifically binds" to a particular target or antigen with higher affinity, higher binding activity, faster and/or more consistently than other substances.

The term "binding affinity" or " _KD " as used herein refers to the equilibrium dissociation constant for the interaction of an antibody with a particular antigen. K _D is the ratio of the rate of dissociation (also called the "release rate" or "k _d ") to the rate of binding, or "association rate" or " _ka (association rate constant)". Thus, K _D is k _d /k _a expressed as molar concentration (M). It follows that the lower the _KD , the higher the binding affinity. Thus, a KD _of 1 μM indicates lower binding affinity than a KD _of 1 nM. The K _D value of an antibody can be determined using a method widely known in the art. One method for determining the K _D of an antibody typically uses surface plasmon resonance using a biosensor system such as the Biacore® system.

The term "vector" includes a nucleic acid molecule capable of delivering another nucleic acid linked to it. One type of vector is a "plasmid", which is a circular loop of double-stranded DNA into which an additional piece of DNA can be ligated. Another type of vector is a viral vector, in which case an additional piece of DNA can be attached to the viral genome. Some vectors are capable of self-replication in the host cells into which they are introduced (eg, a bacterial vector having a bacterial replicator and an episomal vector for mammalian cells). Other vectors (eg, a non-episomal vector for mammalian cells) can be integrated into the genome of host cells when introduced into host cells and thus undergo replication along with the host genome. In addition, some vectors can control the expression of functionally related genes. In this specification, the vectors are referred to as “recombinant expression vectors” (or simply “expression vectors”). Typically, an expression vector useful in recombinant DNA techniques is often present in the form of a plasmid. "Plasmid" and "vector" as used herein are the types of vectors that are most commonly used, and thus can be used interchangeably. However, the present invention includes various types of expression vectors that perform the same functions, such as viral vectors (eg, replication-defective retrovirus, adenovirus, and adeno-associated virus).

The term "host cells" is used to refer to transformed cells or cells transformed with a nucleic acid sequence to express a selected gene of interest. This term includes the descendants of mother cells, regardless of whether they are identical to the original cell in morphological or genetic aspects, provided that the selected gene is present in them.

Beneficial effects

The monoclonal antibody of the present invention has excellent binding affinity for thioredoxin-1, which allows it to bind very specifically to thioredoxin-1, and has very high sensitivity and specificity, which allows it to be effectively used in screening to identify patients with breast cancer. In addition, detection of thioredoxin-1 using the monoclonal antibody that specifically binds to thioredoxin-1 of the present invention, rather than detection using the traditional breast cancer diagnostic biomarker CA15-3, demonstrates exceptionally high sensitivity and specificity, allowing for significantly improved accuracy and reliability of breast cancer diagnosis. The epitope region of the human Trx1 antigen to which the antibody of the present invention binds can be effectively used in the development of an improved antibody to increase the binding affinity of the anti-Trx1 antibody.

DESCRIPTION OF GRAPHIC MATERIALS

In FIG. Figure 1 shows a cleavage map of a recombinant vector expressing the thioredoxin-1 antigen and the result of isotyping the antibody obtained in Example 1. bp - p.o. (base pairs); Ampicillin - ampicillin.

In FIG. 2 shows the amino acid sequences of the light chain (a) and heavy chain (b) of antibody 9G7 (AB1) obtained in Example 1.

In FIG. 3 shows the amino acid sequences of the light chain (a) and heavy chain (b) of antibody 2 B4 (AB2) obtained in Example 1.

In FIG. 4 shows cleavage maps of a recombinant vector expressing the light chain (a) and heavy chain (b) of the high affinity antibody B264.

In FIG. 5 shows cleavage maps of a recombinant vector expressing the light chain (a) and heavy chain (b) of the high affinity antibody B266.

In FIG. 6 shows the results of determination of reduced (+) and unreduced (-) states of antibodies using SDS-PAGE, where (a) shows the result for antibody B264, and (b) shows the result for antibody B266.

In FIG. 7 shows the results of determining the reduced (+) and unreduced (-) states of the B266-1 antibody using SDS-PAGE, where the B266-1 antibody was obtained by modifying the Fc portion of the B266 antibody to the Fc of human IgG1.

In FIG. 8A-8D show gapped IMTG alignment results for the light chain and heavy chain of antibody B266-1 and the light chain and heavy chain of antibody B264 in order.

In FIG. 9 shows the results of the affinity analysis of antibodies B266-1 and B264, wherein (A) shows the interaction score according to the concentration of the antibody, and (B) shows the result of the antibody affinity analysis using the Prism program.

In FIG. 10 is a graph showing the sensitivity and specificity obtained from ROC analysis of ELISA results using antibodies B266-1 and B264.

In FIG. 11 shows a comparison of amino acid sequence homology between human Trx1 and Chrysochloris asiatica Trx1. PREDICTED: thioredoxin-like - predicted sequence: thioredoxin-like; Sequence ID - sequence identifier; Length - length; Number of Matches - number of matches; Query - the sequence in question; human - human; Sbject - comparison sequence; Range - range; Score - indicator; Expect - expectation; Method - method; Identities - identical; Positives - positive; Gaps - gaps.

In FIG. 12 shows the result of electrophoresis to confirm successful cloning through the CaTrx1 cloning plasmid and restriction enzyme digestion (SfiI and XhoI), where lane 1 shows the CaTrx1 cloning plasmid and lane 2 shows the restriction enzyme digestion plasmid.

In FIG. 13 shows the result of analyzing the degree of expression of the CaTrx1 protein secreted by the cell line after transfection of an animal cell with the CaTrx1 plasmid.

In FIG. 14 shows the results of the affinity analysis of antibodies B266-1 and B264 against hTrx1 and CaTrx1.

In FIG. 15A shows a comparison of the amino acid sequences of CaTrx1 and hTrx1. Translation of Chrysochloris asiatica - translation of Chrysochloris asiatica; Translation of human TRX - Translation of human TRX.

In FIG. 15B shows the location of the mutations according to a comparison of the amino acid sequences of CaTrx1 and hTrx1.

In FIG. 15C shows a diagram of the PCR (polymerase chain reaction) fusion to obtain the mutant hTrx1 gene, which is the result of amplification of a DNA fragment, and PCR with overlapping primers carried out one after another.

In FIG. 15D shows the result of amplification of DNA fragments to determine the location of mutations.

In FIG. 15E shows the result of cassettes produced by PCR with overlapping primers using the resulting DNA fragments.

In FIG. 15F shows the expression of 8 types of hTrx1 mutant genes after transformation with 293F plasmids cloning each hTrx1 mutant gene.

In FIG. 16 shows proteins secreted into the culture solution after transduction of human HEK293 cells with genes obtained through the transformation shown in FIG. 15, and their cultivation, revealed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Since the size of Trx1 is approximately 12 kDa, a protein of the appropriate size was detected, confirming the expression of 8 types of transformed genes and their secretion as proteins into the culture solution.

In FIG. 17 shows the result of analysis of the expression level of 8 types of hTrx1 mutant proteins identified in FIG. 16, after cleaning.

In FIG. 18A-18C show the results of binding strength analysis of anti-Trx1 antibody against 8 types of hTrx1 mutant proteins of the present invention.

In FIG. 19 is a diagram showing the general principle of epitope detection using overlapping peptide scanning used in Example 17.

In FIG. 20 shows an image of miniarrays obtained using one of the antibody samples described in Example 17.

In FIG. 21A-21D are a heat map chart showing the degree of interaction of the controls interacting with the antibody samples and all probe peptides, where the y-axis shows the library peptide sequences and the x-axis shows the concentrations of the applied antibody samples. MMC2 values are shown by color code, which ranges from white (0 or low intensity), yellow (medium intensity), and red (high intensity). Mouse IgG - mouse IgG; Control-Spot - control point; Control mouse IgG - control mouse IgG; ug/mL - μg/ml.

In FIG. 22A-22F are images confirming the location of the epitope in the three-dimensional structure of the hTrx1 protein.

OPTIONS FOR IMPLEMENTING THE INVENTION

Next, the present invention will be described in detail.

As described above, in previous studies, the inventors confirmed that thioredoxin-1 is expressed in normal breast tissue at a low level, but in breast cancer tissue its expression level is very high. Thus, thioredoxin-1 has been proven to be useful as a marker for the diagnosis of breast cancer.

Therefore, in subsequent studies, the inventors developed a monoclonal antibody that binds to thioredoxin-1 very specifically and is useful in screening to identify patients with breast cancer. The monoclonal antibody of the present invention binds very specifically to thioredoxin-1 due to excellent binding affinity to thioredoxin-1 and has very high sensitivity and specificity, such that it can be effectively used in screening to identify patients with breast cancer. In addition, detection of thioredoxin-1 using a monoclonal antibody of the present invention that specifically binds to thioredoxin-1, rather than detection of CA15-3, another biomarker traditionally used for the diagnosis of breast cancer, demonstrates excellent sensitivity and specificity, thereby allowing significant improve the accuracy and reliability of breast cancer diagnosis. In addition, the epitope region of the human Trx1 antigen to which the antibody binds can be effectively used in the development of an improved antibody to increase the binding affinity of the anti-Trx1 antibody.

The present invention provides a monoclonal antibody that binds to thioredoxin-1 (Trx1), or an antigen-binding fragment thereof.

The monoclonal antibody of the present invention can be produced using a variety of methods known in the art, such as hybridoma, recombination and phage display technologies, and combinations of these methods. For example, a monoclonal antibody can be produced using hybridoma techniques known in the art. The term "monoclonal antibody" as used herein is not limited to an antibody produced using a hybridoma technique. The term "monoclonal antibody" refers to an antibody derived from any one eukaryotic, prokaryotic or phage clone, but does not refer to the method of its preparation.

The method of producing and screening a specific antibody using hybridoma technique is common and well known in the art. By way of non-limiting example, a mouse can be immunized with the target antigen or cells expressing it. When an immune response is detected, such as detection of an antigen-specific antibody in mouse serum, the spleen is removed from the mouse to isolate spleen cells. The spleen cells are then fused in a known manner with any suitable myeloma cells, for example P3U1, P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2/0-Ag14 or P3X63-Ag8-653. The hybridoma is selected and cloned using the limiting dilution method. The hybridoma clone is then assessed by a method known in the art for its ability to be a cell that secretes an antibody capable of binding to the antigen. Typically, ascites fluid containing high levels of antibodies can be obtained by injecting positive hybridoma clones into the peritoneal cavity of a mouse. In a typical embodiment of the present invention, the Trx1 antigen is produced by transfecting E. coli with a recombinant vector, the cleavage map of which is shown in FIG. 1(a). The spleen of an antigen-immunized rat was then isolated and cells fused to myeloma cells (sp2/0) to produce a Trx1-interacting antibody were determined by ELISA.

An exemplary monoclonal antibody of the present invention or an antigen binding fragment thereof may comprise (a) or (b) as follows, and may be referred to as B264 or B266-1, respectively:

(a) a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 6; or

(b) a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 7, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 8, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 9, and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 11. 12.

The term “complementarity determining region (CDR)” as used herein refers to the amino acid sequence of the hypervariable region of the heavy chain or light chain of an immunoglobulin. Each heavy chain (CDRH1, CDRH2 and CDRH3) and light chain (CDRL1, CDRL2 and CDRL3) has three CDRs, and these CDRs provide key contact residues when an antibody binds an antigen or epitope.

An exemplary monoclonal antibody of the present invention or an antigen binding fragment thereof may comprise (c) or (d) as follows, and may be referred to as B264 or B266-1, respectively:

(c) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 14; or

(d) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16.

An exemplary monoclonal antibody of the present invention or an antigen binding fragment thereof may comprise (e) or (f) as follows and may be referred to as B264 or B266, respectively:

(e) a light chain consisting of the amino acid sequence of SEQ ID NO: 17, and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18; or

(f) a light chain consisting of the amino acid sequence of SEQ ID NO: 19, and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 20.

An exemplary monoclonal antibody of the present invention is referred to as B264, B265, B266, B267, B268 or B269 and, most preferably, B264 or B266-1. B266-1 is a monoclonal antibody in which the Fc portion of B266 is modified to the Fc of human IgG1.

The structural unit of a naturally occurring antibody typically contains a tetramer. A tetramer typically consists of two pairs of identical polypeptide chains, and each pair has one full-length light chain (typically having a molecular weight of about 15 kDa) and one full-length heavy chain (typically having a molecular weight of about 50 to 70 kDa). The amino terminus of each light chain and heavy chain typically contains a variable region of approximately 100-110 or more amino acids involved in antigen recognition. The carboxy terminus of each chain defines a constant region that is typically involved in effector function. Human light chains are usually classified as κ and λ light chains. Heavy chains are generally classified as μ, δ, γ, α, and ε heavy chains, which define antibody isotypes such as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has, but is not limited to, certain subclasses including IgG1, IgG2, IgG3 and IgG4. IgM has, but is not limited to, subclasses including IgM1 and IgM2. Similarly, IgA is classified, without limitation, into subclasses including IgA1 and IgA2. In full-length light and heavy chains, the variable and constant regions are typically connected by a “J region” of about 12 or more amino acids, and the heavy chain also contains a “D region” of about 10 or more amino acids. The variable region of each light chain/heavy chain pair typically forms the antigen binding site. According to one exemplary embodiment of the present invention, in the monoclonal antibody of the present invention, the heavy chain may be an isotype of IgG1, IgG2a, IgG2b, IgG3, IgA or IgM, and the light chain may be a κ chain or a λ chain, and κ light chain and heavy chain are preferred. IgG1 chain.

In a monoclonal antibody of the present invention or an antigen-binding fragment thereof, "antigen-binding fragment thereof" refers to a fragment having the function of binding to an antigen and includes a Fab, F(ab'), F(ab') ₂ , Fv or a single chain antibody molecule. Among antigen-binding antibody fragments, Fab is a structure having light and heavy chain variable regions, a light chain constant region and a first heavy chain constant region (CH1), and contains one antigen binding site. F(ab') differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab') ₂ is formed through a disulfide bond between the cysteine residues of the Fab' hinge regions. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Such an antibody fragment can be produced using a protease, preferably using gene recombination technology. For example, a Fab can be produced by, for example, digesting the whole antibody with papain, and an F(ab') ₂ fragment can be produced by digesting the whole antibody with pepsin.

A typical antibody of the present invention may be a chimeric antibody, a humanized antibody, or a fully human antibody.

A chimeric antibody can be produced by recombinantly combining light chain and heavy chain variable domains (VL and VH) derived from one type of antibody-producing cell and light chain and heavy chain constant domains derived from another type of antibody. Typically, a chimeric antibody uses a rodent or rabbit variable domain and a human constant domain to produce an antibody typically having a human domain. The preparation of such a chimeric antibody is well known in the art and can be accomplished by standard means. It should also be appreciated that the human constant region of the chimeric antibody of the present invention may be selected from the constant region of IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG1l, IgG12, IgG13, IgG14, IgG15, IgG16 , IgG17, IgG18 or IgG19.

The humanized antibody is designed to contain an immunoglobulin domain that is even more similar to that of a human and contains the complementarity-determining region of an animal-derived antibody. This is achieved by carefully studying the sequence of the hypervariable loop of the variable region of a monoclonal antibody and adapting the sequence to the chain structure of the human antibody.

A fully human antibody is an antibody molecule containing a CDR such that both the light chain and heavy chain sequences are entirely derived from a human gene.

The present invention also provides nucleic acid molecule(s) encoding the heavy chain and/or light chain of a monoclonal antibody of the present invention or an antigen binding fragment thereof.

The term "nucleic acid molecule" as used herein includes DNA (gDNA and cDNA) and RNA molecules, and in a nucleic acid molecule, the nucleotide, which is its basic unit, also includes an analogue in which the sugar or basic moiety is modified, as well as the natural nucleotide. The sequences of the nucleic acid molecules encoding the heavy chain and light chain variable regions of the present invention may be modified. Modification includes additions, deletions, or non-conservative or conservative substitutions of nucleotides.

It should be explained that the nucleic acid molecule of the present invention also includes a nucleotide sequence substantially identical to the nucleotide sequence described above. Substantial identity refers to a nucleotide sequence exhibiting at least 80% homology, in one specific example, at least 90% homology, or, in another specific example, at least 95% homology when aligning the nucleotide sequence according to the present the invention with a different sequence to match them as closely as possible, and analyze the aligned sequences using an algorithm commonly used in the art.

According to one exemplary embodiment of the present invention, a nucleic acid molecule encoding the light chain of a monoclonal antibody of the present invention may consist of the nucleotide sequence of SEQ ID NO: 21, and a nucleic acid molecule encoding the heavy chain of a monoclonal antibody of the present invention may consist of the nucleotide sequence SEQ ID NO: 22.

According to another exemplary embodiment of the present invention, a nucleic acid molecule encoding the heavy chain of a monoclonal antibody of the present invention may consist of the nucleotide sequence of SEQ ID NO: 23, and a nucleic acid molecule encoding the light chain of a monoclonal antibody of the present invention may consist of the nucleotide sequence SEQ ID NO: 24.

According to another exemplary embodiment of the present invention, a nucleic acid molecule encoding the light chain of a monoclonal antibody of the present invention may consist of the nucleotide sequence of SEQ ID NO: 27, and a nucleic acid molecule encoding its heavy chain may consist of the nucleotide sequence of SEQ ID NO: 27. 28.

The present invention also provides a recombinant vector containing a nucleic acid molecule encoding a heavy chain, a nucleic acid molecule encoding a light chain of a monoclonal antibody, or both of these nucleic acid molecules.

The recombinant vector system of the present invention can be constructed by various methods known in the art. Typically, the vector of the present invention can be constructed as a cloning vector or an expression vector. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as the host. For example, the vector of the present invention is an expression vector, and when using prokaryotic cells as a host, the vector usually contains a strong promoter capable of performing transcription (for example, tac promoter, lac promoter, lacUVS promoter, lpp promoter, pLλ promoter, pPλ promoter, rac5 promoter , amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter), ribosome binding site for translation initiation and transcription/translation termination sequences. When E. coli is used as a host cell (eg, HB101, BL21, DH5α, etc.), promoter and operator regions of the E. coli tryptophan biosynthesis pathway and the pLλ promoter can be used as regulatory regions. When Bacillus is used as the host cell, the Bacillus thuringiensis toxic protein gene promoter or any promoter capable of expression in Bacillus can be used as the regulatory region.

At the same time, the recombinant vector of the present invention can be obtained by manipulating a plasmid used in the field (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79 , pIJ61, pLAFRl, pHV14, pGEX series, pET series or pUC19), phage (eg λgt4 λB, λ-Charon, λΔz1 or M13) or virus (eg SV40).

When the vector of the present invention is an expression vector and eukaryotic cells are used as the host, the vector typically has a promoter derived from the genome of mammalian cells (for example, a metallothionein promoter, a β-actin promoter, a human hemoglobin promoter, or a 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, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, LTR HIV promoter (virus human immunodeficiency virus), Moloney virus promoter, Epstein-Barr virus (EBV) promoter, or Rous sarcoma virus (RSV) promoter), and a polyadenylation sequence as a transcription termination sequence.

The recombinant vector of the present invention can be fused to another sequence to facilitate purification of the antibody expressed from the recombinant vector. The fusion sequence may be, for example, glutathione S-transferase (Amersham Pharmacia Biotech, USA); maltose binding protein (NEB, USA); FLAG (IBI, USA); a tag sequence such as 6x His (hexahistidine; Qiagen, USA), Pre-S1 or c-Myc; or a leader sequence such as ompA or pelB. In addition, since the protein expressed from the vector of the present invention is an antibody, the expressed antibody can be easily purified using a Protein A column without an additional purification sequence.

At the same time, the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin or tetracycline.

The vector expressing the antibody of the present invention may be a vector system expressing both a light chain and a heavy chain using a single vector, or a vector system expressing a light chain and a heavy chain using two vectors, respectively. In the latter case, two vectors are introduced into host cells through cotransformation and targeted transformation. Cotransformation is a method of selecting cells expressing both a light chain and a heavy chain after introducing vector DNAs respectively encoding the light chain and the heavy chain into host cells. Targeted transformation is a method of selecting cells transformed with a vector containing a light chain (or a heavy chain), transforming the selected cells expressing a light chain with a vector containing a heavy chain (or a light chain), and finally selecting cells expressing both a light chain and a vector containing a light chain. chain and heavy chain.

The present invention also provides host cells containing a recombinant vector of the present invention. Host cells are cells transformed with the recombinant vector of the present invention. Host cells capable of stable and continuous cloning and expression of the vector of the present invention can be any host cells known in the art and include prokaryotic host cells, for example Bacillus sp., strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (eg Pseudomonas putidd), Proteus mirabilis or Staphylococcus (eg Staphylococcus carnosus), but the present invention is not limited to these host cells.

Suitable eukaryotic host cells for the vector include multicellular fungi such as strains of Aspergillus sp. belonging to the division Ascomycota and Neurospora crassa, and unicellular fungi containing enzymes such as yeasts such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces, other lower eukaryotic cells, higher eukaryotic cells such as cells of insect origin, and cells of plant or mammalian origin.

The term "transfection" as used herein refers to the introduction of a gene of interest into host cells using a recombinant vector of the present invention and is used in the same sense as "transformation". Thus, "transfection" and/or "transformation" of host cells can be carried out using suitable standard technology known in the art, in accordance with the host cells, including methods of introducing nucleic acid into the body, cells, tissue or organ. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, silicon carbide fiber agitation, Agrobacterium mediated transformation, PEG (polyethylene glycol) mediated transformation, dextran sulfate, lipofectamine and drying/inhibition, but the present invention is not limited to these methods.

The present invention also provides an epitope of the human Trx1 antigen consisting of any amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34 and 172-176.

The inventors confirmed that hTrx1 and CaTrx1 have 82% amino acid homology; the two types of anti-hTrx1 antibodies of the present invention do not bind to CaTrx1 (FIGS. 11 and 15A). Accordingly, eight parts in which the amino acid sequences of hTrx1 and CaTrx1 differ were identified (FIG. 15B), and gene cassettes for expression of hTrx1 mutant proteins were obtained for gene cloning (FIG. 15C-15F). The N293F cell line was transformed with cloned genes, the expression of 8 types of hTrx1 mutant proteins was confirmed (FIG. 16) and each mutant protein was purified (FIG. 17), followed by confirmation of the binding strength to the B266-1 antibody (hTrx1-hlgG1) and to the B264 antibody ( hTrx1-mlgG1).

As shown in FIG. 18A-18C, a decrease in binding of antibody B266-1 and mutant protein M4 (YSNVIFGNMV) compared to hTrx1 and binding of antibody B264 and mutant proteins Ml (QIESKTAEIEGKED), M2 (QEALDAHAALSS) and M4 compared to hTrx1 was confirmed. Thus, it was confirmed that antibodies B266-1 and B264 most likely share a common M4 binding site.

In addition, microarray analysis was carried out using 108 peptides obtained by overlaying the amino acid sequence of the hTrx1 protein with a shift of one amino acid residue (FIGS. 19 and 20), and the epigones of antibodies B266-1 and B264 were determined by heat map analysis, as shown in Table 25 (FIGS. 21A-21D and 22A-22F).

The present invention also provides a nucleic acid molecule encoding the above-described Trx1 antigen epitope, a recombinant vector containing said nucleic acid molecule, and a host cell containing said recombinant vector.

The Trx1 antigen epitope nucleic acid molecule of the present invention may consist of any amino acid sequence selected from the group consisting of SEQ ID NOs: 32-34 and 172-176.

Descriptions of a nucleic acid molecule encoding the above-described epitope, a recombinant vector containing said nucleic acid molecule, and a host cell containing said recombinant vector are the same as those relating to the antibody of the present invention given above and will therefore be omitted .

The present invention also provides a method for producing a monoclonal antibody that specifically binds to thioredoxin-1 or an antigen-binding fragment thereof, comprising culturing host cells.

Cultivation of host cells to obtain an antibody or an antigen-binding fragment thereof can be carried out in a suitable medium known in the art under culture conditions. One skilled in the art can easily adapt the culture technique to the strain used. Cell culture is classified as suspension culture or adherent culture according to the culture method, and as batch culture, fed culture or continuous culture according to the culture method. The medium used for cultivation must suitably meet the requirements for the specific strains.

The media used in culturing animal cells contains various carbon sources, nitrogen sources and trace elements. Examples of carbon sources used herein include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; lipids such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerin and ethanol; and organic acids such as acetic acid. These carbon sources can be used independently of each other or in combination. Examples of nitrogen sources used herein include organic nitrogen sources such as peptones, yeast extracts, meat broths, malt extracts, corn slurry (CSL) and soybean meal; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources can be used independently or in combination. The medium may contain potassium dihydrogen phosphate, potassium hydrogen phosphate and appropriate sodium salts as a source of phosphorus. In addition, the medium may contain metal salts such as magnesium sulfate or ferrous sulfate. In addition, amino acids, vitamins and suitable precursors may be included.

During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be suitably added to the cell culture to adjust the pH of the cell culture. In addition, bubble formation during culture can be reduced by adding an antifoam agent such as a polyglycol fatty acid ester. In addition, to maintain the aerobic state of the cell culture, oxygen or an oxygen-containing gas (for example, air) can be introduced into it. The cell culture temperature is usually from 20 to 45°C and preferably from 25 to 40°C.

The antibody obtained by culturing host cells can be used without purification or can be used through purification to a high degree of purity using various conventional methods such as dialysis, salt precipitation and chromatography. Of these methods, chromatography is the most widely used, and according to the characteristics of the antibody or culture method, the types and order of columns for ion exchange chromatography, size exclusion chromatography, or affinity chromatography can be selected.

According to the present invention, there is provided a kit for the diagnosis of breast cancer, containing a monoclonal antibody of the present invention or an antigen binding fragment thereof, and a method of providing information necessary for the diagnosis of breast cancer using it.

The term "diagnosis" as used herein refers to confirmation of the presence or sign of a pathological condition. For purposes of the present invention, diagnosis is performed to confirm whether breast cancer is present or not.

Thioredoxin-1 protein is a diagnostic marker for breast cancer, the expression of which is high in breast cancer tissue compared to normal breast tissue.

According to one exemplary embodiment of the present invention, the breast cancer diagnostic kit may be an enzyme-linked immunosorbent assay (ELISA) kit and preferably one or more selected from the group consisting of direct ELISA, indirect ELISA, direct sandwich ELISA and indirect sandwich ELISA. In a typical embodiment of the present invention, the two types of antibodies included in the sandwich ELISA kit include monoclonal antibody B266-1 as a coating antibody and monoclonal antibody B264 as a detection antibody.

The breast cancer diagnostic kit of the present invention may further contain, in addition to the anti-Trx1 antibody, an agent or reagent known in the art for use in an immunoassay.

Here, an immunoassay can be carried out by any of the methods that assess the binding of an antibody to an antigen. Such methods are known in the art and include, for example, Western blotting, ELISA, radioimmunoprecipitation, radial immunodiffusion, immunofluorescence assay, immunoblotting, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunochromatographic assay, FACS and analysis. on protein chips, but the present invention is not limited to these methods.

The agent or reagent used in the immunoassay may include a suitable carrier or support, a marker capable of producing a detectable signal, a solubilizer, a clearing agent, or a stabilizer. When the marker is an enzyme, suitable carriers include a substrate capable of measuring enzyme activity, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate or an arresting agent, but the present invention is not limited to these options.

The anti-Trx1 antibody included in the kit of the present invention is preferably fixed to a suitable carrier or support using various methods disclosed herein, and examples of suitable carriers and supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, agarose , cellulose, nitro -cellulose, dextrax, sephadex, separosis, liposoma, carboximethyl cellulose, polyacrylamide, polystyrene, gabbro, filter paper, ion exchange tar, plastic wrapper, plastic tube, potassium -polyminmethylvinylvinyl acid, and maleic acid, an amino -watering coper, and an amino acid cocker. ethylene and maleic acid, nylon , metal, glass, glass granule and magnetic particle. Other solid supports include cell culture plate, ELISA plate, tube and polymer film. The support can be any possible shape, such as spherical (pellet), cylindrical (test tube or well interior), or flat (sheet or test strip) shape.

A marker capable of producing a detectable signal allows qualitative or quantitative assessment of antigen-antibody complex formation and may be, for example, an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, or a radioisotope. The enzyme may be β-glucuronidase, β-D-glucosidase, urease, peroxidase (for example, horseradish peroxidase), alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase or luciferase. Fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin or fluorescein isothiocyanate can be used as the fluorescent substance. A biotin derivative can be used as a ligand, and acridinium ester or luciferin can be used as a luminescent substance. The microparticle may be colloidal gold or colored latex, and the redox molecule may be ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone or hydroquinone. ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I or ¹⁸⁶ Re can be used as a radioisotope. However, in addition to the substances listed above, any other substance that can be used in an immunoassay can be used.

As a chromogenic enzyme substrate, for example, when choosing horseradish peroxidase (HRP) as an enzyme marker, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine can be used , 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'tetramethylbenzidine, o-dianisidine or 3,3-dimethoxybenzidine. In addition, when choosing alkaline phosphatase as an enzyme marker, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium or n-nitrophenyl phosphate can be used as a substrate. In addition, when choosing p-D-galactosidase as an enzyme marker, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be used as a substrate. In addition to these, various other enzymes and chromogenic enzyme substrates known in the art may be used.

According to one typical embodiment of the present invention, the method of providing information necessary for the diagnosis of breast cancer according to the present invention can be carried out through the following steps:

(a) contacting any one type of monoclonal antibody of the present invention or an antigen binding fragment thereof with a biological sample isolated from a subject suspected of having breast cancer;

(b) measuring the level of expression of thioredoxin-1 protein that binds to a monoclonal antibody or an antigen-binding fragment thereof in said biological sample through the formation of an antigen-antibody complex; And

(c) comparing the level of thioredoxin-1 protein expression measured in step (b) with the control and, if the protein expression level is higher than the control, designating the subject as a patient with breast cancer.

According to another typical embodiment of the present invention, the method of providing information necessary for the diagnosis of breast cancer according to the present invention can be carried out through the following steps:

(a) coating a solid support with a monoclonal antibody or an antigen binding fragment thereof comprising a light chain CDR1-CDR3 and a heavy chain CDR1-CDR3 of an antibody B266 or B266-1, a monoclonal antibody or an antigen binding fragment thereof comprising a light chain variable region and a heavy chain variable region of the antibody B266 or B266-1, or a B266 or B266-1 antibody or an antigen-binding fragment thereof;

(b) applying a biological sample isolated from a subject suspected of having breast cancer onto a coated solid support;

(c) removing the unbound sample;

or its antigen-binding fragment onto a solid support;

(e) removing the unbound monoclonal antibody or antigen-binding fragment thereof;

(f) measuring the expression level of Trx1 protein; And

(g) comparing the level of Trx1 protein expression measured in step (e) with the control and, if the protein expression level is higher than the control, designating the subject as a patient with breast cancer.

The term "isolated biological sample" as used herein includes tissue (breast tissue), cells (breast cells), whole blood, plasma, serum, blood, saliva, synovial fluid, urine, sputum, lymph, cerebrospinal fluid, autopsy specimen tissue (brain, skin, lymph nodes, spinal cord or the like), cell culture supernatant or lysed eukaryotic cells, where the level of expression of the breast cancer marker protein Trx1 is different and includes a sample obtained from a primary lesion or a metastatic lesion . These biological samples, with or without previous manipulations, can be reacted with a monoclonal antibody of the present invention to confirm the level of expression of the Trx1 protein.

The term "subject" as used herein includes mammals, including cow, pig, sheep, chicken, dog and human, birds, etc., and, without limitation, any subject suspected of having breast cancer.

Hereinafter, the present invention will be described in detail with reference to examples to facilitate understanding of the present invention. However, the examples of the present invention can be modified into many different forms, and the following examples should not be construed as limiting the scope of the present invention. Examples of the present invention are provided to more fully explain the present invention to those skilled in the art.

EXAMPLES Example 1

Preparation of human thioredoxin-1 (Trx1) antigen

1-1. Preparation of the Trx1 expression vector

Based on codon usage in E. coli, a gene was synthesized to express the gene encoding the human thioredoxin-1 protein in E. coli. The sequence of the synthesized human thioredoxin-1 gene is shown in Table 1 below.

The sequence of primers used to amplify the human thioredoxin-1 gene is shown in Table 2 below.

table 2

Polymerase chain reaction (PCR) was performed to amplify the gene cloned into the plasmid. We mixed 10 pmol of the synthesized gene as a template, 10 pmol of each of the primers (hTrx1-For and hTrx1-Rev), dNTPs (2.5 mM each), taq polymerase Exprime and a buffer solution. This solution was reacted for 35 cycles at 95°C for 2 minutes, 95°C for 30 seconds, 55°C for 30 seconds and 70°C for 20 seconds with an additional reaction at 70°C for 2 minutes, after which the reaction was stopped. The amplified gene was purified and then to clone the EcoRY site present in the multiple cloning site (MCS) of plasmid pUC57, the plasmid was treated with an appropriate restriction enzyme and purified. The plasmid, treated with the purified gene and restriction enzyme, ligase and buffer solution were mixed and reacted. To transform E. coli DH5α with the resulting plasmid, a competent cell line of E. coli DH5α was heated at 4°C, mixed with the solution with which the plasmid was mixed, and reacted at 4°C for 30 minutes. After interaction, cells were heat shocked at 42°C for 30 seconds, stabilized at 4°C for 2 minutes, spread on solid Luria-Bertani (LB) medium containing antibiotic (50 μg/ml ampicillin) for uniform uptake and cultured at 37°C for 16 hours or more. Colonies grown on the culture medium were screened for a plasmid containing the human thioredoxin-1 gene.

1-2. Expression and purification of Trx1

The plasmid having the human thioredoxin-1 gene identified by screening was purified and then, for protein expression, E. coli strain BL21 was transformed with the purified plasmid according to the method described above. To express the thioredoxin-1 protein by the transformed strain, this strain was cultured in liquid LB medium containing the antibiotic to an OD ₆₀₀ (optical density at 600 nm) of 0.5 at 37°C, after which it was cultured for another 3 hours with the addition of isopropyl-β- D-thiogalactopyranoside (IPTG) to a concentration of 1 mM. SDS-PAGE was then performed to confirm protein expression. For protein purification, the resulting cell line was lysed using ultrasound and then centrifuged (12,000 rpm, 30 min, 4°C), thereby obtaining the supernatant. A commercially available anti-thioredoxin-1 antibody (LF-MA0055, Abfrontier) was added to the resulting supernatant to bind to expressed thioredoxin-1, and protein A/G agarose (Protein A/G PLUS-agarose, sc-2003, Santa Cruz) was added. , which contacted the antibody for their interaction, after which centrifugation and purification were carried out. The purity and molecular weight of the obtained product were then confirmed by SDS-PAGE.

Example 2

Preparation and purification of a monoclonal antibody specific for Trx1

2-1. Mouse immunization

Purified human thioredoxin-1 protein was mixed with adjuvant, then administered to mice (BALB/c), and mouse blood was collected and subjected to ELISA to confirm antibody formation. After two immunizations, the required increase in antibody titer was confirmed (1:5000).

2-2. Cell fusion and hybridoma production

B lymphocytes were isolated from the spleen obtained from an immunized mouse and fused with cultured myeloma cells (sp2/0). The fused cells were cultured in a medium (HAT medium) containing hypoxanthine, aminopterin and thymidine, and selective culture of cells (hybridoma) obtained from the fusion of only a myeloma cell and a B lymphocyte was carried out.

2-3. Selection of hybridoma cells producing Trx1-specific antibody

Using ELISA, three types of antibodies interacting with the human protein thioredoxin-1 were confirmed in the resulting hybridoma cells. The selection of a hybridoma that forms an antibody that interacts with the antigen from ELISA-positive cells was carried out using the limiting dilution method.

2-4. Preparation and purification of monoclonal antibody

The resulting three types of hybridomas were injected into mice, after which ascitic fluid was obtained from each mouse and purified using protein A affinity chromatography. The purified antibody was determined by SDS-PAGE.

Example 3

Monoclonal antibody isotype determination

The isotypes of the three antibodies obtained in Example 2 were confirmed using a rapid ELISA Mouse mAbs Isotyping Kit, Pierce, catalog no. 37503).

As a result, as shown in FIG. 1(b), it was confirmed that the heavy chain of monoclonal antibody 2 B4 is IgG1, the heavy chain of monoclonal antibody 8F3 is IgG2a, the heavy chain of monoclonal antibody 9G7 is IgG2b, and all the light chains are of kappa type.

Example 4

Analysis of amino acid sequences of monoclonal antibodies 9G7(AB1 and 2B4(AB2)

The amino acid sequences of the heavy chains and light chains of monoclonal antibodies 9G7(AB1) and 2B4(AB2) from three types of monoclonal antibodies obtained in Example 2 were analyzed. The amino acid sequence was determined as a sequence capable of fusion with the Fc region suitable for reverse translation and recombinant expression. The sequence determined by the gapped IMTG alignment was aligned and hypermutated, and full-length CDR3 regions were detected using a hypermutation table. Sequences were determined using precise peptide mass maps (FIGS. 2 and 3), and hypermutation and CDR3 were confirmed using MS/MS spectra.

Example 5

Affinity comparison and antibody detection using ELISA

In the amino acid sequence obtained in the manner described above, the position available for hypermutation was determined, and thus genes were synthesized by changing the amino acid sequences of four types (B266, B297, B268 and B269) of 9G7(AB1) and two types (B264 and B265) 2B4(AB2). The six types of antibodies produced above (B264-B269) were expressed and then the affinity of each antibody for the antigen was confirmed by ELISA (the numbers after the “T” in Tables 3-5 reflect the corresponding lot numbers received).

Affinities for three types of antigens, i.e., free Trx1, Fc-bound Trx1 (Trx1-Fc), and His-tagged Trx1 (Trx1-His), were determined by direct ELISA, and the results are shown sequentially in Tables 3-5. As shown in Tables 3-5, B264 in the form of IgG1(κ) and B266 in the form of IgG2b(κ) showed the highest affinity for the three types of antigens.

The amino acid sequences of high affinity antibodies B264 and B266 are shown in Table 6 below.

Example 6

Obtaining antibodies B264 and B266 6-1. Preparation of plasmids expressing antibodies B264 and B266

Since the amino acid sequences of antibodies B264 and B266 are determined as shown in Table 6, chemical synthesis of genes corresponding to the light chains and heavy chains of the corresponding antibodies is possible. The synthesized gene sequences are shown in Table 7 below. The synthesized genes were cloned into pcDNA3.0.

6-2. Expression and purification of antibodies B264 and B266

The HEK293 cell line was cotransfected with pcDNA3-SSJl 1-L and pcDNA3-SSJl1-H to express the B264 antibody or pcDNA3-SSJ12-L and pcDNA3-SSJ12-H to express the B266 antibody and cultured for 7 days. The cell line was cultured and the recombinant monoclonal antibodies secreted into the culture medium were collected and purified by Protein A chromatography. The eluent containing the recombinant monoclonal antibodies was concentrated by ultrafiltration and highly pure antibodies were obtained using a sterile 0.2 μm filter.

The purity and size of the purified antibodies were determined by SDS-PAGE. According to the results of SDS-PAGE, as shown in FIG. 6, the expression of antibodies B264 and B266 was confirmed with sizes of, for example, 47 kDa for the heavy chain and 25 kDa for the light chain under reducing conditions and 150 kDa under non-reducing conditions, indicating that the sizes of the expressed antibodies correspond to the calculated sizes.

Example 7

Confirmation of pairing of two types of monoclonal antibodies by sandwich ELISA

100 μl of coating buffer (0.015 M Na ₂ CO ₃ 0.035 M NaHCO ₃ , 0.003 M NaN ₃ , pH 9.6) and 100 ng of coating antibody (B266) were mixed, added to each well and an O/N reaction was performed (in overnight) at 4°C. 200 μl of PBS (phosphate buffered saline) containing 1% BSA (PBSA; blocking buffer) was added to each well and reacted at room temperature for 60 minutes. Then 20 μl of antigen (50, 25, 12.5 or 0 ng), 80 μl of detection antibody (B264, labeled with biotin; B264-B) were added and the resulting mixture was reacted at 37°C for 90 minutes. The reaction solution was removed and washed by adding 200 μl of PBS containing 0.05% Tween 20 (PBST; wash buffer) to each well. The steps described above were carried out three times.

100 μl of streptavidin-HRP, diluted 1:200, was added to each well, and the reaction was carried out at 37°C for 30 minutes. After removing the reaction solution, washing was carried out by adding 200 μl of PBS containing 0.05% Tween 20 (PBST; wash buffer) to each well. The steps described above were carried out three times.

100 μl of TMB (3,3',5,5'-tetramethylbenzidine) solution was added to each well and the reaction was carried out in the dark at room temperature for 10 minutes, 100 μl of 2.5 M sulfuric acid solution (H ₂ SO) was added ₄ ; stopping buffer) into each well and the result was confirmed at 450 nm.

As a result, as shown in Table 8, the interaction rate increases in accordance with the concentration of the antigen, demonstrating the detection of the antigen by these antibodies. However, due to the high OD in the absence of antigen, antibodies need to be experimented with to improve their performance.

Example 8

Changing the Fc isotype to improve antibody performance

Since the antibody expression system is a transient transfection using a recombinant plasmid rather than a hybridoma, among these recombinant plasmids, a plasmid having a heavy chain was cotransfected with a plasmid having a heavy chain of a different isotype. That is, cotransfection was carried out with a plasmid having a gene encoding a different heavy chain than pcDNA3-SSJ12H from pcDNA3-SSJ12-L and pcDNA3-SSJ12-H used for expression of 9G7(AB1).

Using the method described above, an antibody (B266-1) was obtained in which the Fc portion of B266 was replaced by the Fc of human IgG1. Antibody characteristics were determined by SDS-PAGE (FIG. 7).

The CDR sequences of the ultimately selected monoclonal antibodies B264 and B266-1 were determined by fusion to the Fc region suitable for reverse translation and recombinant expression.

The gapped IMTG alignment is an alignment of the IMTG database and a “defined sequence,” and the database search identified the closest germline sequence and hypermutation. The gapped IMTG alignment results obtained for the light chain and heavy chain of each of antibodies B266-1 and B264 are shown in FIG. 8A-8D, the amino acid sequences of the light chain CDR1 to CDR3 and the heavy chain CDR1 to CDR3 are shown in Table 9, and the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in Table 10. In addition, the amino acid and gene sequences of the light chain and heavy chain B266-1 circuits are shown in Table 11.

Example 9

Confirmation of pairing of monoclonal antibodies B266-1 and B264 obtained by sandwich ELISA

100 μl of coating buffer and 100 ng of coating antibody (B266-1) were mixed, added to each well and reacted O/N (overnight) at 4°C. Washing was carried out by adding 200 μl of washing buffer. The steps described above were carried out twice.

200 µl of PBSA was added to each well and reacted at room temperature for 120 minutes, and then 20 µl of antigen (25 or 0 ng) was added, 80 µl of detection antibody (B264-B) was added and reacted at 37°C for for 90 minutes. The reaction solution was removed and then washed by adding 200 μl of washing buffer to each well. The steps described above were carried out three times.

100 μl of streptavidin-HRP, diluted 1:200, was added to each well and the reaction was carried out at 37°C for 30 minutes, the reaction solution was removed and then washed by adding 200 μl of washing buffer to each well. The steps described above were carried out three times.

100 μl of TMB solution was added to each well, the reaction was carried out in the dark at room temperature for 10 minutes, 100 μl of stopping buffer was added to each well, and the result was confirmed at 450 nm.

As a result, as shown in Table 12, it was confirmed that the antibodies reacted appropriately with the antigens, and the value of the negative control was reduced compared to the antibodies used in Example 6.

Example 10

Monoclonal antibody antigen affinity assay

Two types of monoclonal antibodies specifically acting on the Trx1 antigen were expressed using a transient transfection system using a plasmid, which ensured their stable production. To confirm the affinity for the antigen, an ELISA assay was performed (FIG. 9(a)).

100 μl of coating buffer and 100 ng of Trx1 were mixed and added to each well, followed by reaction at 4°C for 16 hours or more. After removing the reaction solution, 200 μl of PBSA was added to each well and the reaction was carried out at 37°C for 120 minutes. After removing the reaction solution, the resulting antibody B266-1 or B264 was diluted 1/5 of 0.1 μM and added to each well in a volume of 100 μl, after which the interaction was carried out at 37°C for 120 minutes. After removing the reaction solution, washing was carried out by adding 200 μl of washing buffer to each well. The steps described above were carried out twice.

The reaction was carried out using 100 μl of anti-human IgG-HRP (1:4000 dilution) for antibody B266-1 and 100 μl of anti-mouse IgG-HRP (1:4000 dilution) for antibody B264 at 37°C for 60 minutes. After removing the reaction solution, washing was carried out by adding 200 μl of washing buffer to each well. The steps described above were carried out three times.

100 μl of TMB solution was added to each well, the reaction was carried out in the dark at room temperature for 10 minutes, 100 μl of stopping buffer was added to each well, and the result was confirmed at 450 nm. The resulting values were analyzed using Prism (Graphpad) (FIG. 9(b)).

The affinity analysis of the coating antibody B266-1 and the detection antibody B264 confirmed the high negative control value due to the reactivity of B266-1, but the degree of binding of B266-1 and B264 increases as the antigen concentration increases. This indicates binding of B266-1 and B264 to the antigen. When calculating the value of the equilibrium dissociation constant (K _D ) within the framework of the analysis carried out using the Prism program, K _D B266-1 was 1.1 × 10 ^-11 , and K _D B 264 was 1.3 × 10 ^-10 . With a K _D value of 10 ^-10 to 10 ^-12 , the antibody was assessed to have a picomolar (pM) level of sensitivity to the antigen, indicating a high level of sensitivity of B266-1 and B264 to the antigen.

Example 11

Sandwich ELISA of serum from a patient with breast cancer

Sandwich ELISA using coating antibody (B266-1) was performed as follows.

A coating antibody solution was prepared by mixing 100 ml of coating buffer and 0.1 ml of 1 mg/ml B266-1. 100 μl of the resulting coating antibody solution was added to each well of a 96-well plate and the reaction was carried out at 4°C for 12 hours. The antibody solution was removed and washed by adding 200 μl of 0.05% PBST to each well. Washing was carried out three times. 200 μl of PBSA was added to each well and the interaction (blocking process) was carried out at 4°C for 4 hours. PBSA was removed and then the 96-well plate was dried in a thermohygrostat (20°C, 30% relative humidity) for 3 hours.

The detection antibody (B264) was then biotinylated as described below.

Dimethyl sulfoxide (DMSO) was mixed with 20 mg/ml biotin-7-NHS to obtain 2 mg/ml biotin-7-NHS. 15 μl (30 μg) of 2 mg/ml biotin-7-NHS was added to antibody B264, 1 mg/ml, and reacted at 15-25°C for 2 hours. The reaction solution was added to AMICON Ultra-15 (Millipore), filled with PBS solution to the final volume and centrifuged at 3600 × g to a residual volume of 0.5 ml. These actions were carried out three times. The antibody solution (biotinylated B264; B264-B) remaining in the AMICON filter was transferred to a 1.5 mL tube and filled with PBSA to a final concentration of 0.3 mg/mL.

Detection of human Trx1 antigen in breast cancer patient serum was then performed as described below.

A standard antigen solution was added to the first column of a 96-well plate with a coating antibody. 20 μl of serum obtained from a patient with breast cancer was added, followed by 80 μl (0.3 mg/ml) of B264-B solution. Then, after reacting at 37°C for 60 minutes, the antigen-antibody reaction solution was removed, followed by washing with 200 μl of PBST in each well. Washing was carried out three times. 100 μl of streptavidin-HRP (R&D Systems) was added at a dilution of 1:400 and the reaction was carried out at 37°C for 30 minutes. After the reaction, the reaction solution was removed and washed by adding 200 μl of PBST to each well. Washing was carried out three times. 100 μl of TMB solution (Sure Blue) was added and the reaction was carried out at room temperature for 15 minutes in the dark. Added 100 μl of 2 N. H ₂ SO ₄ solution and measured the absorbance at 450 nm using a microplate reader.

Finally, ROC analysis was performed as described below.

Sensitivity and specificity were calculated by analyzing the ELISA result using monoclonal antibodies B266-1 and B264 against Trx1. At a cut-off value of 10.8 ng/ml, the sensitivity was 93.0%, and the sensitivity was 97.4% (FIG. 10).

Example 12

Comparative analysis using another ELISA kit for breast cancer diagnosis

In this example, to evaluate the performance characteristics of recombinant monoclonal antibodies B266-1 and B264, a comparative analysis was performed with another ELISA kit to detect another biomarker CA15-3 for the diagnosis of breast cancer (Table 13).

As a result, as shown in Table 13, when using monoclonal antibodies that specifically bind to Trx1, the sensitivity and specificity were much higher than those of CA15-3.

Example 13

Expression of Chrysochloris asiatica Trx1 protein

13-1. Sequence comparison of human Trx1 (hTrx1) and Chrysochloris asiatica Trx1 (CaTrx1)

By comparing the amino acid sequences of hTrx1 and Chrysochloris asiatica Trx1, which are structurally similar but have low amino acid sequence similarity, they were confirmed to have 82% homology (FIG. 10).

Using the known base sequence of CaTrx1 (NCBI accession number XM 006863001.1), the gene was synthesized and cloned into the pUCIDT-AMT plasmid for storage in E. coli. The plasmid for gene cloning was treated with restriction enzymes Sfi I and Xho I, followed by electrophoresis. As a result, as shown in FIG. 12, in the plasmid treated with restriction enzymes (lane 2), a DNA fragment of 357 bp in size was identified, cleaved from the plasmid, indicating successful synthesis of the CaTrx1 gene (arrow in FIG. 12).

13-2. CaTrx1 protein expression

After transfecting an animal cell with the CaTrx1 plasmid obtained in Example 13-1, CaTrx1 secreted by the cell line was purified and the protein was confirmed by 15% SDS-PAGE; the result is shown in FIG. 13. In FIG. 13 in lanes 1 and 2, the total amount of protein in the cell culture solution of the CaTrx1 transformant was 5 μg and 10 μg, respectively; in lane 3, the amount of control protein (BSA; bovine serum albumin) was 3 μg, confirming that the productivity of the purified CaTrx1 protein was 28.75 mg/ml.

Example 14

Confirmation of the affinity of two types of antibodies for hTrx1 and CaTrx1

In this example, the binding affinity of two types of antibodies, B266-1 (Trx1-hlgG1) and B264 (Trx1-mlgG1), to CaTrx1 was analyzed.

To confirm the binding affinity of B266-1 (Trx1-hIgG1) and B264 (Trx1-mlgG1) to hTrx1 and CaTrx1, a 96-well ELISA plate was coated with 200 ng of hTrx1 or 10 μg of CaTrx1, and 200 μl of blocking buffer was added to each well (4 % skim milk / 1x PBS) followed by reaction for 1 hour at 37°C. After removing the reaction solution, 100 μl of each of B266-1 (Trx1-hIgG1) and B264 (Trx1-mlgG1) was added to each well coated with the appropriate antigen and allowed to react at 37°C for 2 hours. The reaction solution was removed, followed by washing five times with 200 μl lx PBST. In each of the wells treated with B266-1 (Trx1-hIgG1) or B264 (Trx1-mlgG1), 100 μl of anti-human Fc antibody conjugated to HRP and anti-mouse antibody conjugated to HRP were added at a dilution of 1:4000, respectively. , followed by interaction at 37°C for 2 hours. The reaction solution was removed and washed five times using 200 µl lx PBST. 100 μl of the staining reagent was added to each well, and after 10 minutes of interaction, 50 μl of 2.5 M H ₂ SO ₄ was added to each well. After color development, the degree of staining was assessed using an ELI S A-plate reader.

By confirming the binding affinity of B266-1 (Trx1-hIgG1) and B264 (Trx1-mlgG1) to hTrx1, as shown in Table 14 below, for 200 ng of antigen, it was confirmed that the K _D for B266-1 was 2.1 x 10 ^-10 M, and the affinity for B264 was determined at a K _D of 1.7×10 ^-10 M. In addition, as can be seen in FIG. 14A, compared to B266-1, when bound to B264, the interaction rate (OD ₄₉₀ ) was lower and amounted to approximately 50%.

By confirming the binding affinity of antibodies B266-1 and B264 with 10 μg of CaTrx1, as shown in FIG. 14B and Table 14, it was observed that neither of these two types of antibodies bind to CaTrx1.

Example 15

Preparation of mutant hTrx1 antigen

15-1. Determining the location of mutations by amino acid sequencing of hTrx1 and CaTrx1

The known amino acid sequence of hTrx1 (NCBI accession number NP 003320.2) was compared with the amino acid sequence of CaTrx1 (NCBI accession number ХР_006863063.1). As shown in FIG. 15A, although the amino acid sequence homology of hTrx1 and CaTrx1 was 82%, the binding affinity of the antibodies to the antigen varied significantly, and thus there were 8 different parts in which hTrx1 and CaTrx1 have different amino acid sequences (FIG. 15B).

15-2. PCR fusions and cloning for expression of mutant proteins

A) PCR fragments

In the 8 portions in which the amino acid sequences of hTrx1 and CaTrx1 differ, determined in Example 15-1, the hTrx1 sequence was replaced with the CaTrx1 sequence, and then the cassette DNA fragment was amplified to obtain a mutant (FIG. 15D).

Specifically, to obtain the gene for expression of each mutant protein, it is necessary to amplify two DNA fragments for fusion PCR. Thus, two types of primers (F2 and R1; FIG. 15C and Table 15) containing the portion requiring base sequence mutation were prepared, and DNA fragments were amplified using two pairs of primers, F1 with R1 and F2 with R2. To amplify DNA fragments, template DNA (100-200 ng) and 1 µl of each of the forward and reverse primers (10 pmol each) were added to 25 µl of a mixture of 2 x EF-Taq PCR Smart (0.5X Band Doctor) (Solgent, SEF02 -M50h), brought to the final volume with sterile purified water, after which they were thoroughly mixed and amplified using a PCR device (T100 thermal cycler).

PCR was carried out under conditions of 1 cycle of preliminary denaturation at 95°C for 2 min, 30 cycles of 3-step amplification at 95°C for 20 sec, 62°C for 40 sec and 72°C for 1 min and 1 cycle final extension at 72°C for 5 min, after which the reaction was stopped. The amplified DNA fragment was confirmed using a 1% agarose gel (FIG. 15D). Gene purification was performed using the QIAquick Gel Extraction Kit (QIAGEN, 28704) following the manufacturer's protocol.

B) Fusion PCR to fuse two types of DNA fragments and purify the PCR product

To fuse the amplified DNA fragments, PCR was performed using two DNA fragments and primers F1 and R2 (FIG. 15C). The PCR mixture for fusion PCR was prepared by adding 100 to 150 ng of each of the two types of DNA fragments and 1 μl of each of the forward and reverse primers (10 pmol each) to 25 μl of a mixture of 2 x EF-Taq PCR Smart (0.5X Band Doctor (Solgent, SEF02-M50h), made up to final volume with sterile purified water, then mixed thoroughly and amplified using a PCR device. PCR was carried out under conditions of 1 cycle of preliminary denaturation at 95°C for 2 min, 30 cycles of 3-step amplification at 95°C for 20 sec, 62°C for 40 sec and 72°C for 1 min and 1 cycle final extension at 72°C for 5 min, after which the reaction was stopped. After the reaction stopped, the PCR product was confirmed using a 1% agarose gel (FIG. 15E).

Upon completion of the fusion PCR, the resulting PCR product was purified using ethanol precipitation. 3 M sodium acetate (pH 5.2) and 100% ethanol were added to the amplified PCR product in an amount of 1/10 and 2 of the total volume of the PCR product, respectively, mixed thoroughly and the interaction was carried out at -70°C in a low-temperature freezer at for 10 minutes. After this, the resulting mixture was centrifuged at 13,000 rpm for 10 minutes, the supernatant was removed, 1 ml of 70% ethanol was added, and then the resulting mixture was stirred, followed by centrifugation at 13,000 rpm for 10 minutes. The supernatant was removed, the remaining ethanol was removed by reacting in a thermostat at 70°C for 3 minutes, and the precipitated DNA was thoroughly dissolved in 50 ml of distilled water.

C) Cloning the PCR product

To clone the purified PCR product into the N293F plasmid, restriction enzyme treatment was performed. Specifically, 7 μl of Kpn I and 8 μl of 10x buffer were added to 50 μl of PCR product and plasmid N293F and the total volume was adjusted to 80 μl. After thorough stirring, the resulting mixture was reacted at 37°C in a water bath for 3 hours. Upon completion of the reaction, the resulting mixture was purified using ethanol precipitation. After this, the purified mixture was treated with 7 μl of Xho I and 8 μl of 10x buffer and the total volume was adjusted to 80 μl. After thorough mixing, the resulting product was reacted at 37°C in a water bath for 3 hours. To purify post-reaction DNA, an experiment was performed using the QIAquick Gel Extraction Kit (QIAGEN, 28704) following the manufacturer's protocol.

To clone a purified DNA fragment into N293F, 20 ng of N293F treated with DNA fragment (100 ng for 1 kb (kb) or less, 300 ng for 3 kb or less) and restriction enzymes, treated with 1 μl of T4 DNA ligase (Thermo Scientific, EL0011) and added 1 μl of 10x buffer and adjusted the total volume to 10 μl with distilled water. The resulting mixture was reacted for 16 hours at 22°C. Upon completion of the interaction, competent E. coli DH5 cells to be transformed were isolated and dissolved on ice. 2 μl of the ligation product was thoroughly mixed with competent DH5α cells and then reacted on ice for 30 minutes. The reaction product was then reacted at 42°C in a water bath for 90 seconds and further reacted on ice for 3 minutes. 500 μl of SOC medium (20 g Bacto tryptone, 5 g Bacto yeast extract and 0.5 g NaCl per liter) was added to the reaction product and incubated at 37°C in a shaker incubator for 30 minutes. After incubation, 100 μl of the reaction product was nebulized and distributed into LB medium supplemented with 100 μg/ml ampicillin (10 g Bacto tryptone, 5 g Bacto yeast extract and 10 g NaCl per liter) and incubated at 37°C in an incubator for 12 16 hours.

D) Colony PCR and sequencing to confirm transformation

Colony PCR was performed to confirm the presence or absence of the cloning plasmid. The PCR mixture for fusion PCR was prepared by adding 0.5 µl of each of the forward and reverse primers (10 pmol each) to 12.5 µl of a mixture of 2 x EF-Taq PCR Smart (0.5X Band Doctor) (Solgent, SEF02- M50h), brought to the final volume with sterile distilled water, after which they were thoroughly mixed and amplified by PCR. PCR was carried out under conditions of 1 cycle of preliminary denaturation at 95°C for 2 min, 25 cycles of 3-step amplification at 95°C for 20 sec, 62°C for 40 sec and 72°C for 1 min and 1 cycle final extension at 72°C for 5 min, after which the reaction was stopped.

After the reaction stopped, the amplified product was confirmed using a 1% agarose gel (FIG. 15F). The amplified product was purified and Neoprobe Corp. was used for sequencing. The data obtained from sequencing is shown in Table 16 below. Mutated sequences are highlighted in underlined and bold font.

E) Obtaining plasmids (Midi acquisition)

Colonies containing sequenced plasmids were inoculated in 100 ml of 2 x YT medium (17 g tryptone, 10 g yeast extract and 5 g NaCl per liter) containing 100 μg/ml ampicillin and incubated at 37°C and 210 rpm for for 16 hours. Incubated bacteria were obtained by centrifugation at 4500 rpm for 8 minutes. NucleoBond® Xtra Midi (Macherey-Nagel, cat. no. 740410.100) was used to obtain the purified plasmid and the experiment was performed following the manufacturer's protocol.

F) Animal cell culture

19.4 g of Freestyle™ 293 Expression Medium AGT™ powder (AG100009, Thermo Scientific) was dissolved in 1 L of deionized water and sterilized. 35 ml of Freestyle™ 293 Expression Medium AGT™, which was heated in a water bath at 37°C for 30 minutes, was placed in a 125 ml Erlenmeyer flask (CC-431143, Corning). After thawing the frozen 293F cell line (510029, Invitrogen) in a 37°C water bath for approximately 1-2 minutes, the thawed cell line was mixed with 5 ml of Freestyle™ 293 Expression Medium AGT™ and added to an Erlenmeyer flask containing 35 ml of medium. , followed by incubation in a shaker incubator at 8% CO ₂ , 37°C and 85 rpm. After 2-3 days of incubation, 10 μl of the cell line was mixed with 10 μl of trypan blue, 10 μl of the resulting mixture was added to the Luna cell counting chip (L12002, Biosystems) and cell viability and number were confirmed using a Luna™ automatic cell counter (L10001, Biosystems ). After suspending 4-7×10 ⁵ cells/ml in 40 ml of medium, the resulting suspension was centrifuged at 100×g for 5 minutes to remove the supernatant. After removing the supernatant, the cell pellet was mixed with 10 ml of medium to resuspend the pellet, and then 30 ml of medium was added to a 125 ml Erlenmeyer flask. The cells were incubated in a shaker incubator at 8% CO ₂ , 37°C and 85 rpm and the above steps were repeated two or more times.

G) Transfection of animal cells

An aliquot of cells, 5.5×10 ⁵ cells/ml, with a volume of 40 ml was placed in a tube and centrifuged at 100×g for 5 minutes. After removing the culture solution, the pellet was suspended using 10 ml of Freestyle™ 293 Expression Medium AGT™ and added to a 125 ml Erlenmeyer flask. Cells were incubated in a shaker incubator at 8% CO ₂ , 37°C and 85 rpm. Using a Luna™ automated cell counter, cell count and viability were confirmed to be 1×10 ⁶ cells/mL and 90% or more, respectively. Based on a culture solution volume of 40 mL, 25 μg of transfection DNA and 100 μg of PEI (23966, Polysciences) were vortexed, followed by centrifugation at 10,000 rpm for 1 second. DNA and PEI mixed with 800 µl of Freestyle™ 293 Expression Medium AGT™ were mixed and allowed to react at room temperature for 20 minutes. After interaction, the DNA-PEI mixture was added to a 125 ml flask in which the cell line was incubated. After 24 hours, additives were added up to 5 g/l. After this, the cells were further incubated for 5 days and the culture solution was collected.

H) Experiment to confirm expression in culture medium

After 5 days of cultivation, 500 μl of the selected culture solution was added to test tubes. One of the tubes was placed in a centrifuge tube rack for 20 minutes, the supernatant (the sample that was not centrifuged) was used, and the other tubes were centrifuged at 10,000 rpm for 2 minutes to remove cells and only the supernatant (the sample that was centrifuged) was used. 10 μl of 5x sample recovery buffer was mixed with 40 μl of supernatant, followed by boiling at 100°C for 5 minutes. The resulting sample was confirmed by 15% SDS-PAGE using Mini- ^PROTEAN® Tetra Cell (BR165-8029, Bio-Rad) (FIG. 16).

I) Purification using affinity chromatography (Ni-NTA)

The transformed cell line was incubated for 6 days and centrifuged at 4800 rpm for 30 minutes to remove the supernatant. The PolyPrep column (731-1553, Bio-Rad) was washed with 10 mM imidazole wash buffer (pH 7.4) and packed with Ni-Sepharose™ 6 Fast Flow beads (17-5318-02, GE Healthcare). The column was then washed with wash buffer containing 10 mM imidazole (pH 7.4) twice. When approximately 2-3 ml of 10 mM imidazole wash buffer (pH 7.4) remained in the column, the column was washed again with 20 ml of 10 mM imidazole wash buffer (pH 7.4). Medium was added to the washed column. The beads were washed with wash buffer containing 10 mM imidazole (pH 7.4) and eluted with elution buffer containing 500 mM imidazole (pH 7.4). 10 µl of sample was mixed with 200 µl of Coomassie Plus™ Protein Detection Reagent (1856210, Thermo Scientific) and eluted until the sample turned blue. The eluted protein purification solution was concentrated using an Amicon® ultracentrifuge (UFC901096, Millipore) and the buffer was exchanged, repeating the reconcentration with PBS solution at least twice. Protein concentration was determined using Nano-drop and diluted to 0.3-0.5 mg/ml. 3 μg of each protein was confirmed by SDS-PAGE (FIG. 17).

In addition, the concentrations and productivity of 8 types of hTrx1 mutant proteins were evaluated, and the results obtained are shown in Table 17 below. Table 17 shows that 8 types of hTrx1 mutant proteins are expressed in the concentration range from 3.15 to 5.31 mg/ml.

Example 16

ELISA to confirm binding affinity

In this example, the binding affinity of each of the B266-1 and B264 antibodies to the 8 types of hTrx1 mutant proteins obtained in Example 15 was confirmed.

8 types of mutant hTrx1 proteins obtained in Example 3 were dissolved in coating buffer (DPBS; LB001-02, Welgene) at a concentration of 2 μg/ml, thus obtaining antigen solutions, the solution of each antigen was added to a 96-well plate in 100 μl The well and plate were covered with sealing tape, followed by interaction at 4°C for 16 hours. After removing the antigen solution, 200 μl of blocking buffer (1 x PBS with 4% skim milk) was added to each well and the plate was covered with sealing tape, followed by interaction in an incubator at 37°C for 1 hour. Upon completion of the interaction, the blocking buffer was removed, 100 μl of an antibody solution diluted to a certain concentration was added to each well, and the plate was covered with sealing tape, followed by interaction in an incubator at 37°C for 2 hours. The antibody solution was removed and the process of adding and removing 200 μl of wash buffer solution (1 x PBST) per well was repeated a total of 5 times. HRP-conjugated antibodies (HRP-conjugated anti-human Fc, anti-B266; HRP-conjugated anti-mouse Fc, anti-B264) were diluted 1:4000 in antibody dilution solution (1 x PBS with 1% stripped milk), 100 μl of the resulting solution was added to each well and the plate was covered with sealing tape, followed by interaction in an incubator at 37°C for 2 hours. The antibody solution was removed and the process of adding and removing 200 μl of wash buffer solution (1 x PBST) was repeated a total of five times. 10 µl H ₂ O ₂ was added to the staining reagent (one OPD (orthophenylenediamine) tablet, 10 ml PC buffer (5.1 g C ₆ H ₈ O ₇ ⋅H ₂ O and 7.3 g Na ₂ HPO ₄ per liter)) and then 100 μl of the resulting mixture was added to each well, followed by interaction in the dark for 10 minutes. 50 μl of stopping buffer (2.5 M H ₂ SO ₄ ) was added to each well and OD was measured at 490 nm.

As a result, as shown in FIG. 18A, it was confirmed that antibody B266-1 (hTrx1-hIgG1) had a reduced binding strength to a protein having an M4 site mutation (YSNVIFGNMV), and antibody B264 (hTrx1-mlgG1) had a reduced binding strength to a protein having a site mutation M1, M2 and M4 (M1: QIESKTAEIEGKED; M2: QEALDAHAALSS; and M3: YSNVIFGNMV).

Tables 18 and 19 below show the original amino acid sequences and base sequences of the M1, M2 and M4 sites mutated in hTrx1.

Based on the above results, it was confirmed that antibody B266-1 and antibody B264 likely share the M4 portion (YSNVI) of the antigen binding site.

Example 17

Antibody Profiling Using Peptide Microarrays

In this example, the exact amino acid sequence was determined by epitope mapping analysis of the Trx1 antigen using antibodies B266-1 and B264. Specifically, PepStar™ peptide microarray technology (JPT Peptide Technologies (Germany)) was used, as shown in FIG. 19, and epitopes were identified using overlapping peptide scanning.

17-1. Sequences

Antibody profiling experiments were performed using a peptide library consisting of 108 peptides. The complete list of peptides is shown in Tables 20-22 below. Here, SEQ ID NO: 64-81, corresponding to peptides 001-018 (Peptide OOl to Peptide_018), are not native forms and contain regions of recombinant inserts. The sequence with GenBank accession number AAF87085.1 was used as the known amino acid sequence of the hTrx1 protein.

Full-length mouse IgG was also immobilized onto the microarray panel as an analytical control, and additional sequence was included in the peptide library via JPT as an internal process control.

17-2. Analysis conditions

Profiling experiments were performed using a total of two antibody samples (B266-1 and B264) diluted in blocking buffer (Pierce International, Superblock TBS T20, order number 37536). Serial dilutions of 5, 1, 0.2, 0.04, 0.008 and 0.0016 μg/ml were incubated on one multiwell microarray panel at 30°C for 1 hour. The panel contains 21 separate miniarrays (one miniarray for each sample dilution).

After incubation with the samples, 1 μg/ml of fluorescently labeled secondary anti-mouse IgG antibody (anti-mouse IgG(H+L) (Thermo 84545)) was added to the appropriate wells, followed by interaction for 1 hour. The label used was DyLight 650. False-positive binding to peptides was assessed by performing one additional control incubation in which only the secondary antibody was applied to the same microarray panel. Before each step, microarrays were washed with washing buffer.

After washing and drying, the panel was scanned using a high-resolution 635 nm laser scanner (Axon GenePix Scanner 4300 SL50) to obtain fluorescence intensity profiles, and the resulting image was quantified using GenePix spot detection software, calculating the average pixel value for each peptide. For each point, the average signal intensity was obtained (light units from 0 to 65535).

17-3. Image of processed matrices

A typical image showing the fluorescence of the miniarray cultured with one of the antibody samples is shown in FIG. 20. Low background values were observed for all samples. Black indicates no signal, a red shadow indicates an increase in the intensity of the detected signal, white indicates saturation of the detector, and each individual subarray is highlighted in green.

17-4. Heat Map Analysis

Heat map plots were calculated to visualize the results and compare binding regions for individual crops, as shown in FIG. 21A-21D. In FIG. 21A-21D the fluorescence intensity is shown by color code, white indicating no binding and red indicating strong binding. In all analyses, the MMC2 value of the average pixel fluorescence was calculated for each peptide.

MMC2 is the average of all three points on the microarray unless the coefficient of variation (CV), which is the ratio of the standard deviation to the mean, is greater than 0.5. In this case, the average of the two closest values (MC2) is taken as MMC2. The thick black line in the heat map shows the control culture using only the anti-mouse IgG secondary antibody. Cultures of individual antibody samples are indicated by a thin blue line.

In the case of antibody B266-1, as shown in Table 23, the highest signal, approximately 8 times the average background level, was detected for peptides 004 and 005 (Peptide_004 and Peptide_005) (SEQ ID NO: 67 and 68). However, since peptides 004 and 005 are not native forms, these peptides were excluded from the group of possible epitopes.

Antibody B264 showed a concentration-dependent signal profile and very strong interactions with some peptides. The most significant binding was obtained with the peptides listed in Table 24 below, particularly at the two highest sample concentrations.

As shown in Table 24, the highest signal, approximately 7 times the average background level, was measured for peptides 012 and 018 (Peptide_012 and Peptide_018) (SEQ ID NO: 75 and 81). However, since peptides 012 and 018 are not native forms, they were excluded from the group of possible epitopes.

Subsequently, peptides SEQ ID NO: 82-88, corresponding to peptides 019-025 (Peptide_019 to Peptide_025), for which the strongest signal was measured, were expected to be the binding sites of antibody B264, and finally, the epitope of antibody B264 was determined to be "VKQIESKTAFQEALDAAGDKL" (SEQ ID NO: 179).

Peptides SEQ ID NO: 109-120, corresponding to peptides 046-057 (Peptide_046 to Peptide_057), for which the strongest signal was measured, were subsequently expected to be binding sites for antibody B264, which has the same epitope as the binding site for B266-1 .

No significant binding was detected in the control secondary antibody culture. Strong signals, up to saturation levels, were obtained from the full-length mouse IgG challenge in all cultures, indicating excellent assay performance.

The epitope regions obtained by the method described above are shown in Table 25 below, and from the results of 3D modeling with confirmation of tertiary (3D) structures by loading the hTrx1 NMR sequence confirmed against the Protein Database (PDB) as shown in FIG. 22A-22F, in relation to native forms, their sequences are located on the surface of these structures, which confirms the possibility of these peptides functioning as epitopes.

INDUSTRIAL APPLICABILITY

The monoclonal antibody of the present invention can bind very specifically to Trx1 due to its excellent binding affinity and can be effectively used in screening for breast cancer patients due to its exceptionally high sensitivity and specificity. In addition, the accuracy and reliability of breast cancer diagnosis can be significantly improved since detection by the monoclonal antibody of the present invention specifically binding to Trx1, rather than detection by CA15-3, another conventional diagnostic biomarker for breast cancer, exhibits exceptionally high sensitivity and specificity. The epitope region of the human Trx1 antigen to which the antibody of the present invention binds can be effectively used in the development of an improved antibody to increase the binding affinity of the anti-Trx1 antibody.

--->

SEQUENCE LISTING

<110> E&S Healthcare Co., Ltd.

<120> Thioredoxin-1 epitope and monoclonal antibody, specific

contacting him

<130> 1064685

<150> KR 10-2017-0132536

<151> 2017-10-12

<160> 181

<170> Patent In version 3.2

<210> 1

<211> 11

<212>PRT

<213> Artificial

<220>

<223> CDR1 light chain B264

<400> 1

Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 2

<211> 3

<212>PRT

<213> Artificial

<220>

<223> CDR2 light chain B264

<400> 2

Lys Val Ser

1

<210> 3

<211> 10

<212>PRT

<213> Artificial

<220>

<223> CDR3 light chain B264

<400> 3

Cys Phe Gln Gly Ser His Val Pro Tyr Thr

1 5 10

<210> 4

<211> 8

<212>PRT

<213> Artificial

<220>

<223> Heavy chain CDR1 B264

<400> 4

Gly Tyr Thr Phe Thr Ser Tyr Thr

15

<210> 5

<211> 9

<212>PRT

<213> Artificial

<220>

<223> Heavy chain CDR2 B264

<400> 5

Ile Asn Pro Thr Ser Asp Tyr Thr Asn

15

<210> 6

<211> 14

<212>PRT

<213> Artificial

<220>

<223> Heavy chain CDR3 B264

<400> 6

Phe Cys Ala Ser Glu Gly Gly Phe Leu Tyr Tyr Phe Asp Tyr

1 5 10

<210> 7

<211> 5

<212>PRT

<213> Artificial

<220>

<223> CDR1 light chain B266-1

<400> 7

Ser Arg Ile Ser Tyr

15

<210> 8

<211> 3

<212>PRT

<213> Artificial

<220>

<223> CDR2 light chain B266-1

<400> 8

Asp Thr Ser

1

<210> 9

<211> 10

<212>PRT

<213> Artificial

<220>

<223> CDR3 light chain B266-1

<400> 9

Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

1 5 10

<210> 10

<211> 8

<212>PRT

<213> Artificial

<220>

<223> Heavy chain CDR1 B266-1

<400> 10

Gly Phe Asn Ile Lys Asp Thr Phe

15

<210> 11

<211> 8

<212>PRT

<213> Artificial

<220>

<223> Heavy chain CDR2 B266-1

<400> 11

Ile Asp Pro Ala Asn Gly Asn Thr

15

<210> 12

<211> 11

<212>PRT

<213> Artificial

<220>

<223> Heavy chain CDR3 B266-1

<400> 12

Cys Ala Leu Leu Gln Tyr Ser Ala Met Asp Tyr

1 5 10

<210> 13

<211> 112

<212>PRT

<213> Artificial

<220>

<223> Light chain variable region B264

<400> 13

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 14

<211> 119

<212>PRT

<213> Artificial

<220>

<223> Heavy chain variable region B264

<400> 14

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Thr Ser Asp Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Ser Glu Gly Gly Phe Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 15

<211> 105

<212>PRT

<213> Artificial

<220>

<223> Light chain variable region B266-1

<400> 15

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Arg Ile Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 16

<211> 117

<212>PRT

<213> Artificial

<220>

<223> Heavy chain variable region B266-1

<400> 16

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Phe Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Leu Gln Tyr Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 17

<211> 219

<212>PRT

<213> Artificial

<220>

<223> Light chain B264

<400> 17

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 18

<211> 443

<212>PRT

<213> Artificial

<220>

<223> Heavy chain B264

<400> 18

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Thr Ser Asp Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Ser Glu Gly Gly Phe Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Thr Pro Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 19

<211> 212

<212>PRT

<213> Artificial

<220>

<223> Light chain B266

<400> 19

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Arg Ile Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr

100 105 110

Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala

115 120 125

Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val

130 135 140

Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser

145 150 155 160

Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr

165 170 175

Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys

180 185 190

Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn

195 200 205

Arg Asn Glu Cys

210

<210> 20

<211> 452

<212>PRT

<213> Artificial

<220>

<223> Heavy chain B266

<400> 20

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Phe Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Leu Gln Tyr Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu

115 120 125

Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys

130 135 140

Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser

145 150 155 160

Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser

165 170 175

Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp

180 185 190

Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr

195 200 205

Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn

210 215 220

Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu

225 230 235 240

Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val

245 250 255

Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val

260 265 270

Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val

275 280 285

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

290 295 300

Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met

305 310 315 320

Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser

325 330 335

Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro

340 345 350

Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp

355 360 365

Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser

370 375 380

Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr

385 390 395 400

Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu

405 410 415

Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn

420 425 430

Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser

435 440 445

Arg Ser Pro Gly

450

<210> 21

<211> 660

<212> DNA

<213> Artificial

<220>

<223> Light chain B264

<400> 21

gacgtgctga tgacacagac accactcagc ctccctgtga gcctgggcga ccaggcctct

60

atttcttgcc ggtctagcca gagcatcgtg cactccaacg gcaacacata cttggagtgg

120

tatctacaga agcccggcca gtcccctaag ctgctgatat acaaggtgtc taaccgcttc

180

tccggcgtgc ccgacaggtt ctctggcagc ggctctggca ccgacttcac cctcaaaata

240

tctagggtgg aggccgagga cctgggcgtg tactactgct tccagggctc ccacgttcca

300

tacacattcg gcggcggcac aaagttggaa attaagcgcg ctgacgcagc cccaacagtg

360

agcatctttc ctccatcctc tgaacaactt acctctggag gagcctctgt ggtgtgtttc

420

ctgaacaact tctacccaaa ggacatcaat gtgaagtgga agattgatgg ctctgagaga

480

cagaatggag tgctgaactc ctggacagac caggacagca aggacagcac ctacagtatg

540

agtagcaccc tgaccctgac caaggatgaa tatgagagac acaactccta cacttgtgag

600

gctacccaca agaccagcac cagcccaatt gtcaaatcct tcaacaggaa tgagtgttaa

660

<210> 22

<211> 1332

<212> DNA

<213> Artificial

<220>

<223> Heavy chain B264

<400> 22

caggtgcagc tccagcagtc cggcgccgaa ctggccagac ctggcgccag cgtgaagatg

60

agctgcaagg cctccggcta cacattcaca tcttacacca tgcactgggt gaagcagaga

120

cccggccagg gcctggagtg gattggctac attaacccaa catccgacta cacaaactac

180

aaccagaagt tcaaggacaa ggccacactc accgccgaca agtcttctag cacagcctac

240

atgcagctgt ctagcctgac aagcgaggac tctgccgtgt acttctgcgc ctctgaggc

300

ggcttcctgt actacttcga ctactggggc cagggcacca ccctgaccgt gtcctctgcc

360

aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc

420

atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg

480

aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc

540

tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc

600

tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat

660

tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc

720

ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta

780

gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg

840

cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt

900

gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac

960

agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag

1020

gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt

1080

ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat

1140

gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac

1200

ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc

1260

tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct

1320

cctggtaaat aa

1332

<210> 23

<211> 639

<212> DNA

<213> Artificial

<220>

<223> Light chain B266

<400> 23

cagatcgtgc tcacacagtc tccagccatc atgagcgcct ctcctggcga gaaggtgaca

60

atgacctgct ctgcctctag ccgcatttct tacatgtact ggtatcagca gaagccaggc

120

acctccccta agaggtggat atacgacaca tccaagctgg cctccggcgt gcccgcccgg

180

ttcagcggct ctggcagcgg cacaagctac tccctgacaa ttagcacgat ggaggccgag

240

gacgccgcca catactactg ccaccagcgc tcgtcctacc caacattcgg cgccggcaca

300

aaattggaac tgaagagagc tgacgcagcc ccaacagtga gcatctttcc tccatcctct

360

gaacaactta cctctggagg agcctctgtg gtgtgtttcc tgaacaactt ctacccaaag

420

gacatcaatg tgaagtggaa gattgatggc tctgagagac agaatggagt gctgaactcc

480

tggacagacc aggacagcaa ggacagcacc tacagtatga gtagcaccct gaccctgacc

540

aaggatgaat atgagagaca caactcctac acttgtgagg ctacccacaa gaccagcacc

600

agcccaattg tcaaatcctt caacaggaat gagtgttaa

639

<210> 24

<211> 1359

<212> DNA

<213> Artificial

<220>

<223> Heavy chain B266

<400> 24

gaggtgcagt tacaacagtc cggcgccgag ctagtgaagc caggcgccag cgtgaagctg

60

tcttgcacag ccagcggctt caacattaag gacaccttca tgcactgggt gaagcagaga

120

cctgagcagg gcttagagtg gattggccgg atcgaccccg ccaacggcaa cacaaagtac

180

gacccaaagt tccagggcaa ggccacaatt accgccgaca catcttccaa cacagcctac

240

ctccagctgt cgtctctcac cagcgaggac accgccgtgt actactgcgc cctgctccag

300

tactccgcga tggactactg gggccagggc acatctgtga ccgtgtctag cgccaagacc

360

accccaccat ccgtgtaccc actcgcccca ggctgcggcg acaccacagg ctctagcgtg

420

acactgggct gcctggtgaa gggctacttc cccgagtctg tgacagtgac ctggaactct

480

ggctctctgt ctagctctgt gcacaccttc cccgccctgc tgcaatccgg cctgtacaca

540

atgtcttctt ctgtgacagt gcctagctct acatggccat ctcagacagt gacatgctct

600

gtggcccacc ccgcctctag cacaaccgtg gacaagaagc tggagccatc cggccctatt

660

tctacaatta acccttgccc tccttgcaaa gaatgccaca agtgccccgc cccaaacctg

720

gagggcggcc cttctgtgtt cattttccct cctaacatta aggacgtgct gatgatcagc

780

ctcaccccaa aggtgacatg cgtggtggtg gacgtgtccg aggacgaccc tgacgtgcag

840

atttcttggt tcgtgaacaa cgtggaggtg cacaccgccc agacccagac ccaccgggag

900

gactacaact ccaccattcg ggtggtgtct acactgccta ttcagcacca ggactggatg

960

agcggcaaag agttcaagtg caaggtgaac aacaaggacc tgccatctcc tattgagaga

1020

acaatttcta agattaaggg cctggtgcgc gcccctcagg tgtacattct gcctcctccc

1080

gccgagcagc tgagccggaa ggacgtgtcc ctcacatgcc tcgtggtggg cttcaaccct

1140

ggcgacatta gcgtggagtg gacatctaac ggccacacag aagaaaacta caaggacaca

1200

gcccctgtgc tcgactccga cggctcttac ttcatatact ctaagctgaa catgaaaaca

1260

tctaagtggg aaaagaccga ctctttctct tgcaacgtgc ggcacgaggg cctgaagaac

1320

tactacctca agaaaaccat tagcagaagt ccaggctaa

1359

<210> 25

<211> 211

<212>PRT

<213> Artificial

<220>

<223> Light chain B266-1

<400> 25

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Arg Ile Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Val Ala Ala Pro Ser Val

100 105 110

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

115 120 125

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

130 135 140

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

145 150 155 160

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

165 170 175

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

180 185 190

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

195 200 205

Gly Glu Cys

210

<210> 26

<211> 447

<212>PRT

<213> Artificial

<220>

<223> Heavy chain B266-1

<400> 26

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Phe Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Leu Leu Gln Tyr Ser Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 27

<211> 633

<212> DNA

<213> Artificial

<220>

<223> Light chain B266-1

<400> 27

cagatcgtgc tcacacagtc tccagccatc atgagcgcct ctcctggcga gaaggtgaca

60

atgacctgct ctgcctctag ccgcatttct tacatgtact ggtatcagca gaagccaggc

120

acctccccta agaggtggat atacgacaca tccaagctgg cctccggcgt gcccgcccgg

180

ttcagcggct ctggcagcgg cacaagctac tccctgacaa ttagcacgat ggaggccgag

240

gacgccgcca catactactg ccaccagcgc tcgtcctacc caacattcgg cgccggcaca

300

aaattggaac tgaaggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag

360

ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc

420

aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca

480

gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca

540

gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc

600

gtcacaaaga gcttcaacag ggggagagtgt tag

633

<210> 28

<211> 1337

<212> DNA

<213> Artificial

<220>

<223> Heavy chain B266-1

<400> 28

gaggtgcagt tacaacagtc cggcgccgag ctagtgaagc caggcgccag cgtgaagctg

60

tcttgcacag ccagcggctt caacattaag gacaccttca tgcactgggt gaagcagaga

120

cctgagcagg gcttagagtg gattggccgg atcgaccccg ccaacggcaa cacaaagtac

180

gacccaaagt tccagggcaa ggccacaatt accgccgaca catcttccaa cacagcctac

240

ctccagctgt cgtctctcac cagcgaggac accgccgtgt actactgcgc cctgctccag

300

tactccgcga tggactactg gggccagggc acatctgtga ccgtgtctag accaagggcc

360

catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg

420

gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc

480

tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc tactccctca

540

gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga

600

atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgacaaaa

660

ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct

720

ttcccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg

780

tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg

840

aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg

900

tcagcgtcct caccgtcctg caccaggact ggctgaatgg caagggagtac aagtgcaagg

960

tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc

1020

cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc aagaaccagg

1080

tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga

1140

gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac tccgacggct

1200

ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag gggaacgtct

1260

tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc

1320

tgtctccggg taaatga

1337

<210> 29

<211> 315

<212> DNA

<213> Artificial

<220>

<223> Trx1 gene

<400> 29

atggtcaaac agatcgaatc aaaaaccgca tttcaagaag ccctggacgc cgctggtgac

60

aaactggtcg tggtggactt tagtgctacc tggtgcggcc cgtgtaaaat gattaaaccg

120

tttttccata gcctgtctga aaaatacagt aacgttatct ttctggaagt ggatgttgat

180

gactgccagg acgtcgcgag cgaatgcgaa gtgaaatgta tgccgacgtt ccagtttttc

240

aaaaaaggtc aaaaagtcgg tgaatttagc ggtgccaaca aagaaaaact ggaagccacg

300

attaacgaac tggtg

315

<210> 30

<211> 22

<212> DNA

<213> Artificial

<220>

<223> Primer hTrx1-For

<400> 30

taatggtcaa acagatcgaa tc

22

<210> 31

<211> 31

<212> DNA

<213> Artificial

<220>

<223> Primer hTrx1-Rev

<400> 31

caccagttcg ttaatcgtgg taatgaaagc t

31

<210> 32

<211> 7

<212>PRT

<213> Artificial

<220>

<223> hTrx1 M1

<400> 32

Gln Ile Glu Gly Ser Thr Ala

15

<210> 33

<211> 6

<212>PRT

<213> Artificial

<220>

<223> hTrx1 M2

<400> 33

Gln Glu Ala Leu Asp Ala

15

<210> 34

<211> 5

<212>PRT

<213> Artificial

<220>

<223> hTrx1 M4

<400> 34

Tyr Ser Asn Val Ile

15

<210> 35

<211> 21

<212> DNA

<213> Artificial

<220>

<223> hTrx1 M1

<400> 35

cagatcgaat caaaaaccgc a

21

<210> 36

<211> 18

<212> DNA

<213> Artificial

<220>

<223> hTrx1 M2

<400> 36

caagaagccc tggacgcc

18

<210> 37

<211> 15

<212> DNA

<213> Artificial

<220>

<223> hTrx1 M4

<400> 37

tacagtaacg ttatc

15

<210> 38

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M1

<400> 38

gtcaaagaga tcgaaggcaa agaagatttt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 39

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M2

<400> 39

gtcaaacaga tcgaatcaaa aaccgcattt catgctgccc tgagcagtgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 40

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M3

<400> 40

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

tatcatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 41

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M4

<400> 41

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atttggcaac atggtgttcc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 42

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M5

<400> 42

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatga taacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 43

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M6

<400> 43

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gttttataaa

240

aaaagggaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 44

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M7

<400> 44

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gttaacaaag aaaaactgga agccacgatt

300

aacgaactgg tg

312

<210> 45

<211> 312

<212> DNA

<213> Artificial

<220>

<223> TRX-N-His-M8

<400> 45

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctggtcgtgg tggactttag tgctacctgg tgcggcccgt gtaaaatgat taaaccgttt

120

ttccatagcc tgtctgaaaa atacagtaac gttatctttc tggaagtgga tgttgatgac

180

tgccaggacg tcgcgagcga atgcgaagtg aaatgtatgc cgacgttcca gtttttcaaa

240

aaaggtcaaa aagtcggtga atttagcggt gccaacaaag aaaaactgga agccatcatt

300

aacgaactgt

312

<210> 46

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Primer Vector-F

<400> 46

ggcgtgtacg gtgggaggt

19

<210> 47

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Primer Vector-R

<400> 47

agcagcgtat ccacatagcg

20

<210> 48

<211> 57

<212> DNA

<213> Artificial

<220>

<223> Primer TRX M1-F

<400> 48

catcacgtca aagagatcga aggcaaagaa gattttcaag aagccctgga cgccgct

57

<210> 49

<211> 58

<212> DNA

<213> Artificial

<220>

<223> Primer TRX M1-R

<400> 49

ggcttcttga aaatcttctt tgccttcgat ctctttgacg tgatgatgat gatgatga

58

<210> 50

<211> 52

<212> DNA

<213> Artificial

<220>

<223> Primer TRX M2-F

<400> 50

aaaaccgcat ttcatgctgc cctgagcagt gctggtgaca aactggtcgt gg

52

<210> 51

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Primer TRX M2-R

<400> 51

tttgtcacca gcactgctca gggcagcatg aaatgcggtt tttgattcga tctg

54

<210> 52

<211> 58

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M3-OV-F

<400> 52

attaaaccgt tttatcatag cctgtctgaa aaatacagta acgttatctt tctggaag

58

<210> 53

<211> 50

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M3-OV-R

<400> 53

agacaggcta tgataaaacg gtttaatcat tttacacggg ccgcaccagg

50

<210> 54

<211> 65

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M4-OV-F

<400> 54

ctgtctgaaa aatttggcaa catggtgttc ctggaagtgg atgttgatga ctgccaggac

60

gtcgc

65

<210> 55

<211> 73

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M4-OV-R

<400> 55

atccacttcc aggaacacca tgttgccaaa tttttcagac aggctatgga aaaacggttt

60

aatcatttta cac

73

<210> 56

<211> 55

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M5-OV-F

<400> 56

gtgaaatgta tgataacgtt ccagtttttc aaaaaaggtc aaaaagtcgg tgaat

55

<210> 57

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M5-OV-R

<400> 57

aaactggaac gttatcatac atttcacttc gcattcgctc gcgacgtcc

49

<210> 58

<211> 65

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M6-OV-F

<400> 58

acgttccagt tttataaaaa aagggaaaaa gtcggtgaat ttagcggtgc caacaaagaa

60

aaact

65

<210> 59

<211> 64

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M6-OV-R

<400> 59

ttcaccgact ttttcccttt ttttataaaa ctggaacgtc ggcatacatt tcacttcgca

60

ttcg

64

<210> 60

<211> 84

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M7-Xho-R

<400> 60

gaattctcga gctatcacac cagttcgtta atcgtggctt ccagtttttc tttgttaaca

60

ccgctaaatt caccgacttt ttga

84

<210> 61

<211> 59

<212> DNA

<213> Artificial

<220>

<223> Primer TRX-M8-Xho-R

<400> 61

gaattctcga gctatcaaca cagttcgtta atgatggctt ccagtttttc tttgttggc

59

<210> 62

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Primer N293F-colo-F

<400> 62

ggcgtgtacg gtgggaggt

19

<210> 63

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Primer N293F-colo-R

<400> 63

agcagcgtat ccacatagcg

20

<210> 64

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_001

<400> 64

Val Ala Thr Ala Ala Asp Val His Ser Gln His His His His

1 5 10 15

<210> 65

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_002

<400> 65

Ala Thr Ala Ala Asp Val His Ser Gln His His His His His

1 5 10 15

<210> 66

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_003

<400> 66

Thr Ala Ala Asp Val His Ser Gln His His His His His His

1 5 10 15

<210> 67

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_004

<400> 67

Ala Ala Asp Val His Ser Gln His His His His His His His

1 5 10 15

<210> 68

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_005

<400> 68

Ala Asp Val His Ser Gln His His His His His His Val

1 5 10 15

<210> 69

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_006

<400> 69

Asp Val His Ser Gln His His His His His His His Val Lys

1 5 10 15

<210> 70

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_007

<400> 70

Val His Ser Gln His His His His His His Val Lys Gln

1 5 10 15

<210> 71

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_008

<400> 71

His Ser Gln His His His His His His Val Lys Gln Ile

1 5 10 15

<210> 72

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_009

<400> 72

Ser Gln His His His His His His Val Lys Gln Ile Glu

1 5 10 15

<210> 73

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_010

<400> 73

Gln His His His His His His Val Lys Gln Ile Glu Ser

1 5 10 15

<210> 74

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_011

<400> 74

His His His His His His His Val Lys Gln Ile Glu Ser Lys

1 5 10 15

<210> 75

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_012

<400> 75

His His His His His His Val Lys Gln Ile Glu Ser Lys Thr

1 5 10 15

<210> 76

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_013

<400> 76

His His His His His Val Lys Gln Ile Glu Ser Lys Thr Ala

1 5 10 15

<210> 77

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_014

<400> 77

His His His His Val Lys Gln Ile Glu Ser Lys Thr Ala Phe

1 5 10 15

<210> 78

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_015

<400> 78

His His His His Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln

1 5 10 15

<210> 79

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_016

<400> 79

His His His Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu

1 5 10 15

<210> 80

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_017

<400> 80

His His Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala

1 5 10 15

<210> 81

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_018

<400> 81

His Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu

1 5 10 15

<210> 82

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_019

<400> 82

Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp

1 5 10 15

<210> 83

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_020

<400> 83

Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala

1 5 10 15

<210> 84

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_021

<400> 84

Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala

1 5 10 15

<210> 85

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_022

<400> 85

Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly

1 5 10 15

<210> 86

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_023

<400> 86

Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp

1 5 10 15

<210> 87

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_024

<400> 87

Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys

1 5 10 15

<210> 88

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_025

<400> 88

Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys Leu

1 5 10 15

<210> 89

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_026

<400> 89

Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys Leu Val

1 5 10 15

<210> 90

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_027

<400> 90

Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys Leu Val Val

1 5 10 15

<210> 91

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_028

<400> 91

Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys Leu Val Val Val

1 5 10 15

<210> 92

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_029

<400> 92

Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp

1 5 10 15

<210> 93

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_030

<400> 93

Glu Ala Leu Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe

1 5 10 15

<210> 94

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_031

<400> 94

Ala Leu Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser

1 5 10 15

<210> 95

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_032

<400> 95

Leu Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala

1 5 10 15

<210> 96

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_033

<400> 96

Asp Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr

1 5 10 15

<210> 97

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_034

<400> 97

Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp

1 5 10 15

<210> 98

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_035

<400> 98

Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys

1 5 10 15

<210> 99

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_036

<400> 99

Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly

1 5 10 15

<210> 100

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_037

<400> 100

Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro

1 5 10 15

<210> 101

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_038

<400> 101

Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys

1 5 10 15

<210> 102

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_039

<400> 102

Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys

1 5 10 15

<210> 103

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_040

<400> 103

Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys Met

1 5 10 15

<210> 104

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_041

<400> 104

Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys Met Ile

1 5 10 15

<210> 105

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_042

<400> 105

Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys

1 5 10 15

<210> 106

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_043

<400> 106

Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys Pro

1 5 10 15

<210> 107

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_044

<400> 107

Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe

1 5 10 15

<210> 108

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_045

<400> 108

Ser Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe

1 5 10 15

<210> 109

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_046

<400> 109

Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His

1 5 10 15

<210> 110

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_047

<400> 110

Thr Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser

1 5 10 15

<210> 111

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_048

<400> 111

Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu

1 5 10 15

<210> 112

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_049

<400> 112

Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser

1 5 10 15

<210> 113

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_050

<400> 113

Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu

1 5 10 15

<210> 114

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_051

<400> 114

Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys

1 5 10 15

<210> 115

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_052

<400> 115

Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr

1 5 10 15

<210> 116

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_053

<400> 116

Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr Ser

1 5 10 15

<210> 117

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_054

<400> 117

Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr Ser Asn

1 5 10 15

<210> 118

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_055

<400> 118

Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr Ser Asn Val

1 5 10 15

<210> 119

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_056

<400> 119

Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr Ser Asn Val Ile

1 5 10 15

<210> 120

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_057

<400> 120

Pro Phe Phe His Ser Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe

1 5 10 15

<210> 121

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_058

<400> 121

Phe Phe His Ser Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe Leu

1 5 10 15

<210> 122

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_059

<400> 122

Phe His Ser Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe Leu Glu

1 5 10 15

<210> 123

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_060

<400> 123

His Ser Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe Leu Glu Val

1 5 10 15

<210> 124

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_061

<400> 124

Ser Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe Leu Glu Val Asp

1 5 10 15

<210> 125

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_062

<400> 125

Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val

1 5 10 15

<210> 126

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_063

<400> 126

Ser Glu Lys Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp

1 5 10 15

<210> 127

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_064

<400> 127

Glu Lys Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp

1 5 10 15

<210> 128

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_065

<400> 128

Lys Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys

1 5 10 15

<210> 129

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_066

<400> 129

Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln

1 5 10 15

<210> 130

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_067

<400> 130

Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp

1 5 10 15

<210> 131

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_068

<400> 131

Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val

1 5 10 15

<210> 132

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_069

<400> 132

Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val Ala

1 5 10 15

<210> 133

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_070

<400> 133

Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val Ala Ser

1 5 10 15

<210> 134

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_071

<400> 134

Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val Ala Ser Glu

1 5 10 15

<210> 135

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_072

<400> 135

Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val Ala Ser Glu Cys

1 5 10 15

<210> 136

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_073

<400> 136

Glu Val Asp Val Asp Asp Cys Gln Asp Val Ala Ser Glu Cys Glu

1 5 10 15

<210> 137

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_074

<400> 137

Val Asp Val Asp Asp Cys Gln Asp Val Ala Ser Glu Cys Glu Val

1 5 10 15

<210> 138

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_075

<400> 138

Asp Val Asp Asp Cys Gln Asp Val Ala Ser Glu Cys Glu Val Lys

1 5 10 15

<210> 139

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_076

<400> 139

Val Asp Asp Cys Gln Asp Val Ala Ser Glu Cys Glu Val Lys Cys

1 5 10 15

<210> 140

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_077

<400> 140

Asp Asp Cys Gln Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met

1 5 10 15

<210> 141

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_078

<400> 141

Asp Cys Gln Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro

1 5 10 15

<210> 142

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_079

<400> 142

Cys Gln Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr

1 5 10 15

<210> 143

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_080

<400> 143

Gln Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe

1 5 10 15

<210> 144

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_081

<400> 144

Asp Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln

1 5 10 15

<210> 145

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_082

<400> 145

Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe

1 5 10 15

<210> 146

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_083

<400> 146

Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe

1 5 10 15

<210> 147

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_084

<400> 147

Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys

1 5 10 15

<210> 148

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_085

<400> 148

Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys Lys

1 5 10 15

<210> 149

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_086

<400> 149

Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys Lys Gly

1 5 10 15

<210> 150

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_087

<400> 150

Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln

1 5 10 15

<210> 151

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_088

<400> 151

Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys

1 5 10 15

<210> 152

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_089

<400> 152

Lys Cys Met Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val

1 5 10 15

<210> 153

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_090

<400> 153

Cys Met Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly

1 5 10 15

<210> 154

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_091

<400> 154

Met Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu

1 5 10 15

<210> 155

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_092

<400> 155

Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe

1 5 10 15

<210> 156

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_093

<400> 156

Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe Ser

1 5 10 15

<210> 157

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_094

<400> 157

Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly

1 5 10 15

<210> 158

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_095

<400> 158

Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala

1 5 10 15

<210> 159

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_096

<400> 159

Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn

1 5 10 15

<210> 160

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_097

<400> 160

Phe Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys

1 5 10 15

<210> 161

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_098

<400> 161

Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu

1 5 10 15

<210> 162

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_099

<400> 162

Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys

1 5 10 15

<210> 163

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_100

<400> 163

Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu

1 5 10 15

<210> 164

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_101

<400> 164

Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu Glu

1 5 10 15

<210> 165

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_102

<400> 165

Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu Glu Ala

1 5 10 15

<210> 166

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_103

<400> 166

Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu Glu Ala Thr

1 5 10 15

<210> 167

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_104

<400> 167

Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu Glu Ala Thr Ile

1 5 10 15

<210> 168

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_105

<400> 168

Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu Glu Ala Thr Ile Asn

1 5 10 15

<210> 169

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_106

<400> 169

Phe Ser Gly Ala Asn Lys Glu Lys Leu Glu Ala Thr Ile Asn Glu

1 5 10 15

<210> 170

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_107

<400> 170

Ser Gly Ala Asn Lys Glu Lys Leu Glu Ala Thr Ile Asn Glu Leu

1 5 10 15

<210> 171

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Peptide_108

<400> 171

Gly Ala Asn Lys Glu Lys Leu Glu Ala Thr Ile Asn Glu Leu Val

1 5 10 15

<210> 172

<211> 26

<212>PRT

<213> Artificial

<220>

<223> Epitope B264 and B266-1

<400> 172

Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser

1 5 10 15

Leu Ser Glu Lys Tyr Ser Asn Val Ile Phe

20 25

<210> 173

<211> 15

<212>PRT

<213> Artificial

<220>

<223> Epitope B266-1

<400> 173

Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe

1 5 10 15

<210> 174

<211> 21

<212>PRT

<213> Artificial

<220>

<223> Epitope B264

<400> 174

Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala

1 5 10 15

Ala Gly Asp Lys Leu

20

<210> 175

<211> 17

<212>PRT

<213> Artificial

<220>

<223> Epitope B264

<400> 175

Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys Lys

1 5 10 15

Gly

<210> 176

<211> 20

<212>PRT

<213> Artificial

<220>

<223> Epitope B264

<400> 176

Pro Thr Phe Gln Phe Phe Lys Lys Gly Gln Lys Val Gly Glu Phe Ser

1 5 10 15

Gly Ala Asn Lys

20

<210> 177

<211> 78

<212> DNA

<213> Artificial

<220>

<223> Epitope B264 and B266-1

<400> 177

gctacctggt gcggcccgtg taaaatgatt aaaccgtttt tccatagcct gtctgaaaaa

60

tacagtaacg ttatcttt

78

<210> 178

<211> 45

<212> DNA

<213> Artificial

<220>

<223> Epitope B266-1

<400> 178

ccgacgttcc agtttttcaa aaaaggtcaa aaagtcggtg aattt

45

<210> 179

<211> 63

<212> DNA

<213> Artificial

<220>

<223> Epitope B264

<400> 179

gtcaaacaga tcgaatcaaa aaccgcattt caagaagccc tggacgccgc tggtgacaaa

60

ctg

63

<210> 180

<211> 51

<212> DNA

<213> Artificial

<220>

<223> Epitope B264

<400> 180

agcgaatgcg aagtgaaatg tatgccgacg ttccagtttt tcaaaaaagg t

51

<210> 181

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Epitope B264

<400> 181

ccgacgttcc agtttttcaa aaaaggtcaa aaagtcggtg aatttagcgg tgccaacaaa

60

<---

Claims (3)

1. The use of an epitope of a human thioredoxin-1 (Trx1) antigen consisting of the amino acid sequence of SEQ ID NO: 174 for specific binding to a monoclonal antibody or an antigen-binding fragment thereof, wherein said monoclonal antibody comprises a light chain variable region comprising a light chain CDR1 consisting of of the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region consisting of a heavy chain CDR1 consisting of of the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6. 2. Use according to claim 1, wherein said antibody comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 14. 3. Use according to claim 1, wherein said antibody comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 17 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18.