RU2791264C2 - Monoclonal antibody specifically binding to the thioredoxin-1 epitope, its preparation and application - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: group of inventions relates to immunology. A monoclonal antibody specifically binding to thioredoxin-1 (Trx1) or its antigen-binding fragment was proposed, including a variable region of the light chain containing CDR1 with SEQ ID NO: 7, CDR2 with SEQ ID NO: 8, CDR3 with SEQ ID NO: 9, and a variable region of the heavy chain containing CDR1 with SEQ ID NO: 10, CDR2 with SEQ ID NO: 11, CDR3 with SEQ ID NO: 12. Nucleic acid molecules encoding light and heavy chains of a monoclonal antibody or its antigen-binding fragment, a recombinant expression vector, a host cell for obtaining a monoclonal antibody or its antigen-binding fragment, a set and methods for the diagnosis of breast cancer are also proposed.

EFFECT: monoclonal antibody has excellent binding affinity with thioredoxin-1, has high sensitivity and specificity, which significantly improves the accuracy and reliability of breast cancer diagnosis.

25 cl, 40 dwg, 25 tbl, 17 ex

Description

This work is 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 antigen (Trx1) and a monoclonal antibody that specifically binds to it, and more specifically, to an epitope, a monoclonal antibody that specifically binds to it, its antigen-binding fragment, 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 for providing information necessary for diagnosis breast cancer.

PRIOR ART

Thioredoxin (Trx) is a small redox protein of approximately 12 kDa that is present in a reduced state by 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 affects the death of cells and tumors and therefore plays a key role in the regulation of cancer cell growth, and also cleaves the disulfide bond in another oxidized protein, helping to maintain the activity in the reduced state. Thioredoxin-1- and thioredoxin-2-reductases remove nitric oxide from cysteines in mammalian cells, which affects cell death, and has potential significance in various diseases, including inflammatory disease, heart attack, and cancer. In addition, immunohistochemical analysis using an anti-thioredoxin antibody demonstrates the expression of thioredoxin in human cancer tissues, including the liver, colon, pancreas and cervix, and such expression indicates a possible role of thioredoxin in the development of tumors.

Under these circumstances, the inventors have studied a marker for the diagnosis of breast cancer, which allows the diagnosis of breast cancer or its early prediction; thioredoxin-1 expression 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 (Korean Patent No. 10-1058230).

To develop in vitro diagnostics (IVD) based on enzyme-linked immunosorbent assay (ELISA) with high accuracy and high precision, a pair of antibodies to the same antigenic protein is needed, having different sites with different affinity values. Moreover, a system for obtaining antibodies with a certain affinity is needed, which does not require large expenditures. In the present invention, in order to detect thioredoxin-1 (Trx1) present in human serum, two types of highly effective recombinant monoclonal antibodies have been developed that bind to thioredoxin-1 with very high specificity and thus can be useful for screening aimed at detecting patients with breast cancer. In addition, by determining 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 has been proposed to solve the above problems and is directed to providing a monoclonal antibody or antigen-binding fragment thereof, which 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 said 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 a 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 breast cancer diagnostic kit containing a monoclonal antibody as described above, 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 its antigennegative fragment.

Technical solution

To solve the problems described above, the present invention provides a monoclonal antibody that specifically binds to Trx1, or an antigen-binding fragment thereof, containing a light chain variable region containing CDR1 (complementarity determining region 1) of the light chain, consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 light chain, 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, containing a heavy chain CDR1, consisting of the amino acid sequence of 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 to 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 of 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: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 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 another exemplary embodiment of the present invention, the antibody may comprise an IgG1 heavy chain and a kappa (κ) light chain.

According to 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 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 an antibody described above or an antigen-binding fragment thereof.

According to one exemplary embodiment of the present invention, a nucleic acid molecule encoding a 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 comprising 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 a human Trx1 antigen epitope 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 kit for the diagnosis of breast cancer, containing the above-described antibody or antigennegative fragment.

According to one exemplary 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 one selected from the group consisting of direct ELISA, indirect ELISA, direct sandwich ELISA, and indirect sandwich ELISA.

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

In addition, according to the present invention, a method for providing information necessary for diagnosing breast cancer is provided, comprising: (a) coating a solid support with a monoclonal antibody or antigen-binding fragment containing 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 a B266 or B266-1 antibody, 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 to a coated solid support; (c) removing the unbound sample; (d) applying a monoclonal antibody or antigen-binding fragment thereof comprising the light chain CDR1-CDR3 and heavy chain CDR1-CDR3 of antibody B264, a monoclonal antibody or antigen-binding fragment thereof comprising the light chain variable region and the heavy chain variable region of a B264 antibody or B264 antibody, or its antigen-binding fragment on a solid support; (e) removing unbound monoclonal antibody or antigen-binding fragment; (e) measuring the expression level of the Trx1 protein; and (g) comparing the expression level of the Trx1 protein measured in step (e) with a control and, if the expression level of the protein is greater than the control, identifying the subject as a breast cancer patient.

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

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 they are commonly understood by a person skilled in the art to which this invention pertains. In general, the nomenclature used here is well known and widely used in the art.

In this application, the following definitions of key terms are used.

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 its molecule. 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), eg, Fab, Fab', F(ab')2, Fd, Fv, Fc, and so on; complementarity determined region (CDR); bispecific antibody; a heteroconjugated antibody or mutant thereof; an antibody or fusion protein having an antigen-binding fragment (eg, a single domain antibody); a single chain variable fragment (ScFv) and a single domain antibody (eg, shark and camelid antibodies); maxi-body, mini-body, intra-body, dia-body, tri-body, tetra-body, 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 antibody variants, antibody amino acid sequence variants, 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 (e.g., the Trx1 protein) is a term commonly understood in the art to which this invention pertains, and methods for determining such specific binding are also well known in the art. A particular molecule is considered to have "specific binding" when it interacts with or binds to a particular cell or substance more frequently, faster, more consistently and/or with higher affinity than it does 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 when bound to other substances.

The term "binding affinity" or "K _D ", as used herein, refers to the equilibrium dissociation constant when an antibody interacts with a specific antigen. K _D is the ratio of the rate of dissociation (also called "rate of release" or "k _d ") to the rate of binding, or "rate of association" or "k _a (rate constant of association)". Thus, K _D is k _d /k _a expressed as molar concentration (M). It follows that the lower the K _D , the higher the binding affinity. Thus, a K _D of 1 μM indicates a lower binding affinity than a K _D of 1 nM. The K _D value of an antibody can be determined using a method well 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 associated with 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 added to the viral genome. Some vectors are capable of self-replication in 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 mammalian vector) can be integrated into the genome of host cells when introduced into the host cells and thus replicate along with the host genome. In addition, some vectors can direct the expression of operably linked 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" used here are the types of vectors that are used most often, 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, whether they are morphologically or genetically identical to the parent cell, as long as they contain the selected gene.

Beneficial effects

The monoclonal antibody of the present invention has an excellent binding affinity for thioredoxin-1, which allows it to bind very specifically to thioredoxin-1, and has a very high sensitivity and specificity, which allows it to be effectively used in screening aimed at identifying patients with breast cancer. In addition, detection of thioredoxin-1 using the thioredoxin-1 specific binding monoclonal antibody of the present invention, rather than detection using the traditional CA15-3 breast cancer diagnostic biomarker, exhibits exceptionally high sensitivity and specificity, which can significantly improve 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 GRAPHICS

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

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

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

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

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

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

FIG. 7 shows the results of the reduced (+) and unreduced (-) state of the B266-1 antibody using SDS-PAGE, where the B266-1 antibody is obtained by modifying the Fc portion of the B266 antibody with a human IgG1 Fc.

FIG. 8A-8D show the results of IMTG discontinuity alignments for the B266-1 light chain and heavy chain and the B264 light chain and heavy chain in order.

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

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

FIG. 11 shows a comparison of the amino acid sequence homology of human Trx1 and Trx1 of Chrysochloris asiatica. PREDICTED: thioredoxin-like - putative 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 - waiting; Method - method; Identities - identical; Positives - positive; Gaps - gaps.

FIG. 12 shows the result of electrophoresis to confirm successful cloning with the CaTrx1 cloning plasmid and restriction enzyme digestion (Sfi I and Xho I), where lane 1 shows the CaTrx1 cloning plasmid and lane 2 shows the restriction enzyme-treated plasmid.

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

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

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.

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

FIG. 15C shows a PCR (polymerase chain reaction) fusion scheme for obtaining a mutant hTrx1 gene resulting from the amplification of a DNA fragment and PCR with overlapping primers performed one after the other.

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

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

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

FIG. 16 shows proteins secreted into the culture solution after transduction of human HEK293 cells with genes obtained by 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 corresponding size was detected, confirming the expression of 8 types of transformed genes and their secretion as proteins into the culture solution.

FIG. 17 shows the result of analysis of the degree of expression of 8 types of mutant hTrx1 proteins detected in FIG. 16 after cleaning.

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

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

FIG. 20 is a miniarray image obtained using one of the antibody samples described in Example 17.

FIG. 2A-21D is a heatmap chart showing the degree of interaction of controls interacting with antibody samples and all probe peptides, where the y-axis shows the peptide sequences of the library, and the x-axis shows the concentrations of the applied antibody samples. The MMC2 values are shown by color code, the range of which includes 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 - mcg/ml.

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

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereinafter, 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 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 aimed at identifying patients with breast cancer. The monoclonal antibody of the present invention binds very specifically to thioredoxin-1 due to its excellent binding affinity to thioredoxin-1, and has very high sensitivity and specificity, so that it can be effectively used in screening aimed at detecting patients with breast cancer. In addition, the detection of thioredoxin-1 using the monoclonal antibody of the present invention, which specifically binds to thioredoxin-1, and not the detection of CA15-3, another biomarker traditionally used for diagnosing breast cancer, demonstrates excellent sensitivity and specificity, which can significantly improve the accuracy and reliability of breast cancer diagnosis. In addition, the epitope region of the human Trx1 antigen to which the specified 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 a combination of these methods. For example, a monoclonal antibody can be prepared using a hybridoma technique 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 obtaining it.

The method of obtaining and screening a particular antibody using hybridoma technique is common and well known in this field. As a non-limiting example, a mouse can be immunized with the target antigen or cells expressing it. When an immune response is detected, for example, when an antibody specific for an antigen is detected 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 by the method of limiting dilutions. The hybridoma clone is then evaluated for its ability to be a cell secreting an antibody capable of binding to the antigen, using a method known in the art. Typically, ascitic fluid containing high levels of antibodies can be obtained by injecting positive hybridoma clones into the abdominal cavity of mice. In a typical embodiment of the present invention, the Trx1 antigen is obtained by transfection of E. coli with a recombinant vector, the cleavage map of which is shown in FIG. 1(a). The spleen of a rat immunized with the antigen is then isolated and cells fused with myeloma cells (sp2/0) to obtain an antibody interacting with Trx1 are determined by ELISA.

An exemplary monoclonal antibody of the present invention, or an antigen-binding fragment thereof, may comprise (a) or (b) as indicated below 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 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 comprising a heavy chain CDR1 consisting 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; or

(b) 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 of 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: 12.

The term "complementarity determining region (CDR)" as used herein refers to the amino acid sequence of a hypervariable region of an immunoglobulin heavy chain or light chain. 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 to an antigen or epitope.

An exemplary monoclonal antibody of the present invention, or an antigen-binding fragment thereof, may comprise (c) or (d) as indicated below 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 (e) as indicated below 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

(e) 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 with the Fc of human IgG1.

The structural unit of a naturally occurring antibody usually contains a tetramer. The tetramer usually consists of two pairs of identical polypeptide chains, and each pair has one full-length light chain (typically having a molecular weight of approximately 15 kDa) and one full-length heavy chain (typically having a molecular weight of approximately 50 to 70 kDa). The amino terminus of each light chain and heavy chain typically contains a variable region of about 100-110 or more amino acids involved in antigen recognition. The carboxy terminus of each chain defines a constant region that is normally involved in effector function. Human light chains are generally 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, without limitation, several subclasses including IgG1, IgG2, IgG3 and IgG4. IgM has, without limitation, 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 an 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 IgG1, IgG2a, IgG2b, IgG3, IgA, or IgM isotype and the light chain may be a κ chain or a λ chain, and the κ light chain and the heavy chain are preferred. IgG1 chain.

In a monoclonal antibody of the present invention or an antigen-binding fragment thereof, "antigen-binding fragment thereof" means a fragment that performs an antigen-binding function and includes 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 heavy chain first 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 via 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 obtained by, for example, cleavage of the whole antibody with papain, and an F(ab') ₂ fragment can be obtained by cleavage of the whole antibody with pepsin.

An exemplary antibody of the present invention may be a chimeric antibody, a humanized antibody, or a fully human antibody.

A chimeric antibody can be made 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 production of such a chimeric antibody is well known in the art and can be accomplished by standard means. It should also be noted that the human constant region of the chimeric antibody of the present invention can be selected from the constant region of IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16 , IgG17, IgG18 or IgG19.

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

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 both DNA (gDNA and cDNA) and RNA molecules, and in a nucleic acid molecule, the nucleotide that is its basic unit also includes the analog in which the sugar or backbone has been modified, as well as the natural nucleotide. The sequences of nucleic acid molecules encoding the heavy chain and light chain variable regions of the present invention may be modified. The modification includes additions, deletions, or non-conservative or conservative nucleotide substitutions.

It should be clarified 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 showing at least 80% homology, in one specific example at least 90% homology, or in another specific example at least 95% homology when a nucleotide sequence is aligned according to the present invention with a different sequence for their best possible match, and analysis of the aligned sequences using an algorithm commonly used in the art.

According to one exemplary embodiment of the present invention, the nucleic acid molecule encoding the light chain of the monoclonal antibody of the present invention may consist of the nucleotide sequence of SEQ ID NO:21, and the nucleic acid molecule encoding the heavy chain of the 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, the nucleic acid molecule encoding the heavy chain of the monoclonal antibody of the present invention may consist of the nucleotide sequence of SEQ ID NO:23, and the nucleic acid molecule encoding the light chain of the 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, the nucleic acid molecule encoding the light chain of the monoclonal antibody of the present invention may consist of the nucleotide sequence of SEQ ID NO:27, and the nucleic acid molecule encoding its heavy chain may consist of 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 molecule encoding a light chain of a monoclonal antibody, or both of these nucleic acid molecules.

The recombinant vector system of the present invention may be constructed by various methods known in the art. Typically, the vector of the present invention may be designed 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 transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter , amp promoter, recA promoter, SP6 promoter, trp promoter, or T7 promoter), a ribosome binding site for translation initiation, and transcription/translation termination sequences. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the E. coli tryptophan biosynthetic pathway promoter and operator regions 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 manipulation of a plasmid used in the field (for example, pCL, pSC101, pGV1106, pACYC177, Co1E1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79 , pIJ61, pLAFR1, pHV14, pGEX series, pET or pUC19 series), 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 usually 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, HIV LTR promoter) human immunodeficiency virus), a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter), and a polyadenylation sequence as a transcription termination sequence.

The recombinant vector of the present invention can be fused with another sequence to facilitate purification of the antibody expressed from the recombinant vector. The fused 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 selectable marker, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin or tetracycline resistance gene.

The vector expressing an 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 via co-transformation and directed transformation. Co-transformation is a method of selecting cells expressing both the light chain and the heavy chain after introducing vector DNAs encoding the light chain and the heavy chain, respectively, 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 light chain), and finally selecting cells expressing both a light chain chain and heavy chain.

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

As eukaryotic host cells suitable for the vector, multicellular fungi such as Aspergillus sp. cerevisiae and Schizosaccharomyces, other lower eukaryotic cells, higher eukaryotic cells such as cells derived from insects and cells derived from plants or mammals.

The term "transfection" as used herein refers to the introduction of a gene of interest into host cells using the 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 for introducing a nucleic acid into an organism, cells, tissues, or organ. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, silicon carbide 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 a human Trx1 antigen epitope 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 an amino acid homology of 82%, the two types of anti-hTrx1 antibodies of the present invention do not bind to CaTrx1 (FIGS. 11 and 15A). Accordingly, eight portions in which the amino acid sequences of hTrx1 and CaTrx1 differ were identified (FIG. 15B), and gene cassettes for expression of mutant hTrx1 proteins were generated for gene cloning (FIG. 15C-15F). The cloned genes transformed the N293F cell line, confirmed the expression of 8 types of mutant hTrx1 proteins (FIG. 16) and purified each mutant protein (FIG. 17), followed by confirmation of the strength of binding to the B266-1 antibody (hTrx1-hIgG1) and to the B264 antibody ( hTrx1-mIgG1).

As shown in FIG. 18A-18C, reduced binding of B266-1 antibody and mutant M4 protein (YSNVIFGNMV) compared to hTrx1 and binding of B264 antibody and mutant proteins M1 (QIESKTAEIEGKED), M2 (QEALDAHAALSS) and M4 compared to hTrx1 were confirmed. Thus, it was confirmed that antibodies B266-1 and B264 most likely share a common M4 binding site.

In addition, microarray analysis was performed using 108 peptides obtained by superimposing the amino acid sequence of the hTrx1 protein with a shift of one amino acid residue (FIGS. 19 and 20), and the epitopes of B266-1 and B264 antibodies 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 Trx1 antigen epitope described above, 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 be composed of any amino acid sequence selected from the group consisting of SEQ ID NOS:32-34 and 172-176.

The descriptions of the nucleic acid molecule encoding the above epitope, the recombinant vector containing the said nucleic acid molecule, and the host cell containing the said recombinant vector are the same as the descriptions given above for the antibody of the present invention, and therefore will 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 the host cells to produce the antibody or 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 readily 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-batch culture or continuous culture according to the culture method. The medium used for cultivation should suitably meet the requirements for particular strains.

The medium used in culturing animal cells contains various carbon sources, nitrogen sources, and trace elements. Examples of carbon sources used here may be 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 glycerol and ethanol; and organic acids such as acetic acid. These carbon sources may be used independently or in combination. Examples of nitrogen sources used here include organic nitrogen sources such as peptones, yeast extracts, meat broths, malt extracts, corn extract liquid (CSL) and soy flour; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used independently or in combination. The medium may contain potassium dihydrogen phosphate, potassium hydrogen phosphate and the corresponding sodium salts as a source of phosphorus. In addition, the medium may contain metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and suitable precursors may be included.

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

The antibody obtained by culturing host cells can be used without purification, or can be used by purification to a high degree of purity using various conventional methods, such as dialysis, salt precipitation and chromatography. Of these methods, chromatography is 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, a kit for diagnosing breast cancer is provided, containing a monoclonal antibody of the present invention or an antigen-binding fragment thereof, and a method for providing information necessary for diagnosing 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, diagnostics are performed to confirm whether breast cancer is present or not.

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

According to one exemplary embodiment of the present invention, a 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 an exemplary embodiment of the present invention, the two types of antibodies included in the sandwich ELISA kit include B266-1 monoclonal antibody as a coating antibody and B264 monoclonal antibody as a detection antibody.

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

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

As an agent or reagent used in an immunoassay, a suitable carrier or support, a marker capable of producing a detectable signal, a solubilizer, a cleaning agent, or a stabilizer may be included. When the marker is an enzyme, suitable carriers include a substrate to measure enzyme activity, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, or an agent to stop the reaction, but the present invention is not limited to these embodiments.

The anti-Trx1 antibody included in the kit of the present invention is preferably fixed on a suitable carrier or support using various methods disclosed in the document, and examples of suitable carriers and supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, agarose , cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polystyrene, gabbro, filter paper, ion exchange resin, plastic film, plastic vial, polyamine methyl vinyl ether maleic acid copolymer, amino acid copolymer, ethylene maleic acid copolymer, nylon , metal, glass, glass granule and magnetic particle. Other solid supports include a cell culture plate, an ELISA plate, a test tube, and a polymer film. The support may be of any possible shape, such as spherical (bead), cylindrical (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 the formation of an antigen-antibody complex and may be, for example, an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, or a radioisotope. As the enzyme, 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 can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin or fluorescein isothiocyanate can be used. A biotin derivative can be used as a ligand, and acridinium ester or luciferin can be used as a luminescent substance. Colloidal gold or colored latex can be used as the microparticle, and ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone or hydroquinone can be used as the redox molecule. As a radioisotope, 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. 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, nitrosine tetrazolium or p-nitrophenyl phosphate can be used as a substrate. In addition, when choosing β-D-galactosidase as an enzyme marker, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside. In addition to these, various other enzymes and chromogenic enzyme substrates known in the art may be used.

According to one exemplary 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) bringing any one type of monoclonal antibody of the present invention, or antigennegative fragment thereof, into contact with a biological sample isolated from a subject with suspected breast cancer;

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

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

According to another exemplary 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 the light chain CDR1-CDR3 and 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 an antibody heavy chain variable region B266 or B266-1, or B266 or B266-1 antibody or antigen-binding fragment thereof;

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

(c) removing the unbound sample;

(d) applying a monoclonal antibody or antigen-binding fragment thereof comprising the light chain CDR1-CDR3 and heavy chain CDR1-CDR3 of antibody B264, a monoclonal antibody or antigen-binding fragment thereof comprising the light chain variable region and the heavy chain variable region of a B264 antibody or B264 antibody, or its antigen-binding fragment on a solid support;

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

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

(g) comparing the expression level of the Trx1 protein measured in step (e) with the control and, if the expression level of the protein is greater than the control, identifying 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 sample tissue (brain, skin, lymph nodes, spinal cord, or the like), cell culture supernatant, or lysed eukaryotic cells, where the expression level of the Trx1 protein, which is a breast cancer marker, is different and includes a sample obtained from a primary site or a metastatic site . These biological samples, after manipulation or without previous manipulation, can be subjected to interaction with the 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, and so on, and, without limitation, any subject with suspected 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 may 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 antigen (Trx1)

1-1. Obtaining the Trx1 expression vector

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

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

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

1-2. Expression and purification of Trx1

A 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 by the method described above. For the expression of the thioredoxin-1 protein by the transformed strain, this strain was cultured in liquid LB medium containing the antibiotic to OD ₆₀₀ (optical density at 600 nm) 0.5 at 37°C, after which it was cultivated 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. To purify the protein, the resulting cell line was lysed using ultrasound and then centrifuged (12,000 rpm, 30 min, 4°C), thereby obtaining a supernatant. A commercially available antibody against thioredoxin-1 (LF-MA0055, Abfrontier) was added to the resulting supernatant to bind to expressed thioredoxin-1, agarose with protein A/G (Protein A/G PLUS-agarose, sc-2003, Santa Cruz) was added , which contacted the antibody, for their interaction, after which centrifugation and purification were performed. The purity and molecular weight of the resulting product was then confirmed by SDS-PAGE.

Example 2

Production and purification of a monoclonal antibody specific for Trx1

2-1. Mouse Immunization

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

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 cultivation of cells (hybridoma) obtained from the fusion of only a myeloma cell and a B lymphocyte was performed.

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

Three types of antibodies interacting with the human protein thioredoxin-1 were confirmed by ELISA in the resulting hybridoma cells. The selection of hybridoma, forming an antibody that interacts with the antigen, from ELISA-positive cells was performed by the method of limiting dilutions.

2-4. Obtaining and Purifying 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. Purified antibody was determined by SDS-PAGE.

Example 3

Determination of the isotype of a monoclonal antibody

The isotypes of the three antibodies produced in Example 2 were confirmed using a Rapid ELISA Mouse mAbs Isotyping Kit, Pierce cat. no. 37503.

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

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 the monoclonal antibodies 9G7(AB1) and 2B4(AB2) from the three types of monoclonal antibodies obtained in Example 2 were analyzed. The amino acid sequence was determined as a sequence capable of fusion with an Fc region suitable for reverse translation and recombinant expression. Spent alignment and hypermutation of the sequence determined by IMTG-discontinuity alignment, and full-length regions of CDR3 were found using the hypermutation table. Sequences were determined using exact 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 above manner, a 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 obtained above (B264-B269) were expressed and then the affinity of each antibody for the antigen was confirmed by ELISA (the numbers after "T" in Tables 3-5 indicate the corresponding numbers of the batches obtained).

Affinities for three types of antigens, ie, free Trx1, Fc-bound Trx1 (Trx1-Fc) and His-tagged Trx1 (Trx1-His), were determined by direct ELISA and the results are sequentially shown 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 the high affinity B264 and B266 antibodies are shown in Table 6 below.

Example 6

Obtaining antibodies B264 and B266

6-1. Obtaining 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 respective 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-SSJ11-L and pcDNA3-SSJ11-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 high purity antibodies were obtained using a sterile 0.2 μm filter.

The purity and size of the purified antibodies were determined by SDS-PAGE. Based on the SDS-PAGE results, as shown in FIG. 6, the expression of B264 and B266 antibodies was confirmed to be eg 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 corresponded 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 interaction ( overnight) at 4°C. 200 μl of PBS (phosphate buffered saline) containing 1% BSA (PBSA; blocking buffer) was added to each well, and interaction was carried out 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 above steps were carried out three times.

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

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

As a result, as shown in Table 8, the interaction index increases in accordance with the concentration of the antigen, demonstrating the detection of the antigen by these antibodies. However, due to the high OD value in the absence of antigen, it is necessary to experiment with antibodies to improve their performance.

Example 8

Altering the isotype of the Fc portion to improve antibody performance

Since the antibody expression system is a transient transfection using a recombinant plasmid and not a hybridoma, among these recombinant plasmids, a plasmid having a heavy chain was co-transfected with a plasmid having a heavy chain of a different isotype. That is, co-transfection was performed with a plasmid having a gene encoding a different heavy chain than pcDNA3-SSJ12H from pcDNA3-SSJ12-L and pcDNA3-SSJ12-H used to express 9G7(AB1).

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

The CDR sequences of the ultimately selected monoclonal antibodies B264 and B266-1 were determined by fusion with an 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 searching the database determined the closest germline sequence and hypermutation. The results of the gapped IMTG alignment obtained for the light chain and heavy chain of each of the B266-1 and B264 antibodies are shown in FIG. 8A-8D, the amino acid sequences of the light chain CDR1-CDR3 and the heavy chain CDR1-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 light chain and heavy chain amino acid and gene sequences 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 an O/N interaction (overnight) was performed at 4°C. Washing was carried out by adding 200 µl of wash buffer. The steps described above were carried out twice.

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

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

100 μl of TMB solution was added to each well, interaction 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

Analysis of the affinity of a monoclonal antibody against an antigen

Two types of monoclonal antibodies specific for 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 was analyzed by ELISA (FIG. 9(a)).

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

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

100 μl of TMB solution was added to each well, interaction 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 obtained values were analyzed using Prism (Graphpad) (FIG. 9(b)).

Affinity analysis of the B266-1 coat antibody and B264 detection antibody confirmed the high negative control value due to B266-1 reactivity, but the degree of binding of B266-1 and B264 increases with increasing antigen concentration. This indicates the binding of B266-1 and B264 to the antigen. When calculating the value of the equilibrium dissociation constant (K_D) as part of the analysis carried out using the Prism program, K_D B266-1 was 1.1x10^-eleven, a K_D B264 was 1.3x10^-10. With a value of K_D from 10^-10 to 10^-12 the antibody was judged 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 the serum of a patient with breast cancer

Sandwich ELISA using a 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 interaction 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. The PBSA was removed and then the 96-well plate was dried in a thermohygrostat (20° C., 30% relative humidity) for 3 hours.

This was followed by biotinylation of the detection antibody (B264) as described below.

Dimethyl sulfoxide (DMSO) was mixed with 20 mg/ml biotin-7-NHS to give 2 mg/ml biotin-7-NHS. 15 μl (30 μg) 2 mg/ml biotin-7-NHS was added to the B264 antibody, 1 mg/ml, and reacted at 15-25°C for 2 hours. The reaction solution was loaded into an AMICON Ultra-15 (Millipore), filled with PBS to final volume, and centrifuged at 3600 x g to a residual volume of 0.5 ml. These steps 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.

The human Trx1 antigen was then detected in the serum of a breast cancer patient as described below.

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

Finally, ROC analysis was performed as described below.

Sensitivity and specificity were calculated by analyzing the result of an ELISA using monoclonal antibodies B266-1 and B264 against Trx1. At a cutoff 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 the diagnosis of breast cancer

In this example, to evaluate the performance of recombinant monoclonal antibodies B266-1 and B264, a comparative analysis was performed with another ELISA kit to detect another biomarker CA15-3 for diagnosing 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 for CA15-3.

Example 13

Chrysochloris asiatica Trx1 protein expression

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

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

Using the known base sequence of CaTrx1 (NCBI registration number XM_006863001.1), a gene was synthesized and the gene was 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 a plasmid treated with restriction enzymes (lane 2), a 357 bp DNA fragment cleaved from the plasmid was identified, indicating successful synthesis of the CaTrx1 gene (arrow in FIG. 12).

13-2. Expression of the CaTrx1 protein

After transfection of an animal cell with the plasmid CaTrx1 obtained in Example 13-1, the 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 CaTrx1 transformant cell culture solution 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 against hTrx1 and CaTrx1

In this example, the binding affinity of two types of antibodies, B266-1 (Trx1-hIgG1) and B264 (Trx1-mIgG1), to CaTrx1 was analyzed.

To confirm the binding affinity of B266-1 (Trx1-hIgG1) and B264 (Trx1-mIgG1) 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 (4 % skimmed milk / 1x PBS) followed by interaction for 1 hour at 37°C. After removing the reaction solution, 100 μl of each of B266-1 (Trx1-hIgG1) and B264 (Trx1-mIgG1) was added to each well coated with the appropriate antigen and allowed to interact at 37°C for 2 hours. The reaction solution was removed followed by washing five times with 200 μl of 1x PBST. To each of the wells treated with B266-1 (Trx1-hIgG1) or B264 (Trx1-mIgG1), 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 with 200 μl 1x PBST. 100 μl of staining reagent was added to each well, and after 10 minutes of interaction, 50 μl of 2.5 MH ₂ SO ₄ was added to each well. After color development, the degree of staining was assessed using an ELISA plate reader.

As a result of confirming the binding affinity of B266-1 (Trx1-hIgG1) and B264 (Trx1-mIgG1) to hTrx1, as shown in Table 14 below, for 200 ng of antigen, it was confirmed that the K _D for B266-1 was 2.1x10 ^-10 M, and the affinity for B264 was determined at K _D 1.7x10 ^-10 M. In addition, as seen in FIG. 14A, compared to B266-1, when bound to B264, the interaction index (OD ₄₉₀ ) was lower at about 50%.

As a result of confirmation of 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

Obtaining mutant hTrx1 antigen

15-1. Determination of 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 CaTrx1 amino acid sequence (NCBI accession number XP_006863063.1). As shown in FIG. 15A, although the amino acid sequence homology of hTrx1 and CaTrx1 was 82%, the binding affinity of antibodies to antigen was significantly different, and thus there were 8 different parts in which hTrx1 and CaTrx1 have different amino acid sequences (FIG. 15B).

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

A) PCR fragments

In the 8 portions in which the amino acid sequences of hTrx1 and CaTrx1 differ, as 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 a gene for the 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 for which a base sequence mutation is required were prepared, and the DNA fragments were amplified using two primer pairs, 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, then thoroughly mixed and amplification was carried out using a PCR device (T100 thermal cycler).

PCR was performed under conditions of 1 cycle of pre-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 elongation at 72°C for 5 min, after which the reaction was stopped. The amplified DNA fragment was verified using a 1% agarose gel (FIG. 15D). Gene purification was performed using the QIAquick Gel Extraction Kit (QIAGEN, 28704) following the manufacturer's protocol.

C) 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). PCR mix for PCR fusion 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 2 x EF-Taq PCR Smart (0.5X Band Doctor) (Solgent, SEF02-M50h), brought to final volume with sterile purified water, then thoroughly mixed and amplified using a PCR device. PCR was performed under conditions of 1 cycle of pre-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 elongation at 72°C for 5 min, after which the reaction was stopped. After the reaction was terminated, the PCR product was verified using a 1% agarose gel (FIG. 15E).

Upon completion of the PCR fusion, 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 the amount of 1/10 and 2 total volumes of the PCR product, respectively, thoroughly mixed and reacted at -70°C in a low-temperature freezer on for 10 minutes. Thereafter, 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 interacting 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 the PCR product and plasmid N293F, and the total volume was adjusted to 80 µl. After thorough mixing, the resulting mixture was subjected to interaction at 37°C in a water bath for 3 hours. Upon completion of the interaction, the resulting mixture was purified using ethanol precipitation. Thereafter, 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 subjected to interaction at 37°C in a water bath for 3 hours. To purify the DNA obtained at the end of the reaction, an experiment was performed using the QIAquick Gel Extraction Kit (QIAGEN, 28704) following the manufacturer's protocol.

To clone a purified DNA fragment in N293F, 20 ng of N293F treated with a 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 1 μl of 10x buffer was added and the total volume was adjusted to 10 μl with distilled water. The resulting mixture was subjected to interaction 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 DH5α* competent 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 sprayed and distributed in 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. A fusion PCR mix was prepared by adding 0.5 µl of each of the forward and reverse primers (10 pmol each) to 12.5 µl of 2 x EF-Taq PCR Smart (0.5X Band Doctor) mix (Solgent, SEF02- M50h), brought to the final volume with sterile distilled water, then thoroughly mixed and carried out amplification by PCR. PCR was performed under conditions of 1 cycle of pre-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 elongation at 72°C for 5 min, after which the reaction was stopped.

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

E) Obtaining plasmids (Midi-obtaining)

Colonies containing sequenced plasmids were seeded 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, catalog number 740410.100) was used to prepare a purified plasmid and the experiment was carried out 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 a frozen 293F cell line (510029, Invitrogen) in a water bath at 37°C 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 a Luna cell count chip (L12002, Biosystems) and viability and cell count were confirmed using a Luna™ automatic cell counter (L10001, Biosystems ). After suspending 4-7 x 10 ⁵ cells/ml in 40 ml of medium, the resulting suspension was centrifuged at 100 xg for 5 minutes to remove the supernatant. After removal of the supernatant, the cell pellet was mixed with 10 ml of medium to resuspend the pellet, and then 30 ml of the medium was added to a 125 ml Erlenmeyer flask. Cells were incubated in a shaker incubator at 8% CO ₂ , 37° C. and 85 rpm and the above steps were performed two or more times.

G) Transfection of animal cells

An aliquot of cells, 5.5x10 ⁵ cells/ml, 40 ml was placed in a tube and centrifuged at 100 xg 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 the automatic cell counter Luna™, it was confirmed that the number and viability of the cells were 1x10 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 (polyethyleneimine) (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 interact 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 that, the cells were additionally 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 (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 (sample that was centrifuged) was used. 10 μl of 5x reconstitution sample buffer was mixed with 40 μl of the supernatant, followed by boiling at 100° C. for 5 minutes. The resulting sample was confirmed by 15% SDS-PAGE using a 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. A PolyPrep column (731-1553, Bio-Rad) was washed with 10 mM imidazole wash buffer (pH 7.4) and loaded with Ni-Sepharose™ 6 Fast Flow beads (17-5318-02, GE Healthcare). The column was then washed with 10 mM imidazole wash buffer (pH 7.4) twice. When approximately 2-3 ml of wash buffer with 10 mM imidazole (pH 7.4) remained in the column, the column was washed again with 20 ml of wash buffer with 10 mM imidazole (pH 7.4). Medium was added to the washed column. The beads were washed with 10 mM imidazole wash buffer (pH 7.4) and eluted with 500 mM imidazole elution buffer (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 buffer exchanged, repeating the reconcentration with PBS solution at least two times. The 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 are shown in Table 17 below. Table 17 shows that 8 types of mutant hTrx1 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 hTrx1 mutant 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, each antigen solution was added to a 96-well plate at 100 μl the well and the 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% skimmed 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 a 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-B264 anti-mouse Fc) were diluted 1:4000 in antibody diluent (1 x PBS with 1% milk), 100 μl of the resulting solution was added to each well and the plate was covered with a 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 tablet OPD (orthophenylenediamine), 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 stop buffer (2.5 MH ₂ SO ₄ ) was added to each well and the OD was measured at 490 nm.

As a result, as shown in FIG. 18A, it was confirmed that the B266-1 antibody (hTrx1-hIgG1) had a reduced binding strength against a protein having an M4 site mutation (YSNVIFGNMV), and the B264 antibody (hTrx1-mIgG1) had a reduced binding strength towards a protein having site mutations. M1, M2 and M4 (M1: QIESKTAEIEGKED; M2: QEALDAHAALSS; and M3: YSNVIFGNMV).

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

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

Example 17

Antibody profiling using peptide microarrays

In this example, the exact amino acid sequence was determined by Trx1 antigen epitope mapping analysis 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 detected using overlapping peptide scanning.

17-1. Sequences

Antibody profiling experiments were performed using a peptide library of 108 peptides. A complete list of peptides is shown in Tables 20-22 below. Here, SEQ ID NOs: 64-81 corresponding to peptides 001-018 (Peptide_001 to Peptide_018) are not native forms and contain regions of recombinant inserts. The sequence with registration number in GenBank AAF87085.1 was used as the known amino acid sequence of the hTrx1 protein.

Full-length mouse IgG was also immobilized as an analytical control on the microarray panel, and an 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 individual miniarrays (one miniarray for each sample dilution).

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

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

17-3. Image of processed matrices

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

17-4. Heat map analysis

To visualize the results obtained and compare the binding regions for individual cultures, heat map charts were calculated as shown in FIG. 21A-21D. FIG. 21A-21D, fluorescence intensity is shown by color code, white indicates no binding and red indicates strong binding. In all analyzes, the MMC2 value of the average pixel fluorescence was calculated for each peptide.

MMC2 is the average of all three points on the microarray, except when 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 on 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 the B266-1 antibody, 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 NOS: 67 and 68). However, since peptides 004 and 005 are not native forms, these peptides were excluded from the group of possible epitopes.

ID - identifier; Control IgG - control IgG; blank-control - negative control; Control-Spot - control point.

The B264 antibody 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, in particular at the two highest sample concentrations.

As shown in Table 24, the highest signal, approximately 7 times the mean background level, was measured for peptides 012 and 018 (Peptide 012 and Peptide 018) (SEQ ID NOS:75 and 81). However, since peptides 012 and 018 are not native forms, they were excluded from the group of possible epitopes.

Subsequently, the peptides of 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 the B264 antibody, and finally, the epitope of the B264 antibody was determined as "VKQIESKTAFQEALDAAGDKL" (SEQ ID NO:179).

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

ID - identifier; Control IgG - control IgG; blank-control - negative control; Control-Spot - control point.

No significant binding was found in culture of the control secondary antibody. Strong signals, up to saturation level, were obtained at the control point containing full-length mouse IgG in all cultures, indicating excellent assay performance.

The epitope regions obtained by the method described above are shown in Table 25 below, and, based on the results of 3D modeling with confirmation of tertiary (3D) structures by loading the NMR sequence of hTrx1, confirmed by a 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 the diagnosis of breast cancer can be greatly improved, since the detection of the monoclonal antibody of the present invention, which specifically binds to Trx1, and not the detection of CA15-3, another traditional diagnostic biomarker of breast cancer, demonstrates 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 Specifically

Binding thereto

<130> 1064685

<150> KR 10-2017-0132536

<151> 2017-10-12

<160> 181

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<213> Artificial

<220>

<223> Light chain CDR2 B266-1

<400> 8

Asp Thr Ser

1

<210> 9

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Light chain CDR3 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 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 ctctgagggc 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

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 tgcactggggt 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

540

gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc 600

gtcacaaaga gcttcaacag gggagagtgt 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 tgcactggggt 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

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 caaggagtac aagtgcaagg 960

tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc 1020

ccggagaacc 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

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 gt 312

<210> 46

<211> 19

<212> DNA

<213> Artificial

<220>

<223> Vector-F Primer

<400> 46

ggcgtgtacg gtgggaggt 19

<210> 47

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Vector-R Primer

<400> 47

agcagcgtat ccacacatagcg 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

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 ccacacatagcg 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 (35)

1. A monoclonal antibody that specifically binds to thioredoxin-1 (Trx1), or an antigen-binding fragment thereof, containing a light chain variable region containing a light chain CDR1 (complementarity determining region 1), consisting of the amino acid sequence of SEQ ID NO: 7, 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 of SEQ ID NO: 9, and a heavy chain variable region containing 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: 12. 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises 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. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises 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. 4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises 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. 5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody comprises an IgG1 heavy chain and a kappa (κ) light chain. 6. Monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-4, where the antigen-binding fragment is a Fab, F(ab'), F(ab') ₂ , Fv or a single chain antibody molecule. 7. Monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-4, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody. 8. Nucleic acid molecule encoding the light chain of a monoclonal antibody or its antigen-binding fragment according to claim 1. 9. Nucleic acid molecule according to claim 8, consisting of the nucleotide sequence of SEQ ID NO: 23 or 27. 10. A nucleic acid molecule encoding the heavy chain of a monoclonal antibody or its antigen-binding fragment according to claim 1. 11. Nucleic acid molecule according to claim 10, consisting of the nucleotide sequence of SEQ ID NO: 24 or 28. 12. A recombinant expression vector containing a nucleic acid molecule encoding a light chain according to claim 8 and a nucleic acid molecule encoding a heavy chain according to claim 10. 13. A host cell for obtaining a monoclonal antibody or its antigen-binding fragment according to claim 1, containing a recombinant vector according to claim 12. 14. Kit for the diagnosis of breast cancer, containing a monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-4 and the reagent used in the immunoassay. 15. The kit according to claim 14, which is a kit for enzyme-linked immunosorbent assay (ELISA). 16. The kit of claim 15 wherein the ELISA is one or more selected from the group consisting of direct ELISA, indirect ELISA, direct sandwich ELISA, and indirect sandwich ELISA. 17. Method for providing information necessary for the diagnosis of breast cancer, including (a) bringing the monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-4 in contact with a biological sample isolated from a subject with suspected breast cancer; (b) measuring the level of expression of a thioredoxin-1 (Trx1) 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 expression level of the Trx1 protein measured in step (b) with the control and, if the expression level of the protein is greater than the control, identifying the subject as a patient with breast cancer. 18. The method of claim 17, wherein the level of Trx1 protein expression is measured by any method selected from the group consisting of Western blotting, ELISA, sandwich ELISA, radioimmunoassay, radioimmunoassay, Ouchterlony immunodiffusion, immunoprecipitation assay, complement fixation assay, immunochromatographic analysis, FACS (fluorescence-activated cell sorting) and analysis on protein chips. 19. The method of claim 17 wherein the isolated biological sample is any one or more selected from the group consisting of whole blood, serum, plasma, breast tissue, and breast cells. 20. Method for providing information necessary for the diagnosis of breast cancer, including (a) coating a solid support with a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-4; (b) applying a biological sample isolated from a subject suspected of having breast cancer to a coated solid support; (c) removing the unbound sample; (d) applying a monoclonal antibody that specifically binds to thioredoxin-1 (Trx1), or an antigen-binding fragment thereof, on a solid support, wherein said monoclonal antibody contains a light chain variable region containing a light chain CDR1 consisting 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 comprising a heavy chain CDR1 consisting 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; (e) removing unbound monoclonal antibody or antigen-binding fragment; (e) measuring the expression level of the thioredoxin-1 (Trx1) protein; And (g) comparing the expression level of the Trx1 protein measured in step (e) with the control and, if the expression level of the protein is greater than the control, identifying the subject as a patient with breast cancer. 21. The method of claim 20 wherein the level of Trx1 protein expression is measured by any method selected from the group consisting of Western blotting, ELISA, sandwich ELISA, radioimmunoassay, radioimmunoassay, Ouchterlony immunodiffusion, immunoprecipitation assay, complement fixation assay, immunochromatographic analysis, FACS and analysis on protein chips. 22. The method of claim 20 wherein the isolated biological sample is any one or more selected from the group consisting of whole blood, serum, plasma, breast tissue, and breast cells. 23. A method for producing a monoclonal antibody that specifically binds to thioredoxin-1 (Trx1), or its antigen-binding fragment, including culturing the host cell according to claim 13. 24. The method of claim 20, wherein the antibody from step (d) 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. 25. The method of claim 20, wherein the antibody from step (d) 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.

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