KR20200098338A - An antibody or antigen-binding fragment thereof specifically binding to BAG2 polypeptide or fragment thereof - Google Patents

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof specifically binding to a BAG2 polypeptide or a fragment thereof capable of causing antigen-antibody reactions with BAG2 polypeptides of various lengths or fragments thereof.

Description

Antibody or antigen-binding fragment thereof specifically binding to BAG2 polypeptide or fragment thereof {An antibody or antigen-binding fragment thereof specifically binding to BAG2 polypeptide or fragment thereof}

It relates to an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.

The co-chaperone Bcl-2-associated athanogene (BAG) protein family is a variety of physiological processes including intracellular protein folding, stress response, neural differentiation, cell death, and cell proliferation. And functionally binds to various cooperative proteins. Among them, BAG2, one of the members of the BAG domain family with anti-apoptotic activity, is a negative regulator of the C-terminus of Hsc70-interacting protein (CHIP), a chaperone-related ubiquitin ligase, Hsc70-interacting protein. Is known. The main role of BAG2 in the regulation of proteins through inhibition of CHIP activity is associated with neurodegenerative diseases and autosomal recessive disorders through stabilization of chaperone-related proteins such as PINK1 and CFTR. Expression of BAG2 increases proteasome suppressor-induced apoptosis, and BAG2 knockdown partially inhibits apoptosis when exposing thyroid carcinoma cells to MG132, a proteasome inhibitor, so that BAG2 is anti-cell death. It has been suggested that it may have pro-apoptotic activity. Apart from the formation of the BAG2-Hsp70 complex, the BAG protein functionally interacts with various binding partners and modulates various cellular processes such as stress signaling, cell division, cell death, and cell differentiation. On the other hand, it has been suggested that in various mutant K-Ras-induced tumors, overexpression of BAG2 can promote the stabilization of the STK33 protein, a strong oncogene, thereby promoting tumor development. In addition, it has been suggested that the expression and location of the BAG2 protein may vary according to the histopathological and molecular genetic pathological characteristics of cancer cells in the process of breast cancer progression or metastasis. It was found that the BAG2 protein is secreted out of cells through the interaction with the proteolytic enzyme cadexin B in the process of cancer, and it was confirmed that the loss of the BAG2 protein completely inhibited the formation of cancer and metastasis to the lungs in the breast cancer animal model. As a result of this, the development of BAG2 inhibitors will contribute to the treatment and survival rate of cancer patients.

Despite these findings, the role of BAG2 in various cancer progression and metastasis has not been clearly elucidated. In addition, no specific studies have been conducted on BAG2 monoclonal antibodies. Accordingly, there is a need for development of an antibody or antigen-binding fragment thereof that specifically binds to the BAG2 polypeptide or fragment thereof.

One aspect provides an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.

Another aspect provides a polynucleotide comprising a polynucleotide encoding the antibody or antigen binding fragment thereof.

Another aspect provides a host cell comprising the polynucleotide.

Another aspect provides a method of producing the antibody or antigen-binding fragment thereof.

One aspect provides an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.

Each of the BAG2 polypeptides may be derived from mammals. The mammal may be a human ( Homo sapiens ), a mouse ( Mus musculus ), a monkey, a cow or a horse. BAG2 may include the amino acid sequence of SEQ ID NO: 69. SEQ ID NO: 69 amino acid sequence is a sequence corresponding to the NCBI reference SEQ ID NO: NM_004282.4. The BAG2 protein does not match the amino acid sequence of SEQ ID NO: 69, but also includes a variant having biologically equivalent activity to the BAG2 protein. The BAG2 polypeptide contains an amino acid sequence having sequence identity of 60% or more, for example, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% of the sequence of SEQ ID NO: 69. It can be. In addition, the BAG2 protein is at least one amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, or at least 7 amino acid residues in the sequence of SEQ ID NO: 69. It may be a polypeptide having a sequence. In the present specification, the'polypeptide' may be used interchangeably with the'protein'.

The antibody refers to a specific immunoglobulin directed against an antigenic site. The antibody refers to a polypeptide or a combination of polypeptides that specifically binds to a BAG2 polypeptide or a fragment thereof. The form of the antibody includes polyclonal antibodies, monoclonal antibodies, or recombinant antibodies such as ScFv fragments, diabodies, single-chain antibodies, and the like, and all immunoglobulin antibodies may be included. The antibody does not have the structure of an intact antibody having two full-length light chains and two full-length heavy chains, as well as a complete form having two light chains and two heavy chains, but is directed against the antigenic site. A functional fragment of an antibody molecule having a specific antigen-binding site, that is, a binding domain, and retaining an antigen-binding function may also be included.

The antigen-binding fragment is a fragment thereof for the entire structure of an immunoglobulin, and refers to a polypeptide including a portion to which an antigen can bind. For example, the antigen binding fragment can be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, or a combination thereof.

There are five types of heavy chains, γ, δ, α, μ, and ε, and the heavy chain determines the type of antibody. α and γ can be composed of 450 amino acids, and μ and ε can be composed of 550 amino acids. The heavy chain may have two regions: a variable region and a constant region.

The light chain has two types, λ and κ, and may consist of approximately 211 to 217 amino acids. The light chain may have a constant region and a variable region.

The antibody or antigen-binding fragment thereof is a complementarity determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45. A heavy chain variable region comprising a; And a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63. A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46; And VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 64, and SEQ ID NO: 35 A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 41, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; And a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 65, SEQ ID NO: 36 A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 42, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48; And VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 60, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 37 A heavy chain variable region including VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 43, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49; And a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67, the antibody or The antigen-binding fragment thereof includes a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 44, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50; And a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68, or It can be a combination.

The 6th and 7th Xaa in SEQ ID NO: 39 may be glycine (Gly) or alanine (Ala). The second Xaa in SEQ ID NO: 35 may be tyrosine (Tyr) or histidine (H). The eighth Xaa in SEQ ID NO: 41 may be serine (Ser) or threonine (Thr). The 12th Xaa in SEQ ID NO: 47 may be tyrosine (Tyr) or histidine (His). The third Xaa in SEQ ID NO: 53 may be methionine (Met) or isoleucine (Ile). The second Xaa in SEQ ID NO: 59 may be alanine (Ala) or serine (Ser).

The antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 26; A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 32; Or it may include the heavy chain variable region and the light chain variable region.

The antibody or antigen-binding fragment thereof may include a heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 27; The heavy chain variable region of SEQ ID NO: 22 and the light chain variable region of SEQ ID NO: 28; The heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 29; The heavy chain variable region of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO: 30; The heavy chain variable region of SEQ ID NO: 25 and the light chain variable region of SEQ ID NO: 31; Or it may be those including the heavy chain variable region of SEQ ID NO: 26 and the light chain variable region of SEQ ID NO: 32, or a combination thereof.

The 56th and 57th Xaa in SEQ ID NO: 21 may be glycine (Gly) or alanine (Ala). In SEQ ID NO: 23, the first Xaa may be glutamine (Gln) or glutamate (Glu), the 7th Xaa may be serine (Ser) or proline (Pro), and the 12th Xaa may be valine (Val) or alanine ( Ala), the 27th Xaa may be tyrosine (Tyr) or histidine (His), the 58th Xaa may be serine (Ser) or threonine (Thr), and the 61st Xaa may be asparagine (Asn) or serine (Ser), the 74th Xaa may be arginine (Arg) or lysine (Lys), the 83rd Xaa may be phenylalanine (Phe) or leucine (Leu), and the 92nd Xaa may be glycine (Gly) or It may be alanine (Ala), and the 108th Xaa may be histidine (His) or tyrosine (Tyr). The 53rd Xaa in SEQ ID NO: 27 may be isoleucine (Ile) or phenylalanine (Phe). In SEQ ID NO: 29, the 23rd Xaa may be methionine (Met) or isoleucine (Ile), the 45th Xaa may be alanine (Ala) or serine (Ser), and the 73rd Xaa may be glutamate (Glu) or aspart It may be an acid (Asp), and the 100th Xaa may be methionine (Met) or isoleucine (Ile).

The antibody or antigen-binding fragment thereof includes a plurality of antibodies or antigen-binding fragments thereof, and includes one of one of the first set and one of the second set; One of set 1 and one of set 3; And it may be a combination of antibodies selected from the group consisting of one of the 2nd set and the 3rd set. The first set may be an antibody or antigen-binding fragment thereof that binds to the intermediate region of the BAG2 protein comprising the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 27, and the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 28. The second set may be an antibody or antigen-binding fragment thereof that binds to the N-terminus of the BAG2 protein comprising the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 29, and the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 30. . The third set may be an antibody or antigen-binding fragment thereof that binds to the C-terminus of the BAG2 protein comprising the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 31, and the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 32. .

The antibody or antigen-binding fragment thereof may be a monoclonal antibody.

The antibody or antigen-binding fragment thereof may be labeled with a detectable label or a label capable of releasing a detectable signal. The label refers to a detectable compound or composition that has been conjugated directly or indirectly to an antibody to produce a labeled antibody or antigen-binding fragment thereof. The label may be detectable by itself and may catalyze chemical modification of the detectable substrate compound or composition. The label may be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radiolabel, an epitope tag, avidin/biotin, a colloidal gold particle, a colored particle, a magnetic particle, a chromophore label, an ECL label, an enzyme, or the like.

The antibody or antigen-binding fragment thereof is from the group consisting of hybridoma cells deposited with accession numbers KCTC 13737BP, KCTC 13738BP, KCTC 13739BP, KCTC 13740BP, KCTC 13741BP, KCTC 13742BP, KCTC 13743BP, KCTC 13744BP, KCTC 13745BP and KCTC 13746BP. It may be produced by the hybridoma cell of choice. The hybridoma cell refers to a tumorigenic hybrid cell made by artificially fusing two types of cells, and is generally cultivated by fusion with B cells and plasmacytoma cells isolated from immunized individuals. Can be used to continuously produce The hybridoma cells may be mixed with hybrid cells or fused cells.

The present inventors have a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 27 (2A11, 4C2, 8C4); A VH of SEQ ID NO: 22 and a VL of SEQ ID NO: 28 (3B5); A VH of SEQ ID NO: 23 and a VL of SEQ ID NO: 29 (9B3, 9B12, 3B10); A VH of SEQ ID NO: 24 and a VL of SEQ ID NO: 30 (10H7); A VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 31 (3G8); And an anti-BAG2 antibody comprising the VH of SEQ ID NO: 26 and VL (3F12) of SEQ ID NO: 32, and hybridoma cells producing the same were identified. It was confirmed that the identified anti-BAG2 antibody exhibited an antigen-antibody reaction in breast cancer cells.

Another aspect provides a polynucleotide comprising a polynucleotide encoding the antibody or antigen binding fragment thereof.

The polynucleotide may be any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 10 encoding the heavy chain variable region and any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 20 encoding the light chain variable region. have.

The polynucleotide may be a vector. The vector may be one capable of replicating and/or expressing the 　 polynucleotide in a cell. The cells may be eukaryotic cells or prokaryotic cells. The eukaryotic cells may be mammalian cells, plant cells, yeast cells, or insect cells. The mammal may be human, monkey, rabbit, rat, hamster or mouse. The prokaryotic cell may be a bacterial cell. The bacteria may be E. coli. The vector may be an expression vector. The expression vector may be one in which the polynucleotide is operably linked to an appropriate regulatory region so that the polynucleotide can be expressed in a host cell. The regulatory region may be a promoter, enhancer, or terminator. The vector may also contain a selection marker. The vector may be a phage, plasmid, cosmid, mini-chromosome, viral or retroviral vector. The vector may include 　 each containing a polynucleotide encoding a heavy chain variable region or a light chain variable region of the 　 antibody, or a polynucleotide encoding a heavy chain variable region or a light chain variable region.

The polynucleotide may be a detectable label or a label capable of releasing a detectable signal conjugated. The label may be a fluorescent dye, a phosphorescent dye, a radioisotope, a chromophore, a quantum dot, a quencher, a magnetic bead nanoparticle, a gold nanoparticle, a nanophosphor, a silicon nanoparticle, or a semiconductor fine particle having luminescence. For example, the fluorescent dyes are cyanine (cyanine 2), amidomethyl coumarin, fluorocein, indian carbocyanine (cyanine 3), cyanine 3.5, tetramethyl rhodamine, rhodamine red, Texas red, indian carbocy It may be an amine (cyanine 5), cyanine 5.5, cyanine 7, Oyster, and the like. For example, the semiconductor fine particles may be cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphorus (InGaP), silver indium zinc sulfide (AgInZnS), or the like. The method of conjugating the label may be a method of binding a labeling substance to the 3'end of the polynucleotide, a method of binding a labeling substance to the 5'end, and a method of including the nucleotide to which the labeling substance is bound in the polynucleotide. In the method of conjugating a label to the 3'or 5'end, an enzymatic reaction may be used, and the enzyme may be a T4 RNA ligase, a terminal transfer enzyme, a polyA polymerase, or the like. The label can be detected by a fluorescence microscope, a scanning electron microscope, a transmission electron microscope, a computed tomography, magnetic resonance imaging, or the like.

Another aspect provides a host cell comprising the polynucleotide.

The polynucleotide is as described above.

The host cell may be a bacterial cell, a yeast cell, a fungal cell, an insect cell, an animal cell, or a plant cell containing the polynucleotide. The bacterial cells may be E. coli, Streptomyces or Salmonella typhimurium. The yeast cells may be Pichia pastoris. The insect cells may be Drozophila or Spodoptera Sf9 cells. The animal cells are Chinese hamster ovary cells (CHO), mouse myeloma (SP2/0), human lymphoblastoid, COS, mouse myeloma (NSO), 293T, Bow melanoma cells, HT- 1080, baby hamster kidney cells (BHK), human embryonic kidney cells (HEK), PERC.6 (human retinal cells).

The polynucleotide may be introduced into the host cell. The introduction refers to a method of delivering a vector containing a polynucleotide encoding the 　 antibody to a host cell. Such introductions include calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electric shock, microinjection, liposome fusion, lipofectamine and protoplast fusion, etc. It can be carried out by several methods known in the art. In addition, transduction refers to delivery of a target object into cells using viral particles by means of infection. In addition, the vector can be introduced into the host cell by gene bombardment or the like. The introduction can be used interchangeably with transformation.

Another aspect provides a method of preparing the antibody or antigen-binding fragment thereof.

The method includes culturing host cells; And isolating the antibody or antigen-binding fragment thereof from the obtained culture.

The host cell is as described above.

The step of culturing may be performed according to a suitable medium and culture conditions known in the art. Those of ordinary skill in the art can easily adjust and use the medium and culture conditions according to the selected microorganism. The culture method may include, for example, batch, continuous and fed-batch culture.

The medium may contain various carbon sources, nitrogen sources, and trace element components.

The carbon source is, for example, glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, fatty acid glycine such as linoleic acid. It may be an alcohol such as relol and ethanol, an organic acid such as acetic acid, or a combination thereof. The nitrogen source is, for example, peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and organic nitrogen sources and urea, such as soybean meal, and inorganic such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. It may include a nitrogen source, or a combination thereof. The medium may contain, as a source of phosphorus, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and a corresponding sodium-containing salt, a metal salt such as magnesium antioxidant or iron antioxidant. In addition, amino acids, vitamins, and suitable precursors may be included in the medium. The medium or individual components may be added to the culture medium in a batch or continuous manner.

In addition, in the culture of the host cells, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the microorganism culture solution in an appropriate manner to adjust the pH of the culture solution. In addition, it is possible to inhibit the generation of air bubbles by using an antifoaming agent such as fatty acid polyglycol ester during the culture of the host cell.

The cells can be cultured in aerobic, microaerobic, or anaerobic conditions. The microaerobic condition refers to a culture condition in which oxygen at a level lower than that of oxygen in the atmosphere is dissolved in a medium. The low level of oxygen may be, for example, 0.1% to 10%, 1% to 9%, 2% to 8%, 3% to 7%, or 4% to 6%. In addition, the microaerobic condition may be, for example, that the dissolved oxygen concentration in the medium is 0.9 ppm to 3.6 ppm. The culture temperature may be, for example, 20 ℃ to 45 ℃ or 25 ℃ to 40 ℃. The incubation period can be continued until the desired antibody or antigen-binding fragment thereof is obtained as desired.

The separating step may be performed by a method known in the art for the separation of antibodies. The separating step may include performing at least one selected from the group consisting of centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractionation, and chromatography.

The method may further include labeling the isolated antibody or antigen-binding fragment thereof. The label may be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radiolabel, an epitope tag, avidin/biotin, a colloidal gold particle, a colored particle, a magnetic particle, a chromophore label, an ECL label, an enzyme, or the like.

The antibody or antigen-binding fragment thereof that specifically binds to the BAG2 polypeptide or fragment thereof according to one aspect may cause an antigen-antibody reaction with the BAG2 polypeptide or fragment thereof of various lengths.

The polynucleotide encoding the antibody or antigen-binding fragment thereof according to another aspect and a host cell comprising the same can be used to produce the antibody or antigen-binding fragment thereof.

According to the method for producing the antibody or antigen-binding thereof according to another aspect, the antibody or antigen-binding fragment thereof can be efficiently produced.

1 is a diagram showing the results of Western blotting of anti-BAG2 antibodies produced from 10 types of mouse hybridoma cells.
2 is a diagram showing the results of Western blotting for the full-length BAG2 polypeptide or fragment thereof of an anti-BAG2 antibody.
3 is a diagram showing a BAG2 domain to which each anti-BAG2 antibody reacts.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Selection of anti-BAG2 antibodies, sequence analysis and identification of antigen-antibody reactions thereof

1. Selection of monoclonal antibodies targeting BAG2 and analysis of amino acid sequence

The present inventors selected antibodies targeting BAG2, analyzed their amino acid sequence, and determined the complementarity determining region (CDR) of each antibody.

Specifically, human BAG2 protein consisting of the amino acid sequence of SEQ ID NO: 69 The gene consisting of the coding nucleotide sequence of SEQ ID NO: 70 was cloned into pCAGGS plasmid and linearized, and then the linearized construct was inoculated into the muscles of five 6-week-old female BALB/c mice by applying electric shock. . The construct was inoculated intramuscularly three times at intervals of 3 weeks, and consisted of 100 ug of DNA in 100 ul of PBS. At this time, the plasmid of the control group was also carried out the above process in the same manner. In order to produce therapeutic and diagnostic antibodies, a DNA vaccine-based immunization strategy was performed that is more efficient than the antigen injection method using protein. Blood was collected from the fundus venous plexus or coccygeal vein of mice, examined by enzyme immunoassay indicating the serum antibody titer, and the spleen was removed 3 days after the final immunization from mice showing sufficient antibody titer. B lymphocytes were isolated from the spleen, and then the isolated B lymphocytes were fused with cultured myeloma cells, that is, the SP2/0-Ag14 cell line of ATCC to obtain fused cells. After culturing the fused cells in HAT medium supplemented with hypoxanthin, aminopterine and thymidine, about 130 clones of hybridoma cells in which only myeloma and B lymphocytes are fused. It was obtained by selecting. Among the hybridoma cells obtained through the selection process using immunoblotting, 10 hybridoma cells producing antibodies that specifically bind to human BAG2 protein were obtained.

Total RNA of anti-BAG2 antibody was prepared from the 5x10 ⁶ hybridoma cells, and 5'-RACE- from 100 ng total RNA using the SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. cDNA was prepared. The heavy chain variable region (VH) and light chain variable region (VL) coding regions were amplified by PCR, and the amplified gene was inserted into a pGEM-T vector (Promega, USA) and cloned, and an automatic gene analyzer (ABI Prism 310). , Applied Biosystem Co.) was used to analyze the nucleotide sequence. The nucleotide sequence of the analyzed gene was confirmed by comparison with the previously reported nucleotide sequence, and the identified nucleotide sequence was artificially translated to determine the complementarity determining regions (VH-CDR1, -CDR2, -CDR3 and VL-CDR1, -CDR2, -CDR3. ) Was used for sequencing. The sequence determination of the complementarity determining region was performed using Kabat's database (http://www.bioinf.org.uk/abs/).

As a result, 10 kinds of anti-BAG2 antibodies that specifically bind to BAG2, that is, 2A11, 4C2, 8C4, 3B5, 9B3, 9B12, 3B10, 10H7, 3G8 and 3F12 antibodies were obtained from the hybridoma cells. In addition, the amino acid sequences of the heavy chain variable region, light chain variable region, and complementarity determining regions thereof as shown in Tables 1 to 3, and the nucleotide sequence of the gene encoding the antibody were determined.

The 2A11, 4C2, and 8C4 antibodies comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 21 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 27. In the 2A11 antibody, the 56th and 57th Xaa in SEQ ID NO: 21 is Gly, and the 53rd Xaa in SEQ ID NO: 27 is Ile. In the 4C2 antibody, the 56th Xaa in SEQ ID NO: 21 is Gly, the 57th Xaa is Ala, and in SEQ ID NO: 27 the 53rd Xaa is Phe. In the 8C4 antibody, the 56th Xaa in SEQ ID NO: 21 is Ala, the 57th Xaa is Gly, and in SEQ ID NO: 27 the 53rd Xaa is Phe.

In addition, VH-CDR1, -CDR2 and -CDR3 of antibodies 2A11, 4C2, and 8C4 consist of amino acid sequences of SEQ ID NOs: 33, 39 and 45, respectively, and VL-CDR1, -CDR2 and -CDR3 are SEQ ID NO: 51, respectively. It consists of the amino acid sequence of 57 and 63. In the 2A11 antibody, the 56th and 57th Xaas in SEQ ID NO: 21 are Gly. 4C2 antibody in SEQ ID NO: 21, the 56th Xaa is Gly, the 57th Xaa is Ala. In the 8C4 antibody, in SEQ ID NO: 21, the 56th Xaa is Ala, and the 57th Xaa is Gly.

Antibodies 9B3, 9B12 and 3B10 comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 23 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 29. In SEQ ID NO: 23, the first Xaa is Glu, the 7th Xaa is Ser, the 12th Xaa is Val, the 27th Xaa is Tyr, the 58th Xaa is Ser, the 61st Xaa is Asn, and 74 The th Xaa is Lys, the 83 th Xaa is Phe, the 92 th Xaa is Ala, and the 108 th Xaa is Tyr. In the 9B3 antibody, in SEQ ID NO: 29, the 23rd Xaa is Ile, the 45th Xaa is Ala, the 73rd Xaa is Glu, and the 100th Xaa is Ile. In SEQ ID NO: 23, the 1st Xaa is Gln, 7th Xaa is Ser, 12th Xaa is Val, 27th Xaa is Tyr, 58th Xaa is Ser, 61st Xaa is Asn, 74 The th Xaa is Arg, the 83 th Xaa is Phe, the 92 th Xaa is Gly, and the 108 th Xaa is His. In SEQ ID NO: 29, the 23rd Xaa is Met, the 45th Xaa is Ala, the 73rd Xaa is Glu, and the 100th Xaa is Met. In SEQ ID NO: 23, the 1st Xaa is Gln, 7th Xaa is Pro, 12th Xaa is Ala, 27th Xaa is His, 58th Xaa is Thr, 61st Xaa is Ser, 74 The th Xaa is Arg, the 83 th Xaa is Leu, the 92 th Xaa is Gly, and the 108 th Xaa is His. In the 3B10 antibody, in SEQ ID NO: 29, the 23rd Xaa is Met, the 45th Xaa is Ser, the 73rd Xaa is Asp, and the 100th Xaa is Ile.

In addition, VH-CDR1, -CDR2 and -CDR3 of antibodies 9B3, 9B12 and 3B10 consist of amino acid sequences of SEQ ID NOs: 35, 41 and 47, respectively, and VL-CDR1, -CDR2 and -CDR3 are respectively SEQ ID NOs: 53 and 59 And the amino acid sequence of 65. In the 9B3 antibody, the second Xaa in SEQ ID NO: 35 is Tyr, the 8th Xaa in SEQ ID NO: 41 is Ser, the 12th Xaa in SEQ ID NO: 47 is Tyr, the third Xaa in SEQ ID NO: 53 is Ile, and SEQ ID NO: 59 The second Xaa in is Ala. In the 9B12 antibody, the second Xaa in SEQ ID NO: 35 is Tyr, the 8th Xaa in SEQ ID NO: 41 is Ser, the 12th Xaa in SEQ ID NO: 47 is His, the third Xaa in SEQ ID NO: 53 is Met, and SEQ ID NO: 59 The second Xaa in is Ala. In the 3B10 antibody, the second Xaa in SEQ ID NO: 35 is His, the 8th Xaa in SEQ ID NO: 41 is Thr, the 12th Xaa in SEQ ID NO: 47 is His, the third Xaa in SEQ ID NO: 53 is Met, and SEQ ID NO: 59 The second Xaa in is Ser.

Antibody name Nucleotide sequence of VH gene Nucleotide sequence of VL gene 2A11 SEQ ID NO: 1 SEQ ID NO: 11 4C2 SEQ ID NO: 2 SEQ ID NO: 12 8C4 SEQ ID NO: 3 SEQ ID NO: 13 3B5 SEQ ID NO: 4 SEQ ID NO: 14 9B3 SEQ ID NO: 5 SEQ ID NO: 15 9B12 SEQ ID NO: 6 SEQ ID NO: 16 3B10 SEQ ID NO: 7 SEQ ID NO: 17 10H7 SEQ ID NO: 8 SEQ ID NO: 18 3G8 SEQ ID NO: 9 SEQ ID NO: 19 3F12 SEQ ID NO: 10 SEQ ID NO: 20

Antibody name Amino acid sequence of VH region Amino acid sequence of the VL region 2A11 SEQ ID NO: 21 SEQ ID NO: 27 4C2 SEQ ID NO: 21 SEQ ID NO: 27 8C4 SEQ ID NO: 21 SEQ ID NO: 27 3B5 SEQ ID NO: 22 SEQ ID NO: 28 9B3 SEQ ID NO: 23 SEQ ID NO: 29 9B12 SEQ ID NO: 23 SEQ ID NO: 29 3B10 SEQ ID NO: 23 SEQ ID NO: 29 10H7 SEQ ID NO: 24 SEQ ID NO: 30 3G8 SEQ ID NO: 25 SEQ ID NO: 31 3F12 SEQ ID NO: 26 SEQ ID NO: 32

Antibody name Amino acid sequence of VH-CDR1 Amino acid sequence of VH-CDR2 Amino acid sequence of VH-CDR3 Amino acid sequence of VL-CDR1 Amino acid sequence of VL-CDR2 Amino acid sequence of VL-CDR3 2A11 SEQ ID NO: 33 SEQ ID NO: 39 SEQ ID NO: 45 SEQ ID NO: 51 SEQ ID NO: 57 SEQ ID NO: 63 4C2 SEQ ID NO: 33 SEQ ID NO: 39 SEQ ID NO: 45 SEQ ID NO: 51 SEQ ID NO: 57 SEQ ID NO: 63 8C4 SEQ ID NO: 33 SEQ ID NO: 39 SEQ ID NO: 45 SEQ ID NO: 51 SEQ ID NO: 57 SEQ ID NO: 63 3B5 SEQ ID NO: 34 SEQ ID NO: 40 SEQ ID NO: 46 SEQ ID NO: 52 SEQ ID NO: 57 SEQ ID NO: 64 9B3 SEQ ID NO: 35 SEQ ID NO: 41 SEQ ID NO: 47 SEQ ID NO: 53 SEQ ID NO: 58 SEQ ID NO: 65 9B12 SEQ ID NO: 35 SEQ ID NO: 41 SEQ ID NO: 47 SEQ ID NO: 53 SEQ ID NO: 59 SEQ ID NO: 65 3B10 SEQ ID NO: 35 SEQ ID NO: 41 SEQ ID NO: 47 SEQ ID NO: 53 SEQ ID NO: 59 SEQ ID NO: 65 10H7 SEQ ID NO: 36 SEQ ID NO: 42 SEQ ID NO: 48 SEQ ID NO: 54 SEQ ID NO: 60 SEQ ID NO: 66 3G8 SEQ ID NO: 37 SEQ ID NO: 43 SEQ ID NO: 49 SEQ ID NO: 55 SEQ ID NO: 61 SEQ ID NO: 67 3F12 SEQ ID NO: 38 SEQ ID NO: 44 SEQ ID NO: 50 SEQ ID NO: 56 SEQ ID NO: 62 SEQ ID NO: 68

2. Confirmation of antigen-antibody reaction of anti-BAG2 antibody in breast cancer cells

1 is a diagram showing the results of immunoblotting of anti-BAG2 antibodies produced from 10 types of mouse hybridoma cells. Specifically, MDA-MB-231 cells, which are human breast cancer cells, were cultured in DMEM (Welgene) medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C. The cells were harvested from the wells and washed with PBS, 1% Brij 97, 5mM EDTA, 0.02M HEPES pH 7.3, 0.15M NaCl, 1mM PMSF, 0.5mM NaF, 10 μg/ml aprotinin, 0.2mM sodium orthovanadate (sodium orthovanadate) was dissolved in a lysis buffer containing. After incubation on ice for 15 minutes, the nuclei were removed by centrifugation and the supernatant was collected. A 2X sample buffer consisting of 20% glycerol, 4.6% SDS, 0.125M Tris, pH 6.8, and 0.1% bromophenol blue was added to the appropriate amount of the supernatant. A 10 ug protein sample was subjected to SDS-PAGE analysis on a 12% gel under standard conditions using a mini-Protean II system (Bio-Rad Hercules, CA). For immunoblotting, the protein was transferred to the PVDF membrane, Millipore. A blocking solution consisting of 0.1% Tween 20 and 5% bovine serum albumin (BSA) added to TBS was reacted for 1 hour. Thereafter, as the primary antibody, the anti-BAG2 antibody extracted from the hybridoma cell culture was used at a dilution of 1/2000, and the goat anti-mouse HRP conjugate (Dako) used as the secondary antibody was used at a dilution of 1/5000. Used. Film photosensitized in the dark using ECL reagent (Amersham Pharmacia Biotech) as a substrate. The photosensitive band was compared with a standard molecular marker to identify a band corresponding to the size of BAG2.

As a result, as shown in Figure 1, compared with the commercial polyclonal anti-BAG2 antibody ab58682 (Abcam) used as a positive control, the antibodies 2A11, 3B5, 3B10, 3F12, 3G8, 4C2, 8C4, 9B3, 9B12 and 10H7 showed antigen-antibody reactions by targeting BAG2.

Next, for domain mapping of the BAG2 antigen to which the 10 antibodies identified in Section 1 bind, a GST-Empty vector having a molecular weight of about 26 kDa (pcDNA3.1+/GST vector, NovoPro Bioscience Inc. China) was introduced as a negative control. GST-Bag Full vector, GST-Bag F1 vector, GST-Bag F2 vector, GST-Bag F3 vector, and GST-Bag F4 comprising a polynucleotide encoding a human BAG2 protein and a polynucleotide encoding a fragment of the BAG2 protein The vector was introduced into cells, and the cells into which the vector was introduced were cultured to express the gene, and then a cell lysate was obtained. Immunoblotting was performed for the cell lysate using each of the above 10 types of antibodies. The polynucleotide encoding the human BAG2 protein has the nucleotide sequence of SEQ ID NO: 70. The GST-Bag F1, -Bag F2, -Bag F3, and -Bag F4 vectors consist of nucleotide sequences of SEQ ID NOs: 71 to 74, respectively.

2 is a diagram showing the results of immunoblotting for full-length BAG2 polypeptide or fragment thereof of an anti-BAG2 antibody. In Figure 2, A schematically shows the vector and BAG2 protein and fragments thereof, and B is the result of immunoblotting. Specifically, the immunoblotting is performed by introducing each vector into HEK293T cells using a lipofectamine transfection (lipofectamine transfection, Thermo Fisher Scientific, Inc., Waltham, MA, USA) method, and the resulting transformed cells At 37° C., cells were isolated after culturing in DMEM (Welgene) medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin for 30 hours. The separated cells were crushed using the same method as in FIG. 1 and subjected to SDS-PAGE analysis on a 12% gel. For immunoblotting, after reacting with the same blocking solution as in FIG. 1 for 1 hour, 10 kinds of purified anti-BAG2 antibodies at a concentration of 2 mg/ml were diluted 1/10000 with the primary antibody and bound I did. The goat anti-mouse HRP conjugate used as the secondary antibody was used at a 1/5000 dilution concentration, and film-sensitized in the dark using ECL reagent (Amersham Pharmacia Biotech) as a substrate. In order to confirm the size of BAG2, the standard molecular marker size was expressed.

As a result, as shown in Fig. 2, each anti-BAG2 antibody differentially bound to the full-length BAG2 polypeptide or fragment thereof. In particular, all of the 10 anti-BAG2 antibodies are commonly detected at about 50 KDa position in the cell lysate into which the GST-Bag Full vector has been introduced, indicating that all of these antibodies can bind to the full-length BAG2 polypeptide. Finally, the domain region of BAG2 to which each anti-BAG2 antibody reacts was confirmed.

3 is a diagram showing a BAG2 domain to which each anti-BAG2 antibody reacts. As shown in Figure 3, 9B3, 9B12, 3B10 and 10H7 antibodies bound to the N-terminus of the BAG2 protein, 2A11, 3B5, 4C2 and 8C4 antibodies bound to the middle region, and 3F12 and 3G8 antibodies bound to the C-terminus. The N-terminus commonly includes a coiled coil region of 21-60 amino acids, and the intermediate region binds to a part of the BNB region of 109-189 amino acids. Therefore, by using a set of antibodies that bind to different sites, the BAG2 protein or fragment thereof present in the sample can be detected with high sensitivity and specificity.

Korea Research Institute of Bioscience and Biotechnology KCTC13737BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13738BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13739BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13740BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13741BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13742BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13743BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13744BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13745BP 20181128 Korea Research Institute of Bioscience and Biotechnology KCTC13746BP 20181128

<110> Medpacto Inc. <120> A Composition comprising antibody specifically binding to BAG2 for the diagnosis of cancer and method using the same <130> PN119594 <160> 74 <170> KoPatentIn 3.0 <210> 1 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> 2A11-VH <400> 1 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgacgctg 60 tcctgcaagg cttcgggcta cacatttact gactatgaaa tgcactgggt gaagcagact 120 cctgtgcatg gcctggaatg gattggagtt attgatcctg aaactggtgc tactgcctac 180 aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tacagcctac 240 atggagctcc gcagcctgac atctgaggac tctgccgtct attactgtac aagagggaaa 300 ttttattact ccggtcggga ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 2 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> 4C2-VH <400> 2 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgacgctg 60 tcctgcaagg cttcgggcta cacatttact gactatgaaa tgcactgggt gaagcagact 120 cctgtgcatg gcctggaatg gattggagtt attgatcctg aaactggtgc tactgcctac 180 aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tacagcctac 240 atggagctcc gcagcctgac atctgaggac tctgccgtct attactgtac aagagggaaa 300 ttttattact ccggtcggga ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 3 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> 8C4-VH <400> 3 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgacgctg 60 tcctgcaagg cttcgggcta cacatttact gactatgaaa tgcactgggt gaagcagact 120 cctgtgcatg gcctggaatg gattggagtt attgatcctg aaactgctgg tactgcctac 180 aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag tacagcctac 240 atggagctcc gcagcctgac atctgaggac tctgccgtct attactgtac aagagggaaa 300 ttttattact ccggtcggga ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360 gtctcctca 369 <210> 4 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> 3B5-VH <400> 4 gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagtta 60 tcctgcaagg cttctggtta ctcattcact gactacacct tttactgggt gaggcagagc 120 catggagaga gccttgagtg gattggatat attgatcctt acaatggtgg taatacttat 180 aaccggaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc 240 atgcatctca acagcctgac atctgaagac tctgcagtct attactgtgc gagagggtac 300 tataggtacg gggggggggg ggactttgac tactggggcc aaggcaccac tctcacagtc 360 tcctca 366 <210> 5 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 9B3-VH <400> 5 gaggtccagc tgcaacaatc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tagttattac 180 aatgagaagg tcaagggcaa ggcaacactg accgcagaca aatcctccag cactgcctac 240 atgcagttca gcagcctgac agctgatgac tctgcggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ctattggggc caaggcacca ctctcacagt ctcctca 357 <210> 6 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 9B12-VH <400> 6 caggtccaac tgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tagttataac 180 aatgagaagg tcaagggcaa ggcaacactg accgcagaca gatcctccag cactgcctac 240 atgcagttca gcagcctgac agctgatgac tctggggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ccattggggc caaggcacca ctctcacagt ctcctca 357 <210> 7 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 3B10-VH <400> 7 caggtccaac tgcagcagcc tggagctgag ctggcaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggcca cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tacttataac 180 agtgagaagg tcaagggcaa ggcaacactg accgcagaca gatcctccag cactgcctac 240 atgcagctca gcagcctgac agctgatgac tctggggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ccattggggc caaggcacca ctctcacagt ctcctca 357 <210> 8 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> 10H7-VH <400> 8 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctggtta taccttcaca gactattcaa ttcactgggt gaggcaggct 120 ccaggaaagg gttttaagtg gatgggctgg ataaacactg agactggtga gccaacatat 180 gcagatgact tcaagggacg gtttgccctc tctttggaaa cctctgccag cactgcctac 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc tagatttgac 300 tacggtacta gttactggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360 tca 363 <210> 9 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 3G8-VH <400> 9 cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta tagcttcaca aagtatggaa tgaactgggt gaagcaggct 120 ccaggaaagg atatcaagtg gatggggtgg ataaacacca acactggaga ggcaacatat 180 ggtgaagagg tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240 ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagattggga 300 ttgaggtacc ttgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 3F12-VH <400> 10 caggtgcaac tgcaggagtc aggacctgac ctggtgaaac cttctcagtc actttcactc 60 acctgcactg tcactgggta ctccatcacc agtggttata gctggcactg gatccggcag 120 tttccaggaa acaaattgga atggatgggc tacatatatt atagaggtag cactaactac 180 aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctgctgttga aatctgtgac tactgaggac acagccacat attactgtgc aagagaggct 300 tactggggcc aagggactct ggtcactgtc tcagca 336 <210> 11 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 2A11-VL <400> 11 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 12 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 4C2-VL <400> 12 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgttct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 13 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 8C4-VL <400> 13 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagtattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgttct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acaggttcct 300 ccgacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 14 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 3B5-VL <400> 14 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatccagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatcc acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggaatt tatttctgct ctcaaaatac acatattcct 300 ccgacgttcg ctggaggcac caagctggaa atcaaa 336 <210> 15 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> 9B3-VL <400> 15 gaggtccagc tgcaacaatc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagg cttctggata cgccttcact aattacatga tagagtggat aaaacagagg 120 cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tagttattac 180 aatgagaagg tcaagggcaa ggcaacactg accgcagaca aatcctccag cactgcctac 240 atgcagttca gcagcctgac agctgatgac tctgcggtct atttctgtcg gatctatggt 300 aactacaagg ggtactttga ctattggggc caaggcacca ctctcacagt ctcctca 357 <210> 16 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 9B12-VL <400> 16 gacattgtga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacatgaat agctatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctacag aatgatgagt ttccattcac gttcggctcg 300 gggacaaagc tggaaatgaa g 321 <210> 17 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 3B10-VL <400> 17 gacattgtga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacatgaat agctatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt tcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggactat 240 gaagatatgg gaatttatta ttgtctacag aatgatgagt ttccattcac gttcggctcg 300 gggacaaagc tggaaataaa a 321 <210> 18 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 10H7-VL <400> 18 gatgtccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagccacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaaccc 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacacta ccccgctcac tttcggtgga 300 ggcaccaagc tggaaatcaa acgtggagga gccagcctcg tggaattcaa g 351 <210> 19 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> 3G8-VL <400> 19 gacattgtga tgacccagtc tcctgcttcc ttagttgtat ctctggggca gagggccacc 60 atctcatgca gggccagcaa aagtgtcagt acatctgact atagttatat gcactggtac 120 caacagaaac caggacagcc acccaaagtc ctcatctatc ttgcatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acaataggga gcttcctccc 300 acgttcggtg ctgggaccaa gctggagctg aaa 333 <210> 20 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> 3F12-VL <400> 20 gatgttttga tgacccaaac tccactcact ttgtcggtta cctttgggca gccagcctcc 60 atctcttgca ggtcaagtca gagcctctta gatagtgatg gagagacata tttgaattgg 120 ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180 tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttccg 300 tacacgttcg gaggggggac caagctggaa ataaaa 336 <210> 21 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VH <220> <221> VARIANT <222> (56)..(57) <223> Xaa is Gly or Ala <400> 21 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asp Pro Glu Thr Xaa Xaa Thr Ala Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Lys Phe Tyr Tyr Ser Gly Arg Asp Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 22 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VH <400> 22 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Phe Tyr Trp Val Arg Gln Ser His Gly Glu Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Asn Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Arg Tyr Gly Gly Gly Gly Asp Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 23 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VH <220> <221> VARIANT <222> (1) <223> Xaa is Glu or Gln <220> <221> VARIANT <222> (7) <223> Xaa is Ser or Pro <220> <221> VARIANT <222> (12) <223> Xaa is Val or Ala <220> <221> VARIANT <222> (27) <223> Xaa is Tyr or His <220> <221> VARIANT <222> (58) <223> Xaa is Ser or Thr <220> <221> VARIANT <222> (61) <223> Xaa is Asn or Ser <220> <221> VARIANT <222> (74) <223> Xaa is Arg or Lys <220> <221> VARIANT <222> (83) <223> Xaa is Phe or Leu <220> <221> VARIANT <222> (92) <223> Xaa is Gly or Ala <220> <221> VARIANT <222> (108) <223> Xaa is His or Tyr <400> 23 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Met Ile Glu Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Tyr Tyr Asn Glu Lys Val 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Phe Ser Ser Leu Thr Ala Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Arg Ile Tyr Gly Asn Tyr Lys Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 24 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VH <400> 24 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Phe Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Leu Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Phe Asp Tyr Gly Thr Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 25 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VH <400> 25 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Lys Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Asp Ile Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu Ala Thr Tyr Gly Glu Glu Val 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Leu Arg Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VH <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Tyr Tyr Arg Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Leu Leu Lys Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 <210> 27 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VL <220> <221> VARIANT <222> (53) <223> Xaa is Ile or Phe <400> 27 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 Xaa 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 Phe Cys Phe Gln Gly 85 90 95 Ser Gln Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 28 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VL <400> 28 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 Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile His 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 Ile Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Ile Pro Pro Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 29 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VL <220> <221> VARIANT <222> (23) <223> Xaa is Met or Ile <220> <221> VARIANT <222> (45) <223> Xaa is Ala or Ser <220> <221> VARIANT <222> (73) <223> Xaa is Glu or Asp <220> <221> VARIANT <222> (100) <223> Xaa is Met or Ile <400> 29 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Xaa Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Xaa Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Xaa Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Asn Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Xaa Lys 100 105 <210> 30 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VL <400> 30 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 31 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VL <400> 31 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Val Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Asp Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Val Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Asn Arg 85 90 95 Glu Leu Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 32 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VL <400> 32 Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Phe Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Glu Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr 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 Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VHCDR1 <400> 33 Gly Tyr Thr Phe Thr Asp Tyr Glu 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VHCDR1 <400> 34 Gly Tyr Ser Phe Thr Asp Tyr Thr 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VHCDR1 <220> <221> VARIANT <222> (2) <223> Xaa is Tyr or His <400> 35 Gly Xaa Ala Phe Thr Asn Tyr Met 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VHCDR1 <400> 36 Gly Tyr Thr Phe Thr Asp Tyr Ser 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VHCDR1 <400> 37 Gly Tyr Ser Phe Thr Lys Tyr Gly 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VHCDR1 <400> 38 Gly Tyr Ser Ile Thr Ser Gly Tyr Ser 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VHCDR2 <220> <221> VARIANT <222> (6)..(7) <223> Xaa is Gly or Ala <400> 39 Ile Asp Pro Glu Thr Xaa Xaa Thr 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VHCDR2 <400> 40 Ile Asp Pro Tyr Asn Gly Gly Asn 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B5-VHCDR2 <220> <221> VARIANT <222> (8) <223> Xaa is Ser or Thr <400> 41 Ile Asn Pro Gly Ser Gly Gly Xaa 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VHCDR2 <400> 42 Ile Asn Thr Glu Thr Gly Glu Pro 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VHCDR2 <400> 43 Ile Asn Thr Asn Thr Gly Glu Ala 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VHCDR2 <400> 44 Ile Tyr Tyr Arg Gly Ser Thr 1 5 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VHCDR3 <400> 45 Thr Arg Gly Lys Phe Tyr Tyr Ser Gly Arg Asp Tyr Ala Met Asp Tyr 1 5 10 15 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VHCDR3 <400> 46 Ala Arg Gly Tyr Tyr Arg Tyr Gly Gly Gly Gly Asp Phe Asp Tyr 1 5 10 15 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VHCDR3 <220> <221> VARIANT <222> (12) <223> Xaa is Tyr or His <400> 47 Arg Ile Tyr Gly Asn Tyr Lys Gly Tyr Phe Asp Xaa 1 5 10 <210> 48 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VHCDR3 <400> 48 Ala Arg Phe Asp Tyr Gly Thr Ser Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VHCDR3 <400> 49 Ala Arg Leu Gly Leu Arg Tyr Leu Asp Tyr 1 5 10 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VHCDR3 <400> 50 Ala Arg Glu Ala Tyr 1 5 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VLCDR1 <400> 51 Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VLCDR1 <400> 52 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 53 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VLCDR1 <220> <221> VARIANT <222> (3) <223> Xaa is Ile or Met <400> 53 Gln Asp Xaa Asn Ser Tyr 1 5 <210> 54 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VLCDR1 <400> 54 Gln Ser Ile Ser Ser Tyr 1 5 <210> 55 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VLCDR1 <400> 55 Lys Ser Val Ser Thr Ser Asp Tyr Ser Tyr 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VLCDR1 <400> 56 Gln Ser Leu Leu Asp Ser Asp Gly Glu Thr Tyr 1 5 10 <210> 57 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VLCDR2 <400> 57 Lys Val Ser One <210> 58 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VLCDR2 <400> 58 Lys Val Ser One <210> 59 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VLCDR2 <220> <221> VARIANT <222> (2) <223> Xaa is Ala or Ser <400> 59 Arg Xaa Asn One <210> 60 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VLCDR2 <400> 60 Ala Ala Ser One <210> 61 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VLCDR2 <400> 61 Leu Ala Ser One <210> 62 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VLCDR2 <400> 62 Leu Val Ser One <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 2A11,4C2,8C4-VLCDR3 <400> 63 Phe Gln Gly Ser Gln Val Pro Pro Thr 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3B5-VLCDR3 <400> 64 Ser Gln Asn Thr His Ile Pro Pro Thr 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 9B3,9B12,3B10-VLCDR3 <400> 65 Leu Gln Asn Asp Glu Phe Pro Phe Thr 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 10H7-VLCDR3 <400> 66 Gln Gln Ser Tyr Thr Thr Pro Leu Thr 1 5 <210> 67 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3G8-VLCDR3 <400> 67 Gln His Asn Arg Glu Leu Pro Pro Thr 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 3F12-VLCDR3 <400> 68 Trp Gln Gly Thr His Phe Pro Tyr Thr 1 5 <210> 69 <211> 211 <212> PRT <213> Homo sapiens <400> 69 Met Ala Gln Ala Lys Ile Asn Ala Lys Ala Asn Glu Gly Arg Phe Cys 1 5 10 15 Arg Ser Ser Ser Met Ala Asp Arg Ser Ser Arg Leu Leu Glu Ser Leu 20 25 30 Asp Gln Leu Glu Leu Arg Val Glu Ala Leu Arg Glu Ala Ala Thr Ala 35 40 45 Val Glu Gln Glu Lys Glu Ile Leu Leu Glu Met Ile His Ser Ile Gln 50 55 60 Asn Ser Gln Asp Met Arg Gln Ile Ser Asp Gly Glu Arg Glu Glu Leu 65 70 75 80 Asn Leu Thr Ala Asn Arg Leu Met Gly Arg Thr Leu Thr Val Glu Val 85 90 95 Ser Val Glu Thr Ile Arg Asn Pro Gln Gln Gln Glu Ser Leu Lys His 100 105 110 Ala Thr Arg Ile Ile Asp Glu Val Val Asn Lys Phe Leu Asp Asp Leu 115 120 125 Gly Asn Ala Lys Ser His Leu Met Ser Leu Tyr Ser Ala Cys Ser Ser 130 135 140 Glu Val Pro His Gly Pro Val Asp Gln Lys Phe Gln Ser Ile Val Ile 145 150 155 160 Gly Cys Ala Leu Glu Asp Gln Lys Lys Ile Lys Arg Arg Leu Glu Thr 165 170 175 Leu Leu Arg Asn Ile Glu Asn Ser Asp Lys Ala Ile Lys Leu Leu Glu 180 185 190 His Ser Lys Gly Ala Gly Ser Lys Thr Leu Gln Gln Asn Ala Glu Ser 195 200 205 Arg Phe Asn 210 <210> 70 <211> 636 <212> DNA <213> Homo sapiens <400> 70 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 gtcaataagt ttctggatga tttgggaaat gccaagagtc atttaatgtc gctctacagt 420 gcatgttcat ctgaggtgcc acatgggcca gttgatcaga agtttcaatc catagtaatt 480 ggctgtgctc ttgaagatca gaagaaaatt aagagaagat tagagactct gcttagaaat 540 attgaaaact ctgacaaggc catcaagcta ttagagcatt ctaaaggagc tggttccaaa 600 actctgcaac aaaatgctga aagcagattc aattag 636 <210> 71 <211> 270 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F1 vector <400> 71 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga 270 <210> 72 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F2 vector <400> 72 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 360 <210> 73 <211> 450 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F3 vector <400> 73 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 gtcaataagt ttctggatga tttgggaaat gccaagagtc atttaatgtc gctctacagt 420 gcatgttcat ctgaggtgcc acatgggcca 450 <210> 74 <211> 540 <212> DNA <213> Artificial Sequence <220> <223> GST-Bag F4 vector <400> 74 atggctcagg cgaagatcaa cgctaaagcc aacgaggggc gcttctgccg ctcctcctcc 60 atggctgacc gctccagccg cctgctggag agcctggacc agctggagct cagggttgaa 120 gctttgagag aagcagcaac tgctgttgag caagagaaag aaatccttct ggaaatgatc 180 cacagtatcc aaaatagcca ggacatgagg cagatcagtg acggagaaag agaagaatta 240 aatctgactg caaaccgttt gatgggaaga actctcaccg ttgaagtgtc agtagaaaca 300 attagaaacc cccagcagca agaatcccta aagcatgcca caaggattat tgatgaggtg 360 gtcaataagt ttctggatga tttgggaaat gccaagagtc atttaatgtc gctctacagt 420 gcatgttcat ctgaggtgcc acatgggcca gttgatcaga agtttcaatc catagtaatt 480 ggctgtgctc ttgaagatca gaagaaaatt aagagaagat tagagactct gcttagaaat 540 540

Claims (14)

An antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.
The method of claim 1, wherein the antibody or antigen-binding fragment thereof
A heavy chain variable region comprising a complementarity determining region (VH-CDR)1 consisting of the amino acid sequence of SEQ ID NO: 33, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; And a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63. Doing,
A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46; And a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 64,
A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47; And a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 65,
A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48; And a light chain variable region VL comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 54, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 60, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 66,
A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49; And a light chain variable region including VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67,
A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 44, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50; And a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68, or An antibody or antigen-binding fragment thereof that is a combination.
The method of claim 1, wherein the antibody or antigen-binding fragment thereof
A heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 26;
A light chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 32;
Or an antibody or antigen-binding fragment thereof comprising the heavy chain variable region and the light chain variable region.
The method according to claim 1, wherein the heavy chain variable region of SEQ ID NO: 21 and the light chain variable region of SEQ ID NO: 27; The heavy chain variable region of SEQ ID NO: 22 and the light chain variable region of SEQ ID NO: 28; The heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 29; The heavy chain variable region of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO: 30; The heavy chain variable region of SEQ ID NO: 25 and the light chain variable region of SEQ ID NO: 31; An antibody or an antigen-binding fragment thereof that includes the heavy chain variable region of SEQ ID NO: 26 and the light chain variable region of SEQ ID NO: 32, or a combination thereof.
The antibody or antigen-binding fragment thereof according to claim 1, which is a monoclonal antibody.
The antibody or antigen-binding fragment thereof according to claim 1, which is labeled with a detectable label or a label capable of emitting a detectable signal.
The method according to claim 1, High selected from the group consisting of hybridoma cells deposited with accession numbers KCTC 13737BP, KCTC 13738BP, KCTC 13739BP, KCTC 13740BP, KCTC 13741BP, KCTC 13742BP, KCTC 13743BP, KCTC 13744BP, KCTC 13745BP and KCTC 13746BP. An antibody or antigen-binding fragment thereof that can be produced by bridoma cells.
The antibody or antigen-binding fragment thereof according to claim 1, comprising a plurality of antibodies or antigen-binding fragments thereof.
A polynucleotide comprising a polynucleotide encoding the antibody of any one of claims 1 to 8 or an antigen-binding fragment thereof.
The polynucleotide of claim 9, which is a vector.
The polynucleotide of claim 10, wherein a detectable label or a label capable of emitting a detectable signal is conjugated.
A host cell comprising the polynucleotide of claim 9.
Culturing the host cell of claim 12; And
A method for producing an antibody or antigen-binding fragment thereof, comprising the step of isolating the antibody or antigen-binding fragment thereof from the obtained culture.
The method of claim 13, further comprising labeling the produced antibody or antigen-binding fragment thereof.

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