Methods for diagnosing cancer using anti-BAG2 antibody

Abstract

The present application discloses a composition for use in diagnosing cancer, the composition comprising: an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof.

Claims

1. A composition for use in diagnosing cancer, the composition comprising: an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 in which 6th Xaa and 7th Xaa are each Gly, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising VL-CDR1 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; and an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a 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 a 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.

2. The composition of claim 1, wherein the antibody or antigen-binding fragment is labeled with a detectable label or a label capable of emitting a detectable signal.

3. The composition of claim 1, wherein the cancer is breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, or sarcoma.

4. A kit for use in diagnosing cancer, the kit comprising: the composition of claim 1.

5. A composition for use in diagnosing cancer, the composition comprising: an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35 in which 2nd Xaa is Tyr, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41 in which 8th Xaa is Ser, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47 in which 12th Xaa is His and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53 in which 3rd Xaa is Met, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59 in which 2nd Xaa is Ala, and VL-CDR3 consisting of amino acid sequence of SEQ ID NO: 65; and an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a VH-CDR 1 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 comprising a 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.

6. The composition of claim 5, wherein the antibody or antigen-binding fragment is labeled with a detectable label or a label capable of emitting a detectable signal.

7. The composition of claim 5, wherein the cancer is breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, or sarcoma.

8. A kit for use in diagnosing cancer, the kit comprising: the composition of claim 5.

9. A composition for use in diagnosing cancer, the composition comprising: an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 33, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 in which 6th Xaa and 7th Xaa are Ala and Gly, respectively, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45 and a light chain variable region comprising VL-CDR1 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; and an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a VH-CDR 1 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 comprising a 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.

10. The composition of claim 9, wherein the antibody or antigen-binding fragment is labeled with a detectable label or a label capable of emitting a detectable signal.

11. The composition of claim 9, wherein the cancer is breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, or sarcoma.

12. A kit for use in diagnosing cancer, the kit comprising: the composition of claim 9.

13. A composition for use in diagnosing cancer, the composition comprising: an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a VH-CDR 1 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 a 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; and an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a VH-CDR 1 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 comprising a 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.

14. The composition of claim 13, wherein the antibody or antigen-binding fragment is labeled with a detectable label or a label capable of emitting a detectable signal.

15. The composition of claim 13, wherein the cancer is breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, or sarcoma.

16. A kit for use in diagnosing cancer, the kit comprising: the composition of claim 13 .

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;

(2) FIG. 1 shows the results of Western blotting of anti-BAG2 antibodies produced from 10 mouse hybridoma cells;

(3) FIGS. 2A and 2B show the results of Western blotting for the full-length BAG2 polypeptide of anti-BAG2 antibody or a fragment thereof;

(4) FIG. 3 shows a BAG2 domain which reacts with respective anti-BAG2 antibodies;

(5) FIG. 4 shows standard curves for BAG2 protein in possible 90 combinations of anti-BAG2 antibodies;

(6) FIG. 5 shows a diagram showing the specific binding force of the selected 14 antibody combinations with respect to the BAG2 protein;

(7) FIGS. 6A and 6B shows differences in BAG2 expression patterns in various cancer cell lines observed using the antibody combination of 2A11-3F12 and the antibody combination of 9B 12-3G8;

(8) FIGS. 7A and 7B shows significant differences in BAG2 protein expression in the serum of luminal type and TNBC type breast cancer patients observed by using the antibody combination of 2A11-3F12 and the antibody combination of 9B12-3G8;

(9) FIGS. 8A and 8B shows significant differences in BAG2 protein expression in the serum of luminal type and TNBC type breast cancer patients observed by using the antibody combination of 8C4-3G8 and the antibody combination of 10H7-3G8; and

(10) FIGS. 9A, 9B, 9C and 9D show the receiver operating characteristic curve (ROC) values and area under the curve (AUC) values of the four antibody combinations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(11) The following examples are offered by way of illustration of the present invention, and not by way of limitation.

EXAMPLES

Example 1: Selection, Sequencing and Antigen-Antibody Reaction of Anti-Bag2 Antibody

(12) 1. Selection of Monoclonal Antibodies Targeting Bag2 and Analysis of Amino Acid Sequences Thereof

(13) The inventors selected antibodies targeting Bag2, analyzed their amino acid sequences, and determined the complementarity determining region (CDR) of each of the antibodies.

(14) In detail, a gene consisting of the nucleotide sequence of SEQ ID NO: 70 encoding the human BAG2 protein consisting of the amino acid sequence of SEQ ID NO: 69 was cloned into a pCAGGS plasmid and linearized, and then the linearized construct was inoculated into the muscles of five 6-week-old female BALB/c mice by the appliance of electroshock. The construct was inoculated intramuscularly three times at three-week intervals, and consisted of 100 ug of DNA in 100 ul of PBS. At this time, the plasmid of the control group was also subjected to the same manner. To produce therapeutic and diagnostic antibodies, a more efficient DNA vaccine-based immunization strategy than protein-based antigen injection was performed. Blood was collected from the fundus vena cava or the caudal vein of the mouse, and examined by enzyme immunoassay showing the serum antibody titer, and spleens were extracted 3 days after the last immunization from the mouse showing sufficient antibody titer. B lymphocytes were isolated from the spleen, followed by fusion with the myeloma cells cultured with the isolated B lymphocytes, that is, the SP2/0-Ag14 cell line of ATCC, thereby obtaining fused cells. After fused cells were cultured in HAT medium containing hypoxanthin, aminopterine, and thymidine, hybridoma cells fused only with myeloma and B lymphocytes were obtained by selecting approximately 130 clones. Of the hybridoma cells obtained through the selection process by immunoblotting, 10 hybridoma cells producing antibodies specifically binding to human BAG2 protein were obtained.

(15) Total RNA of anti-BAG2 antibodies was produced from the 510.sup.6 hybridoma cells, and 5-RACE-cDNA was produced from 100 ng total RNA by using SMART RACE cDNA Amplification kit (Clontech) according to the instructions of the manufacturer. A heavy chain variable region (VH) and a light chain variable region (VL) coding regions were amplified by PCR, and the amplified genes were inserted into the pGEM-T vector (Promega, USA), cloned, and nucleotide sequences thereof were analyzed by using an automated genetic analyzer (ABI Prism 310, Applied Biosystem Co.). The nucleotide sequences of the analyzed genes were identified by comparison with the previously reported nucleotide sequence, and the identified nucleotide sequences were artificially translated for use in determining sequences of complementarity determining regions VH-CDR1, -CDR2, and -CDR3 and VL-CDR1, -CDR2, and -CDR3. The determining the sequences of complementarity determining regions was performed using Kabat's database (http://www.bioinf.org.uk/abs/).

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

(17) 2A11, 4C2, and 8C4 antibodies include a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 21 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 27. In the 2A11 antibody, the 56th and 57th Xaa of SEQ ID NO: 21 are each Gly and the 53rd Xaa of SEQ ID NO: 27 is lie. In the 4C2 antibody, 56th Xaa and 57th Xaa of SEQ ID NO: 21 are Gly and Ala, respectively, and 53rd Xaa of SEQ ID NO: 27 is Phe. In the 8C4 antibody, 56th Xaa and 57th Xaa of SEQ ID NO: 21 are Ala and Gly, respectively, and 53rd Xaa of SEQ ID NO: 27 is Phe.

(18) VH-CDR1, -CDR2 and -CDR3 of 2A11, 4C2, and 8C4 antibodies consist of amino acid sequences of SEQ ID NOS: 33, 39 and 45, respectively, and VL-CDR1, -CDR2 and -CDR3 consist of amino acid sequences of SEQ ID NOS: 51, 57, and 63, respectively. In 2A11 antibody, 56th and 57th Xaa of SEQ ID NO: 21 each are Gly. Regarding 4C2 antibody, in SEQ ID NO: 21, 56th Xaa is Gly and 57th Xaa is Ala. Regarding 8C4 antibody, in SEQ ID NO: 21, 56th Xaa is Ala and 57th Xaa is Gly.

(19) 9B3, 9B12 and 3B10 antibodies include a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 23 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 29. Regarding 9B3 antibody, in SEQ ID NO: 23, 1st Xaa is Glu, 7th Xaa is Ser, 12th Xaa is Val, 27th Xaa is Tyr, 58th Xaa is Ser, 61st Xaa is Asn, 74th Xaa is Lys, 83rd Xaa is Phe, 92nd Xaa is Ala, and 108th Xaa is Tyr. Regarding 9B3 antibody, in SEQ ID NO: 29, 29th Xaa is Ile, 51th Xaa is Ala, 79th Xaa is Glu, and 106th Xaa is Ile. Regarding 9B12 antibody, in SEQ ID NO:23 1st Xaa is Gin, 7th Xaa is Ser, 12th Xaa is Val, 27th Xaa is Tyr, 58th Xaa is Ser, 61st Xaa is Asn, 74th Xaa is Arg, 83rd Xaa is Phe, 92nd Xaa is Gly, and 108th Xaa is His. Regarding 9B12 antibody, in SEQ ID NO: 29, 29th Xaa is Met, 51th Xaa is Ala, 79th Xaa is Glu, and 106th Xaa is Met. Regarding 3B10 antibody, in SEQ ID NO: 23, 1st Xaa is Gln, 7th Xaa is Pro, 12th Xaa is Ala, 27th Xaa is His, 58th Xaa is Thr, 61st Xaa is Ser, 74th Xaa is Arg, 83rd Xaa is Leu, 92th Xaa is Gly, and 108th Xaa is His. Regarding 3B10 antibody, in SEQ ID NO: 29, 29th Xaa is Met, 51th Xaa is Ser, 79th Xaa is Asp, and 106th Xaa is Ile.

(20) Regarding 9B3, 9B12, and 3B10 antibodies, VH-CDR1, -CDR2, and -CDR3 consist of the amino acid sequences of SEQ ID NOS: 35, 41, and 47, respectively, and VL-CDR1, -CDR2, and -CDR3 consist of the amino acid sequences of SEQ ID NOS: 53, 59, and 65, respectively. Regarding 9B3 antibody, 2nd Xaa of SEQ ID NO: 35 is Tyr, 8th Xaa of SEQ ID NO: 41 is Ser, 12th Xaa of SEQ ID NO: 47 is Tyr, 3rd Xaa of SEQ ID NO: 53 is lie, and 2nd Xaa of SEQ ID NO: 59 is Ala. Regarding 9B12 antibody, 2nd Xaa of SEQ ID NO: 35 is Tyr, 8th Xaa of SEQ ID NO: 41 is Ser, 12th Xaa of SEQ ID NO: 47 is His, 3rd Xaa of SEQ ID NO: 53 is Met, and 2nd Xaa of SEQ ID NO: 59 is Ala. Regarding 3B10 antibody, 2nd Xaa of SEQ ID NO: 35 is His, 8th Xaa of SEQ ID NO: 41 is Thr, 12th Xaa of SEQ ID NO: 47 is His, 3rd Xaa of SEQ ID NO: 53 is Met, and 2nd Xaa of SEQ ID NO: 59 is Ser.

(21) TABLE-US-00001 TABLE 1 Antibody Nucleotide sequence Nucleotide Sequence Name of the VH gene of the 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

(22) TABLE-US-00002 TABLE 2 Antibody Amino acid sequence Amino acid sequence Name of the VH region 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

(23) TABLE-US-00003 TABLE 3 Amino Amino Amino Amino Amino Amino acid acid acid acid acid acid sequence sequence sequence sequence sequence sequence Antibody of VH- ofVH- of VH- of VL- of VL- of VL- Name CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 2A11 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 33 NO: 39 NO: 45 NO: 51 NO: 57 NO: 63 4C2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 33 NO: 39 NO: 45 NO: 51 NO: 57 NO: 63 8C4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 33 NO: 39 NO: 45 NO: 51 NO: 57 NO: 63 3B5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 34 NO: 40 NO: 46 NO: 52 NO: 58 NO: 64 9B3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 35 NO: 41 NO: 47 NO: 53 NO: 59 NO: 65 9B12 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 35 NO: 41 NO: 47 NO: 53 NO: 59 NO: 65 3B10 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 35 NO: 41 NO: 47 NO: 53 NO: 59 NO: 65 10H7 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 36 NO: 42 NO: 48 NO: 54 NO: 60 NO: 66 3G8 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 37 NO: 43 NO: 49 NO: 55 NO: 61 NO: 67 3F12 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 38 NO: 44 NO: 50 NO: 56 NO: 62 NO: 68

(24) 2. Identification of Antigen-Antibody Responses of Anti-BAG2 Antibodies in Breast Cancer Cells

(25) FIG. 1 shows the results of immunoblotting of anti-BAG2 antibodies produced from 10 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 g/ml streptomycin at a temperature of 37 C. The cells were harvested from wells and washed with PBS, and dissolved in a lysis buffer solution containing 1% Brij 97, 5 mM EDTA, 0.02M HEPES pH 7.3, 0.15M NaCl, 1 mM PMSF, 0.5 mM NaF, 10 g/rni aprotinin, and 0.2 mM sodium orthovanadate. After 15 minutes of incubation on ice, the nuclei were removed from the cells by centrifugation and the supernatants were collected. 2 sample buffer consisting of 20% glycerol, 4.6% SOS, 0.125M tris, pH 6.8, 0.1% bromophenol blue was added to an appropriate amount of the supernatants. 10 ug protein samples were subjected to SOS-PAGE analysis on a 12% gel under standard conditions by using a mini-Protean II system (Bio-Rad Hercules, Calif.). For immune blotting, the protein was transferred to Millipore, a PVDF membrane. A blocking solution consisting of 0.1% Tween 20 and 5% bovine serum albumin (BSA) in TBS was allowed to react for 1 hour. Subsequently, the primary antibody was a 1/2000 dilution of anti-BAG2 antibody extracted from hybridoma cell culture, and the goat anti-mouse HRP conjugate (Dako) used as the secondary antibody was diluted to 1/5000. Film-photosensing was carried out in the dark using EGL reagent (Amersham Pharmacia Biotech) as a substrate. The photosensitized bands were compared to standard molecular markers to identify the bands corresponding to the size of BAG2.

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

(27) Next, for domain mapping of the BAG2 antigen to which the ten antibodies identified in Section 1 above, cells, to which the GST-Empty vector (pcDNA3.1+/GST vector, NovoPro Bioscience Inc. China) having a molecular weight of about 26 kDa was introduced, was used as a negative control. GST-Bag Full vector, GST-Bag F1 vector, GST-Bag F2 vector, GST-Bag F3 vector, and GST-Bag F4 vector, each including polynucleotides encoding human BAG2 protein and polynucleotides encoding fragments of the BAG2 protein, were introduced into cells, and the cells with the vectors introduced thereinto were cultured to express the genes, and then, cell lysates were obtained. For cell lysates, immunoblotting was performed using each of the 10 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 the nucleotide sequences of SEQ ID NOS: 71 to 74, respectively.

(28) FIGS. 2A and 2B show the results of immunoblotting for the full-length BAG2 polypeptide of anti-BAG2 antibody or a fragment thereof. In FIG. 2, A shows diagrams of the vectors and BAG2 proteins and fragments thereof, and B shows the results of immunoblotting. In detail, the immunoblotting was performed as follows: each of the vectors was introduced into HEK293T cells by lipofectamine transfection (Thermo Fisher Scientific, Inc., Waltham, MA, USA) method, and the obtained transformed cells were cultured at a temperature of 37 C. in DMEM (Welgene) medium containing 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin for 30 hours, and then cells were isolated. The isolated cells were disrupted using the same method as described in connection with FIG. 1 and subjected to SOS-PAGE analysis on a 12% gel. For immunoblotting, after reacting for 1 hour with the same blocking solution as described in connection with FIG. 1, each of the 10 purified anti-BAG2 antibodies having a concentration of 2 mg/ml was used as a primary antibody at a dilution of 1/10000 dilution and bound to the cells. The goat anti-mouse HRP conjugate used as the secondary antibody was used at a 1/5000 dilution concentration, and Film-photosensing was carried out in the dark using EGL reagent (Amersham Pharmacia Biotech) as a substrate. Standard molecular marker sizes were expressed to confirm the size of BAG2.

(29) As a result, as shown in FIG. 2, each anti-BAG2 antibody was differentially bound to the full-length BAG2 polypeptide or fragments thereof. In particular, for each of the 10 anti-BAG2 antibodies, signals were commonly detected at the position of about 50 kDa in GST-Bag Full vector-introduced cell lysates. This result shows that all of these antibodies can bind to full-length BAG2 polypeptides. Finally, the domain region of BAG2 to which each anti-BAG2 antibody reacts was identified.

(30) FIG. 3 shows a BAG2 domain which reacts with respective anti-BAG2 antibodies. As shown in FIG. 3, 9B3, 9B12, 3B10, and 10H7 antibodies were bound to the N-terminus of BAG2 protein, 2A11, 3B5, 4C2, and 8C4 antibodies were bound to a middle region of BAG2 protein, and 3F12 and 3 GB antibodies were bound to the C-terminus of BAG2 protein. The N-terminus commonly includes a coiled coil region of 21-60 amino acids, and the middle region is bound to a portion 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 its fragments present in the sample may be detected with high sensitivity and specificity.

Example 2: Screening of Combinations of Anti-BAG2 Antibodies and Confirmation of Cancer Diagnostic Efficacy

(31) 1. Screening of Combinations of Anti-BAG2 Antibodies Useful for Cancer Diagnosis

(32) FIG. 4 shows standard curves for BAG2 protein in possible 90 combinations of anti-BAG2 antibodies. As a result, as shown in FIG. 4, from among possible combinations of anti-BAG2 antibodies, 14 antibody combinations showing a standard curve of high slope, 2A11-3F12, 10H7-3G8, 9B12-3G8, 10H7-3F12, 9B8-3G8, 3G8-3F12, 3B5-3F12, 3G8-4C2, 3F12-3G8, 3B5-3G8, 3B5-4C2, 3G8-8C4, and 9B3-3F12, were selected.

(33) FIG. 5 shows a diagram showing the specific binding force of the selected 14 antibody combinations with respect to the BAG2 protein. In detail, Myc-tag BAG2 expression vector or Myc-tag empty vector was each transduced into the human breast cancer cell line MDA-MB-231 by using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. Using 14 antibody combinations, relative amounts of BAG2 protein secreted from 30 ug of lysate of control cells into which Myc-tag empty vector was introduced and cells overexpressing BAG2 protein into which Myc-tag BAG2 expression vector were introduced, were determined. Control cells were labeled with EV and BAG2 overexpressing cells were labeled with OE. That is, by confirming the relative amount of the secreted BAG2 protein of BAG2 overexpressing cells and control cells, it is possible to determine the specific binding capacity of the antibody combination with respect to the BAG2 protein.

(34) As a result, as shown in FIG. 5, four antibody combinations, 2A11-3F12, 9B12-3G8, 8C4-3G8, and 10H7-3F12, having high specific binding ability to BAG2 protein were selected.

(35) 2. Identification of Efficacy of Selected Anti-BAG2 Antibody Combinations in Diagnosis of Various Cancer

(36) In order to confirm the cancer diagnostic efficacy of the antibody combinations selected in Section 1, the antigen-antibody reaction of the antibody combinations with BAG2 was confirmed. In detail, human BAG2 protein having 6 histidine attachments to N-terminus produced and purified from human 293T cells was obtained as an antigen.

(37) Sandwich enzyme-linked immunosorbent assay (Sandwich ELISA) was performed using each of the 2A11-3F12, and 9B12-3G8 antibody combinations.

(38) In detail, Sandwich ELISA was performed through the following process. Each of antibodies 3F12 and 3G8 was reacted in an amount of 1 mg with 27 ul of 10 mM NHS-biotin (Succinimidyl Biotin, Thermo scientific, Cat #21435) to prepare a biotin-bound detecting antibody. Each of antibodies 2A11, 9B12, 8C4, and 10H7 as a capturing antibody was diluted to a concentration of 3 ug/ml by using an ELISA plate-coating buffer (R&D system, DY006), and spread in amount of 100 ul in each well of a 96 well plate, and then, coated at the temperature of 4 C. overnight. Antibody-coated plates were blocked with 1% bovine serum albumin (BSA) buffer (R&D system, DY995) for 1 hour at room temperature. As a BAG2 standard material, human His-tagging BAG2 recombinant proteins (Ybiologics, Korea) produced in human-derived cell line HEK293T and purified were prepared at the concentration of 100 ng/ml using the 1% BSA buffer, and then, diluted 4 fold to prepare 0.02, 0.097, 0.31, 1.56, 6.25, and 25 ng/ml of standard solutions. For blood samples, 8 ml or more of blood was collected from each of 14 healthy people, 20 patients diagnosed with luminal breast cancer, and 38 patients diagnosed with TNBC type breast cancer and then mixed by gently shaking and allowed to stand at room temperature for 20-30 minutes. Then, the blood was centrifuged at 2,500 rpm for 10 minutes. Serum stored below 4 C. was diluted 1/2 fold using the 1% BSA buffer. Into each well of the plate from which the blocking solution was removed, 0, 0.02, 0.097, 0.39, 1.56, 6.25, 25, and 100 ng/ml of the BAG2 standard solutions and 100 ul of the patient sample diluted 1/2 fold were dispensed and reacted at room temperature for 1 hour, and then, washed with a washing buffer (R&D system, WA126). 100 ul of streptavidin-HRP (Pierce, 21130) diluted 1:50000 by using 1% BSA buffer was dispensed into each well and reacted for 30 minutes at room temperature, followed by washing with a washing buffer. 100 ul of 3,3,5,5-tetramethylbenzidine (TMB, R &D system, DY999) was dispensed into each well, reacted for 15 minutes at room temperature in female cow, and then, a stop solution was added thereto to stop the reaction. The concentration of BAG2 protein in the patient sample was calculated from the standard curve of the antibody obtained by measuring the absorbance of the reaction solution at 450 nm.

(39) FIG. 6 shows differences in BAG2 expression patterns in cancer cell lines including breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, and diffuse giant B cell lymphoma which were observed by using anti-BAG2 antibody combinations. FIG. 6A shows the results of sandwich ELISA of 2A11-3F12 antibody combination, and FIG. 6B shows the results of sandwich ELISA of 9B12-3G8 antibody combination.

(40) As illustrated in FIGS. 6A and 6B, when 2A11-3F12, and 9B12-3G8 antibody combinations were used, BAG protein was expressed at high levels in various cancer cell lines including MDA-MB-231 and Hs578T which are breast cancer cell lines, SNU2564 and PANC1 which are pancreatic cancer cell lines, U251MG and T98G which are glioblastoma multiforme (GBM) cell lines, SNU1 and SNU484 which are gastric cancer cell lines, SKOV3 and A2780 which are ovarian cancer cell lines, and SU-DHL4 and SU-DHL6 which are diffuse giant B cell lymphoma (DLBCL) cell lines. In contrast, the BAG2 protein in T47D, SNU2469, A172, SNU620, OVCAR3, and U2932, which are normal cell lines, was not expressed at all or were expressed little. In other words, it was confirmed that the BAG2 protein was significantly highly expressed in cancer cells including breast cancer, pancreatic cancer, glioblastoma multiforme, gastric cancer, ovarian cancer, and diffuse giant B cell lymphoma by using 2A11-3F12, and 9B12-3G8 antibody combinations, compared with normal cells.

(41) Therefore, when the above antibody combinations are used, cancer can be effectively diagnosed by identifying whether the expression pattern of BAG2 protein is similar to that of BAG2 protein showing in cancer cells, compared to normal cells.

(42) 3. Identification of Efficacy of Selected Anti-BAG2 Antibody Combinations in Diagnosis of Breast Cancer

(43) By using the same Sandwich ELISA method described in Section 2, the difference in the expression pattern of BAG2 protein in serum between luminal type and TNBC type breast cancer patients and normal subjects was confirmed. In detail, the serum of healthy volunteers (N=14), luminal type breast cancer patients (N=20) and TNBC type breast cancer patients (N=38) was collected, and the expression pattern of BAG2 protein in the obtained serum was confirmed.

(44) FIGS. 7A and 7B and 8A and 8B show significant differences in BAG2 protein expression in the serum of luminal type and TNBG type breast cancer patients identified by using anti-BAG2 antibody combinations. FIG. 7A shows the case using 9B12-3G8 antibody combination, and FIG. 7B shows the case using 2A11-3F12 antibody combination. FIG. 8A shows the case using 8G4-3G8 antibody combination, and FIG. 8B shows the case using 10H7-3G8 antibody combination.

(45) As shown in FIGS. 7A and 7B and 8A and 8B, when 9B12-3G8, 2A11-3F12, 8G4-3G8, and 10H7-3F12 antibody combinations were used, p-values of the respective antibody combinations were p<0.0001, p=0.0373, p=0.0009, and p=0.0190, indicating that there was a significant difference in BAG2 protein expression patterns in the serum of luminal type and TNBG type breast cancer patients compared to normal subjects. Therefore, by observing significant differences in BAG2 protein expression patterns using the antibody combinations, breast cancer can be effectively diagnosed.

(46) 4. Confirmation of the Usefulness of Selected Anti-BAG2 Antibody Combination in Breast Cancer Diagnosis.

(47) In order to confirm the sensitivity and specificity of cancer diagnosis using each of the four antibody combinations, the results of Section 2 are shown by using ROG curves.

(48) FIGS. 9A, 9B, 9C and 9D show the receiver operating characteristic curves (ROG) and area under the curves (AUG) of respective antibody combinations. FIG. 9A shows the case using 9B12-3G8 antibody combination, FIG. 9B shows the case using 8G4-3G8 antibody combination, FIG. 9C shows the case using 2A11-3F12 antibody combination, and FIG. 9D shows the case using 10H7-3F12 antibody combination.

(49) As shown in FIGS. 9A, 9B, 9C and 9D, when 9B12-3G8, 8G4-3G8, 2A11-3F12, and 10H7-3G8 antibody combinations were used, AUG values were 0.8596, 0.8368, 0.8554, and 0.6736. That is, in the case of all antibody combinations, the AUG values in the ROG curve were around 0.7, and when the 9B12-3G8, 8G4-3G8, and 2A11-3F12 antibody combinations were used, AUG values were measured to be between 0.8 and 0.9. These results show that each antibody combination can be used to diagnose breast cancer by using high sensitivity and specificity. Therefore, the four antibody combinations can be usefully used for diagnosing breast cancer.

(50) A composition for use in diagnosing cancer including an anti-BAG2 antibody or antigen-binding fragment thereof according to one aspect may provide information used for diagnosing cancer.

(51) According to the method of providing information used for diagnosing cancer according to another aspect, unlike the conventional diagnostic method of collecting tissues, the presence or level of BAG2 polypeptide in the blood can be identified or measured from the blood from the blood and diagnostic usefulness is high. Accordingly, various organizations can use the method for the diagnosis of cancer.

(52) [Accession Number]

(53) Depositary: Korea Research Institute of Bioscience and Biotechnology

(54) Accession number: KCTC13737BP

(55) Deposit date: Nov. 28, 2018

(56) Depositary: Korea Research Institute of Bioscience and Biotechnology

(57) Accession number: KCTC13738BP

(58) Deposit date: Nov. 28, 2018

(59) Depositary: Korea Research Institute of Bioscience and Biotechnology

(60) Accession number: KCTC13739BP

(61) Deposit date: Nov. 28, 2018

(62) Depositary: Korea Research Institute of Bioscience and Biotechnology

(63) Accession number: KCTC13740BP

(64) Deposit date: Nov. 28, 2018

(65) Depositary: Korea Research Institute of Bioscience and Biotechnology

(66) Accession number: KCTC13741BP

(67) Deposit date: Nov. 28, 2018

(68) Depositary: Korea Research Institute of Bioscience and Biotechnology

(69) Accession number: KCTC13742BP

(70) Deposit date: Nov. 28, 2018

(71) Depositary: Korea Research Institute of Bioscience and Biotechnology

(72) Accession number: KCTC13743BP

(73) Deposit date: Nov. 28, 2018

(74) Depositary: Korea Research Institute of Bioscience and Biotechnology

(75) Accession number: KCTC13744BP

(76) Deposit date: Nov. 28, 2018

(77) Depositary: Korea Research Institute of Bioscience and Biotechnology

(78) Accession number: KCTC13745BP

(79) Deposit date: Nov. 28, 2018

(80) Depositary: Korea Research Institute of Bioscience and Biotechnology

(81) Accession number: KCTC13746BP

(82) Deposit date: Nov. 28, 2018

(83) All of the references cited herein are incorporated by reference in their entirety.

(84) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein.