ANTIBODY BINDING TO CARBONIC ANHYDRASE AND USE THEREOF

Abstract

Provided is an antibody that recognizes and binds to carbonic anhydrase or antigen-binding fragment, a nucleic acid molecule coding for the antibody or antigen-binding fragment, a vector carrying the nucleic acid molecule, a host cell including the nucleic acid molecule or the vector, and use of the antibody or antigen-binding fragment thereof in the alleviation, prevention, treatment or diagnosis of solid cancers.

Claims

1. An antibody or an antigen-binding fragment thereof, binding to non-catalytic domain of carbonic anhydrase.

2. The antibody or antigen-binding fragment according to claim 1, wherein the epitope of the antibody is a peptide consisting of 7 to 93 consecutive amino acids containing the amino acid sequence of SEQ ID NO: 1 among amino acid sequence of SEQ ID NO: 5.

3. The antibody or antigen-binding fragment according to claim 1, wherein the epitope of the antibody is a peptide consisting of 14 to 93 consecutive amino acids containing the amino acid sequence of SEQ ID NO: 2 among amino acid sequence of SEQ ID NO: 5.

4. The antibody or antigen-binding fragment according to claim 3, wherein the epitope of the antibody is a peptide consisting of 7 to 14 consecutive amino acids containing the amino acid sequence of SEQ ID NO: 1 among amino acid sequence of SEQ ID NO: 2.

5. The antibody or antigen-binding fragment according to claim 1, wherein the epitope of the antibody is a peptide consisting of 14 to 93 consecutive amino acids containing the amino acid sequence of SEQ ID NO: 3 among amino acid sequence of SEQ ID NO: 5.

6. The antibody or antigen-binding fragment according to claim 5, wherein the epitope of the antibody is a peptide consisting of 19 to 93 consecutive amino acids containing the amino acid sequence of SEQ ID NO: 4 among amino acid sequence of SEQ ID NO: 5.

7. The antibody or antigen-binding fragment according to claim 6, wherein the epitope of the antibody is a peptide consisting of 14 to 19 consecutive amino acids containing the amino acid sequence of SEQ ID NO: 3 among amino acid sequence of SEQ ID NO: 4.

8. The antibody or antigen-binding fragment according to claim 6, wherein the epitope of the antibody is a peptide consisting of 7 to 93 consecutive amino acids containing the amino acid sequence of SEQ ID NOs: 1, 2, 3 or 4.

9. The antibody or antigen-binding fragment according to claim 2, wherein the antibody comprises amino acid sequences comprising amino acids of SEQ ID Nos: 6 to 8 of CDRs in V.sub.H and amino acid sequences comprising amino acids of SEQ ID Nos: 9 to 11 of CDRs in V.sub.L.

10. The antibody or antigen-binding fragment according to claim 5, wherein the antibody comprises amino acid sequences comprising amino acids of SEQ ID No: 12 of V.sub.H and amino acid sequences comprising amino acids of SEQ ID No: 13 of V.sub.L.

11. The antibody or antigen-binding fragment according to claim 5, wherein the antibody comprises amino acid sequences comprising amino acids of SEQ ID Nos: 14 to 16 of CDRs in V.sub.H and amino acid sequences comprising amino acids of SEQ ID Nos: 17 to 19 of CDRs in V.sub.L.

12. The antibody or an antigen-binding fragment according to claim 11, wherein the antibody comprises amino acid sequences comprising amino acids of SEQ ID No: 20 of V.sub.H and amino acid sequences comprising amino acids of SEQ ID No: 21 of V.sub.L.

13. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or the antigen-binding fragment is coupled to labeling agents, toxins, or anti-tumor drugs.

14. The antibody or antigen-binding fragment according to claim 13, wherein the labeling agent is selected from the group consisting of a radioisotope, a hapten or a fluorescent, a chromogen, and a dye.

15. The antibody or antigen-binding fragment according to claim 13, wherein the toxin is selected from the group consisting of a radioisotope, a small molecule, a peptide, and a protein.

16. The antibody or antigen-binding fragment according to claim 13, wherein the antibody or antigen-binding fragment is coupled with a toxin to form a fusion protein.

17. The antibody or antigen-binding fragment according to claim 13, wherein the fucoses bound to the antibody or antigen-binding fragment are partly or all removed.

18. A nucleotide molecule encoding the antibody or antigen-binding fragment of the antibody that binds to non-catalytic domain of carbonic anhydrase according to claim 1.

19. A vector introduced by a nucleotide molecule encoding the antibody or antigen-binding fragment of the antibody that binds to non-catalytic domain of carbonic anhydrase according to claim 1.

20. A host expressing the antibody or antigen-binding fragment of the antibody that binds to non-catalytic domain of carbonic anhydrase according to claim 1.

21. The host according to claim 20, wherein the host is a hybridoma cell.

22. The host according to claim 21, wherein the host is a hybridoma cell deposited with accession number KCLRF-BP-00280 or KCLRF-BP-00279, where the hybridoma cell produces the antibody or antigen-binding fragment of the antibody that binds to non-catalytic domain of carbonic anhydrase.

23. A pharmaceutical composition for prevention or treatment of solid cancer, comprising the antibody or antigen-binding fragment of the antibody that binds to non-catalytic domain of carbonic anhydrase according to claim 1.

24. The pharmaceutical composition according to claim 23, wherein the solid cancer is breast cancer, lung cancer, colorectal cancer, stomach cancer, prostate cancer, and liver cancer.

25. The pharmaceutical composition according to claim 24, wherein the solid cancer is a triple-negative breast cancer.

26. The pharmaceutical composition according to claim 23, wherein the antibody or antigen-binding fragment is coupled to a toxin.

27. The pharmaceutical composition according to claim 23, further comprising an anti-tumor chemical drug or an anti-tumor antibody.

28. The pharmaceutical composition according to claim 23, wherein the pharmaceutical composition is administered with radiotherapy.

29. A composition for detecting a solid cancer that comprises the antibody or antigen-binding fragment of the antibody that binds to non-catalytic domain of carbonic anhydrase according to claim 1 and that the a sample is determined as the solid cancer showing a positive reaction to the antibody or antigen-binding fragment.

30. The composition for detecting a solid cancer according to claim 29, wherein the antibody or the antigen-binding fragment is coupled to labeling agents.

31. The composition for detecting a solid cancer according to claim 27, wherein the solid cancer is breast cancer, lung cancer, colorectal cancer, stomach cancer, prostate cancer, and liver cancer.

32. The composition for detecting a solid cancer according to claim 31, wherein the positive reaction is detected by enzyme reaction, fluorescence, luminescence or radiation.

Description

DESCRIPTION OF DRAWINGS

[0069] FIG. 1 shows titers of the solid tumor-specific 27B6 monoclonal antibody in peripheral blood, as measured according to Example 1.

[0070] FIG. 2 shows variable region sequences including CDR sequences of the 27B6 and 4B4 antibodies, as determined according to Example 2,

[0071] FIG. 3 shows the antigen specificity and affinity of the 27B6 chimeric antibody, as measured according to Example 3;

[0072] FIG. 4 illustrates a procedure of screening titers of the 4B4 monoclonal antibody in peripheral blood, as measured according to Example 4,

[0073] FIG. 5 shows the antigen specificity and affinity of the 4B4 chimeric antibody, as measured according to Example 5;

[0074] FIGS. 6a, 6b and 6c show expression patterns of the carbonic anhydrase 12 antigen in various breast cancer cells, as measured according to Example 6,

[0075] FIG. 7 shows electrophoretograms of antigens isolated and purified from the lung adenocarcinomic A549 cell line through columns fabricated with 4B4 and 27B6 monoclonal antibodies;

[0076] FIG. 8 lists proteins identified by LC-MS/MS analysis from purified antigens, as analyzed according to Example 7,

[0077] FIG. 9 shows the amino acid sequence of carbonic anhydrase 12 isoform 1 in which amino acid sequences of the antigens for 4B4 and 27B6 antibodies, detected by LC-MS/MS, are marked, as analyzed according to Example 7,

[0078] FIG. 10 shows the identification of carbonic anhydrase 12 as an antigen for the 4B4 and the 27B6 monoclonal antibody, as analyzed by ELISA assay in Example 7,

[0079] FIG. 11 shows the identification of carbonic anhydrase 12 as an antigen for the 4B4 and the 27B6 monoclonal antibody, as analyzed by Western blotting assay in Example 7,

[0080] FIG. 12 shows the concurrent recognition of carbonic anhydrase 12 by 4B4 and 27B6 monoclonal antibodies as measured by sandwich ELISA assay using 4B4 and 27B6 monoclonal antibodies as capture/detector antibodies in Example 7,

[0081] FIG. 13 shows epitope mapping processes and results of 27B6 and 4B4 monoclonal antibodies, as analyzed according to Example 8,

[0082] FIG. 14 shows the complement-dependent cytotoxic effects of 27B6 and 4B4 antibodies, as analyzed according to Example 9,

[0083] FIG. 15 shows the complement-dependent cytotoxic effects of the 27B6 antibody in triple-negative breast cancer, as analyzed according to Example 9,

[0084] FIG. 16 shows antibody-dependent cell-mediated cytotoxic effects of the 27B6 antibody, as analyzed according to Example 10-1,

[0085] FIG. 17 shows antibody-dependent cell-mediated cytotoxic effects of the 27B6 antibody on triple-negative breast cancer cell lines, as analyzed according to Example 10-2,

[0086] FIG. 18 shows the antibody-dependent cell-mediated cytotoxic effects of the defucosylated 27B6 chimeric antibody, as analyzed according to Example 11-1,

[0087] FIG. 19 shows the antibody-dependent cell-mediated cytotoxic effects of the defucosylated 4B4 and 27B6 chimeric antibodies, as analyzed by a luciferase assay in Example 11-2.

[0088] FIG. 20 shows the expression levels of the CA12 antigen on triple-negative breast cancer cell lines and the binding of 27B6 and 4B4 antibodies to the cell surface of the cell lines, as analyzed according to Example 12.

[0089] FIG. 21 shows the inhibitory activities of 27B6 and 4B4 antibodies against tumor growth in triple-negative breast cancer animal models.

[0090] FIG. 22 shows the inhibitory activity of the 27B6 antibody against triple-negative breast cancer, as analyzed according to Example 12.

[0091] FIG. 23 shows the inhibitory activity of the 4B4 antibody against triple-negative breast cancer, as analyzed according to Example 12.

[0092] FIGS. 24 and 25 show that the binding of the 4B4 antibody alone to tumor cells does not affect the growth of the tumor cells.

[0093] FIG. 26 shows the effect of a combination of the 27B6 antibody and radiotherapy, as analyzed according to Example 14.

MODE FOR INVENTION

[0094] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

Example 1: Production of 27B6 Monoclonal Antibody

[0095] The development of novel antibodies specific for CA12 was achieved in the following experiments. The developed antibodies were observed to be specific for solid tumors, such as adenocarcinoma of the lungs, breast cancer, colorectal cancer, and prostate cancer, as they reacted with antigens expressed specifically in the tumors. They were designated 27B6 and 4B4, respectively.

[0096] 1-1: Design of Target Site for Construction of 27B6 Monoclonal Antibody

[0097] An antibody specific for solid tumor cells was fabricated. For this, mice were immunized directly with solid tumor cells, and monoclonal antibodies were established using a cell fusion technique. Thereafter, an antigen to which the solid tumor cell-specific monoclonal antibody was bound was analyzed and identified.

[0098] 1-2: Preparation of Hybridoma Cell

[0099] In order to develop a monoclonal antibody to an antigen specifically expressed in solid tumors, A549 cells, which are adenocarcinomic human alveolar-basal epithelial cells, were immunized, and a selection was made of an antibody that was positive to the A549 cell line, but negative to the normal cell line L132 during a hybridoma selection process.

[0100] To the end, Balb/c female mice 6 weeks old were each IP (intraperitoneal cavity)-injected with the A549 cell line (ATCC CCL-185) at a dose of 1×10.sup.7 cells three times at regular intervals of three weeks, followed by removing sera from the veins. A dilution of the serum was added to A549 cells. After being left for 30 min at 4° C. to react, the dilution was mixed with 3 ml of PBS and centrifuged for 3 min at 1500 rpm. Unbound antibodies were washed off. A 200-fold dilution of the secondary antibody goat anti-mouse Ig-FITC (Dinona) was used to detect the bound antibodies. After reaction for 15 min at 4° C., the reaction mixture was washed with 3 ml of PBS in the same manner. The sera were measured for antibody titer to A549 cells by flow cytometry. The sera immunized with A549 cells were observed to be highly positive to A549 cells (results not shown). Briefly, three days before a cell fusion experiment, 50 μg of anti-CD40 agonist mAb was added to boost an immune reaction, and A549 (ATCC CCL-185) was injected at a dose of 1×10.sup.7 cells to induce the amplification of an antibody to a surface antigen of A549.

[0101] 1-3: Preparation of Hybridoma Cell

[0102] The spleen was excised from the immunized mice, and a suspension of single splenocytes was obtained and washed twice with RPMI (GIBCO). Viable cells were counted using a 1:1 (v/v) mixture of 0.4% trypan blue (Sigma), which stains only dead cells. The X63 mouse myeloma cell line (ATCC CRL-1580) was employed as a cell fusion partner, and washed and counted in the same manner as the splenocytes.

[0103] The myeloma cells were mixed at a ratio of 1:5 with the splenocytes and centrifuged. The pellet thus obtained was slowly added over 1 min with 1 ml of 50% PEG (polyethylene glycol) 1500 preheated to 37° C. After being incubated for about 1 min, the cell mixture was slowly diluted with an RPMI medium and centrifuged. The resulting cell pellet was resuspended in RPMI (20% FBS) containing 1×HAT (hypoxanthine-aminopterin-thymidine), plated at a volume of 150 μl/well into 96-well plates, and grown in a 37° C. CO.sub.2 incubator. HAT was fed over a predetermined time after the fusion. When a colony was observed in the wells, 150 μl of an HT medium was added to each well, followed by incubation for 48 hrs in a 37° C., 5% CO.sub.2 incubator. Then, three-color immunofluorescence staining was performed before flow cytometry. Briefly, the lung adenocarcinoma cell line A549 and the normal lung cell line L132 were immunologically stained with two different dyes and mixed at a ratio of 1:1. This cell mixture was incubated with 100 μl of a supernatant of the hybridoma cell culture at 4° C. for 30 min and centrifuged, together with 3 ml of PBS, at 1500 rpm for 3 min to remove unbound antibodies. The bound antibodies were detected by incubation with a 200-fold dilution of the secondary antibody goat anti-Mouse Ig-APC (Dinona) at 4° C. for 15 min, followed by washing with 3 ml of PBS in the same manner. Thereafter, the hybridoma cells were measured via flow cytometry.

[0104] An examination was made to see whether the antibody binds to peripheral blood. For this, PBMC (peripheral blood mononuclear cells from the Korean Red Cross Blood Services) was incubated with 100 μl of a hybridoma supernatant at 4° C. for 30 min, and centrifuged, together with 3 ml of PBS, at 1,500 rpm for 3 min to wash off unbound antibodies. A 200-fold dilution of the secondary antibody goat anti-mouse Ig-FITC (Dinona) was used to detect the bound antibodies. After reaction for 15 min at 4° C., the reaction mixture was washed with 3 ml of PBS in the same manner. The antibody titer was measured using flow cytometry, and the results are shown (FIG. 1). FIG. 1 shows titers of the lung adenocarcinoma-specific 27B6 monoclonal antibody in the peripheral blood, as measured via flow cytometry.

[0105] In this manner, the antibody that was positive to the lung cancer cell line A549 and negative to the normal lung cell line L132 and all of granulocytes, lymphocytes and monocytes of the peripheral blood were selected and designated “27B6”. Finally, during a limiting dilution procedure, 27B6 hybridoma cells were diluted and selected for single colony growth.

[0106] The 27B6 hybridoma cell line was deposited on Feb. 14, 2012, with the Korean Cell Line Bank, located at 28, Yongun-Dong, Jongno-Gu, Seoul, Korea, and received Accession No. KCLRF-BP-00280 on Feb. 20, 2012.

Example 2: Analysis of 27B6 Monoclonal Antibody

[0107] 2-1: Determination of Isotype

[0108] The 27B6 monoclonal antibody prepared in Example 1 was analyzed for isotype, using a mouse immunoglobulin isotyping ELISA kit (BD Biosciences, USA). Briefly, isotyping was performed with rabbit anti-murine isotype specific antisera (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa, Lambda) while peroxidase-labeled goat anti-mouse IgG served as a secondary antibody. Color development was induced with ortho-phenylenediamine (OPD) and a hydrogen peroxide substrate. Absorbance at 450 nm was read.

[0109] As a result, the 27B6 monoclonal antibody was identified as mouse IgG1/kappa light chain (results not shown).

[0110] 2-2: 27B6 Antibody CDR Sequence

[0111] An antibody cloning procedure is illustrated in FIG. 2. Heavy and light chain sequences including the CDR sequences of the 27B6 Ab are represented by SEQ ID NOS: 12 and 13, respectively. FIG. 2 shows variable region sequences including CDR sequences of the 27B6 antibody.

TABLE-US-00001 TABLE 1 b V.sub.H QVQLQQSGPQLVWPGASVKISCNTSG CDR1: GYSFTNYW YSFTNYWIHWVKQRPGQGLEWIGMID CDR2: IDPSDSET PSDSETRLNQKFKDKTTLTVDRSSSTA CDR3:  YMQVSSSTSEDSAVYYCTRGIRGGYY TRGIRGGYYAMDY AMDYWGQGTSVTVSS V.sub.L DIQMTQTTSSLSASLGDRVTISCRASQ CDR1: QDISNY DISNYLNWYQQKPEGTVKLLIYYTSRL CDR2: YTS HSGVPSRFSGSGSGTDYSLTISNLEQED CDR3: QQGDTLPRT IATYFCQQGDTLPRTFGEGTKLEIR

Example 3: Development of 27B6 Chimeric Antibody

[0112] When a monoclonal antibody of mouse origin is administered to the human body, the human immune system recognizes the monoclonal antibody as a foreign antigen and thus produces a human anti-mouse antibody (HAMA) to eliminate the mouse antibody from the blood. In addition, the Fc domain of the mouse antibody cannot exert its effective biological functions in the human body. Therefore, not only does the therapeutic effect sharply decrease, but also side effects such as severe allergic reactions and renal dysfunction may be induced. In order to reduce the immunogenicity of the 27B6 antibody upon administration to the human body, a chimeric antibody in which the mouse antibody, except for the variable region, was substituted with the Fc of the human antibody was constructed. The chimeric antibody was observed to be similar in antigen specificity and affinity to the original mouse 27B6 antibody.

[0113] To construct a chimeric antibody, the 27B6-HuIgFc DNA prepared in the above-mentioned manner was transfected into the DHFR DG44 cell line derived from CHO cells, followed by a selective culturing procedure in a selective medium to establish a stable cell line producing a 27B6 recombinant antibody. Details are described as follows.

[0114] First, three hours before transfection, the DG44 cell line (Invitrogen, Cat No. A1100001) was inoculated at a density of 1×10.sup.6 cells/ml into 6-well plates and incubated with 1 ml of GIBCO® CD DG44 Medium (Invitrogen, USA) at 37° C. in a 5% CO.sub.2 atmosphere for 3 hrs. Then, the 27B6-HuIgFc DNA prepared in Example 4-1 was transfected into the competent DC 44 cells using an Effectene transfection reagent kit (QIAGEN, Hilden, Germany).

[0115] Three days post transfection, the supernatant was taken and added to A549 cells which were then incubated at 4° C. for 30 min. Unbound antibodies were removed by centrifugation, together with 3 ml of PBS, at 1500 rpm for 3 min. The bound antibodies were detected by incubation with a 150-fold dilution of the secondary antibody goat anti-Mouse Ig-FITC (Dinona) at 4° C. for 15 min, followed by washing with 3 ml of PBS in the same manner. Thereafter, the antibody titer to A549 cells was measured using flow cytometry. Subsequently, a stable cell line was established. For this, the medium was exchanged with a PowerCHO medium (LONZA, Switzerland) supplemented with 30 nM MTX (Sigma, USA) and 200 μg/ml G418 (Invitrogen, USA), after which clone selection was started. Concentrations of MTX and G418 in the selection medium were increased with the repetition of clone selection rounds. Each round was set to be three weeks. The final round of clone selection was performed in a PowerCHO medium supplemented with 1000 nM MTX and 400 μg/ml G418. Thereafter, the final cell line was established as a single colony through limiting dilution.

[0116] The 27B6 chimeric antibody established in this manner was found to have antigen specificity and affinity to those of the original mouse 27B6 antibody, as measured by flow cytometry (FIG. 3). FIG. 3 shows the antigen specificity and affinity of the 27B6 chimeric antibody.

Example 4: Production of 4B4 Antibody

[0117] 4-1: 27B6 Pairing Antibody

[0118] To develop another antibody which recognizes the same antigen but binds to a different epitope, 27B6 pairing antibody was developed.

[0119] Firstly to explore the possibility of development of 27B6 paring antibody, sandwich ELIS using chimeric 27B6 and mouse serum was established. In the same manner as in section 1-2, balb/c female mice 6 weeks old were each IP (intraperitoneal cavity)-injected with the A549 cell line (ATCC CCL-185) at a dose of 1×10.sup.7 cells three times at regular intervals of three weeks, followed by removing sera from the veins.

[0120] The purified 27B6 chimeric antibody was plated at a concentration of 100 ng/well and incubated at 37° C. for 1 hr. The coated plate was then blocked by incubation with 200 μl of a blocking buffer (Sigma) per well at 37° C. for 1 hr. A548 cells were lysed with 1% NP40 lysis buffer at a concentration of 1×10.sup.7 cells/ml. The A549 lysate was added at a density of 50 μl/well to the 27B6 coated plate and incubated at 37° C. for 1 hr before three rounds of washing with PBS. To each of the washed wells, 100 μl of a 1,000-fold dilution of the previously obtained serum was added. Following 1 hr of incubation, the wells were washed again with PBS. Finally, the bound antibody was detected with the secondary antibody goat anti-Mouse Ig-HRP (Jackson). A 2,000-fold dilution of the secondary antibody was added in an amount of 100 μl to each well, incubated at 37° C., and washed with PBS. Color development was performed by incubation with 50 μl of TMB (3,3′,5,5′-tetramethylbenzidine) at room temperature for 10 min in each well. The reaction was stopped with 2N H.sub.2SO.sub.4 (Sigma). The antibody titer was measured by reading the absorbance at 450 nm.

[0121] As was expected, positive reaction was observed in sandwich ELISA using chimeric 27B6 and mouse serum (data not shown).

[0122] 4-2: Production of Monoclonal Antibody

[0123] Preparation of hybridoma cells from splenocytes of the immunized mice was carried out in the same manner as in Example 1.

[0124] The spleen was excised from the immunized mice, and a suspension of single splenocytes was obtained and washed twice with RPMI (GIBCO). Viable cells were counted using a 1:1 (v/v) mixture of 0.4% trypan blue (Sigma), which stains only dead cells. The X63 mouse myeloma cell line (ATCC CRL-1580) was employed as a cell fusion partner, and washed and counted in the same manner as the splenocytes. The myeloma cells were mixed at a ratio of 1:5 with the splenocytes and centrifuged. To the cell pellet thus obtained, 1 ml of 50% PEG (polyethylene glycol) 1500 preheated to 37° C. was slowly added over 1 min. After being incubated for about 1 min, the cell mixture was slowly diluted with an RPMI medium and centrifuged. The resulting cell pellet was resuspended in RPMI (20% FBS) containing 1×HAT (hypoxanthine-aminopterin-thymidine), plated at a volume of 150 μl/well into 96-well plates, and grown in a 37° C. CO.sub.2 incubator. HAT was fed over a predetermined time after the fusion. When a colony was observed in the wells, 150 μl of an HT medium was added to each well, followed by incubation for 48 hrs in a 37° C., 5% CO.sub.2 incubator. A titer experiment was carried out with 100 μl of the supernatant. As described above, the 27B6 chimeric antibody was plated at a concentration of 100 ng/well and incubated at 37° C. for 1 hr. The coated antibody was then blocked by incubation with 200 μl of a blocking buffer (Sigma) per well at 37° C. for 1 hr. A549 cells were lysed with 1% NP40 lysis buffer at a concentration of 1×10.sup.7 cells/mi. The A549 lysate thus obtained was added at a density of 50 μl/well to the coated antibody and incubated at 37° C. for 1 hr before three rounds of washing with PBS. To each of the washed wells, 100 μl of the previously obtained hybridoma supernatant was added. Following 1 hr of incubation, the wells were washed again with PBS.

[0125] Finally, the bound antibody was detected with the secondary antibody goat anti-Mouse Ig-HRP (Jackson). A 2,000-fold dilution of the secondary antibody was added in an amount of 100 μl to each well, incubated at 37° C., and washed with PBS. Color development was performed by incubation with 50 μl of TMB (3,3′,5,5′-tetramethylbenzidine) at room temperature for 10 min in each well. The reaction was stopped with 2N H.sub.2SO.sub.4 (Sigma). The antibody titer was measured by reading the absorbance at 450 nm. In this screening the positive hybridomas were selected and then evaluated with other additional assays as follows.

[0126] To evaluate of binding activity to A549 cell surface, the hybridoma supernatants were added to A549 cells. After being left for 30 min at 4° C. to react, the reaction mixture was mixed with 3 ml of PBS and centrifuged for 3 min at 1500 rpm. Unbound antibodies were washed off. A 200-fold dilution of the secondary antibody goat anti-mouse Ig-FITC (Dinona) was used to detect the bound antibodies. After reaction for 15 min at 4° C., the reaction mixture was washed with 3 ml of PBS in the same manner. The antibody binding to A549 cells was measured using flow cytometry.

[0127] An examination was made to see whether the antibody binds to peripheral blood. For this, PBMC (peripheral blood mononuclear cells from the Korean Red Cross Blood Services) was incubated with 100 μl of a hybridoma supernatant at 4° C. for 30 min, and centrifuged, together with 3 ml of PBS, at 1,500 rpm for 3 min to wash off unbound antibodies. A 200-fold dilution of the secondary antibody goat anti-mouse Ig-FITC (Dinona) was used to detect the bound antibodies. After reaction for 15 min at 4° C., the reaction mixture was washed with 3 ml of PBS in the same manner. The antibody titer was measured by flow cytometry.

[0128] As a result, the antibody that was positive to the lung cancer cell line A549 and negative to the normal lung cell line L132 and to all of the granulocytes, lymphocytes and monocytes in peripheral blood, like 27B6, was selected and designated “4B4”. Finally, during a limiting dilution procedure, 4B4 hybridoma cells were diluted and selected for single colony growth (FIG. 4). FIG. 4 shows titers of the 4B4 monoclonal antibody in the peripheral blood, as measured by flow cytometry. The 4B4 hybridoma cell line was deposited on Feb. 14, 2012, with the Korean Cell Line Bank, located at 28, Yongun-Dong, Jongno-Gu, Seoul, Korea, and received Accession No. KCLRF-BP-00279 on Feb. 20, 2012.

[0129] 4-3: Analysis of 4B4 Antibody

[0130] An antibody cloning procedure is illustrated in FIG. 2. Heavy and light chain sequences including the CDR sequences of the 4B4 Ab are represented by SEQ ID NOS: 20 and 21, respectively. FIG. 2 shows variable region sequences including CDR sequences of the 27B6 antibody, with the CDR sequences marked in the variable regions (thickly underlined).

TABLE-US-00002 TABLE 2 4B4 Ab  V.sub.H EIQLQQSGPELVKPGASVKISCKASGYS CDR1: GYSYTDYN YTDYNIYWVRQSQGKSLDWIGYIDPAN CDR2: IDPANGDT GDTTYNQKFKGKATLTVDKSSSTAFMH CDR3: ARPIYYGVYWYFDV LNSLTSDGSAVYFCARPIYYGVYWYFD VWGAGTTVTVS V.sub.L DIVMTQAAPSVPVTPGESVSISCRSSKSL CDR1: KSLLHSNGNTY, LHSNGNTYLYWFLQRPGQSPQLLIYRM CDR2: RMS SNLASGVPDRFSGSGSGTAFTLRISRVEA CDR3: MQHLEYPFT EDVGVYYCMQHLEYPFTFGSGTKLEIK

Example 5: Development of 4B4 Chimeric Antibody

[0131] A cloning procedure of 4B4 antibody is illustrated in FIG. 4.

[0132] In order to reduce the immunogenicity of the 4B4 antibody upon administration to the human body, a chimeric antibody in which the mouse antibody, except for the variable region, was substituted with the Fc of the human antibody was constructed. The chimeric antibody was observed to be similar in antigen specificity and affinity to the original mouse 4B4 antibody.

[0133] To construct a chimeric antibody, the 4B4-HuIgFc DNA prepared in the above-mentioned manner was transfected into the DHFR DG44 cell line derived from CHO cells, followed by a selective culturing procedure in a selective medium to establish a stable cell line producing a 4B4 recombinant antibody. Details are described as follows.

[0134] First, three hours before transfection, the DG44 cell line (Invitrogen, Cat No. A1100001) was inoculated at a density of 1×10.sup.6 cells/ml into 6-well plates and incubated with 1 ml of GIBCO® CD DG44 Medium (Invitrogen, USA) at 37° C. in a 5% CO.sub.2 atmosphere for 3 hrs. Then, the 4B4-HuIgFc DNA prepared in Example 6-1 was transfected into competent DC 44 cells using an Effectene transfection reagent kit (QIAGEN, Hilden, Germany).

[0135] Three days post transfection, the supernatant was taken and added to A549 cells, which were then incubated at 4° C. for 30 min. Unbound antibodies were removed by centrifugation, together with 3 ml of PBS, at 1500 rpm for 3 min. The bound antibodies were detected by incubation with a 150-fold dilution of the secondary antibody goat anti-Mouse Ig-FITC (Dinona) at 4° C. for 15 min, followed by washing with 3 ml of PBS in the same manner. Thereafter, the antibody titer to A549 cells was measured by flow cytometry. Subsequently, a stable cell line was established. For this, the medium was exchanged with a PowerCHO medium (LONZA, Switzerland) supplemented with 30 nM MTX (Sigma, USA) and 200 μg/ml G418 (Invitrogen, USA), after which clone selection was started. Concentrations of MTX and G418 in the selection medium were increased with the repetition of clone selection rounds. Each round was set to be three weeks. The final round of clone selection was performed in a PowerCHO medium supplemented with 1000 nM of MTX and 400 μg/ml of G418. Thereafter, the final cell line was established as a single colony through limiting dilution.

[0136] The 4B4 chimeric antibody established in this manner was found to have antigen specificity and of similar to those of the original mouse 4B4 antibody, as measured by flow cytometry (FIG. 5). FIG. 5 shows the antigen specificity and affinity of the 4B4 chimeric antibody.

Example 6: Analysis of Antibody Expression in Various Cell Lines

[0137] 6-1: Antibody Expression in Various Cell Lines

[0138] 27B6 and 4B4 monoclonal antibodies were analyzed for binding to various cell lines obtained from KCLB (Korean Cell Line Bank) and SNU (Seoul National University) using flow cytometry. Briefly, various cell lines were obtained from KCLB (Korean Cell Line Bank) and SNU (Seoul National University). At 37° C. under a 5% CO.sub.2 atmosphere, L-132, SW-900, DU145, LNCap, MCF-7, Huh7, and Hs-578T were cultured in Dulbecco's MEM (GIBCO, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Invitrogen) and A549, NCI-H460, NCI-H417, DLD-1, HCT116, HT-29, SW-480, SW-620, LS174T, PC-3, SNU1, SNU638, SNU719, MKN1, MKN28, MKN45, MKN74, NCI-N87, SK-BR3, MDA-MB231, and MDA-MB453 were cultured in RPMI 1640 (GIBCO, Invitrogen) supplemented with 10% heat-inactivated FBS. In addition, incubation was carried out at 37° C. under a 5% CO.sub.2 atmosphere in Eagle's MEM (GIBCO, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Invitrogen) for Calu-3, Hep3B, SK-HEP-1, C3A, Hep G2, PLC/PRF/5, and BT-20, in IMDM (GIBCO, Invitrogen) supplemented with 20% heat-inactivated fetal bovine serum (FBS; GIBCO, Invitrogen) for KATO III, and in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Invitrogen) for SW480 and MDA-MB468.

[0139] The cultured cancer cell lines were incubated with the 27B6 or the 4B4 monoclonal antibody of the present disclosure at 4° C. for 30 min, washed with PBS, and treated with FITC-conjugated goat anti-mouse IgG (DiNona Inc, Korea) at 4° C. for 15 min. The cell lines were washed again with PBS before analysis by FACScaliber (Becton Dickinson, USA). The results are summarized in Table 3, below. Also, titers of the 27B6 and the 4B4 antibody were measured in various solid tumor cell lines.

[0140] As can be seen in Table 3, the 27B6 and the 4B4 monoclonal antibody according to the present disclosure were found to strongly bind to the lung adenocarcinomic cell line A549 and to some of colorectal cancer, stomach cancer, liver cancer, and breast cancer cell lines, but to bind either weakly or not at all to the normal cell line L-132, the small cell carcinoma cell line NCI-H417, 4 colon cancer cell lines including HT-29, 3 prostate cell lines, 7 gastric cell lines, and 5 liver cell lines. The results of PBMC indicate that normal blood cells are negative to both antibodies.

TABLE-US-00003 TABLE 3 Origin Cell line 27B6 4B4 Lung A549 +++ +++ NCl-H460 ++ ++ Colon HCT116 + − HT-29 + + LS174T +++ +++ Prostate LNCap + + PBMC Lymphocyte − − Monocyte − − Granulocyte − − Gastric SNU 719 + ++ MKN 45 + +++ Liver Huh-7 − ++ Hep3B − + PLC/PRF/5 +++ +++ Breast MCF-7 + + SK-BR3 +++ +++ MDAMB231 +++ +++ MDAMB453 +++ ++ BT20 + − (The percentages of 27B6 and 4B4 positive cells among 5,000 cells were calculated by FACS analysis −: less than 20% of positive cells, +: 20-30%, ++: 40-70%, +++: 60-100%)

[0141] 6-2: Expression Pattern in Breast Cancer Cell

[0142] 27B6 and 4B4 were observed to have positive responses to all ER-, PR-, and HER2-positive breast cancer cells. Accordingly, both antibodies can be used as therapeutic agents for various breast cancer subtypes including triple-negative breast cancer.

[0143] The binding of the 27B6 and the 4B4 monoclonal antibody to three different phenotype breast cancer cell lines was examined via flow cytometry. Cell culturing was carried out at 37° C. under a 5% CO.sub.2 atmosphere for MCF-7 cells in Dulbecco's MEM (GIBCO, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Invitrogen) and for MDA-MB231 and SK-BR-3 cells in RPMI 1640 (GIBCO, Invitrogen) supplemented with 10% heat-inactivated FBS.

[0144] The cultured cancer cell lines were incubated at 4° C. for 30 min with the 27B6 or the 4B4 monoclonal antibody of the present disclosure, washed with PBS, and treated at 4° C. for 15 min with FITC-conjugated goat anti-mouse IgG (DiNona Inc, Korea). The cell lines were washed again with PBS before analysis by FACScaliber (Becton Dickinson, USA). The results are summarized in Table 3.

[0145] 6-3: IHC (ImmunoHistoChemistry)

[0146] Antigens to which 27B6 and 4B4 monoclonal antibodies bind were analyzed for distribution in normal tissues of the human body by immunohistochemistry (IHC). Normal thymus and tonsil tissues of the human body were obtained from the Chungbuk National University Hospital and prepared into cryosections in the department of pathology in the Chungbuk National University Hospital.

[0147] The prepared cryosections were subjected to immunohistochemical staining with 27B6 and 4B4 monoclonal antibodies of the present disclosure as follows. Thymus and tonsil cryosections stored at −20° C. or lower were dried at room temperature for 30-60 min, and immersed in 1×PBS for 60 min. Then, the tissues were treated at room temperature for 10 min with 3% H.sub.2O.sub.2 to suppress the activity of endogenous peroxidase, washed with flowing water, and blocked at room temperature for 30 min with a goat immunoglobulin-containing serum to exclude non-specific staining with mouse antibodies. Then, the tissues were incubated at room temperature for 60 min with the primary antibody (27B6, 4B4). Each antibody was used at a concentration of 10 μg/ml. Thereafter, the tissues were washed three times with 1×PBS for 5 min, incubated at room temperature for 30 min with an HRP-conjugated goat anti-mouse antibody (Dako, Denmark), and then washed three times with 1×PBST (0.05% Tween20, 1× PBS) for 5 min. The color was developed with diaminobenzidine (DAB), followed by washing for 5 min with flowing water. The tissues were counterstained with hematoxylin and then washed for 7 min with flowing water. After staining, the slides were dehydrated and sealed. The staining results were analyzed by microscopy and are shown in Table 4, below.

[0148] As shown in Table 4, the antigens that the 27B6 and the 4B4 monoclonal antibody of the present disclosure recognize are distributed neither in normal thymus nor in normal tonsil tissues. Particularly, nowhere are the antigens expressed in normal mature or immature T cells or B cells. The 27B6 antibody was weakly stained in the basal layer of the tonsil, which, however, seemed to result from non-specific binding.

TABLE-US-00004 TABLE 4 27B6 4B4 Thymus Cortex − − Medulla − − Tonsil Inter follicular T cell − − B cell − − Germinal center − − Basal layer +

Example 7: Analysis of Antigen for Monoclonal Antibody

[0149] 7-1: Isolation and Purification of 4B4 and 27B6 Monoclonal Antibodies

[0150] The lung adenocarcinomic cell line A549 that had been used to develop the 4B4 and the 27B6 monoclonal antibody was cultured. Then, 1×10.sup.8 cells were suspended in 50 ml of a lysis buffer (1% Nonidet P-40; NP-40 in 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, and 1 mM phenyl-methyl-sulfonyl-fluoride; PMSF) and lysed for 15 min. After centrifugation, the cell debris was removed, and a cell lysate was obtained as a supernatant. The cell lysates was used to separate antigens that were recognized by 4B4 or 27B6 antibodies.

[0151] Five mg of each of purified 4B4 and 27B6 monoclonal antibodies were dialyzed against a binding buffer (0.2 M sodium bicarbonate, 0.5M sodium chloride, pH 8.3) to afford two different antibody solutions. A 5-ml column packed with 2 ml of NHS-activated sepharose 4 Fast Flow resin (GE Healthcare) was washed with 20 ml of 1 mM HCl and then with 20 ml of a binding buffer (20 mM sodium bicarbonate, 0.5 M sodium chloride, pH 8.3) so as to allow the prepared antibodies to bind to the column. The column was blocked at the outlet thereof, loaded with either of the two different antibody solutions, and blocked at the inlet thereof. Incubation was performed at room temperature for 4 hrs. Then, 20 ml of a washing buffer (20 mM Sodium acetate, 0.5M sodium chloride, pH 5.4) was made to flow through the column so as to remove excess antibodies that were not bound to the resin. Again, the column was washed with 50 ml of a blocking buffer (0.1 M ethanolamine, 0.5 M sodium chloride, pH 8.3) to remove remaining reaction groups. The two columns were washed with 20 ml of a stock buffer (20 mM Tris-HCl, 150 mM NaCl, 0.02% sodium azide, pH 8.0), and refrigerated until use.

[0152] The prepared columns were applied to FPLC (Acta FPLC) so that the antibodies bound to the resin could recognize antigens and thus could allow for the separation of the antigens. The lung adenocarcinomic A549 cell line lysates was loaded to the column coupled to FPLC and used as an antigenic source that was recognized by 4B4 and 27B6 monoclonal antibodies. Antigen separation was performed in a four-step process: equilibrium; sample loading; washing and second washing; elution. An equilibrium buffer and a wash buffer have the same composition: 0.5% Tween-80, 20 mM Sodium phosphate, 150 mM sodium chloride, pH 7.4. This buffer was used in an amount of 10 ml for equilibrium and in an amount of 20 ml for washing. An elution buffer contained 0.3 M Glycine, 0.1 M sucrose, 0.1 M Mannitol, 1.0 M urea, and 0.5% Tween-80, had a pH of 3.0, and was used in an amount of 20 ml for washing. For the second washing, a mixture in which the elution buffer was mixed at a ratio of 25% with the washing buffer was employed. 5 ml of TCA was added to 20 ml of the eluted solution obtained during the antigen separation and stored for 30 min in a refrigerator. After centrifugation, the pellet was further washed twice with acetone. The finally obtained pellet was suspended in 1×SDS-PAGE sample buffer, subjected to electrophoresis, and stained with Coomassie blue. As described above, antigens that were isolated and purified through the columns respectively fabricated with 4B4 and 27B6 antibodies are shown in FIG. 7. FIG. 7 shows electrophoretograms of antigens isolated and purified from the lung adenocarcinomic A549 cell line through columns fabricated with 4B4 and 27B6 monoclonal antibodies.

[0153] 7-2: Identification of Antigen for 4B4 and 27B6 Monoclonal Antibodies

[0154] The antigens isolated and purified from the resin coupled with the 4B4 and the 27B6 monoclonal antibody were visualized as shown in FIG. 7. The two main protein bands indicated by the arrows at about 58 kDa were analyzed in Seoul Pharma Laboratories. For identification, peptides were prepared via in-gel digestion and analyzed using LC-MS/MS, followed by processing the MS/MS spectra with PLGS (Waters) and MASCOT (Matrix Science). A series of analyses was conducted as follows.

[0155] Gel pieces containing proteins were dehydrated using 100% CAN (acetonitrile) and completely dried in a Speed-vac. The proteins in the dried gel pieces were digested for 15 min with trypsin. The tryptic peptides were extracted with 60% CAN and 0.1% TFA. The pooled extracts were dried in a Speed-vac. The samples were dissolved in 5% CAN, 0.2% TFA (Trifluoroacetic acid) 20 μl prior to LC-MS/MS analysis. Peptides were eluted from the LC column nanoACQUITY UPLC BEH C18 (1.7 μm, 300 Å, 2.1 mm×150 mm I.D.), with a gradient of a mobile phase buffer A (0.1% TFA in 100% DW) to a mobile phase buffer B (0.1% TFA in 100% ACN) in a LC-MS/MS analysis. The separated peptides were analyzed online in a positive survey scan mode on a nano-ESI-Q-TOF instrument. Subsequently, the spectral data were processed with PLGS and MASCOT.

[0156] A series of the analysis processes afforded the final identification results given in FIG. 8. Of the analysis results, carbonic anhydrase 12 was identified in common from the antigens purified by both the 4B4 and the 27B6 monoclonal antibody, as expected, and was found to exist on cell surfaces. The other proteins cannot be antigens for the 4B4 and the 27B6 monoclonal antibody because they are intracellular proteins. Thus, they seemed to be impurities that were included due to imperfect separation and purification. Four peptides were separated by 27B6: QFLLTNNGHSVK (SEQ ID NO: 22), WTYFGPDGENSWSK (SEQ ID NO: 23), GQEAFVPGFNIEELLPER (SEQ ID NO: 24), and YKGQEAFVPGFNIEELLPER (SEQ ID NO: 25). Three peptides were separated by 4B4: QFLLTNNGHSVK (SEQ ID NO: 22), EMINNFR (SEQ ID NO: 26), and GVIYKPATK (SEQ ID NO: 27). Of them, the sequence QFLLTNNGHSVK was analyzed in common in both 4B4 and 27B6. FIG. 9 shows the amino acid sequence of carbonic anhydrase 12 precursor isoform 1, with the analyzed peptide sequence expressed in bold. FIG. 8 lists proteins identified by LC-MS/MS analysis from purified antigens. FIG. 9 shows the amino acid sequence of carbonic anhydrase 12 isoform 1, in which amino acid sequences of the antigens for 4B4 and 27B6 antibodies, detected by LC-MS/MS, are marked.

[0157] 7-3: Assay of Antigen for 4B4 and 27B6 Monoclonal Antibodies (ELISA)

[0158] To evaluate the antigen identification results obtained by LC-MS/MS, the reactivity of the 4B4 and the 27B6 monoclonal antibody to the recombinant protein carbonic anhydrase 12 (R&D Systems) were examined by ELISA and Western blotting assay.

[0159] The recombinant protein CA12 was plated at a density of 100 ng/well into Maxisrop ELISA plates and incubated at 37° C. for 1 hr. To each of the antigen-coated wells, 200 μl of a 1× blocking buffer (Sigma) was added, followed by incubation at 37° C. for 1 hr for blocking. 4B4, 27B6, and an anti-CA12 monoclonal antibody (R&D Systems) were plated, together with 100 μl of PBS, into the plates. After incubation for 1 hr at 37° C., the plates were washed with PBS to remove unbound antibodies. Subsequently, a dilution of goat anti-mouse IgG-HRP (Jackson) was added to the wells, reacted for 30 min, and washed with PBS. Color development was accomplished for 10 min with 50 μl of TMB in each well, and stopped with 50 μl of sulfuric acid. Absorbance at 450 nm was read. Although the reactivity of the 27B6 monoclonal antibody to the recombinant carbonic anhydrase 12 was low, reactivity signals of 4B4, 27B6, and anti-CA12 monoclonal antibody (R&D Systems) against the recombinant antigen are shown in FIG. 10. FIG. 10 shows the identification of carbonic anhydrase 12 as an antigen for the 4B4 and the 27B6 monoclonal antibody, as analyzed by ELISA assay.

[0160] 7-4: Assay of Antigen for 4B4 and 27B6 Monoclonal Antibodies (Western Blotting)

[0161] The recognition of carbonic anhydrase 12 as an antigen by the 4B4 and the 27B6 monoclonal antibody, proven in the previous experiment, was confirmed by Western blotting. The recombinant carbonic anhydrase 12 was boiled for 3 min, loaded into an 8% separating sodium dodecyl sulfate-polyacrylamide gel, and run by electrophoresis. The separated proteins were transferred to a nitrocellulose membrane which was then blocked with 5% skim milk (Sigma) and treated with the 4B4, 27B6, or anti-CA12 monoclonal antibody (R&D Systems) (27B6: lanes 1 and 2, 4B4: lanes 3 and 4, anti-CA12 monoclonal antibody: lanes 5 and 6). After three rounds of washing with a wash buffer (0.1% Tween-20 in PBS), the antibody was coupled with peroxidase-conjugated goat anti-mouse IgG (Sigma, Saint Louis, USA). After the nitrocellulose membrane was washed with a wash buffer, bands were visualized using an enhanced chemiluminescence detection system (ECL, Amersham, Sweden). The results are shown in FIG. 11. FIG. 11 shows the identification of carbonic anhydrase 12 as an antigen for the 4B4 and the 27B6 monoclonal antibody, as analyzed by Western blotting assay. The recombinant CA12 was read at 40 kDa by all of the 4B4, 27B6, and anti-CA12 monoclonal antibodies (R&D Systems).

[0162] 7-5: Assay of Antigen for 4B4 and 27B6 Monoclonal Antibodies (Sandwich ELISA)

[0163] ELISA and WB assays demonstrated that 4B4 and 27B6 monoclonal antibodies recognize carbonic anhydrase 12 as an antigen, but the detection signal of the 27B6 monoclonal antibody was relatively low. To compensate for the relatively low signal, Sandwich ELISA was conducted as follows. The chimeric 4B4 or 27B6 monoclonal antibody was plated at a concentration of 100 ng/well into Maxisrop ELISA plates and incubated at 37° C. for 1 hr. To each of the antigen-coated wells, 200 μl of 1× blocking buffer (Sigma) was added, followed by incubation at 37° C. for 1 hr for blocking. Two-fold serial dilutions of the recombinant carbonic anhydrase 12 staring from 100 ng/ml were added to wells, incubated at 37° C. for 1 hr, and washed with PBS to remove unbound antigens. Subsequently, the 4B4 monoclonal antibody and the 27B6 monoclonal antibody were added at a concentration of 100 ng/well to chimeric 27B6-coated wells and chimeric 4B4-coated wells, respectively. Following 1 hr of incubation at 37° C., the wells were washed with PBS to remove unbound antibodies. In addition, the bound antibodies were incubated with a dilution of goat anti-Mouse IgG-HRP (Jackson) for 30 min and washed with PBS. Color development was accomplished for 10 min with 50 μl of TMB in each well and stopped with 50 μl of sulfuric acid. The absorbance at 450 nm was read. When chimeric 27B6 and 4B4 were used as a capture antibody and a detector antibody, respectively, high reaction signals were read, as shown in FIG. 2. Both of the 4B4 and 27B6 monoclonal antibodies were therefore proven to recognize carbonic anhydrase 12 as an antigen.

[0164] FIG. 12 shows the concurrent recognition of carbonic anhydrase 12 by the 27B6 and 4B4 monoclonal antibodies, as measured by sandwich ELISA assay using the 27B6 and 4B4 monoclonal antibodies as capture/detector antibodies.

Example 8: Epitope Mapping

[0165] To analyze an epitope, as shown in FIG. 13, recombinant antibodies, with or without the epitope determined in Example 8, were constructed, and analyzed for immune reactions.

[0166] 8-1: Construction of CA12 Mutant Recombinant Gene

[0167] The recombinant vector pSec-Tag-CA 12 full-hFc was digested with BamHI and HindIII to prepare CA12 mutant recombinant genes. A recombinant gene in which a full base sequence of CA12 antigen was fused to hFc was inserted into pSec-Tag which was then allowed to express a recombinant fusion protein containing the full length of CA12 plus hFc. As seen in FIG. 13, deletion mutant-hFc constructs having various lengths within a range from the N terminus to amino acid 300 were prepared.

[0168] 8-2: Expression of CA12 Mutant Recombinant Genes

[0169] Respective pSec-Tag vectors carrying the CA12 full-hFc and five different deletion mutant-hFc constructs were introduced into CHO cells with the aid of ViaFect (Promega).

[0170] Briefly, one day before transfection, CHO cells were plated and incubated. After the medium was exchanged with a fresh one, a complex of the vector and ViaFect was applied to the CHO cells and incubated for 48 hrs. Two days after transfection, the culture supernatant was collected and analyzed for the expression of the gene by detecting human Fc (hFc) through sandwich ELISA.

[0171] 8-3: Assay of Epitope of Monoclonal Antibody

[0172] In order to examine a CA12 epitope recognized by the monoclonal antibodies of the present disclosure, 50 ng of an anti-human Ig antibody (Jackson Laboratory) was added to each well and incubated at 37° C. for 1 hr. The antibody fixed to the well, which would serve as a capture antibody, was blocked via incubation with 200 μl of a 1× blocking buffer (Sigma) at 37° C. for 1 hr in each well. Each of the respective cultures containing the CA12 full-hFc and the five different deletion mutant-hFc constructs was added at a concentration of 100 μl/well to the plates. Following 1 hr of incubation at 37° C., the wells were washed with PBS to remove unbound antibodies. Subsequently, a dilution of anti-mouse Ig, Fc specific-HRP (Jackson Laboratory) was added to the wells, reacted for 30 min, and washed with PBS. Color development was accomplished for 10 min with 50 μl of TMB in each well, and stopped with 50 μl of sulfuric acid. The absorbance at 450 nm was read. The presence of CA12 mutant-hFc proteins in the culture supernatants was examined using Capture & Detect Sandwich ELISA, with an anti-human Ig antibody serving as a control. The results are given in FIG. 13.

[0173] As can be seen in FIG. 13, the epitopes were located in a site from a.a. 25 to a.a. 57, which is a non-catalytic domain. Hence, the antibodies of the present disclosure do not bind to the catalytic domain of CA-XII, so they do not inhibit the enzymatic activity of CA-XII.

[0174] In detail, the epitope specific for the 27B6 antibody was found to have the amino acid sequence APVNGSKWTYFGPD of SEQ ID NO: 2 (the span from a.a. 25 to a.a. 38 on SEQ ID NO: 5), as analyzed by the deletion method. A three-dimensional crystal structure of CA-12 confined the epitope into 7 consecutive amino acids WTYFGPD (SEQ ID NO: 1) on the amino acid sequence of SEQ ID NO: 2. Further, the epitope specific for the 4B4 antibody was found to have the amino acid sequence GENSWSKKYPSCGGLLQSP of SEQ ID NO: 4 (the span from a.a. 39 to a.a. 57 on SEQ ID NO: 5) while a three-dimensional crystal structure of CA-12 confined the epitope into 14 consecutive amino acid sequence GENSWSKKYPSCGG of SEQ ID NO: 3 on the amino acid sequence of SEQ ID NO: 4.

Example 9: Therapeutic Effect of Antibody on Solid Tumor (CDC)

[0175] 9-1: CDC Effect in Lung Adenocarcinomic Cell Line

[0176] The lung adenocarcinomic cell line A549 cells were plated at a density of 5×10.sup.3 cells/well into 96-well plates and cultured for 20-24 hrs in a 37° C., CO.sub.2 incubator. After removal of the culture medium from each well, an RPMI medium, free of fetal bovine serum, was mixed with 10% human serum and the chimeric 27B6 antibody was added at a final concentration of 10 μg/ml to a mixture. This solution was plated at a concentration of 100 μl/well into the plates. The 4B4 antibody was also treated in the same manner. Following 3 hrs of incubation in a 37° C. CO.sub.2 incubator, Ez-CyTox agent (DOGEN, KOREA) was added in an amount of 10 μl to each well. Incubation for 3.5 hrs in a 37° C., CO.sub.2 incubator was followed by measuring absorbance at 450 nm on a plate reader. The results are given in FIG. 14. FIG. 14 shows the complement-dependent cytotoxic effects of the 27B6 antibody.

[0177] As can be seen in FIG. 14, the 27B6 and 4B4 monoclonal antibodies of the present disclosure exhibit complement-dependent cytotoxicity.

[0178] 9-2: CDC Effect in Triple-Negative Breast Cancer

[0179] As a target cell, the lung adenocarcinomic cell line A549 was plated at a density of 1×10.sup.4 cells/well into 96-well plates and cultured for 20-24 hrs in a 37° C., CO.sub.2 incubator. After removal of the culture medium from each well, an RPMI medium free of fetal bovine serum was mixed with 10% human serum, and the antibody was added at a final concentration of 10 μg/ml to a mixture. This solution was plated at a concentration of 100 μl/well into the plates. The 4B4 antibody was also treated in the same manner. Following 3 hrs of incubation in a 37° C. CO.sub.2 incubator, an Ez-CyTox viability kit (Daeil Lab, Seoul, Korea) was added in an amount of 10 μl to each well. Incubation for 4 hrs in a 37° C., CO.sub.2 incubator was followed by reading absorbance at 450 nm. As shown in FIG. 15, the 27B6 monoclonal antibody of the present disclosure exhibited complement-dependent cytotoxicity against lung adenocarcinomic tumors (FIG. 15).

Example 10: Therapeutic Effect of Antibody in Solid Tumor (ADCC)

[0180] 10-1: Assay for Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC-LDH Assay)

[0181] In order to prepare effector cells, Ficoll was added to a human blood sample (blood:Ficoll=1:2), followed by centrifugation at 2000 rpm for 20 min to obtain PBMCs (Peripheral Blood Mononuclear Cells). The PBMCs were stored at 37° C. in a 5% FBS-supplemented RPMI medium. The antibody-dependent cell-mediated cytotoxicity assay was conducted in conjunction with an LDH assay or a Luciferase assay.

[0182] As targets, various solid tumor cell lines—HT29 (colorectal cancer), A549 (lung adenocarcinoma), NCI-H460 (lung adenocarcinoma), and MCF7 (breast cancer)—were each plated at a density of 1×10.sup.4 cells/well into 96-well plates and cultured for 18-20 hrs in a 37° C., CO.sub.2 incubator. After removal of the culture medium from each well, the chimeric antibody was added at a concentration of 0 μg/mL, 0.1 μg/mL, or 1 μg/mL to a culture medium supplemented with 5% FBS, and then plated at a concentration of 100 μl/well into the plates, followed by incubation for 30 min in a 37° C. CO.sub.2 incubator. Thereafter, the effector cells prepared above were plated at a density of 5×10.sup.5 cells/well (50 times as many as the target cells), and cultured for 24 hrs in a 37° C. CO.sub.2 incubator. For a positive control, a lysis buffer was added before incubation at 37° C. for 24 hrs. Following 24 hrs of incubation, the cell culture was centrifuged at 2500 rpm for 5 min. The supernatant thus obtained was measured for LDH (lactate dehydrogenase) activity to calculate the cell lysis (Promega assay kit). As shown in FIG. 16, the 27B6 monoclonal antibody of the present disclosure exhibited antibody-dependent cell-mediated cytotoxicity in various solid tumors (FIG. 16). FIG. 16 shows the antibody-dependent cell-mediated cytotoxic effects of the 27B6 antibody.

[0183] 10-2: Antibody-Dependent Cell-Mediated Cytotoxicity Assay in Triple-Negative Breast Cancer (ADCC-LDH Assay)

[0184] In addition, the 27B6 antibody was found to exhibit high antibody-dependent cell-mediated cytotoxicity in triple-negative breast cancer cell lines for which no therapeutic agents had yet been developed (FIG. 17). FIG. 17 shows the antibody-dependent cell-mediated cytotoxic effects of the 27B6 antibody on triple-negative breast cancer cell lines.

[0185] 10-3: Antibody-Dependent Cell-Mediated Cytotoxicity Assay (ADCC-Luciferase Assay)

[0186] Effector cells were prepared in the same manner as in Example 9-2.

[0187] As target cells, the breast cancer cell lines MDAMB231 and SK-BR3 were each plated at a density of 1.25×10.sup.4 cells/well into 96-well plates and cultured for 20-24 hrs in a 37° C. CO.sub.2 incubator. After removal of the culture medium from each well, 25 μl of an RPMI medium containing 4% low IgG FBS was added to each well in which the cells were plated. 27B6 and 4B4 antibodies were 3-fold diluted in serial from 10 μg/ml to 1.2 ng/ml in an RPMI medium containing 4% low IgG FBS. The serial antibody dilutions were each added in an amount of 25 μl/well, and the plates were covered with respective lids and left on a clean bench. ADCC reporter cells (ADCC Reporter Bioassay, Promega) were harvested from the cell culture and suspended at a concentration of 3×10.sup.6 cells/ml in an RPMI medium containing 4% low IgG FBS. To each well was added 25 μl of the suspension of ADCC reporter cells, followed by 24 hrs of incubation in a 37° C. CO.sub.2 incubator. Before the plates were withdrawn, a frozen luciferase substrate was thawed in a water bath. The plates were removed from the clean bench and left at room temperature for 15 min. The luciferase substrate was added at a concentration of 75 μl/well to the plates and reacted for 30 min in a dark condition, followed by measuring luminescence with a luminometer.

[0188] As seen in FIG. 19, 27B6 and 4B4 antibodies, after being defucosylated by treatment with kifunensine, exerted greater antibody-dependent cell-mediated cytotoxicity on MDAMB231 and SK-BR-3 than did corresponding fucosylated ones. FIG. 19 shows the antibody-dependent cell-mediated cytotoxicity of defucosylated 4B4 and 27B6 chimeric antibodies.

Example 11: Therapeutic Effect of Defucosylated Antibody in Solid Tumor (ADCC)

[0189] 11-1: Assay for ADCC of Defucosylated Chimeric 27B6 Antibody-Colon, Lung, Breast Cancer

[0190] Cells producing the 27B6 or the 4B4 chimeric antibody were incubated with 100 ng/ml kifunensine to induce the defucosylation of antibody proteins. With regard to ADCC effects, the defucosylated antibodies were compared to corresponding fucosylated antibodies.

[0191] Assay for ADCC of Kifunensine-treated, ADCC-Enhanced, Chimeric 27B6 Antibody-Colon, Lung, Breast

[0192] As can be seen in FIG. 18, the antibodies defucosylated by kifunensine were increased in antibody-dependent cell-mediated cytotoxicity against various solid tumor cell lines. FIG. 18 shows the antibody-dependent cell-mediated cytotoxicity of the defucosylated 27B6 chimeric antibody.

[0193] 11-2: Assay for ADCC of Defucosylated Chimeric 27B6 Antibody—Triple-Negative Breast Cancer

[0194] Using a luciferase ADCC assay, antibody-dependent cell-mediated cytotoxicity against the triple-negative breast cancer cell line MDAMB231 and the HER2 receptor-positive breast cancer cell line SK-BR3 was analyzed. Antibodies, after being defucosylated by treatment with kifunensine, exerted greater antibody-dependent cell-mediated cytotoxicity on MDAMB231 and SK-BR-3 than did corresponding fucosylated ones. FIG. 19 shows the antibody-dependent cell-mediated cytotoxicity of defucosylated 4B4 and 27B6 chimeric antibodies, as measured by a luciferase assay.

Example 12: Therapeutic Effect of 27B6 and 4B4 Antibodies in Mouse Models

[0195] 12-1: Cell Line Establishment

[0196] Animal models with human breast cancer were established using the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-453. First, MDA-MB-231 or MDA-MB-453 was subcutaneously injected at a dose of 1.5×10.sup.8 cells (in RPMI: Matrigel mixture) into the right flank of mice. The injected mice were randomly classified into test and control groups.

[0197] FIG. 20 further shows the binding of 27B6 and 4B4 antibodies to the surface of MDA-MB231 cells utilized in the animal experiment, and FIG. 21 shows the results of the animal experiment using the antibodies, demonstrating that the antibodies suppress the growth and size of MDA-MB231-induced tumor.

[0198] As test materials, the 27B6 fucosylated chimeric antibody, 27B6 defucosylated chimeric antibody, 4B4 fucosylated chimeric antibody, and 4B4 defucosylated chimeric antibody were inoculated into breast cancer cells. Three days later, the cells were intraperitoneally injected at a dose of 12 mg/kg to each mouse. Injection was conducted twice a week for three weeks. Tumor sizes were measured just before injection. The inhibitory activity of the anti-CA12 antibodies against breast cancer was expressed as the tumor volume calculated according to the following formula: (long axis×short axis.sup.2)/2.

[0199] 12-2: Inhibitory Activity of Anti CA12 Antibodies Against Triple-Negative Breast Cancer

[0200] Targeting a CA12 epitope specifically expressed on triple-negative breast cancer, anti-CA12 antibodies (27B6, 4B4) were assayed for inhibitory activity against triple-negative breast cancer (FIGS. 22 and 23).

[0201] Breast tumors were decreased in volume by 27B6, and the fucosylated antibody was superior in inhibitory activity against tumor growth to the corresponding defucosylated antibody. The inhibitory activity of the 27B6 fucosylated antibody against the growth of breast cancer tumors was found in both MDA-MB-231 and MDA-MB-453. The antibodies inhibited tumor growth at a rate of 98% in the MDA-MB-453 model and at a rate of 55% in the MDA-MB-231 model (FIG. 22). FIG. 22 shows the inhibitory activity of 27B6 antibodies against triple-negative breast cancer.

[0202] In addition, 4B4 antibodies, whether fucosylated or defucosylated, were found to inhibit tumor growth. Further, 4B4 antibodies were superior to 27B6 antibodies with regard to inhibitory activity against tumor growth. Still higher inhibitory activity was detected in the defucosylated form than in the fucosylated form. Particularly, complete remission was observed in the MDA-MB-453 model as the tumor did not grow further after day 21 (FIG. 23). FIG. 23 shows the inhibitory activity of the 4B4 antibodies against triple-negative breast cancer.

Example 13: Effect of Antibody on Cell Survival

[0203] When the antibodies were applied to CA12-positive cancer cells, the effects of the antibodies on cell growth and survival were examined. To this end, cells were plated at a density of 3×10.sup.4 cells/well into 96-well flat bottom plates one day before application (10% RPMI). After 24 hrs, the RPMI was removed, and fresh 5% RPMI containing the antibody was added in an amount of 100 μl to each well.

[0204] After 24 hrs, a CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, Cat.# G1780) was plated at a concentration of 50 μl/well and incubated for 30 min at room temperature. Cell viability was measured using a spectrophotometer. Twenty four hours after the antibody was applied to MDA-MB231 cells, the cell viability was measured.

[0205] The measurements are shown in FIG. 24. The administration of the antibodies neither promoted nor degraded cell viability. The antibodies did not inhibit CA12 enzymatic activity, and had no influences on tumor cell growth. Therefore, the antibodies according to the present disclosure were found to exhibit anti-tumor activity via ADCC and CDC through the immune system.

[0206] Cell viability was measured 24 hrs, 48 hrs and 72 hrs after the administration of the antibody to A549. No significant changes in cell viability were observed compared to the cells to which no antibodies were administered. FIGS. 24 and 25 show that the binding of the 4B4 antibody alone to tumor cells does not affect the growth of the tumor cells.

[0207] The 4B4 antibody, as an anti-CA12 antibody, had no influence on cell growth only when the antibody was bound to cells. This seems to be attributable to the fact that the 4B4 antibody does not affect the enzymatic activity of CA12 because it binds to an N-terminal non-enzymatic region of the CA12 antigen.

Example 14: Combination of Antibody Therapy and Radiotherapy

[0208] An examination was made to see whether or not a combination of the antibody of the present disclosure and radiotherapy could bring about an increased anticancer effect.

[0209] Briefly, the 27B6 antibody of the present disclosure was used in combination with 5 μg/ml cisplatin, 2 Gy radiation, or 4 Gy radiation, and A549 cells were analyzed for CA12 expression via flow cytometry. As a result, both cisplatin and radiation were found to increase the expression of CA12 on cell surfaces, with the maximum expression level induced by 4 Gy radiation. This indicates that a combination of the anti-CA12 antibody of the present disclosure with radiotherapy is able to affect the growth of tumor cells (upper diagram in FIG. 26).

[0210] To assay the effect of the combined therapy on the growth of tumor cells, as shown in the lower diagram of FIG. 26, the viability of the cancer cell line A549 was measured via an MTT assay after it was treated with a combination of the 27B6 antibody and radiotherapy. In FIG. 26, the lower graph shows the effects of a combination of the 27B6 antibody and radiotherapy on cell viability. As can be seen in FIG. 26, a combination of 27B6 and radiotherapy induced cell death at higher rates, compared to the antibody alone or a combination of an isotype control antibody and radiotherapy.