antibody to human trop-2, monoclonal antibody to human trop-2, antibody-derived antibody fragment, h

专利摘要:
ANTI-HUMAN ANTIBODY TROP-2 HAVING ANTI-TUMOR ACTIVITY IN VIVO The present invention provides: an antibody, which specifically reacts with hTROP-2 and has anti-tumor activity in vivo; a hybridoma, which produces the antibody already mentioned; an already mentioned antibody complex and a drug; a pharmaceutical composition for diagnosing or treating a tumor; a method to detect a tumor and a kit to detect or diagnose a tumor.
公开号:BR112012029281B1
申请号:R112012029281-1
申请日:2011-05-17
公开日:2020-12-08
发明作者:Koji Nakamura；Kentaro Okamura；Maki Tamura；Hiroyuki Yanai；Toru Kanke
申请人:Livtech, Inc；
IPC主号:

专利说明:

Technical Field
[0001] The present invention relates to a human anti-TROP-2 antibody having anti-tumor activity and particularly, a human anti-TROP-2 antibody having anti-tumor activity in vivo. In addition, the present invention relates to a hybridoma, which produces the antibody already mentioned and a use of the antibody already mentioned. Fundamental Technique
[0002] Human TROP-2 (Tacstd2, GA733-1 and EGP-1) (also referred to below as “hTROP-2”) is a single transmembrane type 1 cell membrane protein consisting of 323 amino acid residues (see SEQ ID NO: 2) and this protein has been known to be overexpressed in various types of epidermal cell carcinomas. The presence of a cell membrane protein associated with immune resistance, which is commonly expressed in both human trophoblasts and cancer cells, has been suggested (Document other than Patent 1). An antigen molecule recognized by mouse monoclonal antibodies (16225.3, 162-46.2) reacts with the cell membrane protein of a human choriocarcinoma cell line BeWo has been identified. This antigen molecule was considered as one of the molecules expressed in human trophoblasts and was named as Trop-2 (Document other than Patent 2). Then, the same molecule was discovered by other researchers. That is to say, a tumor antigen recognized by a mouse monoclonal antibody GA733 that is obtained by immunization with stomach cancer cells SW948 has been termed as GA733-1 (Document other than Patent 3) and an epithelial glycoprotein recognized by a monoclonal antibody human RS7-3G11 that is obtained by immunization with non-small cell lung cancer cells has been termed as epithelial antigen / carcinoma, EGP-1 (Non-Patent Document 4). In 1995, the Trop-2 gene was cloned and, as a result, it was confirmed that these are the same molecules (Document other than Patent 5). In addition, it has been clarified that the molecule has a function to amplify intracellular calcium signals in cancer cells (Non-Patent Document 6) and therefore has also been referred to as tumor-associated calcium signal transducer 2 (TACSTD2) .
[0003] The hTROP-2 gene is mapped on chromosome 1p32 and this constitutes a family of TACSTD genes together with GA733-2 having approximately 50% homology to this (which was known as “TACSTD1,” “EGP-2 epithelial glycoprotein , ”“ EpCAM ”or“ Trop-1 ”) (Document other than Patent 7). The hTROP-2 protein (323 amino acid residues; SEQ ID NO: 2) has a molecular weight of approximately 36K Dalton and this protein consists of a hydrophilic signal peptide (1 ° to 26 ° amino acids), an extracellular domain (27 ° to 274 ° amino acids), a transmembrane domain (275 ° to 297 ° amino acids) and an intracellular domain (298 ° to 323 ° amino acids). The extracellular domain has four heterogeneous N-linked glycosylation sites and its evident molecular weight is increased by 11 to 13K Dalton due to the addition of sugar chains (Non-Patent Document 5). The TACSTD gene family is considered to have a characteristic thyroglobulin repeat (TY) sequence in the extracellular domain and is associated with cancer cell proliferation, invasion and metastasis.
[0004] To date, a physiological ligand for hTROP-2 has not been identified and its molecular function has not been clarified. However, hTROP-2 has been reported to transmit a calcium signal in tumor cells (Non-Patent Document 6). Furthermore, from the facts that intracellular serine 303 is phosphorylated by Ca2 + dependent protein kinase C (Non-Patent Document 4) and that hTROP-2 has a PIP2 binding sequence in its intracellular domain, it has been suggested that hTROP-2 has a signaling function in tumor cells (Non-Patent Document 8).
[0005] As a result of in vitro diagnostic studies such as immunohistochemistry (IHC) and flow cytometry analysis, hTROP-2 overexpression in many types of epithelium-derived carcinomas, such as stomach cancer, lung cancer, cancer colon cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer and esophageal cancer have been reported. In contrast, the expression of hTROP-2 in normal tissues is limited to cells in the epithelial region and the level of expression of hTROP-2 in normal cells is lower than in cancer cells. In this way, the association of TROP-2 with tumor formation is suggested (Patent Documents 1 to 3 and 9).
[0006] In addition, in analyzing an expression level of hTROP-2 used as a biomarker in clinical samples, it has been shown that when hTROP-2 is highly expressed, correlating with colon cancer malignancy (Documents that non-Patent 10 and 11), pancreatic cancer (Document other than Patent 12) or oral cancer (Document other than Patent 13), the possibility of metastasis or recurrence and such cancer is significantly high. In addition, in a large-scale gene expression analysis using a cDNA micro-series technique, hTROP-2 has been identified as a genetic group, which is overexpressed at the highest level in severe papillary carcinoma of the ovary, compared to normal ovarian epithelium (Non-Patent Document 14).
[0007] Still, in recent years, an important role of hTROP-2 in tumor formation has been demonstrated in models using colon cancer cells (Non-Patent Document 15). Since hTROP-2 expression promotes cell proliferation independent of tumor cell anchorage and is required for tumor formation and proliferation of subcutaneously transplanted cancer cells in immunodeficient mice, this increased the possibility that hTROP-2 should act as a functional tumor antigen and should be used as a new therapeutic target.
[0008] To date, studies regarding the anti-tumor effects of several anti-hTROP-2 antibodies have been reported. An RS7 antibody (Patent Document 1) was examined using in vivo models, in which radiolabeled antibodies were used and anti-tumor activity was demonstrated in nude mouse xenograft models. However, anti-tumor effects by antibody alone (a naked antibody) has not been reported.
[0009] In addition, cytotoxicity of monoclonal antibody bound by anti-hTROP BR110 cytokine (Patent Document 2) in human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981 in in vitro experiments have been reported. However, the cytotoxicity of a naked antibody or a BR110 immune conjugate in vivo has not been disclosed.
[0010] In recent years, it has been reported that an isolated monoclonal antibody, which was produced from an AR47A6.4.2 or AR52A301.5 hybridoma cell line obtained by, immunizing mice with human ovarian cancer tissues, linked to hTROP-2 and that, for the first time, presented, as a naked antibody, anti-tumor activity in nude mouse xenograft models, as well as in vitro cytotoxicity (Patent Documents 3 and 4). In these Patent Documents, the antibody already mentioned showed anti-tumor effects by treatment with antibody only in mouse xenograft models, in which the pancreatic cancer cell lines BxPC-3 and PL45, a PC- prostate cancer cell line 3, an MCF-7 breast cancer cell line and a Colo205 colon cancer cell line were transplanted. The therapeutic effects of the antibody appeared in the models, in which BxPC-3 cells were translated. Other than this, formation and proliferation were only partially (approximately 40% to 60%) suppressed by preventive administration of the antibody and an extremely large amount (approximately 20 mg / kg) of the antibody was required to suppress formation and proliferation tumor.
[0011] Based on the previous findings mentioned above, the potential use of anti-hTROP-2 antibodies as an anti-tumor antibody has been suggested. However, not all anti-hTROP-2 antibodies show anti-tumor effects by treatment with antibody as bare antibodies in vivo only. The antibodies have different functions for hTROP-2, depending on a binding site, affinity and the profile of a monoclonal antibody.
[0012] Patent Document 1: U. S. Patent No. 6653104
[0013] Patent Document 2: U. S. Patent No. 5840854
[0014] Patent Document 3: U. S. Patent No. 7420040
[0015] Patent Document 4: U. S. Patent No. 7420041
[0016] Document other than Patent 1: Faulk WP, et al., Proc. Natl. Acad. Sci. U.S.A., 75 (4), pp. 1947-1951 (1978)
[0017] Document other than Patent 2: Lipinski M, et al., Proc. Natl. Acad. Sci. U.S.A., 78 (8), pp. 5147-5150 (1981)
[0018] Non-Patent Document 3: Linnenbach AJ, et al., Proc. Natl. Acad. Sci. U.S.A., 86 (1), pp. 27-31 (1989)
[0019] Document other than Patent 4: Basu A, et al., Int. J. Cancer, 62 (4), pp. 472-479 (1995)
[0020] Document other than Patent 5: Fornaro M, et al., Int. J. Cancer, 62 (5), pp. 610-618 (1995)
[0021] Document other than Patent 6: Ripani E, et al., Int. J. Cancer, 76 (5), pp. 671-676 (1998)
[0022] Non-Patent Document 7: Calabrese G, et al., Cell Genet., 92 (12), pp. 164-165 (2001)
[0023] Non-Patent Document 8: El Sewedy T et al., Int. J. Cancer, 75 (2), pp. 324-330 (1998)
[0024] Document other than Patent 9: Cubas R, et al., Biochim. Biophys. Acta., 1796 (2), pp. 309-314 (2009)
[0025] Document other than Patent 10: Ohmachi T et al., Clin. Cancer Res., 12 (10), pp. 3057-3063 (2006)
[0026] Non-Patent Document 11: Fang YJ, et al., Int. J. Colorectal Dis., 24 (8), pp. 875-884 (2009)
[0027] Non-Patent Document 12: Fong D, et al., Br. J. Cancer, 99 (8), pp. 1290-1295 (2008)
[0028] Document other than Patent 13: Fong D, et al., Mod. Pathol., 21 (2), pp. 186-191 (2008)
[0029] Non-Patent Document 14: Santin AD, et al., Int. J. Cancer, 112 (1), pp. 14-25 (2004)
[0030] Non-Patent Document 15: Wang J, et al., Mol. Cancer Ther., 7 (2), pp. 280-285 (2008) Summary of the invention
[0031] Under the circumstances already mentioned, it was desired to develop an anti-hTROP-2 antibody (an anti-hTROP-2 monoclonal antibody) having high anti-tumor activity in vivo and particularly, an anti-hTROP-2 antibody or similar, which has an anti-tumor effect as a naked antibody only in vivo and still, having the anti-tumor effect at a low dose.
[0032] The present invention has been completed, while taking into account the circumstances already mentioned. The present invention provides an anti-hTROP-2 antibody (an anti-hTROP-2 monoclonal antibody), a hybridoma, which produces the antibody, an antibody fragment, an antibody complex (an immunoconjugate) or the like, and a drug, a pharmaceutical composition to diagnose or treat a tumor, a method to detect a tumor, a kit to detect or diagnose a tumor and others, which will be described below.
[0033] (1) An antibody against human TROP-2, which has antitumor activity in vivo.
[0034] Examples of the antibody according to (1) above include: an antibody that exhibits tumor growth inhibitory activity of 50% or more at a dosage of 5 to 20 mg / kg body weight; an antibody whose frequency of administration to show tumor growth inhibitory activity is mostly once a week; an antibody that exhibits tumor growth inhibitory activity of 50% or more by a simple administration of 10 mg / kg body weight; an antibody having anti-tumor activity on two or more types of human tumor cell lines and an antibody having a dissociation constant (Kd value) of 1.0 x 10-10 M or less. Here, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer and particularly, human pancreatic cancer. In addition, the tumor also includes recurrent cancer and metastatic cancer. In addition, the tumor cell line is, for example, at least two types selected from the group consisting of a human pancreatic cancer cell line PK-59, a human pancreatic cancer cell line BxPC-3, a human cancer cell line pancreatic cancer KP-3L, a human pancreatic cancer cell line KP-2, a human pancreatic cancer cell line PK-1, a human pancreatic cancer cell line PK-45H, a human pancreatic cancer cell line PK-45P, a human pancreatic cancer cell line TCC-PAN2, a human pancreatic cancer cell line SUIT-2, a human colon cancer cell line CACO-2, a human colon cancer cell line SW480, a human cancer cell line colon DLD-1, a human colon cancer cell line HCT 116, a human breast cancer cell line JIMT-1, a human breast cancer cell line HCC1143, a human breast cancer cell line MCF-7, a human cancer cell line rostrate DU145 and a human prostate cancer cell line PC-3 and particularly, a human pancreatic cancer cell line PK-59 and a human pancreatic cancer cell line BxPC-3. Examples of the antibody according to (1) above include: an antibody in which the CDR amino acid sequences 1 to 3 of the V region of the H chain of the antibody are shown in SEQ ID NO: 36 to 38, respectively and / or the amino acid sequences of CDR 1 to 3 of the V region of the L chain of the antibody are shown in SEQ ID NO: 41 to 43, respectively; an antibody in which the CDR amino acid sequences 1 to 3 of the V region of the antibody H chain are shown in SEQ ID NO: 46 to 48, respectively and / or the CDR amino acid sequences 1 to 3 of the V region of antibody L chains are shown in SEQ ID NO: 51 to 53, respectively; an antibody in which the amino acid sequences of CDR 1 to 3 of the V region of the H chain of the antibody are shown in SEQ ID NO: 56 to 58, respectively and / or the amino acid sequences of CDR 1 to 3 of the V region of L chains of the antibody are shown in SEQ ID NO: 61 to 63, respectively, and an antibody in which the CDR amino acid sequences 1 to 3 of the V region of the H chain of the antibody are shown in SEQ ID NO: 66 to 68 , respectively and / or the CDR amino acid sequences 1 to 3 of the V region of the L chain of the antibody are shown in SEQ ID NO: 71 to 73, respectively.
[0035] An example of the antibody according to (1) above is a monoclonal antibody.
[0036] An example of the antibody according to (1) above is a genetically recombinant antibody and more specific examples include a chimeric antibody, a humanized antibody and a human antibody.
[0037] Here, examples of the chimeric antibody described above include: an antibody where the chimeric antibody's H chain V region consists of the amino acid sequence shown in SEQ ID NO: 35 and / or the L chain V region thereof. consists of the amino acid sequence shown in SEQ ID NO: 40; an antibody in which the H chain V region of the chimeric antibody consists of the amino acid sequence shown in SEQ ID NO: 45 and / or the L chain V region thereof consists of the amino acid sequence shown in SEQ ID NO: 50; an antibody in which the H chain V region of the chimeric antibody consists of the amino acid sequence shown in SEQ ID NO: 55 and / or the L chain V region thereof consists of the amino acid sequence shown in SEQ ID NO: 60 and an antibody in which the H chain V region of the chimeric antibody consists of the amino acid sequence shown in SEQ ID NO: 65 and / or the L chain V region thereof consists of the amino acid sequence shown in SEQ ID NO: 70.
[0038] (2) A monoclonal antibody against human TROP-2, which is produced by a hybridoma having accession No. FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346 or FERM BP-11254.
[0039] Examples of the antibody according to (1) and (2) above include: an antibody, which binds to a site, to which a monoclonal antibody produced by a hybridoma having accession No. FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346 or FERM BP-11254 and an antibody, which binds to a portion comprising at least one region (for example, any region) selected from the group consists of a region that consists of amino acids at positions 43 through 65, a region consisting of amino acids at positions 152 through 165, a region consisting of amino acids at positions 171 through 183, a region consisting of amino acids at positions 109 through 120, a region consisting of amino acids at positions 43 to 56 and a region consisting of amino acids at positions 193 to 206, in the human TROP-2 amino acid sequence shown in SEQ ID NO: 2.
[0040] (3) An antibody fragment derived from the antibody according to (1) or (2) above.
[0041] Examples of the antibody fragment according to (3) above include: an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 36 to 38 and / or the amino acid sequences shown in SEQ ID N °: 41 to 43, for example, an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 35 and / or the amino acid sequence shown in SEQ ID NO: 40; an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 46 to 48 and / or the amino acid sequences shown in SEQ ID NO: 51 to 53, for example, an antibody fragment comprising the sequence of amino acid shown in SEQ ID NO: 45 and / or the amino acid sequence shown in SEQ ID NO: 50; an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 56 to 58 and / or the amino acid sequences shown in SEQ ID NO: 61 to 63, for example, an antibody fragment comprising the sequence of amino acid shown in SEQ ID NO: 55 and / or the amino acid sequence shown in SEQ ID NO: 60 and an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 66 to 68 and / or the sequences amino acid numbers shown in SEQ ID NO: 71 to 73, for example, an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 65 and / or the amino acid sequence shown in SEQ ID NO: 70.
[0042] (4) A hybridoma, which produces the antibody according to (1) or (2) above.
[0043] (5) A hybridoma that produces a monoclonal antibody against human TROP-2, which has Accession No. FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346 or FERM ABP-11254.
[0044] (6) An antibody-drug conjugate, comprising the antibody according to (1) or (2) above and a substance having anti-tumor activity and / or cell death activity.
[0045] (7) An antibody-drug conjugate, comprising the antibody according to (3) above and a substance having anti-tumor activity and / or cell death activity.
[0046] With respect to the conjugate according to (6) and (7) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and cancer human breast cancer and particularly, human pancreatic cancer. In addition, the tumor also includes recurrent cancer and metastatic cancer.
[0047] (8) A pharmaceutical composition, comprising at least one type selected from the group consisting of the antibody according to (1) and (2) above, The antibody fragment according to (3) above and the conjugate of according to (6) and (7) above.
[0048] An example of the composition according to (8) above is a composition used in the treatment of tumor and particularly, a composition, which does not cause weight reduction as a side effect. Another example of the composition is a composition used in the diagnosis of tumor. Here, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer and particularly, human pancreatic cancer. In addition, the tumor also includes recurrent cancer and metastatic cancer.
[0049] (9) A tumor therapeutic agent, comprising at least one type selected from the group consisting of the antibody according to (1) and (2) above, The antibody fragment according to (3) above and the conjugate according to (6) and (7) above.
[0050] An example of the therapeutic agent according to (9) above is a therapeutic agent, which does not cause weight reduction as a side effect.
[0051] (10) A tumor diagnostic agent, comprising at least one type selected from the group consisting of the antibody according to (1) and (2) above, the antibody fragment according to (3) above and the conjugated according to (6) and (7) above.
[0052] With respect to the therapeutic agent according to (9) above and the diagnostic agent according to (10) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, cancer of human prostate, human colon cancer and human breast cancer and particularly, human pancreatic cancer. In addition, the tumor also includes recurrent cancer and metastatic cancer.
[0053] (11) A method for detecting a tumor, comprising: allowing at least one type selected from the group consisting of the antibody according to (1) and (2) above, The antibody fragment according to (3) above and the conjugate according to (6) and (7) above, to react with a sample collected from a living body and then detect a signal from the reacted antibody and / or antibody fragment.
[0054] With respect to the method according to (11) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer and particularly, human pancreatic cancer. In addition, the tumor also includes recurrent cancer and metastatic cancer.
[0055] (12) A kit to treat, diagnose or detect a tumor, comprising at least one type selected from the group consisting of the antibody according to (1) and (2) above, The antibody fragment according to ( 3) above and the conjugate according to (6) and (7) above.
[0056] With respect to the kit according to (12) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer and particularly, human pancreatic cancer. In addition, the tumor also includes recurrent cancer and metastatic cancer. Brief Description of Drawings
[0057] Figure 1 shows the measurement of antigen binding affinity (Kd: dissociation constant) of an anti-hTROP-2 monoclonal antibody (K5-70). Abt: Antibody (total); Agf: Antigen (free).
[0058] Figure 2 shows the reactivity of a hybridoma culture supernatant that produces an anti-hTROP-2 monoclonal antibody, with HuH-7 cells (TROP-2-negative) and HuH-7-hTROP-2 cells. the filled histogram indicates HuH-7 cells and the open histogram indicates HuH-7-hTROP-2 cells.
[0059] Figure 3 shows the reactivity of an anti-hTROP-2 monoclonal antibody with a human pancreatic cancer cell line (PK-59 cells), which endogenously express hTROP-2 on the cell surface. The filled histogram indicates the cell line reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell line reaction with each anti-hTROP monoclonal antibody.
[0060] Figure 4 shows the reactivity of an anti-hTROP-2 monoclonal antibody with a human pancreatic cancer cell line (BxPC-3 cells), which endogenously express hTROP-2 on the cell surface. The filled histogram indicates the cell line reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell line reaction with each anti-hTROP monoclonal antibody.
[0061] Figure 5 shows the reactivity of an anti-hTROP-2 monoclonal antibody (K5-70) with human pancreatic cancer cell lines. The filled histogram indicates the cell line reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell line reaction with each anti-hTROP monoclonal antibody.
[0062] Figure 6 shows the reactivity of an anti-hTROP-2 monoclonal antibody (K5-70) with human colon cancer cell lines (Colo320, CACO2, SW480, DLD1, CW2 and HCT 116), cancer cell lines human breast cells (JIMT-1 and HCC1143) and human prostate cancer cell lines (PC-3 and DU145). The filled histogram indicates the cell line reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell line's reaction with the anti-hTROP monoclonal antibody.
[0063] Figure 7 shows the cross-reactivity of anti-hTROP-2 monoclonal antibodies with mouse TROP-2. Cells prepared by allowing a mouse TROP-2 gene to be transiently expressed in CHO-K1 cells were used and a T2-102 antibody (mouse IgG1) that cross-reacts with mouse TROP-2 were used as an antibody positive control. The filled histogram indicates the cell's reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell's reaction with each anti-hTROP monoclonal antibody.
[0064] Figure 8 shows the cross-reactivity of anti-hTROP-2 monoclonal antibodies with human EpCAM / TROP-1. Cells prepared by allowing a human EpCAM / TROP-1 gene to be transiently expressed in CHO-K1 cells were used and a PE-labeled anti-human EpCAM monoclonal antibody (Becton, Dickinson and Company) was used as an antibody control positive. The filled histogram indicates the cell's reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell's reaction with each anti-hTROP monoclonal antibody.
[0065] Figure 9 shows the cell development inhibitory activity of anti-hTROP-2 antibodies (T6-16, T5-86, K5-70 and K5-107) in a human pancreatic cancer cell line (PK-59 cells ). mIgG indicates an antibody control (mouse IgG) and YY01 indicates a commercially available anti-hTROP-2 antibody (Santa Cruz). White column: 0 μ g / ml; gray column: 0.1 μ g / ml; black column: 1 μ g / ml. The level of activity was expressed as a reason for the value in the case of no antibody addition (0 μ g / ml). The error bar indicates a standard deviation. * P <0.05, ** P <0.01 (by Student's t test).
[0066] Figure 10 shows a scratch test of a human pancreatic cancer cell line (PK-59 cells) in the presence of anti-hTROP antibodies (T6-16 and K5-70).
[0067] Figure 10A shows representative examples of photographs of the PK-59 cell scratch regions. Day 0 shows a representative example immediately after the scratch. mIgG (day 1) shows a photograph taken on the 1st day (24 hours) after the scratch and then add an antibody control (mouse IgG, 1 μ g / ml) to the medium. K5-70 (day 1) shows a photograph taken 1 day (24 hours) after scratching and then adding a K5-70 antibody (1 μ g / ml) to the medium. T6-16 (day 1) shows a photo taken 1 day (24 hours) after scratching and then adding a T6-16 antibody (1 μ g / ml) to the medium. Each arrow in each photograph indicates the extent of a scratch region.
[0068] Figure 10B. The area of a scratch region was analyzed using image analysis software (Scion Image) and based on the value obtained, the values of various types of other samples were calculated using Day 0 of the control mIgG addition group as a standard value of 1. * P <0.05, ** P <0.01 (by Student's t test).
[0069] Figure 11 shows the FACS analysis using a stem cell marker, which was performed on a human pancreatic cancer cell line PK-59. Figure 11A is a view illustrating FACS showing the expression of EpCAM in PK-59 cells. The filled histogram indicates the cell's reaction with only a secondary antibody (PE-labeled anti-mouse IgG) and the open histogram indicates the cell's reaction with an anti-human EpCAM antibody (Becton, Dickinson and Company). Figures 11B and C are aspects that illustrate the FACS that shows the expression of P-glycoprotein / MCR1 from PK-59 cells (Figure 11B) or the expression of ABCG2 in PK-59 cells (Figure 11C). The blue histogram indicates a cell reaction with only a secondary antibody and the red histogram indicates a cell reaction with an anti-human P-glycoprotein / MDR1 antibody (BD Biosciences Pharmingen) (Figure 11B) or with a human ABCG2 antibody (BD Biosciences Pharmingen) (Figure 11C). Figure 11D shows the FACS analysis, in which PK-59 cells were double stained pancreatic cancer stem cell markers, an anti-human CD24 antibody labeled with FITC (BD Biosciences Pharmingen) and an anti-human CD44 antibody labeled with PE (BD Biosciences Pharmingen). Each number in Figure 11D indicates the ratio of cells in each fraction.
[0070] Figure 12 shows the evaluation of the anti-tumor activity of a new anti-hTROP K5-70 monoclonal antibody clone (mouse IgG2a) in xenograft treatment models using PK-59 cells.
[0071] Figure 12A shows the tumor development time course of a control group (mouse IgG) and a K5-70 administration group (a mean value ± standard deviation). The arrow indicates an antibody administration period. * P <0.05, ** P <0.01 (by Student's t test).
[0072] Figure 12B shows the plotted tumor weight of each mouse at the time of the 21st day (Day 21) (the final day of the experiment) in the test of Figure 12A. The numerical value in each plot indicates an average value ± standard deviation. ** P <0.01 (by Student's t test).
[0073] Figure 13 shows the evaluation of the anti-tumor activity of a K5-107 clone (A), a T6-16 clone (B) and a K5-116-2-1 clone (C) in treatment models of xenograft using PK-59 cells. The “•” symbol indicates a control group (mouse IgG) and the “o” symbol indicates an anti-hTROP-2 antibody administration group. The arrow on the graph indicates a period of antibody administration and the numerical value on each plot indicates an average value ± standard deviation. * P <0.05 (by Student's t test).
[0074] Figure 14 shows the evaluation of the anti-tumor activity of a K5-70 clone (A), a T6-16 clone (B) and a K5-116-2-1 (C) clone in prevention models xenograft using PK-59 cells. The “•” symbol indicates a control group (mouse IgG) and the “o” symbol indicates an anti-hTROP-2 antibody administration group. The arrow on the graph indicates a period of antibody administration and the numerical value on each plot indicates an average value ± standard deviation. ** P <0.01 (by Student's t test).
[0075] Figure 15 shows the evaluation of anti-tumor activity of a K5-70 clone in xenograft prevention and treatment models using BxPC-3 cells. Figure 15A shows the tumor development time course of a control group (mouse IgG) and a K5-70 administration group in prevention models (a mean value ± standard deviation). The arrow indicates an antibody administration period. ** P <0.01 (by Student's t test). Figure 15B shows the tumor development time course of a control group (mouse IgG) and a K5-70 administration group in treatment models (a mean value ± standard deviation). The arrow indicates an antibody administration period. * P <0.05 (by Student's t test).
[0076] Figure 16 shows the dose-dependent anti-tumor activity of a K5-70 clone in xenograft prevention models using PK-59 cells. The volume of a tumor is expressed as a mean value ± standard deviation.
[0077] Figure 16A shows the tumor development time course of a control group and K5-70 administration groups at different doses (a mean value ± standard deviation). The arrow indicates an antibody administration period. * P <0.05 (by Student's t test), ** P <0.01 (by Student's t test).
[0078] Figure 16B shows the plotted tumor weight of each mouse at the time of the 17th day (Day 17) (the final day of the experiment) in the test of Figure 16A. The numerical number on each plot indicates an average value ± standard deviation. ** P <0.01 (by Student's t test).
[0079] Figure 17 is a schematic view of a chimeric human / mouse TROP-2 protein used in the experiment. SP: signal sequence; TY domain: type 1 thyroglobulin region; TM: transmembrane region; C: intracellular region, where the filled region is a polypeptide derived from hTROP-2, whereas the open region is a polypeptide derived from mouse TROP-2. The number at the top of the schematic view of the chimeric protein indicates the amino acid number of a mouse TROP-2 protein and the number at its lowest position indicates the amino acid number of an hTROP-2 protein.
[0080] Figure 18 shows the results obtained by the identification of an anti-hTROP-2 monoclonal antibody binding region, using human / mouse chimeric TROP-2. Using HEK293 cells, which constantly express human / mouse chimeric TROP-2-C (hmTROP-2-C) or human / mouse chimeric TROP-2-D (mhTROP-2-D) proteins, reactivity with antibodies monoclonal anti-hTROP-2 shown in the Figure was studied. As a negative control, mouse Ig22b was used.
[0081] Figure 19 shows the results obtained by identifying the antibody binding region of an anti-hTROP-2 monoclonal antibody.
[0082] An hTROP-2 gene and each chimeric human / mouse TROP-2 gene were introduced into HEK293 cells and the FACS analysis was then performed using the cells, in which the genes were transiently expressed. In Figure 19 (A), the reactivity of antibodies K5-70, K5-107, T5-86 and K5-116-2-1 with hTROP-2 (upper box), with hmTROP-2-A (median box) and with hmTROP-2-B (lower box) was studied. As a negative control, mouse Ig22b was used. In Figure 19 (B), the reactivity of antibodies T6-4 and T6-16 with hTROP-2 (upper box), with mhTROP-2-E (middle box) and with mhTROP-2-F (lower box) was studied . As a negative control, mouse Ig22b was used.
[0083] Figure 20 shows the expression of hTROP-2 in normal human tissues. The assays of normal human tissues were immunoted with an anti-hTROP-2 monoclonal antibody clone K5-63-17. (A) skin, (B) esophagus, (C) kidney (cortex), (D) kidney (marrow), (E) pancreas, (F) prostate, (G) bladder, (H) amygdala, (I) heart , (J) liver (magnification: x 200)
[0084] Figure 21 shows the expression of hTROP-2 in cancerous tissues. The human cancerous tissue assays were immunoted with an anti-hTROP-2 monoclonal antibody clone K5-63-17. (A) breast cancer, (B) lung cancer, (C) esophageal cancer, (D) stomach cancer, (E) pancreatic cancer, (F) colon cancer, (G) bladder cancer, (H ) prostate cancer, (I) ovarian cancer (magnification: x 100)
[0085] Figure 22 shows the anti-tumor activity of a K5-70 clone by simple administration in xenograft prevention models using PK-59 cells.
[0086] Figure 22A shows the tumor formation time course in a control group (mouse IgG •) and in a K5-70 (o) administration group (a mean value ± standard deviation). The arrow indicates administration of antibody. * P <0.05 (by Student's t test), ** P <0.01 (by Student's t test).
[0087] Figure 22B shows the plotted tumor weight of each mouse at the time of the 28th day (Day 28) (the final day of the experiment) in the test of Figure 22A. ** P <0.01 (by Student's t test).
[0088] Figure 22C shows the course of tumor formation time in each mouse in a control group (mouse IgG •) and in a K5-70 (o) administration group. The arrow indicates administration of antibody.
[0089] Figure 23 shows the anti-tumor activity of a K5-70 clone in xenograft treatment models using human colon SW480 cells.
[0090] Figure 23A shows the tumor formation time course in a control group (mouse IgG •) and in a K5-70 (o) administration group (a mean value ± standard deviation). The arrow indicates an antibody administration period. ** P <0.01 (by Student's t test).
[0091] Figure 23B shows the plotted tumor weight of each mouse at the time of the 44th day (Day 44) (the final day of the experiment) in the test of Figure 23A. ** P <0.01 (by Student's t test).
[0092] Figure 24 shows the anti-tumor activity of a K5-116-2-1 clone in xenograft treatment models using SW480 cells.
[0093] Figure 24A shows the tumor formation time course in a control group (mouse IgG •) and in a T6-16 (o) administration group (a mean value ± standard deviation). The arrow indicates an antibody administration period. ** P <0.01 (by Student's t test).
[0094] Figure 24B shows the plotted tumor weight of each mouse at the time of the 42nd day (Day 42) (the final day of the experiment) in the test of Figure 24A. ** P <0.01 (by Student's t test).
[0095] Figure 25 shows the anti-tumor activity of a T6-16 clone in models of xenograft treatment using SW480 cells.
[0096] Figure 25A shows the course of tumor formation time in a control group (mouse IgG •) and in a T6-16 (o) administration group (a mean value ± standard deviation). The arrow indicates an antibody administration period. * P <0.05 (by Student's t test).
[0097] Figure 25B shows the tumor weight plotted for each mouse at the time of the 42nd day (Day 42) (the final day of the experiment) in the test of Figure 25A. * P <0.05 (by Student's t test).
[0098] Figure 26 shows the dose-dependent anti-tumor activity of a K5-70 clone in xenograft treatment models using SW480 cells.
[0099] Figure 26A shows the tumor formation time course in a control group (mouse IgG •) and in a K5-70 administration group (o: 1 mg / kg, △: 5 mg / kg, □: 10 mg / kg) (an average value ± standard deviation). The arrow indicates an antibody administration period. * P <0.05 (by Student's t test).
[0100] Figure 26B shows the plotted tumor weight of each mouse at the time of the 42nd day (Day 42) (the final day of the experiment) in the test of Figure 26A. * P <0.05 (by Student's t test).
[0101] Figure 27 shows the anti-tumor activity of a K5-70 clone in xenograft treatment models using SW480 cells.
[0102] Figure 27A shows the anti-tumor activity of a K5-70 antibody at administration intervals of one week. The time course of tumor formation in a control group (•: mouse IgG) and in a K5-70 administration group (o: 10 mg / kg) is shown (a mean value ± standard deviation). The upper arrow (days 10, 17, 24, 31 and 38) indicates the administration of a K5-70 antibody. * P <0.05 by Student's t test.
[0103] Figure 27B shows the tumor formation time course by administering a K5-70 antibody at administration intervals once every ten days (■: q10d) or once every two weeks (•: q14d ). The closed circle (O) indicates a control group (mouse IgG, 10 mg / kg) (a mean value ± standard deviation). The filled arrows (▼: days 9, 19 and 29) and open arrows (▽: Days 9, 23 and 37) indicate the administration of a K5-70 antibody. * P <0.05, ** P <0.01 by Student's t test.
[0104] Figure 28 shows the dose-dependent anti-tumor activity of a T6-16 clone in xenograft treatment models using SW480 cells.
[0105] Figure 28A shows the tumor formation time course in a control group (•: mouse IgG) and in a T6-16 administration group (o: 1 mg / kg, △: 5 mg / kg, □: 10 mg / kg) is shown (an average value ± standard deviation). The arrow indicates an antibody administration period. ** P <0.01 (by Student's t test).
[0106] Figure 28B shows the plotted tumor weight of each mouse at the time of the 43rd day (Day 43) (the final day of the experiment) in the test of Figure 28A. ** P <0.01 (by Student's t test).
[0107] Figure 29 shows the anti-tumor activity of a T6-16 clone in models of xenograft treatment using SW480 cells. Time course of tumor formation in a control group (•: mouse IgG, 10 mg / kg) and in a T6-16 administration group (10 mg / kg) (o: q7d, △: q10d) is shown (a mean value ± standard deviation). Arrows (days 10, 17, 24, 31 and 38) and arrows (days 10, 20, 30 and 40) indicate administration of a T6-16 antibody. Administration was performed once every three days to the control group. * P <0.05, ** P <0.01 by Student's t test.
[0108] Figure 30 shows the anti-tumor activity of a K5-70 clone in xenograft prevention models using DU-145 human prostate cells.
[0109] Figure 30A shows the tumor formation time course in a control group (•: mouse IgG) and in a K5-70 (o) administration group (a mean value ± standard deviation). The arrow indicates an antibody administration period. * P <0.05 (by Student's t test). Figure 30B shows the plotted tumor weight of each mouse at the time of the 40th day (Day 40) (the final day of the experiment) in the test of Figure 30A. * P <0.05 (by Student's t test).
[0110] Figure 31 shows the metastasis inhibiting activity of a K5-70 clone in metastatic liver models using PK-59 cells.
[0111] Figures 31A and 31B show the image of the cut liver from a control group (mouse IgG) (A) and a K5-70 administration group (B), which were removed 6 weeks after cell transplantation. The arrows indicate the metastatic foci in the liver.
[0112] Figure 32 shows the anti-tumor activity of K5-70 in models and xenograft using SW480 cells, which are recurrent models after the administration of irinotecan hydrochloride. This Figure shows the time course of tumor formation in an untreated group (♦), in an irinotecan hydrochloride administration group (40 mg / kg) + K5-70 (o: 10 mg / kg) and in an irinotecan hydrochloride administration group (40 mg / kg) + mouse IgG (•: 10 mg / kg) (a mean value ± standard deviation). The arrows (days 11, 14 and 17) indicate the administration of irinotecan hydrochloride. K-70 antibody or mouse IgG was administered once every three days from Day 20. The arrow indicates an antibody administration period. * P <0.05, ** P <0.01 by Student's t test.
[0113] Figure 33 shows the cDNA nucleotide sequence of a K5-70 H (VH) chain variable region (SEQ ID NO: 34) and the deduced amino acid sequence (SEQ ID NO: 35). A signal peptide is shown in italics. Double-underscore (Q) glutamine indicates the N-terminal amino acid residue of a mature peptide. The CDR sequences (underlined; IYWIN, NIYPSDSYTNYNQKFKD and TSMADY) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequences of CDR 1 to 3 of clone K5-70 VH are shown in SEQ ID NO: 36 to 38, respectively.
[0114] Figure 34 shows the cDNA nucleotide sequence of a variable region of the K5-70 L clone chain (VL) (SEQ ID NO: 39) and the deduced amino acid sequence (SEQ ID NO: 40) . A signal peptide is shown in italics. Double underlined aspartic acid (D) indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; RASQSIGTSIH, YASESIS, and QQSNSWPFT) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone K5-70 VL are shown in SEQ ID NO: 41 to 43, respectively.
[0115] Figure 35 shows the cDNA nucleotide sequence of a variable region of the K5-107 H (VH) clone chain (SEQ ID NO: 44) and the deduced amino acid sequence (SEQ ID NO: 45) . A signal peptide is shown in italics. Double-underscore (Q) glutamine indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; SYWMH, NIYPGGGYTNYDEKFKS, and SSVFDY) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone K5-107 VH are shown in SEQ ID NO: 46 to 48, respectively.
[0116] Figure 36 shows the cDNA nucleotide sequence of a variable region of clone chain K5-107 L (VL) (SEQ ID NO: 49) and the deduced amino acid sequence (SEQ ID NO: 50) . A signal peptide is shown in italics. Double underlined aspartic acid (D) indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; RASQNIGTSIH, YASESIS, and QQSNSWPFT) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone K5-107 VL are shown in SEQ ID NO: 51 to 53, respectively.
[0117] Figure 37 shows the cDNA nucleotide sequence of a variable region of the K5-116-2-1 H (VH) clone chain (SEQ ID NO: 54) and the deduced amino acid sequence (SEQ ID N °: 55). A signal peptide is shown in italics. Double-underscore (Q) glutamine indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; SYWIT, NIYPSDSYTNYNQKFRD and LFDY) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone K5-116-2-1 VH are shown in SEQ ID NO: 56 to 58, respectively.
[0118] Figure 38 shows the cDNA nucleotide sequence of a variable region of clone chain K5-116-2-1 L (VL) (SEQ ID NO: 59) and the deduced amino acid sequence (SEQ ID N °: 60). A signal peptide is shown in italics. Double underlined aspartic acid (D) indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; RASQSIGTSIH, YASESIS, and QQSNSWPFT) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone K5-116-2-1 VL are shown in SEQ ID NO: 61 to 63, respectively.
[0119] Figure 39 shows the cDNA nucleotide sequence of a variable region of the T6-16 H (VH) clone chain (SEQ ID NO: 64) and the deduced amino acid sequence (SEQ ID NO: 65) . A signal peptide is shown in italics. Glutamic acid with a double underline (E) indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; DYNMH, YIYPYNGGTGYNQRFKS and EDYGSSPSYAMDY) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone T6-16 VH are shown in SEQ ID NO: 66 to 68, respectively.
[0120] Figure 40 shows the cDNA nucleotide sequence of a T6-16 L clone chain variable region (VL) (SEQ ID NO: 69) and the deduced amino acid sequence (SEQ ID NO: 70) . A signal peptide is shown in italics. Double underlined aspartic acid (D) indicates the N-terminal amino acid residues of a mature peptide. The CDR sequences (underlined; RSSQSLVHGNGNTYLH, KVSNRFS, and SQTTHVPT) were determined according to the definitions by Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991). The amino acid sequence of CDR 1 to 3 of clone T6-16 VL are shown in SEQ ID NO: 71 to 73, respectively. Mode for Carrying Out the Invention
[0121] In the following, the present invention will be described in detail. The following descriptions are not intended to limit the scope of the present invention. Other than in the following examples, the present invention can be modified and can be carried out, where appropriate, within a range that does not impair the intent of the present invention.
[0122] This specification includes all content as disclosed in the specification of Japanese Patent Application No. 2010-113302 (filed on May 17, 2010), which is a priority document of this application.
[0123] In addition, all publications cited in this specification, which include prior art documents and Patent Documents such as order publications open to the public and patent publications, are hereby incorporated by reference in their entirety. 1. Summary of the present invention
[0124] As mentioned above, human TROP-2 (hTROP-2) is a single transmembrane type 1 membrane protein having a total length of 323 amino acid residues. It has been known that an hTROP-2 gene and a gene product thereof are expressed in various types of cancer cells.
[0125] As mentioned above, it was desired to develop an anti-human hTROP-2 antibody (an anti-human hTROP-2 monoclonal antibody) or the like having high anti-tumor activity in vivo. Under such circumstances, the present inventor performed an evaluation using an extremely large number of clones and as a result, the inventor was successful in obtaining a clone having high anti-tumor activity in vivo. Specifically, the present invention provides a monoclonal antibody, which specifically recognizes the extracellular region of hTROP-2 in vivo and particularly, a monoclonal antibody that exhibits high affinity in an order of picomol (pM). The antibody of the present invention is extremely useful in that it is an anti-hTROP-2 monoclonal antibody, which has significant tumor development inhibitory activity at a lower dose than that of the existing anti-hTROP-2 antibody (for example, in a 1/20 dosage) when it is administered solely as a naked antibody and which also exhibits significant tumor development inhibitory activity in tumor-bearing mouse treatment models, in which multiple types of human cancer cells are used. 2. Production of anti-hTROP-2 antibody
[0126] (1) Antigen preparation
[0127] Information regarding the hTROP-2 amino acid sequence (SEQ ID NO: 2) is disclosed under “Accession number NP 002344” on the website of, for example, NCBI (GenBank) (http: // www. ncbi.nlm.nih.gov/). Information regarding the nucleotide sequence (SEQ ID NO: 1) encoding an hTROP-2 amino acid sequence is disclosed under “Accession number NM 002353” on the same website as described above.
[0128] As an antigen, a polypeptide or peptide (which is also simply referred to as a peptide) comprising at least a portion (the total or a portion) of the hTROP-2 amino acid sequence can be used and preferably, a peptide comprising at least a portion (the total or a portion) of the amino acid sequence of an extracellular region of hTROP-2 can be used. The extracellular region (including a signal peptide) of hTROP-2 indicates a region comprising amino acids at positions 1 to 274 from the amino acid sequence shown in SEQ ID NO: 2 (the signal peptide: amino acids at positions 1 to 274) 26). Here, with respect to a peptide used as an antigen, the above description "at least a portion of the amino acid sequence" is not particularly limited in terms of length. For example, a region comprising amino acids at positions 1 to 145 from the amino acid sequence shown in SEQ ID NO: 2, a region comprising amino acids at positions 146 to 274 of the same amino acid sequence as described above and others are preferable.
[0129] A peptide used as an antigen can be produced by chemical synthesis or by synthesis according to a genetic engineering method using Escherichia coli or similar. A method well known to persons skilled in the art can be applied.
[0130] When a peptide is produced by chemical synthesis, it can be synthesized by a well-known peptide synthesis method. In addition, a solid phase synthesis method or a liquid phase synthesis method can be applied to peptide synthesis. A commercially available peptide synthesizer (for example, PSSM-8 manufactured by Shimadzu Corporation, etc.) can also be used.
[0131] When a peptide is synthesized by a genetic engineering method, first, DNA encoding the peptide is designed and synthesized. The design and synthesis of such DNA can be carried out according to a PCR method, using a vector comprising a full-length or similar hTROP-2 gene as a standard and also using the primers designed to be able to synthesize a desired DNA region. The DNA is then linked to a suitable vector to obtain a recombinant vector used for protein expression and this recombinant vector is then introduced into a host so that a gene of interest can be expressed here, thereby obtaining a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001).
[0132] As a vector, a phage or plasmid capable of replicating autonomously in a host microorganism is used. In addition, an animal virus vector or an insect vector and virus can also be used. For the production of a recombinant vector, a purified DNA can be cleaved with suitable restriction enzymes and the portion of DNA thus cleaved can then be inserted into the restriction enzyme or similar site of a suitable vector DNA in order to link to the vector. The type of host used in the transformation is not particularly limited, so that it is capable of expressing a gene of interest. Examples of such a host include bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, animal cells (COS cells, CHO cells, etc.), insect cells and insects. A mammal, such as a goat, can also be used as such a host. One method of introducing a recombinant vector into a host is publicly known.
[0133] The transformant described above is cultured and a peptide used as an antigen is then collected from the culture. The term "culture" is used here to mean either (a) a culture supernatant and (b) cultured cells, a cultured cell mass or a disintegrated product thereof.
[0134] After completion of the culture, when a peptide of interest is produced in a cell mass or cells, the peptide is extracted by disintegrating the cell mass or cells. On the other hand, when a peptide of interest is produced outside a cell mass or external cells, a culture solution is used directly or the cell mass or cells are removed from the culture solution by centrifugation or the like. Next, the peptide of interest can be isolated and purified by a simple use of biochemical methods commonly used in peptide isolation and purification, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography and affinity chromatography or appropriately. combining the biochemical methods already mentioned.
[0135] In the present invention, a peptide used as an antigen can also be obtained by in vitro translation using a cell-free synthesis system. In this case, two methods, that is, a method using RNA as a standard and a method of using DNA as a standard (transcription / transduction) can be applied. As such, a cell-free synthesis system, a commercially available system, such as ExpresswayTM (Invitrogen), PURESYSTEM (registered trademark; Post Genome Institute) or TNT system (registered trademark; Promega) can be used.
[0136] The peptide thus obtained can bind to a suitable carrier protein, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), human thyroglobulin or chicken Y-globulin.
[0137] In addition, the antigen may be a peptide consisting of an amino acid sequence that comprises a deletion, substitution or addition of one or more amino acids with respect to the amino acid sequence of hTROP-2 (SEQ ID NO: 2) or a partial sequence of this as described above. For example, a peptide consisting of an amino acid sequence can be used, in which one or more (preferably one or more (for example, 1 to 10 and, more preferably 1 to 5)) amino acids are deleted, or one or more (preferably one or more (for example, 1 to 10 and, more preferably 1 to 5)) amino acids are replaced with other amino acids or one or more (preferably one or more (for example, 1 to 10 and, more preferably 1 to 5)) other amino acids are added, with respect to the hTROP-2 amino acid sequence or a partial sequence thereof.
[0138] In the present invention, a gene to be introduced into a cell or the like is a gene encoding an hTROP-2 protein or a partial fragment thereof or a mutant protein thereof or a fragment thereof. As such a gene, a gene having the nucleotide sequence shown in SEQ ID NO: 1 or a partial sequence thereof can be used, for example.
[0139] In addition, as a gene to be introduced into a cell or the like, a nucleotide sequence that hybridizes under stringent conditions to a sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 and which encodes a protein having hTROP-2 activity or a partial sequence thereof.
[0140] The description “stringent conditions” is used here to mean washing conditions after completion of hybridization. As such stringent conditions, a concentration of salt (sodium) in buffer is 10 to 500 mM and a temperature is 42 ° C to 72 ° C and preferably, the salt condition already mentioned is 50 to 300 mM and a temperature from 55 ° C to 68 ° C.
[0141] The mutation can be introduced into a gene according to a known method such as a Kunkel method or a slit duplex method, using, for example, a mutation introduction kit that uses site-directed mutagenesis , such as the GeneTailorTM site-directed mutagenesis system (manufactured by Invitrogen) or the TaKaRa site-directed mutagenesis system (Mutan-K, Mutan-Super Express Km, etc., manufactured by Takara Bio Inc.).
[0142] (2) Production of polyclonal antibody
[0143] The prepared antigen is administered to a mammal for immunization. The type of such a mammal is not particularly limited. For example, a mouse, a mouse and a rabbit can be used and among these, a mouse is preferable.
[0144] The antigen dosage per animal can be determined, when appropriate, depending on the presence or absence of an adjuvant. Examples of such an adjuvant include a complete Freund's adjuvant (FCA), an incomplete Freund's adjuvant (FIA) and an aluminum hydroxide adjuvant. Immunization can be performed mainly by injecting the antigen into an animal's vein, paw pad, subcutis or abdominal cavity. In addition, the immunization interval is not particularly limited and immunization is carried out at intervals of several days to several weeks, preferably at intervals of 1 week, 1 to 10 times and preferably 2 or 3 times. Three to seven days after the day of final immunization, an antibody titrator is measured by enzyme immunoassay (ELISA or EIA), radioimmunoassay (RIA) or other methods. A date when a desired antibody titrator is obtained, blood can be collected to obtain antiserum. In the method described above of collecting an antibody, if it is necessary to purify the antibody, the antibody can be purified by selecting a suitable method from known methods, such as ammonium sulfate precipitation, ion exchange chromatography, filtration chromatography in gel and affinity chromatography or by combining the methods mentioned above, where appropriate. Next, the reactivity of a polyclonal antibody in the antiserum is measured by the ELISA method or similar.
[0145] (3) Production of monoclonal antibody
[0146] (3-1) Collection of cells that produce antibody
[0147] The anti-hTROP-2 antibody of the present invention is not limited, but is preferably a monoclonal antibody.
[0148] The prepared antigen is administered to a mammal, such as a rat, mouse or rabbit for immunization, such as a rat, mouse or rabbit, for immunization. The dosage of the antigen per animal can be determined, when appropriate, depending on the presence or absence of an adjuvant. The same adjuvants as described above can be used here. Also, the same methods of immunization as described above can be applied here. From one to six days and preferably from one to fourteen days after the day of final immunization, cells that produce antibodies are collected. Examples of such cells that produce antibodies include splenic cells, lymph node cells and peripheral blood cells. Of these, lymph node cells and splenic cells are preferable.
[0149] (3-2) Cell fusion
[0150] In order to obtain hybridomas (a cell line that produces antibody), cell fusion of cells that produce antibodies with myeloma cells is performed. Myeloma cells to be fused with cells that produce antibodies, the established cells commonly available from animals as they can be used. The cell line used here is preferably a cell line, which has drug selectivity, cannot survive in an unfused state in a HAT selective medium (containing hypoxanthine, aminopterin and thymidine) and can survive only in a state fused with cells that produce antibodies.
[0151] Examples of myeloma cells include mouse myeloma cell lines such as P3-X63-Ag8.653, P3-X63-Ag8 (X63), P3-X63-Ag8.U1 (P3U1), P3 / NS I / 1-Ag4-1 (NS1) and Sp2 / 0-Ag14 (Sp2 / 0). Myeloma cells can be selected, while taking into account compatibility with cells that produce antibodies, when appropriate.
[0152] Subsequently, myeloma cells are fused with cells that produce antibodies. For such cell fusion, cells that produce antibodies (1 x 106 to 1 x 107 cells / ml) are mixed with myeloma cells (2 x 105 to 2 x 106 cells / ml) in a medium for animal cells, such as DMEM or an RPMI-1640 medium containing no serum. The cellular ratio between cells that produce antibody and myeloma cells (cells that produce antibodies: myeloma cells) is not limited. In general, the cell ratio is preferably 1: 1 to 10: 1 and, more preferably 3: 1. Next, a fusion reaction is carried out in the presence of a cell fusion promoter. As such, a cell fusion promoter, polyethylene glycol having an average molecular weight of 1,000 to 6,000 Dalton (D) or the like can be used. In addition, it is also possible to fuse cells that produce antibodies with myeloma cells, which uses a commercially available cell fusion mechanism that uses electrical stimulation (for example, electroporation).
[0153] (3-3) Selection and cloning of hybridomas
[0154] Hybridomas of interest are selected from cells after cell fusion treatment. As a method of selecting hybridomas, the cell suspension is appropriately diluted with, for example, a medium of RPMI-1640 containing fetal bovine serum and the diluted solution is then dispersed in a microtiter plate. Then, a selective medium is added to each reservoir. As long as the selective medium is appropriately exchanged for a fresh one, culture is carried out. As a result, approximately 14 days after the start of culture in the selective medium, cells that develop from the selective medium can be obtained as hybridomas.
[0155] Subsequently, the evaluation is carried out to examine whether or not an antibody that reacts with hTROP-2 is present in a hybridoma development culture supernatant. The evaluation of hybridomas can be carried out according to a common method and, therefore, the evaluation method is not particularly limited. For example, an aliquot collected from the culture supernatant of the developmental hybridomas contained in the reservoir and the evaluation is then carried out by ELISA, EIA, RIA or similar.
[0156] The cloning of the fused cells can be performed by a limiting or similar dissolution method. An antibody that shows high reactivity with hTROP-2 is determined by flow cytometry or the like and a hybridoma that produces this antibody is then selected. The selected hybridoma is established as a clone.
[0157] (3-4) Monoclonal antibody collection
[0158] As a method of cultivating the established hybridoma and then collecting a monoclonal antibody from the culture obtained, a common cell culture method, an ascites formation method or the like can be adopted. The term "culture" is used here to mean allowing hybridomas to grow in a culture dish or a culture bottle or to allow hybridomas to grow in an animal's abdominal cavity, as described below.
[0159] In the case of the cell culture method, hybridomas are grown hybridomas are grown in a medium for animal cells, such as an RPMI-1640 medium containing 10% fetal bovine serum, a MEM medium or a serum-free medium, under common culture conditions (for example, 37 ° C, 5% CO2 concentration) for 7 to 14 and then an antibody can be obtained from the culture supernatant.
[0160] In the case of the ascites formation method, approximately 1 x 107 hybridomas are administered into the abdominal cavity of an animal of the same species as the mammal whose myeloma cells are derived, so that large amounts of hybridomas are allowed to proliferate. Then, 2 to 3 weeks later, ascites are preferably collected.
[0161] In the antibody collection methods described above, if it is necessary to purify the antibody, the antibody can be purified appropriately by selecting a suitable method from known methods such as ammonium sulfate precipitation, ion exchange chromatography, filtration in gel and affinity chromatography or by combining the methods mentioned above.
[0162] (3-5) selection of clones having anti-tumor activity
[0163] The anti-hTROP-2 antibody of the present invention is an antibody having anti-tumor activity in vivo.
[0164] The term "anti-tumor activity" is used here to mean tumor cell killing activity (cancer cells) or activity to inhibit tumor development. Preferred examples of such anti-tumor activity include activity to inhibit cancer cell development and activity to inhibit tumor angiogenesis. The type of human tumor (tumor cells), in which the antibody of the present invention is capable of exhibiting anti-tumor activity, includes several types of known human tumors, in which the expression of hTROP-2 has been confirmed. The type of such a human tumor is not particularly limited. For example, one or two or more of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer are preferable and human pancreatic cancer is more preferable.
[0165] In addition, the tumor described above may be a recurrent cancer or a metastatic cancer. The antibody of the present invention is also able to exhibit excellent anti-tumor activity against these tumor types.
[0166] The presence of anti-tumor activity in vivo can be confirmed, for example by using a tumor-bearing mouse (a mouse xenograft model), in the subcutis whose desired tumor cells have been transplanted and then by administration of the antibody as obtained above for the mouse. In this case, the antibody can be administered immediately after transplantation of tumor cells (prevention models). Alternatively, this can be administered after confirming that the transplanted tumor has reached a predetermined volume (treatment models). The method of administration is not limited. For example, the antibody can be administered once every three days, every week, every ten days, or every two weeks or by a simple administration (only one period), in a dosage of 5 to 20 mg / kg of body weight, through intraperitoneal administration or similar. In the case of prevention models, the presence or absence of anti-tumor activity and their level can be assessed based on the frequency of tumor formation and tumor volume. In the case of models and treatment, the presence or absence of anti-tumor activity and their level can be assessed based on the volume of the tumor.
[0167] In the present invention, a preferred example of the anti-hTROP-2 antibody having anti-tumor activity in vivo is an antibody in which the CDR amino acid sequences 1 to 3 of the H chain V region of these are shown in SEQ ID No.: 36 to 38, respectively and / or the amino acid sequences of CDR 1 to 3 of region V of the L chain of these are shown in SEQ ID NO: 41 to 43, respectively. A preferred example of the H chain V region is a H chain V region consisting of the amino acid sequence shown in SEQ ID NO: 35. A preferred example of the L chain V region is a L chain V region consisting of a sequence amino acid shown in SEQ ID NO: 40.
[0168] As another embodiment of the anti-hTROP-2 antibody of the present invention, a preferred example is an antibody in which the CDR amino acid sequences 1 to 3 of region V of the H chain of these are shown in SEQ ID NO: : 46 to 48, respectively and / or the amino acid sequences of CDR 1 to 3 of region V of the L chain of these are shown in SEQ ID NO: 51 to 53, respectively. A preferred example of the H chain V region is a H chain V region consisting of an amino acid sequence shown in SEQ ID NO: 45. A preferred example of the L chain V region is a L chain V region consisting of of an amino acid sequence shown in SEQ ID NO: 50.
[0169] Likewise, as a further embodiment of the anti-hTROP-2 antibody of the present invention, a preferred example is an antibody in which the CDR amino acid sequences 1 to 3 of the H chain V region thereof are shown in SEQ ID NO: 56 to 58, respectively and / or the amino acid sequences of CDR 1 to 3 of region V of the L chain of these are shown in SEQ ID NO: 61 to 63, respectively. A preferred example of the H chain V region is a H chain V region consisting of an amino acid sequence shown in SEQ ID NO: 55. A preferred example of the L chain V region is a L chain V region consisting of of an amino acid sequence shown in SEQ ID NO: 60.
[0170] Likewise, as a further embodiment of the anti-hTROP-2 antibody of the present invention, a preferred example is an antibody in which the CDR amino acid sequences 1 to 3 of the H chain V region of these are shown in SEQ ID NO: 66 to 68, respectively and / or the amino acid sequences of CDR 1 to 3 of the V region of the L chain of these are shown in SEQ ID NO: 71 to 73, respectively. A preferred example of the H chain V region is a H chain V region which consists of an amino acid sequence shown in SEQ ID NO: 65. A preferred example of the L chain V region is a L chain V region consisting of of an amino acid sequence shown in SEQ ID NO: 70.
[0171] In the present invention, more specifically, preferred examples of an anti-hTROP-2 antibody having anti-tumor activity in vivo include: an anti-hTROP-2 monoclonal antibody (clone name: K5-70) produced by a hybridoma with accession number FERM BP-11251; an anti-hTROP-2 monoclonal antibody (clone name: K5-107) produced by a hybridoma with accession number FERM BP-11252; an anti-hTROP-2 monoclonal antibody (clone name: K5-116-2-1) produced by a hybridoma with accession number FERM BP-11253; an anti-hTROP-2 monoclonal antibody (clone name: T6-16) produced by a hybridoma with accession number FERM BP-11346 and an anti-hTROP-2 monoclonal antibody (clone name: T5-86) produced by a hybridoma with accession number FERM BP-11254.
[0172] Whereas, the hybridoma with accession number FERM BP-11251 was named as “Mouse-mouse hybridoma K5-70” and was deposited on 12 May 2010; the hybridoma with accession number FERM BP-11252 was named as “Mouse-mouse hybridoma K5-107” and was deposited on 12 May 2010; the hybridoma with accession number FERM BP-11253 was named “Mouse-mouse hybridoma K5-116-2-1” and was deposited on 12 May 2010; the hybridoma with accession number FERM BP-11346 was named “Mouse-mouse hybridoma T6-16” and was deposited on March 1, 2011 and the hybridoma with accession number FERM BP-11254 was named “Hybridido T5- Mouse-mouse 86 ”and was deposited on 12 May 2010. All of these hybridomas were deposited with the International Patent Organism Depositary (IPOD), the National Institute of Advanced Industrial Science and Technology, an Independent Administrative Institution under the Ministry of Economy , Trade and Industry (the AIST, Tsukuba Central 6, Higashi 11-1, Tsukuba, Ibaraki, Japan, postal code: 305-8566).
[0173] Yet another preferred example of the anti-hTROP-2 antibody of the present invention is an anti-hTROP-2 antibody that binds to a site (for example an epitope), to which a monoclonal antibody produced by the hybridoma having accession No. FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346 or FERM BP-11254 connects (recognizes).
[0174] Preferred examples of such an epitope of such an epitope will be given in (36) below.
[0175] (3-6) Anti-hTROP-2 antibody epitope
[0176] The type of an epitope (an antigenic determinant) of the anti-hTROP-2 antibody of the present invention is not limited, as long as it is at least a portion of hTROP-2 as an antigen. For example, such an epitope is preferably at least a portion of the region formed by removing a region consisting of amino acids at positions 252 to 260 from the hTROP-2 amino acid sequence shown in SEQ ID NO: 2, more preferably at least a portion of a region consisting of amino acids in positions 1 to 69 or at least a portion of a region consisting of amino acids in positions 100 to 274 (except for a region consisting of amino acids in position 252 to 260) and further preferably at least a portion of a region consisting of amino acids at positions 27 to 69 or a region consisting of amino acids at positions 109 to 206. Particularly preferred examples of the epitope described above include a region consisting of amino acids at positions 43 to 65 , a region consisting of amino acids at positions 152 to 165, a region consisting of amino acids at positions 171 to 183, a region consisting of amino acids at positions 109 to 120, a region ion consisting of amino acids at positions 193 to 206, a region consisting of amino acids at positions 43 to 56 and a portion comprising such a region, in the hTROP-2 amino acid sequence shown in SEQ ID NO: 2. Yet particular preferred examples include a region consisting of amino acids at positions 43 through 65, a region consisting of amino acids at positions 152 through 165, a region consisting of amino acids at positions 171 through 183, a region consisting of amino acids at positions 109 through 65 120 and a portion comprising such a region. An anti-hTROP-2 antibody, which recognizes the regions described above (binds to the regions or portions described above that comprise such regions), has high internalizing activity in tumor cells, for example and is therefore extremely useful as a immunoconjugate as described last.
[0177] (3-7) Characteristics of anti-hTROP-2 antibody
[0178] As described above, the anti-hTROP-2 antibody of the present invention is an antibody having high anti-tumor activity in vivo at a lower dosage. Specifically, it is preferable that the anti-hTROP-2 antibody present exhibits tumor growth inhibitory activity of 50% or more (preferably 80% or more, more preferably 90% or more, still preferably 95% or more and particularly preferably almost 100 % (e.g. 98% or more, or 99% or more)) at a dosage (as a naked antibody) of 20 mg / kg body weight or less (preferably 10 mg / kg body weight or less, more preferably 5 mg / kg of body weight or less and still preferably 1 mg / kg of body weight or less) with respect to the animal model that carries the tumor.
[0179] Since, tumor growth inhibiting activity (%) can be calculated, for example, by the following formula:
[0180] Tumor development inhibitory activity (%) = 100 - [(tumor volume or tumor weight of the antibody administration group) / (tumor volume or tumor weight of the control group)] x 100
[0181] In addition, the anti-hTROP-2 antibody of the present invention preferably has anti-tumor activity on two or more types of human tumor cell lines. The type of such a human tumor cell line is not limited. For example, such human tumor cell lines are at least two types selected from the group consisting of several types of human pancreatic cancer cell lines, human prostate cancer cell lines, human colon cancer cell lines and breast cancer cell lines human. Specifically, preferred examples of such human tumor cell lines include at least two types selected from the group consisting of a human pancreatic cancer cell line PK-59, a human pancreatic cancer cell line BxPC-3, a human pancreatic cancer cell line KP -3L, a human pancreatic cancer cell line KP-2, a human pancreatic cancer cell line PK-1, a human pancreatic cancer cell line PK-45H, a human pancreatic cancer cell line PK-45P, a human cell line pancreatic cancer human cell TCC-PAN2, a SUIT-2 human pancreatic cancer cell line, a CACO-2 human colon cancer cell line, a SW480 human colon cancer cell line, a DLD human colon cancer cell line -1, a human colon cancer cell line HCT 116, a human breast cancer cell line JIMT-1, a human breast cancer cell line HCC1143, a human breast cancer cell line MCF-7, a li a human prostate cancer cell DU145 and a human prostate cancer cell line PC-3. Of these, as two or more types described above of human tumor cell lines, the human pancreatic cancer cell line PK-59 and the human pancreatic cancer cell line BxPC-3 are more preferable.
[0182] In addition, the anti-hTROP-2 antibody of the present invention has a dissociation constant (Kd value) of preferably 1.0 x 10-10 M or less, more preferably 1.0 x 10-11 M or less and still preferably 1.0 x 10 -12 M or less. Since, the binding capacity (affinity) of the antibody can be measured in the form of the dissociation constant (Kd value), a dissociation rate constant (Kdiss [1 / Sec]) or an association rate constant (Kass [ 1 / M.Sec]), for example, by Scatchard analysis or surface plasmon resonance sensor called Biacore. Like Biacore devices, Biacore 3000, Biacore 2000, Biacore X, Biacore J and Biacore Q (all of which were manufactured by Biacore) can be used, for example. It is preferable that the antibody has a dissociation constant (Kd value) that is as small as possible because it has high binding capacity (affinity). The Kd value is determined based on the two parameters of Kdiss and Kass and this can be expressed in the formula: Kd [M] = Kdiss / Kass. As a method of calculating the Kd value, the method described in the examples as described later (specifically, Example 10) can preferably be adopted.
[0183] (4) Genetically recombinant antibody and antibody fragment
[0184] (4-1) Genetically recombinant antibody
[0185] In a preferred embodiment of the anti-hTROP-2 antibody of the present invention, a genetically recombinant antibody is provided herein. The type of such a genetically recombinant antibody is not limited. Examples include a chimeric antibody, a humanized antibody, and a human antibody.
[0186] A chimeric antibody (i.e., a humanized chimeric antibody) is an antibody formed by binding (conjugating) the variable region of a mouse-derived antibody to the constant region of a human-derived antibody (referring to Proc. Natl. Acad. Sci. USA 81, 6851-6855, (1984), etc.). When such a chimeric antibody is produced, the antibody bound in this way can be easily constructed by a genetic recombination technique.
[0187] When a humanized antibody is produced, a complementary determination region (CDR) is transplanted from the variable region of a mouse antibody into the variable region of a human antibody, thereby producing a reconstructed variable region, in which a structure region (FR) is derived from human and CDR is derived from mouse (which is called CDR graft (CDR transplant)). Subsequently, humanized in this way, the reconstructed human variable region is linked to a human constant region. Such a method for producing a humanized antibody is well known in the present technical field (referring to see Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); JP Patent Publication (Kohyo) No. 4-502408 A (1992) (Japanese Patent No. 2828340; Queen et al.) , etc.).
[0188] In general, in the case of a human antibody (a complete human antibody), its structure comprising a hypervariable region which is the antigen binding site of region V, other parts of region V and a constant region is the same as the antibody structure of a human. However, such a hyper-variable site can also be derived from other animals. A technique that produces a human antibody is publicly known and a method for producing the gene sequences that are common in humans by genetic engineering has been established. A human antibody can be obtained, for example, by a method using a human antibody that produces mice that have the chromosomal fragments that comprise the H chain and L chain genes of the human antibody (referring to Tomizuka, K.et al ., Nature Genetics, (1977) 16, 133-143; Kuroiwa, Y. et. Al., Nuc. Acids Res., (1998) 26, 3447-3448; Yoshida, H. et. Al., Animal Cell Technology : Basic and Applied Aspects, (1999) 10, 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers; Tomizuka, K. et. Al., Proc. Natl. Acad. Sci. USA, (2000) 97, 722-727, etc.), or by a method to obtain a human antibody derived from phage display selected from the human antibody library (referring to Wormstone, IM et. Al, Investigative Ophthalmology & Visual Science., (2002) 43 (7), 2301-8; Carmen, S. et. Al., Briefings in Functional Genomics and Proteomics, (2002) 1 (2), 189-203; Siriwardena, D et. al., Opthalmology, (2002) 109 (3), 427-431, etc.).
[0189] In the case of the aforementioned chimeric antibody, the humanized antibody and human antibody, the N-linked glycoside sugar chain in the antibody Fc region is preferably a sugar chain, where fucose does not bind N-acetylglucosamine in the reduction terminal. A specific example is an antibody consisting of antibody genetically recombinant molecules, which has, in the Fc region of the antibody molecules, the sugar chain where position 1 of the fucose does not bind to position 6 of N-acetylglucosamine at the reduction terminal of the N-glycoside linked sugar chain via an α link. Such an antibody is capable of significantly improving ADCC activity. This point (the characteristics of the N-glycoside linked sugar chain in the antibody Fc region) is also preferable for the aforementioned monoclonal antibody and monoclonal antibody.
[0190] (4-2) Antibody fragment
[0191] The anti-hTROP-2 antibody fragment (partial fragment) of the present invention is included in the antibody of the present invention. Since, the antibody fragment of the present invention has hTROP-2 binding activity (called, it is capable of binding to hTROP-2) and also has anti-tumor activity in vivo, as in the case of the anti-hTROP antibody -2 of the present invention.
[0192] The antibody fragment means a region of a portion of an anti-hTROP-2 polyclonal antibody or anti-hTROP monoclonal antibody (referred to as an antibody fragment derived from the anti-hTROP-2 antibody of the present invention). Examples of such an antibody fragment include peptides that comprise at least a portion thereof, Fab, Fab ', F (ab') 2, Fv (variable antibody fragment), a single stranded antibody (an H chain, an L chain , a H chain V region and an L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (a disulfide stabilized V region) and a complementary determination region (CDR).
[0193] Fab is an antibody fragment with a molecular weight of approximately 50,000 having antigen binding activity, which is formed by the attachment of about one half at the N-terminus of the H chain to the entire L chain via the binding disulfide, among the fragments obtained by treating the antibody molecules with a protease, papain. In addition, it is also possible to produce such a Fab by inserting DNA encoding the Fab of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and then introducing a vector into a prokaryote or eukaryote so as to allow DNA from express in this.
[0194] OF (ab ') 2 is an antibody fragment with a molecular weight of approximately 100,000 having antigen binding activity, the size of which is slightly larger than Fab which binds to Fab via the disulfide bond in the junction region, between fragments obtained by treating the antibody molecules with a protease, pepsin. In addition, it is also possible to produce such F (ab ') 2 by the thioether bond or Fab disulfide bond, as described later.
[0195] Fab 'is an antibody fragment with a molecular weight of approximately 50,000 having antigen binding activity, which is formed by cleavage of the disulfide bond in the aforementioned F (ab') 2 region. In addition, it is possible to produce such Fab 'by inserting the DNA encoding the Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote so as to allow the DNA to express in this.
[0196] scFv is an antibody fragment having antigen binding activity, which is a VH-P-VL or VL-P-VH polypeptide formed by binding a single H chain V (VH) region to a V region single-chain (VL) using a suitable peptide linker (P). Such scFv can be produced to obtain cDNA encoding the VH and VL of an antibody, constructing DNA encoding scFv, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote so as to allow the DNA to express in this.
[0197] The diabody is an antibody fragment formed by dimerization of scFv, which has bivalent antigen binding activities. Such bivalent antigen binding activities may be identical in each other, or they may also be different from each other. Such a diabody can be produced to obtain the cDNA encoding the VH and VL of an antibody, constructing DNA encoding scFv such that the length of the P amino acid sequence is 8 residues or less, by inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector and then introducing the vector into a prokaryote or a eukaryote in this way to allow the DNA to express in it.
[0198] dsFv is an antibody fragment formed by the binding of polypeptides, in which an amino acid residue in each of VH and VL has been replaced with a cysteine residue in each other through the disulfide bond between the residues of cysteine. The amino acid residue to be replaced with the cysteine residues can be selected based on the estimate of the three-dimensional structure of the antibody according to the method of Reiter et al. (Protein Engineering, 7, 697704, 1994). Such dsFv can be produced to obtain the cDNA encoding the VH and VL of an antibody, constructing the DNA encoding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector and then introducing the vector into a prokaryote or a eukaryote in this way to allow the DNA to express in this.
[0199] A peptide comprising a CDR comprises at least one region of VH CDRs (CDR 1 to 3) and VL CDRs (CDR 1 to 3). More preferred examples of such a peptide include a peptide that comprises all of the VH CDRs and a peptide that comprises all of the VL CDRs. A particularly preferred example of the peptide is a peptide that comprises all of the VH and VL CDRs (6 total regions). Preferred examples of the amino acid sequence of such a CDR include an amino acid sequence shown in SEQ ID NO: 36 to 38, 41 to 43, 46 to 48, 51 to 53, 56 to 58, 61 to 63, 66 to 68 and 71 to 73, as described above. A peptide comprising multiple CDRs can be linked to one another, either directly or via a suitable peptide linker. Such a peptide comprising CDR can be produced by constructing DNA encoding the VH and VL of an antibody, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector and then producing the expression vector in a prokaryote or a eukaryote in this way to allow the DNA to express in this. In addition, such a peptide comprising CDR can also be produced by chemical synthesis methods such as a Fmoc method (a fluorenylmethyloxycarbonyl method) and a tBoc method (a t-butyloxycarbonyl method).
[0200] The antibody fragment of the present invention, as it is, can be an antibody fragment, comprising a part or the entire antibody Fc region in which fucose does not bind to N-acetylglucosamine in reducing the end of a chain of sugar linked by N-glycoside. Otherwise, the antibody fragment of the present invention can also be a fusion protein, wherein the antibody already mentioned the fragment is fused to a part of or the entire antibody Fc region in which fucose does not bind to N- acetylglucosamine at the reduction end of a N-glycoside linked sugar chain. Such an antibody fragment is capable of significantly improving ADCC activity and is therefore preferable.
[0201] Next, in the descriptions of the present specification, the antibody already mentioned fragments are also included in the anti-hTROP-2 antibody of the present invention. 3. Preparation of the antibody drug conjugate
[0202] As an immunoconjugate prepared using the aforementioned anti-hTROP-2 antibody of the present invention, an antibody-drug conjugate comprising the aforementioned antibody and a substance (a compound, etc.) having anti -tumor and / or cell death activity. It is noted that a conjugate formed by the prior preparation of each of the aforementioned antibody molecule and the aforementioned substance having anti-tumor activity and / or cell death activity, separately and then combining these, is generally referred to as an immunoconjugate. Otherwise, a conjugate by binding a protein toxin used as a substance having anti-tumor activity and / or cell death activity to an antibody gene in a gene according to a genetic recombination technique, thereby allowing expression as a simple protein (the fusion protein), it is generally referred to as an immunotoxin.
[0203] Examples of a substance having anti-tumor activity include doxorubicin, calicheamicin, mitomycin C, Auristatin E and radioactive isotope (IR). Examples of a substance having cell death activity include saporin, lysine, pseudomonas exotoxin, diphtheria toxin and radioactive isotope (IR). Of these, saporin and pseudomonas exotoxin are preferably used. The type of IR having anti-tumor activity and / or cell death activity is not particularly limited and examples of such IR include 90Y, 111In, 125I, 3H, 35S, 14C, 186Re, 188Re, 189Re, 177Lu, 67Cu, 212Bi, 213Bi, 211At, 198Au, 224Ac, 126I, 133I, 77Br, 113mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 94mTc, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 111Ag, 197PP, 109Pd, 32PP, 109Pd, 32P 33P, 47Sc, 153Sm, 177Lu, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 18F, 75Se, 201Tl, 225Ac, 76Br, 86Y, 169Yb, 166Dy, 212Pb and 223Ra.
[0204] A method of producing an antibody drug conjugate is not limited. For example, a method of binding an antibody to a drug via disulfide bond or hydrazone bond is applied.
[0205] The previously mentioned anti-hTROP-2 antibody of the present invention is excellent in terms of internalizing activity in target tumor cells that express hTROP-2. Thus, by the previous combination of a substance having anti-tumor activity and cell death activity with the anti-hTROP-2 antibody, it becomes possible to allow such a substance directly and highly selective to act on the tumor cells. The antibody drug conjugate of the present invention is extremely excellent in terms of its ability to deliver the agent to target tumor cells.
[0206] Internalization activity in cells can be assessed by fluorescently labeling an antibody with rhodamine or the like and then observing the migratory behavior and location of the antibody using a fluorescence microscope or similar.
[0207] In addition, in the present invention, in addition to the antibody already mentioned in the drug conjugate, a fragment and antibody-drug conjugate can also be provided, in which the antibody already mentioned the fragment is used instead of an antibody. With respect to the details of such a fragment and antibody-drug conjugate, descriptions of the antibody already mentioned in the drug conjugate can be applied, as appropriate.
[0208] Next, in the descriptions of the present specification, such a fragment and antibody-drug conjugate is also included in the antibody drug conjugate of the present invention. 4. Pharmaceutical composition
[0209] The anti-hTROP-2 antibody and antibody drug conjugate of the present invention are useful as active ingredients contained in a pharmaceutical composition.
[0210] The pharmaceutical composition is useful as a pharmaceutical composition for the treatment and / or diagnosis of a tumor. In particular, since the anti-hTROP-2 antibody of the present invention and an antibody-drug conjugate comprising the antibody already mentioned has excellent tumor development inhibitory activity as well as anti-tumor activity, these are preferably used in the treatment of tumor. That is to say, the anti-hTROP-2 antibody and antibody drug conjugate of the present invention are useful as active ingredients contained in a tumor therapeutic agent and a tumor diagnostic agent. It is noted that the treatment described above of tumor includes inhibition of tumor development and suppression of tumor development. Specifically, if this is a tumor therapeutic agent, examples of the tumor therapeutic agent include a tumor development inhibitor and a tumor development suppressor.
[0211] It is preferable to provide the pharmaceutical composition of the present invention in the form of a pharmaceutical composition which comprises the anti-hTROP-2 antibody and / or antibody drug conjugate of the present invention as active ingredients and yet which comprises a pharmacologically acceptable carrier . In addition, the pharmaceutical composition of the present invention can be used in combination with known anti-tumor agents. By such combined use, a greater anti-tumor effect can be obtained.
[0212] The target diseases (tumors), in which the pharmaceutical composition of the present invention is applied, include: the various previously mentioned types of known human tumors, in which the expression of hTROP-2 has been previously confirmed. Among others, one or more of the types selected from human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer are particularly preferable. Such a target disease can be a single disease, or two or more diseases can be developed in combination. In addition, the target tumor may be a recurrent cancer or a metastatic cancer. The pharmaceutical composition of the present invention (furthermore, the anti-hTROP-2 antibody and / or antibody drug conjugate of the present invention) can be effectively used as a therapeutic agent and a diagnostic agent for recurrent cancer or metastatic cancer.
[0213] Examples of the "pharmaceutically acceptable carrier" include an excipient, a diluent, an extender, a disintegrant, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a coloring agent, a sweetener, a thickener, a corrigent, a solubilizer and other additives. Using one or more types of such carriers, a pharmaceutical composition can be prepared in the form of an injection, a liquid agent, a capsule, a suspension, an emulsion, a syrup, etc. These pharmaceutical compositions can be administered orally or parenterally. Another form of parenteral administration is, for example, an injection comprising one or more active ingredients, which is prepared by a common method. Such an injection can be produced by dissolving or suspending the present antibody in a pharmacologically acceptable carrier such as a normal saline solution or a commercially available distilled water used by the injection.
[0214] In particular, when an antibody fragment derived from the anti-hTROP-2 antibody of the present invention (particularly, a low molecular weight antibody fragment) is administered to a living body, a colloidal dispersion system can be used in addition to the previously mentioned components. Such a colloidal dispersion system is anticipated to have an effect of enhancing the stability of a compound (an antibody fragment) in a living body or an efficient transport effect of such a compound in a specific organ, tissue or cell. The type of such a colloidal dispersion system is not limited, as long as it is commonly used. Examples of such a colloidal dispersion system include dispersion systems comprising, as bases, polyethylene glycol, a macromolecular conjugate, a macromolecular aggregate, a nanocapsule, microsphere, pearls and lipids including in an oil-in-water emulsifier, micelle, mixed micelle and liposome. Preferred examples of such a colloidal dispersion system include multiple liposomes and artificial membrane vesicles, which have an efficient transport effect of such a compound to a specific organ, tissue or cell (Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. Et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698) .
[0215] The dosage of the pharmaceutical composition of the present invention differs depending on the age, sex, body weight and symptoms of a patient, therapeutic effects, a method of administration, a time of treatment, the types of the anti-hTROP-2 antibody and antibody drug conjugate of the present invention contained in the pharmaceutical composition, etc. In general, the present pharmaceutical composition can be administered within the range between 600 μ g and 6,000 mg per adult by administration. However, the dosage is not limited to the aforementioned range.
[0216] In a case where the pharmaceutical composition is administered in the form of an injection, for example, it can be administered in a dosage of 100 μ g to 100 mg, by administration, by the body weight of a human patient, once or divided into several administrations, as an average daily dosage. Preferably, the pharmaceutical composition can be administered once every three days, once a week, once every ten days, or once every two weeks, or by a simple administration (where the total number of administrations is 1) . Examples of the dosage form include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection. Of these, intravenous injection is preferable. In addition, such an injection can be prepared in the form of a non-aqueous diluent (for example polyethylene glycol, vegetable oil such as olive oil, alcohols such as ethanol, etc.), the suspension, or an emulsion. Such an injection can be sterilized by mechanical sterilization using a filter, a mixture of a microbicide, etc. The injection can be produced in the form of an injection to be prepared before use. That is, a sterile solid composition is prepared by a freeze drying method or the like and the composition is then dissolved in the sterile distilled water used by the injection or other solvents before it is used, so that it can then be used.
[0217] The present invention provides the use of the aforementioned anti-hTROP-2 antibody and / or antibody drug conjugate of the present invention in the production of a pharmaceutical agent (a medicament) for the treatment and / or diagnosis of the tumor. In addition, the present invention provides the aforementioned anti-hTROP-2 antibody and / or antibody drug conjugate of the present invention, which are used for the treatment and / or diagnosis of a tumor.
[0218] In addition, the present invention provides a method for the treatment and / or diagnosis of a tumor, which is characterized in that it comprises the use (called, administering to patients) the aforementioned anti-hTROP-2 antibody and / or antibody drug conjugate of the present invention. In addition, the present invention also provides the use of the aforementioned anti-hTROP-2 antibody and / or antibody drug conjugate of the present invention in the treatment and / or diagnosis of the tumor. 5. Method for the detection of the tumor
[0219] The method for detecting the tumor of the present invention is characterized in that it comprises allowing the previously mentioned anti-hTROP-2 antibody of the present invention to react with a sample collected from a living body (hereinafter referred to as a biological sample) ) and detect a signal from the reacted antibody.
[0220] As described above, hTROP-2 has been confirmed to be specifically expressed in several types of tumor cells. Thus, hTROP-2 and particularly, free hTROP-2 (a portion of the extracellular region of hTROP-2) can be used as a marker for various types of tumors. In particular, such hTROP-2 can preferably be used as a marker for human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer.
[0221] Since, the anti-hTROP-2 antibody of the present invention is allowed to react with a biological sample and a signal from the reacted antibody is then detected, in order to detect a tumor. The obtained antibody signal can be used as an indicator of the amount of an antigen in the biological sample (i.e., an hTROP-2 amount or a free hTROP-2 amount). In the detection of a tumor using the antibody of the present invention, first, a biological sample collected as a patient analyte, such as a section of tissue or blood used as a tested target, is allowed to bind to the antibody of the present invention by a reaction of antigen antibody. Subsequently, based on the results of measuring the amount of the binding antibody, the amount of an antigen of interest contained in the biological sample is measured. This measurement can be performed according to known immunoassay methods. For example, an immunoprecipitation method, an immunoagglutination method, radioimmunoassay, immunonephelometry, a Western blot method, flow cytometry and others can be used. In the radioimmunoassay, a labeled antibody is used and thus an antibody signal is expressed as the amount of the labeled antibody that is directly detected. Otherwise, an antibody whose known concentration or antibody titrator can be used as a standard solution and thus a signal from the target antibody can be expressed as a relative value. That is, both the standard solution and the analyte can be measured using a measuring device and an antibody signal in a biological sample can be expressed as a value relative to the standard solution value as a criterion. Examples of such radioimmunoassays include the ELISA method, the EI method, the RIA method, fluorescence immunoassay (FIA) and luminescence immunoassay. Of this, the ELISA method is particularly preferable in that it is simple and highly sensitive.
[0222] In the present invention, the state of the tumor can be assessed or diagnosed, using the detection result obtained by the aforementioned detection method as an indicator. For example, when the detection result exceeds a predetermined standard value, the state of a tumor is defined as a positive tumor and when the detection result is less than the predetermined standard value, it is defined as a negative tumor. In the case of the positive tumor, it is determined that certain types of tumors have been developed and in this way the tumor status can be assessed. The term "the state of a tumor" is used here to mean the presence or absence of the development of a tumor, or the degree of its progression. Thus, specific examples of the state of a tumor include the presence or absence of tumor development, the degree of progression of the tumor, the degree of harmfulness, the presence or absence of metastasis and the presence or absence of recurrence.
[0223] In the aforementioned assessment, as a tumor state to be assessed, only one state can be selected from previously mentioned examples, or multiple examples can be combined and selected. The presence or absence of a tumor can be assessed by determining whether or not the tumor was developed, with reference to the predetermined standard value used as a threshold, based on the obtained detection result. The degree of harmfulness is used as an indicator that indicates the degree of progression of a cancer. Based on the detection result, the target tumor can be classified at certain stages of the disease and it can be evaluated. Otherwise, early cancer and advanced cancer can be distinguished from each other and then this can be assessed. For example, it is also possible to determine the target tumor as either an early cancer or an advanced cancer, using the detection result as an indicator. Tumor metastasis can be assessed by determining whether or not neoplasm appeared at the site apart from a position of the initial lesion, using the detection result as an indicator. Recurrence can be assessed by determining whether or not the detection result has exceeded the predetermined standard value against after the stage or interval remission. 6. Kit for the detection or diagnosis of tumor
[0224] The anti-hTROP-2 antibody of the present invention can be provided in the form of a kit to detect or diagnose a tumor. The kit of the present invention comprises a labeling substance, a solid phase reagent in which the labeled antibody or antibody has been immobilized, etc., considering the antibody already mentioned. A labeling substance that labels the antibody means a substance labeled with an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, etc. The kit of the present invention can also comprise other reagents used to carry out the detection of the present invention, in addition to the aforementioned constitutional elements. For example, when such a labeling substance is an enzyme labeling substrate, the kit of the present invention may comprise an enzyme substrate (a chromogenic substrate, etc.), an enzyme substrate dissolving solution, a enzyme reaction, a diluent used by the analytes, etc. In addition, the present kit may also comprise various types of buffers, sterile water, various types of cell culture vessels, various types of reactors (an Eppendorf tube, etc.), a blocking agent (a serum component such as albumin of bovine serum (BSA), skim milk, or goat serum), a washing agent, a surfactant, various types of plates, an antiseptic such as sodium azide, an experimental operation manual (instruction), etc.
[0225] The kit of the present invention can be effectively used to carry out the aforementioned detection method of the present invention and is therefore extremely useful.
[0226] In the following, the present invention will be more specifically described in the following examples. However, these examples are not intended to limit the scope of the present invention. [Example 1] [Cloning of the hTROP-2 gene]
[0227] A full-length hTROP-2 gene was isolated from the human fetal liver (10-week-old embryo) according to the RT-PCR method. First, the following PCR primers were designed based on the sequence of an hTROP-2 gene (Genbank Accession No. NM 002353).
[0228] Advanced starter: 5’-ttcctccgccccaccatggc-3 ’(SEQ ID NO: 3)
[0229] Reverse initiator: 5’-ctcgagcaagctcggttcctttctc-3 ’(SEQ ID NO: 4)
[0230] When these primers were designed, a sequence digested by the restriction enzyme XhoI except for a stop codon was added to the reverse primer. The cDNA was synthesized from the total RNA (TAKARA) prepared from the human fetal liver (10-week-old embryo). Using this cDNA as a model, a PCR reaction was performed with the aforementioned primers. Then, development by agarose gel electrophoresis and extraction of an interest group were performed and it was then cloned into a pCRII (Invitrogen) vector (pCRII-hTROP-2). The cloned hTROP-2 cDNA was confirmed by sequencing.
[0231] An expression vector was constructed by cleaving an EcoRI / XhoI fragment that comprises a pCRII-hTROP-2 hTROP-2 gene and then inserting the fragment into the EcoRI / XhoI site of a pcDNA4 / myc-His © A vector (Invitrogen) (pcDNA4-hTROP-2-myc / His). In addition, a HindIII / PmeI fragment comprising an hTROP-2 gene was cut from pcDNA4-hTROP-2-myc / His (where the HindIII cleavage portion was blunt end) and the fragment was then inserted into a PmeI site of a pcDNA3.1 (+) vector (Invitrogen), thus to construct an expression vector comprising a neomycin resistance gene (pcDNA3.1-hTROP-2-myc / His). [Example 2] [Construction of the cell line capable of stably expressing the hTROP-2 gene]
[0232] The expression vector (pcDNA3.1-hTROP-2-myc / His) encoding the hTROP-2 full-length cDNA, which was produced by the method described above, was introduced into HEK293 cells (RIKEN) cells HuH-7 (HSRRB), 7E2-C cells (described in WO 2005/052156) and CHO-K1 cells (HSRRB), using a lipofectamine 2000 reagent (Invitrogen) and the selection was then carried out using a G418 antibiotic (geneticin; GIBCO BRL). Then, a cell line, which is stably expressed hTROP-2, was established and obtained. [Example 3] [Production of the recombinant protein from the hTROP-2 extracellular region]
[0233] A fragment of the gene encoding a portion of the hTROP-2 extracellular region (specifically, a region consisting of amino acids at positions 1 through 263 from the amino acid sequence shown in SEQ ID NO: 2) was amplified by a PCR method. The following primers were used in the amplification.
[0234] Advanced starter: 5’-ttcctccgccccaccatggc-3 ’(SEQ ID NO: 3)
[0235] Reverse initiator: 5’-ctcgagctcgtccaggtaatagatgagcg-3 ’(SEQ ID NO: 5)
[0236] In this operation, a sequence digested by restriction enzyme XhoI was added to the reverse primer. The DNA fragment amplified by the PCR method was developed by agarose gel electrophoresis and was then purified using QIAquick (trademark) gel extraction kit (QIAGEN). The purified DNA fragment was subcloned into a pCR Blunt (Invitrogen) vector (pCRB-hTROP-2 EC) and the gene sequence was confirmed. Subsequently, an EcoRI / XhoI fragment comprises the fragment of the gene encoding the extracellular region of hTROP-2 was cut from pCRB-hTROP-2 EC and was then inserted into the EcoRI / XhoI site of a pcDNA4 / myc-His © A vector (Invitrogen) (pcDNA4mH-hTROP-2 EC). In addition, in order to produce a NruI restriction cleavage site, the following oligonucleotides were associated and inserted into the BamHI / EcoRI site of pcDNA4mH-hTROP-2 EC.
[0237] Oligonucleotide 1: 5'-gatccactagtcgcgagtggtgg-3 '(SEQ ID NO: 6)
[0238] Oligonucleotide 2: 5'-aattccaccactcgcgactagtg-3 '(SEQ ID NO: 7)
[0239] Likewise, a pBgl II linker (TAKARA) was inserted into the PmeI site of pcDNA4mH-hTROP-2 EC (pcDNA4mH-NB-hTROP-2 EC). In order to produce the recombinant protein using baculovirus, a NruI / BglII fragment comprises the fragment of the gene encoding the hTROP-2 extracellular region that was cut from pcDNA4mH-NB-hTROP-2 EC and was then inserted into the NruI / BglII site of a pPSC8 vector (Nosan Corporation) (pPSC8-hTROP-2 EC). The production of recombinant protein from the hTROP-2 extracellular region using baculovirus has been delegated to Nosan Corporation.
[0240] The recombinant protein from the hTROP-2 extracellular region was purified as follows. Ni Sepharose 6 Fast Flow (GE Healthcare Biosciences) was added to a culture supernatant comprising the recombinant protein, as long as these were allowed to bind to each other at 4 ° C for 2 hours. Then, the resultant was washed with a phosphate buffer containing 20 mM imidazole, using EconoColum (BIO RAD) and this was then eluted with a phosphate buffer containing 300 mM imidazole, as long as it was purified. [Example 4] [Isolation of human EpCAM cDNA and expression vector construction]
[0241] A full-length human EpCAM gene was isolated from the human fetal liver (10-week-old embryo) according to the RT-PCR method. First, the following PCR primers were designed based on the sequence of a human EpCAM gene (Genbank Accession No. NM 002354).
[0242] Advanced starter: 5’-tcctcgtgtcccactcccgg-3 ’(SEQ ID NO: 8)
[0243] Reverse initiator: 5’-ctcgagtgcattgagttccctatgc-3 ’(SEQ ID NO: 9)
[0244] When these primers were designed, an XhoI restriction enzyme-digested sequence except for a stop codon was added to the reverse primer. The cDNA was synthesized from total RNA (TAKARA) from the human fetal liver (10-week-old embryo). Using this cDNA as a model, a PCR reaction was performed with the aforementioned primers. Then, development by agarose gel electrophoresis and extraction of an interest group was performed and it was then cloned into a pCRII (Invitrogen) vector (pCRII-hEpCAM). The cloned human EpCAM cDNA was confirmed by sequencing.
[0245] An expression vector was constructed by cleaving an EcoRI / XhoI fragment comprising a human EpCAM gene from pCRII-hEpCAM and then inserting the fragment into the EcoRI / XhoI site of a pcDNA4 / myc-His © A vector (Invitrogen) (pcDNA4-hEpCAM-myc / His). In addition, a HindIII / PmeI fragment comprising a human EpCAM gene was cut from pcDNA4-hEpCAM-myc / His (where the HindIII cleavage portion was blunt-ended) and the fragment was then inserted into the PmeI site of a pcDNA3 vector .1 (+) (Invitrogen), thus to construct an expression vector that comprises a neomycin resistance gene (pcDNA3.1-hEpCAM-myc / His). [Example 5] [Production of anti-hTROP-2 monoclonal antibody]
[0246] As immunogens, cell lines capable of stably expressing hTROP-2 (HEK293-hTROP-2 cells, CHO-K1- hTROP-2 cells and 7E2-C-hTROP-2 cells) were used here; human pancreatic cell line endogenously expressing a hTROP-2 protein on the cell surface (PK-59, RCB1901; purchased from RIKEN cell bank); and the recombinant protein from the hTROP-2 extracellular region produced by the method described above.
[0247] In the case of cell lines capable of stably expressing hTROP-2, 1 x 107 cells were used and in the case of the recombinant hTROP-2 protein, 20 μ g of the protein was used. The cell lines or recombinant protein was mixed with a TiterMax Gold adjuvant (Funakoshi Corporation) in a 1: 1 mixing ratio, in order to prepare an emulsion. The emulsion was then injected into the two pad feet or abdominal cavity of a mouse (C57 / BL6, Balb / c) (initial immunization). When the immunization was performed by the injection of two pad feet for a short period of time, an intensifier was performed for three to ten days of initial immunization. The day after the final immunization, lymph nodes were collected from both the knees and lymphocytes were then prepared. When immunization was performed by injection into the abdominal cavity for a long period of time, the intensifiers were performed at intervals of once a week after the initial immunization (in which the intensifiers were performed for 1 to 2 months). Then, the shape of the B cells isolated from the spleen according to the common method. In the case of immunization using cells as immunogens, a cell suspension that was PBS containing 5 x 106 cells was used by the enhancers. In the case of using a protein as an immunogen, 5 μg of a PBS solution was used.
[0248] The prepared lymphocytes were mixed with a mouse myeloma cell line (P3-X63-Ag8.653) at a 3: 1 mixing ratio and cell fusion was then performed according to the polyethylene glycol method. Then, the fused cells were cultured for 7 to 28 days in a cellulose metal medium (trade name: ClonaCell-HY Cloning Medium D; Stem Cell), which contains HAT (hypoxanthine, aminopterin and thymidine). The single colonies of hybridoma development were each selected and placed on a 96-well flat-bottom plate and using a liquid selective medium containing HAT, the hybridomas were grown in a 5% CO2 incubator. A culture supernatant from the development of hybridomas from simple colonies was subjected to a primary evaluation using the ELISA cell (described above) and then a secondary evaluation using the FACS analysis using HuH-7-hTROP-2 cells, PK-59, therefore establishing 300 types of hybridomas, which produce anti-hTROP-2 monoclonal antibodies recognizing the hTROP-2 proteins expressed on the cell surface of living cells. [Example 6] [Primary evaluation using ELISA Cell]
[0249] CHO-K1 cells (hTROP-2 negative control; purchased from Japan Health Sciences Foundation) and CHO-K1-hTROP-2 cells (or HUH-7 cells (hTROP-2 negative control; purchased from Japan Health Sciences Foundation ) and HuH-7-hTROP-2 cells) were alternatively inoculated into a 96 well culture plate (BD Falcon) at a cell density of 3 x 104 cells / well and the cells were then cultured in an atmosphere of 5% CO2 at 37 ° C for 1 to 2 days. The cell culture medium was removed by decantation. Then, the cells were washed with ice-cold PBS and were then treated with 4% paraformaldehyde-PBS for 5 minutes, so that the cells were immobilized. The cells were washed with PBS which was cooled on ice and an ELISA plate was then prepared. Then, ELISA was performed according to the common method. Specific procedures will be described below.
[0250] First, blocking with a 2% PBS solution of skimmed milk was carried out at room temperature for 30 minutes to 1 hour. Subsequently, the culture hybridoma supernatant was added to it and these were then reached at room temperature for 1 hour. Then, the resultant was washed with a 0.1% Tween20-PBS solution three times. As a secondary antibody, anti-mouse IgG labeled by horseradish peroxidase (HRP) (GE Healthcare Biosciences), which was diluted 1000 times with a blocking solution, was added to the resultant and these were then reached at room temperature for 1 hour . Then, the resultant was washed with a 0.1% Tween20-PBS solution three times. A TMB substrate solution (3.3 ', 5.5'-tetramethylbenzidine: SIGMA) was added to the reaction solution to perform a color reaction and the reaction was then terminated by adding 1 M sulfuric acid. Then, absorbance (405 nm) was measured using the microplate reader model 550 (BIO RAD). The hybridomas corresponding to a culture hybridoma supernatant exhibiting a high absorbance value to the negative control were subjected to a large scale culture on a 24-well flat bottom plate and were then subjected to a secondary evaluation using FACS analysis. [Example 7] [Secondary evaluation using FACS Analysis]
[0251] Hybridomas, which were political in the primary assessment described above using the ELISA Cell, were subjected to a secondary assessment using FACS Analysis. In evaluating the hybridoma cells, HuH-7 cells, which were human liver cancer cells that do not express hTROP-2, were used as the negative control cells and the reactivity with HuH-7-hTROP-2 cells, which hTROP-2 were stably expressed, it was used as an indicator. Then, the evaluation was carried out based on the reactivity with the PK-59 cells (RCB1901; acquired from RIKEN cell bank), which were endogenously human pancreatic cancer cells that express an hTROP-2 protein on the cell surface.
[0252] The cells were removed from the culture disc by a trypsin treatment and a cell suspension was then prepared (cell density: 2 x 106 cells / ml). The culture hybridoma supernatant, which inhibited positive in the primary evaluation using an ELISA cell, was reacted with 100 μL of the cell suspension at 4 ° C for 20 minutes. The reaction mixture was washed with PBS and it was then reacted with PE-labeled mouse IgG (BD Pharmingen) (0.1 µg) (4 ° C, 30 minutes). Then, the reaction mixture was analyzed using FACSCalibur (Becton, Dickinson and Company).
[0253] Eventually, approximately 300 types of hybridomas, which produce an anti-hTROP-2 monoclonal antibody recognizing an hTROP-2 protein expressed on the cell surface of living cells, were established. [Example 8] [Isotype identification]
[0254] The isotype of the anti-hTROP-2 monoclonal antibody produced was identified using MOUSE ANTIBODY MONOCLONAL ISOTYPING TEST KIT (Serotec) according to a method included with the kit mentioned above. [Example 9] [Ascites formation and TROP-2 antibody purification]
[0255] The hybridoma clones produced by the method described above were administered at a density of 3 x 106 clones in the abdominal cavity of a nude BALB / c mouse, where 2,6,10,14-tetramethylpentadecane (pristane) previously (seven previous days) was administered. Two weeks later, ascites was collected. In addition, these ascites were subjected to caprylic acid precipitation and then affinity purification using a protein G column (HiTrap protein G; GE Healthcare Biosciences) or a protein A column (HiTrap protein A; GE Healthcare Biosciences), from this method for obtaining anti-hTROP-2 monoclonal antibodies from the individual hybridoma clones. [Example 10] [Antigen binding affinity measurement (Kd value measurement)]
[0256] The antigen binding affinity (Kd value) of the generated anti-hTROP-2 monoclonal antibody was calculated by a method using ELISA (Djavadi-Ohaniance L. et al (1996), In Antibody Engineering, Chapter 4, pp. 77-97, IRL Press, Oxford).
[0257] Specifically, the purified recombinant hTROP-2 protein (0.1 μ g / ml) was added to a 96 well culture plate (Corning) so that the antigen was solid phase (at room temperature for 1 hour) , or at 4 ° C overnight). Subsequently, the resultant was washed with PBS three times and 2% skimmed milk (PBS solution) was then added to it to block it (at room temperature for 1 hour). The resultant was washed twice with PBS. Then, an antigen antibody complex that was previously mixed by an antigen solution (a purified hTROP-2 protein; 50, 25, 12.5, 6.25, or 3.125 nM) with each clone (0.5 nM ) of the anti-hTROP-2 monoclonal antibody and then balancing the mixture, was added to the ELISA plate described above and these were reacted (at room temperature for 1 hour). The reaction product was washed with PBS three times and it was then reacted with HRPIgG labeled anti-mouse (final concentration: 1 μ g / ml) (GE Healthcare Biosciences) diluted with a blocking solution (at room temperature for 1 hour ). Subsequently, the reaction product was washed with a 0.1% solution of Tween20-PBS three times and a solution of TMB substrate (3.3 ', 5.5'-tetramethylbenzidine: SIGMA) was then added to the resultant for perform a color reaction. Then, 1 M of sulfuric acid was added to the reaction product to end the reaction. Using the 550 microplate reader model (BIO RAD), absorbance was measured.
[0258] The following calculation expressions were used to measure the dissociation constant (Kd).
[0259] According to the law of mass action, an antibody-antigen reaction is represented by the following expressions. Ag (Antigen) + Ab (Antibody) «■ Ag-Ab (antigen-antibody complex) ■■■■ (1) Kd = k2 / k1 = AgfxAbf / Ag-Ab = AgfxAbf / x (2)
[0260] In expression (2), Agf represents the concentration of a free antigen, Abf represents the concentration of a free antibody and Ag-Ab represents the concentration of an antibody-antigen complex. If Ag-Ab = x, the concentration of free antibody is represented by the following expression. Abf = Abt - x (3)
[0261] The above expression (2) can therefore be Kd = Agf x (Abt — x) / x (4)
[0262] In both terms of expression (4) are multiplied by x / KdxAbt, x / Abt = Agfx (1 — x / Abt) x1 / Kd x / Abtx1 / Agf = (1 — x / Abt) x1 / Kd ■■■■■■■ (5) If X = x / Abt and Y = x / AbtxAgf in expression (5), Y = (1-X) x1 / Kd ■■■■■■■ (6)
[0263] Based on expression (6), the Kd value was calculated.
[0264] The Kds values of the monoclonal antibody generated by anti-hTROP-2 monoclonal antibody 300 clones were measured by the method described above. As a result, there are 133 clones displaying a Kd value of 1 x 10-10 (M) or less, 59 clones displaying a Kd value of 1 x 10-11 (M) or less and 2 clones displaying a Kd value of 1 x 10-12 (M) or less.
[0265] Among the anti-hTROP-2 monoclonal antibodies, which exhibited tumor development inhibitory activity in vivo, the Kds values of 5-70 (mouse IgG2a), T6-16 (mouse IgG2a), K5-107 (Mouse IgG1), K5-116-2-1 (mouse IgG1) and T5-86 (mouse IgG1) were found to be 6.8 x 10-12 (M), 4.3 x 10-12 (M ), 4.7 x 10-12 (M), 2.69 x 10-11 (M) and 8.49 x 10-11 (M), respectively (Figure 1 and Table 1). Table 1 Kds values of anti-hTROP-2 monoclonal antibodies
[Example 11] [Reactivity of anti-hTROP-2 monoclonal antibodies to human cancer cell lines]
[0266] The human cancer cell lines (human tumor cell lines) used in these studies were purchased from Health Science Research Resources Bank (HSRRB), RIKEN cell bank (RIKEN), ATCC (American Type Culture Collection), ECACC (European Collection of Cell Cultures) and DSMZ (German Collection of Microorganisms and Cell Cultures). Specifically, the following cancer cell lines were used.
[0267] huH-1 (HSRRB), HUH-6 (HSRRB), HuH-7 (HSRRB), JHH-5 (HSRRB), JHH-6 (HSRRB), JHH-7 (HSRRB), HLE (HSRRB), HLF (HSRRB), HepG2 (HSRRB), Alexander (HSRRB), KP-1N (HSRRB), KP-1NL (HSRRB), KP-2 (HSRRB), KP-3 (HSRRB), KP-3L (HSRRB), PK-1 (RIKEN), PANC-1 (RIKEN), MIA PaCa-2 (HSRRB),
[0268] PK-59 (RIKEN), PK-45H (RIKEN), PK-45P (RIKEN), BxPC-3 (ATCC), SUIT-2 (HSRRB), TCC-PAN2 (HSRRB), SW480 (ATCC), DLD-1 (HSRRB), LoVo (HSRRB), COLO-320 (RIKEN), CACO-2 (RIKEN), CW-2 (RIKEN), HCT 116 (ATCC), HCC-56 (HSRRB), MCF-7 ( HSRRB), JIMT-1 (DSMZ), HCC1143 (ATCC), A549 (HSRRB), DU145 (RIKEN) and PC-3 (HSRRB).
[0269] The cancer cells were removed from the culture disc by trypsin treatment and the cell suspension was then prepared (cell density: 2 x 106 cells / ml). An anti-hTROP-2 monoclonal antibody (0.1 μ g) was added to 100 μL of the cell suspension and these were then reacted at 4 ° C for 20 minutes. The reaction solution was washed with PBS and this was then reacted with PE-labeled anti-mouse IgG (BD Biosciences Pharmingen) (0.1 μ g) (at 4 ° C for 30 minutes). Then, the resultant was analyzed by FACSCalibur (Becton, Dickinson and Company).
[0270] All of the anti-hTROP-2 antibodies generated do not bind to a human liver cancer cell line HuH-7, which does not endogenously express hTROP-2. Otherwise, anti-hTROP-2 antibodies bound to HuH-7-hTROP-2 cells, in which an hTROP-2 gene was stably expressed (Figure 2). Subsequently, the reactivity of the anti-hTROP-2 monoclonal antibodies generated with human cancer cell lines (in which an hTROP-2 protein was endogenously expressed on the cell surface) was examined by FACS Analysis. As a result, the 300 types of anti-hTROP-2 monoclonal antibodies generated all linked to human pancreatic cancer cell lines (PK-59 and BxPC-3). In particular, antibodies K5-70, T6-16, K5-107, K5-116-2-1 and T5-86, exhibited tumor development inhibiting activity in vivo, all binding to human cancer cell lines at high levels . For example, when compared to a case in which cancer cell lines were reacted with only PE-labeled anti-mouse IgG (BD Biosciences Pharmingen), the previously mentioned antibodies exhibited the following ability to bind PK-59 cells and BxPC cells -3 at the average fluorescence intensity: K5-70 (44 times), T6-16 (59 times), K5-107 (89 times), K5-116-2-1 (122 times) and T5-86 (15 times ) (PK-59 cells; Figure 3); and K5-70 (45 times), T6-16 (25 times), K5-107 (90 times), K5-116-2-1 (121 times) and T5-86 (10 times) (BxPC-3 cells ; Figure 4).
[0271] With respect to human cancer cell lines other than PK-59 and BxPC-3, among 12 types of pancreatic cancer cell lines, monoclonal anti-hTROP-2 antibodies bind to KP-2, KP-3L, PK-1, PK-45H, SUIT-2 and TCC-PAN2 and does not bind to KP-1N, KP-1NL, KP-3, PANC-1 and MIA-PaCa2 (Figure 5). Among human colon cancer cell lines, anti-hTROP-2 monoclonal antibodies bind to CACO-2, SW480, DLD-1 and HCT 116 and do not bind COLO-320 and CW-2 (Figure 6). In addition, anti-hTROP-2 monoclonal antibodies bind to JIMT-1 and HCC1143 (which were both human breast cancer cell lines) and to PC-3 and DU145 (which were both human prostate cancer cell lines). Thus, these endogenously recognized hTROP-2 proteins express on the cell surface of many types of human cancer cell lines (Figure 6). [Example 12] [Cross-reactivity with mouse TROP-2 protein and human TROP-1 / EpCAM protein]
[0272] For the purpose of examining the specificity of the generated anti-hTROP-2 monoclonal antibodies, the antibody reactivity with a mouse TROP-2 protein showing 80% homology at the level of the amino acid sequence with the hTROP-2 protein and with a human TROP-1 / EpCAM protein showing 50% homology at the level of the amino acid sequence with the hTROP-2 protein, was examined by FACS analysis.
[0273] Specifically, each of the mouse expression vectors (TROP-2- pcDNA3.1 (+), purchased by the Institute of Molecular and Cellular Biosciences, the University of Tokyo) comprises the full-length cDNA of a TROP- Mouse (Genbank Accession No. NM 020047, Y08830) and an expression vector (pcDNA3.1-hEpCAM-myc / His) comprises the full-length cDNA of a human TROP-1 / EpCAM gene (Genbank Accession No. NM 002354), was randomly introduced into CHO-K1 cells, using Lipofectamine 2000 reagent (Invitrogen). Then, 24 to 48 hours later, the cells were removed from the culture disc by treatment with trypsin and the cell suspension was then prepared. In this way the prepared cell suspension was successively reacted with the produced anti-hTROP-2 monoclonal antibody (0.1 µg) and with PE labeled anti-mouse IgG and this was then analyzed by FACSCalibur.
[0274] A T2-102 antibody (mouse IgG1) used as a positive control, which showed cross-reactivity with mouse TROP-2, exhibited high binding capacity to CHO-K1 cells in which the TROP-2 gene of mouse was randomly expressed. Otherwise, antibodies K5-70, T6-16, K5-107, K5-116-2-1 and T5-86 do not show such cross-reactivity with mouse TROP-2 (Figure 7).
[0275] Similarly, an anti-human EpCAM monoclonal antibody (BD Biosciences Pharmingen) used as a positive control exhibited high binding capacity to CHO-K1 cells in which human EpCAM / TROP-1 was randomly expressed. Otherwise, antibodies K5-70, T6-16, K5-107, K5-116-2-1 and T5-86 do not show such cross reactivities with human EpCAM / TROP-1 (Figure 8).
[0276] The previously mentioned results demonstrate that the generated anti-hTROP-2 monoclonal antibodies and in particular, antibodies K5-70, T6-16, K5-107, K5-116-2-1 and T5-86, which exhibited the anti-tumor activity in vivo, specifically bind to hTROP-2. [Example 13] [Measurement of cell development inhibiting activity]
[0277] As a method of examining the activity of the monoclonal anti-hTROP-2 antibody to inhibit hTROP-2 function, the influence of the antibody on the cell development of human cancer cells, which endogenously express hTROP-2 on the cell surface, was assessed by measuring the number of living cells using TetraColor ONE (Seikagaku Corporation). Specifically, PK-59 cells were resuspended in RPMI1640 medium containing 0.5% fetal bovine serum (manufactured by BioWest) at a cell concentration of 2 x 105 cells / ml and 100 μL of the prepared cell suspension was then added to each well of a 96 well culture plate. Subsequently, mouse IgG (negative control) and anti-hTROP-2 monoclonal antibodies (final concentrations: 0.1 and 1 μ g / ml) were added to the reservoirs and the mixtures were then cultured at 37 ° C in a 5 ° C incubator. % CO2 for 72 hours. As a control, a commercially available anti-hTROP-2 monoclonal antibody (clone YY01, Santa Cruz) was used. TetraColor ONE (Seikagaku Corporation) was added to the reservoirs and they were then reacted in a 5% CO2 incubator for 1 to 2 hours. After completion of the reaction, the 96 well culture plate was directly subjected to absorbance measurement at a wavelength of 490 nm (control wavelength: 655 nm), using a plate reader. The experiment was carried out using 3 reservoirs for each group. A significant difference test was performed according to the Student's t-test and P <0.05 was determined to be statistically significant.
[0278] Among the anti-hTROP-2 monoclonal antibodies, which were generated by their own company, approximately 160 clones were examined by the method described above, in terms of their effect on the cell development of PK-59 cells. As a result, T6 -16, T5-86, K5-70 and K5-107, which have tumor development inhibitory activity exhibited in vivo, have been confirmed to have 20% to 40% cell development inhibitory activity when compared to mouse IgG (negative control). It is clear that these anti-hTROP-2 antibodies have activity to bind hTROP-2 proteins, which have been expressed on the surface of human cancer cells, to neutralize hTROP-2 proteins and to inhibit the development of cancer cells ( Figure 9). [Example 14] [Scratch test]
[0279] The effect of an anti-hTROP-2 monoclonal antibody on the migratory capacity of human cancer cells was assessed by a scratch test. PK-59 cells were resuspended in an RPMI1640 medium containing 10% fetal bovine serum at a cell concentration of 3 x 105 cells / ml and 100 μL of the prepared cell suspension was then added to each well of the culture dish of 96 reservoirs. When the cells become confluent, a portion of the cells cultured by monolayers was peeled, such that the plaque was scratched in a longitudinal direction with the end of a tip. A monoclonal anti-hTROP-2 antibody and mouse IgG used as a negative control were added to the final concentrations of 0.1 and 1 μ g / ml, respectively, and culture was then performed for 24 hours. Before adding the antibody (Day 0) and 24 hours after culture (Day 1), the scratched region of the cell was photographed and the distance between the cells was then measured. In addition, the area of such a scratched region was quantified using the Scion Image software. The experiment was carried out using 8 reservoirs in each group. A significant difference test was performed according to the Student's t-test and P <0.05 was determined to be statistically significant.
[0280] The effect of an hTROP-2 antibody on the migratory capacity of cells that invades the scratched region was examined. As with the cell development inhibition assay, antibodies having the beneficial effects were evaluated. as an evaluation method, cells were photographed on Day 0 (when the antibody was added) and on Day 1 (24 hours after adding the antibody) and the migratory distance (μ m) and the area of a scratched region were determined image analysis. As a result, as shown in Figure 10, clear differences were observed in terms of the cells' migratory capacity. The T6-16 and K5-70 antibodies, which were used in the present test, have significant inhibitory activity on cell development when compared to the control. Still in a reproducibility test, the same trend was observed. In particular, T6-16 has a result of P <0.01 (by Student's t test) and the correlation with the in vivo test was observed. [Example 15] [Evaluation of the beneficial effects of the anti-hTROP-2 monoclonal antibody on mice that carry the tumor]
[0281] Prevention model
[0282] Pancreatic cancer cell lines (PK-59 and BxPC-3), which hTROP-2 expressed, were removed by treating the cells with trypsin and PBS was added to prepare the cell suspension having a concentration of 1 x 108 cells / ml. In this way the prepared cell suspension was mixed with an equal amount of Matrigel (BD Biosciences Pharmingen) on ice. Using a 26 G syringe, 100 μL of the obtained mixture (5 x 106 cells) was injected into the subcutis of the right flank of each of the 6-week-old female nude mouse (Balb / c, nu / nu). On the day of cancer cell transplantation (Day 1), the mice were divided into groups and administration of the antibody (1, 5 or 10 mg / kg body weight, intraperitoneal administration) was started. Then, administration of the antibody was continued at intervals once every three days. Anti-tumor activity was assessed based on the frequency of tumor formation and tumor volume. The tumor volume was calculated using the following formula.
[0283] Tumor volume (mm3) = (minor axis) 2 x (major axis) x π / 6
[0284] Treatment model
[0285] Pancreatic cancer cell lines (PK-59 and BxPC-3), which hTROP-2 expressed, were removed by treating the cells with trypsin and PBS was added to prepare the cell suspension having a concentration of 1 x 108 cells / ml. In this way the prepared cell suspension was mixed with an equal amount of Matrigel (BD Biosciences Pharmingen) on ice. Using a 26 G syringe, 100 μL of the obtained mixture (5 x 106 cells) was injected into the subcutis of the right flank of each of the 6-week-old female nude mouse (Balb / c, nu / nu). Five to six days after the transplantation of cancer cells, the mice whose tumor volume has increased from 50 to 150 mm3 (mean value: approximately 100 mm3) were divided into groups. The day on which the mice were divided into groups was defined as a first day (Day 1) and administration of the antibody was started. The antibody was administered intraperitoneally at intervals of once every three days (10 mg / kg body weight). Anti-tumor activity was assessed by measuring tumor volume. A significant difference test was performed according to the Student's t-test and P <0.05 was determined to be statistically significant. [Example 16] [In vivo anti-tumor activity analysis of the anti-hTROP-2 monoclonal antibody in the human pancreatic cancer cell xenograft model]
[0286] It is essential for an antibody used for the treatment of cancer, which targets hTROP-2, to have the activity of specifically killed tumor tissues expressing hTROP-2 or inhibiting tumor development.
[0287] Anti-hTROP-2 monoclonal antibodies (approximately 160 clones), which were re-produced in the present invention, were evaluated using the xenograft treatment models of a pancreatic cancer cell line PK-59. PK-59 cells express EpCAM on its surface (Figure 11A) acting as a pancreatic cancer stem cell marker (Chenwei Li, et al. Cancer Res 2007; 67: (3). 1030-1037) and also express P -glycoprotein / MDR1 (Figure 11B) and ABCG2 / CDw338 (Figure 11C) (Chen, CJ et al. Cell 47 (3), 381-389 (1986), Allikmets, R., et al. Hum. Mol. Genet. 5 (10), 1649-1655 (1996)), which are ABC transporters associated with drug resistance. In addition, PK-59 cells contain a cell fraction (8.93%) (Figure 11D) positive for both CD24 and CD44, which is characteristic of pancreatic cancer stem cells and these are assumed to be a highly malignant human pancreatic cancer cell line. (Chenwei Li, et al. Cancer Res 2007; 67: (3). 1030-1037, Jane E. Visvader and Geoffrey J. Lindeman. Nat Rev Cancer. Vol. 8 (10): 755-68, 2008).
[0288] More than approximately 160 clones re-generated do not exhibit the beneficial effects of PK-59 cell xenograft treatment models. Among such clones, clones exhibit significant tumor development inhibiting activity, called clones K5-70, T6-16, K5-107, T5-86 and K5-116-2-1 must be obtained.
[0289] In a group of administration of the clone K5-70 (mouse IgG2a), the rate of tumor development is statistically significantly inhibited. On the 21st day after the initiation of administration (day 21), the tumor volume of a control group (N = 14) was 1200.8 ± 377.3 mm3, considering the tumor volume of the administration group of clone K5 -70 was 748.7 ± 162.9 mm3 (P <0.01 by Student's t test) (Figure 12A). When the tumor volume at the time of initiation of antibody administration was set to 1.0, the tumor volume on the 21st day (Day 21) was 7.8 in the K5-70 clone administration group, considering the volume of tumor in the control group was 12.5 (Figure 12A). The weight of the taxed tumor was 0.43 ± 0.14 g (P <0.01 by Student's t test) in the administration group of clone K5-70, whereas the control group was 0.73 ± 0.26 g. Thus, clone K5-70 exhibited approximately 60% inhibitory activity (Figure 12B).
[0290] Similarly, tumor growth rate was statistically significantly inhibited further in a group of administration of clone K5-107 (mouse IgG1) (N = 8), a group of administration of clone T6-16 (mouse IgG2a) (N = 8), a T5-86 clone administration group (mouse IgG1) and a K5-116-2-1 clone administration group (mouse IgG2a) (N = 8). On the 17th day after initiation of administration (Day 17), the tumor volumes of the administration group of clone K5-107 (N = 8) and the administration group of clone T6-16 (N = 8) were 698.2 ± 175.9 mm3 (P <0.05 by Student's t-test) and 707.2 ± 254.5 mm3 (P <0.05 by Student's t-test), respectively, considering the tumor volume of the control group was 1039.3 ± 271.6 mm3. Likewise, on the 16th day after initiation of administration (Day 16), the tumor volume of the administration group for clone K5-116-2-1 (N = 8) was 508.5 ± 225.2 mm3 (P <0 , 05 by Student's t test), considering the tumor volume of the control group (N = 8) was 797.0 ± 172.9 mm3 (Figure 13).
[0291] Otherwise, in the case of clone T5-86, on the 15th day after initiation of administration (Day 15), the tumor in the administration group of clone T5-86 (N = 8) was 744.1 ± 289 , 1 mm3, considering the tumor volume of the control group (N = 8) was 1033.2 ± 319.4 mm3. Thus, there was no significant difference in terms of tumor volume. However, when comparing the tumor weight, which was performed on the same day, the tumor weight of the T5-86 clone administration group was 0.44 ± 0.13 g (P <0.05 by Student's t test) , considering the tumor weight of the control group was 0.62 ± 0.14 g. Thus, clone T5-86 exhibited significant inhibitory activity.
[0292] Furthermore, in terms of both the tumor volume and the tumor weight, the ratio (T / C) of each clone antibody administration group to the control group on the final day of the experiment is shown in Table 2 below. As shown in Table 2, each clone antibody exhibited significant inhibitory activity (T / C = 62% to 72%) on each antibody in the clone administration group. Table 2

[0293] In addition, the anti-tumor activity of each of the clones K5-70, T6-16 and K5-116-2-1 in the xenograft prevention models of the pancreatic cancer cell line PK-59 was analyzed. After completion of the administration of each antibody clone, tumor development was inhibited in all subjects (N = 8). On the 18th day after initiation of administration (Day 18), the tumor volume of the K5-7O clone administration group (1O mg / kg body weight) was 62.4 ± 8O, 4 mm3 (P <O, O1 by Student's t test), considering the tumor volume of the control group (N = 8) was 88O, 8 ± 2O6.4 mm3. Thus, clone K5-7O exhibited 92.9% tumor development inhibitory activity. On the 28th day after initiation of administration (Day 28), the tumor volume of the T6-16 clone administration group (10 mg / kg body weight) was 152.4 ± 122.3 mm3 (P <O, O1 by Student's t test), considering the tumor volume of the control group (N = 8) was 992.3 ± 25O, 8 mm3. Thus, the T6-16 clone exhibited the tumor development inhibiting activity of 84.6%. On the 20th day after initiation of administration (Day 20), the tumor volume of the K5-116-2-1 clone administration group (10 mg / kg body weight) was 207.7 ± 319.2 mm3 ( P <0.01 by Student's t test), considering the tumor volume of the control group (N = 8) was 1159.4 ± 413.3 mm3. Thus, clone K5-116-2-1 exhibited tumor development inhibitory activity of 82.1% (Figure 14 and Table 3). Furthermore, in all of the experiments, there is no significant difference between the control group and each hTROP-2 anti-antibody administration group in terms of the change in average body weight throughout the tested period.
[0294] In terms of both tumor volume and tumor weight, the ratio (T / C) of each clone antibody administration group to the control group on the final day of the experiment is shown in Table 3 below. As shown in Table 3, inhibition of significant tumor development was observed in each antibody in the clone administration group and in particular, a significant effect such as T / C = 10% or less was confirmed in the K5- clone administration group. 70. Table 3

[0295] The well-known anti-TROP-2 antibody AR47A6.4.2 (US Patent No. 7420041) has exhibited the effect of inhibiting tumor development, at a dosage of 20 mg / kg, in xenograft prevention models using various human cancer cell lines. This anti-TROP-2 antibody AR47A6.4.2 has inhibited the tumor development of a human pancreatic cancer cell line PL45 by almost 100%. However, this antibody has the effect of tumor inhibition on a pancreatic cancer cell line BxPC-3 by a percentage of approximately 50%, on a prostate cancer cell line PC-3 by a percentage of approximately 40%, on a cell line cancerous breast cancer MCF-7 in a percentage of approximately 60% and in a colon cancer cell line Colo205 in a percentage of approximately 40%. In contrast, the anti-hTROP-2 antibody of the invention of the present application exhibited a greater tumor development inhibitory effect at a dosage of half the previously mentioned dosage (10 mg / kg). [Example 17] [Analysis of anti-tumor activity in xenograft models (prevention models and treatment models) of the human pancreatic cancer cell line BxPC-3]
[0296] As in the case of the use of the aforementioned xenograft treatment models of the human pancreatic cancer cell line PK-59, the antitumor activity of the K5-70 clone in the xenograft prevention models and xenograft treatment models of the cancer cell line pancreatic human BxPC-3 was analyzed.
[0297] When compared to a control group (N = 8), the tumor development of the administration group of clone K-70 was significantly inhibited. On the 52nd day (Day 52), the tumor volume of the K5-70 clone administration group (N = 8) was 236.0 ± 136.4 mm3, considering the tumor volume of the control group (N = 8 ) was 616.3 ± 266.8 mm3. Thus, clone K-70 exhibited a 61.7% tumor inhibitory effect (P <0.01 by Student's t test) (Figure 15).
[0298] From the results previously mentioned, it is clear that the anti-hTROP-2 monoclonal antibody exhibits significant inhibitory activity of tumor development in vivo in at least two species of cancer cells. [Example 18] [Dosage-dependent anti-tumor activity of hTROP-2 anti-antibody (clone K5- 70) in hTROP-2 xenograft prevention models that express the pancreatic cancer cell line (PK-59 cells)]
[0299] For the purpose of further analyzing the in vivo tumor development inhibitory activity of the anti-hTROP-2 antibody, a dose-dependent test was performed. As shown in Figure 16, tumor development of PK-59 cells was dependent on the dosage inhibited by administration of K5- 70. On the 21st day after administration of the antibody (Day 21), the tumor volume of the control group (N = 7) was 937.8 ± 295.3 mm3. Otherwise, the tumor volume of the K5-70 administration group (1 mg / kg) (N = 8) was 493.5 ± 305.1 mm3, showing a 50% inhibitory rate and the tumor volume of the group of administration K5-70 (5 mg / kg) (N = 8) was 124.7 ± 89.0 mm3, showing an inhibitory rate of 90%. Thus, it is clear that when compared to the known anti-TROP-2 antibody AR47A6.4.2 (US Patent No. 7420041), the anti-hTROP-2 antibody of the present invention exhibits in vivo a development of the equivalent of tumor inhibitory effect that of the anti-TROP-2 antibody AR47A6.4.2 at a dosage of one twentieth of the anti-TROP-2 antibody AR47A6.4.2 and that this exhibits a greater than 90% inhibitory effect at a dosage of one quarter of this. [Example 19] [Epitope assay]
[0300] Preparation of the mouse / human chimeric TROP-2 protein
[0301] A mouse / human TROP-2 gene was prepared according to the PCR method. The PCR primers as shown below were designed based on a human TROP-2 gene sequence and a mouse TROP-2 gene sequence (Genbank Accession No. NM 020047).
[0302] TROP-2C mouse / human primers
[0303] Y606 (advanced location): 5’-cctgagcctacgctgcgacgaagtggtgcg-3 ’(SEQ ID NO: 10)
[0304] Y607 (reverse location): 5'-cgcaccacttcgtcgcagcgtaggctcagg-3 '(SEQ ID NO: 11)
[0305] Mouse / human TROP-2A primers
[0306] Y612 (advanced location): 5’-gactgctccacgctgacttccaagtgcctg-3 ’(SEQ ID NO: 12)
[0307] Y613 (reverse location): 5'-caggcacttggaagtcagcgtggagcagtc-3 'SEQ ID NO: 13)
[0308] Human / mouse TROP-2B
[0309] Y614 (advanced location): 5’-ctcgtggacaacgatggcctctacgacccg-3 ’(SEQ ID NO: 14)
[0310] Y615 (reverse location): 5’-cgggtcgtagaggccatcgttgtccacgag-3 ’(SEQ ID NO: 15)
[0311] Mouse / human TROP-2-D primers
[0312] Y608 (advanced location): 5’-ccaaagcctgcgctgcgatgagctggtgcgc-3 ’(SEQ ID NO: 16)
[0313] Y609 (reverse location): 5’-gcgcaccagctcatcgcagcgcaggctttgg-3 ’(SEQ ID NO: 17)
[0314] Mouse / human TROP-2-E primers
[0315] Y616 (advanced location): 5’-agcttcctatccgcggtgcactacgagcag-3 ’(SEQ ID NO: 18)
[0316] Y617 (reverse location): 5’-ctgctcgtagtgcaccgcggataggaagct-3 ’(SEQ ID NO: 19)
[0317] Mouse / human TROP-2-F primers
[0318] Y618 (advanced location): 5’-gacattaaaggcgagtctctattccagggc-3 ’(SEQ ID NO: 20)
[0319] Y619 (reverse location): 5’-gccctggaatagagactcgcctttaatgtc-3 ’(SEQ ID NO: 21)
[0320] TROP-2 mouse primers
[0321] Advanced starter: 5-ctactccaccccaccctggcg-3 ’(SEQ ID NO: 22)
[0322] Reverse initiator: 5’-ctcgagcaagctaggttcgcttctc-3 ’(SEQ ID NO: 23)
[0323] To the TROP-2 of mouse reverse primer, a sequence digested by restriction enzyme XhoI except for a stop codon was added. The schematic view of the mouse / human chimeric TROP-2 protein is shown in Figure 17.
[0324] The chimeric protein hmTROP-2-A is a chimeric protein, consisting of a polypeptide ranging from the N-terminal to the amino acid at position 69 of the hTROP-2 protein and a polypeptide ranging from the amino acid at position 64 to the C-terminal of the TROP protein -2 of mouse. The chimeric hmTROP-2-B protein is a chimeric protein, consisting of a polypeptide ranging from the N-terminal to the amino acid at position 101 of the hTROP-2 protein and a polypeptide ranging from the amino acid at position 96 to the C-terminal of the TROP-2 protein of mouse. The chimeric hmTROP-2-C protein is a chimeric protein, consisting of a polypeptide ranging from the N-terminal to the amino acid at position 145 of the hTROP-2 protein and a polypeptide ranging from the amino acid at position 140 to the C-terminal of the TROP-2 protein of mouse. The chimeric protein mhTROP-2-D is a chimeric protein, consisting of a polypeptide ranging from the N-terminal to the amino acid at position 139 of the mouse TROP-2 protein and a polypeptide ranging from the amino acid at position 146 to the C-terminal of the hTROP- protein two. The chimeric protein mhTROP-2-E is a chimeric protein, consisting of a polypeptide ranging from the N-terminal to the amino acid at position 187 of the mouse TROP-2 protein and a polypeptide ranging from the amino acid at position 194 to the C-terminal of the hTROP- protein two. The chimeric protein mhTROP-2-F is a chimeric protein, consisting of a polypeptide ranging from the N-terminal to the amino acid at position 227 of the mouse TROP-2 protein and a polypeptide ranging from the amino acid at position 234 to the C-terminal of the hTROP- protein two.
[0325] The expression vector used in the preparation of the chimeric proteins described above were specifically constructed by the following methods. In order to prepare a chimeric hmTROP-2-A gene, the hTROP-2 gene was used as a template and PCR was performed using the advanced hTROP-2 primer and the mouse / human Y613 TROP-2A primer. Likewise, the mouse TROP-2 gene was used as a template and PCR was performed using the mouse / human TROP-2A primer Y612 and the mouse TROP-2 reverse primer. A DNA fragment amplified by PCR was developed using acrylamide gel and an interest group was then recovered by extraction. Subsequently, the types of DNA fragment extracts were mixed to prepare a model and PCR was then performed using the advanced hTROP-2 primer and the mouse TROP-2 reverse primer. A PCR product was developed by agarose gel electrophoresis and a DNA fragment of interest was then extracted. The extracted DNA fragment was cloned into a pCR (trademark) -Blunt (Invitrogen) vector (pCRB-hmTROP-2-A) and a gene sequence was then confirmed. An expression vector by the animal cells was produced by removing the hTROP-2 gene from pcDNA3.1-hTROP-2-myc / His by digesting EcoRI / XhoI and then inserting an EcoRI / XhoI fragment containing a chimeric hmTROP-2- gene into it The preparation of pCRB-hmTROP-2-A (pcDNA3.1-hmTROP-2-A-myc / His). In addition, the following chimeric genes were prepared by the same method as described above and expression vectors were constructed: hmTROP-2-B (using an advanced human TROP-2 primer, a mouse / human Y615 TROP-2B primer, a TROP primer -2B mouse / human Y614 and a mouse TROP-2 reverse primer), hmTROP-2-C (using an advanced human TROP-2 primer, a mouse / human TROP-2C primer Y607, a TROP-2C primer from mouse / human Y606 and a mouse TROP-2 reverse primer), mhTROP-2-D (using an advanced mouse TROP-2 Primer, a mouse / human Y609 TROP-2-D primer, a TROP-2- primer Mouse / human D Y608 and a human TROP-2 reverse primer), mhTROP-2-E (using an advanced mouse TROP-2 primer, a mouse / human TROP-2-E primer Y617, a TROP-2 primer -E mouse / human Y616 and a human TROP-2 reverse primer), mhTROP-2-F (using an advanced mouse TROP-2 Primer, u m mouse TROP-2-F primer / human Y619, a mouse TROP-2-F primer / human Y618 and a human TROP-2 reverse primer) (pcDNA3.1-hmTROP-2-B-myc / His, pcDNA3 .1- hmTROP-2-C-myc / His, pcDNA3.1-mhTROP-2-D-myc / His, pcDNA3.1-mhTROP-2-E-myc / His and pcDNA3.1-mhTROP-2-F -myc / His).
[0326] Establishment of HEK293 cell lines, which constitutively expresses hTROP-2, mouse / human TROP-2C chimeric proteins and mouse / human TROP-2-D
[0327] The expression vectors described above pcDNA3.1-hTROP-2-myc / His, pcDNA3.1-hmTROP-2-C-myc / His and pcDNA3.1-mhTROP-2-D-myc / His were each one introduced into HEK293 cells. Selection was performed using a G418 antibiotic (Calbiochem) and HEK293 cell lines constitutively expressing the hTROP-2 protein, the hmTROP-2-C chimeric protein and the mhTROP-2-D chimeric protein were established.
[0328] The binding regions of the anti-hTROP-2 monoclonal antibodies K5-70, T5-86, K5-107, T6-4, T6-16 and K5-116-2-1, which exhibited the beneficial effects on the models of xenograft treatment of the pancreatic cancer cell line PK-59, were identified. First, the reactivity of anti-hTROP-2 monoclonal antibodies exhibited the beneficial effects with HEK293 cells, which constantly express the chimeric proteins hmTROP-2-C and mhTROP-2-D, were examined by FACS analysis (Figure 18). As a result, it was observed that K5-70, K5-107, T5-86 and K5-116-2-1 reacted with hmTROP-2-C, but that these antibodies do not react with mhTROP-2-D. Otherwise, T6-4 and T6-16 reacted with mhTROP-2-D, but they do not react with hmTROP-2-C. From these results, the binding region of each of K5-70, K5-107, T5-84 and K5-116-2-1 was limited to a region ranging from the N-terminal to the amino acid at position 145 of hTROP-2 and the binding region of each of T6-4 and T6-16 was limited in a region ranging from the amino acid at position 146 to the amino acid at position 274 of hTROP-2 (Figure 18).
[0329] In order to analyze the binding regions in more detail, vectors used in the expression of chimeric proteins hmTROP-2-A, hmTROP-2-B, mhTROP-2-E and mhTROP-2-F were prepared and the reactivity of chimeric proteins with anti-hTROP-2 monoclonal antibodies exhibiting beneficial effects was analyzed (Figure 19). The newly prepared expression vectors, which were used in the expression of the chimeric proteins, were each introduced into the HEK293 cells and FACS analysis was then performed, using the cells that randomly expressed the chimeric proteins. K5-70, K5-107, T5-86 and K5-116-2-1 reacted with hmTROP-2-A, but does not react with mhTROP-2-B. The 6 types of monoclonal antibodies examined were all reacted with hTROP-2. These results clearly show that the binding region of K5-70, K5-107, T5-86 and K5-116-2-1 is present in a region ranging from the N-terminal to the amino acid at position 69 of hTROP-2. In addition, T6-4 and T6-16 reacted with mhTROP-2-E or mhTROP-2-F. This suggests that these antibodies recognize a region ranging from the amino acid at position 146 to the amino acid at position 193 of hTROP-2. [Example 20] [Immunohistochemistry] <Materials / Method>
[0330] The following cancer and normal tissue assays have been used in immunohistochemistry.
[0331] Test of normal human tissue:
[0332] Normal human organs in duplicates (Catalog No .: AB1, Super Bio Chips)
[0333] Normal fabrics more than simple stains (Catalog No .: A103 (VI), ISU ABXIS)
[0334] Lung cancer tissue assay:
[0335] Metastasis of normal human lung cancer (Catalog Nr .: CCA3, Super Bio Chips)
[0336] Human lung carcinoma tissue with extremity tissue, 2 location cores (Catalog No.: OD-CT-RsLug03-002, Shanghai Outdo Biotech)
[0337] Pancreatic cancer tissue test:
[0338] Tissues of human pancreatic carcinoma with mono-pathological type of 60 cases, 2 nuclei of location (Catalog No.: OD-CT-DgPan03-001, Shanghai Outdo Biotech)
[0339] Lung cancer tissue test:
[0340] Hepatocellular carcinoma, grades I to III with normal tissue controls, 63 cases of tissue tests (Catalog No.: CS03-01-002U, Cybrdi)
[0341] Human lung carcinoma tissue with mono-pathological type of 30 cases, 2 nuclei of location (Catalog No.: OD-CT-DgLiv02-002, Shanghai Outdo Biotech)
[0342] Colon cancer tissue test:
[0343] Colorectal cancer, human (Catalog No .: CD3, Super Bio Chips)
[0344] Human colon carcinoma with extremity tissue, 2 localization nuclei (Catalog Nr .: OD-CT-DgCol03-002, Shanghai Outdo Biotech)
[0345] Colon cancerous lymph node metastasis and liver metastasis tissue assays:
[0346] Colorectal cancer (colon and rectum) with punctate lymph node metastasis assay, 44 cases / 99 nuclei, assay slide (Catalog No. CO991t, Biomax us)
[0347] Colorectal cancer (colon and rectum) with punctate lymph node metastasis and normal adjacent tissue assay, 43 cases / 99 nuclei (Catalog Nr .: CO992, Biomax us)
[0348] Liver metastasis_ colon cancer tissues (Catalog No .: A203 (IV), ISU ABXIS)
[0349] Breast cancer tissue assay:
[0350] Normal metastasis of breast cancer, human (Catalog Nr .: CBA3, Super Bio Chips)
[0351] Human breast carcinoma with extremity tissue, 2 location cores (Catalog No.: OD-CT-RpBre03-002, Shanghai Outdo Biotech)
[0352] Stomach cancer tissue assay:
[0353] Stomach cancer, human (Catalog No .: CQ1, Super Bio Chips)
[0354] Human gastric carcinoma with extremity tissue, 2 nuclei of localization (Catalog No.: OD-CT-DgStm03-002, Shanghai Outdo Biotech)
[0355] Esophageal cancer tissue assay:
[0356] Esophageal cancer, human (Catalog No .: CR1, Super Bio Chips)
[0357] Human esophageal carcinoma with extremity tissue, 2 location cores (Catalog No.: OD-CT-DgEso03-002, Shanghai Outdo Biotech)
[0358] Ovarian cancer tissue assay:
[0359] Ovarian cancer, human (Catalog No .: CJ1, Super Bio Chips)
[0360] Prostate cancer tissue test:
[0361] Human prostate cancer, normal (Catalog No .: CA3, Super Bio Chips)
[0362] Bladder cancer tissue assay:
[0363] Bladder carcinoma / transitional cell carcinoma, grades I to III with normal tissue assays (Catalog No.: CC12-01-001U, Cybrdi)
[0364] Patient information and clinical information regarding the tissue tests described above were obtained from the data sheets linked to these and to the homepages of the individual companies.
[0365] Immunohistochemistry method
[0366] After completing a dewaxing treatment, the tissue assay sheets from normal human tissues and cancerous tissues were subjected to a protease treatment with pepsin at 37 ° C for 5 minutes. Then, the sections were used in immunoting with an anti-hTROP-2 monoclonal antibody. A color reaction was performed using DAB (3,3'-diaminobenzidine) as a substrate and as a counter dye, nuclear dyeing was then carried out using hematoxylin.
[0367] More specifically, these operations were carried out as follows. A paraffin-soaked section was subjected to a dewaxing treatment and was then subjected to a protease treatment with pepsin (DAKO) at 37 ° C for 5 minutes. After antigen activation, the section was treated at room temperature for 20 minutes using a solution prepared by adding a solution of hydrogen peroxide to methanol in a final concentration of 0.3%, so that the endogenous peroxidase activity was deleted. The resultant was washed with PBS at room temperature for 5 minutes twice and this was then blocked at room temperature for 30 minutes using a PBS solution containing 1.5% normal horse serum (DAKO), in this way as to perform a operation to block non-specific binding in tissues. Subsequently, the resultant was reacted with the anti-hTROP-2 monoclonal antibody clone K5-63-17 (final concentration: 10 μ g / ml), which was diluted with a PBS solution containing 1.5% horse serum normal, at room temperature for 1 hour and was then washed with PBS at room temperature for 5 minutes three times. Then, a biotinylated anti-mouse IgG antibody (Vector), which was diluted 200 times with a PBS solution containing 1.5% normal horse serum, was reacted at room temperature for 30 minutes. The reaction product was washed with PBS at room temperature for 5 minutes three times and a Vectastain ABC kit reagent (Vector) was mixed according to the instruction manual included in it, in order to prepare an ABC complex. This ABC complex was reacted at room temperature for 30 minutes. The reaction product was washed with PBS at room temperature for 5 minutes three times and color development was then carried out using DAB solution of simple dyeing of histofin peroxidase substrate (Nichirei Biosciences). After completion of color development, the reaction product was washed with deionized water for 5 minutes and the core was dyed with Mayer's hematoxylin solution (Wako Pure Chemical Industries, Ltd.). Then, dehydration was performed with alcohol, followed by penetration with xylene and assembly in Entellan New (Merck Japan). <Results>
[0368] Expression of hTROP-2 in normal human tissues
[0369] The model of hTROP-2 expression in normal human tissues was analyzed using the anti-hTROP-2 monoclonal antibody clone K5-63-17. An assay of normal human tissue (Catalog No .: AB1, Super Bio Chips) was dewaxed and was then subjected to a hydrophilic treatment. Then, the antigen was activated with a protease, pepsin and immunostaining was then performed using the anti-hTROP-2 monoclonal antibody clone K5-63-17 (Figure 20). As a result, dyeing was observed in the skin, esophagus, kidney (cortex and medulla), pancreas, prostate, bladder and tonsil. A majority of the stained images located on the cell membrane (Figure 20A, B, C, D, F, G and H), but the expression hTROP-2 was partially observed in the cytoplasm (Figure 20E and H). Otherwise, such dyeing was not observed in the heart, liver, stomach, small intestine, large intestine, skeletal muscle, lung, spleen, thymus gland and others (Figure 20I and J).
[0370] Expression of hTROP-2 in human cancerous tissues
[0371] In order to examine the expression of hTROP-2 (positive rate hTROP-2) in human cancerous tissues, assays of cancerous tissue from various human cancerous species were immunoted using the anti-hTROP-2 monoclonal antibody clone K5- 63-17. A section of tissue, in which 10% or more of the cancer cells were stained, was defined as hTROP-2-positive. The dyeing results are shown in Table 4. Table 4

[0372] Representative tinted images are shown in Figure 21. Among the species of cancers, with respect to which hTROP-2 expression was analyzed, prostate cancer has the highest positive rate (92.1%) and also, cancer of lung (65.4%), esophageal cancer (76.7%), bladder cancer (71.2%) and others have high positive rates. Liver cancer has the lowest positive rate (7.61%). It was observed from the stained images that, as with normal cells, hTROP-2 was highly localized on the cell membrane even in the case of cancer cells (Figure 21 A to F, H and I). In addition, hTROP-2 has also been located in the cytoplasm in some cases (Figure 21A, B, E and G).
[0373] The positive hTROP-2 rate in pancreatic cancer was 41.9%. The relationship between this hTROP-2 positive rate and the degree (degree of differentiation) of pancreatic cancer was analyzed. As a result, hTROP-2 was expressed at high frequency in pancreatic cancer with a high degree, termed, with a lower degree of differentiation (Table 5). Table 5
[Example 21] [Anti-tumor activity of the K5-70 antibody by simple administration in the human pancreatic cancer cell line xenograft prevention models PK-59]
[0374] The strong anti-tumor activity in vivo of a K5-70 clone (mouse IgG2a) was further exhibited by a simple administration of K5-70 at a dosage of 10 mg / kg body weight to xenograft prevention models using a human pancreatic cancer cell line PK-59. In a control group (mouse IgG, 10 mg / kg body weight, N = 3), tumor formation was observed in all of the subjects and the tumor volume on the 28th day after cell transplantation (Day 28) was 781.7 ± 74.5 mm3. Otherwise, in a group where K5-70 was only administered once a day for cancer cell transplantation (Day 1) (10 mg / kg body weight, N = 3), the tumor volume on Day 28 was 144.4 ± 176.9 mm3 (P <0.05 by Student's t-test), showing tumor inhibitory activity of 81.5% (Figure 22A). Regarding the tumor weight, the tumor weight of the control group on Day 28 was 0.59 ± 0.06 g. In contrast, the tumor weight of the K5-70 clone administration group was 0.07 ± 0.10 g (P <0.01 by Student's t test), showing an inhibitory activity of 88% (Figure 22B). With respect to both tumor volume and tumor weight, tumor formation was completely inhibited in 2 out of 3 subjects in the K5-70 administration group at a dosage of 10 mg / kg body weight by administration (Figure 22C). [Example 22] [Anti-tumor activity of the anti-human TROP-2 monoclonal antibody in the xenograft treatment models of the human colon cancer cell line SW480]
[0375] The anti-tumor activity of each of the anti-human TROP-2 antibodies (clones K5-70, K5-116-2-1 and T6-16) was examined with the xenograft treatment models using a cell line cancerous human colon SW480. SW480 cells (5 x 106 cells) were subcutaneously transplanted on the right flank of each of the 6-week-old female NOD-scid mice (Day 1). When the average tumor volume reached 100 mm3, clustering was performed (Day 7 or Day 10). From Day 7 or Day 10, intraperitoneal administration of the antibody was performed at the administration intervals once every three days. The anti-tumor activity of clones K5-70 and the anti-tumor activities of clones K5-116-2-1 and T6-16 were evaluated by independent studies, separately. In the study of the evaluation of the anti-tumor activity of K5-70, the tumor volume of a control group (mouse IgG (10 mg / kg body weight), N = 8) on the 44th day after cell transplantation carcinogenic (Day 44) was 365.4 ± 214.6 mm3. Otherwise, the tumor volume of a K5-70 administration group (10 mg / kg body weight) was 27.4 ± 29.4 mm3 (P <0.01 by Student's t test) and thus , tumor formation was significantly inhibited in the K5-70 administration group (inhibitory rate: 92.5%) (Figure 23A). Regarding tumor weight, the tumor weight of the control group was 0.11 ± 0.07 g, whereas the tumor weight of the K5-70 administration group was 0.005 ± 0.007 (g) (P <0.01 Student's t test), showing an inhibitory rate of 95.5% (Figure 23B). In particular, in two of the eight individual mice in the K5-70 administration group, tumor formation was completely inhibited and the presence of a tumor should not be confirmed.
[0376] In the study of the evaluation of the anti-tumor activities of K5-116-2-1 and T6-16, which was performed separately, the tumor volume of the control group on Day 42 was 713.8 ± 354.5 mm3 (N = 8). In contrast, the tumor volume of the K5-116-2-1 administration group (10 mg / kg body weight) was 188.9 ± 97.4 mm3 (N = 8, P <0.01 by the t test of Student) (Figure 24A) and the tumor volume of the T6-16 administration group (10 mg / kg body weight) was 292.8 ± 199.7 mm3 (N = 8, P <0.01 by the t test of Student) (Figure 25A). Thus, the two administration groups above showed inhibitory rates of 73.5% and 59.0%, respectively. Regarding the tumor weight as well, the tumor weight of the control group was 0.39 ± 0.19 g. In contrast, the tumor weight of the K5-116-2-1 administration group was 0.10 ± 0.07 g (P <0.01 by Student's t-test) and the tumor weight of the T6- administration group 16 was 0.17 ± 0.14 g (P <0.05 by Student's t test). Thus, the two administration groups above showed inhibitory rates of 72.2% and 56.4%, respectively (Figure 24B and Figure 25B). [Example 23] [Dosage-dependent anti-tumor activity of clone K5-70 in xenograft treatment models using human colon cancer cell line SW480]
[0377] Subsequently, the dose-dependent anti-tumor activity of clone K5-70 (mouse IgG2a) was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5 x 106 cells) were subcutaneously transplanted on the right flank of each of the 6-week-old female NOD-scid mice. Ten days after transplantation (Day 10) in which the mean tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg / kg body weight in the administration group, N = 8 , 104.4 ± 17.6 mm3), a K5-70 administration group (1 mg / kg body weight) (N = 8, 104.3 ± 16.1 mm3), a K5-70 administration group ( 5 mg / kg body weight) (N = 8, 104.6 ± 15.9 mm3) and a K5-70 administration group (10 mg / kg body weight) (N = 8, 104.8 ± 14, 9 mm3). Then, intraperitoneal administration was performed at intervals of administration once every three days. On day 42, the tumor volume of the control group was 713.8 ± 354.5 mm3. Otherwise, in the K5-70 administration group, dose-dependent tumor formation inhibitory activity was observed. That is, the tumor volume of 1 mg / kg of body weight in the administration group was 485.0 ± 207.3 mm3 (inhibitory rate: 32.1%), the tumor volume of 5 mg / kg of weight of the administration group was 339.5 ± 253.2 mm3 (inhibitory rate: 52.4%) and the tumor volume of 10 mg / kg of body weight of the administration group was 355.4 ± 202.8 mm3 ( inhibitory rate: 50.2%, P <0.05 by Student's t test) (Figure 26A). Likewise, with respect to tumor weight on day 42, the tumor weight of the control group was 0.39 ± 0.19 g. Otherwise, the tumor weight of the K5-70 administration group (1 mg / kg body weight) was 0.24 ± 0.11 g (inhibitory rate: 37.8%), the tumor weight of 5 mg / kg body weight of the administration group was 0.17 ± 0.14 g (inhibitory rate: 55.8%, P <0.05 by Student's t test) and the tumor weight was 10 mg / kg of weight body weight of the administration group was 0.20 ± 0.13 g (inhibitory rate: 47.1%). In this way, the dose-dependent anti-tumor activity was confirmed (Figure 26B). [Example 24] [Analysis of administration intervals of clone K5-70 to xenograft treatment models using human colon cancer cell line SW480]
[0378] Subsequently, in order to analyze the optimal administration intervals of clone K5-70 (mouse IgG2a), the anti-tumor activity of clone K5-70 when it was administered once a week (once every 7 days) has been examined with xenograft treatment models using a SW480 human colon cancer cell line. SW480 cells (5 x 106 cells) were subcutaneously transplanted on the right flank of each of 6-week-old female NOD-scid mice. Ten days after transplantation (Day 10) in which the average tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg / kg of body weight in the administration group, N = 8, 104.4 ± 17.6 mm3) and a K5-70 administration group (10 mg / kg body weight, once a week) (N = 8, 104.3 ± 16.1 mm3). Then, intraperitoneal administration was performed once every 7 days. On day 42, the tumor volume of the control group was 713.8 ± 354.5 mm3, whereas the tumor volume of the K5-70 administration group (once a week) was 323.3 ± 239.9 mm3 (inhibitory rate: 55%, P <0.05 by Student's t test) (Figure 27A). In addition, when the administration interval was increased to once every 10 days and once every two weeks, the tumor volume of the control group on day 39 was 956.9 ± 367.8 mm3. Otherwise, the tumor volume of the K5-70 administration group (administered once every 10 days) on day 39 was 525.4 ± 180.6 mm3 (inhibitory rate: 45.1%, P <0.01 by Student's t test) and the tumor volume of the K5-70 administration group (administered once every 14 days) was 459.4 ± 217.6 mm3 (inhibitory rate: 52.0%, P <0.01 Student's t test) (Figure 27B). In previous techniques (US 7420040 and US 7420041), when antibodies were administered to xenograft treatment models using a pancreatic cancer cell line (BxPC-3) at a dosage of 20 mg / kg body weight, three times a week ( at 2-day administration intervals), antibodies exhibited anti-tumor activity at an inhibitory rate of 50% to 60%. In contrast, the K-70 antibody exhibited anti-tumor activity equivalent to that of the prior art, at a dosage of half of the prior art (10 mg / kg body weight), once every 2 weeks (at 12-hour administration intervals days). If taking into account simple dosing and administration intervals, it becomes clear that the K5-70 antibody exhibited significant anti-tumor activity at a total dosage of at least a tenth of those of the prior art. [Example 25] [Dosage-dependent anti-tumor activity of clone T6-16 in xenograft treatment models using human colon cancer cell line SW480]
[0379] Subsequently, the dosage-dependent anti-tumor activity of clone T6-16 (mouse IgG2a) was examined with the xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5 x 106 cells) were subcutaneously transplanted on the right flank of each of the 6-week-old female NOD-scid mice. Ten days after transplantation (Day 10) in which the average tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg / kg of body weight in the administration group, N = 8, 105.8 ± 9.9mm3), a T6-16 administration group (1 mg / kg body weight) (N = 8, 104.4 ± 13.3 mm3), a T6-16 administration group (5 mg / kg body weight) (N = 8, 104.7 ± 13.0 mm3) and a T6-16 administration group (10 mg / kg body weight) (N = 8, 104.8 ± 12.4 mm3) . Then, intraperitoneal administration was performed at administration intervals once every three days. On day 43, the tumor volume of the control group was 473.5 ± 137.0 mm3. Otherwise, in the T6-16 administration group, dose-dependent tumor formation inhibitory activity was observed. That is, the tumor volume of 1 mg / kg of body weight in the administration group was 397.9 ± 97.5 mm3 (inhibitory rate: 16.0%), the tumor volume of 5 mg / kg of body weight in the administration group was 195.9 ± 89.7 mm3 (inhibitory rate: 58.7%, P <0.01 by Student's t test) and the tumor volume of 10 mg / kg of body weight in the administration group was 190.2 ± 56.5 mm3 (inhibitory rate: 59.8%, P <0.01 by Student's t test) (Figure 28A). Likewise, with respect to tumor weight on day 43, the tumor weight of the control group was 0.19 ± 0.07 g. Otherwise, the tumor weight of the T6-16 administration group (1 mg / kg body weight) was 0.20 ± 0.08 g, the tumor weight of 5 mg / kg body weight of the administration group was 0.08 ± 0.04 g (inhibitory rate: 57.9%, P <0.01 by Student's t test) and the tumor weight of 10 mg / kg body weight in the administration group was 0.09 ± 0.04 g (inhibitory rate: 52.6%, P <0.01 by Student's t test). Thus, dose-dependent anti-tumor activity was confirmed (Figure 28B). [Example 26] [Analysis of administration intervals of clone T6-16 to xenograft treatment models using human colon cancer cell line SW480]
[0380] Subsequently, in order to analyze optimal administration intervals for clone T6-16 (mouse IgG2a), the anti-tumor activity of clone T6-16 when it was administered at administration intervals once a week (once every 7 days) and once every 10 days was examined with xenograft treatment models using a SW480 human colon cancer cell line. The SW480 cells (5 x 106 cells) were subsequently transplanted into the right flank of each of 6 week old female NOD-scid mice. Ten days after transplantation (Day 10) in which the average tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg / kg of body weight in the administration group, N = 8, 105.8 ± 9.9 mm3), a T6-16 administration group (10 mg / kg body weight, once a week) (N = 8, 105.0 ± 11.6 mm3), an administration group T6-16 (10 mg / kg body weight, once every 10 days) (N = 5, 130.8 ± 2.4 mm3). Then, the administration started. On day 43, the tumor volume of the control group was 473.5 ± 137.0 mm3. Otherwise, the tumor volume in the T6-16 administration group (once a week) was 243.7 ± 65.3 mm3 (inhibitory rate: 48.5%, P <0.01 by Student's t test) and the tumor volume of the T6-16 administration group (once every 10 days) was 297.8 ± 54.4 mm3 (inhibitory rate: 37.1%, P <0.05 by Student's t test) ( Figure 29). In prior techniques (US 7420040 and US 7420041), when antibodies were administered to xenograft treatment models using a pancreatic cancer cell line (BxPC-3) at a dosage of 20 mg / kg body weight, three times a week ( at 2-day administration intervals), antibodies exhibited anti-tumor activity at an inhibitory rate of 50% to 60%. In contrast, the T6-16 antibody was observed to exhibit significant anti-tumor activity when it was administered at a dosage of half of the prior techniques once every 10 days (at the 8-day administration intervals). If taking into account a simple dosage and administration intervals, it becomes clear that the T6-16 antibody exhibited significant anti-tumor activity at a total dosage of at least one eighth of those of the prior art. [Example 27] [Analysis of the anti-tumor activity of the K5-70 clone in xenograft prevention models using human prostate cancer cell line DU-145]
[0381] The anti-tumor activity of clone K5-70 in human prostate cancer was evaluated with xenograft prevention models using DU-145 cells (RIKEN Cell Bank, RCB2143). DU-145 cells (5 x 106 cells) were subcutaneously transplanted into each of the 6 week old female nude mice (Balb / c, nu / nu). The day on which the transplant was performed was defined as Day 1. The mice were divided into a control group (N = 8) and a K5-70 administration group (N = 8). From day 1, K-70 was administered intraperitoneally to the mice at a frequency of once every 3 days at a dosage of 10 mg / kg of body weight. On day 40, the tumor volume of the control group was 368.2 ± 307.8 mm3. Otherwise, the tumor volume of the K5-70 administration group was 30.6 ± 29.6 mm3 (P <0.05 by Student's t test), showing the tumor formation of the inhibitory activity of approximately 90% ( Figure 30A). Regarding tumor weights, significant anti-tumor activity was also observed. The tumor weight of the control group on day 40 was 0.18 ± 0.18 g. In contrast, in the K5-70 administration group, tumors disappear from all 8 individual mice and thus, tumor formation was completely inhibited (Figure 30B). From the results mentioned above, it is clear that the anti-human TROP-2 clone K5-70 shows strong anti-tumor activity even in human prostate cancer. [Example 28] [K5-70 clone metastasis inhibitory activity in liver metastasis models using human pancreatic cancer cell line PK-59]
[0382] Cancer metastasis is an important factor that influences the clinical prognosis in the treatment of gastrointestinal cancer. The control of metastasis is therapeutically, significantly important. If not only tumor formation but also cancer metastasis from other organs must be suppressed by administering an antibody for use in cancer therapy that targets TROP-2, high clinical utility must be anticipated. Thus, this is a desired property as a therapeutic cancer antibody.
[0383] TROP-2 expression has been confirmed in many types of carcinomas. TROP-2 has been reported to be expressed at a high level particularly in the metastatic focus (Br. J. Cancer (2008); 99: 1290-1295, Clin. Cancer Res. (2006); 12: 30573063, Mod. Pathol. ( 2008); 21: 186-191). In addition, it also relates that when cancer cells introduced by the Trop-2 gene were transplanted into nude mice through transplenic or transpancreatic administration, the incidence of liver metastasis increased (WO 2010/089782, Molecular Cancer (2010); 9 : 253) and thus, the report suggests that the importance of TROP-2 in the process of cancer metastasis. However, to date, they do not exist in the report specifically describing that an antibody targeting TROP-2 has metastatic inhibitory action in vivo.
[0384] The anti-hTROP-2 mouse monoclonal antibody K5-70, which was discovered by the present invention, exhibits high therapeutic effects in xenograft models, in the subcutis of which pancreatic cancer cells have been transplanted. It has been shown that a scratch test performed in vitro that the K5-70 antibody is able to suppress the migration capacity of pancreatic cancer cells PK-59, in addition to the effect of suppressing the development of cancer cells. Thus, it was considered that the K5-70 antibody should inhibit cancer metastasis in vivo. Whereas, the metastasis inhibiting effect of an anti-hTROP-2 mouse monoclonal antibody was examined, using models in which pancreatic PK-59 cancer cells were injected into the spleen of nude mice in this way that liver metastasis was developed.
[0385] The endogenously expressed pancreatic cancer cell line (PK-59) hTROP-2 was collected by trypsin treatment and the 2 x 107 cells / ml cell suspension was then prepared with PBS. The cell suspension was preserved on ice until transplantation. Each of the 6- or 7-week-old female nude mice (Balb / c, nu / nu) was anesthetized by intraperitoneal administration of pentobarbital and 10 to 15 mm of its left flank was taxed under anesthesia. The spleen was removed in the abdominal cavity and 50 μL of the cell suspension (cells 1 x 106) was then injected into the spleen using a 26G syringe. Four minutes after cell injection, the spleen hilum was ligated with 5-0 silk sutures and the spleen was then taxed. The peritoneum cut was closed with 5-0 silk sutures and the surgical site was then closed with Wound Clips (AUTOCLIP 9 mm, Becton Dickinson). The day before the cancer cell transplant, the mice were divided into groups. An anti-hTROP-2 monoclonal antibody (K5-70) or a control antibody (purified mouse IgG) was intraperitoneally administered to the mice at a dosage of 10 mg / kg of body weight. In addition, seven days after the cancer cell transplantation, such an antibody was administered in the same manner as described above. Four to six weeks after the cancer cell transplant, the mice were euthanized by cervical dislocation. Then, the liver was taxed for each mouse and the presence or absence of the metastatic focus was confirmed.
[0386] In the control group in which the mouse IgG was administered to the mice, in 4 out of the 6 mice in which the PK-59 cells were transplanted, the apparent metastatic focus (2 to 7 foci) was observed around the lobe of the liver four to six weeks after transplantation (Figure 31A, incidence of metastasis: 67%, Table 6). In contrast, in four mice in the K5-70 administration group, in which PK-59 cells were also transplanted, such a metastatic focus was not seen in the liver in all of the mice and thus, an incidence of metastasis was 0% (Figure 31B, Table 6). Table 6 Effect of suppression of metastasis from clone K5-70 in liver metastasis models produced by transplenic transplantation of PK-59 cells in nude mice


[0387] From the results, it becomes clear that the anti-antibody hTROP-2 K5-70 has extremely strong inhibitory action on the metastasis of pancreatic cancer cells PK-59. [Example 29] [Antitumor activity of the K5-70 antibody in xenograft models using colon cancer cell line SW480, which are models of recurrent cancer after administration of irinotecan hydrochloride]
[0388] In recent years, many chemotherapeutic drugs for suppressing the development of cancer cells have been developed as therapeutic cancer drugs. These drugs have achieved certain treatment results. However, these chemotherapeutic drugs were problematic in terms of side effects associated with the suppression of development of this in normal cells other than cancer cells and the recurrence of cancer after the suspension of treatment. Consequently, if tumor recurrence after completion of treatment with chemotherapeutic drugs is to be suppressed by administering a therapeutic carcinogenic antibody targeting TROP-2, high clinical utility should be anticipated. Thus, this is a desired property as a therapeutic carcinogenic antibody.
[0389] As therapeutic agents for colon cancer, in addition to medicines containing 5-FU and platinum, irinotecan hydrochloride (Topotecin, Daiichi Sankyo Co., Ltd.) having a topoisomerase inhibiting effect has recently been applied in clinical sites. With respect to animal models as well, the anti-tumor effect of irinotecan hydrochloride in mouse models, in which various types of human tumor cells including cancer as a typical example has been transplanted, has been related (Cancer Chemother Pharmacol. (1991); 28 (3): 192-8). Thus, the effect prevents the recurrence of the anti-antibody clone hTROP-2 K5-70 (mouse IgG2a) in the recurrent tumor after the administration of irinotecan hydrochloride was examined with the xenograft models using a human colon cancer cell line. SW480. SW480 cells (5 x 106 cells) were subcutaneously transplanted on the right flank of 8-week-old female NOD-scid mice. Eleven days after transplantation (Day 11) in which the average tumor volume reached 100 mm3, the mice were divided into an untreated group (normal saline administration group, N = 8, 130.7 ± 16.2 mm3 ) and an irinotecan hydrochloride administration group (CPT-11, Topotecin, Daiichi Sankyo Co., Ltd.) (N = 16, 123.0 ± 21.4 mm3). Then, irinotecan hydrochloride was intraperitoneally administered to the mice at a dosage of 40 mg / kg of body weight, once every 3 days, 3 times total (Days 11, 14 and 17). On the third day after the final administration of irinotecan hydrochloride (Day 20), the tumor volume of the untreated group reached 232.1 ± 21.1 mm3. Otherwise, the tumor volume of the irinotecan hydrochloride administration group was 126.6 ± 26.6 mm3 (P <0.01 by Student's t-test) and thus, an effect that suppresses the apparent tumor was observed. At this stage, the irinotecan hydrochloride administration group was divided into two groups based on tumor size. One group was defined as a K5-70 administration group (10 mg / kg body weight) (N = 8, tumor volume on day 20: 126.0 ± 28.0 mm3) and the other group was defined as a administration group Mouse IgG (10 mg / kg body weight) (N = 8, tumor size on day 20: 127.2 ± 27.0 mm3). Intraperitoneal administration and tumor volume measurement were performed in each group once every 3 days, so that tumor recurrence was assessed (Figure 32). In the mouse IgG administration group, on the 18th day after the final administration of irinotecan hydrochloride (Day 35), several mice having an apparent recurrent tumor with a tumor volume greater than 300 mm3 were observed. On the 30th day after the final administration of irinotecan hydrochloride (Day 47), a tumor with a tumor volume greater than 300 mm3 was observed in 5 out of the 8 mice (mean tumor volume: 401.7 ± 172.7 mm3). In contrast, in the K5-70 administration group, tumor recurrence was significantly suppressed and the mean tumor volume was 180.5 ± 142.1 mm3 (P <0.05 by Student's t test) (Figure 32). In particular, in the K5-70 administration group, the tumor volume on day 47 becomes less than the tumor volume when the mice were divided into groups (126.0 ± 28.0 mm3). The tumor volume becomes less than 100 mm3 in 4 out of the 8 mice. From these results, it becomes clear that the anti-antibody hTROP-2 K5-70 has an extremely strong suppressive action even on the recurrent tumor after the administration of irinotecan hydrochloride. [Example 30] [Epitope mapping using CLIPS technology] <Materials and methods>
[0390] Peptide synthesis
[0391] 15-mer and 30-mer of linear peptides derived from TROP-2 extracellular domains, which were used in the present experiment, were obtained by solid phase synthesis according to an Fmoc method (9-Fluorenylmethoxycarbonyl). In addition, for discontinuous epitope analysis, a 17-mer peptide derived from the TROP-2 extracellular domain, at both ends of which cysteine residues were added, was synthesized and a conformation having one or two arc structures was reconstructed by the technology CLIPS (Chemically Linked Peptides on Scaffolds technology). When another of the cysteine residue was present next to the added cysteine residue, it was replaced with alanine.
[0392] ELISA epitope evaluation
[0393] 5034 types of synthesized peptides were covalently bound to PEPSCAN cards (455 peptides / cards) and the binding of the synthesized peptides to antibodies was then analyzed by the ELISA method. PEPSCAN cards were allowed to react with anti-human TROP-2 monoclonal antibodies (K5-70, K5-107, K5-116-2-1, T5-86 and T6-16) which were diluted to a concentration of 1 μ g / ml with a blocking buffer (a phosphate buffer containing 4% horse serum, 5% ovalbumin and 1% Tween). After washing, the resultant was allowed to react with a 1000-fold secondary antibody peroxidase complex diluted at 25 ° C for 1 hour. After washing, a substrate solution (a solution containing 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μL of 3% hydrogen peroxide solution) was added to the reaction solution, followed by a chromogenic reaction for 1 hour. Antibody binding activity was quantified by photography with a CCD camera and then performing an image analysis. <Results>
[0394] The anti-human TROP-2 monoclonal antibodies K5-70, K5-107, K5-116- 2-1, T5-86 and T6-16, which exhibited the beneficial effects, were subjected to epitope analysis using CLIPS (Chemically Linked Peptides on Scaffolds) technology. It is noted that the term "amino acid number" is used in the present examples to mean the number of amino acids in the amino acid sequence shown in SEQ ID NO: 2 (hTROP-2 protein (323 amino acid residues)).
[0395] The result of the analysis by the K5-70 antibody is shown in Table 7 below. As a result, it was observed that 33 peptides exhibit strong binding activity to the K5-70 antibody. In these 33 peptides, a sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65) (peptides No. 1-7 and 9 in Table 7), a sequence comprising HHILIDLRHRPTAG (amino acid numbers 152-165) (peptide No. 152-165). 14, 22-25, 28, 29, 31 and 33 in Table 7), a sequence comprising VHYEQPTIQIELRQ (amino acid numbers 193-206) (peptide No. 8, 10, 12, 13, 15, 18, 20, 21, 23, 26, 28, 30 and 32 in Table 7) and the sequence comprising DAELRRLFRER (amino acid numbers 171-183) (peptide No. 11, 12, 14, 16, 18, 19, 21, 22, 25, 29 and 31 in Table 7) repeatedly appears. The K5-70 antibody particularly strongly binds to the sequence comprising VCSPDGPGGRCQCRALGSGMAVD. From these results, it has been suggested that, in the hTROP-2 protein, the 4 types mentioned above of the regions of the peptide sequence are also epitopes of the K5-70 antibody. Table 7 CLIPS peptides derived from human TROP-2 extracellular domains

[0396] The test result for the K5-107 antibody is shown in Table 8 below. As a result, it was observed that the sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65) was comprised of 10 out of 20 peptides (peptides No. 1-6, 8, 13,14 and 17 in Table 8) (Table 8).
[0397] Consequently, it has been suggested that, in the hTROP-2 protein, the previously mentioned peptide sequence region consisting of VCSPDGPGGRCQCRALGSGMAVD may be an epitope of the K5-107 antibody. Table 8 Binding of K5-107 antibody to CLIPS peptides derived from human TROP-2 extracellular domains

[0398] The result of the analysis by the K5-116-2-1 antibody is shown in Table 9 below. In this analysis, three types of peptide sequences, named, a sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65) (peptide No. 1-7, 15 and 25 in Table 9), a sequence comprising HHILIDLRHRPTAG (numbers of amino acids 152-165) (peptides No. 8-11, 16, 17, 19, 20, 22-24 and 27-29 in table 9) and a sequence comprising DLDAELRRLFRER (amino acid numbers 171-183) (N peptides 11-14, 17, 19, 21, 23 and 29 in table 9) appears several times (Table 9). Consequently, it has been suggested that, in the hTROP-2 protein, these three types of peptide sequence regions may be epitopes of the K5-116-2-1 antibody. Table 9

[0399] The results of the analysis by antibodies T5-86 and T6-16 are shown in Table 10 and Table 11 below, respectively. In these analyzes, the antibodies strongly bind to a peptide that comprises a sequence consisting of DPEGRFKARQCN (amino acid numbers 109-120). The peptide sequence mentioned above comprises linking 22 out of 26 peptides to the T5-86 antibody (peptides No. 1-3, 5-8, 10-13, 15-19 and 21-26 in table 10) and was comprised of the binding of 4 out of the 26 peptides to the T6-16 antibody (peptides No. 1, 2, 9 and 13 in table 11) (Table 10 and Table 11). In addition, in the analysis with respect to the T5-86 antibody, other than the sequence comprising DPEGRFKARQCN (amino acid numbers 109-120), a sequence comprising VCSPDGPGGRCQCR (amino acid numbers 43-56) (peptides No. 4, 14 and 20 in table 10) appears several times. In addition, in the analysis with respect to the T6-16 antibody as well, another sequence comprising HHILIDLRHRPTAG (amino acid numbers 152-165) (peptides No. 4-8, 10-12, 19, 21, 23, 25 and 26 in table 11) was observed several times. Consequently, it has been suggested that in the hTROP-2 protein, two types of peptide sequence regions, called, DPEGRFKARQCN (amino acid numbers 109-120) and VCSPDGPGGRCQCR (amino acid numbers 43-56), may be epitopes of the K5- antibody. 86. It has also been suggested that, in the hTROP-2 protein, two types of the peptide sequence regions, called, DPEGRFKARQCN (amino acid numbers 109-120) and HHILIDLRHRPTAG (amino acid numbers 152-165), may be T6-16 antibody epitopes . Table 10
Table 11 Binding of T6-16 antibody to CLIPS peptides derived from human TROP-2 extracellular domains
[Example 31] [Sequencing of the variable regions of the mouse anti-human TROP-2 antibody antibody genes (clones K5-70, K5-107, K5-116-2-1 and T6-16)]
[0400] Total RNA was extracted from 3 x 106 hybridomas that produces the mouse anti-TROP-2 monoclonal antibody, using TRIzol reagent (Invitrogen). With respect to clone K5-70, clone K5-107 and clone K5-116-2-1, cDNA was synthesized using the SMARTerTM RACE cDNA amplification kit (Clontech) according to the method included with the kit, using a primer mouse IgG H chain specific (5'-TCCAKAGTT-3 '(SEQ ID NO: 24)) and a mouse IgG L chain specific primer (5'-GCTGTCCTGATC-3' (SEQ ID NO: 25)). Regarding clone T6-16, cDNA was synthesized using the GeneRacer kit (Invitrogen) according to the method included with the kit, using an oligo dT primer. The genes encoding the variable regions (VH, VL) of the H and L chains of clone K5-70 (mouse IgG2a), clone K5-107 (mouse IgG1) and clone K5-116-2-1 (mouse IgG2a) ) were each cloned by a PCR method using the cDNA synthesized above as a template. In this operation, 10 x Universal Primer A Mix (UPM) included with the SMARTerTM RACE cDNA amplification kit was used as a 5 'primer. Otherwise, as a 3 'primer by VH amplification, a primer having a specific sequence for the mouse IgG H chain was used and as a 3' primer for VL amplification, a primer having a specific sequence for the L chain of H Mouse IgG was used.
[0401] 5'-initiator (10 x Universal Primer A Mix (UPM)):
[0402] Long (0.4 μM)
[0403] 5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT- 3 ’(SEQ ID NO: 26)
[0404] Short (2 μ M)
[0405] 5’-CTAATACGACTCACTATAGGGC-3 ’(SEQ ID NO: 27)
[0406] 3'-primer (primer R):
[0407] VH: 5’-GGGAARTARCCCTTGACCAGGCA-3 ’(SEQ ID NO: 28)
[0408] 1. 5’-GGGAARTAGCCTTTGACAAGGCA-3 ’(SEQ ID NO: 29)
[0409] VL: 5’-CACTGCCATCAATVTTCCACTTGACA-3 ’(SEQ ID NO: 30)
[0410] Using each of the initiators described above, PCR was performed under the following composition of the reaction solution and reaction conditions. In addition, an R primer for the amplified VH cDNA was prepared by mixing two sequences above with each other in an equimolar ratio and was then used. <Composition of the reaction solution>
[0411] cDNA model: 2.5 μL
[0412] Buffer 5 x PrimeSTAR (Mg2 + plus): 10 μL
[0413] 2.5 mM dNTP: 4 μL
[0414] PrimeSTAR HS DNA polymerase (2.5 U / μ L): 0.5 μL
[0415] 10 x Universal Primer A Mix (UPM):
[0416] Initiator R (10 μ M):
[0417] Sterile water:
[0418] Total: <Reaction conditions>
[0419] A reaction was carried out at 94 ° C (10 seconds) and then a cycle consisting of “heat denaturation / dissociation at 98 ° C (10 seconds) ^ annealing at 60 ° C (5 seconds) ^ synthesis / stretching at 72 ° C (60 seconds) ”was performed 30 times in total. Finally, the reaction was carried out at 72 ° C (3 minutes).
[0420] The synthesized VH and VL cDNAs were subcloned into a pMD20-T vector (Takara Bio Inc.) and their nucleotide sequences were determined. The nucleotide sequences of a plurality of the decoded VH and VL clones and the nucleotide sequences specific to the mouse H and L chain variable regions were identified. Figure 33 and Figure 34 show the VH and VL K5-70 consensus cDNA nucleotide sequences and putative amino acid sequences. Figure 35 and Figure 36 show the VH and VL K5-107 consensus cDNA nucleotide sequences and putative amino acid sequences. Figure 37 and Figure 38 shows the consensual VH and VL cDNA nucleotide sequences of K5-116-2-1 and putative amino acid sequences.
[0421] The genes encoding the variable regions (VH, VL) of the H and L chains of the T6-16 clone were cloned by a PCR method using the cDNA synthesized above as a model. In this operation, a primer included with the GeneRacer kit was used as a 5 'primer. Otherwise, as a 3 'primer by VH amplification, a primer having a mouse IgG H chain specific sequence was used and as a 3' primer for VL amplification, a primer having a specific IgG L chain sequence of mouse was used.
[0422] 5'-primer (primer F):
[0423] 5’-CGACTGGAGCACGAGGACACTGA-3 ’(SEQ ID NO: 31)
[0424] 3'-primer (primer R):
[0425] VH: 5’- GCCAGTGGATAGACAGATGG-3 ’(SEQ ID NO: 32)
[0426] VL: 5’- GATGGATACAGTTGGTGCAGC-3 ’(SEQ ID NO: 33)
[0427] Using each of the initiators described above, PCR was performed under the following composition of the reaction solution and reaction conditions. <Composition of the reaction solution>
[0428] cDNA model: 1.0 μL
[0429] Buffer 5 x PrimeSTAR (Mg2 + plus): 10 μL
[0430] 2.5 mM dNTP: 4 μL
[0431] PrimeSTAR HS DNA polymerase (2.5 U / μ L): 0.5 μL
[0432] Initiator F (10 μ M): 3 μL
[0433] Initiator R (10 μ M): 1.0 μL
[0434] Sterile water: 30.5 μL
[0435] Total: 50 μ L <Reaction conditions>
[0436] A cycle consisting of “heat denaturation / dissociation at 98 ° C (10 seconds) ^ annealing at 57 ° C (10 seconds) ^ synthesis / stretching at 72 ° C (60 seconds)” was performed 35 times in the total.
[0437] The synthesized VH and VL cDNAs were subcloned into a pCR4Blunt-TOPO vector (Invitrogen) and their nucleotide sequences were determined. The nucleotide sequences of a plurality of VH clones and VL clones were decoded and the nucleotide sequences specific to the mouse H chain and L chain variable regions were identified. Figure 39 and Figure 40 show the VH and VL T6-16 consensus cDNA nucleotide sequences and putative amino acid sequences.
[0438] Industrial Applicability
[0439] The present invention is capable of providing an antibody, which specifically reacts with hTROP-2 and has high anti-tumor activity in vivo and particularly, a monoclonal antibody having high anti-tumor activity in vivo at a low dosage. In addition, the present invention is capable of providing a hybridoma, which produces the antibody, a fragment of the antibody, an antibody complex or other and various types of drugs, a pharmaceutical composition for diagnosing or treating a tumor, a method for detecting a tumor and a kit to detect or diagnose a tumor. [Free text from the Sequence Listing]
[0440] SEQ ID NO: 3 Synthetic DNA
[0441] SEQ ID NO: 4 Synthetic DNA
[0442] SEQ ID NO: 5 Synthetic DNA
[0443] SEQ ID NO: 6 Synthetic DNA
[0444] SEQ ID NO: 7 Synthetic DNA
[0445] SEQ ID NO: 8 Synthetic DNA
[0446] SEQ ID NO: 9 Synthetic DNA
[0447] SEQ ID NO: 10 Synthetic DNA
[0448] SEQ ID NO: 11 Synthetic DNA
[0449] SEQ ID NO: 12 Synthetic DNA
[0450] SEQ ID NO: 13 Synthetic DNA
[0451] SEQ ID NO: 14 Synthetic DNA
[0452] SEQ ID NO: 15 Synthetic DNA
[0453] SEQ ID NO: 16 Synthetic DNA
[0454] SEQ ID NO: 17 Synthetic DNA
[0455] SEQ ID NO: 18 Synthetic DNA
[0456] SEQ ID NO: 19 Synthetic DNA
[0457] SEQ ID NO: 20 Synthetic DNA
[0458] SEQ ID NO: 21 Synthetic DNA
[0459] SEQ ID NO: 22 Synthetic DNA
[0460] SEQ ID NO: 23 Synthetic DNA
[0461] SEQ ID NO: 24 Synthetic DNA
[0462] SEQ ID NO: 25 Synthetic DNA
[0463] SEQ ID NO: 26 Synthetic DNA
[0464] SEQ ID NO: 27 Synthetic DNA
[0465] SEQ ID NO: 28 Synthetic DNA
[0466] SEQ ID NO: 29 Synthetic DNA
[0467] SEQ ID NO: 30 Synthetic DNA
[0468] SEQ ID NO: 31 Synthetic DNA
[0469] SEQ ID NO: 32 Synthetic DNA
[0470] SEQ ID NO: 33 Synthetic DNA

权利要求:
Claims (13)
[0001]
1. Antibody against human TROP-2, characterized by the fact that it has antitumor activity in vivo, comprising: (a) the amino acid sequences of CDRs 1 to 3 of the heavy chain variable region defined by SEQ ID NO: 36 to 38 , respectively and the amino acid sequences of CDRs 1 to 3 of the light chain variable region defined by SEQ ID NO: 41 to 43, respectively; optionally wherein the antibody comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID NO: 35 and the antibodies comprise the amino acid sequences of the light chain variable region consisting of SEQ ID No. 40; or (b) the amino acid sequences of CDRs 1 to 3 of the variable region of heavy chain defined by SEQ ID NO: 46 to 48, respectively, and the amino acid sequences of CDRs 1 to 3 of the variable region of light chain defined by SEQ ID NO: 51 to 53, respectively; optionally wherein the antibody comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID NO: 45 and the antibodies comprise the amino acid sequence of the light chain variable region consisting of SEQ ID NO: 50; or (c) the amino acid sequence of CDRs 1 to 3 of the heavy chain variable region defined by SEQ ID NO: 56 to 58, respectively, and the amino acid sequence of CDRs 1 to 3 of the light chain variable region defined by SEQ ID NO: 61 to 63, respectively; optionally wherein the antibody comprises the heavy chain variable region amino acid sequence consisting of SEQ ID NO: 55 and the antibodies comprise the light chain variable region amino acid sequences consisting of SEQ ID No. 60; or (d) the amino acid sequences of CDRs 1 to 3 of the heavy chain variable region defined by SEQ ID NO: 66 to 68, respectively, and the amino acid sequence of CDRs 1 to 3 of the light chain variable region defined by SEQ ID NO: 71 to 73, respectively; optionally wherein the antibody comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID NO: 65 and the antibodies comprise the amino acid sequences of the light chain variable region consisting of SEQ ID No. 70.
[0002]
2. Antibody according to claim 1, characterized by the fact that (a) the antibody has antitumor activity in two or more types of human tumor cell lines, optionally in which the tumor cell lines are at least two types selected from the group consists of a human pancreatic cancer cell line PK-59, a human pancreatic cancer cell line BxPC-3, a human pancreatic cancer cell line KP-3L, a human pancreatic cancer cell line KP-2, a PK-1 human pancreatic cancer cell line, a PK-45H human pancreatic cancer cell line, a PK-45P pancreatic cancer human cell line, a TCC-PAN2 human pancreatic cancer cell line, a lineage pancreatic cancer human cell line SUIT-2, a CACO-2 human colon cancer cell line, a SW480 colon cancer human cell line, a DLD-1 human colon cancer cell line, a human colon cancer cell line HCT 116, a human breast cancer cell line JIMT-1, a human breast cancer cell line HCC1143, a human breast cancer cell line MCF-7, a DU145 human prostate cancer cell line and PC-3 human prostate cancer cell line, and preferably the tumor cell lines are the PK-59 human pancreatic cancer cell line and the human cancer cell line pancreatic BxPC-3 .; and / or (b) the dissociation constant (Kd value) is 1.0 x 10-10 M or less; and / or (c) the antibody is a monoclonal antibody; and / or (d) the tumor is at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer; and preferably it is human pancreatic cancer; and / or (e) the tumor is a recurrent cancer or a metastatic cancer.
[0003]
Antibody according to any one of claims 1 to 2, characterized in that the antibody is a genetically recombinant antibody, which is preferably a chimeric antibody, a humanized antibody or a human antibody.
[0004]
Antibody according to any one of claims 1 to 3, characterized by the fact that the antibody binds: (a) to a site to which the monoclonal antibody produced by the hybridoma having accession number FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346 or FERM BP-11254 binds; or (b) a portion comprising at least one region selected from the group consists of a region consisting of amino acids at positions 43 through 65, a region consisting of amino acids at positions 152 through 165, a region consisting of amino acids at positions 171 to 183, a region consisting of amino acids at positions 109 to 120, a region consisting of amino acids at positions 43 to 56, and a region consisting of amino acids at positions 193 to 206, in the human TROP-2 amino acid sequence shown SEQ ID NO: 2; or (c) a portion comprising a region consisting of the amino acids at positions 43 to 65 in the human TROP-2 amino acid sequence shown in SEQ ID NO: 2; or (d) a portion comprising a region consisting of amino acids at positions 152 to 165 in the human TROP-2 amino acid sequence shown in SEQ ID NO: 2; or (e) a portion comprising a region consisting of amino acids at positions 171 to 183 in the human TROP-2 amino acid sequence shown in SEQ ID NO: 2; or (f) a portion comprising a region consisting of the amino acids at positions 109 to 120 in the human TROP-2 amino acid sequence shown in SEQ ID NO: 2.
[0005]
5. Monoclonal antibody against human TROP-2, characterized by the fact that it is produced by a hybridoma having access number FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346, or FERM BP-11254 .
[0006]
An antibody-derived antibody fragment as defined in any one of claims 1 to 5, characterized in that it comprises, (a) the amino acid sequences of the heavy chain variable region of CDRs 1 to 3 defined by SEQ ID NO: : 36 to 38 respectively and the amino acid sequences of the light decadeia variable region of CDRs 1 to 3 defined by SEQ ID NO: 41 to 43 respectively; optionally wherein the antibody fragment comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID NO: 35, and the amino acid sequences of the light chain variable region consisting of SEQ ID NO: 40; or (b) the amino acid sequences of the heavy chain variable region CDRs 1 to 3 defined by SEQ ID NO: 46 to 48, respectively and the amino acid sequences of the light chain variable region of CDRs 1 to 3 defined by SEQ ID No. 51 to 53 respectively, optionally wherein the antibody fragment comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID No.: 45 and the amino acid sequences of the light chain variable region consisting of SEQ ID No. : 50; or (c) the amino acid sequences of the heavy chain variable region of CDRs 1 to 3 defined by SEQ ID NO: 56 to 58, respectively and the amino acid sequences of the light chain variable region of CDRs 1 to 3 defined by SEQ. ID NO: 61 to 63, respectively, optionally wherein the antibody fragment comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID NO: 55, and the amino acid sequences of the light chain variable region consisting of the SEQ ID NO: 60; or (d) the amino acid sequences of the heavy chain variable region of CDRs 1 to 3 defined by SEQ ID NO: 66 to 68, respectively and the amino acid sequences of the light chain variable region of CDRs 1 to 3 defined by SEQ. ID NO: 71 to 73, respectively, optionally wherein the antibody fragment comprises the amino acid sequences of the heavy chain variable region consisting of SEQ ID NO: 65 and the amino acid sequences of the light chain variable region consisting of SEQ ID NO: 70.
[0007]
7. Hybridoma, characterized by the fact that it produces an antibody as defined in any one of claims 1 to 4, or that it has accession number FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346, or FERM BP-11254.
[0008]
8. Conjugate, characterized by the fact that it comprises an antibody as defined in any one of claims 1 to 4 or an antibody fragment as defined in claim 6, and a substance having antitumor activity and / or cell death activity, wherein, optionally: (a) the tumor is at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer; or (b) the tumor is human pancreatic cancer; and / or (c) the tumor is a recurrent cancer or a metastatic cancer.
[0009]
9. Pharmaceutical composition, characterized by the fact that it comprises at least one selected from the group consisting of an antibody as defined in any one of claims 1 to 4, an antibody fragment as defined in claim 6 and a conjugate as defined in claim 8 and a pharmacologically acceptable carrier.
[0010]
10. Use of pharmaceutical composition as defined in claim 9, characterized by the fact that it is in: a method of diagnosing a tumor.
[0011]
11. Use of a composition according to claim 10, characterized by the fact that: (a) the tumor is at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and breast cancer human; and / or (b) the tumor is a recurrent cancer or a metastatic cancer.
[0012]
12. Method for detecting a tumor, characterized in that it comprises: - allowing at least one selected from the group consisting of an antibody as defined in any one of claims 1 to 4, an antibody fragment as defined in claim 6, and a conjugate as defined in claim 8, reacting with a sample collected from a living body; and - then detecting a signal from the reacted antibody and / or antibody fragment; optionally in which the tumor is at least one type selected from the group consists of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer.
[0013]
13. Kit for treating, diagnosing or detecting a tumor, characterized in that it comprises at least one selected from the group consisting of an antibody as defined in any one of claims 1 to 4, an antibody fragment as defined in claim 6 and a conjugate as defined in claim 8; optionally, where the tumor is at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colon cancer and human breast cancer.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

IL162181A|1988-12-28|2006-04-10|Pdl Biopharma Inc|A method of producing humanized immunoglubulin, and polynucleotides encoding the same|

---

DE69632333T2|1995-10-19|2004-12-30|Bristol-Myers Squibb Co.|MONOCLONAL ANTIBODY BR110 AND ITS APPLICATION|

---

US6653104B2|1996-10-17|2003-11-25|Immunomedics, Inc.|Immunotoxins, comprising an internalizing antibody, directed against malignant and normal cells|

---

EP3483183B1|2002-03-01|2021-04-21|Immunomedics, Inc.|Immunoconjugate comprising humanised rs7 antibodies|

---

ES2345493T3|2003-11-28|2010-09-24|Kanagawa Academy Of Science And Technology|METHOD OF DETECTION OF CANCER OF LIVER, DIAGNOSIS OF CANCER OF LIVER AND REMEDY FOR CANCER.|

---

US20080131428A1|2006-02-24|2008-06-05|Arius Research, Inc.|Cytotoxicity mediation of cells evidencing surface expression of TROP-2|

---

US7420041B2|2006-02-24|2008-09-02|Arius Research Inc.|Cytotoxicity mediation of cells evidencing surface expression of TROP-2|

---

CA2750038C|2009-02-05|2016-10-04|Oncoxx S.R.L.|Anti-trop-2 monoclonal antibodies and uses thereof in the treatment and diagnosis of tumors|

---

EA031043B1|2010-05-17|2018-11-30|Ливтех, Инк.|Anti-human trop-2 antibody having antitumor activity in vivo|

---

JP2015095020A|2013-11-11|2015-05-18|株式会社デンソーアイティーラボラトリ|Image-added two-dimensional code generation device, image-added two-dimensional code generation method, and image-added two-dimensional code generation program|US9770517B2|2002-03-01|2017-09-26|Immunomedics, Inc.|Anti-Trop-2 antibody-drug conjugates and uses thereof|

---

US8435529B2|2002-06-14|2013-05-07|Immunomedics, Inc.|Combining radioimmunotherapy and antibody-drug conjugates for improved cancer therapy|

---

EP3483183B1|2002-03-01|2021-04-21|Immunomedics, Inc.|Immunoconjugate comprising humanised rs7 antibodies|

---

US9745380B2|2002-03-01|2017-08-29|Immunomedics, Inc.|RS7 antibodies|

---

JP4959136B2|2002-12-13|2012-06-20|イミューノメディクス、インコーポレイテッド|Immunoconjugates with cleavable linkages in cells|

---

EA031043B1|2010-05-17|2018-11-30|Ливтех, Инк.|Anti-human trop-2 antibody having antitumor activity in vivo|

---

US9427464B2|2011-11-22|2016-08-30|Chiome Bioscience Inc.|Anti-human TROP-2 antibody having an antitumor activity in vivo|

---

ITCH20120008A1|2012-05-16|2013-11-17|Saverio Alberti|USE OF TROP-2 AS A PREDICTIVE MARKER FOR ANSWERING ANTI-TUMOR THERAPY BASED ON INHIBITORS OF CD9, AKT AND RELATED SIGNAL PATHWAYS|

---

CN102827282B|2012-09-04|2015-02-11|林红|Human resistant Trop-2 genetically engineered antibody immunoglobulin Gand application thereof|

---

US11253606B2|2013-07-23|2022-02-22|Immunomedics, Inc.|Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer|

---

US10137196B2|2012-12-13|2018-11-27|Immunomedics, Inc.|Dosages of immunoconjugates of antibodies and SN-38 for improved efficacy and decreased toxicity|

---

US9931417B2|2012-12-13|2018-04-03|Immunomedics, Inc.|Antibody-SN-38 immunoconjugates with a CL2A linker|

---

WO2014092804A1|2012-12-13|2014-06-19|Immunomedics, Inc.|Dosages of immunoconjugates of antibodies and sn-38 for improved efficacy and decreased toxicity|

---

US10206918B2|2012-12-13|2019-02-19|Immunomedics, Inc.|Efficacy of anti-HLA-DR antiboddy drug conjugate IMMU-140in HLA-DR positive cancers|

---

US9492566B2|2012-12-13|2016-11-15|Immunomedics, Inc.|Antibody-drug conjugates and uses thereof|

---

US9707302B2|2013-07-23|2017-07-18|Immunomedics, Inc.|Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer|

---

US10195175B2|2015-06-25|2019-02-05|Immunomedics, Inc.|Synergistic effect of anti-Trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or PARP inhibitors|

---

US10058621B2|2015-06-25|2018-08-28|Immunomedics, Inc.|Combination therapy with anti-HLA-DR antibodies and kinase inhibitors in hematopoietic cancers|

---

EP3044323A4|2013-09-13|2017-07-05|F.Hoffmann-La Roche Ag|Compositions and methods for detecting and quantifying host cell protein in cell lines and recombinant polypeptide products|

---

CA2921999A1|2013-09-13|2015-03-19|Genentech, Inc.|Methods and compositions comprising purified recombinant polypeptides|

---

SG10201902571VA|2013-12-25|2019-04-29|Daiichi Sankyo Co Ltd|Anti-trop2 antibody-drug conjugate|

---

MX2016010683A|2014-02-21|2017-05-11|Ibc Pharmaceuticals Inc|Disease therapy by inducing immune response to trop-2 expressing cells.|

---

JP2018500930A|2014-12-04|2018-01-18|アブルッツォ・セラノスティックス・エッセ・エッレ・エッレ|Humanized anti-TROP-2 monoclonal antibody and use thereof|

---

JP6746845B2|2015-04-22|2020-08-26|イミューノメディクス、インコーポレイテッドＩｍｍｕｎｏｍｅｄｉｃｓ， Ｉｎｃ．|Isolation, detection, diagnosis and/or characterization of circulating TROP-2 positive cancer cells|

---

US11173213B2|2015-06-29|2021-11-16|Daiichi Sankyo Company, Limited|Method for selectively manufacturing antibody-drug conjugate|

---

EP3413914A4|2016-02-10|2019-10-16|Immunomedics, Inc.|Combination of abcg2 inhibitors with sacituzumab govitecanovercomes resistance to sn-38 in trop-2 expressing cancers|

---

MX2019008059A|2017-01-17|2019-12-11|Daiichi Sankyo Co Ltd|Anti-gpr20 antibody and anti-gpr20 antibody-drug conjugate.|

---

EP3600283A4|2017-03-27|2020-12-16|Immunomedics, Inc.|Treatment of trop-2 expressing triple negative breast cancer with sacituzumab govitecan and a rad51 inhibitor|

---

EP3606964A4|2017-04-03|2020-12-09|Immunomedics, Inc.|Subcutaneous administration of antibody-drug conjugates for cancer therapy|

---

TW201900683A|2017-05-15|2019-01-01|日商第一三共股份有限公司|anti-CDH6 antibody and anti-CDH6 antibody-drug conjugate|

---

CN108680750A|2017-12-29|2018-10-19|广西壮族自治区人民医院|The ELISA detection method and kit of TROP2 expressing quantities|

---

EP3842546A1|2018-08-23|2021-06-30|Daiichi Sankyo Company, Limited|Sensitivity marker for antibody-drug conjugate|

---

AU2020205100A1|2019-01-04|2021-08-19|Trio Pharmaceuticals, Inc.|Multi-specific protein molecules and uses thereof|

---

AU2020285681A1|2019-05-29|2022-01-27|Daiichi Sankyo Company, Limited|Dosage of an antibody-drug conjugate|

---

WO2021188896A1|2020-03-20|2021-09-23|Immunomedics, Inc.|Biomarkers for sacituzumab govitecan therapy|

---

WO2021226440A1|2020-05-08|2021-11-11|Bolt Biotherapeutics, Inc.|Elastase-substrate, peptide linker immunoconjugates, and uses thereof|

法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-16| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-10| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-08-18| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-10-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/05/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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JP2010113302|2010-05-17|

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JP2010-113302|2010-05-17|

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PCT/JP2011/061709|WO2011145744A1|2010-05-17|2011-05-17|Anti-human trop-2 antibody having antitumor activity in vivo|

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