WO2008105560A1 - 抗grp78抗体を有効成分として含む医薬組成物 - Google Patents
抗grp78抗体を有効成分として含む医薬組成物 Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
Definitions
- composition comprising anti-GRP78 antibody as active ingredient
- the present invention relates to a method for treating cancer and an anticancer agent.
- GRP protein (glucose-regulated proteins) is a molecular chaperone localized in ER.
- a protein family whose expression is induced in response to various ER stresses that cells receive from inside and outside, such as when glucose is deficient or when proteins with abnormal conformations accumulate in the endoplasmic reticulum (ER) (Non-Patent Document 1).
- GRP78 is a GRP protein having a molecular weight of 78 kDa, and is also known as BiP (immunoglobulin binding protein).
- BiP immunoglobulin binding protein
- GRP78 overexpression experiments or antisense knockdown experiments have shown that GRP78 is involved in its defense response against cell death caused by ER stress (Non-patent Document 1). Cancerous cells or in vivo environments that have changed due to canceration are considered to be constantly exposed to ER stress, such as hyponutrition, hypoxia, and low pH. In order to support this, increased expression of GRP78 protein has been confirmed in several cancer cell lines and clinical cancer samples (Non-patent Documents 2 to 5).
- Non-Patent Documents 6 and 7 show that breast cancer patient groups in which GRP78 expression is enhanced are less effective in chemotherapy with adriamycin than groups in which GRP78 expression is low.
- Non-patent Document 1 discloses that enhanced expression of GRP78 plays a role in the mechanisms of cancer cell survival, malignancy, and anticancer drug resistance.
- GRP78 is known to be a molecular chaperone localized in ER.
- GRP78 protein is localized on the cell surface in some cancer cells.
- antitumor agents targeting GRP78 whose localization has been changed on the cell surface has been shown in several completely different ways.
- Tg thaps igargin
- this report shows a transient phenomenon at the time of cell death induction by Tg treatment, and is not data showing a permanent localization of GRP78 in cancer cells.
- the antibody used is a commercially available goat-derived polyclonal antibody, its epitope is also unknown.
- Non-patent Document 11 a peptide was synthesized by adding an apoptosis-inducing peptide sequence (KLAKLAK) 2 (Non-patent Document 11) to this GRP78-binding peptide.
- KLAKLAK apoptosis-inducing peptide sequence
- This peptide not only induces cell death in vitro against DU145 cells, but also mice It has been reported that it exhibits an antitumor effect in an in vivo test using a transplant model (Non-patent Document 10).
- K5 Kringle 5
- GRP78 GRP78 protein expressed in
- Non-Patent Document 1 Lee AS. Trends Biochem Sci. 2001, 26, 504-10
- Non-Patent Document 2 Pat ierno, et al. 1998, Cancer Res. 47, 6220-24
- Non-Patent Document 3 Bini et al. 1997, Elec trophores is. 18, 2832-41
- Non-Patent Literature 4 Gaz it et al. 1999, Breast Cancer Res. Treat. 54, 135-46 Non-Patent Literature 5 Fernandez et al. 2000, Breast Cancer Res. Treat. 59, 15-26 Non-Patent Literature 6 Reddy et al, J. Bio. Chem. 2003, 278, 20915-24
- Non-Patent Document 7 Dong et al, 2005, Cancer Res. 65, 5785-5791
- Non-Patent Document 8 Lee et al. 2006, Cancer Res. 66, 7849-7853
- Non-Patent Document 9 De lpi no et al. 1998, Mol ecul ar Membrane Biology, 15, 21-26 Non-Patent Document 1 0 Arap et al. 2004, CANCER CELL. 6, 275-284
- Non-Patent Literature 1 Javadpour et al. 1996, J. Med. Chem. 39, 3107-3113
- Non-Patent Document 1 3 Mi sra et al. 2002, J. Biol. Chem. 277, 42082-42087
- Non Patent Literature 1 4 Mintz et al. 2003, Nat Biotech. 21, 57-63
- Non-patent literature 1 5 Gonzalez-Gronow et al. 2006, Cancer Res. 66, 11424-11431
- Non-patent literature 1 6 Davi doson et al., 2005, Cancer Res. 65, 4663-4672
- An object of the present invention is to provide a novel pharmaceutical composition using an anti-GRP78 antibody. More specifically, the present invention aims to provide a novel method for treating cancer using an anti-GRP78 antibody, a novel cytostatic and anticancer agent containing an anti-GRP78 antibody, and a novel anti-GRP78 antibody.
- the present inventor has attempted to produce an antitumor antibody targeting GRP78 whose localization in cancer cells has changed to the cell membrane. To that end, it was first necessary to identify amino acids that could be antibody epitopes exposed on the cell surface in cancer cells. Therefore, we purified the GRP78 protein, immunized it with mice, and selected only antibodies that stained cancer cells. As a result, an anti-GRP78 antibody that specifically binds to the cell surface of cancer cells was successfully obtained. Next, an attempt was made to identify the sequence recognized by the obtained antibody. This analysis revealed that this antibody specifically recognizes 40 amino acids of GRP78 376-415. That is, it was revealed that the 376-415 amino acid region of GRP78 was exposed outside the cell.
- a pharmaceutical composition comprising an antibody that binds to glucose-regulated protein 78 (GRP78).
- composition according to (1) which is an anticancer agent.
- composition according to (1) or (2), wherein the antibody is a monoclonal antibody.
- CDR1 has the amino acid sequence described in SEQ ID NO: 8
- CDR2 has the amino acid sequence described in SEQ ID NO: 9
- CDR3 has the amino acid sequence described in SEQ ID NO: 10.
- CDR1 has the amino acid sequence described in SEQ ID NO: 18
- CDR2 has the amino acid sequence described in SEQ ID NO: 19
- CDR3 has the amino acid sequence described in SEQ ID NO: 20. It has the heavy chain variable region, the amino acid sequence described in SEQ ID NO: 21 as CDR1, the amino acid sequence described in SEQ ID NO: 22 as CDR2, and the amino acid sequence described in SEQ ID NO: 23 as CDR3
- An antibody having a light chain variable region having:
- CDR1 has the amino acid sequence described in SEQ ID N0: 61
- CDR2 has the amino acid sequence described in SEQ ID NO: 62
- CDR3 has the amino acid sequence described in SEQ ID NO: 63 It has the heavy chain variable region, the amino acid sequence described in SEQ ID NO: 64 as CDR1, the amino acid sequence described in SEQ ID N0: 65 as CDR2, and the amino acid sequence described in SEQ ID NO: 66 as CDR3.
- CDRl has the amino acid sequence described in SEQ ID NO: 71
- CDR2 has the amino acid sequence described in SEQ ID NO: 72
- CDR3 has the amino acid sequence described in SEQ ID NO: 73 It has the heavy chain variable region, the amino acid sequence described in SEQ ID NO: 74 as CDR1, the amino acid sequence described in SEQ ID NO: 75 as CDR2, and the amino acid sequence described in SEQ ID NO: 76 as CDR3
- An antibody having a light chain variable region having:
- CDRl has the amino acid sequence described in SEQ ID NO: 81
- CDR2 has the amino acid sequence described in SEQ ID NO: 82
- CDR3 has the amino acid sequence described in SEQ ID NO: 83 It has the heavy chain variable region, the amino acid sequence described in SEQ ID NO: 84 as CDR1, the amino acid sequence described in SEQ ID NO: 85 as CDR2, and the amino acid sequence described in SEQ ID NO: 86 as CDR3.
- An antibody having a light chain variable region having:
- CDRl has the amino acid sequence described in SEQ ID NO: 91
- CDR2 has the amino acid sequence described in SEQ ID NO: 92
- CDR3 has the amino acid sequence described in SEQ ID NO: 93
- It has the heavy chain variable region, the amino acid sequence described in SEQ ID NO: 94 as CDR1, the amino acid sequence described in SEQ ID NO: 95 as CDR2, and the amino acid sequence described in SEQ ID NO: 96 as CDR3.
- An antibody having a light chain variable region having: The antibody according to any one of (8) to (10), which recognizes the same epitope as that recognized by.
- An anti-GRP78 antibody conjugated with a labeling substance (27) An anti-GRP78 antibody conjugated with a labeling substance. (28) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or a fragment thereof.
- the present invention is used for treating various tumors and cancers in which GRP78 is exposed on the cell surface by providing a novel antibody having binding activity to GRP78 and capable of being internalized. It was shown that a novel pharmaceutical composition that can be provided can be provided. In addition, by using an antibody having such characteristics, it is possible to provide diagnostic methods for various tumors and cancers.
- FIG. 1 is an analysis of the binding activity of the obtained antibody to GRP78 by Western plotting. Lane 1 was washed with cell lysate prepared from DU145 cells (cell lysate), and Lane 2 was washed with GST-fused GRP78 protein purified from E. coli and stained with each antibody. AS (ant serum) is a mouse antiserum collected before cell fusion.
- FIG. 2 is a view obtained by analyzing the binding activity of the obtained anti-GRP78 antibody to the cell surface of DU145 cells by FACS.
- FIG. 3 is an analysis of the binding activity of GA-20 antibody to the cell surface of various cancer cells by FACS.
- Figure 4 shows (A) the analysis of the binding activity of GA-20 antibody to the cell surface of various immortal cell lines by FACS; and (B) the expression of GRP78 protein in various immortal cell lines. It is the figure which analyzed by Western blotting using -20.
- FIG. 5 shows the analysis of the internalization activity of GA-20 and GA-21 antibodies into cells by FACS. Each antibody was reacted with DU145 cells, incubated for 2 hours at O or 37, and then the antibody bound to the cell surface was detected with a secondary antibody (FITC-labeled anti-mouse IgG antibody).
- FIG. 6 is an analysis of the cellular uptake of GA-20 antibody that binds to the cell surface of DU15 and GA-31 antibody that does not bind to the cell surface by cell immunostaining. Each antibody was added to the cultured DU145 cells, and the culture was continued at 37 t: for 3 hours. Thereafter, the cells were processed according to the scheme shown below, and the antibodies incorporated into the cells were detected.
- FIG. 7 is a Western plot showing the epitope of each antibody binding to GRP78.
- FIG. The upper row is a schematic diagram of the GST-fused GRP78 protein (1-6) used for the epitope analysis.
- the lower panel shows the results of Western blot analysis in which each GST-fused GRP78 protein (1-6) was subjected to SDS-PAGE and the reactivity of each antibody against each protein was analyzed.
- FIG. 8 is a diagram showing the results of a Western plot performed to further narrow down the EPs in GRP78 of GA-20 and GA-21.
- the upper row is a schematic diagram of the GST-fused GRP78 protein limited to a narrower range.
- the lower panel shows the results of Western blot analysis in which each GST-fused GRP78 protein (1-5) was subjected to SDS-PAGE and the reactivity of GA-20 and GA-21 antibodies against each protein was analyzed.
- Figure 9 shows the fraction eluted with toxin-labeled GA-20 scFv antibody (GA20-PE40) purified from Escherichia coli using an ELISA system with the binding activity to GRP78 as an indicator. The results are shown.
- the upper diagram shows a schematic diagram showing an ELISA system that detects the binding activity of GA20-PE40 to GRP78.
- the lower diagram shows the elution fraction binding activity obtained as a result of ELISA.
- “Ini ti al” is the E. coli lysate that induced GA20-PE40 expression
- “pass” is the column passage fraction of the lysate applied to the HisTrap column
- “wash” is the column wash fraction
- “E lute 1” to “e lute 7” indicate the fractions eluted from the column.
- FIG. 10 shows the cytotoxic activity of purified GA20-PE40 against DU145 cells (FIG. 10A), 22Rvl cells (FIG. 10B), or DG44 cells (FIG. 10C).
- the elution fraction (elute 2, 3, 4) was added to each cell at a concentration of 10%, and then the number of viable cells was measured, and the ratio to the number of cells in the PBS addition group was quantified.
- FIG. 11 is a view obtained by analyzing the binding activity of the obtained anti-GRP78 antibody to the cell surface of 22Rvl cells by FACS.
- FIG. 12 shows GA-20 antibody and 4 types of newly obtained antibodies by re-immunization (GC-18 antibody, GC-20 antibody, GD-4 antibody, GD-17 antibody) )
- GC-18 antibody, GC-20 antibody, GD-4 antibody, GD-17 antibody Is a schematic diagram of the GST-fused GRP78 protein used for epithelial analysis.
- Figure 12 (B) shows that each GST-fused GRP78 protein is induced to express in E. coli by adding IPTG, and confirmed by SDS-PAGE and CBB staining. It is a result. '
- FIG. 13 is a view obtained by analyzing the epitope of each antibody by Western plotting. SDS-PAGE of each GST-fused GRP78 protein and the results of Western blot analysis of the reactivity of each antibody are shown. The table below shows the epitope of each antibody from the results of the Western plot.
- FIG. 14 is a diagram showing purified GD17scFv-PE40 stained with CBB after SDS-PAGE in order to examine the purity of purified GD17scFv-PE40.
- FIG. 15 shows the binding activity of purified GD17scFv-PE40 to the GRP78 protein and the results of EL I SA performed to analyze the stability of the protein.
- 4t Storage, preserved at 37, and freeze-thawed GD17scFv-PE40 were diluted to various concentrations, and binding activity to GST-GRP78 was analyzed by ELISA.
- the table below shows the EC 5 binding activity of each preparation to the GRP78 protein. It is indicated by value.
- FIG. 16A shows the results of cytotoxic activity of purified GD17scFv-PE40 on various cell lines.
- GD17scFv-PE40 was diluted to various concentrations, added to a cancer cell line (Fig. 16A), cultured for several days, and the number of viable cells was analyzed. The concentration of the antibody that gives 50% of the maximum activity (EC 5 value) is shown in the table.
- FIG. 16B shows the results of cytotoxic activity of purified GD17scFv-PE40 on various cell lines.
- GD17scFv-PE40 was diluted to various concentrations, added to a normal cell line (FIG. 16B), cultured for several days, and the number of viable cells was analyzed.
- the antibody concentration (EC 5. value) that gives 50% of the maximum activity is shown in the table.
- FIG. 17 shows the analysis of GRP78 protein expression in various cell lines by Western plotting using GD-17 antibody.
- FIG. 18 shows the results of analysis of the antitumor activity of GD17scFv-PE40 in an in vivo mouse transplantation model. After transplantation of 22Rvl (dayO), dayl 7, day21, day23, day26, and day29 were administered PBS (vehicle) or GD17scFv-PE40 at 0.5 mg / kg (each arrow), and then tumor volume over time was measured.
- the anti-GRP78 antibody of the present invention may be an antibody that binds to the GRP78 protein (SEQ ID N0: 2).
- the origin mouse, rat, human, etc.
- type monoclonal antibody, polyclonal antibody
- shape modified antibody, low molecular weight antibody, modified antibody, etc.
- the anti-GRP78 antibody used in the present invention preferably specifically binds to GRP78.
- the anti-GRP78 antibody used in the present invention is preferably a monoclonal antibody.
- GRP78 is known to be localized on the cell membrane in cancer cells and the like.
- One preferred embodiment of the anti-GRP78 antibody used in the present invention is an antibody that recognizes a region exposed to the outside of the cell when GRP78 is localized on the cell membrane.
- Such an antibody can be produced, for example, by preparing an antibody using the GRP78 protein (SEQ ID N0: 2) as an immunogen, and cancer cells that express GRP78 on the cell membrane from the prepared antibodies (for example, prostate cancer cells). For example, by selecting an antibody that can bind to the strain DU145). More specifically, an antibody that recognizes a region exposed to the outside of the cell when GRP78 is localized on the cell membrane can be obtained by the method described in the Examples.
- the region exposed to the outside of the cell when GRP78 is localized on the cell membrane is preferably a region where the growth promoting action of ⁇ 2 macroglopurin is not mimicated when an antibody binds to the region.
- a region other than the 98th to 115th region of GRP78 is preferred.
- the region from position 376 to position 415 (SEQ ID NO: 3) in the amino acid sequence of SEQ ID NO: 2 is listed. be able to.
- an antibody that recognizes the region exposed to the outside of the cell when GRP78 is localized on the cell membrane is an antibody that recognizes the region from position 376 to position 415 of GRP78.
- antibodies that recognize the region from amino acids 376 to 415 of the GPR78 protein include, but are not limited to, amino acids 384 to 391 (ie, amino acids 9 to 16 in SEQ ID NO: 3)
- An antibody that recognizes amino acids 392 to 407 ie, amino acids 17-32 of SEQ ID NO: 3
- amino acids 400 to 415 ie, amino acids 25-40 of SEQ ID NO: 3
- an antibody to be recognized Whether or not the antibody recognizes the target epitope is determined by methods known to those skilled in the art. For example, it can be confirmed by the method described in the examples.
- Another preferred embodiment of the antibody used in the present invention is an antibody having an internalizing activity.
- an antibody having an internalizing activity refers to an antibody that is transported into the cell (cytoplasm, vesicle, other organelle, etc.) when bound to GRP78 localized on the cell surface. means.
- an anti-GRP78 antibody conjugated with a labeling substance is expressed as a cell that expresses GRP78 (for example, a prostate cancer cell line). DU145, etc.) to check whether the labeling substance has been taken up into the cell, contact a cell expressing GRP78 with an anti-GRP78 antibody conjugated with a cytotoxic substance, and cell death in the GRP78-expressing cell It can be confirmed by a method of confirming whether or not the user has been guided. More specifically, it can be confirmed whether or not the antibody has an internalizing activity by the method described in Examples.
- a particularly preferred antibody includes an antibody that recognizes a region exposed to the outside when GRP78 is localized on the cell membrane and has an internalizing activity.
- an antibody that recognizes a region exposed to the outside of the cell when GRP78 is localized on the cell membrane is selected by the above-described method, and then has an internalizing activity from the selected antibody. It can be obtained by further selecting antibodies.
- preferable antibodies used in the present invention include the following antibodies (a) to (s).
- An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 as CDR1, the amino acid sequence of SEQ ID NO: 9 as CDR2, and the amino acid sequence of SEQ ID NO: 10 as CDR3.
- An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 as CDR1, the amino acid sequence of SEQ ID NO: 19 as CDR2, and the amino acid sequence of SEQ ID NO: 20 as CDR3.
- An antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 21 as CDR1, the amino acid sequence of SEQ ID NO: 22 as CDR2, and the amino acid sequence of SEQ ID NO: 23 as CDR3.
- An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 61 as CDR1, the amino acid sequence of SEQ ID NO: 62 as CDR2, and the amino acid sequence of SEQ ID NO: 63 as CDR3.
- An antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 64 as CDR1, the amino acid sequence of SEQ ID NO: 65 as CDR2, and the amino acid sequence of SEQ ID N0: 66 as CDR3.
- An antibody comprising the heavy chain variable region of (j) and the light chain variable region of (k).
- An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID N0: 81 as CDR1, the amino acid sequence of SEQ ID N0: 82 as CDR2, and the amino acid sequence of SEQ ID NO: 83 as CDR3.
- An antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 86 as the amino acid sequence, CDR3.
- An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 91 as CDR1, the amino acid sequence of SEQ ID N0: 92 as CDR2, and the amino acid sequence of SEQ ID NO: 93 as CDR3.
- An antibody that recognizes the same epitope as a certain antibody can be obtained, for example, by the following method.
- test antibody shares an epitope with an antibody can be confirmed by competition for the same epitope.
- ⁇ Competition between antibodies is detected by cross-blocking assay.
- competitive ELISA assembly is a preferred cross-blocking assembly.
- the GRP78 protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competitive antibody, and then the anti-antibody of the present invention.
- GRP78 antibody is added.
- the amount of the anti-GRP78 antibody of the present invention bound to the GRP78 protein in the well is indirectly related to the binding ability of a candidate competitive antibody (test antibody) that competes for binding to the same epitope.
- the amount of binding of the anti-GRP78 antibody of the present invention to the well coated with the GRP78 protein decreases, and the GRP78 protein of the test antibody binds to the well. Increases the amount of binding to the trapped well.
- the amount of antibody bound to the well can be easily measured by labeling the antibody in advance.
- a piotin-labeled antibody can be measured by using an avidin peroxidase conjugate and an appropriate substrate.
- Peroxidase The cross-blocking assay using which enzyme label is called competitive ELISA assay.
- the antibody can be labeled with other labeling substances that can be detected or measured. Specifically, radiolabels or fluorescent labels are known.
- the amount of the antibody bound to the well is measured by a labeled antibody that recognizes the constant region of the antibody. You can also. Alternatively, even if the antibodies are of the same species, if the classes are different, the amount of antibody bound to the well can be measured by an antibody that identifies each class.
- the candidate antibody is at least 20%, preferably at least 20-50%, more preferably at least 50% of the anti-GRP78 antibody compared to the binding activity obtained in a control test performed in the absence of the candidate competing antibody. If the binding can be blocked, the candidate competitive antibody is an antibody that binds to, or competes for, binding to substantially the same epitope as the anti-GRP78 antibody of the invention.
- an antibody bound with a cytotoxic substance can be mentioned. When an antibody to which a cytotoxic substance is bound is taken into the cell, the cytotoxic substance can induce killing action or cell death to the cell that has taken in the antibody. Therefore, it is preferable that the antibody bound with the cytotoxic substance further has an internalizing activity.
- the anti-GRP78 antibody to which the cytotoxic substance of the present invention is bound for example, an antibody having a cytotoxic activity or inducing cell death against a cancer cell expressing GRP78 (DU145, 22RvK MCF7, etc.) Can be mentioned.
- the cytotoxic substance used in the present invention may be any substance as long as it can induce killing action or cell death in cells, and examples thereof include toxins, radioactive substances, and chemotherapeutic agents.
- These cytotoxic substances in the present invention include a prodrug that is converted into a cytotoxic substance that is active in vivo. Activation of the prodrug may be enzymatic conversion or non-enzymatic conversion.
- the toxin means various proteins, polypeptides, etc. exhibiting cytotoxicity derived from microorganisms, animals or plants. Examples of the toxin used in the present invention include the following.
- FEBS letter 195, 1-8, 1986 Dodecandrin (StirpeF., Barbieri L "FEBS letter 195, 1- Tritin (Stirpe F., Barbieri L., FEBS letter 195, 1-8, 1986); Luff in (Stirpe F., Barbieri L. FEBS letter 195 1-8, 1986 5 ); Trichokirin (Casellas P., et al., Eur. J. Biochem. 176, 581-588, 1988; B perfumesi A., et al., Clin. Exp. Immunol., 89, 341-346) , 1992).
- the radioactive substance refers to a substance containing a radioisotope.
- the radioactive isotope is not particularly limited, and any radioactive isotope may be used.
- 32 P 14 C 1 25 I 13I I 18 3 ⁇ 4e 188 Re can be used.
- the chemotherapeutic agent means a substance having cytotoxic activity other than the above-mentioned toxins and radioactive substances, and includes a cytostatic agent, an antitumor agent, an enzyme and the like.
- the chemotherapeutic agent used in the present invention is not particularly limited, but a low molecular weight chemotherapeutic agent is preferable. If the molecular weight is small, it is unlikely that it will interfere with the function of the antibody even after binding to the antibody.
- the low molecular weight chemotherapeutic agent usually has a molecular weight of 100 2000, preferably 200 1000.
- the following chemotherapeutic agents can be used.
- Melphalan Radar GF, et al., Nature 255, 487-488, 1975
- Cis-plat inum Hurwitz E. and Haimovich J. Method In Enzymology 178, 369-375, 1986
- IL-2 interleukin 2
- TNF a tumor necrosis factor alpha
- INF interferon
- Carboxypeptidase carboxypeptidase
- Alkaline phosphatase Alkaline Phosphatase
- beta-lactamase / 3-lactamase
- cytidine deaminase One type of cytotoxic substance may be used in the present invention, or two or more types of cytotoxic substances may be used in combination.
- the anti-GRP78 antibody can be bound to the above-mentioned cytotoxic substance by a covalent bond or a non-covalent bond. Methods for producing antibodies bound with these cytotoxic substances are known.
- the anti-GRP78 antibody and the cytotoxic substance may be directly bound via a linking group or the like possessed by themselves, or indirectly via another substance such as a linker or an intermediate support. May be combined.
- Examples of the linking group when the anti-GRP78 antibody and the cytotoxic substance are directly bonded include a disulfide bond using an SH group. Specifically, the intramolecular disulfide bond in the Fc region of the antibody is reduced with a reducing agent such as dithioleitol, and the disulfide bond in the cytotoxic substance is reduced in the same manner to convert both into disulfide bonds.
- an antibody or a cytotoxic substance may be activated with an activation promoter such as Ellman's reagent to promote the formation of a disulfide bond between the two.
- an activation promoter such as Ellman's reagent
- Other methods for directly binding the anti-GRP78 antibody and cytotoxic substance include, for example, a method using a Schiff base, a calpositimide method, an active ester method (N-hydroxysucccinimide method), and a method using Mixed anhydride. , Gia "/ Ole can be used for example.
- the method of binding the anti-GRP78 antibody to the cytotoxic substance can also be indirectly linked through other substances.
- Other substances for indirectly binding are not particularly limited, for example, any one or a combination of two or more of amino group, carboxyl group, mercapto group, etc.
- 15 may include compounds having two or more compounds, peptide linkers, compounds having binding ability to anti-GRP78 antibodies, and the like.
- An example of a compound having two or more amino groups, strong loxyl groups, mercapto groups, etc. in combination of one or more is, for example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP : N-Succinimidyl 3- (2-pyridylditio) propinate) (Wawrzynczak EJ, et al., Cancer Res., 50,
- succinimidyl 6-3- [2-pyridyldithio] propionamide) hexanoe IX SP- Succinimidyl 6-3- [2-pyridyldi tioj pro i nam ide) hexanoate (Hermanson GT, BI0C0NJUGATE Techniques, 230-232, 1996); sulfosuccinimidyl 6-3- [2-pyridyl dithio] propionamide) (Sulfo- LC- SPDP: Sulfosuccinimidyl 6-3-
- succinimidyloxycarbonyl -a- (2-pyridyl Dithio) Toluene (SMPT: Succinimidyloxycarbonyl-a- (2-pyr idyldi tio) toruene) (Thorpe PE, etal., Cancer Res., 47, 5924-5931, 1987); Succinimidyl 6- ( ⁇ -methyl) -[2-pyridyldithio] toluamide) hexanoate (LC-SMPT: Succinimidyl 6- ⁇ -methyl-L 2-pyr idyldi tio] toruamide) hexanoate) (Hermanson GT, BIOCONJUGATE Techniques, 232-235, 1996); Sulfosuccinimidylol 6- ( ⁇ -methyl- [2-pyridyldithio] toluamide) hexanoate (Sulfo-LC
- anti-GRP78 antibody and cytotoxic substances include, for example, peptides, antibodies, poly L-glutamic acid (PGA), force lupoxymethyl dextran, dextran, aminodextran, avidin / piotine, cis ⁇ Aconitic acid, dartamic acid dihydrazide, human serum albumin (HSA), etc.
- PGA poly L-glutamic acid
- HSA human serum albumin
- proteinaceous cytotoxic substances bind to antibodies using genetic engineering techniques. It is also possible to do. Specifically, for example, it encodes the above cytotoxic peptide
- a recombinant vector can be constructed by fusing DNA encoding the anti-GRP78 antibody in-frame and incorporating it into an expression vector.
- a transformed cell obtained by introducing the vector into an appropriate host cell is cultured, and the incorporated DNA is expressed, whereby an anti-GRP78 antibody to which a toxic peptide is bound can be obtained as a fusion protein.
- a proteinaceous drug or toxin is generally placed on the C-terminal side of the antibody.
- a peptide linker can be interposed between the antibody and the proteinaceous drug or toxin.
- the anti-GRP78 monoclonal antibody of the present invention can be obtained using known means.
- a mammal-derived monoclonal antibody is particularly preferable.
- Monoclonal antibodies derived from mammals include those produced by hyperpridoma and those produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques.
- a monoclonal antibody-producing hybridoma can be prepared using a known technique, for example, as follows. First, GRP78 protein is used as a sensitizing antigen, and this is immunized according to a normal immunization method. Immune cells obtained from an immunized animal are fused with known parental cells by a conventional cell fusion method to obtain a hyperidoma. Furthermore, from this high-pridoma, a high-pridoma that produces an anti-GRP78 antibody can be selected by screening cells that produce the target antibody by an ordinary screening method. Specifically, the production of a monoclonal antibody is performed as shown below, for example.
- a GRP78 protein used as a sensitizing antigen for antibody acquisition can be obtained.
- the base sequence of the human GRP78 gene is already known (SEQ ID NO: Do, that is, the gene sequence encoding GRP78 is inserted into a known expression vector and transformed into an appropriate host cell.
- the target human GRP78 protein can be purified from the host cell or culture supernatant by known methods, and purified natural GRP78 protein can also be used in the same manner. Set up your mouth matography one or more times It can be produced by combining or using alone.
- a fusion protein obtained by fusing a desired partial polypeptide of GRP78 protein with a different polypeptide can also be used as an immunogen.
- an antibody Fc fragment or a peptide tag can be used.
- a vector that expresses a fusion protein should be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into the expression vector as described above. Can do.
- the method for manufacturing a fusion protein is Mo l ecu l ar Cl oning 2nd ed. (Sambrook, J. etal., Mo l ecul ar C l oning 2 nd ed., 9. 47-9. 58, Co ld Spr ing Harbor Lab Press, 1989).
- the GRP78 protein thus purified can be used as a sensitizing antigen used for immunization against mammals.
- a partial peptide of GRP78 can also be used as a sensitizing antigen.
- the following peptides can be sensitized antigens:
- GRP78 used as a partial peptide are not limited.
- a preferred region can be selected from the region from 376 to 415 of GRP78 (SEQ ID NO: 3).
- the number of amino acids constituting the peptide to be sensitized antigen is preferably at least 3 or more, for example, 5 or more, or 6 or more. More specifically, a peptide having 8 to 50, preferably 10 to 30 residues can be used as a sensitizing antigen.
- Mammals immunized with the sensitizing antigen are not particularly limited. In order to obtain a monoclonal antibody by the cell fusion method, it is preferable to select an immune animal in consideration of compatibility with the parent cell used for cell fusion. In general, rodent animals are preferred as immunized animals. Specifically, mice, rats, hamsters, or rabbits can be used as immunized animals. In addition, monkeys can be used as immunized animals.
- the above animals can be immunized with a sensitizing antigen according to known methods.
- mammals can be immunized by injecting the sensitizing antigen intraperitoneally or subcutaneously.
- the sensitizing antigen is administered to mammals several times every 4 to 21 days.
- the sensitizing antigen is diluted with PBS (Phosphate-Buf fered Saline) or physiological saline at an appropriate dilution ratio and used for immunization.
- a sensitizing antigen can be administered with Ajuban.
- it can be mixed with Freund's complete adjuvant and emulsified to give a sensitizing antigen.
- An appropriate carrier can be used for immunization with the sensitizing antigen.
- a partial peptide having a low molecular weight is used as a sensitizing antigen, it is desirable to immunize the sensitizing antigen peptide by binding it to a carrier protein such as albumin or albumin to keyhole limpet.
- immune cells are collected from the mammal and subjected to cell fusion.
- spleen cells can be used as preferred immune cells.
- Mammalian myeloma cells are used as the cells fused with the immune cells.
- the myeloma cells are preferably provided with an appropriate selection marker for screening.
- a selectable marker is a trait that can (or does not) survive under certain culture conditions.
- Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase-deficient (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency).
- HGPRT deficiency hypoxanthine-guanine-phosphoribosyltransferase-deficient
- TK deficiency thymidine kinase deficiency
- Cells with HGPRT or TK deficiency have sensitivity to hypoxanthine-aminopterin-thymidine (hereinafter abbreviated as HAT sensitivity).
- HAT-sensitive cells are unable to synthesize DNA in HAT selection medium and die, but when fused with normal cells, they can continue to synthesize DNA using the salvage circuit of normal cells. Above all, it will proliferate.
- HGPRT-deficient and TK-deficient cells can be selected in media containing 6 thioguanine, 8 azaguanine (hereinafter abbreviated as 8 AG), or 5 'promodoxyuridine, respectively.
- 8 AG 8 azaguanine
- 5 'promodoxyuridine normal cells die because they incorporate these pyrimidine analogs into DNA, but cells deficient in these enzymes cannot survive these pyrimidine analogs and can survive in selective media.
- G418 resistance is a 2-deoxystreptin antibiotic based on the neomycin resistance gene. Confer resistance to (mycin analogs).
- Various myeloma cells suitable for cell fusion are known. For example, P3 (P3x63Ag8.653) (J. I ⁇ unol.
- the cell fusion between the immune cells and myeloma cells is performed according to a known method, for example, the method of Kohler and Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It is possible to do.
- the cell fusion can be performed in a normal nutrient culture medium in the presence of a cell fusion promoter.
- a cell fusion promoter for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used.
- an auxiliary agent such as dimethyl sulfoxide can be added as desired to increase the fusion efficiency.
- the usage ratio of immune cells and myeloma cells can be set arbitrarily.
- the number of immune cells is preferably 1 to 10 times that of the myeloma cell.
- the culture medium used for the cell fusion for example, RPMI 1640 culture medium suitable for growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. it can .
- serum supplements such as fetal calf serum (FCS) can be added to the culture medium.
- a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and the target fusion cell (eight hybridoma) is formed by mixing the PEG solution that has been pre-warmed to about 37. Is done.
- PEG having an average molecular weight of about 1000 to 6000 can be added usually at a concentration of 30 to 60% (w / v).
- cell fusion agents and the like unfavorable for the growth of the hyperidoma are removed by repeating the operation of adding the appropriate culture solution listed above and centrifuging to remove the supernatant.
- the resulting hyperidoma has the myeloma used for cell fusion. Can be selected by using a selective culture medium according to the selection marker
- cells having HGPRT or TK deficiency can be selected by culturing in a HAT culture solution (a culture solution containing hypoxanthine, aminobuterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can be selectively proliferated in the HAT medium. Incubation with the HAT medium is continued for a period of time sufficient to kill cells (non-fusion cells) other than the target hybridoma.
- a target hyperidoma can be selected by culturing for several days to several weeks.
- an antibody that recognizes GRP78 can be prepared by the method described in International Publication W003 / 104453.
- Screening and single cloning of the target antibody can be preferably performed by a screening method based on a known antigen-antibody reaction.
- the antigen is bound to a carrier such as a bead made of polystyrene or a commercially available 96-well microtie plate and reacted with the culture supernatant of Hypridoma.
- a secondary antibody labeled with an enzyme is reacted. If the target supernatant that reacts with the sensitizing antigen is contained in the culture supernatant, the secondary antibody binds to the carrier via this antibody. By detecting the secondary antibody finally bound to the carrier, it can be determined whether or not the target antibody is present in the culture supernatant.
- GRP78 protein that is practically homogeneous, including those used for immunization, can be preferably used as the antigen.
- a target antibody can be obtained by sensitizing human lymphocytes with an antigen.
- human lymphocytes are first sensitized with GRP78 protein in the in vitro mouth.
- the immunized lymphocytes are then fused with an appropriate fusion partner.
- the fusion partner for example, a myeloma cell derived from a human and having a permanent division ability can be used (see Japanese Patent Publication No. 59878).
- the anti-GRP78 antibody obtained by this method is GRP78. It is a human antibody having binding activity to protein.
- an anti-GRP78 human antibody can be obtained by administering GRP78 protein as an antigen to a transgenic animal having all repertoires of human antibody genes.
- Antibody-producing cells of immunized animals can be immortalized by cell fusion with an appropriate fusion partner or Epstein-Barr virus infection. It is also possible to isolate human antibodies against the GRP78 protein from the immortalized cells thus obtained (see International Publications W0 94/25585, W093 / 12227, W0 92/03918, W0 94/02602) .
- W0 94/25585, W093 / 12227, W0 92/03918, W0 94/02602 see International Publications W0 94/25585, W093 / 12227, W0 92/03918, W0 94/02602 .
- By cloning the immortalized cells it is possible to clone cells that produce an antibody having the desired reaction specificity.
- the immune system of the animal recognizes human GRP78 as a foreign substance. Therefore, a human antibody against human GRP78 can be easily obtained.
- the hyperpridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution.
- the hybridoma can be stored for a long time in liquid nitrogen.
- the hyperidoma can be cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant.
- a hybridoma can be administered to a compatible mammalian J3 ⁇ 4 product to proliferate, and a monoclonal antibody can be obtained as its ascites.
- the former method is suitable for obtaining a highly pure antibody.
- an antibody encoded by an antibody gene cloned from an antibody-producing cell can also be used.
- the cloned antibody gene can be expressed as an antibody by incorporating it into a suitable vector and introducing it into a host. Methods for isolation of antibody genes, introduction into vectors, and transformation of host cells have already been established (eg, Vandamme, AM et ah, Eur. J. Biochem. (1990) 192, 767-775).
- cDNA encoding the variable region (V region) of an anti-GRP78 antibody can be obtained from a hyperidoma cell that produces the anti-GRP78 antibody.
- total RNA is usually extracted from the hybridoma first.
- Methods for extracting mRNA from cells include, for example, guanidine ultracentrifugation (Ch i rgwin, JM et al., Biochemi st ry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159) and the like can be used.
- the extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GE Healthcare Bioscience).
- kits for extracting total mRNA directly from cells such as QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Bioscience) are also commercially available. Using such a kit, total mRNA can also be obtained from eight hybridomas.
- cDNA encoding the antibody V region can be synthesized using reverse transcriptase.
- cDNA can be synthesized by AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation).
- the desired cDNA fragment is purified from the obtained PCR product and then ligated with the vector DNA.
- a recombinant vector is prepared, introduced into E. coli and the like and a colony is selected, a desired recombinant vector can be prepared from the E. coli that has formed the colony. Whether or not the recombinant vector has the target cDNA base sequence can be confirmed by a known method such as the dideoxynucleotide chain termination method.
- a PCR method using primers for variable region gene amplification can also be used.
- cDNA is synthesized using the extracted mRNA as a saddle shape to obtain a cDNA library. It is convenient to use a commercially available kit for the synthesis of the cDNA library. Actually, the amount of mRNA obtained from only a few cells is extremely small, so the yield is low when purified directly. Therefore, it is usually purified after adding carrier RNA that is apparently free of antibody genes. Alternatively, if a certain amount of RNA can be extracted, it is possible to efficiently extract only RNA from antibody-producing cells. For example, RNA extraction from 10 or more, or 30 or more, preferably 50 or more antibody-producing cells, The addition of carrier RNA may not be necessary.
- cDNA libraries are amplified by PCR.
- Primers for amplifying antibody genes by PCR are known. For example, based on the disclosure of a paper (J. Mol. Biol. (1991) 222, 581-597), a primer for amplification of a human antibody gene can be designed. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, when using a cDNA library with an unknown subclass as a saddle, PCR should be performed in consideration of all possibilities.
- a primer capable of amplifying genes encoding a1 to a5 as a heavy chain and / chain and a ⁇ chain as a light chain is selected. Can be used.
- a primer that anneals to the region corresponding to the hinge region is generally used as the 3 'primer.
- primers suitable for each subclass can be used as the 5 ′ primer.
- the PCR products from the primers for gene amplification of the heavy chain and light chain subclasses should be independent libraries.
- the library thus synthesized it is possible to reconstruct an immunoglopurin consisting of a combination of a heavy chain and a light chain.
- the target antibody can be screened using the binding activity of the reconstituted immunoglobulin to GRP78 as an index.
- the binding of the antibody of the present invention to GRP78 is specific.
- An antibody that binds to GRP78 can be screened, for example, as follows.
- a step of selecting an antibody that binds to GRP78 Methods for detecting the binding between the antibody and GRP78 are known. Specifically, fixed to the carrier A test antibody is reacted with GRP78, and then a labeled antibody that recognizes the antibody is reacted. If the labeled antibody on the carrier is detected after washing, the binding of the test antibody to GRP78 can be proved.
- enzyme active proteins such as peroxidase and / 3-galactosidase, or fluorescent substances such as FITC can be used.
- a fixed specimen of cells expressing GRP78 can also be used.
- a vanning method using a phage vector can also be used.
- the antibody gene is obtained as a library of heavy chain and light chain subclasses as described above, a screening method using a phage vector is advantageous.
- the gene encoding the variable region of the heavy chain and the light chain can be made into a single chain Fv (scFv) by ligating with an appropriate linker sequence. If a gene encoding scFv is inserted into a phage vector, a phage expressing scFv on the surface can be obtained.
- the polynucleotide encoding the antibody may encode the full length of the antibody, or may encode a part of the antibody.
- a part of an antibody refers to any part of an antibody molecule.
- an antibody fragment may be used as a term indicating a part of an antibody.
- a preferred antibody fragment in the present invention contains a complementary chain determining region (CDR) of an antibody. More preferably, the antibody fragment of the present invention comprises all three CDRs constituting the variable region.
- the cDNA is digested with a restriction enzyme that recognizes restriction enzyme sites inserted at both ends of the cDNA.
- a preferred restriction enzyme recognizes and digests a base sequence that is unlikely to appear in the base sequence constituting the antibody gene.
- a restriction enzyme that provides a sticky end is preferred.
- a chimeric antibody can be obtained by fusing the gene encoding the antibody constant region (C region) and the gene encoding the V region in-frame.
- the term “chimeric antibody” means that the organism from which the constant region and the variable region originate is different. Therefore, in addition to a heterologous chimeric antibody such as mouse-human, baboon-human homologous chimeric antibody is also included in the chimeric antibody of the present invention.
- a chimeric antibody expression vector can also be constructed by inserting the V region gene into an expression vector having a constant region in advance.
- a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene is placed on the 5 ′ side of an expression vector holding DNA encoding a desired antibody constant region (C region).
- a chimeric antibody expression vector is constructed by digesting the two with the same combination of restriction enzymes and fusing them in in-frame.
- the antibody gene can be incorporated into an expression vector so as to be expressed under the control of the expression control region.
- the expression control region for expressing an antibody includes, for example, an enhancer promoter.
- a recombinant cell expressing DNA encoding the anti-GRP78 antibody can be obtained.
- DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) can be incorporated into different expression vectors. By simultaneously transforming a vector into which the H chain and L chain have been incorporated into the same host cell (co-tranffect), an antibody molecule having the H chain and L chain can be expressed.
- host cells may be transformed by incorporating DNAs encoding H and L chains into a single expression vector (see International Publication W0 94/1 1 523).
- Many combinations of a host and an expression vector for producing an antibody by isolating an antibody gene once and introducing it into a suitable host are known. Any of these expression systems can be applied to the present invention.
- animal cells, plant cells, or fungal cells can be used.
- mammalian cells CH0, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc.
- amphibian cells such as Xenopus laevis oocytes
- insect cells sf9, sf2K Tn5, etc.
- an antibody gene expression system using cells derived from the genus Nicotiana such as Nicotiana tabacum is known.
- Callus cultured cells can be used for transformation of plant cells.
- fungal cells include yeast (Saccharomyces genus such as Saccharomyces serevisiae), Pichia genus such as methanol-utilizing yeast (Pichia pastoris), and filamentous fungi (Aspergi 1 lus niger). Aspergillus genus) can be used.
- antibody gene expression systems using prokaryotic cells are also known.
- bacterial cells such as E. coli and Bacillus subtilis can be used in the present invention.
- an expression vector in which a useful promoter commonly used, an antibody gene to be expressed, and a poly A signal are functionally linked downstream of the 3 'side.
- a useful promoter commonly used an antibody gene to be expressed, and a poly A signal are functionally linked downstream of the 3 'side.
- human cytomegalovirus immediate early promoter / enhancer can be mentioned.
- Other promoter enhancers that can be used for expression of the antibody of the present invention include viral promoter / enhancer or promoter / enhancer derived from mammalian cells such as human longion factor-1 (HEFla).
- viruses that can use the promoter Z enhancer include retrovirus, poliovirus, adenovirus, and simian virus 40 (SV40).
- the method of Mulligan et al. (Nature (1979) 277, 108) can be used.
- HEF1 ⁇ promoter no enhancer can be easily detected by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322). It can be used for target gene expression.
- the gene can be expressed by functionally combining a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed.
- promoters include lacZ promoter and araB promoter.
- lacZ promoter the method of Ward et al. (Nature (1989) 341, 544-546; FASEBJ. (1992) 6, 2422-2427) can be used.
- the araB promoter can be used for expression of the target gene by the method of Better et al. (Science (1988) 240, 1041-1043).
- the pelB signal sequence (Lei, SP et a., J. Bacter iol. (1987) 169, 4379) may be used when it is produced in the periplasm of E. coli. After separating the antibody produced in periplasm, the structure of the antibody is reconfigured to have the desired binding activity by using a protein denaturant such as guanidine hydrochloride of urea. (Refolded).
- a protein denaturant such as guanidine hydrochloride of urea.
- Refolded As the origin of replication inserted into the expression vector, those derived from SV40, poliovirus, adenovirus, ushipapilloma virus (BPV) and the like can be used.
- selectable markers can be inserted into expression vectors for gene copy number amplification in host cell systems. Specifically, use of a selection marker such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthating guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc. can do.
- APH aminoglycoside transferase
- TK thymidine kinase
- Ecogpt E. coli xanthating guanine phosphoribosyltransferase
- dhfr dihydrofolate reductase
- DMEM fetal calf serum
- FCS fetal calf serum
- the antibodies expressed and produced as described above are used in normal protein purification. Purification can be achieved by using known methods alone or in appropriate combinations. For example, antibodies can be separated and purified by appropriately selecting and combining affinity columns such as protein A columns, chromatographic strength rams, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manua, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
- transgenic animals can also be used for the production of recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the target antibody has been introduced.
- an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein that is uniquely produced in milk.
- a protein secreted in milk for example, goat casein can be used.
- the DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat.
- the desired antibody can be obtained as a fusion protein with milk protein from milk produced by a transgene goat (or its offspring) born from the goat that received the embryo.
- holmon can be used as appropriate in transgene goats to increase the amount of milk containing the desired antibody produced by transgene goats (Ebert, KM et al., Bio / Technology (1994 ) 12, 699-702).
- a C region derived from an animal antibody can be used.
- CaK Ca2, C ⁇ force, and C ⁇ can be used as the L chain C region of the mouse antibody H chain C region.
- animal antibodies such as rat, rabbit, goat, hidge, camel and monkey can be used. These sequences are known.
- the C region can be modified to improve the stability of the antibody or its production.
- an antibody when administered to a human, it can be a genetically modified antibody that has been artificially modified for the purpose of, for example, reducing the heterologous antigenicity to humans.
- the recombinant antibodies include, for example, chimeric antibodies, humanized antibodies and the like, and these modified antibodies can be produced using known methods.
- a chimeric antibody refers to an antibody in which different variable regions and constant regions derived from each other are linked.
- mouse antibody The antibody consisting of the heavy chain and light chain variable regions and the human antibody heavy and light chain constant regions is a mouse-hepatic-heterologous chimeric antibody.
- a recombinant vector expressing the chimeric antibody By ligating the DNA encoding the variable region of the mouse antibody with the DNA encoding the constant region of the human antibody and incorporating it into the expression vector, a recombinant vector expressing the chimeric antibody can be prepared.
- the chimeric antibody produced during the culture can be obtained by culturing recombinant cells transformed with the vector and expressing the integrated DNA.
- a chimeric antibody is composed of a V region of an antibody derived from a non-human animal and a C region derived from a human antibody.
- humanized antibodies have a complementarity determining region (CDR) for non-human animal-derived antibodies, a framework region (FR) for human antibody-derived regions, and C region derived from human antibody.
- CDR complementarity determining region
- FR framework region
- Human antibodies are useful as an active ingredient of the therapeutic agent of the present invention because of their reduced antigenicity in the human body.
- variable region of an antibody is usually composed of three complementarity-determining regions (CDRs) sandwiched between four frames (FR).
- CDR is a region that substantially determines the binding specificity of an antibody.
- the amino acid sequence of CDR is rich in diversity.
- the amino acid sequence constituting FR often shows high homology among antibodies having different binding specificities. Therefore, it is generally said that the binding specificity of one antibody can be transplanted to another antibody by CDR grafting.
- Humanized antibodies are also referred to as reshaped human antibodies. Specifically, non-human animals, for example, humanized antibodies obtained by grafting mouse antibody CDRs into rabbit antibodies are known. General genetic recombination techniques for obtaining humanized antibodies are also known.
- overlap extension PCR is known as a method for transplanting mouse antibody CDRs into human FRs.
- a base sequence encoding the CDR of the mouse antibody to be transplanted is added to primers for synthesizing human antibody FRs.
- Primers are prepared for each of the four FRs.
- selection of human FRs having high homology with mouse FRs is said to be free in maintaining CDR function. That is, generally, it is preferable to use human FR comprising an amino acid sequence highly homologous to the FR amino acid sequence adjacent to the mouse CDR to be transplanted.
- the base sequences to be linked are designed so that they are connected to each other in a frame.
- Human FRs are synthesized individually with each primer.
- a product in which DNA encoding mouse CDR is added to each FR is obtained.
- the nucleotide sequences encoding the mouse CDRs of each product are designed to overlap each other.
- the complementary CDR synthesis reaction is performed by annealing the overlapping CDR portions of the products synthesized using the human antibody gene as a saddle type. By this reaction, human FRs are linked via the mouse CDR sequence.
- the V region gene in which 3 CDRs and 4 FRs are ligated is amplified by a primer that is annealed to the 5 'end and 3' end and added with an appropriate restriction enzyme recognition sequence.
- a vector for human antibody expression can be prepared. After introducing the integration vector into a host to establish a recombinant cell, the recombinant cell is cultured, and the humanized antibody is expressed by expressing the DNA encoding the humanized antibody. Produced in cell culture (see European Patent Publication EP 239400, International Publication W0 96/02576).
- the CDR By qualitatively or quantitatively measuring and evaluating the binding activity of the humanized antibody prepared as described above to the antigen, the CDR binds to a favorable antigen-binding site when linked via CDR.
- FRs of human antibodies that can be formed can be suitably selected. If necessary, FR amino acid residues can be substituted so that CDRs of the reshaped human antibody form an appropriate antigen-binding site.
- No acid sequence mutations can be introduced. Specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to FR. Base sequence mutations are introduced into FR synthesized with such primers.
- a mutant FR sequence having a desired property can be selected by measuring and evaluating the antigen-binding activity of a mutant antibody substituted with an amino acid by the above method (Sato, L et al., Cancer Res, 1993). , 53, 851-856).
- a method for obtaining a human antibody is also known.
- human lymphocytes are sensitized in vitro with the desired antigen or cells expressing the desired antigen.
- a desired human antibody having an antigen-binding activity can be obtained by fusing sensitized lymphocytes with human myeloma cells (see Japanese Patent Publication No. 1-59878).
- U266 can be used as a human myeloma cell that is a fusion partner.
- a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (International Publications W0 93/12227, W0 92/03918, W0 94/02602, W0 94/25585, W0 96/34096, W0 96/33735).
- a technique for obtaining a human antibody by panning using a human antibody library is also known.
- the V region of a human antibody can be expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and the phage that binds to the antigen can be selected.
- the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of scFv that binds to the antigen, the V region sequence is fused in-frame with the sequence of the desired human antibody C region, and then inserted into an appropriate expression vector, whereby an expression vector can be prepared.
- the human antibody can be obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding the human antibody. These methods are already known (International Publications W0 92/01047, W0 92/20791, W0 93/06213, W0 93/11236, W0 93/19172, W0 95/01438, W0 95/15388).
- the antibody of the present invention includes not only a bivalent antibody typified by IgG but also a monovalent antibody or a multivalent antibody typified by IgM as long as it binds to the GRP78 protein.
- Multivalent of the present invention Antibodies include multivalent antibodies that all have the same antigen binding site, or multivalent antibodies that have some or all different antigen binding sites.
- the antibody of the present invention is not limited to the full-length molecule of the antibody, and may be a low molecular weight antibody or a modified product thereof as long as it binds to the GRP78 protein.
- the low molecular weight antibody includes an antibody fragment in which a part of a full-length antibody (such as whole IgG) is missing. As long as it has the ability to bind to the GRP78 antigen, partial deletion of the antibody molecule is tolerated.
- the antibody fragment in the present invention preferably contains either or both of a heavy chain variable region (VH) and a light chain variable region (VL).
- VH heavy chain variable region
- VL light chain variable region
- the amino acid sequence of VH or VL can include substitutions, deletions, additions and Z or insertions.
- VH or VL can include substitutions, deletions, additions and Z or insertions.
- the variable region may be hatched if chimerized.
- the antibody fragment include Fab, Fab ′, F (ab ′) 2, Fv and the like.
- Specific examples of the low molecular weight antibody include, for example, Fab, Fab ', F (ab') 2, Fv, scFv (single chain Fv), Diabody, sc (Fv) 2 (single chain ( Fv) 2). Multimers of these antibodies (for example, dimer, trimer, tetramer, polymer) are also included in the low molecular weight antibody of the present invention.
- Antibody fragments can be obtained by treating an antibody with an enzyme to produce antibody fragments.
- an enzyme that produces an antibody fragment for example, papain, pepsin, or plasmin is known.
- genes encoding these antibody fragments can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, Co, MSet al., J. Immunol. (1994) 152, 2968—2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Plueckthun, A. SSkerra, A. Methods in Enzymology (1989) 178, 476-496, Lamoyi, E.
- a digestive enzyme cleaves a specific position of an antibody fragment to give an antibody fragment having a specific structure as follows. Any part of the antibody can be deleted using genetic engineering techniques for such enzymatically obtained antibody fragments: Papain digest: F (ab) 2 or Fab;
- Diapody refers to a bivalent antibody fragment constructed by gene fusion (Hol iger P et a, Proc. Nat 1. Acad. Sci. USA 90: 6444-6448 (1993), EP404 , 097, W093 / 11161).
- a diapody is a dimer composed of two polypeptide chains.
- the polypeptide chains constituting the dimer are linked to each other in the same chain by VL and VH.
- the linker in a diapody is generally so short that VL and VH cannot bond to each other.
- the amino acid residue constituting the linker is, for example, about 5 residues. Therefore, VL and VH encoded on the same polypeptide chain cannot form a single chain variable region fragment, but form a dimer with another single chain variable region fragment. As a result, the diapody has two antigen binding sites.
- scFv is obtained by linking the H chain V region and L chain V region of an antibody.
- the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Hus ton, JS et al., Proc. Natl. Acad. Sci. US A, 1988, 85, 5879-5883.).
- the H chain V region and the L chain V region in scFv may be derived from any antibody described as an antibody in the present specification.
- There are no particular restrictions on the peptide linker that connects the V regions For example, any single chain peptide consisting of about 3 to 25 residues can be used as the linker.
- V regions can be ligated by, for example, the PCR method as described above.
- the entire DNA sequence that codes for the H chain or H chain V region of the antibody and the L chain or L chain V region of the antibody are used as a cage.
- DNAs encoding the V regions of the H chain and the L chain are respectively amplified.
- DNA encoding a part of the peptide linker is prepared.
- Encodes a peptide linker DNA can also be synthesized using PCR.
- a PCR reaction is performed using each DNA of [H chain V region DNA]-[peptide linker DNA]-[L chain V region DNA] and a primer for assembly PCR.
- the assembly primer for PCR consists of a combination of a primer that anneals to the 5 ′ side of the [H chain V region DNA] and a primer that anneals to the 3 ′ side of the [L chain V region DNA]. That is, the assembly-one primer for PCR is a set of primers that can amplify DNA encoding the full-length sequence of scFv to be synthesized.
- nucleotide sequences that can be linked to each V region DNA are added to [peptide linker DNA]. As a result, these DNAs are ligated and the scFv full length is finally generated as an amplification product by the primers for assembly PCR.
- an expression vector containing them and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. Further, the scFv can be obtained by culturing the resulting recombinant cell and expressing the DNA encoding the scFv.
- sc (Fv) 2 is a low molecular weight antibody in which two VHs and two VLs are combined with a linker to form a single chain (Hudson et aU JI thigh unol. Methods 1999; 231: 177-189) .
- sc (Fv) 2 can be prepared, for example, by linking scFv with a linker.
- VHs and two VLs are based on the N-terminal side of the single-stranded polypeptide.
- VH, VL, VH, VL ([VH] Linker [VL] Linker [VH] Linker [VL])
- An antibody characterized by being arranged in the following order is preferable.
- the order of the two VHs and the two VLs is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangements can also be mentioned.
- linker I [VL] Linker I [VH] Linker I [VL] Linker I [VH]
- the linker that binds the variable region of an antibody is disclosed in any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996).
- a linker that can be used can be used.
- a peptide linker is preferred.
- the length of the peptide linker is not particularly limited, and can be appropriately selected by those skilled in the art depending on the purpose.
- the amino acid residues constituting the peptide linker are 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, particularly preferably 12 to 18 amino acids (for example, 15 amino acids).
- the amino acid sequence constituting the peptide linker can be any sequence as long as it does not inhibit the scFv binding action.
- the V regions can be linked using a synthetic chemical linker (chemical crosslinker).
- a crosslinking agent usually used for crosslinking of peptide compounds and the like can be used in the present invention.
- a crosslinking agent usually used for crosslinking of peptide compounds and the like can be used in the present invention.
- the preferred low molecular weight antibody in the present invention is Diabody or sc (Fv) 2.
- an antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or DNA encoding these antibody fragments is constructed and expressed as an expression vector.
- an enzyme such as papain or pepsin to generate antibody fragments, or DNA encoding these antibody fragments is constructed and expressed as an expression vector.
- the antibody of the present invention can also be used as a modified antibody conjugated with various molecules such as polyethylene glycol (PEG).
- PEG polyethylene glycol
- Such a modified antibody can be obtained by chemically modifying the antibody of the present invention. Antibody modification methods are already established in this field.
- the antibody of the present invention may be a bispecific antibody.
- a bispecific antibody is an antibody having a variable region that recognizes different epitopes in the same antibody molecule, but the epitope may exist in different molecules or the same molecule. It may be present inside. That is, in the present invention, the bispecific antibody can also have an antigen binding site that recognizes a different epitope on the GRP78 molecule. It is also possible to use a dual-characteristic antibody in which one recognition site recognizes GRP78 and the other recognition site recognizes a cytotoxic substance.
- the “antibody” in the present invention includes these antibodies.
- a bispecific antibody that recognizes an antigen other than GRP78 can also be combined. For example, a bispecific antibody that recognizes an antigen that is specifically expressed on the cell surface of the target cancer cell as in GRP78 and is different from GRP78 can be combined.
- bispecific antibodies can be produced by combining two antibodies with different recognition antigens.
- the antibodies to be bound may be 1 Z2 molecules each having an H chain and an L chain, or may be 1 Z4 molecules consisting of only an H chain.
- bispecific antibody-producing fused cells can be produced by fusing hybridomas producing different monoclonal antibodies.
- Bispecific antibodies can be produced by genetic engineering techniques. Antibody binding activity
- a known means can be used to measure the antigen binding activity of the antibody (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
- ELISA enzyme-linked immunosorbent assay
- EIA enzyme immunoassay
- RIA radioimmunoassay
- fluorescent immunoassay can be used.
- a technique for measuring the binding activity of an antibody to an antigen expressed in a cell for example, the method described on pages 359 to 420 in the Antibodies A Laboratory Manual can be mentioned.
- a method for measuring the binding between an antigen expressed on the cell surface suspended in a buffer solution or the like and an antibody against the antigen a method using a flow cytometry can be preferably used.
- test GRP78 antibody As an example of a suitable method for measuring the binding activity of the test GRP78 antibody to the antigen, it is stained with a FITC-labeled secondary antibody that recognizes the test antibody reacted with cells expressing GRP78, and then FACSCalibur (BD).
- FACSCalibur One example is a method of measuring and analyzing the fluorescence intensity using CELL QUEST Software (BD). Antiproliferative activity
- the following method is preferably used.
- a method for evaluating or measuring the cell growth inhibitory activity in a test tube a method is used in which the uptake of labeled thymidine added to the medium by living cells is measured as an indicator of DNA replication ability.
- a dye exclusion method in which the ability to exclude a dye such as trypan blue is removed under a microscope or an MTT method is used.
- MTT (3- ( 4, 5-d ime thy l th i azoto 2-yl) -2, 5-d ipheny l te t razo li um bromi de) is used to convert to blue formazan product . More specifically, after adding a test antibody together with a ligand to a culture medium of a test cell for a certain period of time, the MTT solution is added to the culture medium and allowed to stand for a certain period of time, whereby MTT is taken into the cell. Make it. As a result, MTT, a yellow compound, is converted into a blue compound by succinate dehydrogenase in the mitochondria in the cell.
- the blue product is dissolved and colored, and the absorbance is measured to obtain an index of the number of viable cells.
- reagents such as MTS, XTT, and WST-K WST-8 are also commercially available (such as nacalai esque) and can be suitably used.
- a control antibody can be used.
- a tumor-bearing mouse model can be used as a method for evaluating or measuring the cell growth inhibitory activity in vivo. For example, after transplanting a cancer cell expressing GRP78 into the skin or subcutaneous of a non-human test animal, the test antibody is administered intravenously or intraperitoneally every day or every several days from that day or the next day.
- the cytostatic activity can be evaluated by measuring the size of the tumor over time.
- Control antibody is administered in the same manner as in vitro, and the cytostatic activity can be determined by the tumor size in the anti-GRP78 antibody administration group being significantly smaller than the tumor size in the control antibody administration group .
- a nude (nu / nu) mouse in which the thymus is genetically deleted and the function of the T lymphocyte is deleted can be preferably used. By using the mouse, it is possible to exclude the involvement of T lymphocytes in the test animal in the evaluation and measurement of the cytostatic activity of the administered antibody.
- the present invention provides a method for inhibiting the growth of cells by contacting cells expressing GRP78 with the antibodies of the present invention.
- the antibody of the present invention is as described above as an antibody that binds to the GRP78 protein contained in the cell growth inhibitor of the present invention.
- the cell to be contacted with the anti-GRP78 antibody is not particularly limited as long as it expresses GRP78, but GRP78 is preferably a cell localized on the cell membrane, and is a cell associated with a disease. Are preferred.
- a preferable example of a cell associated with a disease is a cancer cell. This also includes vascular endothelial cells (tumor blood vessels) present in malignant tumors.
- the target cancer types are not particularly limited, and examples include prostate cancer, breast cancer, knee cancer, liver cancer, lung cancer, esophageal cancer, melanoma, colon cancer, stomach cancer, ovarian cancer, bladder cancer, and brain tumor. Delivery method using anti-GRP78 antibody
- the present invention relates to a method for transferring a cytotoxic substance into cells using an anti-GRP78 antibody.
- the antibody used in this method is an anti-GRP78 antibody to which the above-mentioned cytotoxic substance is bound. In that case, an antibody having an internalizing activity is preferable.
- the cytotoxic substance can be delivered by bringing a cell expressing GRP78 into contact with an anti-GRP78 antibody bound with the cytotoxic substance.
- the cell to which the cytotoxic substance is delivered is not particularly limited, but is preferably a cell in which GRP78 is localized on the cell membrane, and is preferably a cell associated with a disease. Examples of cells associated with the disease include cancer cells.
- vascular endothelial cells present in malignant tumors.
- the target cancer types are not particularly limited, and examples include prostate cancer, breast cancer, knee cancer, liver cancer, lung cancer, esophageal cancer, melanoma, colon cancer, stomach cancer, ovarian cancer, bladder cancer, and brain tumor.
- the contact may be performed in vitro or in vivo.
- a solid or the like obtained by solution or lyophilization can be used as appropriate.
- an aqueous solution it may be an aqueous solution containing purely antibodies, for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents.
- It may be a solution containing a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, a fluidity promoter, a corrigent and the like.
- concentration to be added is not particularly limited, but the final concentration in the culture solution is preferably in the range of 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg / ml. Preferably 1 g / ml to 1 mg / ml can be suitably used.
- “contact” in vivo refers to a non-human grafted GRP78-expressing cell.
- the administration method can be either oral or parenteral. Particularly preferred is a parenteral administration method, and specific examples of the administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration.
- the pharmaceutical composition cell growth inhibitor and anticancer agent of the present invention can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
- the administration method can be appropriately selected depending on the age and symptoms of the test animal.
- aqueous solution When administered as an aqueous solution, it may be an aqueous solution containing pure antibody alone, for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, It may be a solution containing a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, a fluidity promoter, a corrigent and the like.
- the dose for example, the dose can be selected in the range of O. OOO lmg to l OOOmg per kg body weight per administration. Alternatively, for example, the dose can be selected in the range of 0.001 to 100000 mg / body per patient.
- the antibody dose of the present invention is not limited to these doses.
- the invention features a pharmaceutical composition containing an antibody that binds to GRP78 protein.
- the present invention is characterized by a cell growth inhibitor containing an antibody that binds to the GRP78 protein, particularly an anticancer agent.
- the cytostatic agent and anticancer agent of the present invention are preferably administered to a subject suffering from or possibly suffering from cancer.
- the cell growth inhibitor containing an antibody that binds to the GRP78 protein is a method for inhibiting cell growth including a step of administering an antibody that binds to the GRP78 protein to a subject, or in the production of a cell growth inhibitor. It can also be expressed as the use of an antibody that binds to the GRP78 protein.
- the anticancer agent containing an antibody that binds to the GRP78 protein is a method for preventing or treating cancer comprising the step of administering an antibody that binds to the GRP78 protein to a subject.
- it can be expressed as the use of an antibody that binds to the GRP78 protein in the production of an anticancer agent.
- the antibody contained in the pharmaceutical composition of the present invention (for example, cell growth inhibitor, anticancer agent) is not particularly limited as long as it binds to GRP78 protein, and any of the antibodies exemplified herein can be used.
- the pharmaceutical composition of the present invention can be administered either orally or parenterally.
- an administration method by parenteral administration is particularly preferred, and specific examples of the administration method include injection administration, nasal administration, pulmonary administration, and transdermal administration.
- the pharmaceutical composition of the present invention can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.
- an appropriate administration method can be selected depending on the patient's age and symptoms.
- the dosage for example, the dosage can be selected in the range of O. OOOlmg to l OOOmg per kg body weight per administration.
- the dose can be selected in the range of 0.001 to 100000 mg / body per patient.
- the pharmaceutical composition of the present invention is not limited to these doses.
- the pharmaceutical composition of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaciutical Science, Latestedition, Mark Publishing Company, Easton, USA)
- a conventional method for example, Remington's Pharmaciutical Science, Latestedition, Mark Publishing Company, Easton, USA
- the agent include binders, binders, disintegrants, lubricants, fluidity promoters, and corrigents, but are not limited thereto, and other conventional carriers can be used as appropriate.
- ⁇ L starch, carmellocalcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polypropylene lugetila
- examples include minoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid tridalylide, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.
- the present invention further provides a method for producing a pharmaceutical, particularly an anticancer agent, comprising the following steps.
- Method for producing pharmaceutical composition comprising the following steps
- the anti-GRP78 antibody and cytotoxic substance described above can also be used for the anti-GRP78 antibody and cytotoxic substance. Diagnosis of cancer
- the present invention provides a method for diagnosing diseases, particularly a method for diagnosing cancer, using an anti-GRP78 antibody.
- the diagnostic method of the present invention can be performed by detecting an anti-GRP78 antibody incorporated into cells.
- the anti-GRP78 antibody used in the present invention preferably has an internalizing activity, and is preferably labeled with a labeling substance.
- a preferred embodiment of the diagnostic method of the present invention includes a diagnostic method using an anti-GRP78 antibody labeled with a labeling substance and having an internalizing activity.
- the anti-GRP78 antibody that binds the labeling substance the above-mentioned anti-GRP78 antibody can be used.
- the labeling substance that binds to the anti-GRP78 antibody is not particularly limited.
- a labeling substance known to those skilled in the art such as a fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance, and a radioactive substance can be used.
- radioisotopes 32P, 14C, 1251, 3H, 1311, etc.
- fluorescein 32P, 14C, 1251, 3H, 1311, etc.
- fluorescein 32P, 14C, 1251, 3H, 1311, etc.
- fluorescein rhodamine
- danssil mouth lid umbelliferone
- luciferase peroxidase
- alkaline phosphatase alkaline phosphatase
- 3-galactosidase ⁇ -glucosidase
- examples thereof include horseradish peroxidase, darcoamylase, lysozyme, saccharide oxidase, microperoxidase, and piotin.
- avidin conjugated with an enzyme such as Alri-Liphosphaisase.
- known methods such as the dartal aldehyde method, maleimide method, pyridyl disulfide method, and periodic acid method
- Binding of the labeling substance to the antibody can be performed by methods known to those skilled in the art.
- the target cancer type is not particularly limited.
- prostate cancer breast cancer, knee cancer, liver cancer, lung cancer, esophageal cancer, melanoma, colon cancer, stomach cancer, Ovarian cancer, bladder cancer, brain tumor.
- the diagnosis in the present invention may be performed in vivo or may be performed in vitro.
- diagnosis is performed in vitro, it can be performed, for example, by a method including the following steps.
- the sample to be collected is not particularly limited, and examples thereof include cells and tissues collected from subjects.
- secondary samples obtained from a test sample such as a sample in which tissues or cells collected from the body of an organism are fixed, or a cell culture solution, are also included in the sample of the present invention.
- diagnosis is performed in vivo, for example, it can be performed by a method including the following steps.
- the dose of anti-GRP78 antibody can be appropriately determined by those skilled in the art depending on the type of labeling substance, the type of disease to be diagnosed, and the like.
- the labeled anti-GRP78 antibody may be formulated by the method described above.
- the present invention further provides a method for producing a diagnostic agent, particularly a cancer diagnostic agent, comprising the following steps: (a) providing an anti-GRP78 antibody;
- the anti-GRP78 antibody and labeling substance described above can also be used for the anti-GRP78 antibody and labeling substance.
- the present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (positions 376 to 415 of GRP78) or a fragment thereof.
- a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (positions 376 to 415 of GRP78) or a fragment thereof can be used to evaluate the binding activity of the immunogen and the prepared antibody in producing the antibody of the present invention.
- the fragment is a fragment of at least 5 amino acids, preferably 10 amino acids or more, more preferably 15 amino acids or more.
- polypeptide fragment consisting of the amino acid sequence of SEQ IN NO: 3 is not particularly limited, but is a fragment consisting of the amino acid sequence of positions 384 to 391 of GRP78 (SEQ ID N0: 9 of 9 From 392 to 407 of GRP78 (fragment consisting of amino acids 17 to 32 of SEQ ID N0: 3), from 400 to 415 of GRP78 (Fragments consisting of amino acid sequences 25 to 40 of SEQ ID N0: 3) and the like.
- GRP78 E. coli expression vector the GRP78 gene was first cloned as follows. First, human colon adenocarc inoma cDNA (MTC Multiple Ti ssue cDNA Panel, Clontech) was used as a cage, and RT-PCR was performed using Pyrobest Taq polymerase (Yukara) under the following conditions to clone the full-length GRP78 gene.
- GRP-1 tgaagctct ccctggtggc (SEQ ID NO: 26)
- GRP-2 ctacaactca tctttttctg ctgta (SEQ ID NO: 27)
- the obtained PCR product is converted into a saddle shape and PCR is performed again under the following conditions.
- the GRP78 gene fragment of nucleotide numbers 55 to 1965 is BamHK at the 5 'end and 3' end, respectively.
- a GRP78 cDNA fragment to which an Xhol cleavage sequence was added was obtained.
- GRP-GST-1 aaaggatccg aggaggagga caagaaggag gacgtggg (SEQ ID NO: 28)
- GRP-GST-2 tttctcgagc tacaactcat ctttttctgc tgtatcctc (SEQ ID NO: 29)
- GRP78 protein was prepared as an immunogen for obtaining a GRP78-binding antibody.
- E. coli (BL21) was transformed with pGEX-GRP78-full. This was cultured in LB medium (300 ML), 0D 61. When the value reached 0.5 or more, IPTG was added to 0.5 mM to induce protein expression. After culturing for 5 hours, E. coli was collected by centrifugation.
- the collected E. coli was suspended in 30 ml of B-BER (Pierce) and solubilized. Next, this solubilized Escherichia coli lysate was diluted 10-fold with PBS, Dartathon Sepharose 4B (Amersham Farmacia) equilibrated with PBS was added thereto, and incubated at 4.about.4. After washing with PBS several times, the unadsorbed protein was removed, and PreScissuion protease (Amersham Almacia) in protease reaction solution (50 mM Tris-HCK 150 mM NaCK 1 mM EDTA, 1 mM DTT, pH 7.5). Was reacted with 4 ⁇ .
- B-BER B-BER
- mice For the first immunization, create a GRP78 protein emulsion with COMPLETE AMUBANT (DIFCO: DF263810) and the second and subsequent IMC0MPLETE ADJUBANT (DIFCO: DF263910), and use this for each mouse [(MRL / lpr, male , 4 weeks old) (Balb / c, female, 6 weeks old): All purchased from Nippon Chisursuriba] 3 mice were immunized by subcutaneous injection (1 mL of thermosiline, needle 26G). Two weeks after the first immunization, the second immunization was performed, and immunization was performed 4 to 5 times every other week thereafter. In the final immunization, GRP78 (50 / z g) was suspended in 100/1 PBS and immunized by tail vein injection, and cell fusion was performed 3 days later.
- COMPLETE AMUBANT DIFCO: DF263810
- IMC0MPLETE ADJUBANT DIF
- Cell fusion was performed as follows. The spleen was aseptically removed from the mouse and ground in medium 1 (RPMI 1640 + PS) to give a single cell suspension. This was passed through a 70 // m nylon mesh (Falcon) to remove adipose tissue and the number of cells was counted. The resulting B cells were mixed with mouse mye.Roma cells (P3U1 cells) to a cell number ratio of approximately 2: 1, and 1 mL of 50% PEG (Roche, cat #: 783 641) was added. Cell fusion was performed.
- medium 1 RPMI 1640 + PS
- Falcon 70 // m nylon mesh
- the reactivity to the prostate cancer cell line (DU145) was analyzed by FACS using the culture supernatant of the positive well.
- DU145 (obtained from ATCC) was cultured and passaged with EMEM (invitrogen) containing 10% FCS, ImM sodium pyruvate, and 0.1 lmMNEM. DU145 was peeled off with 1 mM EDTA / PBS, the culture supernatant of hyperidoma was reacted with this, and incubated at 4T: for 1 hour. Then, FITC-labeled anti-mouse IgG antibody (BECKMAN COULTER: PN IM0819) was added and incubated at 4 for 30 minutes. Subsequently, the binding activity of each hybridoma culture supernatant to the DU145 cell surface was analyzed by FACS (Becton Dickinson). ⁇
- the GA-19 antibody, GA-23 antibody, GA-28 antibody, and GA-31 antibody were obtained from 50 mL of the supernatant of the single clone hybridoma culture obtained from Hi Trap Protein G HP 1 mL column (Amersham Biosciences # 17 -0404-01), and for Ig-20 and IgG3 GA-20 antibody and GA-21 antibody, 1 ml of Protein L-agarose (SIGMA) was packed in an open column to purify the antibody.
- SIGMA Protein L-agarose
- the hybridoma supernatant was adsorbed at a flow rate of 1 mL / min, washed with 20 mL of 20 mM phosphate buffer (pH 7.0), and eluted with 3.5 mL of 0.1 M Glycine-HC1 (pH 2.7). . Elution fractions were collected in 0.5 ml each in an Eppendorf tube to which 50 ml of 1M Tris-HC1 (pH 9.0) was added in advance. 0D 28 .
- nn is measured, the fractions containing the antibody are collected, PBS (-) is added to make a total volume of 2.5 mL, and then PBS (-is added using PD-10 column (Amersham Biosciences # 17-085 ⁇ 01). ) Buffer was replaced.
- the purified antibody was passed through a 0.22 fi filter (MILLIPORE # SLGV033RS), and the properties of each purified antibody were examined in detail below.
- Example 3 Analysis of GRP78 antibody
- DU145 cells detached with 1 mM EDTA were incubated with each antibody (10 zg / ml) at 4 for 1 hour in FACS buffer. After that, FITC-labeled anti-mouse IgG antibody (BECKMAN COULTER: PN IM0819) was added and incubated at 4 for 30 minutes. Subsequently, the binding activity of DU145 cells to GRP78 was analyzed by FACS (Becton Dickinson).
- the base sequence of the heavy chain variable region of the GA-20 antibody is SEQ ID NO: 4
- the amino acid sequence of the heavy chain variable region is SEQ ID NO: 5
- the base sequence of the light chain variable region is SEQ ID NO: 6
- the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7, respectively.
- the amino acid sequence of CDR1 of the heavy chain variable region of the GA-20 antibody is SEQ ID NO: 8
- the amino acid sequence of CDR2 is SEQ ID NO: 9
- the amino acid sequence of CDR3 is SEQ ID NO: 10.
- the amino acid sequence of CDR1 of the chain variable region is described in SEQ ID NO: 11
- the amino acid sequence of CDR2 is described in SEQ ID NO: 12
- the amino acid sequence of CDR3 is described in SEQ ID NO: 13.
- the base sequence of the heavy chain variable region of the GA-21 antibody is SEQ ID NO: 14
- the amino acid sequence of the heavy chain variable region is SEQ ID NO: 15
- the base sequence of the light chain variable region is SEQ ID NO: 16
- the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17, respectively.
- amino acid sequence of CDR1 of the heavy chain variable region of the GA-21 antibody is SEQ ID N0: 18
- amino acid sequence of CDR2 is SEQ ID NO: 19
- amino acid sequence of CDR3 is SEQ ID NO: 20
- the light chain The amino acid sequence of CDR1 of the variable region is described in SEQ ID NO: 21, the amino acid sequence of CDR2 is described in SEQ ID NO: 22, and the amino acid sequence of CDR3 is described in SEQ ID NO: 23, respectively.
- Ovarian cancer cell lines (ES-2, SK0V3), breast cancer cell lines (MCF7), colon cancer cell lines (LoVo), prostate cancer cell lines (DU145, LNcap, 22RvK PC3) are purchased from ATCC and recommended by ATCC Culture was performed under conditions. These cells were stained with GA-20 antibody (lO ⁇ g / ml) as described above and subjected to FACS analysis. As a result, it was confirmed that the GA-20 antibody stains not only DU145 but also multiple cancer cells such as LoVo, LNcap, and 22Rvl (Fig. 3).
- DU145 cells are peeled off with ImMEDTA, and one is allowed to react with each antibody (10 ig / ml) for 2 hours in FACS buffer (2% FCS, 0.05% NaN 3 in PBS). Medium (RPI1640 containing 10% FCS) was incubated with each antibody ( ⁇ zg / ml) for 2 hours at 37 ° C. Subsequently, antibodies remaining on the cell surface were detected by FACS analysis using FITC-labeled mouse IgG.
- GRP-GST-1 SEQ ID NO: 28
- GRP-GST-3 SEQ ID NO: 30
- GRP-GST-4 SEQ ID NO: 31
- GRP-GST-2 SEQ ID NO: 29
- cDNA fragment encoding GRP78 encoding amino acids 289-654 with BamHI and Xhol cleavage sequences added to the 5 'and 3' ends respectively. Obtained.
- pGEX-GRP78-ful l is in a saddle shape and GRP-GST-4 (SEQ ID NO: 31) is used as the sense primer and antisense primer GRP-GST-3 (SEQ ID NO: 30) is used at 94 to 30 Second, 72 30 seconds X 25 cycles were used to obtain a cDNA fragment encoding GRP78 (encoding amino acids 289-350) with BamHI and Xhol cleavage sequences added to the 5 'and 3' ends, respectively.
- pGEX-GRP78-ful l is in a saddle shape and GRP-GST-4 (SEQ ID NO: 31) is used as a sense primer and antisense primer GRP-GST-5 (SEQ ID NO: 32) is used at 30 in 94 PCR was performed in 30 seconds x 25 cycles at seconds 72 and 72 to obtain a cDNA fragment encoding GRP78 (encoding amino acids 289-445) with BamHI and Xhol cleavage sequences added to the 5 'end and 3' end, respectively.
- GRP-GST-7 (SEQ ID NO: 34) is used as the sense primer and antisense primer GRP-GST-8 (SEQ ID 0: 35) is used at 94 to 30 PCR is performed in seconds, 64 at 30 seconds, 72 at 30 seconds x 25 cycles, and a cDNA fragment encoding GRP78 (encoding amino acids 345-385) with BamHI and Xhol cleavage sequences added to the 5 'and 3' ends, respectively. Obtained.
- GRP-GST-9 SEQ ID NO: 36
- GRP-GST-10 SEQ ID NO: 37
- GRP-GST-11 SEQ ID NO: 38
- GRP-GST-5 SEQ ID 0: 32
- Seconds 64:30 seconds, 72 seconds 30 seconds x 25 cycles to obtain a cDNA fragment encoding GRP78 (encoding amino acids 406-445) with BamHI and Xhol cleavage sequences added to the 5 'and 3' ends, respectively. It was.
- GRP-GST-7 (SEQ ID NO: 34) as the sense primer and antisense primer GRP-GST-5 (SEQ ID NO: 32) as 94 " 30 seconds, 64 seconds 30 seconds, 72 seconds 30 seconds X 25 cycles of PCR, and a GRP78 (encoding amino acids 345-445) fragment with BamHI and Xhol cleavage sequences added to the 5 'and 3' ends, respectively.
- GRP- -GST- -1 aaaggatccg aggaggagga caagaaggag gacgtggg (SEQ ID NO: 28)
- GRP- -GST- -2 tttctcgagc tacaactcat ctttttctgc tgtatcctc (SEQ ID NO: 29)
- GRP- -GST- -3 tttctcgagc taatcagaat cttccaacac tttctggacg ggc (SEQ ID NO: 29)
- GRP- -GST- -3 tttctcgagc taatcagaat cttccaacac tttctggacg ggc (SEQ ID NO:
- GRP- -GST- -4 aaaggatccc ggcgcgaggt agaaaaggcc aaac (SEQ ID NO: 31)
- GRP- -GST- -5 ttctcgagct aggtaggcac cactgtgttc cttgg (SEQ ID NO: 32)
- GRP- -GST- -6 ttctcgagct agatttcttc aggtgtcagg cgatt (SEQ ID NO: 33)
- GRP- -GST- -7 tttggatccg tgttggaaga ttctgatttg aaga (SEQ ID NO: 34)
- GRP- -GST- -8 ttctcgagct aggatggttc cttgccattg aagaa (SEQ ID NO: 35)
- GRP- -GST- -9 aaaggatcca aagagttctt caatggcaag ga (SEQ ID NO: 36)
- GRP-GST-10 ttctcgagct ataccaggtc acctgtatct tgatc (SEQ ID NO: 37)
- GRP-GST-11 aaaggatcct ctggtgatca agatacaggt gac (SEQ ID NO: 38) 4-1-2. Induction of each GST-fused GRP78 protein expression
- E. coli expression vectors were transformed into E. coli BL21 strain. These E. coli were cultured in LB medium (1 ml each), and IPTG (final 1 ⁇ ) was added in the logarithmic growth phase to induce protein expression. After 4 to 5 hours, E. coli was recovered and dissolved in SDS sample buffer (0.5 ml) to obtain a lysate. Of these, 51 was plotted on SDS-PAGE, followed by a PVDF membrane according to a standard method, and used for Western plotting.
- GRP78 protein prepared above was subjected to Western blotting using the GST fusion protein, and it was examined which region of the GRP78 protein each anti-GRP78 antibody obtained recognized.
- the GA-19 antibody that does not stain cells with FACS shows the N-terminal half (19-350), and the GA-23 antibody, GA-28 antibody, and GA-31 antibody The region of 538-654 at the C end was recognized.
- the GA-20 antibody and GA-21 antibody that stain cells with FACS clearly showed that the region that spans about 100 amino acids of 350-445 was recognized from the staining pattern of the Western plot. .
- a GST fusion protein was prepared by dividing this 350-445 region into three regions, and Western blotting was performed in the same manner as described above to identify GA-20 antibody and GA-21 antibody binding regions.
- the epitope of GA-20 antibody and GA-21 antibody is 40 amino acids of amino acid number 376-415 in GRP78 protein (FIG. 8).
- Example 5 Production of cell death inducer using anti-GRP78 antibody (GA-20 antibody) that recognizes extracellular region
- VL-k ge t cac tgg atg gtg gga aga tg (SEQ ID NO: 39) Primer for cloning heavy chain variable region
- VH-M cca cca gat tc t tat cag aca gg (SEQ ID NO: 40) 94 5 seconds, T 2 minutes, 5 cycles
- an expression vector encoding a toxin labeled antibody (GA20_PE40) in which a toxin (PE40) was added to a single chain Fv (scFv) of the GA-20 antibody was constructed.
- the PE40 gene which is an immunotoxin, was PCR-amplified under the following conditions using a plasmid DNA (pJH8) purchased from ATCC as a saddle type.
- antisense primer (PE-2) with FLAG tag sequence 10 XKOD-Plus buffer, 2 mM dNTPs, 25 mM MgS04, K0D-Plus (manufactured by Yukara Co., Ltd.), 10 seconds at 98 ° C 5 Second, 68T: 4 minutes, 5 cycles
- PE-1 taagaat tcg gtggcgcgcc ggagt tcccg aaccgtcca ccccgccggg t tc t tc tggt t tagagggcg gcagcc tggc cgcgc tg (SEQ ID NO: 41)
- PE-2 acttagcggc cgctcactac agttcgtcttt ttatcgtc gtcatccttg tagtccggcg gtttgccggg ctggc (SEQ ID NO: 42)
- the amplification product of this PCR reaction was inserted into pGEM-T easy using pGEM-T Easy Vector System I (Promega). The sequence was confirmed by ABI3730 sequencer.
- the PE40 gene cloned into pGEM-Teasy by PCR amplification was digested with EcoRI and Notl, excised from an agarose gel, and this gene fragment was inserted between EcoRI-Notl of pET22b_His to prepare pET22b_His-PE40.
- GA20 antibody single-chain Fv that is, a gene encoding the heavy chain variable region and light chain variable region of GA-20 antibody linked by a 15 amino acid linker sequence ((GlyGlyGlyGlySer) 3 ) (SEQ ID NO: 43)
- the fragment was amplified by PCR under the following conditions.
- the GA-20 antibody heavy chain variable region TA-cloned into pCRII-TOPO is made into a saddle type, and the heavy chain variable region is a sense primer (GA20-1; SEQ ID NO: 44), an antisense primer (GA20-2; SEQ ID NO: 45), while the light chain variable region is sense primer (GA20-3; SEQ IDN0: 46) and antisense primer (GA20-4; SEQ ID N0: 47).
- TAKARA # R005 PCR amplification was performed in 25 cycles at 94 min for 30 min and 1T for 30 min.
- the PCR product of the heavy chain and light chain variable region obtained here was purified with an S-300 HR column (Amersham Biosciences # 27-5130-01), and 1 L each was mixed in the same tube. Combined with pyrobest DNA polymerase, the reaction was carried out at 94 minutes for 1 minute, followed by annealing at 94 cycles for 30 minutes and 72 minutes for 30 minutes. The reaction solution after annealing was reacted with pyrobest DNA polymerase under the following conditions using primers GA20-1 (SEQ ID NO: 44) and GA20-4 (SEQ ID NO: 47). PCR amplification was performed in 30 cycles for 30 minutes, 72 minutes for 1 minute, and 25 cycles.
- the amplified fragment was purified with an S-400 HR column (Amersham Biosciences # 27-5140-01), cut with EcoRI-Hindlll, and cut out from an agarose gel. This was inserted between HindIII and EcoR of pET22b-His-PE40 prepared in 5-2- ⁇ 1, the nucleotide sequence was confirmed, and pET22b_His_GA20scFv-PE40 was prepared.
- the primer sequences used are shown below.
- GA20-1 aaaagcttga ggtccagctg caacagtctg g (SEQ ID NO: 44)
- GA20-2 cccgaaccac caccacccga accaccacca cctgaggaga cggtgactga ggttcc (SEQ ID NO: 45)
- GA20-3 tggttcgggt ggtggtggtt cgggtggtgg cggatcggac attgtgatgt cacagtctcc atcct (SEQ ID NO: 46)
- GA20-4 ttgaattctt tgatttccag cttggtgcct c (SEQ ID NO: 47)
- the nucleotide sequence of the obtained GA20_PE40 is shown in SEQ ID NO: 24, and the amino acid sequence defined by the nucleotide sequence is shown in SEQ ID NO: 25. 5-2-2. Purification of toxin-labeled GA-20_—single chain Fv antibody (GA20_PE40)
- E. coli BL21 strain transformed with pET22b_His—GA20scFv-PE40 was plated on LB Agar plate containing 50 g / ml ampicillin. The grown single colonies were picked up and cultured in LB medium (3 ml) containing 50 ig / ml carbenicillin (Cosmo Bio). After culturing for 4 hours, the grown bacteria were expanded to LB medium containing 200 ml of carbenicillin (50 ⁇ g / ml) and cultivation was continued. When the logarithmic growth phase was reached, the culture medium was replaced with fresh LB medium (200 ml containing carbenicillin), and IPTG (final ImM) was added to induce protein expression. After culturing for 5 hours, the bacteria were collected by centrifugation.
- the protein adsorbed on the column was washed with a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4), and elution buffer (20 mM sodium phosphate, 0.5 M NaCK 500 mM imidazole, pH 7.4). ), Elution was carried out by separating a total of 7 fractions of 500 / xl.
- ELISA assay using the binding activity to GRP78 as an index was performed to examine the eluted fraction binding activity.
- ELISA was performed as follows. GST-GRP78 purified from E. coli, or HB-EGF protein (R & D) as a negative control (plate) coated with lzg / ml (NUNC), Diluent buffer (1% BSA, 50 mM Tris, 1 mM MgCl 2. Each fraction diluted 40-fold with 150 mM NaCK 0.05% Tween20) was added.
- the obtained GA20-PE40 was analyzed for follicle death inducing activity.
- Hamster ovary cells DG44 were seeded in 96 well plates at lX10 3 / well and prostate cancer cell lines DU145 and 22Rvl at 6 ⁇ 10 3 / well at 90 1 / well. The next day, GA20-PE40 fraction (Elute 2 3, 4) and 10 1 / wel l of PBS were added and incubated at 37 ° C. Five days later, the number of viable cells was measured with WST-8 (Dojindo Laboratories) reagent, and the ratio of viable cells compared to the control (PBS added group) was quantified and graphed.
- WST-8 Dojindo Laboratories
- Example 6 Obtaining anti-GRP78 antibody by reimmunization
- E. coli was collected and solubilized with 25-1111 8-1 (Pierce). Next, this solubilized E. coli lysate was diluted 10-fold with PBS, and Gluyuthione Sepha mouth 4B (Amersham Almasia) equilibrated with PBS was added thereto. Incubated. Thereafter, dartathione sepharose 4B was washed several times with PBS to remove unadsorbed protein, and the target protein was eluted with 20 mM dull thione.
- the eluted fractions were stained with CBB after SDS-PAGE, and the fractions containing the protein of interest were collected. This sample was further separated from impure protein in PBS using gel filtration chromatography (Superdex 200 16/60, GE Healthcare), and only the target protein was purified to high purity. Use this purified protein as an immunogen for the following experiments. I used it.
- Example 6-1 GST-GRP78 (376-415) purified in Example 6-1 was prepared in the same manner as in Example IV-2, with the mouse [(MRL / lpr, male, 4 weeks old) (Balb / c, female, 6 (Week-aged): Both were immunized by purchase from Nippon Chiyuru River. Hypridoma was produced as described in Example IV-3.
- a fusion protein of GRP78 (amino acids 376 to 415) and maltose binding protein (MBP) (MBP-GRP78 (376-415)) was prepared as follows.
- PGEX-GRP78 (376-415) prepared in Example 4 was digested with BamHI-Sall, and a gene fragment encoding GRP78 (376-415) was excised. This was inserted between BamHI-Sal I of pMAL-c2X (New England BioLabs) to construct MBP-GRP 78 (376-415) expression vector pMAL-c2X-GRP78 (376-415).
- Escherichia coli BL21 strain transformed with this vector was cultured in LB medium (250 ml), and when 0D 61fl reached 0.5 or higher, protein expression was induced by IPTG (1 mM). After culturing for 5 hours, E. coli was collected by centrifugation and solubilized with 25 ml of B-PER (Pierce). Next, this solubilized Escherichia coli lysate was diluted 5-fold with a column buffer (20 mM Tris, H 7.5, 200 mM NaCK 1 mM EDTA) and equilibrated with the column buffer. One resin (New Engl and BioLabs) was added and incubated at 4 ° C.
- amylose resin was washed several times with a column buffer to remove unadsorbed protein, and the target protein was eluted with an elution buffer (a column buffer containing lO mM matrix).
- an elution buffer a column buffer containing lO mM matrix.
- the fractions containing the protein of interest were identified by SDS-PAGE after SDS-PAGE, and the fractions containing the protein of interest were combined, and these were combined into one and buffered and replaced with PBS using a PD10 column.
- the purified MBP-GRP78 (376-415) was used for the following experiment for ELISA screening of the bound antibody. 6-3-2.
- GRP78-binding antibody by ELISA (Primary screening) Using an ELISA plate coated with MBP-GRP78 (376-415) purified in Example 6-3-1 at 1 g / ml. An antibody that binds to the region of amino acids 376 to 415 of the GRP78 protein was screened.
- Example 2-1 The method is as described in Example 2-1.
- the GRP78-binding antibody selected by the primary screening using the binding activity to the MBP-GRP78 (376-415) protein as an index was subsequently subjected to the secondary screening described below.
- 6-3-3 Screening of anti-GRP78 antibody localized on the cell surface by FACS (secondary screening)
- the GRP78-binding antibody obtained in the primary screening was then subjected to secondary screening using the binding activity against prostate cancer cell lines (DU145 and 22Rvl (ATCC CRL-2505)) as an index.
- the method is as described in Example 2-2.
- FACS analysis was performed using a prostate cancer cell line (22Rvl). Cells were stained with each antibody (10 / zg / ml), and then FACS analysis was performed by the method described in Example 3-2-1.
- the four antibodies obtained here are antibodies that recognize the partial region of amino acids 376 to 415 of GRP78 because they are established by immunization with GST-GRP78 (376-415) protein. . Therefore, this region is further divided into four regions (amino acids 376 to 391 (ie, amino acids 1 to 16 of SEQ ID NO: 3), amino acids 384 to 399 (ie, SEQ ID N0) as shown in FIG. 12A. : Amino acids 9-24 of 3), amino acids 392-407 (ie, amino acids 17-32 of SEQ ID NO: 3), amino acids 400-415 (ie, amino acids 25-40 of SEQ ID NO: 3) ) And 4 types of antibodies were examined to determine which sequence among amino acids 376 to 415 of GRP78 was recognized.
- DNA fragments encoding regions consisting of amino acids 376 to 391, amino acids 384 to 399, amino acids 392 to 407, and amino acids 400 to 415 of the GRP78 protein were prepared as follows.
- the DNA fragment encoding GRP78 (376-391) (ie, amino acids 1-16 of SEQ ID NO: 3) is an oligomer (GEP1 / GEP2; SEQ ID N0: 48 and 49, respectively), GRP78 (384-399) (Ie SEQ ID NO: 3 amino acids 9-24) DNA fragments encoding oligomers (GEP3 / GEP4; SEQ ID N0: 50 and 51 respectively), GRP78 (392-407) (ie SEQ ID NO: The DNA fragment encoding 3 amino acids 17-32) is an oligomer (GEP5 / GEP6; it SEQ ID NOs: 52 and 53) and GRP78 (400-415) (ie, amino acids 25-40 of SEQ ID NO: 3) are oligomers (GEP7 / GEP8; SEQ IDN0: 54 and 55) were annealed respectively.
- GEP1 gatccaaaga gttcttcaat ggcaaggaac catcccgtgg cataaaccca gate (SEQ ID NO: 48)
- GEP2 tcgagatctg ggtttatgcc acgggatggt tccttgccat tgaagaactc tttg (SEQ ID NO: 49)
- GEP3 gatccccatc ccgtggcata aacccagatg aagctgtagc gtatggtgct gctc (SEQ ID NO:
- GEP tcgagagcag caccatacgc tacagcttca tctgggttta tgccacggga tggg (SEQ ID NO: 51)
- GEP5 gatccgaagc tgtagcgtat ggtgctgctg tccaggctgg tgtgctctct ggtc (SEQ ID NO: 15 52)
- GEP6 tcgagaccag agagcacacc agcctggaca gcagcaccat acgctacagc ttcg (SEQ ID NO: 53)
- GEP7 gatccgtcca ggctggtgtg ctctctggtg atcaagatac aggtgacctg gtac (SEQ ID NO: 54)
- GEP8 tcgagtacca ggtcacctgt atcttgatca ccagagagca caccagcctg gacg (SEQ ID NO:
- the DNA fragment produced here is inserted downstream of the GST coding region of the E. coli GST fusion expression vector (PGEX-6P-1) cleaved with BamHI and Xhol., And the GRP78-GST fusion protein that encodes each region is expressed.
- Vectors (PGEX-GRP78 (376-391), pGEX-GRP78 (384-399), 25 PGEX-GRP78 (392-407), and pGEX-GRP78 (400-415), respectively) were constructed.
- E. coli expression vectors were transformed into E. coli BL21 Protein expression was induced by the method described in Example 4- ⁇ 2. As a result, as shown in FIG. 12B, it was confirmed that the target protein was expressed in E. coli. Therefore, this protein was used for the epitope map below. 6-4-2-2. Epitope map of each antibody
- the GD-17 antibody has a region spanning amino acids 38 to 391 of GRP78, and the GC-18 and GC-20 antibodies span a region spanning amino acids 392 to 407. It was also found that the GD-4 antibody recognizes the region from amino acid positions 400 to 415. It was also found that the GA-20 antibody obtained early recognizes the same region as the GD-4 antibody (Fig. 13).
- VH-G1 primer SEQ ID NO: 56
- VH-G1 cca cca gat tc t tat cag aca gg (SEQ ID NO: 56)
- the base sequence of the heavy chain variable region of the GC-18 antibody that binds to the region spanning amino acids 392 to 407 of GRP78 is SEQ ID NO: 57
- the base sequence amino acid sequence of the heavy chain variable region is SEQ ID NO: 58
- the light chain variable region base sequence is SEQ ID NO: 59
- the light chain variable region amino acid sequence is Described in SEQ ID NO: 60, respectively.
- the amino acid sequence of CDR1 of the heavy chain variable region of the GC-18 antibody is SEQ ID NO: 61
- the amino acid sequence of CDR2 is SEQ ID NO: 62
- the amino acid sequence of CDR3 is SEQ ID NO: 63.
- the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 64
- the amino acid sequence of CDR2 is shown in SEQ ID NO: 65
- the amino acid sequence of CDR3 is shown in SEQ ID NO: 66, respectively.
- the nucleotide sequence of the heavy chain variable region of the GC-20 antibody that binds to the region spanning amino acids 392 to 407 of GRP78 is SEQ ID NO: 67, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 68.
- the nucleotide sequence of the light chain variable region is shown in SEQ ID N0: 69, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 70.
- the CDR1 amino acid sequence of the heavy chain variable region of the GC-20 antibody is SEQ ID NO: 71
- the amino acid sequence of CDR2 is SEQ ID NO: 72
- the amino acid sequence of CDR3 is SEQ ID NO: 73
- the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 74
- the amino acid sequence of CDR2 is shown in SEQ ID NO: 75
- the amino acid sequence of CDR3 is shown in SEQ ID NO: 76, respectively.
- the nucleotide sequence of the heavy chain variable region of the GD-4 antibody that binds to the region of amino acids 400 to 415 of GRP78 is SEQ ID NO: 77
- the amino acid sequence of the heavy chain variable region is SEQ ID NO: 78
- the light chain is shown in SEQ ID NO: 79
- the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 80, respectively.
- amino acid sequence of CDR1 of the heavy chain variable region of the GD-4 antibody is SEQ ID N0: 81
- the amino acid sequence of CDR2 is SEQ ID NO: 82
- the amino acid sequence of CDR3 is SEQ ID NO: 83
- the light chain The amino acid sequence of CDR1 of the variable region is shown in SEQ ID NO: 84
- the amino acid sequence of CDR2 is shown in SEQ ID NO: 85
- the amino acid sequence of CDR3 is shown in SEQ ID NO: 86, respectively.
- SEQ ID NO: 87 for the nucleotide sequence of the heavy chain variable region of the GD-17 antibody that binds to the region spanning amino acids 384 to 391 of GRP78
- SEQ ID NO: 88 for the heavy chain variable region.
- the nucleotide sequence of the light chain variable region is shown in SEQ ID N0: 89
- the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 90.
- the amino acid sequence of CDR1 of the heavy chain variable region of the GD-17 antibody The column is SEQ ID NO: 91
- the amino acid sequence of CDR2 is SEQ ID NO: 92
- the amino acid sequence of CDR3 is SEQ ID NO: 93
- the amino acid sequence of CDR1 of the light chain variable region is SEQ ID NO: 94.
- the amino acid sequence of CDR2 is described in SEQ ID NO: 95
- the amino acid sequence of CDR3 is described in SEQ ID NO: 96, respectively.
- Example 7 Analysis of drug efficacy with toxin-labeled GDI 7-chain antibody (GD17- scFv-PE40) 7-1. Preparation of GD17_scFv-PE40 expression vector
- GD-17 antibody single-chain Fv that is, a linker sequence consisting of 15 amino acids
- the GD-17 antibody heavy chain variable region TA-cloned into pCRI I-T0P0 is made into a saddle type, and the heavy chain variable region is composed of a sense primer (GD17-1, SEQ ID NO: 97) and an antisense primer (GDI 7- 2, SEQ ID NO: 98), while the light chain variable region uses sense primer (GD17-3, SEQ ID NO: 99) and antisense primer (GDI 7-4, SEQ ID NO: 100)
- sense primer GD17-3, SEQ ID NO: 99
- antisense primer GDI 7-4, SEQ ID NO: 100
- the PCR product of the heavy chain and light chain variable region obtained here was purified with an S-300 HR column (Amersham Biosciences # 27-5130-01), and 1 / L each was placed in the same tube. After mixing and reaction with pyrobest DNA polymerase at 94 for 1 minute, annealing was performed at 94, 30 minutes, 72 30 minutes, and 5 cycles.
- the amplified fragment was purified with an S-400 HR column (Amersham Biosciences # 27-5140-01), cut with EcoRI-Hindlll, and cut out from an agarose gel. This was inserted between HindIII and EcoRI of pET22b-His-PE40 prepared in Example 5-2- ⁇ 1, the nucleotide sequence was confirmed, and pET22b_His GD17scFv-PE40 was prepared.
- the primer sequences used are shown below.
- GD17-1 aaaagcttca ggttcagctc cagcagtctg g (SEQ ID NO: 97)
- GDI 7-2 cccgaaccac caccacccga accaccacca cctgaggaga ctgtgagagt ggtgcct (SEQ ID NO: 98)
- GDI 7-3 tggttcgggt ggtggtggtt cgggtggtgg cggatcggat gttgtgatga cccaaactcc ac (SEQ ID NO: 99)
- GDI 7-4 tlgaattctt tcagctccag cttggtccc (SEQ ID NO: 100)
- SEQ ID NO: 101 The base sequence of the obtained GD17scFv_PE40 is shown in SEQ ID NO: 101, and the amino acid sequence defined by the base sequence is shown in SEQ ID NO: 102.
- the Escherichia coli BL21 strain transformed with pET22b_His_GD17scFv-PE40 was cultured in LB medium containing carbenici 11 in (50 g / ml). When the logarithmic growth phase was reached, IPTG (final dose 1 mM) was added and incubated overnight at room temperature (at 24) for protein induction. Escherichia coli recovered by centrifugation is suspended in a binding buffer (20 mM sodium phosphate, 500 mM NaCK, 20 mM imidazole, pH 7.4), disrupted by sonication, and the soluble fraction is removed from the HisTrapFF crude column ( GE Healthcare).
- a binding buffer (20 mM sodium phosphate, 500 mM NaCK, 20 mM imidazole, pH 7.4
- the target protein is then eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4), diluted about 10 times with TBS buffer, and then filled with M2 agarose (Sigma). I put it on the Affinity Gel.
- the target protein is eluted with M2 elution buffer (0.1 M glycine-HC1, pH. 3.5) using AKTA Explorer (GE Healthcare), and quickly eluted into PBS using a PD10 column (GE Healthcare).
- the purified GD17scFv-PE40 which was obtained by substituting the buffer and used as the final sample, was subjected to SDS-PAGE and CBB staining to confirm that it was purified with 100% purity (Fig. 14).
- GST-GRP78 (l ⁇ g) was prepared by diluting each sample with a dilution buffer (1% BSA, 50 mM Tris, 1 mM MgCl 2 , 150 mM NaCK 0.05% Tween 20) and purifying it from E. coli. / ml) was added to the plate (NUNC) coated. After reaction at room temperature for 1 hour, wash the plate 3 times with TBS-T (TBS-0. 05% Tween 20), add 1 zg / ml of anti-Flag antibody (M2 antibody, Sigma), and incubate at room temperature for 1 hour . After washing again with TBS-T three times, the reaction was performed with alkaline phosphatase-labeled anti-mouse IgG (ZYMED) for 1 hour, and color was developed with 1 mg / ml substrate (Sigma).
- a dilution buffer 1% BSA, 50 mM Tris, 1 mM MgCl 2
- cancer cell lines 22Rvl, LNcap, MCF7, BxPC3, PANCK SK0V3 or human-derived normal cell lines (HUVEC, MRC5), mouse-derived normal cell lines (CH0, NIH3T3, BaF3) were used for analysis.
- the cell lines used in the experiments were purchased from CAMBREX for HUVEC and from ATCC for other cell lines, and cultured according to the instructions from the source.
- GD17scFv-PE40 was diluted with RPMI 1640 medium (Invitrogen) containing 10% FCS and added to the cells. After culturing for 5 days, the number of viable cells was measured with WST-8 (Nacalai).
- the concentration (EC 5. Value) that shows 50% of the maximum activity is about 2 to 20 ⁇ , indicating strong cell death-inducing activity. It was confirmed that it has (Fig. 16 ⁇ ). In particular, for MCF7 and 22Rvl cells, the EC 5fl value was about 2-4 ⁇ , confirming strong cytotoxic activity. On the other hand, for human and mouse normal cells There was no activity at all, or slight cytotoxicity was confirmed when added at a high concentration (FIG. 16B).
- Human prostate cancer cell line 22Rvl cells (ATCC CRL-2505) were collected with 0.02% EDTA solution containing 0.05% trypsin, and then the number of cells was 1X10 7 cells / 0.2 mL HBSS (SIGMA Cat. No. H 9269). , 7 weeks old (CAnN. Cg-Foxnl ⁇ nu> / CrlCr1j (BALB-nu / nu)): Nippon Chiya-Lus Ribaichi Co., Ltd. was transplanted subcutaneously into the abdomen.
- Tumor engraftment was confirmed, and on the 16th day of transplantation (day 16), the tumor volume and body weight were divided into 7 groups (control group 1 group, drug administration group 6 groups), the day after grouping (dayl7), On day 21, day 23, day 26, and day 29, physiological saline was administered to the control group and GD17scFv-PE40 at a dose of 0.5 mg / kg was instantaneously administered intravenously at a dose of 10 mL / kg to the drug administration group. Tumor volume was measured over time, and the final measurement was 2 days after the last administration (day 31).
- Figure 18 shows the results.
- the tumor growth inhibition rate at the final measurement was 47%, and as a result of analyzing the tumor volume by non-parametric Dunnett multiple comparison, a significant tumor growth inhibitory effect was found in the 0.5 mg / kg group. . From this result, GRP78 was targeted
- the present invention provides a novel antibody having binding activity to GRP78 and capable of internalizing, thereby treating various tumors and cancers in which GRP78 is exposed on the cell surface. It has been shown that novel pharmaceutical compositions that can be used can be provided. In addition, by using an antibody having such characteristics, it is possible to provide diagnostic methods for various tumors and cancers.
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Abstract
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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EP08721318.7A EP2130552B1 (en) | 2007-02-27 | 2008-02-27 | Pharmaceutical composition comprising anti-grp78 antibody as active ingredient |
AU2008219972A AU2008219972A1 (en) | 2007-02-27 | 2008-02-27 | Pharmaceutical composition comprising anti-GRP78 antibody as active ingredient |
CA002679266A CA2679266A1 (en) | 2007-02-27 | 2008-02-27 | Pharmaceutical composition comprising anti-grp78 antibody as active ingredient |
JP2009501332A JP5374360B2 (ja) | 2007-02-27 | 2008-02-27 | 抗grp78抗体を有効成分として含む医薬組成物 |
CN2008800024441A CN101951953A (zh) | 2007-02-27 | 2008-02-27 | 含抗grp78抗体作为有效成分的药物组合物 |
US12/449,778 US8192740B2 (en) | 2007-02-27 | 2008-02-27 | Pharmaceutical composition comprising anti-GRP78 antibody as active ingredient |
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JP2007-047534 | 2007-02-27 | ||
JP2007047534 | 2007-02-27 |
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EP (1) | EP2130552B1 (ja) |
JP (1) | JP5374360B2 (ja) |
CN (1) | CN101951953A (ja) |
AU (1) | AU2008219972A1 (ja) |
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JPWO2008105560A1 (ja) | 2010-06-03 |
JP5374360B2 (ja) | 2013-12-25 |
CN101951953A (zh) | 2011-01-19 |
AU2008219972A1 (en) | 2008-09-04 |
EP2130552A4 (en) | 2011-03-02 |
EP2130552A1 (en) | 2009-12-09 |
US8192740B2 (en) | 2012-06-05 |
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EP2130552B1 (en) | 2016-06-15 |
US20100041074A1 (en) | 2010-02-18 |
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