HK1152265A - Pharmaceutical composition comprising anti-grp78 antibody as active ingredient - Google Patents

Description

Pharmaceutical composition containing anti-GRP 78 antibody as active ingredient

Technical Field

The present invention relates to methods of treating cancer and anticancer agents.

Background

The GRP protein (glucose regulating protein) is a chaperone located in the ER. For example, it is known that when glucose is deficient or a protein with an abnormal higher structure accumulates in the vesicle (ER), the expression of cells is induced in response to various ER stresses from the inside and outside of the cell (non-patent document 1).

GRP78 is a GRP protein with a molecular weight of 78kDa, also known as BiP (immunoglobulin-binding protein). It was found that GRP78 is involved in a defense response against cell death caused by ER stress, as a result of an overexpression experiment or antisense suppression (Knock down) experiment of GRP78 (non-patent document 1).

Conditions such as low nutrition, hypoxia, low pH presented by cancerous cells or the altered in vivo environment resulting from the carcinogenesis are often considered to occur with ER stress. In support of this, expression of GRP78 protein was found to be elevated in a variety of cancer cell lines and clinical cancer specimens (non-patent documents 2 to 5). It has been found through experiments that the acquisition of anticancer activity of an anticancer agent having topoisomerase inhibitory activity or angiogenesis inhibitory activity is closely related to the increase in GRP78 protein expression and its anti-apoptotic activity (non-patent documents 6 and 7). It has also been found that the group of breast cancer patients with GRP 78-resistant expression showed lower expression than GRP78, and that chemotherapy with doxorubicin had a lower effect (non-patent document 8).

These reports suggest that expression resistance of GRP78 plays a role in the mechanism of survival and deterioration of cancer cells and resistance to anticancer agents (non-patent document 1).

As described above, GRP78 is known as a chaperone localized in the ER, and GRP78 protein has been reported to be localized on the cell surface in some cancer cells. And the possibility of applying antitumor agents targeting the cell surface localized GRP78 was suggested by several entirely different approaches.

FACS analysis after treatment of rhabdomyosarcoma cell line TE671/RD with thapsigargin (Tg) confirmed that there were a few cell membranes stained with anti-GRP 78 antibody, indicating that GRP78 was located on the cell membrane (non-patent document 9).

However, the reports only indicate transient events in cell death induced by Tg treatment, and are not data indicating a constant change in GRP78 position in cancer cells. And since the antibody used was a commercially available goat-derived polyclonal antibody, the epitope thereof was not clarified.

Thereafter, other groups obtained 2 GRP 78-binding peptides (WIFPWIQL, WDLAWMFRLPVG) by phage binding assay, and reported that the peptides not only bound to GRP78 protein immobilized on the plate, but also adsorbed to the cell surface of prostate cancer cell line DU145 and taken up by the cells (non-patent document 10). This experiment revealed the possibility that GRP78 is located on the cell membrane of cancer cells.

The possibility of using the GRP78 binding peptide as an antitumor agent was also confirmed. That is, it has been reported that an apoptosis-inducing peptide sequence (KLAKLAK) is added to the GRP 78-binding peptide₂The synthesized peptide (non-patent document 11) not only induced cell death of DU145 cells in vitro, but also exhibited an antitumor effect in an in vivo experiment using a mouse transplantation model (non-patent document 10).

In addition, other groups have found that the target of K5 is GRP78 protein expressed by vascular endothelial cells in the process of searching for a target molecule recognized by a domain known as Kringle5(K5) of plasminogen which inhibits angiogenesis (non-patent document 16). It was also found that recombinant K5 not only inhibited angiogenesis but also induced cell death in various cancer cell lines cultured under hypoxic conditions by GRP78 (non-patent document 16).

This series of experiments indicates that peptides that bind to GRP78 expressed on the cell surface of cancer cells or vascular endothelial cells can be used as antitumor agents. However, it is not clear which part of GRP78 is considered to be expressed on the cell surface, and therefore, it is considered to be somewhat difficult to develop a drug using the peptide.

In contrast to this view, another 2 independent groups reported that GRP78 was localized on the cell membrane in a different manner.

A research group found that α 2 macroglobulin (α 2M;) recognizing a receptor functions as a proliferation factor for prostate cancer cell lines (1-LN) (non-patent document 12); thereafter, the group found that the receptor was GRP78 (non-patent document 13). This finding suggests that the GRP78 protein, which has long been recognized as an ER protein, also functions as a receptor for proliferation factors on cell membranes.

The other group found that an antibody recognizing the peptide sequence "CNVSDKSC" (anti-CNVSDKSC antibody) was present in the serum of prostate cancer patients and found in the process of analyzing the antigen protein recognized by the antibody, i.e., GRP78 protein (non-patent document 14). But in fact the sequence "CNVSDKSC" or its analogous sequence is not present in GRP78, it is not clear which part of GRP78 the antibody from prostate cancer patients recognize.

Thereafter, other groups performed tertiary structure analysis on the peptide "CNVSDKSC" and searched for the presence of regions with the same tertiary structure in GRP 78. As a result, Leu was found to be located at GRP78⁹⁸-Leu¹¹⁵The sequence "LIGRTWNDPSVQQDIKFL" of (a) corresponds to the sequence. After rabbit polyclonal antibodies against the sequences were prepared, it was found that the antibodies stained the cell surface of cancer cells such as prostate cancer strain 1-LN, DU145, melanocyte strain DM 413. It was also found that the antibody can increase the intracellular calcium concentration of prostate cancer cells, induce cell proliferation of prostate cancer cells, and exhibit an anti-apoptotic effect on TNF α, as observed when α 2M is added (non-patent document 15). Thus, from Leu to GRP78⁹⁸-Leu¹¹⁵The antibody of (3) mimics the ligand activity of α 2M, and it was found that this region in GRP78 is a binding sequence of α 2M (non-patent document 15).

This report confirmed that GRP78 is present on the cell surface of prostate cancer and the like, and made clear the Leu of GRP78⁹⁸-Leu¹¹⁵(LIGRTWNDPSVQQDIKFL) is exposed extracellularly as α 2M binding sites.

Furthermore, it was found by other means that antibodies directed against the region 98-115 of GRP78 could stain the cell surface of cancer cells, thereby making clear that the region could be an extracellular epitope of GRP 78.

Although elevated expression of GRP78 protein has been found in many cancers as described and its localization to the cell membrane is clear, it is still difficult to exploit this knowledge to prepare new antibody therapeutics targeting GRP 78. The reason is, firstly, that it is not possible to prepare antibodies expected to have the same effect as the GRP78 binding peptide, since it is not known that the peptide binds to that region of the GRP78 protein. There are virtually no monoclonal antibodies that can replace the action of the peptide. Second, although antibodies that bind to the 98-115 region of GRP78 bind to the cell surface of cancer cells, they mimic the proliferation-promoting effect of α 2M, and are not expected to have antitumor activity.

It is therefore considered difficult to inhibit tumor activity using antibodies that bind to GRP 78.

Non-patent document 1: lee AS. trends Biochem Sci.2001, 26, 504-10

Non-patent document 2: patierno, et al.1998, Cancer Re s.47, 6220-24

Non-patent document 3: bini et al 1997, electrophosphoresis.18, 2832-41

Non-patent document 4: gazit et al 1999, Breast Cancer Res. Treat.54, 135-46

Non-patent document 5: fernandez et al.2000, Breast Cancer Res. Treat.59, 15-26

Non-patent document 6: reddy et al, J.Bio.chem.2003, 278, 20915-24

Non-patent document 7: dong et al, 2005, Cancer Res.65, 5785-

Non-patent document 8: lee et al.2006, Cancer Res.66, 7849-

Non-patent document 9: delpino et al 1998, Molecular Membrane biology, 15, 21-26

Non-patent document 10: arap et al 2004, CANCER CELL.6, 275-

Non-patent document 11: javadpour et al 1996, J.Med.chem.39, 3107-

Non-patent document 12: application et al.2000, Archives of biochemistry and biophysics.383, 135-141

Non-patent document 13: misra et al 2002, J.biol.chem.277, 42082-

Non-patent document 14: mintz et al 2003, Nat Biotech.21, 57-63

Non-patent document 15: Gonzalez-Gronow et al 2006, Cancer Res.66, 11424-

Non-patent document 16: davidoson et al, 2005, Cancer Res.65, 4663-

Disclosure of Invention

Problems to be solved by the invention

The present invention aims to provide novel pharmaceutical compositions using anti-GRP 78 antibodies. The present invention more specifically aims to provide a novel cancer treatment method using an anti-GRP 78 antibody, a novel cell proliferation inhibitor and an anticancer agent containing an anti-GRP 78 antibody, and a novel anti-GRP 78 antibody.

Means for solving the problems

The inventors have attempted to prepare anti-tumor antibodies that target GRP78 localized to the cell membrane of cancer cells. For this reason, it is necessary to first identify amino acids that can become epitopes of antibodies exposed to the cell surface of cancer cells. Therefore, the GRP78 protein was purified, mice were immunized with it, and antibodies were screened for staining only cancer cells. As a result, an anti-GRP 78 antibody that specifically binds to the cell surface of cancer cells was successfully obtained. Attempts were then made to identify sequences that recognized the resulting antibodies. The antibody specifically recognizes 40 amino acids 376-415 of GRP78 as found by analysis. That is, the 376-415 amino acid region of GRP78 was found to be exposed extracellularly. It was next confirmed that the antibody recognizing the epitope was rapidly taken up by the cells. The scFv antibody labeled with the toxin specifically kills cancer cells by preparing the scFv antibody to which the toxin is added and analyzing the in vitro effect on cancer cell lines. When the effect of the antibody was analyzed using a xenograft mouse model in which cancer cells were transplanted, it was observed that the administration of the antibody significantly inhibited the proliferation of the transplanted tumor. This result confirmed that the antibody exerts antitumor activity not only in vitro but also in vivo. The above knowledge suggests that antibodies directed against the extracellular region of GRP78 can be used as anti-tumor agents.

The present inventors have recognized based on the above knowledge that the above problems can be solved.

Specifically, the following (1) to (28) are provided.

(1) A pharmaceutical composition comprising an antibody that binds to glucose regulatory protein 78(GRP 78).

(2) The composition of (1), which is an anticancer agent.

(3) The composition of (1) or (2), wherein the antibody is a monoclonal antibody.

(4) The composition of any one of (1) - (3), wherein said antibody is an antibody that binds to GRP78 located on the surface of a cell.

(5) The composition of any one of (1) - (4), wherein said antibody is an antibody that is taken up by a cell expressing GRP 78.

(6) The composition of any one of (1) - (5), wherein the antibody is a monoclonal antibody that binds to SEQ id no: 3.

(7) The composition of any one of (1) to (6), wherein the antibody is an antibody that binds to a wounded cellular substance.

(8) A monoclonal antibody that binds to GRP 78.

(9) The antibody according to (8), which is characterized by binding to GRP78 expressed on the surface of a cell.

(10) The antibody according to (8) or (9), which is characterized by being taken up by a cell expressing GRP 78.

(11) (8) the antibody of any one of (8) to (10), characterized by having the amino acid sequence shown in SEQ ID NO: 3.

(12) The antibody according to any one of (8) to (10), which is characterized by recognizing the same epitope as that recognized by the antibody according to any one of the following (a) to (f):

(a) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 8, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 9, the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in the figure 10,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 11, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 12, or a pharmaceutically acceptable salt thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 13;

(b) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 18, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 19, the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof, wherein,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 21, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 22, or a sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 23;

(c) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 61, or a sequence of amino acids,

CDR2 has the amino acid sequence of SEQ ID NO: 62, or a sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 63, or a fragment thereof,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 64, or a sequence shown in SEQ ID NO,

CDR2 has the amino acid sequence of SEQ ID NO: 65, and (b) an amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 66;

(d) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 71, or a nucleotide sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 72, or a sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 73 with a sequence of an amino acid sequence shown in SEQ ID NO,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 74, or a sequence shown in SEQ ID NO,

CDR2 has the amino acid sequence of SEQ ID NO: 75, the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 76;

(e) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 81, or a sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 82, or a pharmaceutically acceptable salt thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 83 of a sequence of amino acids as shown in SEQ ID NO,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 84, and the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 85, or a pharmaceutically acceptable salt thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 86;

(f) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 91, or a sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 92, or a nucleotide sequence thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 93 (b) to (b) a sequence shown as 93,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 94, or a pharmaceutically acceptable salt thereof,

CDR2 has the amino acid sequence of SEQ ID NO: 95 of the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 96.

(13) The antibody according to any one of (8) to (12), which is characterized by having a cell damaging activity on a cell expressing GRP 78.

(14) The antibody according to (13), which is characterized by binding a cell-damaging substance.

(15) A method of delivering cell-damaging material into a cell using an anti-GRP 78 antibody.

(16) A method of inhibiting cell proliferation by a cell damaging agent that binds to an anti-GRP 78 antibody.

(17) The method of (15) or (16), wherein the cell is a cancer cell.

(18) Use of an anti-GRP 78 antibody for the delivery of a cell damaging substance into a cell.

(19) Use of an anti-GRP 78 antibody having cellular uptake activity for inhibiting cell proliferation.

(20) The use of (18) or (19), wherein the cell is a cancer cell.

(21) Use according to any of (18) to (20), characterized in that the anti-GRP 78 antibody binds to a cell damaging substance.

(22) A method of preparing a pharmaceutical composition comprising:

(a) providing an anti-GRP 78 antibody;

(b) confirming whether the antibody of (a) has an activity of being taken up by cells;

(c) screening for antibodies having cellular uptake activity;

(d) binding of the antibody selected in (c) to the cell damaging material.

(23) The method of (22), wherein the pharmaceutical composition is an anticancer agent.

(24) A method of diagnosing cancer using an anti-GRP 78 antibody.

(25) The method of (24), which is characterized by using an anti-GRP 78 antibody conjugated with a label.

(26) The method of (24) or (25), characterized by detecting an anti-GRP 78 antibody taken into the cell.

(27) An anti-GRP 78 antibody conjugated to a label.

(28) Consisting of SEQ ID NO: 3 or a fragment thereof.

Effects of the invention

The present invention shows that it is possible to provide a novel pharmaceutical composition useful for treating various tumors or cancers exposing GRP78 to the cell surface by providing a novel antibody having GRP78 binding activity and being taken up by cells. Methods of diagnosing various tumors or cancers can also be provided by using antibodies having such characteristics.

Brief Description of Drawings

FIG. 1: the resulting antibody was analyzed by western blot for binding activity to GRP 78. In lane 1, cell lysate prepared from DU145 cells, in lane 2 GST-GRP78 fusion protein prepared from E.coli, stained with each antibody. The antiserum was mouse antiserum collected prior to cell fusion.

FIG. 2: the resulting anti-GRP 78 antibody was analyzed by FACS for binding activity to the cell surface of DU145 cells.

FIG. 3: graphs of the binding activity of the GA-20 antibody to the cell surface of various cancer cells were analyzed by FACS.

FIG. 4: (A) graphs analyzing the binding activity of the GA-20 antibody to the cell surface of various immortalized cell lines by FACS; (B) western blotting using GA-20 was performed to analyze the pattern of GRP78 protein expression for various immortalized cell lines.

FIG. 5: the activities of GA-20 and GA-21 antibodies taken into cells were analyzed by FACS. Each antibody was reacted with DU145 cells, and after incubation at 0 ℃ or 37 ℃ for 2 hours, the antibody bound to the cell surface was detected with a secondary antibody (FITC-labeled anti-mouse IgG antibody).

FIG. 6: a graph was analyzed by cell immunostaining for whether the GA-20 antibody bound to the cell surface of DU145, and the GA-31 antibody not bound to the cell surface of the cells were taken into the cells. Each antibody was added to DU145 cells in culture, and cultured continuously at 37 ℃ for 3 hours. The cells were then processed as shown in the flow chart below fig. 6, and antibodies taken into the cells were detected.

FIG. 7: the epitope of each antibody bound to GRP78 was mapped by western blot analysis. The upper panel shows a schematic diagram of GST-GRP78 fusion proteins (1-6) used in epitope analysis. The lower panel shows the results of SDS-PAGE of each GST-GRP78 fusion protein (1-6) and Western blotting of the reactivity of each antibody against each protein.

FIG. 8: FIG. of the results of Western blot analysis for further purification of the epitopes within GRP78 of GA-20 and GA-21. The top panel is a schematic diagram of a smaller range of GST-GRP78 fusion proteins. The lower panel shows the results of SDS-PAGE of GST-GRP78 fusion proteins (1-5) and Western blotting of the reactivity of GA-20 and GA-21 antibodies against the respective proteins.

FIG. 9: shows the results of analysis of which fraction the toxin-labeled GA-20scFv antibody (GA20-PE40) purified from Escherichia coli was eluted from by ELISA system using the binding activity to GRP78 as an index. The upper panel shows a schematic diagram of an ELISA system for detecting the binding activity of GA20-PE40 to GRP78, and the lower panel shows the binding activity of the eluted fractions obtained from the ELISA results.

"initial fraction" means E.coli lysate subjected to induction of expression of GA20-PE40,

"pass fraction" means the fraction of lysate applied to the HisTrap column that passes directly through the column,

"wash fraction" means the fraction of the washed column,

"elution fraction 1" - "elution fraction 7" means the elution fraction eluted from the column.

FIG. 10: shows the cell-damaging activity of purified GA20-PE40 on DU145 cells (FIG. 10A), 22Rv1 cells (FIG. 10B) or DG44 cells (FIG. 10C). The elution fractions (elution fractions 2, 3, 4) were added to each cell at a concentration of 10%, followed by determination of the number of viable cells and quantification of the ratio of the number of cells relative to the PBS-added group.

FIG. 11: the binding activity of the resulting anti-GRP 78 antibody to the cell surface of 22Rv1 cells was analyzed by FACS.

FIG. 12: FIG. 12A is a schematic diagram of GST-GRP78 fusion proteins used for analyzing epitopes of GA-20 antibody and 4 novel antibodies (GC-18 antibody, GC-20 antibody, GD-4 antibody, and GD-17 antibody) obtained by re-immunization. FIG. 12(B) is a result of confirming that each GST-GRP78 fusion protein was expressed by induction of E.coli by addition of IPTG through SDS-PAGE and CBB staining.

FIG. 13: the epitope of each antibody was analyzed by western blotting. The results of SDS-PAGE of each GST-GRP78 fusion protein and Western blot analysis for reactivity of each antibody are shown. The table below shows the results of the epitopes of each antibody obtained from the western blot results.

FIG. 14: graph of CBB staining after SDS-PAGE of purified GD17scFv-PE40 in order to adjust the purity of purified GD17scFv-PE 40.

FIG. 15: shows the results of ELISA performed to analyze the binding activity of purified GD17scFv-PE40 to GRP78 protein and the stability of the protein. GD17scFv-PE40 stored at 4 ℃ and left to stand overnight at 37 ℃ and GD17scFv-PE40 frozen and thawed were diluted to respective concentrations, and the binding activity to GST-GRP78 was analyzed by ELISA. The following table is given in EC₅₀The values show the binding activity to GRP78 protein for each sample.

FIG. 16A: the results of the cell-damaging activities of purified GD17scFv-PE40 on various cell lines are shown. Diluting GD17scFv-PE40After releasing to each concentration, cancer cell lines were added (fig. 16A), and the number of viable cells was analyzed after several days of culture. The antibody concentrations (EC) giving 50% of the maximal activity are shown in the table₅₀Value).

FIG. 16B: the results of the cell-damaging activities of purified GD17scFv-PE40 on various cell lines are shown. GD17scFv-PE40 was diluted to each concentration, and a normal cell line was added (FIG. 16B), and the number of live cells was analyzed after several days of culture. The antibody concentrations (EC) giving 50% of the maximal activity are shown in the table₅₀Value).

FIG. 17: a graph showing the Western blot analysis of GRP78 protein expression of various cell lines using the GD-17 antibody.

FIG. 18: results of analyzing in vivo antitumor activity of GD17scFv-PE40 in a mouse transplantation model. PBS (vehicle) or GD17scFv-PE40 at 0.5mg/kg was administered at day 17, day 21, day 23, day 26 and day 29 after implantation of 22Rv1 (day 0), and tumor volume was then determined over time.

Detailed Description

The anti-GRP 78 antibody of the present invention is not limited to any kind of antibodies (e.g., mouse, rat, human), species (e.g., monoclonal antibody, polyclonal antibody) and shape (e.g., altered antibody, reduced antibody, modified antibody) as long as it binds to GRP78 protein (SEQ ID NO: 2).

The anti-GRP 78 antibodies used in the present invention preferably specifically bind to GRP 78. And the anti-GRP 78 antibody used in the present invention is preferably a monoclonal antibody.

GRP78 is known to be located on the cell membrane of, for example, cancer cells. One of the preferred embodiments of the anti-GRP 78 antibody used in the present invention may be, for example, an antibody that recognizes the region exposed to the outside of the cell when GRP78 is located on the cell membrane.

For example, an antibody can be obtained by preparing an antibody using GRP78 protein (SEQ ID NO: 2) as an immunogen and screening the prepared antibody for an antibody that binds to a cancer cell expressing GRP78 on the cell membrane (e.g., prostate cancer cell line DU145, etc.). More specifically, an antibody recognizing a region exposed to the outside of a cell when GRP78 is located on the cell membrane can be obtained by the method described in examples.

The region of GRP78 exposed to the outside of the cell when it is located on the cell membrane in the present invention is preferably a region which does not mimic the proliferation promoting effect of α 2 macroglobulin when an antibody binds to the region, and particularly preferably a region other than amino acids 98 to 115 of GRP 78.

Preferred regions of GRP78 that are exposed extracellularly when located on the cell membrane are, for example, SEQ id no: 2(SEQ ID NO: 3) from amino acid 376 to amino acid 415. Therefore, the antibody recognizing the region exposed to the outside of the cell when GRP78 is present on the cell membrane is preferably an antibody recognizing the region of amino acids 376 to 415 of GRP78, for example. The antibody recognizing the region of amino acids No. 376 to 415 of GRP78 is not particularly limited and may be, for example, an antibody recognizing amino acids No. 384 to 391 (i.e., amino acids No. 9 to 16 of SEQ ID NO: 3), an antibody recognizing amino acids No. 392 to 407 (i.e., amino acids No. 17 to 32 of SEQ ID NO: 3), and an antibody recognizing amino acids No. 400 to 415 (i.e., amino acids No. 25 to 40 of SEQ ID NO: 3). Whether an antibody recognizes a target epitope can be confirmed by a method known to those skilled in the art (for example, the method described in examples).

Other preferred embodiments of the antibody used in the present invention are, for example, an antibody having an uptake activity by cells. The term "antibody having cellular uptake activity" as used herein refers to an antibody which, when bound to GRP78 located on the surface of a cell, can be transported into the cell (e.g., into the cytoplasm, vesicle, other organelles).

Whether or not an antibody has an activity of being taken into a cell can be confirmed by a method known to those skilled in the art, for example, a method in which an anti-GRP 78 antibody conjugated with a marker is contacted with a cell expressing GRP78 (for example, prostate cancer cell line DU145 or the like) and whether or not the marker is taken into the cell is confirmed; a method of contacting a cell expressing GRP78 with an anti-GRP 78 antibody to which a cell damaging substance has been bound, and confirming whether or not cell death of the cell expressing GRP78 can be induced, and the like. More specifically, whether or not the antibody has an activity of being taken into cells can be confirmed by the method described in examples.

Particularly preferred antibodies of the present invention may be, for example, antibodies that recognize regions exposed to the outside of cells when GRP78 is located on the cell membrane and have an activity of being taken up by the cells. The antibody can be obtained by first screening an antibody recognizing a region exposed to the outside of the cell when GRP78 is located on the cell membrane according to the above-mentioned method, and then screening the screened antibody again for an antibody having an uptake activity by the cell.

Preferred antibodies used in the present invention may be, for example, antibodies of the following (a) to(s).

(a) An antibody comprising a heavy chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 8;

CDR2 has the amino acid sequence of SEQ ID NO: 9, and (b) the amino acid sequence shown in the figure;

CDR3 has the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

(b) An antibody comprising a light chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 11;

CDR2 has the amino acid sequence of SEQ ID NO: 12;

CDR3 has the amino acid sequence of SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof.

(c) An antibody comprising the heavy chain variable region of (a) and the light chain variable region of (b).

(d) An antibody comprising a heavy chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 18;

CDR2 has the amino acid sequence of SEQ ID NO: 19;

CDR3 has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

(e) An antibody comprising a light chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 21;

CDR2 has the amino acid sequence of SEQ ID NO: 22;

CDR3 has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt thereof.

(f) An antibody comprising the heavy chain variable region of (d) and the light chain variable region of (e).

(g) An antibody comprising a heavy chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 61;

CDR2 has the amino acid sequence of SEQ ID NO: 62;

CDR3 has the amino acid sequence of SEQ ID NO: 63, or a pharmaceutically acceptable salt thereof.

(h) An antibody comprising a light chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 64;

CDR2 has the amino acid sequence of SEQ ID NO: 65;

CDR3 has the amino acid sequence of SEQ ID NO: 66.

(i) An antibody comprising the heavy chain variable region of (g) and the light chain variable region of (h).

(j) An antibody comprising a heavy chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 71;

CDR2 has the amino acid sequence of SEQ ID NO: 72;

CDR3 has the amino acid sequence of SEQ ID NO: 73, or a pharmaceutically acceptable salt thereof.

(k) An antibody comprising a light chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 74;

CDR2 has the amino acid sequence of SEQ ID NO: 75;

CDR3 has the amino acid sequence of SEQ ID NO: 76.

(l) An antibody comprising the heavy chain variable region of (j) and the light chain variable region of (k).

(m) an antibody comprising a heavy chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 81;

CDR2 has the amino acid sequence of SEQ ID NO: 82;

CDR3 has the amino acid sequence of SEQ ID NO: 83 is shown in the figure.

(n) an antibody comprising a light chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 84;

CDR2 has the amino acid sequence of SEQ ID NO: 85;

CDR3 has the amino acid sequence of SEQ ID NO: 86, or a pharmaceutically acceptable salt thereof.

(o) an antibody comprising the heavy chain variable region of (m) and the light chain variable region of (n).

(p) an antibody comprising a heavy chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 91;

CDR2 has the amino acid sequence of SEQ ID NO: 92;

CDR3 has the amino acid sequence of SEQ ID NO: 93, or a pharmaceutically acceptable salt thereof.

(q) an antibody comprising a light chain variable region having the following CDR1-CDR3, wherein

CDR1 has the amino acid sequence of SEQ ID NO: 94;

CDR2 has the amino acid sequence of SEQ ID NO: 95;

CDR3 has the amino acid sequence of SEQ ID NO: 96.

(r) an antibody comprising the heavy chain variable region of (p) and the light chain variable region of (q).

(s) an antibody that recognizes the same epitope as that recognized by the antibody of any one of (a) to (r).

An antibody recognizing the same epitope as a certain antibody can be obtained, for example, by the following method.

The test antibody can be confirmed to share an epitope with an antibody by competitive binding to the same epitope. Competition between antibodies can be detected, for example, by a competition blocking assay. A preferred cross-blocking assay is, for example, a competitive ELISA assay. Specifically, GRP78 protein coated in the wells of a microplate is pre-incubated in the presence or absence of a candidate competitor antibody, followed by addition of an anti-GRP 78 antibody of the invention. The amount of anti-GRP 78 antibody of the invention that binds to GRP78 protein in the well is indirectly related to the binding capacity of the candidate competitor antibody (test antibody) that competes for binding to the same epitope. That is, as the affinity of the test antibody for the same epitope increases, the amount of binding of the anti-GRP 78 antibody of the present invention to the GRP78 protein-coated well decreases, and the amount of binding of the test antibody to the GRP78 protein-coated well increases.

The amount of antibody bound to the wells can be readily determined by pre-labeling the antibody. For example, a biotin-labeled antibody can be assayed by using an avidin peroxidase conjugate and a suitable substrate. Cross-blocking assays that utilize enzyme (e.g., peroxidase) labels are particularly referred to as competitive ELISA assays. The antibody may be labeled with other labels that may be detected or measured. Specifically, emissive markers or fluorescent markers are well known.

The amount of antibody bound to the well may also be determined from a labeled antibody that recognizes the constant region of the antibody when the test antibody and the anti-GRP 78 antibody of the invention have constant regions derived from different species. Even for antibodies derived from the same species, the amount of antibody bound to the well can be determined from the antibodies recognizing each type, as long as the types are different.

A candidate competing antibody is essentially an antibody that binds to the same epitope or can compete for binding to the same epitope as an anti-GRP 78 antibody of the invention, provided that the candidate antibody can block binding of at least 20%, preferably at least 20-50%, more preferably at least 50% of the anti-GRP 78 antibody compared to the binding activity obtained from a control experiment performed in the absence of the candidate competing antibody.

One of the preferred embodiments of the antibody used in the present invention is, for example, an antibody to which a cell-damaging substance is bound. When the antibody bound with the cell-damaging substance is taken into the cell, the killing effect or cell death of the cell taking the antibody can be induced by the cell-damaging substance. Therefore, the antibody to which the cell-damaging substance is bound preferably also has an uptake activity by cells.

A preferred embodiment of the anti-GRP 78 antibody that binds to a cell damaging substance of the present invention may be, for example, an antibody that has a damaging activity or induces cell death to a cancer cell expressing GRP78 (e.g., DU145, 22Rv1, MCF 7).

The cell-damaging substance used in the present invention may be any substance that can induce cell killing action or cell death, and may be, for example, a toxin, a radioactive substance, a chemotherapeutic agent, or the like. These inventive cell-damaging substances include prodrugs that can be converted into cell-damaging substances that are active in vivo. The activation of the prodrug may be either an enzymatic or a non-enzymatic conversion.

The "toxin" according to the present invention refers to various proteins or polypeptides derived from microorganisms, animals or plants, which exhibit cytotoxicity. The toxin used in the present invention may be, for example, the following substances. Diphtheria toxin A chain (Langon J.J., et al., Methods in Enzymology, 93, 307, 308, 1983), Pseudomonas exotoxin (Nature Medicine, 2, 350, 353, 1996), ricin A chain (Fulton R.J., et al., J.biol.Chem., 261, 5314. sub. 5319, 1986; Sivam G., et al., Cancer Res., 47, 3169, 3173, 1987; Cumbera.J.et al., J.Immunol.methods, 135, 15-24, 1990; Wawynczake.J., et al., Cancer Res., 50, 7519. sub. 7562, 1990; Ghee V., et al., J.J.munol.J.J.J.J.J.J., method, 142, 1991, Cancer Res., 50, 7519. sub. 62, 1990; Ghee V. et al., Glycyrne, 31, 19823, 1987, 7519. sub. E.E.J.J.J.J.J.J.J.J.J.19823, 19823. sub. E.E.J.J.J.J.J.142, 19823, 1983, K., 9. sub. toxin J.E.D.S.E.S.E.E.D.D.E.E.D. K, 19823, 1983, K, A. C.E, K, A., 3169 and 3173, 1987; thorpe p.e., et al, Cancer res, 47, 5924-; cumber a.j.et al, j.immunol.methods, 135, 15-24, 1990; wawrzynczak e.j., et al, Cancer res, 50, 7519-; blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), pokeweed antiviral protein (PAP-s) (blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), bronodin (blognesi a., et al, clin.exp.immunol., 89, 341- & 346, 1992), saporin (blognesi a., clin.exp.immunol., 89, 341- & 346, 1992), momordica charantia poison protein (Cumber a.j., im., j.munol.methods, 135, 15-24, 1990; wawrzynczak e.j., et al, Cancer res, 50, 7519-; borognesi A, et al, Clin. exp. Immunol., 89, 341-346, 1992), Momordica cochinchinensis poison protein (Borognesi A, et al, Clin. exp. Immunol., 89, 341-346, 1992), carnation poison protein 32 (Borognesi A, et al, Clin. exp. Immunol., 89, 341-346, 1992), carnation poison protein 30(Stirpe F, Barbieri L., FEBS letter 195, 1-8, 1986), Rhein poison protein II (Modelcin) (Stirpe F, Barbieri L., FEBS letter 195, 1-8, 1986), Viscum poison protein (Stirpe F, Barbieri L., FEI L., FER 195, 1-8, 195), Lorentz poison protein (FEirpe F, Barbieri L., FEI 195, Stirgesbee 195, 1-8, Nearve 195, wheat germ protein (FEirber L., FEirrient F, FEirrient 195, FEirrient L., FEirrient 195, wheat germ protein, FEirrient L., FEirrient 195, FEirrient F, FEirrient 195, FEirrient K195, FE, 1986) soft melon protein (stir f., barbieril l., FEBS letter 195, 1-8, 1986), trichokirin (caselas p., et al, eur.j. biochem.176, 581-588, 1988; bolognesi a., et al., clin. exp. immunol., 89, 341-.

The "radioactive substance" as used herein refers to a substance containing a radioactive isotope. The radioisotope is not particularly limited, and any radioisotope can be used, for example³²P、¹⁴C、¹²⁵I、³H、¹³¹I、¹⁸⁶Re、¹⁸⁸Re, and the like.

The "chemotherapeutic agent" of the present invention refers to a substance having a cell damaging activity other than the toxin and radioactive substance, and includes cytokines, antitumor agents, enzymes, and the like. There is no particular limitation on the chemotherapeutic agent used in the present invention, and a low molecular weight chemotherapeutic agent is preferred. When the molecular weight is small, it is considered that the possibility of interfering with the function of the antibody after binding to the antibody is low. The low molecular weight chemotherapeutic agents of the present invention typically have a molecular weight of 100-2000, preferably 200-1000. In the present invention, although not particularly limited, the following chemotherapeutic agents may be used, for example. American (Rowland G.F., et al., Nature 255, 487-488, 1975), cis-platinum (Hurwitz E.and Haimovich J., Method In Enzymology 178, 369-375, 1986; SchechterB., et al., int.J.cancer 48, 167-172, 1991), carboplatin (Ota, Y., et al., Asia-Oceania J.Obstet.Gyncol.19, 449-FPA, 1993), mitomycin C (Noguchi, A.et al., Bioconjugate Chem.3, 132-137, 1992), doxorubicin (doxorubicin) (Shih, L.B., Imal, Cancer Res.4192-4198, 1991; Zwland Z.261, 1995, J.257, J.072, Z.40, J.072, 267, J.p., Zhu, 267, 40, J.072, 267, J.p., 072, 267, 40, J.072, J.p., 072, 267, J., z., et al, Cancer immunol. Immmother 40, 257-; hudecz, f., et al, Bioconjugate chem.1, 197-; tukaday et al, J.Natl.cancer Inst.75, 721-; yamaguchi t., et al, jpn.j. Cancer res.85, 167-; kulkarni, p.n., et al, Cancer res.41, 2700-; shin, l.b., et al, int.j. cancer 41, 832-; gamett m.c., et al, int.j.cancer 31, 661- "670, 1983), 5-fluorouridine (Shin, l.b., int.j.cancer 46, 1101-.

One kind of cell damaging substance may be used in the present invention, and two or more kinds of cell damaging substances may be used in combination.

The binding of the anti-GRP 78 antibody to the cell injury substance can be covalent or non-covalent. Methods for producing antibodies that bind to these cell damaging substances are well known in the art.

The anti-GRP 78 antibody may be directly linked to the cell-injurious substance via a linker group of the antibody itself, or indirectly linked to the cell-injurious substance via a linker or an intermediate support. The linking group that can directly bind the anti-GRP 78 antibody to the cell injury substance may be, for example, 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 (e.g., dithiothreitol or the like), and the disulfide bond in the cell-damaging substance is also reduced, and then the two are bound with a disulfide bond. Either the antibody or the cell-damaging agent can be activated with an activating agent (e.g., an elman reagent) prior to conjugation, thereby promoting disulfide bond formation between the two. Other methods for directly binding the anti-GRP 78 antibody to the cell-damaging material include, for example, a method using Schiff base, a carbodiimide method, an activated ester method (N-hydroxysuccinimide method), a method using a mixed acid anhydride, and a method using a diazo reaction.

The anti-GRP 78 antibody can also be bound indirectly to the wound cell material by other means. Other substances usable for indirect binding are not particularly limited, and may be, for example, a compound having 2 or more amino groups, carboxyl groups, mercapto groups, etc., either alone or in combination of two or more, a compound having a peptide linker, a compound capable of binding to an anti-GRP 78 antibody, etc. The compound having 2 or more amino groups, carboxyl groups, mercapto groups, etc., either alone or in combination of two or more thereof may be, for example, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Wawrzynczak E.J., et al, Cancer Res., 50, 7519-ion 7562, 1990; Thorpe P.E., et al, Cancer Res., 47, 5924-ion 5931, 1987); succinimidyl-6-3- [ 2-pyridyldithio ] -propionamide) hexanoate (LC-SPDP) (Hermanson G.T., BIOCONJUGATE technologies, 230-propan 232, 1996), thiosuccinimidyl-6-3- [ 2-pyridyldithio ] -propionamide) hexanoate (Sulfo-LC-SPDP) (Hermanson G.T., BIOCONJUGATE technologies, 230-propan 232, 1996), N-succinimidyl-3- (2-pyridyldithio) butyrate (SPDB) (Wawrzynczak E.J., et al, Br. J.cancer, 66, 361-buzzo 366, 1992), succinimidyl-oxocarbyl-alpha- (2-pyridyldithio) toluene (Thorpe.E., et al, Cancer et al, Res.47, 5924, PT 5931, PT 597), succinimidyl-6- (2-pyridyldithio) -toluamide (SMspy-2-pyridyldithio) -propionamide ) (Hermanson G.T., BIOCONJUGATE Techniques, 232-plus 235, 1996), Thiosuccinimidyl-6- (. alpha. -methyl- [ 2-pyridyldithio ] toluamide) -hexanoate (Sulfo-LC-SMPT) (Hermanson G.T., BIOCONJUGATETechniques, 232-plus 235, 1996), succinimidyl-4- (p-maleimidophenyl) -butyrate (SMPB) (Hermanson G.T., BIOCONJUGATE Techniques, 242-plus 243, 1996), Thiosuccinimidyl-4- (p-maleimidophenyl) -butyrate (Sulfo-SMPB) (Hermanson G.T., ATE BIOCONJUGUGH-acids, 242-plus 243, 1996), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) (Hermanson G.T., BIOCONJUG-238, UGI-plus 237, 1996), m-maleimidobenzoyl-N-hydroxythiosuccinimidyl ester (Sulfo-MBS) (Hermanson G.T., BIOCONJUGATE Techniques, 237-.

Other substances used to bind anti-GRP 78 antibodies to cell damaging substances are, for example, peptides, antibodies, poly-L-glutamic acid (PGA), carboxymethyl dextran, aminodextran, avidin-biotin, aconitic acid, glutamic acid dihydrazide, Human Serum Albumin (HSA), and the like.

Protein cell injury substances can also be combined with the antibody by a genetic engineering method. Specifically, for example, a DNA encoding the cell damaging peptide may be fused in frame with a DNA encoding an anti-GRP 78 antibody and incorporated into an expression vector to construct a recombinant vector. The fusion protein of the anti-GRP 78 antibody conjugated to the toxic peptide can be obtained by culturing transformed cells obtained by introducing the vector into an appropriate host cell and expressing the recombinant DNA. When obtaining a fusion protein of an antibody, a protein drug or toxin is generally placed at the C-terminus of the antibody. Antibodies can also be linked to proteinaceous agents or toxins via peptide linkers.

The anti-GRP 78 monoclonal antibody of the present invention can be obtained according to a known method. The anti-GRP 78 antibody of the invention is particularly preferably a monoclonal antibody derived from a mammal. The monoclonal antibody derived from a mammal includes a monoclonal antibody produced by a hybridoma, a monoclonal antibody produced by a host transformed with an expression vector containing an antibody gene by a genetic engineering method, and the like.

Hybridomas producing monoclonal antibodies can be prepared using known techniques (e.g., as follows). GRP78 protein was first used as a priming antigen and immunization was performed according to a conventional immunization method. The hybridoma is obtained by fusing an immune cell obtained from an immunized animal to a known parent cell according to a conventional cell fusion method. Then, cells producing the desired antibody are selected from the hybridomas by a conventional screening method, thereby selecting hybridomas producing anti-GRP 78 antibody.

Specifically, monoclonal antibodies can be prepared as follows. First, GRP78 protein (SEQ ID NO: 2) used as a sensitizing antigen for obtaining an antibody can be obtained by expressing the GRP78 gene. The base sequence of the human GRP78 gene is known (SEQ ID NO: 1). Namely, the desired human GRP78 protein can be purified from the host cells or culture supernatant by a known method, after inserting a gene sequence encoding GRP78 into a known expression vector and transforming the host cells into appropriate ones. Purified native GRP78 protein may also be used as well. The production can be carried out by a single or multiple use of conventional chromatography (e.g., ion chromatography, affinity chromatography, etc.) in combination or alone. As used herein, fusion proteins obtained by fusing a desired portion of the GRP78 protein polypeptide with a different polypeptide can also be used as immunogens. Fusion proteins useful as immunogens can be prepared using, for example, an Fc fragment of an antibody or a peptide tag. Vectors for expressing fusion proteins can be prepared by fusing genes encoding the desired two or more polypeptide fragments in frame and inserting the fused genes into expression vectors as described above. The preparation method of the fusion protein is described in Molecular Cloning2^nd ed.(Sambrook，J.et al.，Molecular Cloning 2^nd ed.，9.47-9.58，Cold Spring Harbor Lab.Press，1989)。

The GRP78 protein purified as described above can be used as a priming antigen for immunization of mammals. Partial peptides of GRP78 may also be used as sensitizing antigens. For example, the following peptides can be used as sensitizing antigens:

a peptide chemically synthesized according to the amino acid sequence of human GRP 78;

a peptide obtained by integrating a part of the human GRP78 gene into an expression vector for expression;

a peptide obtained by decomposing human GRP78 protein with a protease.

There is no particular limitation on the region of GRP78 used as a partial peptide and its size. A preferred region may be selected from amino acid 376 to 415 of GRP78 (SEQ ID NO: 3). The number of amino acids of the peptide constituting the sensitizing antigen is preferably at least 3 or more (e.g., 5 or more, or 6 or more). More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

There is no particular limitation on the mammal immunized with the sensitizing antigen. In order to obtain a monoclonal antibody according to the cell fusion method, it is preferable to select an immunized animal in consideration of its suitability with a parent cell for cell fusion. It is generally preferred to use rodents as the immunizing animal. Specifically, a mouse, rat, hamster, or rabbit can be used as the immunized animal. In addition, monkeys can also be used as immunized animals.

The animal may be immunized with a sensitizing antigen according to known methods. For example, a mammal may be immunized by conventional means- -intraperitoneal or subcutaneous injection of a sensitizing antigen. Specifically, the sensitizing antigen can be administered to the mammal several times daily from day 4 to day 21. The sensitizing antigen can be diluted with PBS (phosphate buffered saline) or physiological saline at an appropriate dilution ratio and used for immunization. Sensitizing antigen can also be co-administered with an adjuvant. For example, it can be used as a sensitizing antigen by mixing with Freund's complete adjuvant and emulsifying. Suitable supports may also be used in immunising with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is preferable to immunize the antigen by binding the sensitizing antigen peptide to a support protein such as albumin or keyhole limpet hemocyanin.

After immunization of the mammal as described above and confirmation of the elevated amount of the desired antibody in the serum, immune cells were collected from the mammal and used for cell fusion. Splenocytes are particularly used as preferred immune cells.

Myeloma cells of mammals can be used as cells that can be fused with the immune cells. Myeloma cells preferably have suitable selection markers for selection. A selectable marker refers to a trait that is viable (or non-viable) under specific culture conditions. Known screening markers include hypoxanthine guanine phosphoribosyl transferase deficiency (hereinafter abbreviated as HGPRT deficiency), thymidine kinase deficiency (hereinafter abbreviated as TK deficiency), and the like. Cells deficient in HGPRT or TK have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter referred to as HAT sensitivity). HAT sensory cells die because they are unable to synthesize DNA in HAT selective medium, but can continue DNA synthesis using the salvage pathway of normal cells after fusion with normal cells, and thus can proliferate in HAT selective medium.

HGPRT-deficient or TK-deficient cells can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter referred to as 8AG), or 5' -bromodeoxyuridine. Cells with these enzyme deficiencies survive in selective media because normal cells will incorporate these pyrimidine analogs into DNA and die, but cells with these enzyme deficiencies will not incorporate these pyrimidine analogs. In addition, a selection marker called G418 resistance is imparted with resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by a neomycin resistance gene. Various myeloma cells suitable for cell fusion are known. For example, P3(P3x63Ag8.653) (J.Immunol. (1979)123, 1548-1550), P3x63Ag8U.1(Current Topicsin microbiology and Immunology (1978)81, 1-7), NS-1(Kohler.G.and Milstein, C.Eur.J.Immunol. (1976)6, 511-519), MPC-11 (Margulies.D.H.et., Cell (1976)8, 405-415), SP2/0(Shulman, M.et., Nature (1978)276, 269-270), FO (St.Groth., S.F.et., J.Immunol.methods (1980)35, 1-21), S194 (Trbrow.I.313), Ex.277, 121-21, myeloma cells (9, 11, 121-21-42-11, 11-11, 150, 23-21, 133, 11, 23-21, III, 11, III, J.I.J.I.J.J.I.J.J.D.D.277, M.D.K.9, M.K.K.K..

Cell fusion of the immune cells and myeloma cells can be performed according to known Methods (e.g., Kohler & Milstein et al) (Kohler.G. and Milstein, C., Methods Enzymol. (1981)73, 3-46).

More specifically, the fusion of the cells can be performed in a conventional nutrient medium in the presence of a fusogenic agent. Fusogenic agents such as polyethylene glycol (PEG), Sendai virus (HVJ) and the like can be used. If necessary, an auxiliary agent such as dimethyl sulfoxide can be added to further improve the fusion efficiency.

The ratio of the immune cells to the myeloma cells to be used can be arbitrarily set. For example, the immune cells are preferably 1 to 10 times as many as myeloma cells. For example, RPMI1640 culture medium, MEM culture medium, and other conventional culture media for culturing such cells, which are suitable for the proliferation of the myeloma cell line, can be used as the culture medium for carrying out the cell fusion. Serum supplements such as Fetal Calf Serum (FCS) may also be added to the culture medium.

Cell fusion can be performed by uniformly mixing predetermined amounts of the immune cells and myeloma cells in the culture solution, and then mixing a PEG solution preheated to about 37 ℃ to form fused cells of interest (hybridomas). In cell fusion, PEG with an average molecular weight of about 1000-6000 can be added at a concentration of typically 30-60% (w/v). The appropriate culture medium is then added in succession, and the operation of removing the supernatant by centrifugation is repeated to remove the cell fusion agent and the like which are unfavorable for the growth of the hybridoma.

The hybridoma obtained as described above can be selected by using a selection medium corresponding to a selection marker possessed by myeloma used for cell fusion. For example, HGPRT or TK deficient cells can be selected by culturing in HAT medium (hypoxanthine, aminopterin and thymidine containing medium). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells successfully fused with normal cells can be selectively proliferated in HAT culture solution. Although cells other than the target hybridoma (non-fused cells) die, there is a sufficient time for the culture to continue using the HAT culture solution. Specifically, the hybridoma of interest can be generally selected by culturing for several days to several weeks. Subsequently, hybridomas producing the antibody of interest can be screened and monocloned by performing conventional limiting dilution. Or an antibody recognizing GRP78 can be prepared according to the method described in International publication WO 03/104453.

The antibody of interest can be suitably screened and monoclonal using a known screening method based on an antigen-antibody reaction. For example, an antigen is bound to a support such as beads made of polystyrene or a commercially available 96-well microplate, and then reacted with a culture supernatant of a hybridoma. The support is then washed and reacted with an enzyme-labeled secondary antibody. If the culture supernatant contains an antibody reactive with the sensitizing antigen, the second antibody will be bound to the support by the antibody. Finally, the presence of the antibody of interest in the culture supernatant can be determined by detecting the secondary antibody bound to the support. It becomes possible to clone a hybridoma producing a desired antibody having a binding ability to an antigen by a limiting dilution method. In this case, an antigen such as that used for immunization may be used, and the same GRP78 protein as that used in the practice may be used.

In addition to the above-described method for obtaining a hybridoma by immunizing an antigen to an animal other than a human, the target antibody can be obtained by antigen-sensitizing lymphocytes. Specifically, human lymphocytes were first sensitized in vitro with GRP78 protein. Next, the immunosensitized lymphocytes are fused with a suitable fusion partner. For example, a human-derived myeloma cell having a permanent division ability can be used as a fusion partner (see Japanese patent publication No. Hei 1-59878). The anti-GRP 78 antibody obtained by the method is a human antibody having a binding activity to GRP78 protein.

An anti-GRP 78 human antibody was obtained by administering GRP78 protein as an antigen to a transgenic animal having a full complement of human antibody genes. The antibody-producing cells of the immunized animal may be immortalized by cell fusion with a suitable conjugate or infection with Epstein-Bar virus, or the like. Human antibodies against the GRP78 protein can be isolated from immortalized cells obtained as described (see International publications WO94/25585, WO93/12227, WO92/03918, WO 94/02602). Cells producing antibodies specific for the reaction of interest can be cloned by further cloning the immortalized cells. When a transgenic animal is used as an immunized animal, the animal's immune system recognizes human GRP78 as a foreign body. Human antibodies against human GRP78 are therefore readily available. The monoclonal antibody-producing hybridoma prepared as described above may be subcultured in a conventional culture medium. Alternatively, the hybridoma may be stored in liquid nitrogen for a long period of time.

The hybridoma can be cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant. Or a monoclonal antibody can be obtained from ascites thereof by administering the hybridoma to a mammal suitable therefor and proliferating. The former method is suitable for obtaining an antibody with high purity.

Antibodies encoded by antibody genes cloned from antibody-producing cells can also be used in the present invention. The antibody can be expressed by introducing the cloned antibody gene into a host after integrating it into an appropriate vector. Methods for isolating antibody genes, introducing vectors, and transforming hosts have been established (see, e.g., Vandamme, a.m.et., eur.j.biochem. (1990)192, 767-775).

For example, a cDNA encoding the variable region (V region) of an anti-GRP 78 antibody can be obtained from a hybridoma cell producing an anti-GRP 78 antibody. For this, total RNA is first extracted from the hybridoma. Methods for extracting mRNA from cells can be used, for example, guanidine ultracentrifugation (Chirgwin, J.M.et al., Biochemistry (1979)18, 5294-.

The extracted mRNA can be purified using, for example, an mRNA purification kit (manufactured by GE Healthcare Bioscience). There are also commercially available kits for directly extracting total mRNA from cells, such as QuickPrep mRNA purification kit (GE Healthcare Bioscience). Total mRNA can also be obtained from hybridomas using a kit as described above. cDNA encoding the V region of the antibody can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV reverse transcriptase first strand cDNA Synthesis kit (manufactured by Biochemical industries, Ltd.). cDNA can be synthesized and amplified by the 5 '-RACE method using 5' -Ampli FINDER kit (manufactured by Clontech) and PCR (Frohman, M.A.et. al., Proc Natl.Acad.Sci.USA (1988)85, 8998-. The following suitable restriction sites may also be introduced at both ends of the cDNA during the synthesis of the cDNA as described above.

The cDNA fragment of interest is purified from the resulting PCR product and subsequently ligated to a vector DNA. A recombinant vector is prepared as described above, introduced into, for example, Escherichia coli, and after selection of colonies, a desired recombinant vector is prepared from the colony-forming Escherichia coli. Whether or not the recombinant vector contains the base sequence of the cDNA of interest can be confirmed by a known method (e.g., dideoxynucleotide chain termination method).

The gene encoding the variable region can be obtained by PCR using primers for amplifying the variable region gene. First, cDNA is synthesized using the extracted mRNA as a template to obtain a cDNA library. It is convenient to use a commercially available kit for synthesizing the cDNA library. In fact, mRNA is obtained only in a very small number of cells, and its direct purification results in a low yield. Purification is usually performed after addition of vector RNA known not to contain antibody genes. Or when a certain amount of RNA can be extracted, RNA of antibody-producing cells can be efficiently extracted. When RNA is extracted from, for example, antibody-producing cells of. gtoreq.10, or. gtoreq.30, preferably. gtoreq.50, it may not be necessary to add carrier RNA.

The antibody gene can be amplified by PCR using the obtained cDNA library as a template. Primers for amplifying antibody genes by the PCR method are known. Primers for amplifying human antibody genes can be designed according to the disclosure of the literature such as J.mol.biol. (1991)222, 581-597, etc. These primers are composed of a base sequence different from the subclass of immunoglobulin. Therefore, when cDNA libraries of unknown subclasses are used as templates, PCR is performed in consideration of all possibilities.

Specifically, when a gene encoding human IgG, for example, is to be obtained, primers that can amplify genes encoding the heavy chain γ 1 to γ 5, the light chain κ chain, and the λ chain can be used. For amplifying the lgG variable region gene, a primer renaturing a portion corresponding to the hinge region can be generally used as a primer on the 3' side. And primers corresponding to each subclass can be used as primers on the 5' side.

PCR products obtained from primers for amplifying genes of each subclass of heavy and light chains were used as independent libraries. Immunoglobulins made from combinations of heavy and light chains can be reconstituted using libraries synthesized as described. The binding activity of the reconstituted immunoglobulin to GRP78 can be used as an index to screen for a target antibody.

The binding of the antibodies of the invention to GRP78 is preferably specific. Antibodies that bind to GRP78 can be screened, for example, by the following procedure.

(1) Contacting an antibody comprising a V region encoded by cDNA obtained from a hybridoma with GRP 78;

(2) detecting binding of GRP78 to the antibody; and

(3) antibodies were screened for binding to GRP 78.

Methods of detecting binding of antibodies to GRP78 are known. Specifically, a test antibody is reacted with GRP78 immobilized on a support, and a labeled antibody that recognizes the antibody is reacted with the test antibody. If the labeled antibody is detectable from the washed support, binding of the test antibody to GRP78 is demonstrated. Proteins having enzymatic activity such as peroxidase or β -galactosidase, or fluorescent substances such as FITC can be used as the label. Antibody binding activity can also be assessed using a fixed sample of GRP 78-expressing cells.

Panning using a phage vector can also be used as an antibody screening method using binding activity as an index. When antibody genes are obtained as libraries of subclasses of heavy and light chains as described above, a screening method using a phage vector is advantageous. The genes encoding the variable regions of the heavy and light chains may be joined by appropriate ligand sequences into a single chain fv (scFv). A phage expressing scFv on its surface can be obtained by inserting a gene encoding scFv into a phage vector. The DNA encoding the scFv having the desired activity can be recovered by contacting the phage with the antigen of interest and recovering the phage that binds to the antigen. This procedure can be repeated as necessary to concentrate the scFv having the desired binding activity.

The polynucleotides of the invention encoding the antibodies may encode the full length of the antibody or may encode a portion of the antibody. A portion of an antibody refers to any portion of an antibody molecule. Hereinafter, a part of the antibody is also referred to as an antibody fragment. Preferred antibody fragments of the invention contain antibody Complementarity Determining Regions (CDRs). The antibody fragment of the present invention more preferably contains 3 CDRs which all constitute the variable region.

After obtaining a cDNA encoding the V region of the desired anti-GRP 78 antibody, the cDNA is digested with restriction enzymes that recognize restriction sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize a nucleotide sequence that is less likely to occur in the nucleotide sequence constituting the antibody gene, and digest it. In order to insert 1 copy of the digested fragment into the vector in the correct orientation, it is preferable to provide a restriction enzyme with an attached end. The antibody expression vector can be obtained by inserting cDNA encoding the V region of the anti-GRP 78 antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by in-frame fusion of a gene encoding a constant region (C region) of an antibody with a gene encoding the V region. The chimeric antibody as described herein refers to a constant region and a variable region derived from different organisms. Therefore, not only mouse-human hetero-chimeric antibodies but also human-human allochimeric antibodies are chimeric antibodies of the present invention. A vector for expressing a chimeric antibody can also be constructed by inserting the V region gene into an expression vector having a constant region in advance.

Specifically, for example, a DNA encoding a desired antibody constant region (C region) may be inserted into the 5' side of an expression vector, and a restriction enzyme recognition sequence of a restriction enzyme digesting the V region gene may be provided. Vectors expressing chimeric antibodies can be constructed by digesting both with the same combination of restriction enzymes and performing in-frame fusion.

To prepare the anti-GRP 78 antibody of the present invention, the antibody gene may be incorporated into an expression vector in a form expressed under the control of an expression regulatory region. Expression regulatory regions for expressing the antibody include, for example, enhancers or promoters. Recombinant cells expressing DNA encoding anti-GRP 78 antibodies can then be obtained by transforming the expression vector into suitable host cells.

For expression of antibody genes, DNAs encoding the heavy chain (H chain) and light chain (L chain) of an antibody may be integrated into respective different expression vectors. The antibody molecule having an H chain and an L chain can be expressed by co-transforming a vector integrating the H chain and the L chain into a host cell. The host cell may also be transformed by incorporating the DNA encoding the H chain and the L chain into a single expression vector (see International publication WO 94/11523).

Combinations of hosts and expression vectors for producing antibodies by introducing isolated antibody genes into appropriate hosts are known. Any of these expression systems can be applied to the present invention. If eukaryotic cells are used as hosts, animal cells, plant cells or fungal cells can be used. Specifically, the animal cell which can be used in the present invention is, for example, a mammalian cell (CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc.), an amphibian cell (Xenopus laevis oocyte, etc.), an insect cell (sf9, sf21, Tn5, etc.).

An antibody gene expression system derived from a plant cell such as a cell of Nicotiana (Nicotiana) such as Nicotiana tabacum (Nicotiana) or the like is known. Transformation of plant cells may utilize cells cultured from callus.

Fungal cells may also be used, such as Saccharomyces (Saccharomyces) such as Saccharomyces (Saccharomyces serevisiae), Pichia (Pichia) such as Pichia pastoris, Aspergillus (Aspergillus) such as Aspergillus niger and the like.

Antibody gene expression systems using prokaryotes are also known. For example, when bacterial cells are used, bacterial cells such as escherichia coli (e.coli), bacillus subtilis, and the like can be used in the present invention.

When mammals are used, an expression vector can be constructed in which a commonly used useful promoter, an antibody gene to be expressed, and a poly A signal downstream of the 3' side thereof are functionally linked. For example, the promoter/enhancer may be exemplified by the human cytomegalovirus immediate early promoter/enhancer.

Still other promoters/enhancers for antibody expression of the present invention are promoters/enhancers derived from mammalian cells, such as viral promoters/enhancers, or human elongation factor 1 α (HEF1 α). Examples of viruses that can utilize the promoter/enhancer include retroviruses, polyoma viruses, adenoviruses, simian virus 40(SV40), and the like.

When the SV40 promoter/enhancer is used, the method of Mullgan et al (Nature (1979)277, 108) can be used. In addition, the HEF1 alpha promoter/enhancer can be easily used for the expression of a target gene according to the method of Mizushima et al (Nucleic acids SRes. (1990)18, 5322).

When Escherichia coli is used, the gene can be expressed by functionally linking a commonly used useful promoter, a signal sequence for secreting an antibody, and the antibody gene to be expressed. Examples of the promoter include lacZ promoter and araB promoter. When the lacZ promoter is used, the method of Ward et al (Nature (1989)341, 544-2427; FASEBJ. (1992)6, 2422-2427) can be used. The araB promoter can also be used for expressing a gene of interest according to the method of Better et al (Science (1988)240, 1041-1043).

When produced in the cytoplasm of E.coli, the pe1B signal sequence (Lei, S.P.et al, J.Bacteriol. (1987)169, 4379) can be used as a signal sequence for antibody secretion. After isolation of the antibody produced in the cytoplasm, the antibody is refolded (refold) into a form with the desired binding activity using a protein denaturing agent such as guanidine hydrochloride.

The origin of replication inserted into the expression vector may be derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), and the like. To increase the copy number of the gene in the host cell, a selection marker may also be inserted into the expression vector. Specifically, a selection marker such as an aminoglycoside transferase (APH) gene, a Thymidine Kinase (TK) gene, E.coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, or the like can be used.

The antibody of interest can be produced by introducing these expression vectors into host cells and culturing the transformed host cells in vitro or in vivo. The host cell can be cultured according to a known method. For example, DMEM, MEM, RPMI1640, IMDM may be used as the culture medium, and a serum supplement such as Fetal Calf Serum (FCS) may be used in combination.

The antibody expressed and produced as described above can be purified by using a known conventional protein purification method alone or in a suitable combination. For example, the antibody can be isolated and purified by suitably selecting and combining an affinity column (e.g., protein A column), a chromatography column, filtration, ultrafiltration, salting out, dialysis, etc. (antibodies in Laboratory Manual. Ed harbor, David Lane, Cold Spring harbor Laboratory, 1988).

In addition, in addition to the above host cells, 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 desired antibody has been introduced. For example, a fusion gene can be constructed by in-frame insertion of an antibody gene into a gene encoding a protein that is inherently produced in an emulsion. Proteins secreted into milk, such as goat beta casein, can be used. The DNA fragment containing the fusion gene into which the antibody gene is inserted may be injected into goat embryos, and the injected embryos are introduced into female goats. The fusion protein of the desired antibody and a milk protein can be obtained from the milk produced by a transgenic goat born from the goat receiving the embryo (or progeny thereof). In order to increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones may be suitably used for the transgenic goat (Ebert, K.M.et. al., Bio/Technology (1994)12, 699-702). The C region derived from an animal antibody can be used as the C region of the recombinant antibody of the present invention. For example, the mouse antibody H chain C region C gamma 1, C gamma 2a, C gamma 2b, C gamma 3, C mu, C delta, C alpha 1, C alpha 2, C epsilon, L chain C region C kappa, C lambda can be used. Animal antibodies other than mouse antibodies, such as those of rat, rabbit, goat, sheep, camel, monkey, and the like, may also be used. The sequences of these antibodies are known. In addition, the C region may be modified to improve the stability of the antibody or its production. When the antibody is administered to a human according to the present invention, the antigenicity against a human can be reduced by artificially modifying a recombinant antibody. Recombinant antibodies include, for example, Chimeric (Chimeric) antibodies, Humanized (Humanized) antibodies.

These altered antibodies can be prepared according to well known methods. A chimeric antibody is an antibody in which variable regions derived from different sources are linked to a constant region. For example, an antibody comprising the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody is a mouse-human-xenochimeric antibody. A recombinant vector for expressing a chimeric antibody can be prepared by ligating a DNA encoding a variable region of a mouse antibody with a DNA encoding a constant region of a human antibody and integrating them into an expression vector. Culturing the recombinant cell transformed with the vector and expressing the integrated DNA to obtain the chimeric antibody produced during the culture. The C region of a human antibody can be used for the C regions of a chimeric antibody and a humanized antibody. For example, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.mu.s, C.delta.C.alpha.1, C.alpha.2 and C.epsilon.can be used as the C region of the H chain. C.kappa.and C.lambda.can also be used as the C region of the L chain. The amino acid sequences of these C regions, and the base sequences encoding them, are known. In addition, the C region of human antibodies may be modified to improve the safety of the antibodies themselves or the antibodies produced.

Chimeric antibodies generally consist of a V region of an antibody derived from an animal other than human and a C region derived from a human antibody. In contrast, a humanized antibody is composed of Complementarity Determining Regions (CDRs) of an antibody derived from an animal other than a human, a Framework Region (FR) derived from a human antibody, and a C region derived from a human antibody. The humanized antibody has low antigenicity in the human body and is therefore useful as an active ingredient of the therapeutic agent of the present invention.

Antibody variable regions are typically composed of 3 Complementarity Determining Regions (CDRs) separated by 4 Framework Regions (FRs). CDRs are regions that substantially determine the specificity of an antibody. The amino acid sequences of the CDRs are diverse. The amino acid sequences constituting the FR are highly identical to each other even among antibodies having different binding specificities. Thus, in general, the binding specificity of one antibody is not reduced by grafting to another antibody due to grafting of the CDRs.

Humanized antibodies are also referred to as reshaped (reshaped) antibodies. Specifically, a humanized antibody obtained by grafting a CDR of an antibody of an animal other than a human (for example, a mouse) to a human antibody is known. General genetic recombination methods for obtaining humanized antibodies are known.

Specifically, as a method for grafting CDRs of a mouse antibody to human FRs, for example, overexpression PCR is known. In the overexpression PCR, a base sequence encoding a CDR of a mouse antibody to be grafted is added to a primer for synthesizing a FR of a human antibody. Primers were used for each of the 4 FRs. When a mouse CDR is grafted to a human FR, a human FR having a high degree of identity with the mouse FR is generally selected irrespective of the function of the CDR. That is, it is generally preferred to use a human FR composed of an amino acid sequence having high identity with the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.

In addition, the base sequences to be ligated are designed so as to be ligated in frame with each other. Human FRs were synthesized from each primer. As a result, a product obtained by attaching DNA encoding mouse CDRs to each FR was obtained. The base sequences of the mouse CDRs encoding the respective products can be designed in a manner overlapping with each other. Next, complementary strand synthesis reaction is carried out by renaturing overlapping CDR portions of the product synthesized using the human antibody gene as a model. The human FRs are linked by the mouse CDR sequences from the reaction.

Finally, the full length of the gene was amplified by primers renatured to the 5 'and 3' ends of the V region gene linking the 3 CDRs and 4 FRs, with the addition of appropriate restriction enzyme recognition sequences. A vector for expressing a human antibody is prepared by inserting the DNA obtained as described above and a DNA encoding a human antibody C region into an expression vector in the form of in-frame fusion. The humanized antibody is produced from a culture of the cultured cells by establishing recombinant cells into which the integration vector has been introduced, culturing the recombinant cells, and expressing a DNA encoding the humanized antibody (see European patent publication EP 239400, International publication WO 96/02576).

The FRs of a human antibody that form a good antigen-binding site when CDRs are ligated can be suitably selected by qualitatively or quantitatively measuring and evaluating the binding activity of the humanized antibody prepared as described above to an antigen. The amino acid residues of the FR may also be substituted as necessary so that the CDRs of the reshaped human antibody form an antigen binding site. For example, amino acid sequence variations can be introduced into the FR by PCR methods used to graft mouse CDRs onto human FRs. Specifically, a primer that hybridizes to FR can be introduced with a mutation in a partial base sequence. The FR synthesized by the primer has a mutation in the base sequence introduced therein. Variant FR sequences having desired properties can be screened by measuring and evaluating the binding activity of the variant antibody having amino acid substitutions to an antigen using the above-described method (Sato, K.et al, Cancer Res, 1993, 53, 851-856).

Methods for obtaining human antibodies are also known. For example, lymphocytes are primed in vitro with a desired antigen or cells expressing a desired antigen. Then, a desired human antibody having an antigen-binding activity is obtained by fusing the sensitized lymphocytes with human myeloma cells (see Japanese examined patent publication (Kokoku) No. 1-59878). The fusion partner or human myeloma cell may be, for example, U266.

In addition, a desired human antibody can be obtained by immunizing a transgenic animal having a full set of human antibody genes with a desired antigen (see International publication Nos. WO93/12227, 92/03918, WO94/02602, WO94/25585, WO96/34096, WO 96/33735). Techniques for obtaining human antibodies by panning using human antibody libraries are also known. For example, human antibody V regions can be expressed as single chain antibodies (scFv) on the surface of a phage by phage display, and the phage selected for binding to the antigen. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by analyzing the selected phage gene. After the DNA sequence of the scFv that binds to the antigen is determined, an expression vector can be prepared by fusing the V region sequence in-frame with the desired human antibody C region sequence, and then inserting into an appropriate expression vector. The human antibody can be obtained by introducing the expression vector into an appropriate expression cell as described above and expressing a gene encoding the human antibody. These methods are known (International publications WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388).

The antibody of the present invention includes not only a bivalent antibody represented by IgG but also a single antibody or a multivalent antibody represented by IgM as long as it can bind to GRP78 protein. Multivalent antibodies of the invention include multivalent antibodies having all the same antigen binding sites, or multivalent antibodies having partially 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 an antibody or a modified product thereof as long as it binds to GRP78 protein.

Reduced antibodies include antibody fragments that lack a portion of a full-length antibody (e.g., a full-length IgG). Partial deletion of the antibody molecule is allowed as long as it binds to the GRP78 antigen. The antibody fragment of the present invention preferably includes either or both of a heavy chain variable region (VH) and a light chain variable region (VL). The amino acid sequence of VH or VL may comprise substitutions, deletions, additions and/or insertions. A portion of either or both VH and VL may be deleted so long as they bind to GRP78 antigen. The variable regions may also be chimerized or humanized. Specific examples of the antibody fragment may be, for example, Fab ', F (ab') 2, Fv, etc. Specific examples of the reduced antibody may be, for example, Fab ', F (ab') 2, Fv, scFv (single chain Fv), Diabody (Diabody), sc (Fv)2 (single chain (Fv)2), and the like. Multimers (e.g., dimers, trimers, tetramers, multimers) of these antibodies also belong to the reduced antibodies of the present invention.

Fragments of an antibody can be obtained by treating the antibody with an enzyme to produce antibody fragments. Enzymes that can produce antibody fragments are known, for example, as papain, pepsin, plasmin, or the like. Genes encoding these antibodies can also be constructed and expressed in suitable host cells after introduction into an expression vector (see, e.g., Co, M.S.et. al., J.Immunol. (1994)152, 2968-2976; Better, M. & Horwitz, A.H.methods in Enzymology (1989)178, 476 496; Plcthun, A. & Skerra, A.methods in Enzymology (1989)178, 476-496; Lamoyi, E., Methods in Enzymology (1989)121, 652-663; Rousseaux, J.et. al., Methods in Enzymology (1989)121, 663-669; R.E.et. Bial., TIECH (1991) 137, 132).

The digestion enzyme cleaves a specific site of the antibody fragment to produce an antibody fragment having a specific structure as described below. For the antibody fragment obtained by enzyme digestion, if a genetic engineering method is adopted, any part of the antibody can be deleted:

and (3) papain digestion: f (ab)2 or Fab;

and (3) pepsin digestion: f (ab ') 2 or Fab';

plasmin digestion: facb.

Diabodies refer to bivalent (bivalent) antibody fragments constructed by gene fusion (Holliger P et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993), EP404,097, WO93/11161, etc.). Diabodies are dimers consisting of 2 polypeptide chains. The VL and VH in the same chain of each polypeptide chain that makes up the dimer are typically connected by a linker. The linker in diabodies is generally short so that the VL and VH do not bind to each other. Specifically, the amino acid residues constituting the linker are, for example, about 5 residues. Thus, VL and VH encoded on the same polypeptide chain do not form single chain variable fragments, but rather form distinct single chain variable fragments and diabodies. As a result, the diabody has 2 antigen binding sites.

scFv can be 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 in the scFv are linked by a linker, preferably a peptide linker (Huston, J.S.et al, Proc.Natl.Acad.Sci.U.S.A, 1988, 85, 5879-. The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described in the present specification. There is no particular limitation on the peptide linker connecting the V regions. Any single chain peptide of, for example, about 3 to about 25 residues may be used as a linker. The V region can be ligated by, for example, the PCR method described above. To link the V regions using the PCR method, first, a DNA sequence encoding the H chain or H chain V region of the antibody and a DNA sequence encoding the full length of the DNA sequence of the L chain or L chain V region of the antibody or a DNA sequence encoding a desired part of the amino acid sequence are used as templates.

The DNA encoding the H chain and L chain V regions can be amplified separately by a PCR method using a primer pair having sequences corresponding to the sequences at both ends of the DNA to be amplified. The DNA encoding the peptide linker moiety is then used. DNA encoding a peptide linker can also be synthesized using PCR. In this case, a nucleotide sequence to which an amplification product of each V region independently synthesized can be ligated is added to the 5' side of the primer used. Next, PCR reaction was carried out using each DNA of [ H chain V region DNA ] - [ peptide linker DNA ] - [ L chain V region DNA ] and primers for assembly PCR. The primers used for the assembly PCR were composed of a combination of a primer renatured to the 5 'side of [ H chain V region DNA ] and a primer renatured to the 3' side of [ L chain V region DNA ]. That is, the primers used for assembling the PCR are a primer set that can amplify the DNA encoding the full-length sequence of the scFv to be synthesized. The "peptide linker DNA" has a nucleotide sequence capable of linking the DNAs of the V regions. As a result, an amplification product of scFv full length is finally generated from the primers used for assembling PCR to which these DNAs are ligated. Once DNA encoding scFv is prepared, an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. Furthermore, the scFv can be obtained by culturing the resulting recombinant cell and expressing a DNA encoding the scFv.

sc (fv)2 is a reduced antibody in which 2 VH and 2 VL are combined into a single chain by a linker or the like (Hudson et al, J.Immunol.methods 1999; 231: 177-189). Sc (fv)2 can be prepared by ligating, for example, an scFv through a linker.

Furthermore, it is preferable that the antibody is characterized in that VH, VL, VH and VL (VH linker [ VL linker [ VH ] VL ]) are arranged in this order from the N-terminal side of the single-chain polypeptide with 2 VH and 2 VL as base points.

The order of 2 VH and 2 VL is not particularly limited to the above arrangement, and may be in any order. For example, the following arrangement is possible.

[ VL ] linker [ VH ] linker [ VL ]

[ VH ] linker [ VL ] linker [ VH ]

[ VH ] linker [ VL ]

[ VL ] linker [ VH ]

[ VL ] linker [ vH ] linker [ VL ] linker [ VH ]

Any peptide linker or synthetic compound linker that can be introduced by genetic Engineering (see, for example, linkers disclosed in Protein Engineering, 9(3), 299-305, 1996) can be used as a linker for binding to the variable region of an antibody. Peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited and may be appropriately selected by those skilled in the art as needed. The amino acid residues constituting the peptide linker are usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (e.g., 15 amino acids).

The amino acid sequence constituting the peptide linker may be any sequence as long as it does not inhibit the binding action of scFv.

Synthetic chemicals (chemical bridging agents) can also be used to link the V regions. The bridging agent conventionally used in a bridged peptide compound and the like can be used in the present invention. For example, N-hydroxysuccinimide (NHS), disuccinimidyl suberic acid (DSS), bis (thiosuccinimidyl) suberic acid (BS3), dithiobis (succinimidyl propionic acid) (DSP), dithiobis (thiosuccinimidyl propionic acid) (DTSSP), ethyleneglycol bis (succinimidyl succinic acid) (EGS), ethyleneglycol bis (thiosuccinimidyl succinic acid) (thio-EGS), disuccinimidyl tartaric acid (DST), dithiosuccinimidyl tartaric acid (thio-DST), bis [2- (succinimidyloxycarbonyloxy) ethyl ] sulfone (BSOCOES), bis [2- (thiosuccinimidyloxycarbonyloxy) ethyl ] sulfone (thio-BSOCOES), and the like can be used.

Typically 3 linkers are required to bind 4 antibody variable regions. Several linkers may be the same or different. The preferred reduced antibodies of the invention are preferably diabodies or sc (fv) 2. The reduced antibody can be obtained by treating an antibody with an enzyme (e.g., papain, pepsin, etc.) to form an antibody fragment, or constructing DNA encoding the antibody fragment, which is then expressed in a suitable host cell after introduction into an expression vector (see, e.g., Co, M.S. et al, J.Immunol (1994)152, 2968-2976; Better, M.and Horwitz, A.H., Methods Enzymol. (1989)178, 476-496; Pluckthun, A.and Skerra, A.s Enzymol. (1989)178, 497-515; Lamoyi, E., Methods Enzymol. (1986)121, 652-663; Roussex, J.et al, Methods Enzymol. (1986)121, 652-663; Roussex, J.137-11, Methods Enzymol. (1986), Tredhol. (1989; Tredhne. 121, Waldhne. W.132).

The antibody of the present invention may be a modified antibody to which various molecules such as polyethylene glycol (PEG) are bound. The modified antibody may be obtained by chemical modification of an antibody of the invention. Methods for modifying antibodies have been established in the art.

The antibodies of the invention can be bispecific antibodies. Bispecific antibodies refer to antibodies having variable regions within the same antibody molecule that recognize different epitopes, which epitopes may be present in different molecules or may be present in the same molecule. That is, the bispecific antibodies of the invention may have antigen binding sites that recognize different epitopes on GRP78 molecules. Bispecific antibodies in which one recognition site recognizes GRP78 and the other recognition site recognizes a cell-damaging agent can also be used. The "antibodies" described herein also include such antibodies.

The invention may also be combined with bispecific antibodies that recognize antigens other than GRP 78. Bispecific antibodies recognizing an antigen other than GRP78, which is also specifically expressed on the cell surface of target cancer cells as GRP78, for example, can be combined.

Methods for making bispecific antibodies are known. For example, bispecific antibodies can be prepared by binding 2 antibodies that differ in the antigen recognized. The antibody to be bound may be 1/2 molecules each having an H chain and an L chain, or 1/4 molecules composed of only an H chain. Alternatively, bispecific antibody-producing fused cells can be prepared by fusing monoclonal antibody-producing hybridomas. Bispecific antibodies can also be prepared by genetic engineering methods.

Binding Activity of antibodies

The antigen binding activity of Antibodies can be determined by well known methods (Antibodies antibody Manual. Ed Harlow, David Lane, Cold Spring harbor laboratory, 1988). For example, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), fluoroimmunoassay, or the like can be used. The method of measuring the binding activity of an antibody to an antigen expressed in a cell can be, for example, the method described in the aforementioned Antibodies A Laboratory Manual, page 359-420.

In addition, a method of measuring binding between an antigen expressed on the surface of a cell suspended in, for example, a buffer and an antibody against the antigen can be particularly applied to a method using a flow cytometer. The flow cytometer used may be, for example, FACSCANTO^TM II、FACSAria^TM、FACSArray^TM、FACSVantage^TM SE、FACSCalibur^TM(all manufactured by BD Biosciences Co., Ltd.) or EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADCEPICS XL ADC, Cell Lab Quanta/Cell Lab Quanta SC (all manufactured by Beckman Coulter Co., Ltd.).

One of the preferred methods for measuring the binding activity of a test GRP78 antibody to an antigen is, for example, staining with a FITC-labeled secondary antibody that recognizes the test antibody to be reacted with GRP 78-expressing cells, measuring with FACSCalibur (BD), and analyzing the fluorescence intensity with CELLQUEST software (BD).

Proliferation inhibitory Activity

The method for evaluating or measuring the cell proliferation inhibitory effect by the anti-GRP 78 antibody may preferably be the following method. The method for evaluating or measuring the activity of inhibiting cell proliferation in vitro may be a method of using living cells to bind the cells added to the medium³H]A method for measuring uptake of labeled thymidine as an indicator of DNA replication ability. A more convenient method is a dye exclusion method or MTT method, which can count the ability of excluding a dye such as trypan blue from cells under a microscope. The latter takes advantage of the ability of living cells to convert the tetraborate MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) to a blue formazan product. More specifically, the test antibody is added to a culture solution of the test cell together with the ligand, and after a lapse of time, an MTT solution is added to the culture solution, and left to stand for a period of time to allow the cell to take up MTT. As a result, the yellow compound MTT is converted into a blue compound by succinate dehydratase in the mitochondria of the cell. This blue compound was dissolved, developed, and then the absorbance thereof was measured to use it as an index of the number of living cells. In addition to MTT, commercially available MTS, XTT, WST-1, WST-8, etc. (nacalaitesque, etc.) may be suitably selected. Control antibodies may also be used in determining activity.

In addition, methods for evaluating or measuring the activity of inhibiting cell proliferation in vivo can utilize tumor-bearing mouse models. For example, a cancer cell expressing GRP78 is transplanted into a non-human test animal intradermally or subcutaneously, and the test antibody is administered intravenously or intraperitoneally every day or every several days from the day or the next day. During which the tumor size is measured to evaluate the activity of known cell proliferation. The control antibody was administered in the same manner as the evaluation performed in vitro, and the activity of inhibiting cell proliferation was judged by significantly smaller tumor size of the group administered with the anti-GRP 78 antibody compared to the tumor size of the group administered with the control antibody. When a mouse is used as a non-human test animal, a nude mouse (nu/nu) whose T lymphocyte is disabled by genetic means due to thymus deficiency can be suitably used. Interference of T lymphocytes in the test animal in the course of evaluating and measuring the cell proliferation-suppressing activity of the administered antibody can be excluded by using the mouse.

Method for inhibiting cell proliferation

The invention provides methods of inhibiting the proliferation of a cell expressing GRP78 by contacting the cell with an antibody of the invention. As described above, the antibody of the present invention binds to GRP78 protein contained in the cell proliferation inhibitor of the present invention. The cell to be contacted with the anti-GRP 78 antibody is not particularly limited as long as it is a cell expressing GRP78, but is preferably a cell in which GRP78 is located on the cell membrane, or preferably a disease-related cell. Preferred examples of the disease-related cells may be cancer cells. Vascular endothelial cells (tumor vessels) present in malignant tumors are also of this type. The kind of the target cancer is not particularly limited, and may be, for example, prostate cancer, breast cancer, pancreatic cancer, lung cancer, esophageal cancer, melanoma, colon cancer, stomach cancer, ovarian cancer, bladder cancer, brain tumor.

Delivery methods using anti-GRP 78 antibodies

The present invention relates to methods of delivering cell damaging substances into cells using anti-GRP 78 antibodies. The antibody used in the present invention is an anti-GRP 78 antibody that binds to the cell-damaging substance. In this case, an antibody having an activity of being taken into cells is preferable. Delivery of the cell-injurious substance may be effected by contacting an anti-GRP 78 antibody that binds to the cell-injurious substance with a cell that expresses GRP 78. The cell to be delivered with the cell-damaging substance in the present invention is not particularly limited, but a cell in which GRP78 is located on the cell membrane or a disease-related cell is preferred. The disease-associated cell can be, for example, a cancer cell. Vascular endothelial cells (tumor vessels) present in malignant tumors are also of this type. The kind of the target cancer is not particularly limited, and may be, for example, prostate cancer, breast cancer, pancreatic cancer, lung cancer, esophageal cancer, melanoma, colon cancer, stomach cancer, ovarian cancer, bladder cancer, brain tumor.

The "contacting" of the present invention may be performed in vitro or in vivo. The form of the antibody to be added at this time may be suitably a solution or a solid form obtained by a method such as lyophilization. May be a solution containing, for example, surfactants, excipients, dyes, fragrances, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binding agents, disintegrating agents, lubricants, glidants, flavoring agents, and the like. The concentration to be added is not particularly limited, but the final concentration in the culture solution is preferably 1pg/ml to 1g/ml, more preferably 1ng/ml to 1mg/ml, and still more preferably 1. mu.g/ml to 1 mg/ml.

Alternatively, the "contacting" in vivo of the present invention may be performed by administering to a non-human animal having cells expressing GRP78 transplanted into the body or an animal including a human having cancer cells endogenously expressing GRP 78. The administration method may be either oral or parenteral. A method of non-oral administration is particularly preferred, and the administration method may specifically be, for example, injection administration, nasal administration, pulmonary administration, transdermal administration, or the like. The cell proliferation inhibitor and the anticancer agent of the pharmaceutical composition of the present invention can be administered systemically or locally by injection (e.g., intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.). The method of administration can be selected as appropriate according to the age and symptoms of the animal to be tested. When administered as an aqueous solution, it may be an aqueous solution containing only the antibody, or a solution containing, for example, a surfactant, an excipient, a dye, an aromatic agent, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, a glidant, a flavoring agent, or the like. The amount administered can be selected, for example, from 0.0001mg to 1000mg/kg per administration. Or alternatively, an amount of, e.g., 0.001 to 100000mg per patient. However, the amount of the antibody of the present invention to be administered is not limited to these amounts.

Pharmaceutical composition

In another aspect, the invention features pharmaceutical compositions containing an antibody that binds to GRP78 protein. The invention also features inhibitors of cell proliferation, particularly anticancer agents, comprising antibodies that bind to GRP78 protein. The cell proliferation inhibitor and the anticancer agent of the present invention are preferably administered to a subject suffering from cancer or a subject having a possibility of suffering from cancer.

The cell proliferation inhibitor containing an antibody binding to GRP78 protein of the present invention may be embodied as a method of inhibiting cell proliferation comprising the step of administering an antibody binding to GRP78 protein to a subject, or as a use of an antibody binding to GRP78 protein for the preparation of a cell proliferation inhibitor.

In addition, the anticancer agent containing an antibody binding to GRP78 protein of the present invention may be embodied as a method for preventing or treating cancer comprising the step of administering an antibody binding to GRP78 protein to a subject, or as a use of an antibody binding to GRP78 protein for the preparation of an anticancer agent.

The antibody contained in the pharmaceutical composition (e.g., cell proliferation inhibitor and anticancer agent) of the present invention is not particularly limited as long as it binds to GRP78 protein, and any antibody exemplified in the present specification can be used.

The method of administering the pharmaceutical composition of the present invention can be performed by either oral or parenteral administration. Particularly preferred is a method of administration by parenteral administration, and specific examples of the method of administration include injection administration, nasal administration, pulmonary administration, and transdermal administration. Examples of injectable administration are systemic or local administration of the pharmaceutical composition of the present invention by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. The method of administration may be suitably selected depending on the age and symptoms of the patient. The amount administered can be selected, for example, from 0.0001mg to 1000mg/kg per administration. Or alternatively, an amount of, e.g., 0.001 to 100000mg per patient. However, the administration amount of the pharmaceutical composition of the present invention is not limited to these administration amounts.

The Pharmaceutical composition of the present invention can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark publishing Company, Easton, U.S. A), or can be formulated together with pharmaceutically acceptable vehicles or additives. The vehicle may be, for example, a surfactant, excipient, dye, aromatic agent, preservative, stabilizer, buffer, suspending agent, isotonic agent, binder, disintegrant, lubricant, glidant, flavoring agent, etc., but is not limited thereto, and other conventional carriers may be suitably used. Specifically, for example, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylamine acetate, polyvinylpyrrolidone, gelatin, medium-grade fatty acid triglyceride, polyoxyethylene (60) hardened castor oil, white sugar, carboxymethylcellulose, corn starch, inorganic salts, and the like can be mentioned.

Process for the preparation of a medicament

The present invention also provides a method for preparing a medicament, particularly an anticancer agent, comprising the following steps.

A process for preparing a pharmaceutical composition comprising the steps of:

(a) providing an anti-GRP 78 antibody;

(b) confirming whether the antibody of (a) has an activity of being taken up by cells;

(c) screening for antibodies having cellular uptake activity;

(d) binding the antibody selected in (c) to the cell-damaging material.

Whether or not it has an activity of being taken into cells can be confirmed by the above-mentioned method. The anti-GRP 78 antibody and the cell-damaging substance may be the anti-GRP 78 antibody and the cell-damaging substance.

Cancer diagnosis

The present invention provides methods of disease diagnosis, particularly cancer diagnosis, using anti-GRP 78 antibodies.

The diagnostic method of the present invention can be carried out by detecting an anti-GRP 78 antibody taken into cells. The anti-GRP 78 antibody for use in the present invention preferably has an uptake activity by cells, and is preferably labeled with a marker.

Therefore, a preferred embodiment of the diagnostic method of the present invention may be a diagnostic method using an anti-GRP 78 antibody labeled with a marker and having an activity of being taken into cells, for example. The anti-GRP 78 antibody conjugated to a label can be the anti-GRP 78 antibody described above.

The label to which the anti-GRP 78 antibody is bound is not particularly limited, and any label known to those skilled in the art, such as a fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance, a radioactive substance, and the like, can be used, and specifically, for example, a radioisotope (32P, 14C, 125I, 3H, 131I, and the like), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β -galactosidase, β -glucosidase, horseradish peroxidase, glucoamylase, lysozyme, carbohydrate oxidase, microperoxidase, biotin, and the like can be used. If biotin is used as the label, avidin to which an enzyme such as alkaline phosphatase is bonded is preferably added after the biotin-labeled antibody is added. The label and the anti-GRP 78 antibody can be bound by a known method such as the glutaraldehyde method, the maleimide method, the pyridine disulfide method, or the peroxoiodine method.

The label may be bound to the antibody by methods well known to those skilled in the art.

When the disease diagnosed by the method according to the present invention is cancer, the kind of the target cancer is not particularly limited, and may be, for example, prostate cancer, breast cancer, pancreatic cancer, lung cancer, esophageal cancer, melanoma, colon cancer, stomach cancer, ovarian cancer, bladder cancer, brain tumor.

The diagnosis of the present invention may be performed in vivo or in vitro.

In vitro diagnosis can be carried out by a method comprising, for example, the following steps:

(a) providing a sample taken from a subject;

(b) contacting the sample of (a) with an anti-GRP 78 antibody that binds to a label;

(c) detecting the antibody taken into the cell.

The sample to be sampled is not particularly limited, but is preferably, for example, a cell, tissue or the like taken from a subject. In addition, a secondary sample obtained from a test sample, such as a specimen collected from a living tissue or a cell fixed sample or a cell culture solution, is also a sample of the present invention.

In vivo diagnosis can be performed by a method comprising, for example, the following steps:

(a) administering to the subject an anti-GRP 78 antibody conjugated to a marker;

(b) detecting the antibody taken into the cancer cell.

The amount of the anti-GRP 78 antibody to be administered is appropriately determined by those skilled in the art depending on the kind of the marker, the kind of the disease to be diagnosed, and the like. The labeled anti-GRP 78 antibody can be formulated according to the methods described above.

The present invention also provides a method for preparing a diagnostic reagent, particularly a cancer diagnostic reagent, comprising the steps of:

(a) providing an anti-GRP 78 antibody;

(b) confirming whether the antibody of (a) has an activity of being taken up by cells;

(c) screening for antibodies having cellular uptake activity;

(d) the antibody screened in (c) binds to a label.

Whether or not it has an activity of being taken into cells can be confirmed by the above-mentioned method. In addition, the anti-GRP 78 antibody and the marker may be the anti-GRP 78 antibody and the marker.

GRP78 partial peptide

The present invention provides a polypeptide consisting of SEQ ID NO: 3 (amino acids 376 to 415 of GRP78), or a fragment thereof. Consisting of SEQ ID NO: 3 (amino acids 376 to 415 of GRP78), or a fragment thereof can be used as an immunogen for producing the antibody of the present invention, or can be used to evaluate the binding activity of the produced antibody. The "fragment" of the present invention is at least 5 amino acids or more, preferably 10 amino acids or more, and more preferably 15 amino acids or more. To a polypeptide consisting of SEQ ID NO: examples of the polypeptide fragment consisting of the amino acid sequence of 3 include, but are not limited to, a fragment consisting of the amino acid sequence of amino acids Nos. 384 to 391 of GRP78 (a fragment consisting of the amino acid sequence of amino acids No. 9 to 16 of SEQ ID NO: 3), a fragment consisting of the amino acid sequence of amino acids Nos. 392 to 407 of GRP78 (a fragment consisting of the amino acid sequence of amino acids No. 17 to 32 of SEQ ID NO: 3), and a fragment consisting of the amino acid sequence of amino acids No. 400 to 415 of GRP78 (a fragment consisting of the amino acid sequence of amino acids No. 25 to 40 of SEQ ID NO: 3).

Examples

Example 1: immunization

1-1 preparation of immunogens

1-1-1 construction of GRP78 E.coli expression vector

To construct the GRP78 E.coli expression vector, the GRP78 gene was first cloned according to the following method. First, RT-PCR was performed using human colon adenocarcinoma cDNA (MTC, multiple-tissue cDNA panel, CloneTech) as a template and Pyrobest Taq polymerase (TAKARA) under the following conditions to clone the full-length GRP78 gene.

GRP-1：atgaagctct ccctggtggc(SEQ ID NO：26)

GRP-2：ctacaactca tctttttctg ctgta(SEQ ID NO：27)

(94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 120 seconds, 27 cycles)

Subsequently, PCR was performed again using the obtained PCR product as a template according to the following conditions to obtain a PCR product represented by SEQ ID NO: 1 nucleotide sequence nt55-1965 GRP78 gene fragment with BamHI and XhoI cleavage sequence GRP78 cDNA fragment added at 5 'end and 3' end respectively.

GRP-GST-1：aaaggatccg aggaggagga caagaaggag gacgtggg(SEQID NO：28)

GRP-GST-2：tttctcgagc tacaactcat ctttttctgc tgtatcctc(SEQID NO：29)

(94 ℃ 30 seconds, 64 ℃ 30 seconds, 72 ℃ 120 seconds, 25 cycles)

This was digested with BamHI and XhoI and inserted into the downstream of GST coding region of E.coli GST fusion expression vector (pGEX-6P-1, Amersham Pharmacia) similarly digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP 78-full length).

1-1-2 expression induction and purification of GST fusion GRP78 protein

Next, GRP78 protein was prepared for use as an immunogen to obtain GRP78 binding antibodies.

Coli (BL21) was first transformed with pGEX-GRP 78-full length. It was cultured in LB medium (300ml) to OD₆₁₀When the concentration reached 0.5 or more, IPTG was added to 0.5mM, whereby inducible expression of the protein was carried out. After 5 hours of culture, E.coli was collected by centrifugation.

The collected E.coli was suspended in 30ml of B-BER (PIAS Co.) for lysis. This lysed E.coli lysate was then diluted 10-fold with PBS and glutathione Sepharose 4B (Amersham pharmacia) equilibrated with PBS was added thereto and incubated overnight at 4 ℃. Thereafter, the reaction mixture was washed several times with PBS to remove the unadsorbed protein, and PreScission protease (Amersham Pharmacia) was reacted in a protease reaction solution (50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM DTT, pH7.5) at 4 ℃ overnight. This operation cleaved the GST protein of the GRP78-GST fusion protein from the GRP78 protein (amino acids 19-654). Subsequently, the GRP78 protein eluted by protease digestion was subjected to gel filtration chromatography using Superdex200HR 1030 column (Amersham Pharmacia), and the objective GRP78 protein (amino acid No. 19-654) was recovered.

1-2. immunization

An emulsion of GRP78 protein was prepared with complete adjuvant (DIFCO: DF263810) at the time of primary immunization and incomplete adjuvant (DIFCO: DF263910) after secondary immunization, and was subcutaneously injected at 50. mu.g/mouse into each mouse [ (MRL/1pr, male, 4 weeks old) (Balb/c, female, 6 weeks old): each was purchased from Charles River, Japan and immunized every 3 times (TERMO syringe 1ml, needle 26G). The secondary immunization was performed 2 weeks after the primary immunization, and thereafter, 4 to 5 immunizations were performed at 1-week intervals. GRP78 (50. mu.g) was suspended in 100. mu.l PBS and the last immunization was performed by tail vein injection and cell fusion was performed 3 days later.

1-3 preparation of hybridomas

Cell fusion was performed as follows. Spleens were aseptically removed from mice and ground into single cell suspensions in culture medium 1(RPMI1640+ PS). Adipose tissues and the like were removed by passing them through a 70 μm nylon mesh (Falcon), and cell counting was performed. The resulting B cells were mixed with mouse myeloma cells (P3U1 cells) at a cell number ratio of about 2: 1, and 1mL of 50% PEG (Roche, cat #: 783641) was added for cell fusion. The fused cells were suspended in culture solution 2[ RPMI1640+ PS, 10% FCS, HAT (Sigma, H0262), 5% BMconditioned H1 (Roche: #1088947) ] and injected into appropriate (10) 96-well plates at 200. mu.L/well, and cultured at 37 ℃. Hybridoma selection was performed with the culture supernatant after 1 week.

Example 2: screening of anti-GRP 78 antibodies recognizing GRP78 localized on cell membranes

2-1 screening by ELISA for GRP 78-binding antibodies (Primary screening)

To obtain anti-GRP 78 antibodies on the cell surface, a screen for antibodies that bind GRP78 was first performed. ELISA was used for the screening of bound antibodies.

The ELISA plate coated with 1. mu.g/ml of GRP78 protein purified from E.coli (NUNC) was reacted with the culture supernatant of the hybridoma and incubated for 1 hour. Then reacted with Alkaline Phosphatase (AP) -labeled anti-mouse IgG (ZYMED: #62-6622) for 1 hour, and 1mg/ml of a substrate (SIGMA: S0942-50TAB) was added thereto to develop color. OD measurement with plate reader (BioRad Co.)₄₀₅And screening ELISA positive holes.

2-2 selection of anti-GRP 78 antibody on cell surface by FACS (Secondary selection)

The reactivity of the culture supernatants of wells positive for the primary screening to the prostate cancer cell line (DU145) was analyzed by FACS.

DU145 (obtained from ATCC) was cultured and passaged using EMEM (invitrogen) containing 10% FCS, 1mM sodium pyruvate, 0.1mM NEAA. DU145 was detached with 1mM EDTA/PBS and reacted with the culture supernatant of hybridoma, followed by incubation at 4 ℃ for 1 hour. Then, an FITC-labeled anti-mouse IgG antibody (BECKMAN COULTER: PN IM0819) was added thereto, and the mixture was incubated at 4 ℃ for 30 minutes. Then, the binding activity of each hybridoma culture supernatant to the cell surface of DU145 cells was analyzed by facs (bd).

2-3. Limiting dilution (Limiting dilution)

Wells that slightly exhibited binding activity to DU145 cells by FACS analysis were selected and subjected to Limiting Dilution (LD) to make hybridomas in the wells monoclonal. I.e., the number of cells in each well was determined, and 96-well plates were seeded at 3 cells/well. After about 10 days of culture, the culture supernatants of the wells in which the colonies were formed were again subjected to ELISA, and antibody-producing monoclonals were screened by using the binding activity as an index. By this sequential operation, 6 clones of GRP 78-binding antibodies (GA-19 antibody, GA-20 antibody, GA-21 antibody, GA-23 antibody, GA-28 antibody, and GA-31 antibody) were obtained.

2-4. determination of subtype

Isotripp (Roche # 1493027) was used to determine the antibody subtype. For subtype determination, culture supernatants of hybridomas diluted 10-fold with PBS (-) were used.

The purified subtypes of each antibody are shown below.

TABLE 1

Antibodies	Subtype of cell
		GA-19	G1

GA-20	M
		GA-21	G3
GA-23	G2a
		GA-28	G1
GA-31	G1

2-5 purification of antibodies

The GA-19 antibody, GA-23 antibody, GA-28 antibody, and GA-31 antibody were purified from the culture supernatant of the resulting 50mL monoclonal hybridoma using a Hi Trap protein G HP 1mL column (Amersham Biosciences # 17-0404-01); in addition, 1ml of protein L-Sepharose (SIGMA) was packed in an open column to purify GA-20 antibody and GA-21 antibody as IgM and IgG 3. Hybridoma supernatant was adsorbed at a flow rate of 1 mL/min, washed with 20mL of 20mM phosphate buffer (pH7.0), and then eluted with 3.5mL of 0.1M glycine-HCl (pH 2.7). The eluted fractions were collected in 0.5ml per tube using Eppendorf tubes to which 50. mu.L of each 1M Tris-HCl (pH9.0) had been previously added. Determination of OD_280nmAfter antibody-containing fractions were pooled and PBS (-) was added to make the total volume 2.5mL, the buffer was changed to PBS (-) using PD-10 column (Amersham Bioscience # 17-0851-01). The purified antibody was passed through a 0.22 μm filter (MILLIPORE # SLGV033RS), described in detail belowThe properties of each purified antibody are discussed.

Example 3: assay for GRP78 antibody

3-1 Western blot analysis

Western blot analysis was performed to confirm that the resulting antibody specifically binds to GRP78(GST-GRP78) purified from E.coli and GRP78 expressed in DU145 cells.

DU145 cells (1X 10) were added to lane 1⁶Cells) cell lysis sample lysed with 300. mu.l NP40 lysis buffer (0.5% NP40, 10mM Tris-HCl, 150mM NaCl, 5mM EDTA, pH7.5), GST-GRP78 fusion protein (0.1. mu.g) purified from E.coli was added to lane 2 and blotted on PVDF membrane according to a protocol.

After reacting with each antibody (2. mu.g/ml) and a secondary antibody (HRP-labeled anti-mouse IgG), the protein was detected with ECL Western blot detection reagent (GE Healthcare).

As a result, it was found that each of the obtained antibodies could recognize not only GST-GRP78 fusion protein expressed by E.coli but also specifically GRP78 protein endogenously expressed in cells (FIG. 1).

FACS analysis

3-2-1. binding Activity to cell surface of prostate cancer Strain (DU145)

Whether each of the purified anti-GRP 78 antibodies stained the cell surface of cancer cells was analyzed by FACS.

DU145 cells stripped with 1mM EDTA were incubated with each antibody (10. mu.g/ml) in FACS buffer for 1 hour at 4 ℃. Then, an FITC-labeled anti-mouse IgG antibody (BECKMAN COULTER: PN IM0819) was added and incubated at 4 ℃ for 30 minutes. The binding activity of DU145 cell surface to GRP78 was then analyzed by facs (bd).

As a result, DU145 cells were stained in 2 clones (GA-20 antibody, GA-21 antibody) among the 6 anti-GRP 78 antibody clones obtained (FIG. 2).

Thus, it was confirmed that the GA-20 antibody and the GA-21 antibody recognize an extracellular epitope of GRP78 molecule expressed in cancer cells.

The base sequence of the heavy chain variable region of the GA-20 antibody is shown in SEQ ID NO: 4, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5, the base sequence of the light chain variable region is shown in SEQ ID NO: 6, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7. and the amino acid sequence of CDR1 of the heavy chain variable region of the GA-20 antibody is shown in SEQ ID NO: 8, the amino acid sequence of CDR2 is shown in SEQ ID NO: 9, the amino acid sequence of CDR3 is shown in SEQ ID NO: 10; the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 11, the amino acid sequence of CDR2 is shown in SEQ ID NO: 12, the amino acid sequence of CDR3 is shown in SEQ ID NO: 13.

the base sequence of the heavy chain variable region of the GA-21 antibody is shown in SEQ ID NO: 14, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, the base sequence of the light chain variable region is shown in SEQ ID NO: 16, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17. and the amino acid sequence of CDR1 of the heavy chain variable region of the GA-21 antibody is shown in SEQ ID NO: 18, the amino acid sequence of CDR2 is shown in SEQ ID NO: 19, the amino acid sequence of CDR3 is shown in SEQ ID NO: 20; the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 21, the amino acid sequence of CDR2 is shown in SEQ ID NO: 22, the amino acid sequence of CDR3 is shown in SEQ ID NO: 23.

3-2-2 evaluation of FACS binding Activity to other tumors

Next, FACS reactivity of GA-20 antibody, an antibody recognizing GRP78 located on the cell surface, to other tumors was investigated. Ovarian cancer cell lines (ES-2, SKOV3), breast cancer cell lines (MCF7), colon cancer cell lines (LoVo), prostate cancer cell lines (DU145, LNcap, 22Rv1, PC3) were purchased from ATCC and cultured according to the culture conditions recommended by ATCC. These cells were stained using GA-20 antibody (10. mu.g/ml) as described above and subjected to FACS analysis. As a result, it was found that not only DU145 but also GA-20 antibody could stain various cancer cells such as LoVo, LNcap, 22Rv1 (FIG. 3).

3-2-3 evaluation of FACS binding Activity on cells other than cancer cells

Next, FACS binding activity to cells other than cancer cells was analyzed. Monkey kidney cells (COS7), human normal fibroblasts (MRC5), mouse primary B cells (Ba/F3), mouse fibroblasts (NIH3T3), and hamster ovary cells (DG44) were purchased from ATCC and cultured in the media prepared by ATCC. These cells were stained with GA-20 antibody (10. mu.g/ml) and FACS analysis was performed. At the same time, the cells were lysed with NP40 lysis buffer and subjected to Western blot analysis with GA-20 antibody (2. mu.g/ml). As a result, it was found that none of these cells were stained by FACS (fig. 4A), although they all expressed GRP78 (fig. 4B). From this, it is presumed that, even though the cell expresses GRP78, it is localized only on the cell surface of a specific cell such as a cancer cell.

3-3 analysis of uptake Activity by cells

3-3-1 analysis by FACS

The following experiment was conducted to confirm whether or not the anti-GRP 78-recognizing antibodies (GA-20 antibody and GA-21 antibody) staining the cell surface had the activity of being taken up into the cells.

DU145 cells were detached with 1mM EDTA in FACS buffer (2% FCS, 0.05% NaN)₃PBS) was allowed to react with each antibody (10. mu.g/ml) at 0 ℃ for 2 hours, while each antibody (10. mu.g/ml) was incubated with each antibody (10. mu.g/ml) in a culture medium (RPMI1640 containing 10% FCS) at 37 ℃ for 2 hours. Thereafter, antibodies remaining on the cell surface were detected with FITC-labeled mouse IgG by performing FACS analysis.

As a result, the antibody on the cell surface was found to disappear by reacting with any antibody at 37 ℃ for 2 hours (FIG. 5).

3-3-2 analysis by cellular immunostaining

To confirm that the phenomenon indicates that the antibody is not released outside the cell, the following experiment was performed. To DU145 cells cultured in the dish, each 20. mu.g/ml GA-20 antibody or GA-31 antibody not bound to the cell surface as a negative control was added, and cultured at 37 ℃ for 3 hours. After washing with PBS, the cells were washed 2 times with glycine buffer (0.1M glycine, pH2.7) to remove the antibody bound to the cell surface. Thereafter, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, followed by 0.1% Triton X100 at room temperature for 5 minutes. It was stained with FITC-labeled mouse IgG, and the antibody taken into the cells was observed with a fluorescence microscope (KEYENCE).

As a result, GA-20 antibody having an activity of binding to the cell surface could be detected in the cells, whereas GA-31 antibody not binding to the cell surface could not be detected in the cells (FIG. 6). As a result, it was confirmed that the GA-20 antibody bound to the cell surface was taken into the cells by culturing at 37 ℃ for 3 hours.

Example 4: epitope analysis

4-1 preparation of GST fusion protein for epitope mapping

4-1-1 preparation of expression vector of GST fusion protein for epitope mapping

To identify the epitope of each antibody, an E.coli expression vector was first constructed as a GST fusion protein that divides the GRP78 protein into various regions.

Construction of GST-GRP78-N (19-350) expression vector

pGEX-GRP 78-full length was used as a template, and PCR was carried out using a forward primer GRP-GST-1(SEQ ID NO: 28) and a reverse primer GRP-GST-3(SEQ ID NO: 30) at 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 120 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids No. 19 to 350) having digestion sequences BamHI and XhoI attached to the 5 'end and 3' end, respectively.

This was digested with BamHI and XhoI and inserted into the downstream of GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI, to thereby construct a GRP78-GST fusion protein expression vector (pGEX-GRP78-N (19-350)).

4-1-1-2 construction of GST-GRP78-C (289-654) expression vector

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-4(SEQ ID NO: 31) and a reverse primer GRP-GST-2(SEQ ID NO: 29) in cycles of 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 120 seconds × 25, to obtain cDNA fragments encoding GRP78 (amino acids 289-654) having digestion sequences BamHI and XhoI added to the 5 '-end and 3' -end, respectively.

It was digested with BamHI and XhoI and inserted into the downstream of GST coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78-C (289-654)).

Construction of GST-GRP78-C (289-350) expression vector

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-4(SEQ ID NO: 31) and a reverse primer GRP-GST-3(SEQ ID NO: 30) at 94 ℃ for 30 seconds and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids 289-350) having enzyme digestion sequences BamHI and XhoI attached to the 5 '-and 3' -ends, respectively.

It was digested with BamHI and XhoI and inserted into the downstream of GST coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78-C (289-350)).

Construction of GST-GRP78-C (289-445) expression vector

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-4(SEQ ID NO: 31) and a reverse primer GRP-GST-5(SEQ ID NO: 32) at 94 ℃ for 30 seconds and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids 289-445) having enzyme digestion sequences BamHI and XhoI attached to the 5 '-and 3' -ends, respectively.

It was digested with BamHI and XhoI and inserted into the downstream of GST coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78-C (289-445)).

Construction of GST-GRP78-C (289-538) expression vector

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-4(SEQ ID NO: 31) and a reverse primer GRP-GST-6(SEQ ID NO: 33) at 94 ℃ for 30 seconds and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids 289-538) having enzyme digestion sequences BamHI and XhoI attached to the 5 '-and 3' -ends, respectively.

It was digested with BamHI and XhoI and inserted into the downstream of GST coding region of E.coli GST fusion expression vector (pGEX-6P-1) which was also digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGST-GRP78-C (289-538)).

Construction of GST-GRP78(345-385) expression vector

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-7(SEQ ID NO: 34) and a reverse primer GRP-GST-8(SEQ ID NO: 35) at 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids Nos. 345 to 385) each having a digestion sequence BamHI and XhoI added to the 5 'end and 3' end, respectively.

The resulting fragment was digested with BamHI and XhoI and inserted into the downstream of GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which had also been digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78 (345-385)).

4-1-1-7 construction of GST-GRP78(376-

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-9(SEQ ID NO: 36) and a reverse primer GRP-GST-10(SEQ ID NO: 37) at 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids No. 376 to 415) having digestion sequences BamHI and XhoI attached to the 5 'end and 3' end, respectively.

The resulting fragment was digested with BamHI and XhoI and inserted into the downstream of GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which had also been digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78 (376. 415)).

4-1-1-8 construction of GST-GRP78(406-

pGEX-GRP 78-full length was used as a template, and PCR was carried out using a forward primer GRP-GST-11(SEQ ID NO: 38) and a reverse primer GRP-GST-5(SEQ ID NO: 32) at 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids No. 406 to 445) having cleavage sequences BamHI and XhoI attached to the 5 'end and 3' end, respectively.

The resulting fragment was digested with BamHI and XhoI and inserted into the downstream of GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which had also been digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78 (406-.

Construction of GST-GRP78(345-

Using pGEX-GRP 78-full length as a template, PCR was carried out using a forward primer GRP-GST-7(SEQ ID NO: 34) and a reverse primer GRP-GST-5(SEQ ID NO: 32) at 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 30 seconds × 25 cycles to obtain cDNA fragments encoding GRP78 (amino acids No. 345 to No. 445) having enzyme digestion sequences BamHI and XhoI attached to the 5 'end and 3' end, respectively.

The resulting fragment was digested with BamHI and XhoI and inserted into the downstream of GST-coding region of E.coli GST fusion expression vector (pGEX-6P-1) which had also been digested with BamHI and XhoI, thereby constructing a GRP78-GST fusion protein expression vector (pGEX-GRP78 (345-445)).

The primer sequences used are listed below.

GRP-GST-1：aaaggatccg aggaggagga caagaaggag gacgtggg(SEQID NO：28)

GRP-GST-2：tttctcgagc tacaactcat ctttttctgc tgtatcctc(SEQID NO：29)

GRP-GST-3：tttctcgagc taatcagaat cttccaacac tttctggacg ggc(SEQ ID NO：30)

GRP-GST-4：aaaggatccc ggcgcgaggt agaaaaggcc aaac(SEQ IDNO：31)

GRP-GST-5：ttctcgagct aggtaggcac cactgtgttc cttgg(SEQ IDNO：32)

GRP-GST-6：ttctcgagct agatttcttc aggtgtcagg cgatt(SEQ IDNO：33)

GRP-GST-7：tttggatccg tgttggaaga ttctgatttg aaga(SEQ IDNO：34)

GRP-GST-8：ttctcgagct aggatggttc cttgccattg aagaa(SEQ IDNO：35)

GRP-GST-9：aaaggatcca aagagttctt caatggcaagga(SEQ ID NO：36)

GRP-GST-10：ttctcgagct ataccaggtc acctgtatct tgatc(SEQ IDNO：37)

GRP-GST-11：aaaggatcct ctggtgatca agatacaggt gac(SEQ IDNO：38)

4-1-2 expression Induction of respective GST fusion GRP78 proteins

Coli BL21 strain was transformed with each of the E.coli expression vectors constructed as described above. These E.coli were grown in LB medium (1 ml each) and IPTG (final 1mM) was added in the logarithmic growth phase to induce protein expression. After 4 to 5 hours, E.coli was recovered, and it was lysed in SDS sample buffer (0.5ml) to form a lysate, after which 5. mu.l of the lysate was subjected to SDS-PAGE according to a standard method and blotted on a PVDF membrane, thereby being used for Western blotting.

4-2 epitope mapping of the respective antibodies

Each region of the GRP78 protein, GST fusion protein, prepared as described above was used for western blotting and each of the resulting anti-GRP 78 antibodies was investigated for which region of the GRP78 protein was recognized.

According to the initial Western blot results (FIG. 7), the GA-19 antibody not FACS-stained with cells recognized the N-terminal side (19-350), while the GA-23 antibody, GA-28 antibody, and GA-31 antibody recognized the 538-654 region of the C-terminal.

And Western blot staining pattern showing that cell FACS-stained GA-20 antibody, GA-21 antibody recognized a region spanning about 100 amino acids from amino acid No. 350 to amino acid No. 445.

The binding regions of the GA-20 antibody and the GA-21 antibody were identified by preparing a GST fusion protein obtained by dividing the amino acid region No. 350 to No. 445 into 3 regions and performing Western blot analysis as described above. As a result, it was found that the epitope of the GA-20 antibody or the GA-21 antibody was 40 amino acids among amino acids 376 to 415 in the GRP78 protein (FIG. 8).

Example 5: preparation of cell death inducer Using anti-GRP 78 antibody (GA-20 antibody) recognizing extracellular region

Cloning of the variable region of the GA-20 antibody and amino acid sequence sequencing thereof

From about 5X 10 with Trizol (#15596-⁶Total RNA was purified from each hybridoma. The full-length cDNA was synthesized from 1. mu.g of the total RNA obtained using SMART RACE cDNA amplification kit (CLONTECH # PT3269-1) according to the attached manual. Using the obtained cDNA as a template, PCR was performed under the following conditions using Advantage 2PCR enzyme system (CLONTECH # PT3281-1) to amplify the genes encoding the heavy chain (VH) and light chain (VL) variable regions of the GA-20 antibody.

Primers for cloning light chain variable regions

General primer mixtures (UPM) -k (VL-k)

UPM: attached to the kit

VL-k：gct cac tgg atg gtg gga aga tg(SEQ ID NO：39)

Primers for cloning heavy chain variable regions

UPM～VH-M

UPM: attached to the kit

VH-M：cca cca gat tct tat cag aca gg(SEQ ID NO：40)

94 ℃ for 5 seconds, 72 ℃ for 2 minutes, 5 cycles

5 cycles of 94 ℃ for 5 seconds, 70 ℃ for 10 seconds, 72 ℃ for 2 minutes

27 cycles of 94 ℃ for 5 seconds, 68 ℃ for 10 seconds, 72 ℃ for 2 minutes

The gene fragment amplified as described above was TA-cloned into pCRII-TOPO (Invitrogen TOPO TA-cloning kit, #45-0640), followed by determination of the base sequence of each insert using an ABI3730 sequencer.

5-2 preparation of toxin-labeled GA-20-Single chain Fv antibody (GA20-PE40)

5-2-1. preparation of GA20_ PE40 expression vector

Preparation of pET22b _ His _ PE40 from 5-2-1

Cell death-inducing antibodies labeled with immunotoxin (PE40) were prepared in an attempt based on GA-20 antibody, an antibody that specifically recognizes GRP78 located on the cell surface.

First, a toxin-labeled antibody (GA20_ PE40) expression vector encoding a toxin (PE40) attached to a single chain fv (scFv) of the GA-20 antibody was constructed. The gene of PE40, which is an immunotoxin, was amplified by PCR using plasmid DNA (pJH8) purchased from ATCC as a template under the following conditions.

Using a forward primer (PE-1) having an EcoRI recognition sequence at the 5 'end, followed by addition of a linker sequence consisting of 18 amino acids, and a reverse primer (PE-2) having a NotI recognition sequence at the 5' end, followed by addition of a stop codon, an ER shift signal sequence (KDEL), and a FLAG tag sequence, the primers were mixed in 10 XKOD-Plus buffer, 2mM dNTP, 25mM MgSO4, KOD-Plus (TAKARA Co.) and the like

10 seconds at 98 ℃, 5 seconds at 72 ℃, 4 minutes at 68 ℃ for 5 cycles

10 seconds at 98 deg.C, 5 seconds at 70 deg.C, 4 minutes at 68 deg.C, 5 cycles

10 seconds at 98 ℃ and 4 minutes at 68 ℃ for 25 cycles

PCR amplification was performed. The primer sequences are shown below.

PE-1：taagaattcg gtggcgcgcc ggagttcccg aaaccgtccaccccgccggg ttcttctggt ttagagggcg gcagcctggc cgcgctg(SEQ ID NO：41)

PE-2：acttagcggc cgctcactac agttcgtctt tcttatcgtcgtcatccttg tagtccggcg gtttgccggg ctggc(SEQ ID NO：42)

The amplification product of the PCR reaction was inserted into pGEM-T Easy using pGEM-T Easy Vector System I (manufactured by Promega). Sequencing was performed by an ABI3730 sequencer.

Then, a His tag sequence, HindIII recognition sequence, EcoRI recognition sequence, and NotI recognition sequence were inserted under the Pe1B signal sequence of pET22b vector (Novagen) which is an E.coli expression vector, to prepare pET22b _ His.

Then, the PE40 gene cloned into pGEM-T easy was amplified by PCR using EcoRI and NotI, separated by agarose gel, and the gene fragment was inserted between EcoRI and NotI of pET22b _ His to prepare pET22b _ His _ PE 40.

Construction of pET22b _ His _ GA20scFv-PE40 from 5-2-1-2

A single-chain Fv encoding the GA20 antibody, i.e., a linker sequence consisting of 15 amino acids ((GlyGlyGlyGlySer)₃) (SEQ ID NO: 43) the gene fragments of the heavy chain variable region and the light chain variable region of the joined GA-20 antibody were amplified by PCR.

First, the heavy chain variable region of GA-20 antibody cloned in pCRII-TOPO was used as a template, the heavy chain variable region was subjected to PCR amplification using a forward primer (GA20-1, SEQ ID NO: 44) and a reverse primer (GA20-2, SEQ ID NO: 45) as the heavy chain variable region, and a forward primer (GA20-3, SEQ ID NO: 46) and a reverse primer (GA20-4, SEQ ID NO: 47) as the light chain variable region with pyrobest DNA polymerase (TAKARA # R005) at 94 ℃ for 1 minute, and then subjected to PCR amplification at 94 ℃ for 30 minutes, 72 ℃ for 30 minutes and 25 cycles.

The PCR products of the heavy and light chain variable regions obtained were then purified using an S-300HR column (Amersham Biosciences #27-5130-01), 1. mu.L of each was mixed in the same tube, and after reaction with pyrobest DNA polymerase at 94 ℃ for 1 minute, PCR amplification was performed at 94 ℃ for 30 minutes, at 72 ℃ for 30 minutes, and at 5 cycles.

mu.L of the reaction mixture after renaturation with the primers GA20-1(SEQ ID NO: 44) and GA20-4(SEQ ID NO: 47) was reacted with pyrobest DNA polymerase at 94 ℃ for 1 minute under the following conditions, and then PCR amplification was carried out at 94 ℃ for 30 minutes, 72 ℃ for 1 minute, and 25 cycles.

The amplified fragment was purified by an S-400HR column (Amersham Biosciences #27-5140-01), digested with EcoRI-HindIII, and separated by agarose gel. This was inserted between HindIII and EcoR of pET22b _ His _ PE40 prepared in 5-2-1-1, and the nucleotide sequence was confirmed to prepare pET22b _ His _ GA20scFv-PE 40.

The primer sequences used are shown below:

GA20-1：aaaagcttga ggtccagctg caacagtctg g(SEQ ID NO：44)

GA20-2：cccgaaccac caccacccga accaccacca cctgaggagacggtgactga ggttcc(SEQ ID NO：45)

GA20-3：tggttcgggt ggtggtggtt cgggtggtgg cggatcggacattgtgatgt cacagtctcc atcct(SEQ ID NO：46)

GA20-4：ttgaattctt tgatttccag cttggtgcct c(SEQ ID NO：47)

the resulting base sequence of GA20_ PE40 is shown in SEQ ID NO: 24, and the corresponding amino acid sequence of the base sequence is shown in SEQ ID NO: 25.

5-2-2 purification of toxin-labeled GA-20_ Single chain Fv antibody (GA20_ PE40)

Coli BL21 transformed with pET22b _ His _ GA20scFv-PE40 was inoculated on LB agar plates containing 50. mu.g/ml ampicillin. The single colony formed was selected and cultured in LB medium (3ml) containing 50. mu.g/ml carbenicillin (CosmoBio). After 4 hours of culture, the resulting bacteria were expanded to LB medium containing 200ml of carbenicillin (50. mu.g/ml) and the culture was continued. When logarithmic growth phase was reached, the culture medium was replaced with fresh LB medium (200 ml of carbenicillin was added) and IPTG (final concentration of 1mM) was added to induce protein expression. After 5 hours of culture, the bacteria were recovered by centrifugation.

Bacteria suspended in 20mlB-BER (PIAS) were lysed in the presence of a protease inhibitor (Roche) completely free of EDTA, and insoluble proteins were removed by centrifugation to prepare a cell lysate. The cell lysate samples were applied to a HisTrap column (hittrap chelating HP 1ml, amersham pharmacia) and GA20_ PE40 was purified according to the product instructions. That is, the protein adsorbed to the column was washed with a binding buffer (20mM sodium phosphate, 0.5M NaCl, 10mM imidazole, pH7.4), and eluted by taking 7 fractions of 500. mu.l each with an elution buffer (20mM sodium phosphate, 0.5M NaCl, 500mM imidazole, pH 7.4).

To confirm from which fraction the toxin-labeled GA20 antibody of interest eluted, the binding activity of the eluted fractions was investigated by performing ELISA assay using GRP78 binding activity as an index. ELISA was performed as follows. To the coated with 1. mu.g/ml GST-GRP78 purified from E.coli or negative control HB-EGF protein (R)&Company D) (NUNC) was added to a plate containing diluted buffer (1% BSA, 50mM Tris, 1mM MgCl)₂150mM NaCl, 0.05% Tween20) 40-fold diluted fractions. After 1 hour of reaction at room temperature, the plate was washed 3 times with TBS-T (TBS-0.05% Tween20), added 1. mu.g/ml anti-Flag antibody (M2 antibody, Sigma), and incubated for 1 hour at room temperature. TBS-T was washed 3 times, reacted with alkaline phosphatase-labeled anti-mouse IgG (ZYMED) for 1 hour, and then developed with 1mg/ml substrate (Sigma).

As a result, GRP78 specific binding activity was detected in the elution fractions 2, 3(Elute2, 3), confirming that GA20-PE40 protein was concentrated in the elution fractions 2, 3 (fig. 9, bottom).

Fractions 2, 3 and 4 were then applied to a PD-10 column (GE Healthcare) and the buffer was changed to PBS according to the instructions. These were used for analysis of wound cell activity after filter sterilization through 0.22 μm filters (Millipore).

5-3 analysis of the antitumor Activity of toxin-labeled anti-GRP 78 antibody (GA20-PE40)

The cell death-inducing activity of the obtained GA20-PE40 was analyzed.

Hamster ovary cells DG44 at 1X 10³Cells/well prostate cancer cell lines DU145, 22Rv1 at 6X 10³Cells/well, 96 well plates were seeded at 90. mu.l/well. The next day, GA20-PE40 fractions (elution fractions 2, 3, 4) and PBS were added at 10. mu.l/well, and cultured at 37 ℃. After 5 days, the number of living cells was measured by WST-8 (Dojindo chemical) reagent, and the ratio of the living cells to the control (PBS-added group) was quantified to prepare a graph.

As a result, in contrast to no detectable effect of GA20-PE40 on DG44 cell growth (fig. 10C), nocellular activity was exhibited by the elution fractions 2, 3 in which GRP78 binding activity was detected in 2 prostate cancer cell lines (DU145, 22Rv1) (see fig. 10A for DU145 and fig. 10B for 22Rv 1).

This result indicates that antibodies directed against the extracellular epitope of GRP78 (350-445) are useful as anti-tumor agents.

Example 6: obtaining anti-GRP 78 antibody by re-immunization

6-1 preparation of immunogen GST-GRP78(376-

From the above analysis described in example 4, it is inferred that the region consisting of amino acids 376 to 415 of GRP78 protein is the extracellular region of GRP 78. Therefore, the regions were re-immunized in the following manner, and an antibody having high affinity to recognize the extracellular region of GRP78 was attempted to be prepared.

First, Escherichia coli (BL21) transformed with the GST-GRP78(376- & gt 415) expression vector (pGEX-GRP78(376- & gt 415)) described in example 4 was cultured in LB medium (250ml) until OD was reached₆₁₀Protein expression was induced with IPTG (1mM) above 0.5. Coli cultured for 5 hours was collected and lysed in 25ml of B-PER (PIAS). Next, the lysed E.coli lysate was diluted 10-fold with PBS and PBS-equilibrated glutathione Sepharose 4B (Amersham pharmacia) was added thereto and incubated overnight at 4 ℃. After that, glutathione Sepharose 4B was washed several times with PBS to remove unadsorbed protein, and the objective protein was eluted with 20mM glutathione.

The eluted fractions were subjected to SDS-PAGE, stained with CBB, and fractions containing the target protein were pooled. The sample was further subjected to gel filtration chromatography (Superdex 20016/60, GEHealthcare) to separate impure protein in PBS and only the target protein was purified with high purity. The purified protein was used as an immunogen in the following experiments.

Immunization with GST-GRP78(376-415) protein

GST-GRP78(376- & ltSUB & gt 415) purified in example 6-1 was used to immunize a mouse [ (MRL/1pr, male, 4-week-old) (Balb/c, female, 6-week-old) according to the same method as in example 1-2: all purchased from Charles River, Japan. Hybridomas were prepared as described in examples 1-3.

6-3 screening of anti-GRP 78 antibodies

Purification of MBP-GRP78(376-

A fusion protein of GRP78 (amino acids 376-415) and Maltose Binding Protein (MBP) (MBP-GRP78(376-415)) was prepared as follows.

pGEX-GRP78(376- & lt415 & gt) prepared in example 4 was digested with BamHI-SalI, and the gene fragment encoding GRP78(376- & lt415 & gt) was excised. It was inserted between BamHI-SalI of pMAL-c2X (New England BioLabs) to construct MBP-GRP78(376-

Then, will be describedColi BL21 strain transformed with the vector was cultured in LB medium (250ml) until OD was reached₆₁₀Protein expression was induced with IPTG (1mM) above 0.5. Coli after 5 hours of culture was collected by centrifugation and lysed in 25ml of B-PER (PIAS). Next, the lysed E.coli lysate was diluted 5-fold with column buffer (20mM Tris, pH7.5, 200mM NaCl, 1mM EDTA) and column buffer equilibrated amylose resin (New England BioLabs) was added thereto and incubated at 4 ℃ overnight. Thereafter, the amylose resin was washed several times with a column buffer to remove unadsorbed proteins, and the objective protein was eluted with an elution buffer (column buffer containing 10mM maltose). After SDS-PAGE of the eluted fractions, fractions containing the target protein were identified by CBB staining, and after pooling the fractions, the buffer was changed to PBS using a PD10 column. This purified MBP-GRP78(376-415) was used in the following experiments for ELISA screening of bound antibodies.

6-3-2 screening by ELISA for GRP 78-binding antibodies (Primary screening)

The antibody binding to the region of amino acids 376 to 415 of GRP78 protein was screened using an ELISA plate coated with 1. mu.g/ml of MBP-GRP78(376-415) purified in example 6-3-1.

The procedure is as described in example 2-1. GRP 78-binding antibodies narrowed by the primary screening using the binding activity to MBP-GRP78(376-415) protein as an indicator were subjected to secondary screening as described below.

6-3-3 screening of anti-GRP 78 antibodies on cell surface by FACS (Secondary screening)

The GRP 78-binding antibody obtained by the primary screening was subjected to secondary screening using the binding activity to prostate cancer cell lines (DU145 and 22Rv1(ATCC CRL-2505)) as an index. The procedure is as described in example 2-2.

As a result, 4 kinds of antibodies that stained the prostate cancer cell line FACS were newly found: GC-18 antibody, GC-20 antibody, GD-4 antibody, GD-17 antibody.

These antibodies were subjected to limiting dilution and made monoclonal according to the method described in examples 2-3. The subtypes of these antibodies were determined according to the methods described in examples 2 to 4. The subtypes of each antibody are shown below.

TABLE 2

Antibodies	Subtype of cell
		GC-18	G1

GC-20	G1
		GD-4	G1
GD-17	G1

The antibody was then purified according to the method described in examples 2 to 5, and the properties of the antibody were analyzed in detail below.

6-4 analysis of antibodies newly obtained by Re-immunization

FACS analysis 6-4-1

In order to confirm that the 4 purified antibodies could stain the cell surface of cancer cells, FACS analysis was performed using a prostate cancer cell line (22Rv 1). The cells were stained with each antibody (10. mu.g/ml), and FACS analysis was performed according to the method described in example 3-2-1.

As a result, these antibodies were found to bind to the cell surface of 22Rv1 cells (fig. 11).

6-4-2 epitope mapping

The 4 antibodies thus obtained were antibodies obtained by immunization with GST-GRP78(376-415), and thus were antibodies recognizing a partial region of amino acids 376 to 415 of GRP 78. Thus, the region was subsequently subdivided into 4 regions as shown in FIG. 12A (amino acids No. 376 to 391 (i.e., amino acids No. 1 to 16 of SEQ ID NO: 3), amino acids No. 384 to 399 (i.e., amino acids No. 9 to 24 of SEQ ID NO: 3), amino acids No. 392 to 407 (i.e., amino acids No. 17 to 32 of SEQ ID NO: 3), and amino acids No. 400 to 415 (i.e., amino acids No. 25 to 40 of SEQ ID NO: 3)), and it was analyzed which of amino acids No. 376 to 415 of GRP78 was recognized by each of the 4 antibodies.

6-4-2-1 preparation of GST fusion protein for epitope mapping

6-4-2-1-1. construction of GST fusion protein expression vector for epitope mapping

First, DNA fragments encoding the regions consisting of amino acids 376 to 391, 384 to 399, 392 to 407, and 400 to 415 of the GRP78 protein were prepared as follows.

Preparing a DNA fragment encoding GRP78(376-391) (i.e., amino acids 1-16 of SEQ ID NO: 3) by renaturing the oligomer (GEP1/GEP2, SEQ ID NO: 48 and 49, respectively); preparing a DNA fragment encoding GRP78(384-399) (i.e., amino acids No. 9-24 of SEQ ID NO: 3) by renaturing the oligomer (GEP3/GEP4, SEQ ID NO: 50 and 51, respectively); preparing a DNA fragment encoding GRP78(392-407) (i.e., amino acids 17-32 of SEQ ID NO: 3) by renaturing the oligomer (GEP5/GEP6, SEQ ID NOS: 52 and 53, respectively); a DNA fragment encoding GRP78(400-415) (i.e., amino acids 25-40 of SEQ ID NO: 3) was prepared by renaturing the oligomer (GEP7/GEP8, SEQ ID NO: 54 and 55, respectively).

The oligomer sequences used are listed below.

GEP1：gatccaaaga gttcttcaat ggcaaggaac catcccgtggcataaaccca gatc(SEQ ID NO：48)

GEP2：tcgagatctg ggtttatgcc acgggatggt tccttgccattgaagaactc tttg(SEQ ID NO：49)

GEP3：gatccccatc ccgtggcata aacccagatg aagctgtagcgtatggtgct gctc(SEQ ID NO：50)

GEP4：tcgagagcag caccatacgc tacagcttca tctgggtttatgccacggga tggg(SEQ ID NO：51)

GEP5：gatccgaagc tgtagcgtat ggtgctgctg tccaggctggtgtgctctct ggtc(SEQ ID NO：52)

GEP6：tcgagaccag agagcacacc agcctggaca gcagcaccatacgctacagc ttcg(SEQ ID NO：53)

GEP7：gatccgtcca ggctggtgtg ctctctggtg atcaagatacaggtgacctg gtac(SEQ ID NO：54)

GEP8：tcgagtacca ggtcacctgt atcttgatca ccagagagcacaccagcctg gacg(SEQ ID NO：55)

The prepared DNA fragment was inserted into the GST coding region downstream of the Escherichia coli GST fusion expression vector (pGEX-6P-1) digested with BamHI and XhoI to construct GRP78-GST fusion protein expression vectors (pGEX-GRP78 (376-.

6-4-2-1-2 expression Induction of respective GST fusion GRP78 proteins

Escherichia coli BL21 was transformed with the E.coli expression vector constructed as described above, and the expression induction of the protein was carried out according to the method described in example 4-1-2. As a result, as shown in FIG. 12B, it was confirmed that the objective protein was expressed in E.coli. The protein was then used for epitope mapping as described below.

Epitope mapping of 6-4-2-2. antibodies

Western blot analysis was performed using GST fusion proteins prepared as described above to analyze which region of the GRP78 protein each anti-GRP 78 antibody obtained recognized.

From the staining pattern of the Western blot (FIG. 13), it was found that the GD-17 antibody recognized the region spanning amino acids 384-391 of GRP78, the GC-18 and GC-20 antibodies recognized the region spanning amino acids 392-407 of GRP78, and the GD-4 antibody recognized the region spanning amino acids 400-415 of GRP 78. Furthermore, it was found that the GA-20 antibody obtained at the initial stage recognized the same region as the GD-4 antibody (FIG. 13).

6-4-3 cloning of variable region and determination of amino acid sequence

The cloning and amino acid sequence determination of the variable regions of the newly obtained antibodies (GC-18 antibody, GC-20 antibody, GD-4 antibody, GD-17 antibody) were carried out in accordance with the method described in example 5-1. However, since these antibodies are all IgG1, the following VH-G1 primer (SEQ ID NO: 56) can be used:

VH-G1：cca cca gat tct tat cag aca gg(SEQ ID NO：56)

the heavy chain variable region was cloned.

The amplified light and heavy chain gene fragments were TA-cloned into pCRII-TOPO (Invitrogen TOPO TA-cloning kit, #45-0640), followed by confirmation of the base sequence of each insert.

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 shown in SEQ ID NO: 57, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 58; the base sequence of the light chain variable region is shown in SEQ ID NO: 59, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60. in addition, the amino acid sequence of CDR1 of the heavy chain variable region of the GC-18 antibody is shown in SEQ ID NO: 61, the amino acid sequence of CDR2 is shown in SEQ ID NO: 62, the amino acid sequence of CDR3 is shown in 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.

the base 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 shown in SEQ ID NO: 67, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 68; the base sequence of the light chain variable region is shown in SEQ ID NO: 69, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 70. in addition, the amino acid sequence of CDR1 of the heavy chain variable region of the GC-20 antibody is shown in SEQ ID NO: 71, the amino acid sequence of CDR2 is shown in SEQ ID NO: 72, the amino acid sequence of CDR3 is shown in 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.

the base sequence of the heavy chain variable region of the GD-4 antibody that binds to the region spanning amino acids 400-415 of GRP78 is shown in SEQ ID NO: 77, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 78, a nitrogen source; the base sequence of the light chain variable region is shown in SEQ ID NO: 79 and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 80. in addition, the amino acid sequence of CDR1 of the heavy chain variable region of the GD-4 antibody is shown in SEQ ID NO: 81, the amino acid sequence of CDR2 is shown in SEQ ID NO: 82, the amino acid sequence of CDR3 is shown in SEQ ID NO: 83; the amino acid sequence of CDR1 of the light chain 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.

the base sequence of the heavy chain variable region of the GD-17 antibody that binds to the region spanning amino acids 384-391 of GRP78 is shown in SEQ ID NO: 87, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 88; the base sequence of the light chain variable region is shown in SEQ ID NO: 89, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 90. in addition, the amino acid sequence of CDR1 of the heavy chain variable region of the GD-4 antibody is shown in SEQ ID NO: 91, the amino acid sequence of CDR2 is shown in SEQ ID NO: 92, the amino acid sequence of CDR3 is shown in SEQ ID NO: 93; the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 94, the amino acid sequence of CDR2 is shown in SEQ ID NO: 95, the amino acid sequence of CDR3 is shown in SEQ ID NO: 96.

example 7: analysis of drug efficacy of toxin-labeled GD17 single-chain antibody (GD17_ scFv-PE40)

7-1, construction of GD17_ scFv-PE40 expression vector

Single-chain Fv encoding GD-17 antibody, i.e., a linker sequence consisting of 15 amino acids ((GlyGlyGlyGlySer)₃) (SEQ ID NO: 43) and PCR amplifying the gene segments of the heavy chain variable region and the light chain variable region of the connected GD-17 antibody.

First, the heavy chain variable region of GD-17 antibody cloned in pCRII-TOPO was used as a template, the heavy chain variable region was amplified by PCR using a forward primer (GD17-1, SEQ ID NO: 97) and a reverse primer (GD17-2, SEQ ID NO: 98) as the templates, and the light chain variable region was amplified by PCR using a forward primer (GD17-3, SEQ ID NO: 99) and a reverse primer (GD17-4, SEQ ID NO: 100) using pyrobest DNA polymerase (TAKARA # R005) at 94 ℃ for 1 minute and at 94 ℃ for 30 minutes, 72 ℃ for 30 minutes and 25 cycles.

The PCR products of the heavy and light chain variable regions obtained were then purified using an S-300HR column (Amersham Biosciences #27-5130-01), 1. mu.L of each was mixed in the same tube, and after reaction with pyrobest DNA polymerase at 94 ℃ for 1 minute, PCR amplification was performed at 94 ℃ for 30 minutes, at 72 ℃ for 30 minutes, and at 5 cycles.

mu.L of the reaction mixture after renaturation was reacted with primers GD17-1(SEQ ID NO: 97) and GD17-4(SEQ ID NO: 100) at 94 ℃ for 1 minute under the following conditions, and then PCR amplification was carried out at 94 ℃ for 30 minutes, 72 ℃ for 1 minute, and 25 cycles.

The amplified fragment was purified by an S-400HR column (Amersham Biosciences #27-5140-01), digested with EcoRI-HindIII, and separated by agarose gel. This was inserted between HindIII and EcoRI of pET22b _ His _ PE40 prepared in 5-2-1-1, and the nucleotide sequence was confirmed to prepare pET22b _ His _ GD17scFv-PE 40.

The primer sequences used are shown below:

GD17-1：aaaagcttca ggttcagctc cagcagtctg g(SEQ ID NO：97)

GD17-2：cccgaaccac caccacccga accaccacca cctgaggagactgtgagagt ggtgcct(SEQ ID NO：98)

GD17-3：tggttcgggt ggtggtggtt cgggtggtgg cggatcggatgttgtgatga cccaaactcc ac(SEQ ID NO：99)

GD17-4：ttgaattctt tcagctcca gcttggtccc(SEQ ID NO：100)

the base sequence of the resulting GD17scFv _ PE40 is shown in SEQ ID NO: 101, and the corresponding amino acid sequence of the base sequence is shown in SEQ ID NO: 102.

7-2 Mass purification of toxin-labeled Single-chain antibodies

Coli BL21 transformed with pET22b _ His _ GD17scFv-PE40 was inoculated on LB agar plates containing carbenicillin (50. mu.g/ml). When logarithmic growth phase was reached, IPTG (final amount of 1mM) was added and the mixture was incubated at room temperature (24 ℃ C.) overnight to induce protein expression. Coli recovered by centrifugation was suspended in binding buffer (20mM sodium phosphate, 500mM NaCl, 20mM imidazole, pH7.4), and after ultrasonication the lysate fraction was applied to a HisTrap FF Crude column (GE Healthcare). The desired protein was then eluted with an elution buffer (20mM sodium phosphate, 500mM NaCl, 500mM imidazole, pH7.4), diluted about 10-fold with TBS and applied to an affinity gel packed with M2 agarose (Sigma). The target protein was eluted with M2 elution buffer (0.1M glycine-HCl, pH3.5) using AKTA explorer (GE Healthcare), and its buffer was rapidly changed to PBS using PD10(GE Healthcare) as a final sample.

The purified GD17scFv-PE40 was CBB stained after SDS-PAGE to confirm whether it was purified to 100% purity (fig. 14).

7-3 assay of binding Activity for GRP78 protein

The purified GD17scFv-PE40 protein was next analyzed for GRP78 binding activity. Binding activity to immobilized GRP78 was compared by ELISA assay in samples stored at 4 ℃, overnight at 37 ℃ and in samples subjected to freeze-thaw.

Diluted with dilution buffer (1% BSA, 50mM Tris, 1mM MgCl)₂150mM NaCl, 0.05% Tween20) were added to plates (NUNC) coated with GST-GRP78 (1. mu.g/ml) purified from E.coli. After 1 hour of reaction at room temperature, the plate was washed 3 times with TBS-T (TBS-0.05% Tween20), added with 1. mu.g/ml anti-Flag antibody (M2 antibody, Sigma) and incubated for 1 hour at room temperature. TBS-T was washed 3 times, reacted with alkaline phosphatase-labeled anti-mouse IgG (ZYMED) for 1 hour, and then developed with 1mg/ml substrate (Sigma).

As a result, it was found that the GD17scFv-PE40 protein binding activity to the GRP78 protein was EC in any of the samples stored at 4 ℃ overnight at 37 ℃ or the samples subjected to freeze-thaw₅₀Around 1.3nM, indicating that the purified sample is a relatively stable protein (fig. 15).

7-4 analysis of Activity to induce cell death in vitro

The cell death-inducing activity of the purified GD17scFv-PE40 was analyzed using cancer cell lines (22Rv1, LNcap, MCF7, BxPC3, PANC1, SKOV3), human-derived normal cell lines (HUVEC, MRC5), mouse-derived normal cell lines (CHO, NIH3T3, BaF 3).

Among the cell lines used in the experiment, HUVEC was purchased from CAMBREX, and other cell lines were purchased from ATCC, and were cultured separately according to the product instructions.

Each cell was seeded on a 96-well plate, and GD17scFv-PE40 diluted to various concentrations with 10% FCS-containing RPMI1640 medium (Invitrogen) was added to the cells the following day. The number of viable cells was measured with WST-8 (NACALA) after 5 days of culture.

As a result, it was found that, although the sensitivity of cancer cells varied from cell to cell, the concentration (EC) showing 50% of the maximum activity was exhibited₅₀Values) were about 2-20nM, indicating strong cell death-inducing activity (fig. 16A). In particular the EC of MCF7 or 22Rv1 cells₅₀Values of about 2-4nM indicate potent wounded cell activity. Whereas normal cells from human and mouse were either completely inactive or slightly cytostatic after addition at high concentrations (FIG. 16B).

To confirm that the differences in sensitivity were not due to the presence or absence of GRP78 protein expression between the cells used in the experiment, western blot analysis was performed using GD-17 antibody. Cell lysates were prepared from cells according to the protocol and subjected to SDS-PAGE and Western blot analysis using GD-17 antibody (2. mu.g/ml). As shown in FIG. 17, a staining pattern specific to the GD-17 antibody was also detectable in a cell line that did not produce a cell damaging activity by GD17scFv-PE40, and it was concluded that the cancer cell-specific cell damaging activity by GD17scFv-PE40 was not due to whether cancer cells and normal cells express GRP78 protein, but due to the presence of GRP78 protein at different positions between the two cells.

7-5 analysis of in vivo antitumor Activity

Human prostate cancer cell line 22Rv1 cells (ATCC CRL-2505) were recovered in 0.02% EDTA solution supplemented with 0.05% trypsin at 1X 10⁷Cells/0.2 ml HBSS (SIGMACAT. No. H9269) transplanted into nude mice (male, 7 weeks old (CAnN. Cg-Foxn 1)<nu>CrlCrlj (BALB-nu/nu)): charles River, japan). Tumor colonization was confirmed and divided into 7 groups (control group 1, drug administration group 6) on day 16 after transplantation according to tumor volume and body weight.

The control group was administered with physiological saline on each of the following days (day 17), day 21, day 23, day 26, and day 29, and the drug administration group was administered with GD17scFv-PE40 at a dose of 0.5mg/kg, and was administered intravenously in an instantaneous manner at an administration rate of 10 ml/kg. Tumor volumes were measured over time, with the last measurement being taken 2 days after the last administration (day 31).

The results are shown in FIG. 18. The tumor proliferation inhibition rate in the last measurement is 47%, and the data of the tumor volume are analyzed through nonparametric dannett multiple comparison, and the result shows that the 0.5mg/kg administration group has a remarkable tumor proliferation inhibition effect. From this result, the in vivo effectiveness of GD17scFv-PE40 targeting GRP78 was confirmed, and it was confirmed that an antibody targeting GRP78 can be used as a cancer therapeutic antibody.

Possibility of industrial application

The present invention shows that it is possible to provide a novel pharmaceutical composition for treating various tumors or cancers exposing GRP78 to the cell surface by providing a novel antibody having GRP78 binding activity and being taken up by cells. Also, a method for diagnosing various tumors or cancers is provided by using the antibody having the characteristics.

Sequence listing

<110>FORERUNNER PHARMA RESEARCH CO.，LTD.

<120> pharmaceutical composition containing anti-GRP 78 antibody as active ingredient

<130>YCT-1342

<150>JP 2007-47534

<151>2007-02-27

<160>102

<170>PatentIn version 3.1

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Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala

1 5 10 15

cgg gcc gag gag gag gac aag aag gag gac gtg ggc acg gtg gtc ggc 96

Arg Ala Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly

20 25 30

atc gac ctg ggg acc acc tac tcc tgc gtc ggc gtg ttc aag aac ggc 144

Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly

35 40 45

cgc gtg gag atc atc gcc aac gat cag ggc aac cgc atc acg ccg tcc 192

Arg Val Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser

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tat gtc gcc ttc act cct gaa ggg gaa cgt ctg att ggc gat gcc gcc 240

Tyr Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala

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aag aac cag ctc acc tcc aac ccc gag aac acg gtc ttt gac gcc aag 288

Lys Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys

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cgg ctc atc ggc cgc acg tgg aat gac ccg tct gtg cag cag gac atc 336

Arg Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp Ile

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aag ttc ttg ccg ttc aag gtg gtt gaa aag aaa act aaa cca tac att 384

Lys Phe Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile

115 120 125

caa gtt gat att gga ggt ggg caa aca aag aca ttt gct cct gaa gaa 432

Gln Val Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu

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att tct gcc atg gtt ctc act aaa atg aaa gaa acc gct gag gct tat 480

Ile Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr

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ttg gga aag aag gtt acc cat gca gtt gtt act gta cca gcc tat ttt 528

Leu Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe

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aat gat gcc caa cgc caa gca acc aaa gac gct gga act att gct ggc 576

Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly

180 185 190

cta aat gtt atg agg atc atc aac gag cct acg gca gct gct att gct 624

Leu Asn Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala

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tat ggc ctg gat aag agg gag ggg gag aag aac atc ctg gtg ttt gac 672

Tyr Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp

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ctg ggt ggc gga acc ttc gat gtg tct ctt ctc acc att gac aat ggt 720

Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly

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gtc ttc gaa gtt gtg gcc act aat gga gat act cat ctg ggt gga gaa 768

Val Phe Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu

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gac ttt gac cag cgt gtc atg gaa cac ttc atc aaa ctg tac aaa aag 816

Asp Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys

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aag acg ggc aaa gat gtc agg aaa gac aat aga gct gtg cag aaa ctc 864

Lys Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu

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cgg cgc gag gta gaa aag gcc aaa cgg gcc ctg tct tct cag cat caa 912

Arg Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln

290 295 300

gca aga att gaa att gag tcc ttc tat gaa gga gaa gac ttt tct gag 960

Ala Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu

305 310 315 320

acc ctg act cgg gcc aaa ttt gaa gag ctc aac atg gat ctg ttc cgg 1008

Thr Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg

325 330 335

tct act atg aag ccc gtc cag aaa gtg ttg gaa gat tct gat ttg aag 1056

Ser Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys

340 345 350

aag tct gat att gat gaa att gtt ctt gtt ggt ggc tcg act cga att 1104

Lys Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile

355 360 365

cca aag att cag caa ctg gtt aaa gag ttc ttc aat ggc aag gaa cca 1152

Pro Lys Ile Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro

370 375 380

tcc cgt ggc ata aac cca gat gaa gct gta gcg tat ggt gct gct gtc 1200

Ser Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val

385 390 395 400

cag gct ggt gtg ctc tct ggt gat caa gat aca ggt gac ctg gta ctg 1248

Gln Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu

405 410 415

ctt gat gta tgt ccc ctt aca ctt ggt att gaa act gtg gga ggt gtc 1296

Leu Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val

420 425 430

atg acc aaa ctg att cca agg aac aca gtg gtg cct acc aag aag tct 1344

Met Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser

435 440 445

cag atc ttt tct aca gct tct gat aat caa cca act gtt aca atc aag 1392

Gln Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys

450 455 460

gtc tat gaa ggt gaa aga ccc ctg aca aaa gac aat cat ctt ctg ggt 1440

Val Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly

465 470 475 480

aca ttt gat ctg act gga att cct cct gct cct cgt ggg gtc cca cag 1488

Thr Phe Asp Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln

485 490 495

att gaa gtc acc ttt gag ata gat gtg aat ggt att ctt cga gtg aca 1536

Ile Glu Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr

500 505 510

gct gaa gac aag ggt aca ggg aac aaa aat aag atc aca atc acc aat 1584

Ala Glu Asp Lys Gly Thr Gly Asn Lys Asn Lys Ile Thr Ile Thr Asn

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gac cag aat cgc ctg aca cct gaa gaa atc gaa agg atg gtt aat gat 1632

Asp Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp

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gct gag aag ttt gct gag gaa gac aaa aag ctc aag gag cgc att gat 1680

Ala Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp

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act aga aat gag ttg gaa agc tat gcc tat tct cta aag aat cag att 1728

Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile

565 570 575

gga gat aaa gaa aag ctg gga ggt aaa ctt tcc tct gaa gat aag gag 1776

Gly Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu

580 585 590

acc atg gaa aaa gct gta gaa gaa aag att gaa tgg ctg gaa agc cac 1824

Thr Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His

595 600 605

caa gat gct gac att gaa gac ttc aaa gct aag aag aag gaa ctg gaa 1872

Gln Asp Ala Asp Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu

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gaa att gtt caa cca att atc agc aaa ctc tat gga agt gca ggc cct 1920

Glu Ile Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro

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ccc cca act ggt gaa gag gat aca gca gaa aaa gat gag ttg tag 1965

Pro Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu

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Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala

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Arg Ala Glu Glu Glu Asp Lys Lys Glu Asp Val Gly Thr Val Val Gly

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Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Gly Val Phe Lys Asn Gly

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Arg Val Glu Ile Ile Ala Asn Asp Gln Gly Asn Arg Ile Thr Pro Ser

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Tyr Val Ala Phe Thr Pro Glu Gly Glu Arg Leu Ile Gly Asp Ala Ala

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Lys Asn Gln Leu Thr Ser Asn Pro Glu Asn Thr Val Phe Asp Ala Lys

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Arg Leu Ile Gly Arg Thr Trp Asn Asp Pro Ser Val Gln Gln Asp Ile

100 105 110

Lys Phe Leu Pro Phe Lys Val Val Glu Lys Lys Thr Lys Pro Tyr Ile

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Gln Val Asp Ile Gly Gly Gly Gln Thr Lys Thr Phe Ala Pro Glu Glu

130 135 140

Ile Ser Ala Met Val Leu Thr Lys Met Lys Glu Thr Ala Glu Ala Tyr

145 150 155 160

Leu Gly Lys Lys Val Thr His Ala Val Val Thr Val Pro Ala Tyr Phe

165 170 175

Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Thr Ile Ala Gly

180 185 190

Leu Asn Val Met Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Ile Ala

195 200 205

Tyr Gly Leu Asp Lys Arg Glu Gly Glu Lys Asn Ile Leu Val Phe Asp

210 215 220

Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Thr Ile Asp Asn Gly

225 230 235 240

Val Phe Glu Val Val Ala Thr Asn Gly Asp Thr His Leu Gly Gly Glu

245 250 255

Asp Phe Asp Gln Arg Val Met Glu His Phe Ile Lys Leu Tyr Lys Lys

260 265 270

Lys Thr Gly Lys Asp Val Arg Lys Asp Asn Arg Ala Val Gln Lys Leu

275 280 285

Arg Arg Glu Val Glu Lys Ala Lys Arg Ala Leu Ser Ser Gln His Gln

290 295 300

Ala Arg Ile Glu Ile Glu Ser Phe Tyr Glu Gly Glu Asp Phe Ser Glu

305 310 315 320

Thr Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Met Asp Leu Phe Arg

325 330 335

Ser Thr Met Lys Pro Val Gln Lys Val Leu Glu Asp Ser Asp Leu Lys

340 345 350

Lys Ser Asp Ile Asp Glu Ile Val Leu Val Gly Gly Ser Thr Arg Ile

355 360 365

Pro Lys Ile Gln Gln Leu Val Lys Glu Phe Phe Asn Gly Lys Glu Pro

370 375 380

Ser Arg Gly Ile Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val

385 390 395 400

Gln Ala Gly Val Leu Ser Gly Asp Gln Asp Thr Gly Asp Leu Val Leu

405 410 415

Leu Asp Val Cys Pro Leu Thr Leu Gly Ile Glu Thr Val Gly Gly Val

420 425 430

Met Thr Lys Leu Ile Pro Arg Asn Thr Val Val Pro Thr Lys Lys Ser

435 440 445

Gln Ile Phe Ser Thr Ala Ser Asp Asn Gln Pro Thr Val Thr Ile Lys

450 455 460

Val Tyr Glu Gly Glu Arg Pro Leu Thr Lys Asp Asn His Leu Leu Gly

465 470 475 480

Thr Phe Asp Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln

485 490 495

Ile Glu Val Thr Phe Glu Ile Asp Val Asn Gly Ile Leu Arg Val Thr

500 505 510

Ala Glu Asp Lys Gly Thr Gly Asn Lys Asn LysIle Thr Ile Thr Asn

515 520 525

Asp Gln Asn Arg Leu Thr Pro Glu Glu Ile Glu Arg Met Val Asn Asp

530 535 540

Ala Glu Lys Phe Ala Glu Glu Asp Lys Lys Leu Lys Glu Arg Ile Asp

545 550 555 560

Thr Arg Asn Glu Leu Glu Ser Tyr Ala Tyr Ser Leu Lys Asn Gln Ile

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Gly Asp Lys Glu Lys Leu Gly Gly Lys Leu Ser Ser Glu Asp Lys Glu

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Thr Met Glu Lys Ala Val Glu Glu Lys Ile Glu Trp Leu Glu Ser His

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Gln Asp Ala Asp Ile Glu Asp Phe Lys Ala Lys Lys Lys Glu Leu Glu

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Glu Ile Val Gln Pro Ile Ile Ser Lys Leu Tyr Gly Ser Ala Gly Pro

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Pro Pro Thr Gly Glu Glu Asp Thr Ala Glu Lys Asp Glu Leu

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Lys Glu Phe Phe Asn Gly Lys Glu Pro Ser Arg Gly Ile Asn Pro Asp

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Glu Ala Val Ala Tyr Gly Ala Ala Val Gln Ala Gly Val Leu Ser Gly

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Asp Gln Asp Thr Gly Asp Leu Val

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catggaaaga gccttgagtg gattggaggt attaatccta acaatggtgg tactagctac 180

aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac 240

atggagctcc gcagcctgac atctgaggat tctgcagtct attactgtgc aagagaaaag 300

tatggtaact actatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360

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<211>120

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<213> mice

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

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Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

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Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>6

<211>336

<212>DNA

<213> mice

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atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctt 300

cggacgttcg gtggaggcac caagctggaa atcaaa 336

<210>7

<211>112

<212>PRT

<213> mice

<400>7

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>8

<211>5

<212>PRT

<213> mice

<400>8

Glu Tyr Thr Met His

1 5

<210>9

<211>17

<212>PRT

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<400>9

Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210>10

<211>11

<212>PRT

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Glu Lys Tyr Gly Asn Tyr Tyr Ala Met Asp Tyr

1 5 10

<210>11

<211>17

<212>PRT

<213> mice

<400>11

Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu

1 5 10 15

Ala

<210>12

<211>7

<212>PRT

<213> mice

<400>12

Trp Ala Ser Thr Arg Glu Ser

1 5

<210>13

<211>8

<212>PRT

<213> mice

<400>13

Lys Gln Ser Tyr Asn Leu Arg Thr

1 5

<210>14

<211>336

<212>DNA

<213> mice

<400>14

caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60

acctgcacag tctctggttt ctcattaact agctatggtg tacactgggt tcgccagtct 120

ccaggaaagg gtctggagtg gctgggagtg atatggagtg gtggaagcac agactataat 180

gaagctttca tatccagatt gagcatcagc aaggacaatt ccaagagcca agttttcttt 240

aaaatgaaca gtctgcaagc taatgacaca gccatatatt actgtgccag aaattgggac 300

tactggggcc aaggcaccac tctcacagtc tcctca 336

<210>15

<211>112

<212>PRT

<213> mice

<400>15

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Glu Ala Phe Ile

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Asn Trp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

100 105 110

<210>16

<211>336

<212>DNA

<213> mice

<400>16

gacattgtga tgtcacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60

atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctt 300

cggacgttcg gtggaggcac caagctggaa atcaaa 336

<210>17

<211>112

<212>PRT

<213> mice

<400>17

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>18

<211>5

<212>PRT

<213> mice

<400>18

Ser Tyr Gly Val His

1 5

<210>19

<211>16

<212>PRT

<213> mice

<400>19

Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Glu Ala Phe Ile Ser

1 5 10 15

<210>20

<211>4

<212>PRT

<213> mice

<400>20

Asn Trp Asp Tyr

1

<210>21

<211>17

<212>PRT

<213> mice

<400>21

Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu

1 5 10 15

Ala

<210>22

<211>7

<212>PRT

<213> mice

<400>22

Trp Ala Ser Thr Arg Glu Ser

1 5

<210>23

<211>8

<212>PRT

<213> mice

<400>23

Lys Gln Ser Tyr Asn Leu Arg Thr

1 5

<210>24

<211>2022

<212>DNA

<213> mice

<220>

<221>CDS

<222>(1)..(2022)

<223>

<400>24

atg aaa tac ctg ctg ccg acc gct gct gct ggt ctg ctg ctc ctc gct 48

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

gcc cag ccg gcg atg gcc atg gat cac cat cac cat cac cat cac cat 96

Ala Gln Pro Ala Met Ala Met Asp His His His His His His His His

20 25 30

cat cac aag ctt gag gtc cag ctg caa cag tct gga cct gag ctg gtg 144

His His Lys Leu Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val

35 40 45

aag cct ggg gct tca gtg aag ata tcc tgc aag act tct gga tac aca 192

Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr

50 55 60

ttc act gaa tac acc atg cac tgg gtg aag cag agc cat gga aag agc 240

Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser

65 70 75 80

ctt gag tgg att gga ggt att aat cct aac aat ggt ggt act agc tac 288

Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr

85 90 95

aac cag aag ttc aag ggc aag gcc aca ttg act gta gac aag tcc tcc 336

Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser

100 105 110

agc aca gcc tac atg gag ctc cgc agc ctg aca tct gag gat tct gca 384

Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala

115 120 125

gtc tat tac tgt gca aga gaa aag tat ggt aac tac tat gct atg gac 432

Val Tyr Tyr Cys Ala Arg Glu Lys Tyr Gly Asn Tyr Tyr Ala Met Asp

130 135 140

tac tgg ggt caa gga acc tca gtc acc gtc tcc tca ggt ggt ggt ggt 480

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

145 150 155 160

tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac att gtg atg tca 528

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser

165 170 175

cag tct cca tcc tcc ctg gct gtg tca gca gga gag aag gtc act atg 576

Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met

180 185 190

agc tgc aaa tcc agt cag agt ctg ctc aac agt aga acc cga aag aac 624

Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn

195 200 205

tac ttg gct tgg tac cag cag aaa cca ggg cag tct cct aaa ctg ctg 672

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu

210 215 220

atc tac tgg gca tcc act agg gaa tct ggg gtc cct gat cgc ttc aca 720

Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr

225 230 235 240

ggc agt gga tct ggg aca gat ttc act ctc acc atc agc agt gtg cag 768

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln

245 250 255

gct gaa gac ctg gca gtt tat tac tgc aag caa tct tat aat ctt cgg 816

Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Asn Leu Arg

260 265 270

acg ttc ggt gga ggc acc aag ctg gaa atc aaa gaa ttc ggt ggc gcg 864

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Phe Gly Gly Ala

275 280 285

ccg gag ttc ccg aaa ccg tcc acc ccg ccg ggt tct tct ggt tta gag 912

Pro Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Leu Glu

290 295 300

ggc ggc agc ctg gcc gcg ctg acc gcg cac cag gct tgc cac ctg ccg 960

Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro

305 310 315 320

ctg gag act ttc acc cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg 1008

Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu

325 330 335

gag cag tgc ggc tat ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg 1056

Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala

340 345 350

gcg cgg ctg tcg tgg aac cag gtc gac cag gtg atc cgc aac gcc ctg 1104

Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu

355 360 365

gcc agc ccc ggc agc ggc ggc gac ctg ggc gaa gcg atc cgc gag cag 1152

Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln

370 375 380

ccg gag cag gcc cgt ctg gcc ctg acc ctg gcc gcc gcc gag agc gag 1200

Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu

385 390 395 400

cgc ttc gtc cgg cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac 1248

Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn

405 410 415

gcc gac gtg gtg agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg 1296

Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala

420 425 430

ggc ccg gcg gac agc ggc gac gcc ctg ctg gag cgc aac tat ccc act 1344

Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr

435 440 445

ggc gcg gag ttc ctc ggc gac ggc ggc gac gtc agc ttc agc acc cgc 1392

Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg

450 455 460

ggc acg cag aac tgg acg gtg gag cgg ctg ctc cag gcg cac cgc caa 1440

Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln

465 470 475 480

ctg gag gag cgc ggc tat gtg ttc gtc ggc tac cac ggc acc ttc ctc 1488

Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu

485 490 495

gaa gcg gcg caa agc atc gtc ttc ggc ggg gtg cgc gcg cgc agc cag 1536

Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln

500 505 510

gac ctc gac gcg atc tgg cgc ggt ttc tat atc gcc ggc gat ccg gcg 1584

Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala

515 520 525

ctg gcc tac ggc tac gcc cag gac cag gaa ccc gac gca cgc ggc cgg 1632

Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg

530 535 540

atc cgc aac ggt gcc ctg ctg cgg gtc tat gtg ccg cgc tcg agc ctg 1680

Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu

545 550 555 560

ccg ggc ttc tac cgc acc agc ctg acc ctg gcc gcg ccg gag gcg gcg 1728

Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala

565 570 575

ggc gag gtc gaa cgg ctg atc ggc cat ccg ctg ccg ctg cgc ctg gac 1776

Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp

580 585 590

gcc atc acc ggc ccc gag gag gaa ggc ggg cgc ctg gag acc att ctc 1824

Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu

595 600 605

ggc tgg ccg ctg gcc gag cgc acc gtg gtg att ccc tcg gcg atc ccc 1872

Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro

610 615 620

acc gac ccg cgc aac gtc ggc ggc gac ctc gac ccg tcc agc atc ccc 1920

Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro

625 630 635 640

gac aag gaa cag gcg atc agc gcc ctg ccg gac tac gcc agc cag ccc 1968

Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro

645 650 655

ggc aaa ccg ccg gac tac aag gat gac gac gat aag aaa gac gaa ctg 2016

Gly Lys Pro Pro Asp Tyr Lys Asp Asp Asp Asp Lys Lys Asp Glu Leu

660 665 670

tag tga 2022

<210>25

<211>672

<212>PRT

<213> mice

<400>25

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Met Asp His His His His His His His His

20 25 30

His His Lys Leu Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val

35 40 45

Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr

50 55 60

Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser

65 70 75 80

Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr

85 90 95

Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser

100 105 110

Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala

115 120 125

Val Tyr Tyr Cys Ala Arg Glu Lys Tyr Gly Asn Tyr Tyr Ala Met Asp

130 135 140

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

145 150 155 160

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser

165 170 175

Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met

180 185 190

Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn

195 200 205

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu

210 215 220

Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr

225 230 235 240

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln

245 250 255

Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Asn Leu Arg

260 265 270

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Phe Gly Gly Ala

275 280 285

Pro Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Leu Glu

290 295 300

Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro

305 310 315 320

Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu

325 330 335

Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala

340 345 350

Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu

355 360 365

Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln

370 375 380

Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu

385 390 395 400

Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn

405 410 415

Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala

420 425 430

Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr

435 440 445

Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg

450 455 460

Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln

465 470 475 480

Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu

485 490 495

Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln

500 505 510

Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala

515 520 525

Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg

530 535 540

Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu

545 550 555 560

Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala

565 570 575

Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp

580 585 590

Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu

595 600 605

Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro

610 615 620

Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro

625 630 635 640

Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro

645 650 655

Gly Lys Pro Pro Asp Tyr Lys Asp Asp Asp Asp Lys Lys Asp Glu Leu

660 665 670

<210>26

<211>20

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying the full-length Grp78 Gene

<400>26

atgaagctct ccctggtggc 20

<210>27

<211>25

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying the full-length Grp78 Gene

<400>27

ctacaactca tctttttctg ctgta 25

<210>28

<211>38

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>28

aaaggatccg aggaggagga caagaaggag gacgtggg 38

<210>29

<211>39

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>29

tttctcgagc tacaactcat ctttttctgc tgtatcctc 39

<210>30

<211>43

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>30

tttctcgagc taatcagaat cttccaacac tttctggacg ggc 43

<210>31

<211>34

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>31

aaaggatccc ggcgcgaggt agaaaaggcc aaac 34

<210>32

<211>35

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>32

ttctcgagct aggtaggcac cactgtgttc cttgg 35

<210>33

<211>35

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>33

ttctcgagct agatttcttc aggtgtcagg cgatt 35

<210>34

<211>34

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>34

tttggatccg tgttggaaga ttctgatttg aaga 34

<210>35

<211>35

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>35

ttctcgagct aggatggttc cttgccattg aagaa 35

<210>36

<211>32

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>36

aaaggatcca aagagttctt caatggcaag ga 32

<210>37

<211>35

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>37

ttctcgagct ataccaggtc acctgtatct tgatc 35

<210>38

<211>33

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying GST-Grp78 fusion gene sequence

<400>38

aaaggatcct ctggtgatca agatacaggt gac 33

<210>39

<211>23

<212>DNA

<213> Artificial

<220>

<223> primers for cloning nucleotide sequence encoding variable region of light chain of GA-20 antibody

<400>39

gctcactgga tggtgggaag atg 23

<210>40

<211>23

<212>DNA

<213> Artificial

<220>

<223> primers for cloning nucleotide sequence encoding heavy chain variable region of GA-20 antibody

<400>40

ccaccagatt cttatcagac agg 23

<210>41

<211>87

<212>DNA

<213> Artificial

<220>

<223> primers for generating nucleotide sequence encoding single-chain Fv derived from GA-20 antibody

<400>41

taagaattcg gtggcgcgcc ggagttcccg aaaccgtcca ccccgccggg ttcttctggt 60

ttagagggcg gcagcctggc cgcgctg 87

<210>42

<211>75

<212>DNA

<213> Artificial

<220>

<223> primers for generating nucleotide sequence encoding single-chain Fv derived from GA-20 antibody

<400>42

acttagcggc cgctcactac agttcgtctt tcttatcgtc gtcatccttg tagtccggcg 60

gtttgccggg ctggc 75

<210>43

<211>15

<212>PRT

<213> Artificial

<220>

<223>Linker polypeptide of scFv sntibody.

<400>43

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>44

<211>31

<212>DNA

<213> Artificial

<220>

<223> primers for amplifying nucleotide sequence encoding single-chain Fv derived from GA-20 antibody

<400>44

aaaagcttga ggtccagctg caacagtctg g 31

<210>45

<211>56

<212>DNA

<213> Artificial

<220>

<223> primers for generating nucleotide sequence encoding single-chain Fv derived from GA-20 antibody

<400>45

cccgaaccac caccacccga accaccacca cctgaggaga cggtgactga ggttcc 56

<210>46

<211>65

<212>DNA

<213> Artificial

<220>

<223> primers for generating nucleotide sequence encoding single-chain Fv derived from GA-20 antibody

<400>46

tggttcgggt ggtggtggtt cgggtggtgg cggatcggac attgtgatgt cacagtctcc 60

atcct 65

<210>47

<211>31

<212>DNA

<213> Artificial

<220>

<223> primers for generating nucleotide sequence encoding single-chain Fv derived from GA-20 antibody

<400>47

ttgaattctt tgatttccag cttggtgcct c 31

<210>48

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(376-391)

<400>48

gatccaaaga gttcttcaat ggcaaggaac catcccgtgg cataaaccca gatc 54

<210>49

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(376-391)

<400>49

tcgagatctg ggtttatgcc acgggatggt tccttgccat tgaagaactc tttg 54

<210>50

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(384-399) region

<400>50

gatccccatc ccgtggcata aacccagatg aagctgtagc gtatggtgct gctc 54

<210>51

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(384-399) region

<400>51

tcgagagcag caccatacgc tacagcttca tctgggttta tgccacggga tggg 54

<210>52

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(392-

<400>52

gatccgaagc tgtagcgtat ggtgctgctg tccaggctgg tgtgctctct ggtc 54

<210>53

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(392-

<400>53

tcgagaccag agagcacacc agcctggaca gcagcaccat acgctacagc ttcg 54

<210>54

<211>54

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(400-415) region

<400>54

gatccgtcca ggctggtgtg ctctctggtg atcaagatac aggtgacctg gtac 54

<210>55

<211>53

<212>DNA

<213> Artificial

<220>

<223> primer for amplifying nucleotide sequence corresponding to GRP78(400-415) region

<400>55

tcgagtacca ggtcacctgt atcttgatca ccagagagca caccagcctg gac 53

<210>56

<211>23

<212>DNA

<213> Artificial

<220>

<223> primers for cloning nucleotide sequences encoding heavy chain variable regions of GC-18, GC-20, GD-4 and GD-17 antibodies

<400>56

ccaccagatt cttatcagac agg 23

<210>57

<211>402

<212>DNA

<213> mice

<400>57

atgagagtgt tgattcttgt gtacctgttg acagcccttc ctggtatctt gtcagatgta 60

cggcttcagg agtcaggacc tggccaggtg aagccttctc agacagtgtc cctcacctgc 120

tctgtcactg gctactctat cactaatggt aatcactggt ggaactggat ccggcaggtt 180

tcaggatcca aactggagtg gatagggtac ataagttcca gtggtagcac tgacagcaat 240

ccatctctca aaagtcgaat ctccatcact agagacactt ccaagaacca gttattcctg 300

cagttgaact ctgtgactac tgaagatata gccacatatt actgtgcaag aggctactac 360

tttgactact ggggccaagg caccactctc acagtctcct ca 402

<210>58

<211>116

<212>PRT

<213> mice

<400>58

Asp Val Arg Leu Gln Glu Ser Gly Pro Gly Gln Val Lys Pro Ser Gln

1 5 10 15

Thr Val Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Asn Gly

20 25 30

Asn His Trp Trp Asn Trp Ile Arg Gln Val Ser Gly Ser Lys Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Ser Ser Gly Ser Thr Asp Ser Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu

65 70 75 80

Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Ile Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210>59

<211>393

<212>DNA

<213> mice

<400>59

atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ctccactggt 60

gacattgtgc tcacccaatc tccagcttct ttggctgtgt ctctagggca gagtgtcacc 120

atctcctgca gagccagtga aagtgttgaa tattatggca ctagtttaat gcagtggtac 180

caacagaaac caggacagcc acccaaactc ctcatctatg ctacatccaa cgtggaatct 240

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 300

cctgtggagg aggatgatat tgcaatgtat ttctgtcagc aaagtaggaa acttccttcg 360

acgttcggtg gaggcaccaa gctggaaatc aaa 393

<210>60

<211>111

<212>PRT

<213> mice

<400>60

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Ser Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Thr Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Leu Pro Ser Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>61

<211>7

<212>PRT

<213> mice

<400>61

Asn Gly Asn His Trp Trp Asn

1 5

<210>62

<211>16

<212>PRT

<213> mice

<400>62

Tyr Ile Ser Ser Ser Gly Ser Thr Asp Ser Asn Pro Ser Leu Lys Ser

1 5 10 15

<210>63

<211>6

<212>PRT

<213> mice

<400>63

Gly Tyr Tyr Phe Asp Tyr

1 5

<210>64

<211>15

<212>PRT

<213> mice

<400>64

Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln

1 5 10 15

<210>65

<211>7

<212>PRT

<213> mice

<400>65

Ala Thr Ser Asn Val Glu Ser

1 5

<210>66

<211>9

<212>PRT

<213> mice

<400>66

Gln Gln Ser Arg Lys Leu Pro Ser Thr

1 5

<210>67

<211>402

<212>DNA

<213> mice

<400>67

atgagagtgt tgattcctgt gtacctgttg acagcccttc ctggtatctt gtctgatgta 60

cgacttcagg agtcaggacc tggcctggtg aagccttctc agacagtgtc cctcacctgc 120

actgtcactg gctactctat cactaatggt aatcactggt ggaactggat ccggcaggtt 180

tcaggaagca aactggagtg gatagggtac ataagctcca gtggtagcac tgacagcaat 240

ccatctctca aaagtcgaat ctccatcact agagacactt ccaagaacca gttattcctg 300

cagttgaact ctgtgactac tgaagatata gccacatatt actgtgcaag aggctactac 360

tttgactact ggggccaagg caccactctc acagtctcct ca 402

<210>68

<211>116

<212>PRT

<213> mice

<400>68

Asp Val Arg Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Val Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Asn Gly

20 25 30

Asn His Trp Trp Asn Trp Ile Arg Gln Val Ser Gly Ser Lys Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Ser Ser Ser Gly Ser Thr Asp Ser Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Leu

65 70 75 80

Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Ile Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210>69

<211>408

<212>DNA

<213> mice

<400>69

atgcatcaga ccagcatggg catcaagatg gaatcacaga ctctggtcct catatccata 60

ctgctctggt tatatggagc tgatgggaac attgtaatga cccaatctcc caaatccatg 120

tccatgtcag taggagagag ggtcaccttg acctgcaagg ccagtgagaa tgtggttact 180

tatgtttcct ggtatcaaca gaaaccagag cagtctccta aactgctgat atacggggca 240

tccaaccggt acactggggt ccccgatcgc ttcacaggca gtggatctgc aacagatttc 300

actctgacca tcagcagtgt gcaggctgaa gaccttgcag attatcactg tggacagggt 360

tacagctatc cgtacacgtt cggagggggg accaagctgg aaataaaa 408

<210>70

<211>107

<212>PRT

<213> mice

<400>70

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>71

<211>7

<212>PRT

<213> mice

<400>71

Asn Gly Asn His Trp Trp Asn

1 5

<210>72

<211>16

<212>PRT

<213> mice

<400>72

Tyr Ile Ser Ser Ser Gly Ser Thr Asp Ser Asn Pro Ser Leu Lys Ser

1 5 10 15

<210>73

<211>6

<212>PRT

<213> mice

<400>73

Gly Tyr Tyr Phe Asp Tyr

1 5

<210>74

<211>11

<212>PRT

<213> mice

<400>74

Lys Ala Ser Glu Asn Val Val Thr Tyr Val Ser

1 5 10

<210>75

<211>7

<212>PRT

<213> mice

<400>75

Gly Ala Ser Asn Arg Tyr Thr

1 5

<210>76

<211>9

<212>PRT

<213> mice

<400>76

Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr

1 5

<210>77

<211>423

<212>DNA

<213> mice

<400>77

atgctgttgg ggctgaagtg ggttttcttt gttgtttttt atcaaggtgt gcattgtgag 60

gtgcagcttg ttgagactgg tggaggattg gtgcagccta aagggtcatt gaaactctca 120

tgtgcagcct ctggattcac cttcaatacc aatgccatga actgggtccg ccaggctcca 180

ggaaagggtt tggaatgggt tgctcgcata agaagtaaaa gtaataatta tgcagcatat 240

tatgccgatt cagtgaaaga caggttcacc atctccagag atgattcaca aagcatgctc 300

tatctgcaaa tgaacaactt gaaaactgag gacacagcca tgtattactg tgtgagagaa 360

ggctacggtt atagcttata ttttgactac tggggccaag gcaccactct cacagtctcc 420

tca 423

<210>78

<211>122

<212>PRT

<213> mice

<400>78

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Asn

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ala Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg Glu Gly Tyr Gly Tyr Ser Leu Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>79

<211>399

<212>DNA

<213> mice

<400>79

atggagtttc agacccaggt actcatgtcc ctgctgctct gcatgtctgg tgcctgtgca 60

gacattgtga tgactcagtc tccaactttc cttgctgtga cagcaagtaa gaaggtcacc 120

attaattgca cggccagtga gagcctttat tcaagcaaac acaaggtgca ctacttggct 180

tggtaccaga agaaaccaga tcaatctcct aaactgctga tatacggggc atccaaccga 240

tacattgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctgacc 300

atcagcagtg tacaggttga agacctcaca cattattact gtgcacagtt ttacagctat 360

cctctcacgt tcggtgctgg gaccaagctg gagctgaaa 399

<210>80

<211>113

<212>PRT

<213> mice

<400>80

Asp Ile Val Met Thr Gln Ser Pro Thr Phe Leu Ala Val Thr Ala Ser

1 5 10 15

Lys Lys Val Thr Ile Asn Cys Thr Ala Ser Glu Ser Leu Tyr Ser Ser

20 25 30

Lys His Lys Val His Tyr Leu Ala Trp Tyr Gln Lys Lys Pro Asp Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Ile Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Val Glu Asp Leu Thr His Tyr Tyr Cys Ala Gln

85 90 95

Phe Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210>81

<211>5

<212>PRT

<213> mice

<400>81

Thr Asn Ala Met Asn

1 5

<210>82

<211>19

<212>PRT

<213> mice

<400>82

Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ala Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Asp

<210>83

<211>11

<212>PRT

<213> mice

<400>83

Glu Gly Tyr Gly Tyr Ser Leu Tyr Phe Asp Tyr

1 5 10

<210>84

<211>17

<212>PRT

<213> mice

<400>84

Thr Ala Ser Glu Ser Leu Tyr Ser Ser Lys His Lys Val His Tyr Leu

1 5 10 15

Ala

<210>85

<211>7

<212>PRT

<213> mice

<400>85

Gly Ala Ser Asn Arg Tyr Ile

1 5

<210>86

<211>9

<212>PRT

<213> mice

<400>86

Ala Gln Phe Tyr Ser Tyr Pro Leu Thr

1 5

<210>87

<211>408

<212>DNA

<213> mice

<400>87

atggaatgta actggatact tccttttatt ctgtcagtaa cttcaggtgt ctactcacag 60

gttcagctcc agcagtctgg ggctgaactg gcaagacctg gggcttcagt gaagttgtcc 120

tgcaaggctt ctggctacac ctttactagc tactggatgc attgggtaaa acagaggcct 180

ggacagggtc tggaatggat tggggctatt tatcctggag atggtgatac taggtacact 240

cagaagttca agggcaaggc cacattgact gcagataaat cctccagcac agcctacatg 300

caactcagca gcttggcatc tgaggactct gcggtctatt actgtgcaag cggaattact 360

gcggtagccg actactgggg ccaaggcacc actctcacag tctcctca 408

<210>88

<211>117

<212>PRT

<213> mice

<400>88

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Ile Thr Ala Val Ala Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210>89

<211>393

<212>DNA

<213> mice

<400>89

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60

gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagag ccttgtacac agtaatggaa acacctattt acattggtac 180

ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca tgttccgctc 360

acgttcggtg ctgggaccaa gctggagctg aaa 393

<210>90

<211>112

<212>PRT

<213> mice

<400>90

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210>91

<211>5

<212>PRT

<213> mice

<400>91

Ser Tyr Trp Met His

1 5

<210>92

<211>17

<212>PRT

<213> mice

<400>92

Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe Lys

1 5 10 15

Gly

<210>93

<211>8

<212>PRT

<213> mice

<400>93

Gly Ile Thr Ala Val Ala Asp Tyr

1 5

<210>94

<211>16

<212>PRT

<213> mice

<400>94

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210>95

<211>7

<212>PRT

<213> mice

<400>95

Lys Val Ser Asn Arg Phe Ser

1 5

<210>96

<211>9

<212>PRT

<213> mice

<400>96

Ser Gln Ser Thr His Val Pro Leu Thr

1 5

<210>97

<211>31

<212>DNA

<213> Artificial

<220>

<223> Forward primer for amplifying nucleotide sequence encoding heavy chain variable region of GD-17 antibody

<400>97

aaaagcttca ggttcagctc cagcagtctg g 31

<210>98

<211>57

<212>DNA

<213> Artificial

<220>

<223> reverse primer for amplifying nucleotide sequence encoding heavy chain variable region of GD-17 antibody

<400>98

cccgaaccac caccacccga accaccacca cctgaggaga ctgtgagagt ggtgcct 57

<210>99

<211>62

<212>DNA

<213> Artificial

<220>

<223> Forward primer for amplifying nucleotide sequence encoding variable region of light chain of GD-17 antibody

<400>99

tggttcgggt ggtggtggtt cgggtggtgg cggatcggat gttgtgatga cccaaactcc 60

ac 62

<210>100

<211>29

<212>DNA

<213> Artificial

<220>

<223> reverse primer for amplifying nucleotide sequence encoding variable region of light chain of GD-17 antibody

<400>100

ttgaattctt tcagctccag cttggtccc 29

<210>101

<211>2013

<212>DNA

<213> mice

<220>

<221>CDS

<222>(1)..(2013)

<223>

<400>101

atg aaa tac ctg ctg ccg acc gct gct gct ggt ctg ctg ctc ctc gct 48

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

gcc cag ccg gcg atg gcc atg gat cac cat cac cat cac cat cac cat 96

Ala Gln Pro Ala Met Ala Met Asp His His His His His His His His

20 25 30

cat cac aag ctt cag gtt cag ctc cag cag tct ggg gct gaa ctg gca 144

His His Lys Leu Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

35 40 45

aga cct ggg gct tca gtg aag ttg tcc tgc aag gct tct ggc tac acc 192

Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr

50 55 60

ttt act agc tac tgg atg cat tgg gta aaa cag agg cct gga cag ggt 240

Phe Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

65 70 75 80

ctg gaa tgg att ggg gct att tat cct gga gat ggt gat act agg tac 288

Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr

85 90 95

act cag aag ttc aag ggc aag gcc aca ttg act gca gat aaa tcc tcc 336

Thr Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser

100 105 110

agc aca gcc tac atg caa ctc agc agc ttg gca tct gag gac tct gcg 384

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala

115 120 125

gtc tat tac tgt gca agc gga att act gcg gta gcc gac tac tgg ggc 432

Val Tyr Tyr Cys Ala Ser Gly Ile Thr Ala Val Ala Asp Tyr Trp Gly

130 135 140

caa ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt 480

Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa act cca 528

Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro

165 170 175

ctc tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga 576

Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg

180 185 190

tct agt cag agc ctt gta cac agt aat gga aac acc tat tta cat tgg 624

Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp

195 200 205

tac ctg cag aag cca ggc cag tct cca aag ctc ctg atc tac aaa gtt 672

Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val

210 215 220

tcc aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca 720

Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

225 230 235 240

ggg aca gat ttc aca ctc aag atc agc aga gtg gag gct gag gat ctg 768

Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu

245 250 255

gga gtt tat ttc tgc tct caa agt aca cat gtt ccg ctc acg ttc ggt 816

Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly

260 265 270

gct ggg acc aag ctg gag ctg aaa gaa ttc ggt ggc gcg ccg gag ttc 864

Ala Gly Thr Lys Leu Glu Leu Lys Glu Phe Gly Gly Ala Pro Glu Phe

275 280 285

ccg aaa ccg tcc acc ccg ccg ggt tct tct ggt tta gag ggc ggc agc 912

Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Leu Glu Gly Gly Ser

290 295 300

ctg gcc gcg ctg acc gcg cac cag gct tgc cac ctg ccg ctg gag act 960

Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr

305 310 315 320

ttc acc cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg gag cag tgc 1008

Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys

325 330 335

ggc tat ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg gcg cgg ctg 1056

Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu

340 345 350

tcg tgg aac cag gtc gac cag gtg atc cgc aac gcc ctg gcc agc ccc 1104

Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro

355 360 365

ggc agc ggc ggc gac ctg ggc gaa gcg atc cgc gag cag ccg gag cag 1152

Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln

370 375 380

gcc cgt ctg gcc ctg acc ctg gcc gcc gcc gag agc gag cgc ttc gtc 1200

Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val

385 390 395 400

cgg cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc gac gtg 1248

Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val

405 410 415

gtg agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg ggc ccg gcg 1296

Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala

420 425 430

gac agc ggc gac gcc ctg ctg gag cgc aac tat ccc act ggc gcg gag 1344

Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu

435 440 445

ttc ctc ggc gac ggc ggc gac gtc agc ttc agc acc cgc ggc acg cag 1392

Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln

450 455 460

aac tgg acg gtg gag cgg ctg ctc cag gcg cac cgc caa ctg gag gag 1440

Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu

465 470 475 480

cgc ggc tat gtg ttc gtc ggc tac cac ggc acc ttc ctc gaa gcg gcg 1488

Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala

485 490 495

caa agc atc gtc ttc ggc ggg gtg cgc gcg cgc agc cag gac ctc gac 1536

Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp

500 505 510

gcg atc tgg cgc ggt ttc tat atc gcc ggc gat ccg gcg ctg gcc tac 1584

Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr

515 520 525

ggc tac gcc cag gac cag gaa ccc gac gca cgc ggc cgg atc cgc aac 1632

Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn

530 535 540

ggt gcc ctg ctg cgg gtc tat gtg ccg cgc tcg agc ctg ccg ggc ttc 1680

Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe

545 550 555 560

tac cgc acc agc ctg acc ctg gcc gcg ccg gag gcg gcg ggc gag gtc 1728

Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val

565 570 575

gaa cgg ctg atc ggc cat ccg ctg ccg ctg cgc ctg gac gcc atc acc 1776

Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr

580 585 590

ggc ccc gag gag gaa ggc ggg cgc ctg gag acc att ctc ggc tgg ccg 1824

Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro

595 600 605

ctg gcc gag cgc acc gtg gtg att ccc tcg gcg atc ccc acc gac ccg 1872

Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro

610 615 620

cgc aac gtc ggc ggc gac ctc gac ccg tcc agc atc ccc gac aag gaa 1920

Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu

625 630 635 640

cag gcg atc agc gcc ctg ccg gac tac gcc agc cag ccc ggc aaa ccg 1968

Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro

645 650 655

ccg gac tac aag gat gac gac gat aag aaa gac gaa ctg tag tga 2013

Pro Asp Tyr Lys Asp Asp Asp Asp Lys Lys Asp Glu Leu

660 665

<210>102

<211>669

<212>PRT

<213> mice

<400>102

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Met Asp His His His His His His His His

20 25 30

His His Lys Leu Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

35 40 45

Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr

50 55 60

Phe Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

65 70 75 80

Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr

85 90 95

Thr Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser

100 105 110

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala

115 120 125

Val Tyr Tyr Cys Ala Ser Gly Ile Thr Ala Val Ala Asp Tyr Trp Gly

130 135 140

Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr Pro

165 170 175

Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg

180 185 190

Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp

195 200 205

Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val

210 215 220

Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

225 230 235 240

Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu

245 250 255

Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Leu Thr Phe Gly

260 265 270

Ala Gly Thr Lys Leu Glu Leu Lys Glu Phe Gly Gly Ala Pro Glu Phe

275 280 285

Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Leu Glu Gly Gly Ser

290 295 300

Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr

305 310 315 320

Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys

325 330 335

Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu

340 345 350

Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro

355 360 365

Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln

370 375 380

Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val

385 390 395 400

Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val

405 410 415

Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala

420 425 430

Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu

435 440 445

Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln

450 455 460

Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu

465 470 475 480

Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala

485 490 495

Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp

500 505 510

Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr

515 520 525

Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn

530 535 540

Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe

545 550 555 560

Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val

565 570 575

Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr

580 585 590

Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro

595 600 605

Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro

610 615 620

Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu

625 630 635 640

Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro

645 650 655

Pro Asp Tyr Lys Asp Asp Asp Asp Lys Lys Asp Glu Leu

660 665

Claims (28)

1. A pharmaceutical composition comprising an antibody that binds to glucose regulatory protein 78(GRP 78).

2. The composition of claim 1, which is an anti-cancer agent.

3. The composition of claim 1 or 2, wherein the antibody is a monoclonal antibody.

4. The composition of any one of claims 1-3, wherein said antibody is an antibody that binds to GRP78 located on the surface of a cell.

5. The composition of any one of claims 1-4, wherein said antibody is an antibody that is taken up by a cell expressing GRP 78.

6. The composition of any one of claims 1-5, wherein the antibody is a heavy chain variable region of SEQ ID NO: 3.

7. The composition of any one of claims 1-6, wherein the antibody is an antibody that binds to a wounded cellular material.

8. A monoclonal antibody that binds to GRP 78.

9. The antibody of claim 8, characterized by binding to GRP78 expressed on the cell surface.

10. The antibody of claim 8 or 9, characterized by being taken up by GRP 78-expressing cells.

11. The antibody of any one of claims 8-10, characterized by having an affinity for SEQ ID NO: 3.

12. The antibody of any one of claims 8-11, characterized by recognizing the same epitope as that recognized by the antibody of any one of the following (a) to (f):

(a) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 8, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 9, the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in the figure 10,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 11, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 12, or a pharmaceutically acceptable salt thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 13;

(b) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 18, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 19, the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof, wherein,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 21, the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 22, or a sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 23;

(c) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 61, or a sequence of amino acids,

CDR2 has the amino acid sequence of SEQ ID NO: 62, or a sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 63, or a fragment thereof,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 64, or a sequence shown in SEQ ID NO,

CDR2 has the amino acid sequence of SEQ ID NO: 65, and (b) an amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 66;

(d) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 71, or a nucleotide sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 72, or a sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 73 with a sequence of an amino acid sequence shown in SEQ ID NO,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 74, or a sequence shown in SEQ ID NO,

CDR2 has the amino acid sequence of SEQ ID NO: 75, the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 76;

(e) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 81, or a sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 82, or a pharmaceutically acceptable salt thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 83 of a sequence of amino acids as shown in SEQ ID NO,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 84, and the amino acid sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 85, or a pharmaceutically acceptable salt thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 86;

(f) an antibody having the following heavy chain variable region and light chain variable region:

wherein the heavy chain variable region has the following CDRs 1-3,

CDR1 has the amino acid sequence of SEQ ID NO: 91, or a sequence shown in the specification,

CDR2 has the amino acid sequence of SEQ ID NO: 92, or a nucleotide sequence thereof,

CDR3 has the amino acid sequence of SEQ ID NO: 93 (b) to (b) a sequence shown as 93,

and wherein the light chain variable region has the following CDRs 1-CDRs 3,

CDR1 has the amino acid sequence of SEQ ID NO: 94, or a pharmaceutically acceptable salt thereof,

CDR2 has the amino acid sequence of SEQ ID NO: 95 of the amino acid sequence shown in the specification,

CDR3 has the amino acid sequence of SEQ ID NO: 96.

13. The antibody of any one of claims 8-12, characterized by having a cell damaging activity against cells expressing GRP 78.

14. The antibody of claim 13, characterized in that a cell damaging substance is bound.

15. Methods of delivering cell damaging substances into cells using anti-GRP 78 antibodies.

16. A method of inhibiting cell proliferation by a cell damaging agent that binds to an anti-GRP 78 antibody.

17. The method of claim 15 or 16, wherein the cell is a cancer cell.

18. Use of an anti-GRP 78 antibody for the delivery of a cell damaging substance into a cell.

19. Use of an anti-GRP 78 antibody having cellular uptake activity for inhibiting cell proliferation.

20. The method of claim 18 or 19, wherein the cell is a cancer cell.

21. Use according to any one of claims 18 to 20, characterised in that the anti-GRP 78 antibody binds wounded cellular material.

22. A method of preparing a pharmaceutical composition comprising:

(a) providing an anti-GRP 78 antibody;

(b) confirming whether the antibody of (a) has an activity of being taken up by cells;

(c) screening for antibodies having cellular uptake activity;

(d) binding of the antibody selected in (c) to the cell damaging material.

23. The method of claim 22, wherein the pharmaceutical composition is an anti-cancer agent.

24. Methods of diagnosing cancer using anti-GRP 78 antibodies.

25. Method according to claim 24, characterized in that an anti-GRP 78 antibody conjugated to a label is used.

26. Method according to claim 24 or 25, characterized in that the uptake of anti-GRP 78 antibodies into the cells is detected.

27. An anti-GRP 78 antibody conjugated to a label.

28. Consisting of SEQ ID NO: 3 or a fragment thereof.