WO2015098112A1 - 食道がんのマーカーおよびその利用 - Google Patents
食道がんのマーカーおよびその利用 Download PDFInfo
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C12Q2600/00—Oligonucleotides characterized by their use
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- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
- G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
Definitions
- the present invention relates to a marker for cancer, particularly esophageal cancer, and related techniques, methods, drugs and the like.
- Esophageal cancer occurs in the esophagus and often has no subjective symptoms in the early stages, while esophageal cancer discovered asymptomatically has a high probability of cure because it is early.
- SCC squamous cell carcinoma associated antigen
- CEA carcinoembryonic antigen
- CA19-9, p53, etc. are used as tumor markers for esophageal cancer, but tumor markers are advanced malignant tumors. It is currently used only to grasp the dynamics of cancer, and definitive tumor markers for esophageal cancer are not currently available, and some cancers do not show abnormal values It is said that.
- the present invention provides a novel marker for esophageal cancer and its applied technology.
- the present inventors have found that the Glypican-1 molecule was significantly more strongly expressed in esophageal cancer cells than normal cells, found that it can be used as a tumor marker, and suppress Glypican-1 Thus, the present invention was completed by finding that esophageal cancer can be treated.
- the present invention provides a detection agent for identifying esophageal cancer, comprising a substance that binds to Glypican-1 or an expression product thereof.
- the present invention provides a method for identifying esophageal cancer using a substance that binds to Glypican-1 or its expression product.
- the detection agent of the present invention may be used for identification.
- the detection agent of the present invention is an antibody or a fragment or functional equivalent thereof, or a nucleic acid.
- the detection agent of the present invention is labeled.
- Glypican-1 is SEQ ID NO: 1 (nucleic acid sequence) or SEQ ID NO: 2 (amino acid sequence) or an equivalent thereof.
- the detection agent of the present invention is a nucleic acid
- the nucleic acid is a probe or a primer.
- the probe or primer used as the detection agent of the present invention may be the nucleic acid sequence shown in SEQ ID NO: 1, its complementary strand, or a fragment thereof.
- the probe or primer used as the detection agent of the present invention may have SEQ ID NO: 25 and / or 26.
- the esophageal cancer targeted by the detection agent of the present invention includes a lymph node metastasis site, squamous cell carcinoma and / or adenocarcinoma.
- the target sample when the expression of Glypican-1 in a target sample is increased compared to that of a normal sample, the target sample is diagnosed as having esophageal cancer. This may be a feature.
- the present invention provides a marker for identifying esophageal cancer comprising Glypican-1 or an expression product thereof or a fragment or derivative thereof.
- the esophageal cancer includes a lymph node metastasis site, squamous cell carcinoma and / or adenocarcinoma.
- the esophageal cancer includes squamous cell carcinoma and / or adenocarcinoma.
- the esophageal cancer comprises squamous cell carcinoma.
- the esophageal cancer may be human.
- the Glypican-1 used in the present invention is SEQ ID NO: 1 (nucleic acid sequence) or a complementary strand thereof, or SEQ ID NO: 2 (amino acid sequence) or an equivalent thereof.
- the present invention provides a method using Glypican-1 or an expression product thereof as an index for identifying esophageal cancer.
- the target sample when the expression of Glypican-1 in a target sample is increased compared to that in a normal sample, the target sample is diagnosed as having esophageal cancer. It is good.
- the identification in the method of the present invention, can be performed using the detection agent of the present invention.
- the esophageal cancer targeted by the method of the present invention includes a lymph node metastasis site, squamous cell carcinoma and / or adenocarcinoma.
- the present invention provides a preventive or therapeutic agent for esophageal cancer, comprising an inhibitor of Glypican-1.
- the esophageal cancer is Glypican-1 positive.
- the esophageal cancer targeted by the preventive or therapeutic agent of the present invention includes a lymph node metastasis site, squamous cell carcinoma and / or adenocarcinoma.
- the esophageal cancer may include squamous cell carcinoma.
- the present invention may be a prophylactic or therapeutic agent for administration to a patient determined to have developed Glypican-1-positive esophageal cancer.
- the Glypican-1 inhibitor of the present invention can be an antibody or a fragment or functional equivalent thereof, or a nucleic acid.
- the Glypican-1 inhibitor of the present invention is a nucleic acid, and the nucleic acid may be siRNA.
- the siRNA comprises SEQ ID NO: 25 and / or SEQ ID NO: 26.
- the antibody is a monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, human antibody, multifunctional antibody, bispecific or oligospecific antibody, single chain antibody, scFV, An antibody selected from diabodies, sc (Fv) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the present invention provides a method for preventing or treating esophageal cancer in a subject comprising administering to a subject in need thereof an effective amount of a Glypican-1 inhibitor.
- the present invention provides a method for preventing or treating esophageal cancer in a subject, comprising the step of examining that the sample of the subject is positive for Glypican-1.
- any embodiment employed in the prophylactic or therapeutic agent of the present invention can be similarly adopted by combining one or more features. Understood.
- the present invention provides a prophylactic or therapeutic agent for esophageal cancer comprising a Glypican-1 binding agent.
- the prophylactic or therapeutic agent of the present invention further comprises a cell killing agent.
- the binding agent of Glypican-1 in the present invention may be an antibody, a fragment or a functional equivalent thereof, or a nucleic acid.
- the Glypican-1 binding agent used in the present invention is an antibody or a fragment or functional equivalent thereof, and may be further bound with a cell-killing agent.
- the esophageal cancer in the present invention is Glypican-1 positive.
- the esophageal cancer targeted by the prophylactic or therapeutic agent using the binding agent of the present invention includes a lymph node metastasis site, squamous cell carcinoma and / or adenocarcinoma.
- the esophageal cancer in the present invention may include squamous cell carcinoma.
- the detection agent, binding agent or inhibitor in the present invention is an antibody or a fragment or a functional equivalent thereof,
- heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are respectively SEQ ID NO: 7 at positions 31-35, 50-66, 99-120, 169-176, An antibody comprising the amino acid sequence shown at positions 193 to 199 and 232 to 242;
- heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are respectively positions 31 to 35 of SEQ ID NO: 8
- heavy chain CDR1, 2, 3, light chain CDR1, 2 An antibody comprising an amino acid sequence represented by positions 31-35, 50-66, 99-120, 169-176, 193-199, and 232-241 of SEQ ID NO: 9, respectively
- h) Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35 of SEQ ID NO: 10, respectively 5
- An antibody comprising an amino acid sequence shown at positions -113, 162-170, 187-193, and 226-236, (n) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3, respectively
- An antibody comprising the amino acid sequence shown by SEQ ID NO: 16 at positions 31-35, 50-66, 99-110, 159-166, 183-189, and 222-237, (o) heavy chain CDR1, 2 , 3, light chain CDR1, 2, and 3 are shown at positions 31-35, 50-65, 98-113, 162-171, 188-194, and 227-237 of SEQ ID NO: 17, respectively.
- (P) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are respectively represented by SEQ ID NO: 18, positions 31-35, 50-66, 99-116, 165- An antibody comprising an amino acid sequence shown at positions 174, 191-197, and 230-240,
- Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 19, positions 31-35, 50-66, 99-115, 164-175, 193-199, and An antibody comprising the amino acid sequence shown at positions 232 to 241;
- (r) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, and 99 of SEQ ID NO: 20;
- the detection agent, binding agent, or inhibitor in the present invention is an antibody or a fragment or functional equivalent thereof, and the antibody has positions 33 to 61, or positions 339 to 358 of SEQ ID NO: 2 and / or It may have 388 to 421 position or 430 to 530 position as an epitope.
- antibodies are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc (Fv ) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the present invention provides the following antibodies: (a) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 65 of SEQ ID NO: 3, respectively.
- An antibody comprising an amino acid sequence shown at positions 98-114, 163-170, 187-193, and 226-235, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3; An antibody comprising an amino acid sequence represented by positions 31 to 35, 50 to 66, 99 to 110, 159 to 166, 183 to 187, and 222 to 231 of SEQ ID NO: 4, respectively,
- (c) heavy chain CDR1 , 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, 99 to 113, 162 to 170, 187 to 193, and 226 to 237 of SEQ ID NO: 5, respectively.
- (D) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 6, positions 31-35, 51-66, 99-112,
- (e) heavy chain CDR1 2, 3, light chain CDR1, 2, and 3 are at positions 31 to 35, 50 to 66, 99 to 120, 169 to 176, 193 to 199, and 232 to 242 of SEQ ID NO: 7, respectively.
- An antibody comprising the amino acid sequence shown, (f) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 8, positions 31-35, 50-65, 98-114, 163, respectively.
- Antibodies comprising amino acid sequences shown at positions -170, 187-193, and 226-235, (g) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are each represented by SEQ ID NO: 31 Antibodies comprising amino acid sequences shown at positions -35, 50-66, 99-120, 169-176, 193-199, and 232-241, (h) heavy chain CDR1, 2, 3, light The strands CDR1, 2, and 3 represent the amino acid sequences represented by positions 31 to 35, 50 to 65, 98 to 113, 162 to 171, 188 to 194, and 227 to 237 of SEQ ID NO: 10, respectively.
- An antibody comprising (i) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 respectively Antibodies comprising amino acid sequences shown at positions -35, 50-65, 98-114, 163-170, 187-193, and 226-235, (j) heavy chain CDR1, 2, 3, light
- the chain CDR1, 2, and 3 represent the amino acid sequences represented by positions 31 to 35, 50 to 66, 99 to 113, 162 to 169, 186 to 192, and 225 to 234 of SEQ ID NO: 12, respectively.
- the antibody comprising, (k) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-116, 165-174, 191 of SEQ ID NO: 13, respectively.
- an antibody comprising the amino acid sequence shown at positions 226 to 236, (n) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35 and positions 50 to 66 of SEQ ID NO: 16, respectively.
- (p) heavy chain CDR1 , 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 18, positions 31-35, 50-66, 99-116, 165-174, 191-197, and 230-240, respectively.
- (Q) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 19, positions 31 to 35, positions 50 to 66, and positions 99 to 115, respectively. Includes amino acid sequences shown at positions 164-175, 193-199, and 232-241
- the antibody, (r) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 20, positions 31-35, 50-66, 99-117, 166-177, 194- An antibody comprising the amino acid sequence shown at positions 200 and 233 to 242;
- (s) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 32 to 36, 51 to 51 of SEQ ID NO: 21, respectively;
- These antibodies may have, as epitopes, positions 33 to 61 of SEQ ID NO: 2, or positions 339 to 358 and / or positions 388 to 421, or positions 430 to 530.
- These antibodies are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc (Fv ) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the present invention relates to an antibody having an epitope at positions 33 to 61, or positions 339 to 358 and / or positions 388 to 421, or positions 430 to 530 of SEQ ID NO: 2, or a fragment or functional equivalent thereof Offer things.
- These antibodies are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc (Fv ) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the present invention provides a therapeutic method, preventive method, use, etc. using the pharmaceutical composition, therapeutic agent or prophylactic agent of the present invention.
- an effective marker for esophageal cancer is provided, and that it can also be used for the treatment or prevention of esophageal cancer. Therefore, esophageal cancer, which has been impossible or difficult in the past, can be diagnosed, treated or prevented at an early stage.
- FIG. 1 shows the results showing expression of Glypican-1 in various cells by Western blot.
- the molecular weight display (kDa) based on the molecular weight marker of the protein is shown at the left end, and samples from the left are HEEEpic, Het1A (or normal cells), TE1, TE5, TE6, TE8, TE9, TE10, TE11, TE14, and TE15 (or more). Esophageal cancer cell line).
- Glypican-1 shown in the upper panel shows a band around 70 kDa.
- ⁇ -actin was used as a control shown in the lower part.
- FIG. 2 shows the result of showing the relative expression level of Glypican-1 (Accession No. P35052) using iTRAQ®.
- FIG. 3 is a diagram showing by FACS experiment that Glypican-1 is expressed on the cell surface of esophageal cancer cells. It is shown as a comparison between PE-A and Glypican-1.
- the cells used were HEEpic and Het1A (first to second strains from the upper left) as normal esophageal epithelial cell lines, and TE1, TE5, TE6, TE8 (three from the upper left) as esophageal cancer cell lines. Eyes to 6 strains), TE9, TE10, TE11, TE14 and TE15) and the lower 5 strains) were used.
- FIG. 3 is a diagram showing by FACS experiment that Glypican-1 is expressed on the cell surface of esophageal cancer cells. It is shown as a comparison between PE-A and Glypican-1.
- the cells used were HEEpic and Het1A (first to second strains from the upper left) as normal esophageal epithelial cell lines,
- FIG. 4A is a photograph showing the expression of Glypican-1 in esophageal squamous cell carcinoma.
- the antibody used is Atlas antibodies: HPA030571.
- the left bar is 200 ⁇ m and the right bar is 20 ⁇ m.
- FIG. 4B is a photograph showing the expression of Glypican-1 in normal esophageal epithelium (left) and esophageal squamous cell carcinoma (right).
- the antibody used is Atlas antibodies: HPA030571.
- the top shows 100 times and the bottom shows 400 times.
- Glypican-1 was shown to be highly expressed in esophageal squamous cell carcinoma.
- FIG. 1 is a photograph showing the expression of Glypican-1 in esophageal squamous cell carcinoma.
- FIG. 4B is a photograph showing the expression of Glypican-1 in normal esophageal epithelium (left) and esophageal squamous
- FIG. 4C is a photograph showing that Glypican-1 is highly expressed even at the lymph node metastasis site of esophageal cancer.
- the left shows normal lymph nodes, and the right shows esophageal cancer lymph node metastasis. The top shows 100 times and the bottom shows 400 times.
- FIG. 4D shows that in patients with esophageal squamous cell carcinoma, the Glypican-1 high expression group has a higher survival rate than the low expression group.
- the horizontal axis indicates the survival period, and the vertical axis indicates the survival rate.
- FIG. 5A shows the results of comparing the expression level of Glypican-1 (GPC1) mRNA level in various normal tissues with esophageal cancer cell line (TE11) by real-time PCR analysis.
- the vertical axis represents the relative expression level of GPC1 mRNA (GADPH or ⁇ -actin was used as a control).
- GPC1 mRNA GPC1 mRNA
- FIG. 5B shows that high expression at the mRNA level of GPC-1 is observed in esophageal cancer tissues as compared to non-cancerous sites. Left is tumor, right is non-tumor. The vertical axis shows the relative amount of GPC1 mRNA based on ⁇ -actin mRNA.
- FIG. 6 shows the results showing serum Glypican-1 concentrations in healthy individuals and patients with esophageal squamous cell carcinoma. It is the result of having measured the density
- the unit of the vertical axis is (pg / ml).
- Samples from the left are HC1, HC2, HC3, HC4, HC5 (healthy human samples), ESCC-10, ESCC-28, ESCC-32, ESCC-55, ESCC-105, ESCC-119, ESCC-125, ESCC- 164 and ESCC-220 (above esophageal squamous cell carcinoma patient sample) blood.
- HC1, HC2, HC3, HC4, HC5 healthy human samples
- ESCC-10 ESCC-28, ESCC-32, ESCC-55, ESCC-105, ESCC-119, ESCC-125, ESCC- 164 and ESCC-220 (above esophageal squamous cell carcinoma patient sample) blood.
- ESCC-10 ESCC-28, ESCC-32, ESCC-55, ESCC-105, ESCC-119, ESCC-125, ESCC- 164 and ESCC-220 (above
- FIG. 7 shows the results of measuring the serum Glypican-1 concentration in healthy individuals and patients with squamous cell carcinoma of the esophagus and performing the Mann-Whitney U test.
- the concentration of GPC-1 in the serum was measured in the serum obtained from the blood sample of each subject, and the Mann-Whitney U test was performed.
- the unit of the vertical axis is pg / ml.
- FIG. 8 shows the result of expression suppression using siRNA against Glypican-1.
- the target cell lines are TE5, TE6, TE8 and TE9 shown from the left in the upper row, and TE14 and TE15 shown from the left in the lower row.
- the vertical axis of each graph shows the relative growth in% relative to the negative control siRNA.
- no siRNA No treatment
- one using negative control siRNA Negative Control siRNA
- GPC-1 siRNA siRNA against Glypican-1
- NS indicates no significant difference
- the numerical value following p indicates statistical significance at the risk rate at the indicated numerical value.
- the growth of esophageal cancer cell lines was suppressed by suppressing the expression using siRNA against GPC-1.
- normal cells HEEpic
- FIG. 9 is a view showing apoptosis by suppressing expression using siRNA against Glypican-1.
- the target cell lines are TE6 and TE8 shown from the left in the upper row, and TE14 shown in the lower row.
- the vertical axis of the graph shows the activity of caspase-3 as an index of apoptosis in terms of A. U. It is shown by.
- three types of experiments were performed: no siRNA (No treatment), one using negative control siRNA (Negative Control siRNA), and one using siRNA against Glypican-1 (GPC-1 siRNA).
- NS indicates no significant difference, and the numerical value following p indicates statistical significance at the risk rate at the indicated numerical value.
- Apoptosis was induced by suppression of expression using siRNA against GPC-1 in esophageal cancer cell lines.
- FIG. 10A shows the results of observing, by Western blot, changes in the activation of serine threonine kinase AKT (another name for ProteinKinaseB) by suppressing expression using siRNA against GPC-1 in an esophageal cancer cell line.
- target esophageal cancer cells TE6, TE8 and TE14 were used.
- FIG. 10B shows that suppression of GPC1 expression in the esophageal cancer cell line shows an increase in the expression of pre-apoptotic protein and a decrease in the expression of anti-apoptotic protein.
- the left panel shows the Western blot of Bak, Puma and GAPDH from the top (24 hours), and the left column shows no treatment, negative control RNA and GPC-1 siRNA. The right panel shows 48 hours later. From the top, Bik, Bim, Bad, Bcl-w and GAPDH bands are shown.
- FIG. 10C shows that inhibition of EGFR phosphorylation is observed by suppressing GPC-1 expression in esophageal cancer cell lines. From above, phosphorylated EGFR (Y1068), EGFR, and GAPDH are shown. From left column, no treatment, negative control, RNA, GPC-1 siRNA are shown.
- FIG. 11 shows the result of confirming the reactivity of the obtained antibody to Glypican-1 by FACS (FITC-A: GPC1). From the results of reactivity, it is understood that these antibodies may be used as diagnostics and therapeutics.
- FIG. 12 shows the calculation result of the dissociation constant by Biacore analysis.
- FIG. 13 shows that GPC-1 monoclonal antibody cross-reacts with mouse GPC-1.
- the upper column shows clones 1-5, 1-12, 1-28, 1-57 (# 2, # 4, # 7, # 10, respectively) from the left side, and the lower column shows clones 2-60, 2 from the left side. -63, 2-70 (# 17, # 18, # 19 respectively). Since the original figure is shown in red and blue, blue is indicated by an arrow.
- the curve indicated by the arrow indicates the reactivity of various clones with 293 cells in which mouse GPC1 was forcibly expressed.
- FIG. 14 shows the anti-GPC-1 antibody # 19 showing TE14 cell growth inhibitory activity.
- the upper column shows clones 1-5, 1-12, 1-28, 1-57 (# 2, # 4, # 7, # 10, respectively) from the left side, and the lower column shows clones 2-60, 2 from the left side.
- -63, 2-70 (# 17, # 18, # 19 respectively).
- the black circle indicates the antibody
- the white circle indicates mIgG2a.
- the horizontal axis indicates antibody concentration ( ⁇ g / ml).
- FIG. 15 shows that anti-GPC-1 mAB (# 7, # 9) has an epitope region at amino acids 33-61.
- the upper column shows clones 1-5, 1-12, 1-28, 1-57 (# 2, # 4, # 7, # 10, respectively) from the left side, and the lower column shows clones 2-60, 2 from the left side. -63, 2-70 (# 17, # 18, # 19 respectively).
- the peak painted in gray is a histogram of 293 cells stained with each clone, and the white is stained with each clone against 293 cells expressing human GPC1 with deletion of amino acids 33-61. A histogram is shown.
- FIG. 16A shows epitope analysis of anti-GPC-1 antibody using human GPC-1 / GPC-3 chimeric protein.
- FIG. 16B shows the result of epitope analysis of the antibody of the present invention.
- the left panel is a figure surrounding the amino acid position of the epitope in GPC-1 (SEQ ID NO: 2).
- the right column shows the amino acid position of the epitope of each antibody.
- the epitope of the antibody of the present invention is at positions 33-61 (# 7, # 19), or 339-358 and / or 388-404 and / or 405-421 (# 4, 388).
- FIG. 17 (AD) shows the epitope analysis results of anti-GPC antibody using human GPC-1 / GPC-3 chimeric protein.
- FIG. 17A shows the results of using anti-FLAG-M2 in the left column and using anti-GPC-1 mAb # 4 in the right column. From the top, pFLAG-CMV3 vector, pFLAG-CMV3hGPC1, pFLAG-CMV3 hGPC3, and human GPC1 / GPC3 chimeric proteins are used. Since the original figure is shown in red and blue, blue is indicated by an arrow.
- FIG. 17 (AD) shows the epitope analysis results of anti-GPC antibody using human GPC-1 / GPC-3 chimeric protein.
- FIG. 17B shows the results using the anti-GPC-1 mAb # 7 in the left column and the anti-GPC-1 mAb # 17 on the right. From the top, pFLAG-CMV3 vector, pFLAG-CMV3hGPC1, pFLAG-CMV3 hGPC3, and human GPC1 / GPC3 chimeric proteins are used. Since the original figure is shown in red and blue, blue is indicated by an arrow.
- FIG. 17 shows the epitope analysis results of anti-GPC antibody using human GPC-1 / GPC-3 chimeric protein.
- FIG. 17C shows the results using anti-GPC-1 mAb # 18, and the right shows the results using anti-GPC-1 mAb # 19. From the top, pFLAG-CMV3 vector, pFLAG-CMV3hGPC1, pFLAG-CMV3 hGPC3, and human GPC1 / GPC3 chimeric proteins are used.
- FIG. 17 shows the epitope analysis results of anti-GPC antibody using human GPC-1 / GPC-3 chimeric protein.
- FIG. 17D shows the results using anti-GPC-1 mAb # 2
- the right shows the results using anti-GPC-1 mAb # 10. From the top, pFLAG-CMV3 vector, pFLAG-CMV3hGPC1, pFLAG-CMV3 hGPC3, and human GPC1 / GPC3 chimeric proteins are used.
- FIG. 18 shows examination of the antitumor effect of anti-GPC1 antibody against esophageal cancer.
- anti-GPC1 antibodies # 4 and # 19 were used and mouse IgG2a (Sigma M 7769) was used.
- An intraperitoneal administration of 10 mg / kg was performed.
- the model used was a SCID female 6 week old mouse. First, 2 ⁇ 10 6 cells were transplanted subcutaneously. Tumor size was approximately 100 mm 3 (10-14 days after transplantation).
- FIG. 19 shows that anti-GPC-1 antibody exhibits an anti-tumor effect against an esophageal cancer xenograft model in SCID mice.
- the horizontal axis indicates the number of days after treatment, and the vertical axis indicates the tumor volume (mm 3 ).
- Statistical significance was confirmed by one-way ANOVA and Dunnett's test. * Indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, and *** indicates p ⁇ 0.001.
- FIG. 19 shows that anti-GPC-1 antibody exhibits an anti-tumor effect against an esophageal cancer xenograft model in SCID mice.
- the horizontal axis indicates the number of days after treatment, and the vertical axis indicates the tumor volume (mm 3 ).
- Diamonds indicate control IgG2
- FIG. 21 shows that anti-GPC-1 antibody # 4 exhibits an anti-tumor effect independent of ADCC against an esophageal cancer xenograft model in NOD / SCID mice.
- FIG. 22 is a diagram showing that anti-GPC antibody # 4 exhibits an anti-tumor effect independent of ADCC against an esophageal cancer xenograft model in NOD / SCID mice.
- FIG. 23 shows examination of the anti-tumor effect of anti-GPC1 antibody against GPC1-negative cell line (LK2 xenograft model). As shown, anti-GPC1 antibodies # 4 and # 19 were used and mouse IgG2a (Sigma M 7769) was used. An intraperitoneal administration of 10 mg / kg was performed. The model used was a SCID female 6 week old mouse.
- FIG. 24 shows that anti-GPC antibodies do not show anti-tumor effects in vivo against GPC1-negative cell lines (LK2 xenograft model).
- the vertical axis shows the tumor volume (mm 3 ).
- FIG. 26 shows the protocol of the anti-GPC1 antibody safety test using mice.
- Mouse IgG2a (Sigma M7769) and anti-GPC-1 antibody # 4 are intraperitoneally administered to C57BL / 6J (8 weeks old) at 1 mg / body weight, and the following items are evaluated on day 7. Select the brain, heart, kidney, liver, lungs, and spleen as the removed organ.
- WBC white blood cells
- RBC red blood cells
- Hb hemoglobin
- Plt total bilirubin
- ALT alkaline phosphatase
- ALP amylase
- BUN Blood urea nitrogen
- Cr chromium
- Ca calcium
- P total protein
- TP total protein
- ALP alkaline phosphatase
- BUN Blood urea nitrogen
- Cr chromium
- P total protein
- TP total protein
- ALP albumin
- K sodium
- Globn glutamine
- Glu glutamine
- Ly lymphocytes
- Mo monocytes
- Gr indicates granulocytes
- Hct indicates a hematocrit value.
- FIG. 28 shows a comparison between control IgG and anti-GPC1 antibody (female). Each value is the same as in FIGS. FIG.
- the abbreviations described with reference to FIG. FIG. 30 shows a comparison between control IgG and anti-GPC1 antibody (female). Each value is the same as in FIGS.
- Glypican-1 glycosylphosphatidylinositol (GPI) anchored cell surface proteoglycans having heparan sulfate. Is. It has been implicated in cell adhesion, migration, lipoprotein metabolism, growth factor activity regulation and blood coagulation inhibition. It is said to bind to several fibroblast growth factors (FGFs), such as FGF-1, FGF-2 and FGF-7. Glypican-1 functions as an extracellular chaperone of VEGF165 and is considered to assist in restoring the receptor binding ability after oxidation.
- FGFs fibroblast growth factors
- Glypican is currently known in six types, Glypican-1 to Glypican-6, but it is always recognized as a cancer marker even if it is a Glypican-family member in relation to cancer Rather, the members seem to be irrelevant.
- Glypican-1 is registered with UniProt as accession number P35052 (see http://www.uniprot.org/uniprot/P35052).
- NCBI has NP_002072.2 (precursor amino acid sequence), In NM_002081.2 (mRNA), EMBL, GenBank and DDBJ, they are registered as X54232.1 (mRNA), BC051279.1 (mRNA), and AC110619.3 (genomic).
- Glypican-1 For the nucleic acid sequence (full length) of human Glypican-1, SEQ ID NO: 1 is a representative example, and for the amino acid sequence, SEQ ID NO: 2 is a representative example.
- Glypican-1 As used herein, “Glypican-1,” “GPC-1,” or “GPC1” refers to a specific SEQ ID NO or accession number as long as it meets the specific purpose of the invention.
- a “derivative”, “analog” or “variant” as used herein is preferably, but not intended to be limited, substantially homologous to a protein of interest (eg, Glypican-1)
- a protein of interest eg, Glypican-1
- Such molecules when compared to sequences aligned in various embodiments over amino acid sequences of the same size or aligned by computer homology programs known in the art.
- Nucleic acids that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical or that encode such molecules are (highly) stringent It can hybridize to sequences encoding component proteins under mild, moderately stringent or non-stringent conditions.
- Glypican-1 In the present invention, humans are mainly discussed for Glypican-1, but chimpanzee (Pantroglodytes) (K7B6W5), rhesus monkey (Macaca mulatta) (F6VPW9), mouse (Mus musculus) (Q9QZF2), rat (Rattus norvegicus) (P35053) , Chickens (Gallus gallus) (F1P150) and other animals other than humans are known to express Glypican-1 protein, so these animals, particularly mammals, are also within the scope of the present invention. Is understood to enter.
- the functional domain of Glypican-1 such as the extracellular domain (approximately 500 amino acids and containing 12 cysteine residues) and the C-terminal hydrophobic region (GPI-anchor domain) are conserved Is preferred.
- a fragment of Glypican-1 is a polypeptide containing an arbitrary region of Glypican-1, and as long as it functions as an object of the present invention (for example, a marker or therapeutic target), it is not necessarily natural Glypican-1 It may not have a biological function.
- the representative nucleotide sequence of Glypican-1 is (A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or a fragment sequence thereof; (B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2, A polynucleotide that encodes a variant polypeptide having functional activity; (D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or a fragment thereof; (E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (F) a
- the biological activity typically means that Glypican-1 can be distinguished from other proteins existing in the same organism as the activity or marker.
- amino acid sequence of Glypican-1 (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2 and having biological activity; (C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1; (D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2; or (e) at least 70% identity to at least 80% identity to any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
- a substance that binds to Glypican-1 “a binding agent for Glypican-1” or “a molecule that interacts with Glypican-1” is a molecule or substance that binds at least temporarily to Glypican-1. It is. For detection purposes, preferably it is possible to indicate that it has been bound (eg, labeled or in a labelable state), and for therapeutic purposes, it is further advantageous that a therapeutic agent is further bound.
- substances that bind to Glypican-1 include antibodies, antisense oligonucleotides, siRNA, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes, and peptidomimetics. .
- a substance that binds to Glypican-1 or “Glypican-1 interacting molecule may be an inhibitor of Glypican-1, eg directed against Glypican-1, especially directed against the active site of Glypican-1
- binding proteins or binding peptides as well as nucleic acids directed against the Glypican-1 gene, such as nucleic acids that inhibit the expression of Glypican-1 gene or the activity of Glypican-1.
- nucleic acids that inhibit the expression of Glypican-1 gene or the activity of Glypican-1 refers to single-stranded or single-stranded DNA or RNA, or modifications or derivatives thereof, and includes but is not limited to antisense nucleic acids, aptamers, siRNAs (small interfering RNAs) and ribozymes.
- Binding protein or “binding peptide” for 1 refers to any protein or peptide that binds to Glypican-1 and is directed against Glypican-1 (eg, If, polyclonal or monoclonal antibodies), including antibody fragments and functional equivalents without limitation.
- protein protein
- polypeptide oligopeptide
- peptide refers to a polymer of amino acids having an arbitrary length.
- This polymer may be linear, branched, or cyclic.
- the amino acid may be natural or non-natural and may be a modified amino acid.
- the term can also encompass one assembled into a complex of multiple polypeptide chains.
- the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
- amino acid is a general term for organic compounds having an amino group and a carboxyl group.
- amino acid sequence may be chemically modified. Any amino acid in the amino acid sequence may form a salt or a solvate. Further, any amino acid in the amino acid sequence may be L-type or D-type.
- the protein according to the embodiment of the present invention includes the above-mentioned “specific amino acid sequence”.
- specific amino acid sequence examples include N-terminal modification (for example, acetylation, myristoylation, etc.), C-terminal modification (for example, amidation, glycosylphosphatidylinositol addition, etc.), or side chain Modifications (for example, phosphorylation, sugar chain addition, etc.) are known.
- the amino acid may be natural or non-natural as long as it satisfies the object of the present invention.
- polynucleotide As used herein, “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably.
- oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides.
- oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond uracil in oligonucleotide is C- Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide substituted with C-5 propynylcytosine Nucleotide derivatives, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is substituted with 2'-O-propylribos
- a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell .Probes 8: 91-98 (1994)).
- nucleic acid is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
- nucleotide may be natural or non-natural.
- gene refers to a factor that defines a genetic trait
- gene may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.
- homology of a gene refers to the degree of identity of two or more gene sequences to each other, and generally “having homology” means that the degree of identity or similarity is high. Say. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes have homology.
- a “homolog” or “homologous gene product” is a protein in another species, preferably a mammal, that performs the same biological function as the protein component of the complex further described herein. Means. Such homologues may also be referred to as “ortholog gene products”. It will be understood that such homologues, homologous gene products, orthologous gene products and the like can be used as long as they meet the objectives of the present invention.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one letter code.
- comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.
- the identity search can be performed, for example, using NCBI's BLAST 2.2.28 (issued 2013.4.2).
- the identity value usually refers to a value when the above BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value. Similarity is a numerical value calculated for similar amino acids in addition to identity.
- “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, and may be any value or less.
- Polypeptides that have undergone deletion, addition, insertion, or substitution with other amino acids of one or several amino acid residues are known to maintain their biological activity (Market al., Proc Natl Acad Sci USA.1984 Sep; 81 (18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science. 1984 Jun 29 ; 224 (4656): 1431-1433.).
- Antibodies with deletions and the like can be prepared by, for example, site-specific mutagenesis, random mutagenesis, or biopanning using an antibody phage library.
- site-specific mutagenesis method for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) Can be used. It is possible to select an antibody having the same activity as that of the wild type from mutant antibodies into which deletion or the like has been introduced by performing various characterizations such as FACS analysis and ELISA.
- “90% or more” may be, for example, 90, 95, 96, 97, 98, 99, or 100% or more, and is within the range of any two values thereof. Also good.
- the above-mentioned “homology” may be calculated according to a method known in the art, based on the ratio of the number of amino acids homologous in two or more amino acid sequences. Before calculating the ratio, the amino acid sequences of the group of amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence when necessary to maximize the ratio of the same amino acids.
- polynucleotide hybridizing under stringent conditions refers to well-known conditions commonly used in the art.
- a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe.
- hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then a 0.1 to 2-fold concentration was obtained.
- polynucleotide that can be identified by washing the filter under conditions of 65 ° C using SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate).
- SSC saline-sodium citrate
- composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate.
- stringent conditions for example, the following conditions can be adopted.
- a polypeptide used in the present invention is encoded by a nucleic acid molecule that hybridizes under highly or moderately stringent conditions to a nucleic acid molecule encoding a polypeptide specifically described in the present invention. Are also included.
- a “purified” substance or biological factor refers to a substance from which at least a part of the factor naturally associated with the substance or biological factor has been removed. .
- the purity of a biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.
- the term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent.
- the substance or biological agent used in the present invention is preferably a “purified” substance.
- an “isolated” substance or biological agent is substantially free of the factors that naturally accompany the substance or biological agent. Say things.
- the term “isolated” as used herein does not necessarily have to be expressed in purity, as it will vary depending on its purpose, but is preferably at least 75% by weight, more preferably if necessary. Means that there is at least 85%, more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological agent.
- the materials used in the present invention are preferably “isolated” materials or biological agents.
- a “corresponding” amino acid or nucleic acid or moiety refers to a predetermined amino acid or nucleotide in a polypeptide or polynucleotide as a reference for comparison in a polypeptide molecule or polynucleotide molecule (eg, Glypican-1).
- a complex molecule it refers to the corresponding part (eg, heparan sulfate).
- an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule.
- Corresponding amino acids for example, cysteinylation, glutathioneation, SS bond formation, oxidation (eg methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc.
- the corresponding amino acid can be an amino acid responsible for dimerization.
- Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain. Such corresponding regions or domains are useful when designing complex molecules in the present invention.
- a “corresponding” gene eg, a polynucleotide sequence or molecule
- a “corresponding” gene has, or is expected to have, in a species, the same effect as a given gene in a species to which comparison is made. It refers to a gene (for example, a polynucleotide sequence or a molecule), and when there are a plurality of genes having such an action, those having the same origin in evolution.
- a gene corresponding to a gene can be an ortholog of that gene.
- each human Glypican-1 can find the corresponding Glypican-1 in other animals (particularly mammals).
- Such corresponding genes can be identified using techniques well known in the art.
- a corresponding gene in a certain animal is a reference gene of the corresponding gene (for example, Glypican-1), and a sequence such as SEQ ID NO: 1 or SEQ ID NO: 2 is used as a query sequence. Can be found by searching a database containing the sequences of the animals.
- fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
- the length of the fragment can be appropriately changed according to the purpose.
- the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits obtain.
- examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
- Non-integer lengths may also be appropriate as a lower limit.
- such a fragment falls within the scope of the present invention as long as the full-length fragment functions as a marker or target molecule as long as the fragment itself also functions as a marker or target molecule. Is understood.
- the term “activity” refers herein to the function of a molecule in the broadest sense. Activity is not intended to be limiting, but generally includes the biological function, biochemical function, physical function or chemical function of a molecule. Activity activates, promotes, stabilizes, inhibits, suppresses, or destabilizes, for example, enzyme activity, the ability to interact with other molecules, and the function of other molecules Ability, stability, and ability to localize to specific intracellular locations. Where applicable, the term also relates to the function of the protein complex in the broadest sense.
- biological function refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto.
- the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance.
- functions of Glypican-1 involved in apoptosis of esophageal cancer cells, caspase-3 cleavage, AKT phosphorylation and the like can be mentioned, but not limited thereto.
- a biological function can be exerted by “biological activity”.
- biological activity refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). For example, an activity in which another molecule is activated or inactivated by interaction with one molecule. When two factors interact, the biological activity can be the binding between the two molecules and the resulting biological change, and for example, one molecule was precipitated using an antibody Sometimes, when other molecules co-precipitate, the two molecules are considered to be linked. Therefore, seeing such coprecipitation is one of the judgment methods. For example, when a factor is an enzyme, the biological activity includes the enzyme activity.
- an agent when an agent is a ligand, the ligand includes binding to the corresponding receptor.
- biological activity can be measured by techniques well known in the art.
- “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus),
- expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form.
- a gene, a polynucleotide or the like is transcribed and translated into a polypeptide form.
- transcription and production of mRNA are also an aspect of expression.
- expression product includes such a polypeptide or protein, or mRNA. More preferably, such polypeptide forms may be post-translationally processed.
- the expression level of Glypican-1 can be determined by any method.
- the expression level of Glypican-1 can be determined by evaluating the amount of mRNA of Glypican-1, the amount of Glypican-1 protein, and the biological activity of Glypican-1 protein. Such measurements can be used in companion diagnostics.
- the amount of mRNA or protein of Glypican-1 can be determined by a method as described in detail elsewhere in this specification or other methods known in the art.
- the “functional equivalent” refers to any object having the same target function but different structure from the target original entity. Accordingly, the functional equivalent of “Glypican-1” or its antibody is not Glypican-1 or its antibody itself, but Glypican-1 or its variant or variant (eg, amino acid sequence variant). Glypican-1 or its antibody has a biological action, and at the time of action, it changes to Glypican-1 or its antibody itself or a variant or variant of this Glypican-1 or its antibody (For example, including nucleic acids encoding Glypican-1 or its antibody itself or variants or variants of Glypican-1 or its antibodies, and vectors, cells, etc. containing the nucleic acids) Understood.
- a functional equivalent of Glypican-1 or an antibody thereof can be used in the same manner as Glypican-1 or an antibody thereof, even if not specifically mentioned.
- Functional equivalents can be found by searching a database or the like.
- search refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods.
- Electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad.
- Bio searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization. It is not limited. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.
- an amino acid sequence having one or more amino acid insertions, substitutions or deletions, or those added to one or both ends can be used.
- “insertion, substitution or deletion of one or a plurality of amino acids in the amino acid sequence, or addition to one or both ends thereof” is a well-known technical method such as site-directed mutagenesis. It means that a modification has been made by substitution of a plurality of amino acids to the extent that can occur naturally by a method or by natural mutation.
- the modified amino acid sequence has, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, further preferably 1 to 5, particularly preferably 1 to 2 amino acid insertions, substitutions or deletions. Lost or added to one or both ends thereof.
- the modified amino acid sequence is preferably an amino acid sequence having one or more (preferably 1 or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of Glypican-1 It may be.
- conservative substitution means substitution of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid.
- non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
- polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
- positively charged (basic) amino acids include arginine, histidine, and lysine.
- negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- inhibitor refers to a substance or factor that inhibits the biological action of a target entity (eg, receptor or cell).
- the inhibitor of Glypican-1 of the present invention is a factor that can temporarily or permanently reduce or eliminate the function of target Glypican-1 or cells expressing Glypican-1.
- factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, nucleic acids in the form of RNAi factors such as antisense and siRNA, and the like.
- agonist refers to a substance that expresses or enhances the biological action of a target entity (for example, a receptor).
- a target entity for example, a receptor
- agonists also called ligands
- synthesized ones and modified ones can be mentioned.
- antagonist refers to a substance that suppresses or inhibits the expression of a biological action of a target entity (for example, a receptor).
- a target entity for example, a receptor
- synthetic antagonists and modified ones can be mentioned.
- those that inhibit or inhibit competitively with agonists (or ligands) there are those that inhibit or inhibit non-competitively. It can also be obtained by modifying the agonist.
- An antagonist can be included in the concept of an inhibitor (inhibitor) or an inhibitory factor because it suppresses or inhibits a physiological phenomenon. Accordingly, herein, an antagonist is used interchangeably with “suppressor”.
- the “anti-Glypican-1 antibody” includes an antibody having binding ability to Glypican-1.
- the method for producing this anti-Glypican-1 antibody is not particularly limited, and for example, it may be produced by immunizing mammals or birds with Glypican-1.
- chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc (Fv) 2 (single chain (Fv) 2 ), scFv-Fc and the like are also included.
- the anti-Glypican-1 antibody according to an embodiment of the present invention is an anti-Glypican-1 antibody that specifically binds to a specific epitope of Glypican-1 from the viewpoint of particularly strongly suppressing the growth of malignant tumors. Is preferred.
- the anti-Glypican-1 antibody according to one embodiment of the present invention may be a monoclonal antibody. If it is a monoclonal antibody, it can be made to act on Glypican-1 more efficiently than a polyclonal antibody. From the viewpoint of efficiently producing an anti-Glypican-1 monoclonal antibody, it is preferable to immunize chickens with Glypican-1.
- the antibody class of the anti-Glypican-1 antibody according to an embodiment of the present invention is not particularly limited, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.
- the anti-Glypican-1 antibody according to an embodiment of the present invention may be an antibody fragment having an antigen binding activity (hereinafter also referred to as “antigen-binding fragment”).
- antigen-binding fragment an antibody fragment having an antigen binding activity
- there are effects such as an increase in stability or antibody production efficiency.
- the anti-Glypican-1 antibody according to one embodiment of the present invention may be a fusion protein.
- This fusion protein may be a polypeptide or oligopeptide bound to the N- or C-terminus of the anti-Glypican-1 antibody.
- the oligopeptide may be a His tag.
- the fusion protein may be a fusion of a mouse, human or chicken antibody partial sequence. Such fusion proteins are also included in one form of the anti-Glypican-1 antibody according to this embodiment.
- the anti-Glypican-1 antibody according to an embodiment of the present invention may be, for example, an antibody obtained through a step of immunizing an organism with purified Glypican-1, Glypican-1-expressing cells, or a Glypican-1-containing lipid membrane. Good. From the viewpoint of enhancing the therapeutic effect on Glypican-1-positive malignant tumors, it is preferable to use Glypican-1-expressing cells for immunization.
- An anti-Glypican-1 antibody according to an embodiment of the present invention is an antibody having a CDR set of an antibody obtained through a step of immunizing an organism with purified Glypican-1, Glypican-1-expressing cells or a Glypican-1-containing lipid membrane It may be. From the viewpoint of enhancing the therapeutic effect on Glypican-1-positive malignant tumors, it is preferable to use Glypican-1-expressing cells for immunization.
- a CDR set is a set of heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3.
- the “Glypican-1-expressing cell” may be obtained, for example, by expressing Glypican-1 after introducing a polynucleotide encoding Glypican-1 into the cell.
- Glypican-1 includes a Glypican-1 fragment.
- the “Glypican-1-containing lipid membrane” may be obtained, for example, by mixing Glypican-1 and a lipid bilayer membrane.
- Glypican-1 includes a Glypican-1 fragment.
- the anti-Glypican-1 antibody according to an embodiment of the present invention is an antibody obtained through a step of immunizing a chicken with an antigen, or a CDR set of the antibody, from the viewpoint of enhancing the therapeutic effect on a Glypican-1-positive malignant tumor.
- An antibody having is preferred.
- the anti-Glypican-1 antibody according to an embodiment of the present invention may have any binding force as long as the object is achieved, for example, at least 1.0 ⁇ 10 6 or more, 2.0 ⁇ 10 6 6 above, 5.0 ⁇ 10 6 or more, there may be mentioned a 1.0 ⁇ 10 7 or more without being limited to, usually, with a K D value (kd / ka) is, 1.0 ⁇ 10 -7 or less It may be 1.0 ⁇ 10 ⁇ 9 (M) or 1.0 ⁇ 10 ⁇ 10 (M).
- the anti-Glypican-1 antibody according to one embodiment of the present invention may have ADCC or CDC activity.
- the anti-Glypican-1 antibody according to an embodiment of the present invention may be an antibody that binds to a wild type or a mutant type of Glypican-1. Variants include those resulting from differences in DNA sequences between individuals.
- the amino acid sequence of wild-type or mutant Glypican-1 is preferably 80% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more with respect to the amino acid sequence shown in SEQ ID NO: 2. Have homology.
- antibody includes a molecule or population thereof that can specifically bind to a particular epitope on an antigen.
- the antibody may be a polyclonal antibody or a monoclonal antibody.
- the antibody can exist in various forms, for example, full-length antibody (antibody having Fab region and Fc region), Fv antibody, Fab antibody, F (ab ′) 2 antibody, Fab ′ antibody, diabody, single Chain (single chain) antibodies (eg, scFv), sc (Fv) 2 (single chain (Fv) 2 ), scFv-Fc, dsFv, multispecific antibodies (eg, oligo-specific antibodies, bivalent-specific antibodies) ), Diabody, peptide or polypeptide having antigen binding property, chimeric antibody (eg, mouse-human chimeric antibody, chicken-human chimeric antibody, etc.), mouse antibody, chicken antibody, humanized antibody, human antibody, or equivalents thereof
- the antibody includes an antibody modified product or an antibody unmodified product.
- an antibody and various molecules such as polyethylene glycol may be bound.
- the modified antibody can be obtained by chemically modifying the antibody using a known technique.
- such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
- the anti-Glypican-1 antibody used in the present invention may be bound to the Glypican-1 protein, and its origin, type, shape, etc. are not limited.
- known antibodies such as non-human animal antibodies (eg, mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used.
- monoclonal or polyclonal antibodies can be used as antibodies, but monoclonal antibodies are preferred.
- the binding of the antibody to the Glypican-1 protein is preferably specific binding.
- the antibody includes an antibody modified product or an antibody unmodified product.
- an antibody and various molecules such as polyethylene glycol may be bound.
- the modified antibody can be obtained by chemically modifying the antibody using a known technique.
- a “polyclonal antibody” refers to, for example, mammals (eg, rats, mice, rabbits, cows, monkeys, etc.), birds, etc. in order to induce the production of polyclonal antibodies specific to the antigen. It can be generated by administering an immunogen containing the antigen of interest. Administration of the immunogen may involve infusion of one or more immunizing agents and, if desired, an adjuvant.
- An adjuvant may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gel (such as aluminum hydroxide), or a surfactant (such as lysolecithin). .
- Immunization protocols are known in the art and may be performed by any method that elicits an immune response, depending on the host organism chosen (Protein Experiment Handbook, Yodosha (2003): 86-91). .).
- a “monoclonal antibody” is an antibody in which the individual antibodies constituting the population substantially correspond to a single epitope, except for antibodies having mutations that can naturally occur in small amounts. Including the case of Alternatively, the individual antibodies that make up the population may be antibodies that are substantially identical except for antibodies that have mutations that can occur naturally in small amounts. Monoclonal antibodies are highly specific and differ from normal polyclonal antibodies, which typically include different antibodies corresponding to different epitopes. In addition to its specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins.
- the form “monoclonal” may be characterized as being derived from a substantially homogeneous population of antibodies, but does not mean that the antibodies must be produced in any particular way.
- the monoclonal antibody may be prepared by a method similar to the hybridoma method described in “KohlerG,“ Milstein® C., “Nature.” 1975 Aug. 7; 256 (5517): 495-497. ”.
- monoclonal antibodies may be made by methods similar to recombinant methods such as those described in US Pat. No. 4,816,567.
- the monoclonal antibody can be identified as "Clacksonet.al., Nature.
- any technique known in the art can be used.
- the construction of a typical mass production system of antibodies and the production of antibodies can be exemplified as follows. That is, CHO cells are transfected with an H chain antibody expression vector and an L chain antibody expression vector, cultured using selection reagents G418 and Zeocin, and cloned by limiting dilution. After cloning, clones that stably express the antibody are selected by ELISA. The selected CHO cells are expanded and cultured, and the culture supernatant containing the antibody is collected. Antibodies can be purified from the collected culture supernatant by Protein A or Protein G purification.
- Fv antibody is an antibody containing an antigen recognition site. This region contains a dimer of one heavy chain variable domain and one light chain variable domain by non-covalent bonds. In this configuration, the three CDRs of each variable domain can interact to form an antigen binding site on the surface of the VH-VL dimer.
- the “Fab antibody” refers to, for example, about half of the N-terminal side of the H chain and the entire L chain, among fragments obtained by treating an antibody containing the Fab region and the Fc region with the proteolytic enzyme papain. Is an antibody bound through some disulfide bonds.
- Fab can be obtained, for example, by treating an anti-Glypican-1 antibody according to an embodiment of the present invention containing a Fab region and an Fc region with the proteolytic enzyme papain.
- ⁇ F (ab ′) 2 antibody '' refers to, for example, 2 sites corresponding to Fab in a fragment obtained by treating an antibody containing Fab region and Fc region with proteolytic enzyme pepsin.
- F (ab ′) 2 can be obtained, for example, by treating an anti-Glypican-1 antibody according to an embodiment of the present invention containing a Fab region and an Fc region with a proteolytic enzyme pepsin.
- it can be prepared by linking the following Fab ′ with a thioether bond or a disulfide bond.
- Fab ′ antibody is, for example, an antibody obtained by cleaving a disulfide bond in the hinge region of F (ab ′) 2 .
- F (ab ′) 2 can be obtained by treating with a reducing agent dithiothreitol.
- the “scFv antibody” is an antibody in which VH and VL are linked via an appropriate peptide linker.
- the scFv antibody obtains cDNA encoding VH and VL of the anti-Glypican-1 antibody according to the embodiment of the present invention, constructs a polynucleotide encoding VH-peptide linker-VL, and uses the polynucleotide as a vector. And can be produced using cells for expression.
- a “diabody” is an antibody having a bivalent antigen binding activity.
- the bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
- diabody constructs a polynucleotide encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less, incorporates the obtained polynucleotide into a vector, and produces it using expression cells. it can.
- “dsFv” is an antibody in which a polypeptide in which a cysteine residue is introduced into VH and VL is bound via a disulfide bond between the cysteine residues.
- the position to be introduced into the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Reiteretal., Protein Eng. 1994 May; 7 (5): 697-704.) .
- an “antigen-binding peptide or polypeptide” is an antibody comprising antibody VH, VL, or CDR1, 2, or 3 thereof. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
- Fv antibody Fab antibody, F (ab ′) 2 antibody, Fab ′ antibody, scFv antibody, diabody, dsFv antibody, peptide or polypeptide having antigen binding properties (hereinafter also referred to as “Fv antibody etc.”)
- Production method is not particularly limited.
- DNA encoding a region such as an Fv antibody in the anti-Glypican-1 antibody according to the embodiment of the present invention can be incorporated into an expression vector and produced using an expression cell. Alternatively, it may be produced by a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBOC method (t-butyloxycarbonyl method).
- the antigen-binding fragment according to an embodiment of the present invention may be one or more of the above Fv antibodies.
- a “chimeric antibody” is, for example, a variable region of an antibody between heterologous organisms and a constant region of the antibody, and can be constructed by a gene recombination technique.
- a mouse-human chimeric antibody can be prepared, for example, by the method described in "Roguskaet al., Proc Natl Acad Sci U S A. 1994Feb 1; 91 (3): 969-973.”
- Basic methods for making mouse-human chimeric antibodies include, for example, encoding the mouse leader and variable region sequences present in the cloned cDNA, and the human antibody constant regions already present in mammalian cell expression vectors.
- the mouse leader sequence and variable region sequence present in the cloned cDNA may be linked to a sequence encoding a human antibody constant region and then linked to a mammalian cell expression vector.
- the fragment of the human antibody constant region can be of any human antibody H chain constant region and human antibody L chain constant region, for example, for human H chain, C ⁇ 1, C ⁇ 2, C ⁇ 3 or C ⁇ 4, For the L chain, C ⁇ or C ⁇ can be mentioned, respectively.
- a “humanized antibody” has, for example, one or more CDRs derived from a non-human species, a framework region (FR) derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin.
- Antibody humanization can be performed using various techniques known in the art (Almagro et al., FRont Biosci. 2008 2008 Jan 1; 13: 1619-1633.).
- FR residues important for antigen binding may be identified by modeling the interaction of CDR and FR residues.
- an unusual FR residue at a particular position may be identified by sequence comparison.
- a “human antibody” is derived from a gene encoding human immunoglobulin, for example, the variable region and the constant region of the heavy chain and the region including the variable region and the constant region of the light chain that constitute the antibody.
- the main production methods include a transgenic mouse method for producing human antibodies, a phage display method, and the like.
- human antibodies having various antigen-binding abilities can be produced instead of mouse antibodies.
- a human monoclonal antibody can be obtained by a conventional hybridoma method.
- phage display method a foreign gene is fused to the N-terminal side of the coat protein (g3p, g10p, etc.) of filamentous phages such as M13 and T7, which are typically E. coli viruses. It is a system for expressing as a protein. For example, it can be prepared by the method described in “Vaughanet al., Nat Biotechnol. 1996 Mar; 14 (3): 309-314.”.
- the antibody can be added to any antibody by CDR-grafting (Ozaki et al., Blood. 1999 Jun 1; 93 (11): 3922-3930.)
- CDR-grafting To the heavy chain CDR of the anti-Glypican-1 antibody according to the embodiment of the present invention.
- it may be prepared by grafting light chain CDRs.
- DNA encoding the heavy chain light chain CDR or light chain CDR of the anti-Glypican-1 antibody according to the embodiment of the present invention and the heavy chain light chain CDR or light chain CDR of a known human or non-human organism-derived antibody are excluded.
- a region-encoding DNA can be obtained by ligation to a vector according to a method known in the art, and then expression using a known cell.
- the region excluding the heavy chain CDR or the light chain CDR may be optimized using a phage display method or the like.
- FR shuffle Damschroderetal., Mol Immunol. 2007 Apr; 44 (11): 3049-3060.
- the “heavy chain” is typically the main component of a full-length antibody.
- the heavy chain is usually linked to the light chain by disulfide bonds and non-covalent bonds.
- the domain on the N-terminal side of the heavy chain has a region called the variable region (VH) where the amino acid sequence is not constant even with antibodies of the same type and the same class.
- VH variable region
- CDR complementarity determining region
- Fv variable region: including heavy chain variable region (VH) and light chain variable region (VL)
- CDR1, CDR2, and CDR3 consisting of about 5 to 30 amino acid residues.
- CDR3 is known to have the highest contribution in binding of an antibody to an antigen.
- Kabat's definition (Sequences of Proteins ofImmunological Interest, 5th ed., Public HealthService, National Institutes ofHealth, Bethesda, MD. (1991)) or Chothia's definition (Chothiaet al., J. Mol.Biol., 1987; 196: 901-917) may be adopted.
- the definition of Kabat is adopted as a preferred example, but is not necessarily limited thereto. In some cases, it may be determined in consideration of both the Kabat definition and the Chothia definition. It can also be a CDR.
- antigen refers to any substrate that can be specifically bound by an antibody molecule.
- immunogen refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response.
- epitope or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well known techniques once the primary sequence of the nucleic acid or amino acid is provided. It will be understood that the antibodies of the present invention can be used in the same manner even if they have the same epitope, even antibodies having other sequences.
- the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
- “means” refers to any tool that can achieve a certain purpose (for example, detection, diagnosis, treatment).
- a certain purpose for example, detection, diagnosis, treatment.
- “means for selectively recognizing” A means by which one object can be recognized differently from another.
- a “marker (substance or gene)” indicates whether or not a certain state (for example, disease state, disordered state, level of malignant state, presence or absence, etc.) is at risk.
- a certain state for example, disease state, disordered state, level of malignant state, presence or absence, etc.
- markers can include genes, gene products, metabolites, enzymes, and the like.
- detection, diagnosis, preliminary detection, prediction or pre-diagnosis for a certain state is an agent, agent, factor or means specific for the marker associated with the state, Or it can implement
- expression product also referred to as gene product refers to a protein or mRNA encoded by a gene.
- a gene product (Glypican-1) that has not been shown to be associated with esophageal cancer can be used as an indicator of esophageal cancer.
- Esophageal cancer is used in a normal sense, and in a broad sense including cancer in the esophagus.
- Esophageal cancer includes, but is not limited to, squamous cell carcinoma, adenocarcinoma, lymph node metastasis, and the like. It is understood that about half of Japanese esophageal cancers start near the center of the esophagus in the chest, and then 1/4 occur in the lower part of the esophagus. .
- the present invention can be used as an index of esophageal cancer as a whole, including those of squamous cell carcinoma, adenocarcinoma, and lymph node metastasis.
- the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (for example, an organism such as a human or a cell, blood, serum, etc. removed from the organism). .
- sample refers to any substance obtained from a subject or the like, and includes, for example, serum. Those skilled in the art can appropriately select a preferable sample based on the description of the present specification.
- drug drug
- drug may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity).
- Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto.
- a factor specific for a polynucleotide is a polynucleotide having complementarity with a certain sequence homology to the sequence of the polynucleotide (eg, 70% or more sequence identity), Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region.
- Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor.
- specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.
- diagnosis identifies various parameters related to a disease, disorder, condition (eg, esophageal cancer), etc. in a subject, and determines the current state or future of such a disease, disorder, or condition. To do.
- conditions within the body can be examined, and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject.
- various parameters such as methods can be selected.
- diagnosis in a narrow sense means diagnosis of the current state, but in a broad sense includes “early diagnosis”, “predictive diagnosis”, “preliminary diagnosis”, and the like.
- the diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method of the present invention can be used from the body and can be performed away from the hands of medical personnel such as doctors.
- diagnosis, prior diagnosis or diagnosis may be referred to as “support”.
- detection agent or “test agent (agent)” refers to any agent that can detect or inspect a target object in a broad sense.
- diagnostic agent refers to any agent that can diagnose a target condition (for example, a disease such as esophageal cancer) in a broad sense.
- treatment refers to prevention of worsening of a disease or disorder when it becomes such a condition or disease (eg, esophageal cancer), preferably maintaining the status quo, More preferably, it means alleviation, more preferably elimination, and includes the ability to exert a symptom improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.
- the term “therapeutic agent (agent)” refers to any agent that can treat a target condition (for example, diseases such as esophageal cancer).
- the “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
- the pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing the active ingredient and the carrier.
- the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient.
- the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution).
- the therapeutic agent for esophageal cancer includes a drug (prophylactic agent) used for the prevention of esophageal cancer or an esophageal cancer cell growth inhibitor.
- prevention refers to preventing a certain disease or disorder (for example, esophageal cancer) from entering such a state before it enters such a state. Diagnosis can be performed using the drug of the present invention, and for example, esophageal cancer can be prevented using the drug of the present invention as necessary, or countermeasures for prevention can be taken.
- a certain disease or disorder for example, esophageal cancer
- prophylactic agent refers to any agent that can prevent a target condition (for example, diseases such as esophageal cancer) in a broad sense.
- interaction refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state. The detection, inspection and diagnosis of the present invention can be realized by utilizing such interaction.
- bond means a physical or chemical interaction between two substances or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like.
- a physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.
- a “factor” (or drug, detection agent, etc.) that interacts (or binds) “specifically” to a biological agent such as a polynucleotide or a polypeptide is defined as that
- the affinity for a biological agent such as a nucleotide or polypeptide thereof is typically equal or greater than the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. Includes those that are high or preferably significantly (eg, statistically significant). Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.
- a first substance or factor interacts (or binds) “specifically” to a second substance or factor means that the first substance or factor has a relationship to the second substance or factor. Interact (or bind) with a higher affinity than a substance or factor other than the second substance or factor (especially other substances or factors present in the sample containing the second substance or factor) That means.
- Specific interactions (or bindings) for substances or factors include, for example, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, etc., nucleic acid and protein reactions, protein-lipid interactions, nucleic acid-lipids Examples include, but are not limited to, interactions.
- the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor.
- both substances or factors are proteins
- the fact that the first substance or factor interacts (or binds) “specifically” to the second substance or factor is, for example, by antigen-antibody reaction Examples include, but are not limited to, interaction by receptor-ligand reaction, enzyme-substrate interaction, and the like.
- the first substance or factor interacts (or binds) “specifically” to the second substance or factor by means of an antibody and its antigen Interaction (or binding) between is included.
- an object in a sample can be detected or quantified.
- detection or “quantification” of polynucleotide or polypeptide expression includes mRNA measurement and immunoassay methods, including, for example, binding or interaction with a detection agent, test agent or diagnostic agent. It can be achieved using any suitable method. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR.
- an immunological measurement method for example, an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a luminescence immunoassay (LIA), an immunoprecipitation method (IP), an immunodiffusion method (SRID), an immune method
- LIA luminescence immunoassay
- IP immunoprecipitation method
- SRID immunodiffusion method
- an immune method examples are turbidimetry (TIA), Western blotting, immunohistochemical staining, and the like.
- the quantification method include ELISA method and RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array).
- the DNA array has been extensively outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and the latest PCR method”). For protein arrays, see NatGenet.
- gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like.
- RT-PCR RT-PCR
- RACE method RACE method
- SSCP method immunoprecipitation method
- two-hybrid system in vitro translation and the like.
- further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Laboratory Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.
- expression level refers to the amount of polypeptide or mRNA that is expressed in a target cell, tissue, or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA, RIA, fluorescent antibody, Western blot, and immunohistochemical staining using the antibody of the present invention.
- the expression level of the peptide at the mRNA level is mentioned.
- “Change in expression level” means expression at the protein level or mRNA level of the polypeptide used in the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Means that the amount increases or decreases. By measuring the expression level of a certain marker, various detection or diagnosis based on the marker can be performed.
- “reduction” or “suppression” or synonyms for activity, expression products (eg, proteins, transcripts (RNA, etc.)) or synonyms are reductions in the quantity, quality or effect of a particular activity, transcript or protein. Or activity to decrease. When “disappears” of the decrease, it means that the activity, the expression product, etc. are below the detection limit, and in particular, may be “disappear”. As used herein, “disappearance” is encompassed by “decrease” or “suppression”.
- “increase” or “activation” of an activity, expression product eg, protein, transcript (RNA, etc.) or a synonym thereof refers to a quantity, quality or effect of a particular activity, transcript or protein. An activity that increases or increases.
- nucleic acid primer refers to a substance necessary for the initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction.
- a nucleic acid molecule for example, DNA or RNA
- the primer can be used as a marker detection means.
- the term “probe” refers to a substance that serves as a search means used in biological experiments such as screening in vitro and / or in vivo.
- a nucleic acid molecule containing a specific base sequence or a specific Examples include, but are not limited to, peptides containing amino acid sequences, specific antibodies or fragments thereof.
- the probe is used as a means for marker detection, inspection or diagnosis.
- the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others.
- a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like.
- the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more.
- Alexa TM Fluor is desirable as a fluorescent substance.
- Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series corresponding to a wide range of fluorescent wavelengths. It is stable, bright and has low pH sensitivity.
- Examples of combinations of fluorescent dyes having a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa TM 555 and Alexa TM 633, a combination of Alexa TM 488 and Alexa TM 555, and the like.
- any nucleic acid can be used as long as it can bind to the base moiety, but cyanine dyes (eg, CyDye TM series Cy3, Cy5, etc.), rhodamine 6G reagent, 2-acetylaminofluorene ( AAF), AAIF (iodine derivative of AAF) and the like are preferably used.
- the fluorescent substance having a difference in maximum fluorescence emission wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like.
- the target object by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.
- a “tag” is a substance for sorting molecules by a specific recognition mechanism such as a receptor-ligand, more specifically, a binding for binding a specific substance.
- a substance that plays the role of a partner eg, having a relationship such as biotin-avidin, biotin-streptavidin
- label can be included in the category of “label”.
- a specific substance to which a tag is bound can be selected by bringing the substrate to which the binding partner of the tag sequence is bound into contact.
- tags or labels are well known in the art.
- Representative tag sequences include, but are not limited to, myc tag, His tag, HA, Avi tag and the like.
- Such a tag may be bound to the marker of the present invention, a marker detection agent, a test agent, or a diagnostic agent (which may be a primer or a probe).
- in vivo refers to the inside of a living body. In a particular context, “in vivo” refers to the location where a target substance is to be placed.
- in vitro refers to a state in which a part of a living body is removed or released “outside the living body” (for example, in a test tube) for various research purposes. A term that contrasts with in vivo.
- ex vivo refers to a series of operations ex vivo when a certain treatment is performed outside the body but is then intended to be returned to the body. Also in the present invention, an embodiment in which cells in the living body are treated with the drug of the present invention and returned to the patient can be envisaged.
- the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, instructions, etc.) usually divided into two or more compartments.
- a portion to be provided eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, instructions, etc.
- This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use.
- Such kits preferably include instructions or instructions that describe how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how the reagents should be processed).
- the kit when the kit is used as a reagent kit, the kit usually contains instructions including usage of test agents, diagnostic agents, therapeutic agents, antibodies, etc. Is included.
- the “instruction sheet” describes the method for using the present invention for a doctor or other user.
- This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given.
- the instructions may include a word indicating that the administration site is oral or esophageal administration (for example, by injection).
- This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) It is clearly stated that it has been received.
- the instruction sheet is a so-called package insert and is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage or an e-mail provided on the Internet). But it can be provided.
- the present invention provides a marker for identifying esophageal cancer comprising Glypican-1 or an expression product thereof or a fragment or derivative thereof.
- Glypican-1 is present in the living body, and its expression is low in healthy individuals or samples derived from it, and it is remarkably high in esophageal cancer, so it can be used as an effective marker for esophageal cancer.
- a marker for identifying esophageal cancer comprising Glypican-1 or an expression product thereof or a fragment or derivative thereof.
- Glypican-1 expression was found to be an index of esophageal cancer. Therefore, according to the present invention, esophageal cancer can be detected or selected by detecting the expression of Glypican-1 in a subject subject or a sample derived therefrom (for example, serum). Therefore, it is understood that a drug for treating esophageal cancer can be detected and screened using the ability of the marker of the present invention to reduce, suppress, increase or activate as an index.
- the present invention provides a detection agent, test agent or diagnostic agent for identifying esophageal cancer, which contains a substance that binds or interacts with Glypican-1.
- the substance binding is preferably specific.
- any substance can be used as the detection agent, test agent, or diagnostic agent as long as it can bind to or interact with Glypican-1, but as a representative example thereof, for example, Examples include factors antibodies or fragments or functional equivalents thereof, or nucleic acids that encode these factors, particularly nucleic acid primers that can amplify Glypican-1, or probes that can bind to or interact with Glypican-1. It is not limited to them.
- the detection agent, test agent or diagnostic agent of the present invention can be used as a detection kit, test kit or diagnostic kit.
- the esophageal cancer targeted by the present invention includes those of lymph node metastasis, squamous cell carcinoma and / or adenocarcinoma, particularly including but not limited to squamous cell carcinoma.
- the esophageal cancer targeted by the present invention is Glypican-1 positive cancer.
- the esophageal cancer targeted by the present invention is human cancer.
- the detection agent, test agent or diagnostic agent of the present invention is a complex in which another substance (for example, a label or the like) is bound to a moiety (for example, an antibody or the like) that enables detection, inspection or diagnosis.
- a moiety for example, an antibody or the like
- it may be a complex molecule.
- “complex” or “complex molecule” means any construct comprising two or more moieties.
- the other part may be a polypeptide or other substance (eg, sugar, lipid, nucleic acid, other hydrocarbon, etc.).
- two or more parts constituting the complex may be covalently bonded, and bonded by other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). May be.
- bonds for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.
- the “complex” includes a molecule formed by linking a plurality of molecules such as a polypeptide, a polynucleotide, a lipid, a sugar, and a small molecule.
- the detection agent, test agent or diagnostic agent of the present invention can take the form of probes and primers.
- the probes and primers of the present invention can specifically hybridize with Glypican-1.
- Glypican-1 expression is an indicator of esophageal cancer and is useful as an indicator.
- the probes and primers according to the present invention can be used to identify esophageal cancer.
- the probe and primer of the present invention need only be capable of detecting the expression of Glypican-1, and are a polymer comprising a plurality of bases or base pairs such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Point to.
- double-stranded cDNA can also be used in tissue in situ hybridization, and the probes and primers of the present invention include such double-stranded cDNA.
- an RNA probe riboprobe
- the present invention can take the form of a primer.
- nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene (eg, SEQ ID NO: 1).
- Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides of at least 9 contiguous nucleotides, more preferably of at least 10 contiguous nucleotides, more preferably of at least 11 contiguous nucleotides.
- Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above Is included.
- a sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).
- the primer according to the present invention can also be used as a primer set composed of two or more of the primers.
- the primer and primer set according to the present invention are known to detect a target gene using a nucleic acid amplification method such as a PCR method, an RT-PCR method, a real-time PCR method, an in situ PCR method, or a LAMP method. In the method, it can utilize as a primer and a primer set according to a conventional method.
- the primer set according to the present invention can be selected so that the nucleotide sequence of a target protein such as Glypican-1 can be amplified by a nucleic acid amplification method such as PCR.
- Nucleic acid amplification methods are well known, and selection of primer pairs in nucleic acid amplification methods is obvious to those skilled in the art.
- one of two primers (primer pair) is paired with the plus strand of the double-stranded DNA of the target protein such as Glypican-1, and the other primer is paired with the minus strand of the double-stranded DNA.
- the primer can be selected so that the other primer is paired with the extended strand extended by one primer.
- the primer of the present invention can be chemically synthesized based on the nucleotide sequence disclosed herein. Preparation of the primer is well known, for example, according to "Molecular Cloning, A Laboratory Manual 2 nd ed.” (Cold Spring Harbor Press (1989)), “Current Protocolsin Molecular Biology” (John Wiley & Sons (1987-1997)) Can be implemented.
- the present invention may take the form of a “probe”.
- nucleic acid molecules that are usually used as probes include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is homologous or complementary to the nucleic acid sequence of the target gene (eg, SEQ ID NO: 1).
- Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides of at least 9 contiguous nucleotides, more preferably of at least 10 contiguous nucleotides, more preferably of at least 11 contiguous nucleotides.
- Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above Is included.
- the detection agent of the present invention may be labeled.
- the detection agent, test agent or diagnostic agent of the present invention may have a tag attached thereto.
- the label or tag used in the present invention can take any of the forms described herein.
- the present invention provides a method for using Glypican-1 as an index for identifying esophageal cancer, or a method for detecting, examining or diagnosing esophageal cancer.
- a step of detecting an expression product of Glypican-1 for example, a protein or mRNA in vivo.
- a detection agent, a test agent or a diagnostic agent containing a substance that binds to an expression product of Glypican-1, such as a protein or mRNA can be used.
- Such a detection agent, test agent or diagnostic agent is described in the present specification, and a person skilled in the art performs the method of the present invention using a technique known in the art as necessary based on the description. It is understood that you can.
- the detection agent, test agent or diagnostic agent of the present invention is brought into contact with a target sample, and there is an expression product of the target target Glypican-1, for example, a protein or mRNA, in the sample Whether or not its level or amount.
- Glypican-1 for example, a protein or mRNA
- contact means that a plurality of substances are arranged so as to cause interaction or binding between the plurality of substances.
- a polypeptide or polynucleotide can be achieved either in direct or indirect physical proximity to a marker of the invention or a sample containing it.
- the polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like.
- Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.
- a specific method for detecting an expression product of Glypican-1, such as protein or mRNA is particularly limited as long as it is a method capable of detecting an expression product of Glypican-1 in a sample (eg, serum, etc.), such as protein or mRNA.
- a sample eg, serum, etc.
- a hybridization method, a nucleic acid amplification method, and an antigen-antibody reaction method can be mentioned.
- the sample used may be any sample that is considered to contain an expression product.
- serum can be used.
- Serum can be obtained by a conventional method.
- the detection, test or diagnosis according to the invention comprises the hybridization of a probe according to the invention with a nucleic acid sample (such as mRNA or complementary DNA (cDNA) transcribed therefrom), ie a hybridization complex, Glypican-1 expression in a cell sample can be detected by detecting the nucleotide duplex directly or indirectly.
- a nucleic acid sample such as mRNA or complementary DNA (cDNA) transcribed therefrom
- cDNA complementary DNA
- Detection of an expression product of Glypican-1 using a hybridization method is performed by, for example, (a) contacting a polynucleotide derived from a test sample with a probe according to the present invention; and (b) hybridization complex It can be carried out by a process of detecting a body.
- step (a) mRNA prepared from the target test sample or complementary DNA (cDNA) transcribed from the mRNA can be brought into contact with the probe as a polynucleotide derived from the test cell sample.
- the probe can be labeled and used.
- radioactivity for example, 32 P, 14 C, and 35 S
- fluorescence for example, FITC, europium
- enzymatic reaction such as chemical coloring (for example, peroxidase, alkaline phosphatase), etc.
- chemical coloring for example, peroxidase, alkaline phosphatase
- a label may be mentioned.
- the detection of the hybridization product can be performed using a well-known method such as Northern hybridization, Southern hybridization, colony hybridization and the like. Since the sample in which the hybridization complex is detected indicates that the tissue of the subject expresses Glypican-1, it can be determined that the subject from which the sample is derived has a high possibility of esophageal cancer. .
- a nucleic acid sample (mRNA or a transcription product thereof) is amplified by a nucleic acid amplification method using the primer or primer set according to the present invention, and the amplified product is detected.
- the expression of Glypican-1 in the sample can be detected, examined or diagnosed using this.
- Detection of expression of Glypican-1 using the nucleic acid amplification method includes, for example, (i) a step of performing a nucleic acid amplification method using a polynucleotide derived from a test sample as a template and a primer or primer set according to the present invention; and ( ii) It can be carried out by detecting the formed amplification product.
- step (i) mRNA prepared from the target test sample or complementary DNA (cDNA) transcribed from the mRNA can be used as a template.
- Detection of the amplification product can be carried out using nucleic acid amplification methods such as PCR, RT-PCR, real-time PCR, and LAMP.
- the detection of the amplification product in this sample indicates that the subject's tissue expresses Glypican-1, so that the subject from which the sample is derived is determined to have a high possibility of esophageal cancer Can do.
- the expression of Glypican-1 in the sample is detected, examined or diagnosed by contacting the antibody according to the present invention with the sample and detecting the antigen-antibody reaction. be able to.
- Detection of Glypican-1 expression using antigen-antibody reaction includes, for example, (I) a step of contacting a protein derived from a test cell sample with an antibody according to the present invention; and (II) a step of measuring an antigen-antibody complex.
- Methods for detecting an antigen-antibody reaction are well known to those skilled in the art.
- Glypican-1 in serum can be detected by an immunological method. Immunological methods include cell tissue samples that have been appropriately treated, such as cell separation and extraction procedures, immunohistochemical staining, enzyme immunoassay, western blotting, agglutination, competition, etc. A known method such as a method or a sandwich method can be applied.
- the immunohistochemical staining method can be performed by, for example, a direct method using a labeled antibody, an indirect method using a labeled antibody against the antibody, or the like.
- the labeling agent known labeling substances such as fluorescent substances, radioactive substances, enzymes, metals, and dyes can be used. Since the sample in which the antigen-antibody complex is detected contains cells expressing Glypican-1, it can be determined that the subject from which the sample is derived has a high possibility of esophageal cancer.
- Each detection step described above can be performed not only once, but also by repeating or combining the steps to improve the accuracy of esophageal cancer diagnosis. Therefore, when such an embodiment is adopted, according to the detection, inspection, or diagnosis method of the present invention, esophageal cancer can be diagnosed with higher accuracy by performing the above steps twice or more. .
- marker genes preferably growth marker genes other than Glypican-1 (eg, known markers such as SCC or CEA) or these factors are used in combination to improve the accuracy of esophageal cancer diagnosis. be able to.
- the preparation procedure of the diagnostic agent and the like of the present invention as a medicine is known in the art, and is described in, for example, the Japanese Pharmacopeia, the US Pharmacopeia, and the pharmacopoeia of other countries. Accordingly, those skilled in the art can determine the embodiment, such as the amount to be used, without undue experimentation as described herein.
- the concentration of the marker can be measured by mass spectrometry.
- mass spectrometry either matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization (ESI) can be applied as the ionization method, but MALDI produces less multivalent ions. Is preferred.
- MALDI-TOF-MS combined with a time-of-flight massspectromer (TOF) can measure the marker concentration more accurately.
- TOF time-of-flight massspectromer
- the test material When measuring the concentration of the marker by electrophoresis, for example, the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target marker, and the gel is stained with an appropriate dye or fluorescent substance. What is necessary is just to measure the density and fluorescence intensity of the band corresponding to the target marker. If separation of markers is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can be used. Furthermore, instead of detecting directly from the gel, Western blotting can be performed to measure the amount of marker on the membrane.
- SDS-PAGE SDS-polyacrylamide gel electrophoresis
- IEF isoelectric focusing
- a method by liquid high performance chromatography can be used. That is, the concentration of the marker in the sample can be measured by subjecting the sample to HPLC to separate the target marker and measuring the peak area of the chromatogram.
- the present invention provides the following antibodies: (a) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 65 of SEQ ID NO: 3, respectively.
- An antibody comprising an amino acid sequence shown at positions 98-114, 163-170, 187-193, and 226-235, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3; An antibody comprising an amino acid sequence represented by positions 31 to 35, 50 to 66, 99 to 110, 159 to 166, 183 to 187, and 222 to 231 of SEQ ID NO: 4, respectively,
- (c) heavy chain CDR1 , 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, 99 to 113, 162 to 170, 187 to 193, and 226 to 237 of SEQ ID NO: 5, respectively.
- (D) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 6, positions 31-35, 51-66, 99-112,
- (e) heavy chain CDR1 2, 3, light chain CDR1, 2, and 3 are at positions 31 to 35, 50 to 66, 99 to 120, 169 to 176, 193 to 199, and 232 to 242 of SEQ ID NO: 7, respectively.
- An antibody comprising the amino acid sequence shown, (f) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 8, positions 31-35, 50-65, 98-114, 163, respectively.
- Antibodies comprising amino acid sequences shown at positions -170, 187-193, and 226-235, (g) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are each represented by SEQ ID NO: 31 Antibodies comprising amino acid sequences shown at positions -35, 50-66, 99-120, 169-176, 193-199, and 232-241, (h) heavy chain CDR1, 2, 3, light The strands CDR1, 2, and 3 represent the amino acid sequences represented by positions 31 to 35, 50 to 65, 98 to 113, 162 to 171, 188 to 194, and 227 to 237 of SEQ ID NO: 10, respectively.
- An antibody comprising (i) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 respectively Antibodies comprising amino acid sequences shown at positions -35, 50-65, 98-114, 163-170, 187-193, and 226-235, (j) heavy chain CDR1, 2, 3, light
- the chain CDR1, 2, and 3 represent the amino acid sequences represented by positions 31 to 35, 50 to 66, 99 to 113, 162 to 169, 186 to 192, and 225 to 234 of SEQ ID NO: 12, respectively.
- the antibody comprising, (k) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-116, 165-174, 191 of SEQ ID NO: 13, respectively.
- an antibody comprising the amino acid sequence shown at positions 226 to 236, (n) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35 and positions 50 to 66 of SEQ ID NO: 16, respectively.
- (p) heavy chain CDR1 , 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 18, positions 31-35, 50-66, 99-116, 165-174, 191-197, and 230-240, respectively.
- (Q) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 19, positions 31 to 35, positions 50 to 66, and positions 99 to 115, respectively. Includes amino acid sequences shown at positions 164-175, 193-199, and 232-241
- the antibody, (r) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 20, positions 31-35, 50-66, 99-117, 166-177, 194- An antibody comprising the amino acid sequence shown at positions 200 and 233 to 242;
- (s) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 32 to 36, 51 to 51 of SEQ ID NO: 21, respectively;
- These antibodies may have, as epitopes, positions 33 to 61 of SEQ ID NO: 2, or positions 339 to 358 and / or positions 388 to 421, or positions 430 to 530.
- These antibodies are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc (Fv ) 2 (single chain (Fv) 2 ), and scFv-Fc. These antibodies can be used in any application described herein.
- the present invention relates to an antibody having an epitope at positions 33 to 61, or positions 339 to 358 and / or positions 388 to 421, or positions 430 to 530 of SEQ ID NO: 2, or a fragment or functional equivalent thereof Offer things.
- These antibodies are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc (Fv ) 2 (single chain (Fv) 2 ), and scFv-Fc. These antibodies can be used in any application described herein.
- kits In one aspect, the present invention provides a kit for detection, testing and / or diagnosis for performing the method for detection, testing and / or diagnosis according to the present invention.
- This kit contains the detection agent, test agent and / or diagnostic agent of the present invention.
- any embodiment described in this specification can be used alone or in combination.
- the detection kit according to the present invention includes a detection kit for carrying out the detection according to the embodiment of the present invention, specifically, a kit for detecting the expression of Glypican-1. And a kit comprising at least the probe according to the present invention. This probe may be labeled.
- This detection kit detects the expression of Glypican-1 by a hybridization method.
- the detection method of the first aspect optionally further comprises various reagents for carrying out the hybridization method, such as substrate compounds, hybridization buffers, instructions, and / or instruments used for detection of the label. Can be included.
- the detection kit of this embodiment according to the present invention is a probe, primer, primer that can detect the expression of an esophageal cancer marker gene other than Glypican-1 (eg, SCC, CEA, etc.) in order to perform highly accurate detection.
- a set or antibody may further be included.
- These probes, primers, primer sets, or antibodies may be labeled.
- This detection kit further detects the expression of marker genes of esophageal cancer other than Glypican-1 by any one of hybridization, nucleic acid amplification, and antigen-antibody reaction.
- the detection kit according to the present invention includes a detection kit for performing the detection of another embodiment according to the present invention, specifically, for detecting the expression of Glypican-1.
- Kits comprising at least a primer according to the present invention or a primer set according to the present invention.
- This detection kit detects the expression of Glypican-1 by a nucleic acid amplification method. Therefore, the detection method of the second aspect includes various reagents for carrying out the nucleic acid amplification method, for example, a buffer, an internal standard indicating that PCR can proceed normally, instructions, and / or instruments, if desired. Can further be included.
- the detection kit of this embodiment according to the present invention further comprises a probe, primer, primer set, or antibody capable of detecting the expression of an esophageal cancer marker gene other than Glypican-1 in order to perform highly accurate detection. May be. These probes, primers, primer sets, or antibodies may be labeled.
- This detection kit further detects the expression of esophageal cancer markers other than Glypican-1 by any one of a hybridization method, a nucleic acid amplification method, and an antigen-antibody reaction method.
- the detection kit according to the present invention includes a detection kit for performing the detection of the further embodiment according to the present invention, specifically, a kit for detecting the protein of Glypican-1. And a kit comprising at least the antibody according to the present invention. This antibody may be labeled.
- This detection kit detects the expression of Glypican-1 by detecting the antigen-antibody reaction.
- the detection method of this embodiment further includes various reagents for carrying out the antigen-antibody reaction, for example, secondary antibodies used in the ELISA method, coloring reagents, buffers, instructions, and / or instruments, if desired. be able to.
- kits, compositions or systems may be markers of the invention (eg, markers in samples from any subject, factors that interact specifically with the markers, so long as Glypican-1 can be identified) It can also be understood that means for selectively recognizing the marker can be used, and thus any equivalent factor or means known in the art, as well as the factor or means specifically described herein. It is understood that means can be used.
- the factor used in the present invention is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof, preferably the factor is a protein or complex.
- a molecule eg, glycoprotein, lipid protein, etc.
- the factor is an antibody (eg, a polyclonal antibody or a monoclonal antibody).
- Such factors are preferably labeled or labelable. This is because it is easy to diagnose.
- the means used are: mass spectrometer, nuclear magnetic resonance analyzer, X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunology Means (eg Western blotting, EIA (enzyme immunoassay), RIA (radioimmunoassay), ELISA (enzyme linked immunosorbent assay)), biochemical means (eg pI electrophoresis, Southern blotting, two-dimensional electrophoresis), Electrophoresis equipment, chemical analysis equipment, fluorescence two-dimensional differential electrophoresis (2DE-DIGE), isotope labeling (ICAT), tandem affinity purification (TAP), physical means, laser microdissection and these Selected from the group consisting of
- the system or kit of the invention further comprises a marker standard.
- a marker standard may be used to confirm whether a marker detection means (such as a factor that specifically interacts with the marker or a means for selectively recognizing the marker) is functioning normally. preferable.
- the present invention may further include a means for purifying a target sample.
- a means for purifying a target sample include chromatography. Since purification can increase the accuracy of the diagnosis, it can be used in preferred embodiments, but this is not essential.
- the factor or means used in the present invention has the ability to quantify the marker of the present invention.
- Such quantification may be a means or factor that can draw a calibration curve properly when a standard curve is drawn.
- Preferable examples include antibodies, mass spectrometry, and chromatographic analysis. Therefore, in one embodiment, the system of the present invention further comprises a quantification means for quantifying the marker.
- the quantification unit includes a determination unit that compares the standard curve with a measurement result to determine whether the marker is within a normal value range.
- determination means can be realized using a computer.
- kits or system of the invention comprises a marker or a composition comprising said factor that specifically interacts with the marker.
- the present invention provides a level of proliferative capacity or differentiation state of a marker in a sample from a subject, a factor that specifically interacts with the marker, or a means for selectively recognizing the marker, or Provided is the use in the manufacture of a medicament for predictive diagnosis, pre-diagnosis, prediction, detection or diagnosis of a disease, disorder or condition related thereto.
- the sample may be acquired by any means. Usually, when a person in charge other than the doctor is engaged in the measurement, it may have been acquired by the doctor in some form.
- the determination of the level of proliferative potential or the state of differentiation, or the associated disease, disorder or condition, or whether it is possible, is abnormal compared to the normal value, relative to each marker It can be implemented by determining. It will be appreciated that in the method of the present invention, the markers used, etc. may have any one or more features described elsewhere in this specification as long as they do not conflict.
- a method for measuring the marker concentration a method generally used for protein quantification can be used as it is as long as the marker concentration can be specifically measured. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.
- One preferred embodiment in the detection or diagnosis of the present invention is to capture a marker on a carrier and measure the concentration of the captured marker. That is, a substance having affinity for the marker is immobilized on the carrier, and the marker is captured on the carrier via the substance having the affinity. According to this embodiment, the influence of the contaminant contained in the sample can be reduced, and the marker concentration can be measured with higher sensitivity and higher accuracy.
- an immunoassay when used as a marker measurement method, it is preferable to use a carrier on which an antibody is immobilized.
- an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed.
- a sandwich EIA system can be constructed by preparing two types of antibodies specific to a marker and having different epitopes, one of which is immobilized on a carrier as a primary antibody and the other of which is enzyme-labeled as a secondary antibody.
- immunoassay systems based on binding inhibition methods and competitive methods can be constructed.
- a substrate when used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of markers can be measured simultaneously, and rapid measurement is possible.
- the marker when mass spectrometry is used as a method for measuring a marker, the marker can be captured on a carrier by ion binding or hydrophobic interaction in addition to the antibody. Ion binding and hydrophobic interactions are not as specific as bioaffinity such as antigens and antibodies, and substances other than markers are captured, but according to mass spectrometry, they are quantified by a mass spectrometer spectrum that reflects molecular weight. No problem.
- the concentration of the marker can be measured more accurately.
- substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges.
- a substrate and a metal ion substrate are preferably used.
- the ion exchanger When the marker is captured on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier.
- both an anion exchanger and a cation exchanger can be used as the ion exchanger, and further, a strong anion exchanger, a weak anion exchanger, a strong cation exchanger, and a weak cation exchanger. Any of these can be used.
- weak anion exchangers include those having weak anion exchange groups such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).
- strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), quaternary ammonium (trimethylammonium). ) (QMA) and the like having a strong anion exchange group.
- weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM).
- strong cation exchanger include those having a strong cation exchange group such as sulfopropyl (SP).
- hydrophobic groups examples include C4 to C20 alkyl groups, phenyl groups, and the like.
- a marker can be captured on a carrier on which metal ions such as Cu 2+ , Zn 2+ , Ni 2+ , Ca 2+ , Co 2+ , and Mg 2+ are immobilized.
- a carrier to be used a known carrier such as a bead, a microtiter plate, or a resin can be used.
- beads and microtiter plates are conventionally used in immunoassays, and the measurement system can be easily constructed.
- a carrier having a planar portion such as a substrate can also be used. In this case, it is preferable to immobilize a substance having affinity for the marker in a part of the flat surface portion.
- An example is a carrier in which a chip is used as a base and an antibody specific for a marker is immobilized in spots on a plurality of spots on the surface.
- the detection, inspection or diagnosis method according to the present invention can be applied to screening of substances effective for the prevention or treatment of esophageal cancer. That is, an effective substance can be screened using the test substance as an indicator of binding or interaction with Glypican-1 or a nucleic acid molecule encoding the same.
- Test substances that can be used include synthetic low molecular weight compounds, proteins, synthetic peptides, purified or partially purified polypeptides, antibodies, bacterial release substances (including bacterial metabolites), nucleic acids (antisense, ribozymes, RNAi, etc.), etc. Preferably, it is a compound or a salt thereof, or a solvate (for example, hydrate) thereof, but is not limited thereto.
- the test substance may be a novel substance or a known substance.
- the substance identified by the screening method according to the present invention can be used as a substance effective for the treatment or prevention of esophageal cancer.
- the present invention provides a composition or medicament (therapeutic or preventive agent) for preventing or treating esophageal cancer, comprising an inhibitor of Glypican-1.
- a composition or medicament for preventing or treating esophageal cancer, comprising an inhibitor of Glypican-1.
- This therapeutic or prophylactic agent is superior from the viewpoint of safety because it uses an antibody.
- the esophageal cancer targeted by the present invention is Glypican-1 positive. In one embodiment, esophageal cancer targeted by the present invention includes those of lymph node metastasis, squamous cell carcinoma and / or adenocarcinoma. In a specific embodiment, the esophageal cancer targeted by the present invention includes squamous cell carcinoma.
- the composition or medicament (such as a therapeutic or prophylactic agent) of the present invention is administered to a patient determined to have developed Glypican-1-positive esophageal cancer. It is formulated assuming that.
- the inhibitor of Glypican-1 used in the present invention is an antibody or fragment or functional equivalent thereof, or a nucleic acid.
- the inhibitor of Glypican-1 used in the present invention is a nucleic acid
- the nucleic acid is an antisense nucleic acid, siRNA, etc.
- the siRNA comprises SEQ ID NO: 23 and / or Or 24, and may include SEQ ID NO: 25 and SEQ ID NO: 26. .
- the inhibitor of Glypican-1 is an antibody or a fragment or functional equivalent thereof, and the antibody has any of the following SEQ ID NOs: 1-22 or a fragment or functional equivalent thereof It is characterized by that.
- the antibody is an antibody or an antigen-binding fragment thereof containing any sequence including CDR of the full-length sequence, or an antibody or an antigen-binding fragment thereof containing a variable region of the following sequence, one in its framework region, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, or 20 or more substitutions, impossible, or Or an antibody or antigen-binding fragment thereof containing the deletion.
- the present invention provides: (a) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are respectively SEQ ID NO: 3 at positions 31-35, 50-65, 98-114, 163 Antibodies comprising the amino acid sequences shown at positions -170, 187-193, and 226-235, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are each represented by SEQ ID NO: 31 Antibodies comprising amino acid sequences shown at positions -35, 50-66, 99-110, 159-166, 183-187, and 222-231, (c) heavy chain CDR1, 2, 3, light The strands CDR1, 2, and 3 represent the amino acid sequences shown in positions 31 to 35, 50 to 66, 99 to 113, 162 to 170, 187 to 193, and 226 to 237 of SEQ ID NO: 5, respectively.
- the antibody comprising, (d) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 6, positions 31-35, 51-66, 99-112, 160-170, 187, respectively.
- Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 8, positions 31-35, 50-65, 98-114, 163-170, 187-193, and
- An antibody comprising the amino acid sequence shown at positions 226 to 235, (g) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31 to 35, 50 to 66, 99 of SEQ ID NO: 9, respectively.
- An antibody comprising an amino acid sequence shown at positions -120, 169-176, 193-199, and 232-241, (h) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3, respectively
- An antibody comprising the amino acid sequence shown at positions 31-35, 50-65, 98-113, 162-171, 188-194, and 227-237 of SEQ ID NO: 10
- (i) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 65, and 98 to 1 of SEQ ID NO: 11, respectively.
- An antibody comprising the amino acid sequence shown at positions 14, 163-170, 187-193, and 226-235, (j) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are sequences An antibody comprising the amino acid sequence of positions 12 to 35, 50 to 66, 99 to 113, 162 to 169, 186 to 192, and 225 to 234 of No. 12, (k) heavy chain CDR1, 2 , 3, light chain CDR1, 2, and 3 are shown at positions 31-35, 50-66, 99-116, 165-174, 191-197, and 230-240 of SEQ ID NO: 13, respectively.
- heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 14, positions 31-35, 50-66, 99-113, 162- An antibody comprising an amino acid sequence shown at positions 169, 186-192, and 225-235, (m) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are each represented by SEQ ID NO: 15 35th, 50-66, 99-113, 162-170, 187-193, and 226-236 (N) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 16, positions 31-35, 50-66, 99-110, 159, respectively.
- An antibody comprising the amino acid sequence shown at positions 166, 183-189, and 222-237, (o) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are each represented by Antibodies comprising amino acid sequences shown at positions 35, 50-65, 98-113, 162-171, 188-194, and 227-237, (p) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 contain the amino acid sequences shown at positions 31-35, 50-66, 99-116, 165-174, 191-197, and 230-240 of SEQ ID NO: 18, respectively.
- the antibody, (q) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 19, positions 31-35, 50-66, 99-115, 164-175, 193-
- (r) heavy chain CDR1, 2 3, light chain CDR1, 2, and 3 are shown at positions 31-35, 50-66, 99-117, 166-177, 194-200, and 233-242 of SEQ ID NO: 20, respectively.
- An antibody comprising an amino acid sequence, (s) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 21, positions 32-36, 51-67, 100-114, 163-171, respectively.
- Antibodies comprising the amino acid sequences shown at positions 188 to 194 and 227 to 236, and (t) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are respectively 31 to 31 of SEQ ID NO: 22
- embodiments described elsewhere in this specification and / or techniques known in the art can be used.
- such an antibody or fragment or functional equivalent thereof preferably has inhibitory activity downstream of Glypican-1 or its signal transduction pathway.
- Such activity can be determined by examining the expression level of Glypican-1 or its activity, or by directly using an esophageal cancer cell line, cell proliferation inhibition, cytotoxicity in antibody-dependent cytotoxicity (ADCC), or It may be confirmed by transplanting into a model animal and observing tumor regression.
- ADCC antibody-dependent cytotoxicity
- These antibodies of the present invention are, in certain embodiments, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies. , ScFV, diabody, sc (Fv) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the present invention provides a method for preventing or treating esophageal cancer in a subject, comprising administering to the subject in need thereof an effective amount of an inhibitor of Glypican-1. It will be understood that any form described elsewhere herein can be utilized as a Glypican-1 inhibitor used in the prevention or treatment methods of the present invention.
- the present invention also provides a composition or medicament (therapeutic or prophylactic agent) for preventing or treating esophageal cancer, comprising a Glypican-1 binding agent.
- a composition or medicament for preventing or treating esophageal cancer, comprising a Glypican-1 binding agent.
- the composition or medicament (such as a therapeutic or prophylactic agent) further comprises a cell killing agent. Accordingly, it can be said that such a composition or medicament (such as a therapeutic or prophylactic agent) can contain a complex molecule.
- the Glypican-1 binding agent is an antibody or fragment or functional equivalent thereof, or a nucleic acid.
- the binding agent of Glypican-1 is an antibody or a fragment or functional equivalent thereof and further bound with a cell-killing agent.
- the “cell-killing drug” is a drug that may dissolve the cell membrane.
- Cytocidal agents are referred to as cytotoxic peptides in the case of peptides, and cytotoxic peptides have various designations in the art, for example, “soluble peptide component”, “cell killing sequence” These are also referred to as “cell-lytic peptide (sequence)” or “cell membrane-lytic peptide (sequence)”, and these are used interchangeably for the purposes of the present invention.
- Representative examples of such cytotoxic agents include Gail D. et al., Cancer Res 2008; 68: 9280-9290 .; Ian Krop and Eric P. Winer, ClinCancer Res; 20 (1); 1-6.
- peptides include, but are not limited to, cell membrane lytic peptides, cell membrane potential destabilizing peptides, cell membrane lytic / nucleic acid binding peptides, and mitochondrial membrane disrupting peptides.
- such a cell-killing drug may be bound to the binding agent of the present invention such as an antibody with a spacer.
- the “spacer” refers to a portion that forms a chemical bond between molecules of a chain polymer so as to form a bridge, and is also referred to as a linker.
- Examples of peptide spacers typically include, but are not limited to, a sequence of 0 to 5 amino acids consisting of G and P. The spacer is not essential and may not be present.
- the combination of the binding agent of the present invention and the cell killing agent can be said to be a complex molecule.
- An example of such a molecule is that a cytotoxic portion corresponding to the explosive portion and a portion responsible for specificity to the cancer cell corresponding to the warhead portion (for example, a receptor highly expressed in cancer cells).
- a spacer is used, it is composed of a cancer cell specific binding agent + spacer + cell killing agent.
- any cancer cell-specific binding agent, any spacer, any cell-killing agent can be arbitrarily combined, and examples of their production and use are described.
- Such molecules are usually synthesized by chemical synthesis, but when composed of peptides, a method of forcibly expressing and purifying by gene recombination, or a method combined therewith is also possible.
- the cancer cells to be treated are examined for the expression of Glypican-1 on the cell surface and the susceptibility of cancer cells to cytotoxic agents. Based on the results, the warhead and explosive are selected and the optimal molecule for the cancer cell is designed.
- a custom-made peptide toxin obtained by chemical synthesis or the like can be combined with a DDS such as atelocollagen as necessary, and can be treated by local administration or systemic administration.
- the esophageal cancer targeted by the complex molecule of the present invention is Glypican-1 positive.
- the esophageal cancer comprises squamous cell carcinoma.
- the Glypican-1 binding agent is an antibody or a fragment or functional equivalent thereof, and the antibody can have any of the sequences described above.
- the administration route of the therapeutic agent is preferably an effective route for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration.
- the administration form may be, for example, an injection, capsule, tablet, granule or the like.
- Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers.
- the aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH.
- the therapeutic agent may contain, for example, a buffer (for example, phosphate buffer), a stabilizer and the like.
- compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present invention comprise a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient.
- pharmaceutically acceptable refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered.
- Such carriers can be sterile liquids, such as water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, minerals Oil, sesame oil, etc. are included.
- Water is a preferred carrier when the drug is administered orally.
- Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously.
- saline solutions and aqueous dextrose and glycerol solutions are used as liquid carriers for injectable solutions.
- Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, chloride Sodium, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardening Castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts and the like are included.
- compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.
- These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. It is also possible to formulate the composition as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. Examples of suitable carriers are E.I. W. Martin, Remington ’s Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A).
- compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
- the formulation must be suitable for the mode of administration.
- surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity Accelerators, flavoring agents and the like may be included.
- various delivery systems are known, and such systems can be used to administer the therapeutic agent of the present invention to an appropriate site (eg, esophagus).
- Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules: the use of recombinant cells capable of expressing therapeutic agents (eg, polypeptides), receptor-mediated endocytosis Use; such as the construction of therapeutic nucleic acids as part of a retroviral vector or other vector.
- Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
- an inhaler or nebulizer can be used with an aerosolizing agent and can be administered with other biologically active agents. Administration can be systemic or local.
- the present invention when used in the esophageal region, it can be further administered by any suitable route, such as by direct injection into the esophagus.
- the nucleic acid is encoded in vivo by in vivo administration by constructing the nucleic acid as part of a suitable nucleic acid expression vector and administering it to be present in a cell. It is also possible to facilitate the expression of the protein, for example by the use of retroviral vectors or by direct injection or by the use of microparticle guns or the nucleic acids with lipids, cell surface receptors or transfection agents. This can be done by coating or by administering a nucleic acid linked to a tag sequence known to enter the nucleus. Alternatively, the nucleic acid therapeutic can be introduced into the cell and taken up by homologous recombination into the host cell DNA for expression.
- the composition can be formulated as a pharmaceutical composition adapted for human administration according to known methods. Such compositions can be administered by injection. Typically, compositions for injection administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In general, the ingredients are supplied separately or mixed together in a unit dosage form, for example in a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product.
- a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product.
- composition is to be administered by infusion
- it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- compositions, medicament, therapeutic agent, and preventive agent of the present invention in a neutral form, salt form, or other prodrug (for example, ester).
- pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine And those formed with free amine groups such as those derived from, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, and the like.
- the amount of the therapeutic agent of the invention effective for the treatment of a particular disorder or condition can vary depending on the nature of the disorder or condition, but can be determined by those skilled in the art by standard clinical techniques based on the description herein. In addition, in some cases, in vitro assays can be used to help identify optimal dosage ranges.
- the exact dose to be used in the formulation can also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, and within the range of any two of them. Also good.
- the dosing interval is not particularly limited, for example, it may be administered once or twice per 1, 7, 14, 21, or 28 days, or once or twice per any two of these ranges Also good.
- the dose, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient.
- the therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exhibits a desired action. If the malignant tumor marker is significantly decreased after administration, it may be determined that there is a therapeutic effect. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
- a “patient” is a human or non-human mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset. , Monkey or chimpanzee).
- the patient may be a patient who is judged or diagnosed as having developed a Glypican-1 positive malignant tumor. At this time, the determination or diagnosis is preferably performed by detecting the protein level of Glypican-1.
- the pharmaceutical composition or therapeutic agent or prophylactic agent of the present invention can be provided as a kit.
- the present invention provides a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention.
- a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention.
- associated with such containers manufactured, used or sold for human administration by a government agency in a manner prescribed by the government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to indicate information indicating authorization.
- the kit of the present invention can also contain an expression vector encoding a protein for use as a composition, therapeutic agent, prophylactic agent or medicament of the present invention, and the protein is biologically expressed after being expressed. Can also be reconstituted to form a complex that is active. Such a kit preferably also contains the necessary buffers and reagents. In some cases, such containers may be accompanied by instructions for use of the kit (package insert) and / or in a form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to provide information indicating the approval of manufacture, use or sale for human administration by a government agency.
- a pharmaceutical composition comprising a nucleic acid of the invention can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present invention, it may be useful to achieve sustained release of nucleic acids using such compositions.
- the present invention provides: (a) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are respectively represented by positions 31 to 35, 50 to 65, and 98 to Antibodies comprising amino acid sequences shown at positions 114, 163-170, 187-193, and 226-235, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are sequences An antibody comprising an amino acid sequence represented by positions 31 to 35, 50 to 66, 99 to 110, 159 to 166, 183 to 187, and 222 to 231 of No.
- heavy chain CDR1, 2 , 3, light chain CDR1, 2, and 3 are shown at positions 31-35, 50-66, 99-113, 162-170, 187-193, and 226-237 of SEQ ID NO: 5, respectively.
- heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are represented by SEQ ID NO: 6, positions 31-35, 51-66, 99-112, 160- An antibody comprising the amino acid sequence shown at positions 170, 187-193, and 226-236,
- Heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 65, 98 to 114, 163 to 170, and 187 to 193 of SEQ ID NO: 8, respectively.
- heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are positions 31 to 35 and 50 to 66 of SEQ ID NO: 9, respectively.
- An antibody comprising an amino acid sequence shown at positions 99-120, 169-176, 193-199, and 232-241,
- heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 An antibody comprising the amino acid sequences shown in SEQ ID NO: 10 at positions 31-35, 50-65, 98-113, 162-171, 188-194, and 227-237, respectively
- heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 are positions 31 to 35 and 50 to 65 of SEQ ID NO: 11, respectively.
- (1) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, and 99 to 113 of SEQ ID NO: 14, respectively.
- An antibody comprising amino acid sequences shown at positions 162-169, 186-192, and 225-235, (m) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 15 respectively. 31-35, 50-66, 99-113, 162-170, 187-193, and 226-23
- An antibody comprising the amino acid sequence shown at position 6, (n) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31-35, 50-66, 99-110 of SEQ ID NO: 16, respectively.
- An antibody comprising the amino acid sequence shown at position 159-166, 183-189, and 222-237, (o) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 17, respectively.
- (P) heavy chain CDR1, 2, 3 comprising an amino acid sequence represented by positions 31-35, 50-65, 98-113, 162-171, 188-194, and 227-237 of .
- Light chain CDR1, 2, and 3 are amino acids represented by positions 31 to 35, 50 to 66, 99 to 116, 165 to 174, 191 to 197, and 230 to 240 of SEQ ID NO: 18, respectively.
- An antibody comprising a sequence, (q) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-115, 164-175 of SEQ ID NO: 19, respectively.
- An antibody comprising an amino acid sequence shown at positions 193 to 199 and 232 to 241;
- (r) heavy chain C DR1,2,3, light chain CDR1,2, and 3 are respectively 31-35, 50-66, 99-117, 166-177, 194-200, and 233-242 of SEQ ID NO: 20
- (S) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 32 to 36, 51 to 67, and 100 to 114 of SEQ ID NO: 21, respectively.
- antibodies comprising amino acid sequences shown at positions 227 to 236, and (t) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3, respectively
- a variant of the antibody wherein the variant contains one or several substitutions, additions or deletions in the framework of the antibody, but the CDR does not contain a mutation.
- An anti-Glypican- One antibody may be sufficient.
- an anti-Glypican-1 antibody comprising at least one set of the set of amino acid sequences of heavy chain CDR1, 2, and 3 listed above.
- antibodies may in certain embodiments be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, It can be an antibody selected from diabodies, sc (Fv) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the anti-Glypican-1 antibody according to one embodiment of the present invention comprises a set of amino acid sequences of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3, and further, heavy chain FR1, 2, 3, 4, light chain FR1, 2, 3, and 4 at least one, preferably 2, 3, 4, 5, 6, 7, or all frameworks of SEQ ID NOs: 1-6 It can be the same or substantially the same as any, or the same except for conservative substitutions.
- One or more antibodies may be used.
- Another embodiment of the invention is an anti-Glypican-1 antibody comprising at least one set of the amino acid sequence sets of heavy chains FR1, 2, 3, and 4 listed above.
- the anti-Glypican-1 antibody according to one embodiment of the present invention may be in the form of scFv, in which case the linker between the heavy chain and the light chain has an amino acid sequence between the heavy chain and the light chain. You may do it.
- amino acid sequences listed above as long as the anti-Glypican-1 antibody has a desired effect, (i) one or several nucleotide sequences are deleted, substituted, inserted or added in the above amino acid sequences
- An amino acid sequence (ii) an amino acid sequence having 90% or more homology to the amino acid sequence, and (iii) a polynucleotide comprising a base sequence complementary to the base sequence encoding the amino acid sequence, It may be one or more amino acid sequences selected from the group consisting of amino acid sequences encoded by polynucleotides that specifically hybridize under stringent conditions.
- a transformant can be prepared by introducing a polynucleotide or vector encoding an anti-Glypican-1 antibody according to an embodiment of the present invention into a cell. By using this transformant, the anti-Glypican-1 antibody according to the embodiment of the present invention can be produced.
- the transformant may be a cell of a human or a mammal other than a human (eg, rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), monkey cells COS-7, and the like. Alternatively, the transformant may be Escherichia genus, yeast or the like.
- vectors examples include plasmids derived from E. coli (eg, pET-Blue), plasmids derived from Bacillus subtilis (eg, pUB110), plasmids derived from yeast (eg, pSH19), and animal cell expression plasmids (eg, pA1-11, pcDNA3.1- V5 / His-TOPO), bacteriophages such as ⁇ phage, virus-derived vectors, and the like can be used.
- These vectors may contain components necessary for protein expression such as promoter, origin of replication, or antibiotic resistance gene.
- the vector may be an expression vector.
- a method for introducing the polynucleotide or vector into a cell for example, calcium phosphate method, lipofection method, electroporation method, adenovirus method, retrovirus method, or microinjection can be used (Revised 4th edition) Xinjiang Genetic Engineering Handbook, Yodosha (2003): 152-179.).
- a production method using antibody cells for example, the method described in "Protein Experiment Handbook, Yodosha (2003): 128-142.” Can be used.
- antibody purification for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion, cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl Apatite chromatography or lectin chromatography can be used (Protein Experiment Handbook, Yodosha (2003): 27-52.).
- nucleic acid can be selected as an inhibitor of the nucleic acid form of the present invention using antisense activity as an index.
- antisense activity refers to an activity capable of specifically suppressing or reducing the expression of a target gene. More specifically, depending on a certain nucleotide sequence introduced into the cell, an activity that can reduce the protein expression level by specifically reducing the mRNA level of a gene having a nucleotide sequence region complementary to that sequence.
- a technique there are a method of directly introducing an RNA molecule complementary to mRNA produced from a target gene into a cell, and a method of introducing a construction vector capable of expressing RNA complementary to a target gene in the cell. Broadly divided.
- Antisense activity is usually achieved by a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the gene of interest.
- a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 19 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths, 17 contiguous nucleotide lengths, 18 contiguous nucleotide lengths, 19 contiguous lengths Nucleotide length, 20 consecutive nucleotide lengths, 21 consecutive nucleotide lengths, 22 consecutive nucleotide lengths, 23 consecutive nucleotide lengths, 24 consecutive nucleotide lengths, 25 consecutive nucleotide lengths Of 40 consecutive
- nucleic acid sequences include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, 95% homologous to the sequences described above.
- antisense activity is preferably complementary to a sequence at the 5 'end of the nucleic acid sequence of the gene of interest.
- antisense nucleic acid sequences also include those having one, several or one or more nucleotide substitutions, additions and / or deletions relative to the sequences described above. Therefore, in the present specification, antisense activity includes, but is not limited to, a decrease in gene expression level.
- RNA interference RNA interference
- RNA interference or “RNAi” is an abbreviation for RNA interference, and is an organism generally known in the art that inhibits or down-regulates gene expression in cells mediated by factors that cause RNAi. Process. For example, a phenomenon in which homologous mRNA is specifically decomposed by introducing a factor causing RNAi such as double-stranded RNA (also referred to as dsRNA) into a cell, and the synthesis of a gene product is suppressed, and a technique used therefor Say.
- RNAi can also be used synonymously with “factor causing RNAi”, “factor causing RNAi”, “RNAi factor” and the like in some cases.
- RNAi see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamore et al. , 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashiretal.
- RNAi is synonymous with other terms used to describe sequence-specific RNA interference such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, and epigenetics. Understood.
- the “factor causing RNAi” may be any as long as it causes “RNAi”.
- the “factor causing RNAi” includes “small interfering nucleic acid”, “siNA”, “small interfering RNA”, “siRNA”, “small interfering nucleic acid molecule”, “small interfering oligonucleotide molecule”. ”Or“ chemically modified small interfering nucleic acid molecules ”and the like, these terms inhibit or down-regulate gene expression or viral replication by mediating RNA interference“ RNAi ”or gene silencing in a sequence-specific manner. Refers to any nucleic acid molecule that can. These terms may also represent individual nucleic acid molecules, a plurality of such nucleic acid molecules, or a pool of such nucleic acid molecules. These molecules can be double stranded nucleic acid molecules comprising self-complementary sense and antisense regions.
- siRNA typically used in the present invention is a double-stranded RNA having a short length, usually about 20 bases (eg, typically about 21 to 23 bases) or less. .
- Such siRNA can be used for treatment, prevention, prognosis, etc. of a disease because it suppresses gene expression by expressing in a cell and suppresses expression of a pathogenic gene targeted by the siRNA.
- the siRNA used in the present invention may take any form as long as it can cause RNAi.
- the antisense region in an agent that causes RNAi such as siRNA, includes a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a part thereof, and a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof
- a sense region having These molecules can be assembled from two separate oligonucleotides, one strand is the sense strand and the other is the antisense strand.
- the antisense strand and the sense strand are self-complementary (i.e., each strand has a nucleotide sequence in the other strand such that the antisense strand and the sense strand form a double-stranded or double-stranded structure).
- Complementary nucleotide sequences are included, where, for example, the double-stranded region is from about 15 to about 30, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.
- the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof
- the sense strand includes a nucleotide sequence corresponding to the target nucleic acid sequence or portion thereof (eg, about 15 to about 25 or more nucleotides of the molecule are present in the target nucleic acid or portion thereof.
- these molecules are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of these molecules are linked by a nucleic acid linker or a non-nucleic acid linker.
- These molecules can be polynucleotides having a double-stranded, asymmetric duplex, hairpin or asymmetric hairpin secondary structure comprising self-complementary sense and antisense regions, where the antisense region is A nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule, or a portion thereof, and a sense region that has a nucleotide sequence corresponding to the target nucleic acid sequence, or a portion thereof, the molecule comprising two or more A ring having a loop structure and a stem comprising a self-complementary sense region and an antisense region
- the antisense region may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
- the circular polynucleotide can be processed in vivo or in vitro to produce an active molecule that can mediate RNAi, these factors being present in the nucleotide sequence or part thereof in the target nucleic acid molecule.
- Single-stranded polynucleotides having nucleotide sequences that are complementary can also be included (eg, these factors do not require a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof to be present in these factors).
- Single-stranded polynucleotides are 5 ′ phosphates (eg, Martinez et Al. , 2002, Cell. 110, 563-574 and Schwarz et al.
- Glypican-1 inhibitors of the present invention comprise separate sense and antisense sequences or regions.
- the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions and / or Alternately non-covalently linked by stacking interactions.
- a Glypican-1 inhibitor of the present invention comprises a nucleotide sequence that is complementary to the nucleotide sequence of a target gene.
- a Glypican-1 inhibitor of the present invention interacts with the nucleotide sequence of a target gene so as to inhibit expression of the target gene.
- inhibitors of Glypican-1 are not necessarily limited to molecules containing only RNA, but also include chemically modified nucleotides and non-nucleotides.
- when the present invention is a small interfering nucleic acid molecule, it may be devoid of 2'hydroxy (2'-OH) containing nucleotides.
- the invention can be a small interfering nucleic acid that does not require the presence of a nucleotide having a 2 'hydroxyl group to mediate RNAi.
- ribonucleotides for example, nucleotides having a 2'-OH group
- a linked linker or other linkage comprising one or more nucleotides with 2′-OH groups Or it may have associated groups, moieties or chains.
- an agent that inhibits Glypican-1 of the present invention may comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
- inhibitors of Glypican-1 are nucleic acid molecules that can mediate sequence-specific RNAi, such as small interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (miRNA), short hairpins. It may be RNA (shRNA), small interfering oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA).
- siRNA small interfering RNA
- dsRNA double stranded RNA
- miRNA microRNA
- shRNA small interfering oligonucleotide
- small interfering nucleic acid small interfering modified oligonucleotide
- chemically modified siRNA small interfering modified oligonucleotide
- ptgsRNA post-transcriptional gene silencing RNA
- factors that cause RNAi include, for example, a sequence having at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions. Examples include, but are not limited to, RNA comprising a double-stranded portion of nucleotide length or a variant thereof.
- the factor preferably comprises a 3 'overhang, more preferably the 3' overhang can be 2 or more nucleotides long DNA (eg, 2 to 4 nucleotides long DNA).
- RNAi used in the present invention include, but are not limited to, a pair of short reverse complementary sequences (for example, 15 bp or more, for example, 24 bp).
- RNAi RNAi to work
- a relatively long (eg, 40 base pairs or more) RNA helicase
- Dicer an RNaseIII-like nuclease having a domain excises the molecule from the 3 ′ end by about 20 base pairs to produce a short dsRNA (also called siRNA).
- siRNA is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in an organism, or about 40
- a protein specific to the siRNA binds to form an RNA-induced-silencing-complex (RISC). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity.
- RISC RNA-induced-silencing-complex
- siRNA sequence and the mRNA sequence cleaved as a target is preferably 100% identical.
- the cleavage activity by RNAi is not completely lost, but a partial activity remains.
- the mutation of the base at the center of siRNA has a large effect, and the cleavage activity of mRNA by RNAi is extremely reduced.
- siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA typically having about 40 bases or more) can be used as such a factor. it can.
- siRNA is synthesized by dsRNA in addition to the pathway described above, where the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP). It is also contemplated that this dsRNA becomes Dicer's substrate again, generating new siRNA and amplifying the action.
- RdRP RNA-dependent RNA polymerase
- siRNA itself and factors that produce siRNA are also useful.
- 35 dsRNA molecules almost completely degrade the mRNA in a cell having 1,000 copies or more, so it is understood that siRNA itself and factors that generate siRNA are useful. Is done.
- the factor causing RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having a protruding portion at the 3 'end.
- shRNA short hairpin structure
- shRNA is a single-stranded RNA that includes a partially palindromic base sequence, and thus has a double-stranded structure within the molecule, resulting in a hairpin-like structure.
- shRNA is artificially chemically synthesized.
- such shRNA can be generated by synthesizing RNA in vitro with T7 RNA polymerase, which has a hairpin structure DNA in which the DNA sequences of the sense strand and antisense strand are ligated in the reverse direction.
- shRNAs are approximately 20 bases in length (typically 21 bases, 22 bases, 23 bases, etc.) in length after being introduced into the cell. It should be understood that it is degraded to cause RNAi as well as siRNA and has the therapeutic effect of the present invention. It should be understood that such effects are exerted in a wide range of organisms such as insects, plants, animals (including mammals). Thus, since shRNA causes RNAi similarly to siRNA, it can be used as an active ingredient of the present invention.
- the shRNA can also preferably have a 3 'overhang.
- the length of the double-stranded part is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more.
- the 3 'protruding end may be preferably DNA, more preferably DNA having a length of at least 2 nucleotides, and further preferably DNA having a length of 2 to 4 nucleotides.
- the factor causing RNAi used in the present invention can be either artificially synthesized (for example, chemical or biochemical) or naturally occurring, and the effect of the present invention can be achieved between the two. There is no essential difference. Those chemically synthesized are preferably purified by liquid chromatography or the like.
- RNAi used in the present invention can also be synthesized in vitro.
- antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused through the mechanism described above, and the effects of the present invention are achieved.
- RNA can be introduced into cells by any appropriate method such as the calcium phosphate method.
- Factors causing RNAi of the present invention also include factors such as single strands that can hybridize to mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present invention.
- One embodiment of the present invention is a therapeutic agent for Glypican-1 positive esophageal cancer comprising an RNAi molecule against Glypican-1 or a polynucleotide encoding the RNAi molecule. If this RNAi molecule or a polynucleotide encoding the RNAi molecule is used, growth of Glypican-1-positive esophageal cancer cells can be suppressed.
- the “polynucleotide” may be a polymer compound having 10 or more nucleotides and in which nucleotides are linearly polymerized.
- the “RNAi molecule” is an RNA strand having an RNAi action, and examples thereof include siRNA, shRNA, miRNA, and small RNA having an RNAi action.
- RNAi is a function of a target gene or mRNA, etc. by one or more of siRNA, shRNA, miRNA, short or long one or double stranded RNA, or a modification thereof Including the phenomenon that is suppressed or silenced.
- siDirect2.0 (Naito et al., BMC Bioinformatics. 2009 Nov 30; 10: 392.) Can be used for RNAi molecule design. Further, it may be entrusted to a trust company (for example, Takara Bio Inc.). The RNAi action can be confirmed by quantifying the expression level of the RNA strand by real-time RT-PCR. Alternatively, it can also be performed by methods such as analysis of RNA strand expression level by Northern blot, analysis of protein amount by Western blot, and observation of phenotype. Moreover, the plasmid which produces siRNA or shRNA with respect to a specific gene can be purchased from a trust company (for example, Takara Bio Inc. etc.), for example.
- siRNA includes an RNA strand capable of inducing RNAi.
- the duplex of siRNA can be divided into a guide strand and a passenger strand, and the guide strand is incorporated into RISC.
- the guide strand incorporated into RISC is used to recognize the target RNA.
- Artificially produced RNAi research is mainly used in RNAi research, but some that exist endogenously in the living body are also known.
- the guide strand may be composed of RNA having 15 or more bases. If it is 15 bases or more, the possibility of binding to the target polynucleotide with high accuracy increases.
- the guide strand may be composed of RNA having 40 bases or less. If it is 40 bases or less, the risk that disadvantageous phenomena such as interferon response occur will be lower.
- shRNA includes a single-stranded RNA strand that can induce RNAi and can form a hairpin-like structure (hairpin-like structure).
- shRNA is cleaved by Dicer in the cell, and siRNA is excised. It is known that target RNA is cleaved by this siRNA.
- the shRNA may be composed of 35 or more nucleotides. If it is 35 or more, the possibility that a hairpin-like structure peculiar to shRNA can be formed with high accuracy increases.
- the shRNA may be composed of RNA of 100 bases or less. If it is 100 bases or less, the risk that disadvantageous phenomena such as interferon response occur will be reduced.
- the length of shRNA is not necessarily 100 bases or less. However, it is thought that it can function as shRNA.
- miRNA includes an RNA strand having a function similar to that of siRNA, and is known to suppress or degrade the translation of a target RNA strand.
- the difference between miRNA and siRNA generally lies in the production pathway and detailed mechanism.
- small RNA refers to a relatively small RNA strand, and examples thereof include siRNA, shRNA, miRNA, antisense RNA, and single or double stranded small RNA.
- the RNAi molecule may contain an overhang consisting of 1 to 5 bases at the 5 'end or 3' end. In this case, it is considered that the efficiency of RNAi increases. This number may be, for example, 5, 4, 3, 2, or 1 base, and may be within the range of any two of them. Further, when the RNAi molecule is double-stranded, mismatch RNA may exist between the RNA strands. The number may be, for example, 1, 2, 3, 4, 5, or 10 or less, and may be in the range of any two of them.
- the RNAi molecule may contain a hairpin loop. The number of bases in the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, and any two values thereof. It may be within the range.
- each base sequence may be deleted, substituted, inserted, or added.
- the notation of each base sequence is the 5 'end on the left side and the 3' end on the right side.
- the length of the RNAi molecule may be, for example, 15, 18, 20, 25, 30, 40, 50, 60, 80, 100, 200, or 400 bases, and within the range of any two of them. It may be. This number is preferably 15 or more or 100 or less from the viewpoint of enhancing the therapeutic effect on Glypican-1 positive malignant tumors.
- the “RNA strand” includes those in which RNA or an equivalent thereof is constituted in a combined form.
- the “DNA strand” includes those in which a plurality of DNAs or their equivalents are combined.
- the RNA strand or DNA strand includes an RNA strand or a DNA strand in the form of a single strand or a plurality of strands (for example, a double strand).
- the RNA strand or DNA strand may be bound to a cell uptake promoting substance (for example, PEG or a derivative thereof), a label tag (for example, a fluorescent label tag), or a linker (for example, a nucleotide linker).
- RNA strand or DNA strand can be synthesized using a nucleic acid synthesizer. In addition, it can also be purchased from a trust company (for example, Invitrogen). In vivo RNA or DNA strands may form salts or solvates. In addition, RNA strands or DNA strands in vivo may be subjected to chemical modification.
- the term RNA strand or DNA strand includes, for example, an RNA strand or DNA strand that forms a salt or solvate, or an RNA strand or DNA strand that has undergone chemical modification.
- the RNA strand or DNA strand may be an RNA strand analog or a DNA strand analog.
- the “salt” includes, for example, an anion salt formed with any acidic (eg, carboxyl) group or a cation salt formed with any basic (eg, amino) group.
- the salts include inorganic salts or organic salts, for example, salts described in Bergeet al., J. Pharm. Sci., 1977, 66, 1-19. Examples thereof include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and the like.
- a “solvate” is a compound formed by a solute and a solvent. As for the solvate, for example, J.
- the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.
- “chemical modification” includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, or the like.
- the RNAi molecule preferably contains a base sequence complementary to a part of the base sequence of Glypican-1 mRNA from the viewpoint of stably exhibiting RNAi action.
- the “part” may be, for example, 5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 50 bases or more, and any two values thereof. It may be within the range.
- the siRNA used in the examples described later includes the base sequence of SEQ ID NO: 25. These base sequences are base sequences complementary to a part of Glypican-1 mRNA, and are considered to be portions that function as guide strands.
- One embodiment of the present invention includes such an RNAi molecule comprising the base sequence of SEQ ID NO: 25.
- the RNAi molecule may further include a base sequence complementary to the base sequence represented by SEQ ID NO: 25 (for example, SEQ ID NO: 26).
- the “complementary base sequence” is a base sequence possessed by another highly complementary polynucleotide capable of hybridizing to one polynucleotide.
- the full-length sense strand of the siRNA used in Examples described later is the nucleotide sequence of SEQ ID NO: 27 (5′-GGGACACGCUCACGGCCAATT-3 ′), and the full-length antisense strand is SEQ ID NO: 28 (5′-UUGGCCGUGAGCGUGUCCCTG-3 ′). Is the base sequence.
- nucleotide sequences are deleted, substituted, inserted or added in the above-mentioned basic acid sequences. It may be an amino acid sequence or (ii) a base sequence encoded by a polynucleotide that specifically hybridizes under stringent conditions to a polynucleotide comprising a base sequence complementary to the above base sequence.
- One embodiment of the present invention is a therapeutic agent for a Glypican-1 positive malignant tumor comprising a Glypican-1 antagonist.
- the Glypican-1 antagonist includes substances that inhibit Glypican-1 expression or function. If this Glypican-1 antagonist is used, the growth of Glypican-1 positive malignant tumor cells can be suppressed.
- the form of the antagonist is not particularly limited as long as it has an action of inhibiting the expression or function of Glypican-1, and may be, for example, an antibody, an RNA chain, a DNA chain, a small organic compound, or a polypeptide. .
- the RNA strand may be an RNAi molecule for Glypican-1.
- As the DNA strand a DNA strand encoding an RNAi molecule for Glypican-1 can be used.
- the form of this DNA strand may be, for example, a vector.
- “inhibiting protein expression” includes, for example, inhibiting a transcription mechanism from a gene to mRNA, or inhibiting a translation mechanism from mRNA to protein. It also includes, for example, reducing protein mass by inducing degradation of a gene, mRNA, or protein.
- “inhibiting the function of a protein” includes causing a structural change in the protein and reducing the activity of the protein. In addition, for example, it includes a decrease in the amount of mRNA or protein produced as a result of inhibiting gene expression.
- the “state in which expression is inhibited” includes a state in which the expression level is significantly reduced as compared with the normal state.
- the expression level may be based on the amount of mRNA or protein.
- “significantly” may include, for example, when statistically significant differences are evaluated using Student's t test (one-sided or two-sided) and p ⁇ 0.05. Or the state in which the difference has arisen substantially is included.
- “the state in which the function is inhibited” includes a state in which the activity is significantly decreased as compared with that in the normal state.
- One embodiment of the present invention is a novel method for treating esophageal cancer.
- This therapeutic method is, for example, a therapeutic method including a step of administering an anti-Glypican-1 antibody to a patient. If this treatment method is used, Glypican-1-positive esophageal cancer can be treated. Moreover, since this therapeutic method uses an antibody, it is excellent from the viewpoint of safety as demonstrated in Examples.
- the subject esophageal cancer includes those at the lymph node metastasis site, squamous cell carcinoma and / or adenocarcinoma, and particularly squamous cell carcinoma. In particular, in the present specification, a remarkable effect is shown in Glypican-1 positive squamous cell carcinoma, and it is understood that it is effective in Glypican-1 positive esophageal cancer.
- the antibodies used in the therapeutic methods of the present invention include (a) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 respectively at positions 31 to 35 of SEQ ID NO: 3, An antibody comprising an amino acid sequence shown at positions -65, 98-114, 163-170, 187-193, and 226-235, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, An antibody comprising an amino acid sequence represented by positions 31 to 35, 50 to 66, 99 to 110, 159 to 166, 183 to 187, and 222 to 231 of SEQ ID NO: 4, respectively (c ) Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 5, positions 31-35, 50-66, 99-113, 162-170, 187-193, and An antibody comprising the amino acid sequence shown at positions 226 to 237, (d) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are positions 31 to 35, 51 to 66, and 99 of SEQ ID NO: 6, respectively.
- Amino acids shown at positions -112, 160-170, 187-193, and 226-236 An antibody comprising a sequence, (e) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, 99 to 120, and 169 to 176 of SEQ ID NO: 7, respectively. An antibody comprising the amino acid sequence shown at positions 193 to 199 and 232 to 242; (f) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35 of SEQ ID NO: 8, respectively.
- An antibody comprising an amino acid sequence shown at positions 50-65, 98-114, 163-170, 187-193, and 226-235, (g) heavy chain CDR1, 2, 3, light chain CDR1, An antibody in which 2 and 3 each include an amino acid sequence represented by positions 31 to 35, 50 to 66, 99 to 120, 169 to 176, 193 to 199, and 232 to 241 of SEQ ID NO: 9, (h) Heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 65, 98 to 113, 162 to 171 and 188 to 194 of SEQ ID NO: 10, respectively.
- an antibody comprising an amino acid sequence shown at positions 227-237, (i) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3
- An antibody comprising an amino acid sequence represented by positions 31 to 35, 50 to 65, 98 to 114, 163 to 170, 187 to 193, and 226 to 235, respectively, of SEQ ID NO: 11,
- (j) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are SEQ ID NO: 12 at positions 31-35, 50-66, 99-113, 162-169, 186-192, and 225-234, respectively.
- (K) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, and 99 to 116 of SEQ ID NO: 13, respectively.
- An antibody comprising the amino acid sequence shown, (p) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 18, positions 31-35, 50-66, 99-116, 165, respectively.
- Antibodies comprising the amino acid sequences shown at positions -174, 191-197, and 230-240, (q) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are each represented by 31 of SEQ ID NO: 19 -35th, 50-66th, 99-115th, 164-175th, 193-199th, and 232-241st
- An antibody comprising the amino acid sequence shown, (r) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 20, positions 31-35, 50-66, 99-117, 166, respectively.
- An antibody comprising the amino acid sequence shown at positions 177, 194 to 200, and 233 to 242 (s) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 is 32 of SEQ ID NO: 21, respectively.
- One or more antibodies selected from the group consisting of, or a variant of the antibody, wherein the variant contains one or several substitutions, additions or deletions in the framework of the antibody To the CDR Does not contain mutations, a mutant may be an anti-Glypican-1 antibody. If this anti-Glypican-1 antibody is used, the proliferation of Glypican-1-positive malignant tumor (eg, esophageal cancer) cells can be particularly effectively suppressed. In addition, Glypican-1-positive malignant tumors (for example, esophageal cancer) can be diagnosed efficiently.
- an anti-Glypican-1 antibody comprising at least one set of the set of amino acid sequences of heavy chain CDR1, 2, and 3 listed above.
- antibodies may in certain embodiments be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, It can be an antibody selected from diabodies, sc (Fv) 2 (single chain (Fv) 2 ), and scFv-Fc.
- Glypican-1 positive and Glypican-1 non-positive patients among esophageal cancer patients There are Glypican-1 positive and Glypican-1 non-positive patients among esophageal cancer patients. Therefore, it is preferable to perform the said treatment method with respect to the patient in whom esophageal cancer was judged to be Glypican-1 positive esophageal cancer among esophageal cancer patients. Thus, it becomes possible to perform more optimal medication by diagnosing the presence or absence of Glypican-1 positivity in advance.
- the above esophageal cancer treatment method preferably includes a step of diagnosing that the patient has developed Glypican-1-positive esophageal cancer.
- this treatment method may include a step of examining that patient-derived esophageal cancer cells express Glypican-1.
- the onset diagnosis of Glypican-1-positive esophageal cancer may be performed by, for example, mRNA expression diagnosis or protein expression diagnosis.
- This diagnosis is preferably a protein expression diagnosis from the viewpoint of realizing a more optimal medication by accurately diagnosing Glypican-1 positive.
- the protein expression diagnosis may be performed using, for example, an anti-Glypican-1 antibody.
- Glypican-1 expression level of patient-derived esophageal cancer cells is significantly larger than normal cells or Glypican-1-negative esophageal cancer cells. You may judge that Alternatively, a total protein obtained from a patient-derived esophageal cancer cell and a total protein obtained from a normal cell or a Glypican-1-negative esophageal cancer cell are subjected to Western blot, and a normal cell or a Glypican-1-negative esophageal cancer cell Compared to the above case, it may be judged that the patient-derived esophageal cancer cells develop Glypican-1-positive esophageal cancer when the band intensity corresponding to Glypican-1 is significantly stronger .
- RT-PCR may be used instead of Western blot.
- serum or plasma obtained from a malignant tumor patient and serum or plasma obtained from a healthy person or a Glypican-1-negative malignant tumor patient are subjected to ELISA using an anti-Glypican-1 antibody, and the healthy person or Glypican-1 negative It may be judged that Glypican-1-positive malignant tumor has developed when serum or plasma derived from a malignant tumor patient has a significantly stronger expression level of Glypican-1 than in a malignant tumor patient .
- Serum and plasma samples themselves may be quantified, or exosomes may be isolated from serum and plasma, and Glypican-1 in the exosomes may be subjected to an ELISA method for analysis.
- the method for treating esophageal cancer may include a step of administering a Glypican-1 antagonist to a patient.
- a Glypican-1 antagonist to a patient.
- an RNAi molecule against Glypican-1 or a polynucleotide encoding the RNAi molecule may be administered to a patient.
- One embodiment of the present invention is a novel diagnostic agent for esophageal cancer comprising an anti-Glypican-1 antibody.
- This diagnostic agent may be, for example, a companion diagnostic agent for treating esophageal cancer targeting Glypican-1, including an anti-Glypican-1 antibody. Since some patients with esophageal cancer are Glypican-1 positive and non-Glypican-1 positive, use this companion diagnostic agent to test whether esophageal cancer is Glypican-1 positive in advance Then, the therapeutic efficacy of esophageal cancer treatment targeting Glypican-1 can be diagnosed.
- “companion diagnosis” includes a diagnosis performed for the purpose of assisting optimal medication by predicting individual differences in drug effects and side effects by examination.
- the diagnostic agent for esophageal cancer is a diagnosis comprising an anti-Glypican-1 antibody for diagnosing the therapeutic efficacy of an anti-Glypican-1 antibody or a Glypican-1 antagonist against esophageal cancer. It may be a medicine. Since some patients with esophageal cancer are positive for Glypican-1 and not positive for Glypican-1, this diagnostic agent is used to test whether esophageal cancer is positive for Glypican-1 in advance. Then, the therapeutic effectiveness of the anti-Glypican-1 antibody or Glypican-1 antagonist for the patient can be diagnosed.
- One embodiment of the present invention is a companion diagnostic method for treating esophageal cancer targeting Glypican-1, which comprises the step of testing that an esophageal cancer sample of an esophageal cancer patient is Glypican-1 positive. Since some patients with esophageal cancer are Glypican-1 positive and non-Glypican-1 positive, this companion diagnostic method is used to test whether esophageal cancer is Glypican-1 positive in advance Then, the therapeutic efficacy of esophageal cancer treatment targeting Glypican-1 can be diagnosed.
- the diagnostic method may further include the step of isolating or extracting an esophageal cancer sample from an esophageal cancer patient.
- the “esophageal cancer sample” may be an esophageal cancer tissue or cell obtained from an esophageal cancer patient.
- One embodiment of the present invention is a method for examining the therapeutic efficacy of an anti-Glypican-1 antibody or a Glypican-1 antagonist against esophageal cancer.
- This method is, for example, a test method including a step of testing that an esophageal cancer sample of an esophageal cancer patient is positive for Glypican-1.
- This test method may include a step of detecting the presence of Glypican-1 in an esophageal cancer sample. This detection method is such that the amount of Glypican-1 in an esophageal cancer sample is normal cells or negative for Glypican-1 The process of detecting that it is significantly larger than an esophageal cancer cell may be included.
- Glypican-1 For detection of Glypican-1, for example, RT-PCR, Western blot, or immunohistochemical staining may be used.
- the evaluation criteria for the presence or absence of Glypican-1 may be the same as in the case of the onset diagnosis of Glypican-1 positive esophageal cancer described above.
- Methods for testing therapeutic effectiveness include methods for testing efficacy for treatment.
- One embodiment of the present invention is an esophageal cancer cell growth inhibitor comprising an anti-Glypican-1 antibody.
- it is a method for inhibiting the growth of esophageal cancer cells, comprising a step of contacting an anti-Glypican-1 antibody with esophageal cancer cells.
- it is an esophageal cancer cell growth inhibitor containing a Glypican-1 antagonist.
- it is a method for inhibiting the growth of esophageal cancer cells, comprising a step of bringing a Glypican-1 antagonist into contact with esophageal cancer cells.
- the esophageal cancer cell growth inhibitor or therapeutic agent according to the embodiment of the present invention has a growth rate, growth amount, or volume of esophageal cancer, compared to when the growth inhibitor is not added or the therapeutic agent is not added. It may be an agent that reduces by 10, 20, 30, 40, 50, or 70% or more. This ratio may be within the range of the two numerical values exemplified here.
- One embodiment of the present invention is an agent for suppressing mitosis of esophageal cancer cells, comprising an anti-Glypican-1 antibody.
- it is a method for inhibiting cell division of esophageal cancer cells, comprising a step of bringing an anti-Glypican-1 antibody into contact with esophageal cancer cells.
- it is a mitotic inhibitor of esophageal cancer cells containing a Glypican-1 antagonist.
- it is a cell division suppression method of an esophageal cancer cell including the process which a Glypican-1 antagonist and an esophageal cancer cell are made to contact.
- the agent for inhibiting mitosis of esophageal cancer cells reduces the division rate of esophageal cancer cells by 10, 20, 30, or 50% or more compared to when no mitosis inhibitor is added. It may be a drug to be caused. This ratio may be within the range of the two numerical values exemplified here.
- One embodiment of the present invention is a therapeutic agent for Glypican-1-dependent esophageal cancer comprising an anti-Glypican-1 antibody.
- Glypican-1-dependent esophageal cancer can be treated.
- One embodiment of the present invention is the use of anti-Glypican-1 antibodies or Glypican-1 antagonists for the production of esophageal cancer therapeutics. Yet another embodiment is the use of an anti-Glypican-1 antibody for the manufacture of a companion diagnostic for the treatment of esophageal cancer targeting Glypican-1.
- One embodiment of the present invention includes a step of introducing a polynucleotide encoding Glypican-1 into a cell, a step of expressing the Glypican-1 in the cell, and a cell expressing the Glypican-1 Immunizing a chicken with an antigen.
- a method for producing an anti-Glypican-1 antibody According to this production method, an excellent anti-Glypican-1 antibody can be efficiently produced for the treatment or diagnosis of Glypican-1-positive esophageal cancer.
- Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, FM (1995) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Innis, MA et al. (1995) .PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJet al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method”, Yodosha, 1997, etc., which are incorporated herein by reference in their entirety (which may be all).
- the oligonucleotide of the present invention can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). is there.
- an automated DNA synthesizer commercially available from Biosearch, Applied Biosystems, etc.
- Stein et al. Steinet al., 1988, Nucl. Acids Res. 16: 3209
- control pore glass polymer supports Sarinet al., 1988
- Proc. Natl. Acad. Sci. USA 85: 7448-7451 can also be used to prepare methylphosphonate oligonucleotides.
- Example 1 Expression of Glypican-1 in various cells by Western blot
- Example 2 Expression of Glypican-1 in various cells by Western blot
- Lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 x protease inhibitorcocktail (Nacalai Tesque), 1 x phosphatase inhibitor cocktail (Nacalai Tesque)) and centrifuged (13,200 The supernatant was recovered as a protein extract by rpm, 4 ° C., 15 min). Protein concentration is determined using a protein quantification kit (DCProtein Assay kit (Bio-Rad Laboratories)) and bovine serum albumin (BSA) was quantified as a standard.
- DCProtein Assay kit Bio-Rad Laboratories
- BSA bovine serum albumin
- Glypican-1 was analyzed by Western blot using a specific antibody. As a result, it was confirmed that Glypican-1 was not expressed in HEEpic and Het1A, but was expressed in an esophageal squamous cell carcinoma cell line.
- Glypican-1 is highly expressed specifically in esophageal cancer cells, not normal esophageal cells.
- Example 2 Relative expression level of Glypican-1 (Accession No. P35052)
- Example 2 Relative expression level of Glypican-1 (Accession No. P35052)
- the relative expression level of Glypican-1 (Accession No. P35052) in normal cells and various esophageal cancer cell lines was examined.
- the cell surface membrane protein containing Gypypican-1 was biotinylated with sulfo-NHS-SS-biotin on 8 types of cell lines cultured in a 150 mm petri dish.
- the extracted protein was purified with Neuroto-avidin beads.
- bovine serum albumin labeled with sulfo-NHS-SS-biotin was added in equal amounts as an internal standard, and used for correction of quantitative results by a mass spectrometer.
- the purified protein was digested with trypsin and labeled with iTRAQ reagent.
- the normal strain was 1.2 units
- the esophageal cancer cell line was more than twice for TE6, TE9, TE10 and TE14, and 1.6 to 1.6 for TE1 and TE8. It showed a high relative ratio of 1.7 and was found to be highly expressed twice or more in 4 types of cancer cell lines among 6 types of esophageal squamous cell carcinoma cell lines. Therefore, it was shown that Glypican-1 is useful as a marker for esophageal cancer.
- Example 3 FACS analysis that Glypican-1 is expressed on the cell surface of esophageal cancer cells.
- FACS analysis The cells were washed twice with PBS (Nacalai Tesque) and peeled off from the dish with 0.02% EDTA solution (Nacalai Tesque). Cells were washed twice with FACSstaining buffer (PBS supplemented with 1% FBS and 0.1% sodium azide) and stained with 5-fold diluted goat anti-human Glypican-1 antibody (R & D Systems, Minneapolis, MN), followed by 50 Stained with PE-labeled anti-goat IgG antibody diluted 1: 2. Stained cells were measured with FACSCanto II (Becton Dickinson, Mountain View, CA, USA) and analyzed using FlowJo software (TreeStar, Stanford, CA, USA).
- FACSCanto II Becton Dickinson, Mountain View, CA, USA
- Example 1-3 it was revealed that Glypican-1 is highly expressed specifically not in normal esophageal cells but in esophageal cancer cells.
- Example 4 Expression by immunostaining of Glypican-1 in esophageal squamous cell carcinoma
- Glypican-1 in tissues (primary and lymph node metastasis) of esophageal squamous cell carcinoma cases was confirmed by immunohistochemical staining.
- FIG. 4A shows the result.
- FIG. 4B shows the result performed in another series.
- Glypican-1 was evaluated for expression of cancer antigen candidate molecules not in cell lines but in cancer tissues by performing immunohistochemical staining on surgical tissues.
- significant enhancement of Glypican-1 expression in esophageal cancer cell lines was also observed by immunohistochemical staining.
- Glypican-1 is highly expressed in the cell membrane of esophageal squamous cell carcinoma tissue.
- Glypican-1 was also expressed at the lymph node metastasis site of esophageal cancer (FIG. 4C)). That is, anti-Glypican-1 antibody is expected to have a therapeutic effect not only on the primary site but also on metastatic lesions. Subsequently, the expression of Glypican-1 was analyzed by immunohistochemical staining in tissues of esophageal squamous cell carcinoma (88) cases. As the primary antibody, Atlas antibodies (HPA030571) was used and stained with Dako ChemMate ENVISION Kit / HRP (DAB) -universal kit (K5007).
- DAB Dako ChemMate ENVISION Kit / HRP
- the intensity of staining was divided into 3 steps from 0 to 2, the area was divided into 4 steps from 0 to 3, and the product was used as the staining score.
- FIG. 5A The result is shown in FIG. 5A.
- glypican-1 expression analysis in various normal tissues was performed by real-time PCR analysis using commercially available RNA from various human normal tissues, and the expression level of glypican-1 was examined as a relative value to GAPDH expression However, it was confirmed that the expression in normal tissues was low. No significant expression of Glypican-1 was observed in any tissue, whereas a significant increase in expression was observed in the esophageal cancer cell line. (FIG. 5A). The same tendency was observed when esophageal cancer tissue, non-cancerous site, and TE14 were added.
- FIG. 5A The same tendency was observed when esophageal cancer tissue, non-cancerous site, and TE14 were added.
- FIG. 5B shows the results of Glypican-1 expression analysis in the esophageal cancer tissue and non-cancerous site of the same patient by real-time PCR analysis.
- Example 6 Comparison of serum Glypican-1 concentrations in healthy subjects and patients with esophageal squamous cell carcinoma
- the serum Glypican-1 concentration was higher than that in healthy human serum (FIGS. 6 to 7).
- Glypican-1 For clinical application of anti-Glypican-1 antibody drugs, selective treatment of esophageal cancer patients that express Glypican-1 is thought to lead to personalized medicine. Therefore, a method for selecting Glypican-1-positive patients is necessary. Although there is a possibility that Glypican-1 in a biopsy tissue of esophageal cancer is examined by immunohistochemical staining, a method with low invasiveness is preferable because it is highly invasive. There is a possibility that Glypican-1 or its extracellular domain expressed in esophageal cancer tissue is free in the blood.
- Glypican-1 in the blood of esophageal cancer patients can be quantified, it is suggested that the expression level of Glypican-1 is high in esophageal cancer tissues at high blood Glypican-1 concentrations.
- Blood samples have the advantage of being less invasive than biopsy.
- the present inventors have succeeded for the first time in detecting esophageal cancer patients whose blood Glypican-1 concentration is higher than that of healthy individuals. It is highly possible that Glypican-1 is highly expressed in these patient tissues, and measurement of blood Glypican-1 concentration is considered to be highly useful as a companion diagnostic agent.
- Example 7 Suppression of expression using siRNA against Glypican-1 and suppression of apoptosis and AKT.
- Esophageal cancer cells were seeded at 2,000 cells / well in a 96-well plate and subjected to cell proliferation assay by WST-8 assay 72 hours after siRNA transfection using lipofectamine2000.
- SiRNA for Glypican-1 and negative control siRNA were obtained from QIAGEN.
- TE6, TE8, and TE14 were washed with ice-cold PBS ( ⁇ ), peeled off with a cell scraper, and cells were collected by centrifugation.
- Cells were treated with RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, phosphatase, 1 x protease inhibitor (Nacalai Tesque) cocktail). After dissolution, the supernatant was recovered as a protein extract by centrifugation (13,200 rpm, 4 ° C., 15 min). The protein concentration was quantified using a protein quantification kit (DC Protein Assay kit (Bio-Rad Laboratories)) with bovine serum albumin (BSA) as a standard.
- DC Protein Assay kit Bio-Rad Laboratories
- BSA bovine serum albumin
- Anti-phospho-AKT (Thr308) antibody, anti-phospho-AKT (Sre473) antibody, anti-AKT antibody and anti-cleaved caspase-3 antibody were purchased from CellSignaling Technology. Membranes were incubated with various primary antibodies for 1 hour at room temperature. After washing 3 times with TBST for 10 minutes, the PVDF membrane was incubated at room temperature for 1 hour using HRP-labeled anti-rabbit antibody (GE healthcare) diluted 5,000 times with TBST. After the PVDF membrane was washed with TBST for 10 minutes three times, the reacted protein was detected by a fluorescence reaction system (PerkinElmer). Anti-GAPDH antibody (Santa Cruz Biotechnology) was used as a loading control.
- Glypican-1 siRNA increases the expression of pro-apoptotic proteins (pro-apoptotic proteins) Puma, Bik, Bim and anti-apoptotic proteins (Anti-apoptotic protein) ) And Bcl-w expression was decreased, and a decrease in EGFR phosphorylation level was also observed.
- the present invention can function as a therapeutic or preventive agent for esophageal cancer, particularly esophageal squamous cell carcinoma.
- Example 8 Antibody production and characterization
- GPC-1 chicken antibody was produced and characterized. The protocol is shown below.
- Human Glypican-1 cDNA was ligated into a mammalian expression vector (pcDNA3.1-V5 / His-TOPO) and cloned into the C-terminus to become a V5 / His tag fusion protein.
- the expression vector was transfected into a chicken lymphoblastoid cell line by electroporation, and 2 mg / ml G418 was added to select the expression cells.
- the obtained LSR-expressing cell line was hyperimmunized to chickens.
- the antibody titer was measured by cell-ELISA.
- As the cell line a Glypican-1-expressing chicken lymphoblastoid cell line was used at 4 ⁇ 10 5 cells / well.
- scFv phage antibody library (Preparation of scFv phage antibody library from immune chicken spleen) After removing the spleen from the immunized chicken, lymphocytes were isolated. RNA was extracted from the obtained lymphocytes, and cDNA was synthesized to prepare a scFv phage antibody library. For the preparation of the phage antibody library, the general procedure described in [nakamura et al., J Vet Med Sci. 2004 Jul; 66 (7): 807-14] was followed.
- anti-Glypican-1 antibody or isotype control antibody (MouseIgG2a, M7769, Sigma) was intraperitoneally administered at a frequency of 10 mg / kg twice a week for a total of 6 times.
- PBS: Matrigel 1: 1: 1).
- anti-Glypican-1 antibody or isotype control antibody (MouseIgG2a, M7769, Sigma) is administered intraperitoneally six times at a frequency of 10 mg / kg twice a week.
- TE-8 transplanted mice are dissected 25 days after the start of antibody administration, and the tumor weight is also measured.
- Tumor volume calculated from major axis x minor axis x height. Since NOD / Scid mice have low NK cell activity, if no anti-tumor effect is observed, it can be said that the anti-tumor effect of the anti-Glypican-1 antibody shown in Scid mice is mainly ADCC activity. If NOD / Scid mice also show an antitumor effect to some extent, it means that the anti-Glypican-1 antibody exerts an antitumor effect by inhibiting the Glypican-1 function itself in addition to the ADCC activity.
- TE14 cells were seeded in a 96-well plate at 2000 cells / well and incubated overnight in a 37 ° C. CO 2 incubator. The cell supernatant of the 96-well plate was discarded, and dilutions of anti-Glypican-1 antibody (0 ⁇ g / ml, 1 ⁇ g / ml, 10 ⁇ g / ml, 100 ⁇ g / ml) were added at 100 ⁇ L / well. Cell proliferation assay was performed 72 hours later by WST-8 assay.
- mouse IgG2 biolegend, 400224, MOPC-173
- MOPC-173 which is a non-anti-LSR antibody
- FIG. Proliferation of esophageal cancer cells (TE14) was suppressed by contacting with anti-Glypican-1 antibody # 19 (2-70).
- Epitope analysis Epitope analysis of various anti-Glypican-1 antibodies was performed. A truncate mutant in which amino acids 33-61 were deleted from the full-length expression vector of human Glypican-1 was prepared. This expression vector was transfected into 293 cells, and the reactivity with various anti-Glypican-1 antibodies was analyzed by FACS. As a result, in two clones # 7 (1-28) and # 19 (2-70), the reactivity in FACS was lost, so # 7 (1-28) and # 19 (2-70) ) Was considered to exist in the 33rd to 61st region (FIG. 16A, FIG. 16B).
- the antigen-antibody complex obtained by immunoprecipitation of the epitope region of anti-Glypican-1 antibody # 4 (1-12) was trypsin digested, and the peptide region bound to the antibody was identified with a mass spectrometer.
- Anti-Glypican-1 antibody # 4 (1-12) or mouse IgG2 (biolegend, 400224, MOPC-173) and recombinant Glypican-1 (R & Dsystems 4519-GP-050) are mixed and immunoprecipitated using protein G sepharose did. Then, trypsin is used for enzymatic digestion in the form of beads, and after washing the beads, the peptide bound to the antibody is eluted with 0.1% formic acid.
- LC-MS / MS analysis and MASCOT search program version 2.4.1; Matrix The peptide was analyzed by database search by Science).
- the anti-GPC1 antibody # 4 (1-12) administration group in the NOD / SCID mice showed a partial but significant inhibition of tumor growth in vivo compared to the control IgG administration group An effect was observed (FIG. 21). There was also a significant difference in tumor weight. Similar results were observed for tumor weight (FIG. 22).
- mice were divided into two groups, and anti-GPC1 antibody (# 1-12) or isotype control antibody (MouseIgG2a, M7769, Sigma) was administered intraperitoneally twice a week for a total of 6 times (FIG. 23). Mice were dissected 24 days after the start of antibody administration, and tumor weights were also measured.
- Tumor volume major axis x minor axis x minor axis x 0.5.
- the anti-GPC1 antibody administration group showed no significant inhibitory effect on the tumor growth in vivo compared to the control IgG administration group (Fig. 24). Similar results were obtained for tumor weight (FIG. 25). This suggests that GPC1 must be expressed in tumor cells in order to exert the anti-tumor effect of the anti-GPC1 antibody.
- mice Since anti-Glypican-1 antibody # 4 (1-12) also cross-reacts with mouse Glypican-1, an acute toxicity test was conducted when administered to mice. 1 mg of mouse IgG2a (Sigma, M7769) or anti-Glypican-1 antibody # 4 (1-12) was intraperitoneally administered to males and females of C57BL / 6J (8w) mice. Then, the heart, kidney, liver, lung, and spleen were removed and subjected to pathological analysis by HE staining. In addition, blood was collected and analyzed using an automatic blood cell counter (VetScanHMII) and an animal biochemical blood analyzer (VetScanVS2) (FIG. 26).
- VetScanHMII automatic blood cell counter
- VetScanVS2 animal biochemical blood analyzer
- Esophageal cancer marker and esophageal cancer control technology are provided, and technology that can be used in industries (reagents, pharmaceuticals, etc.) related to esophageal cancer diagnosis, treatment and prevention technology is provided.
- SEQ ID NO: 1 human Glypican-1 nucleic acid sequence (NM_002081.2) Sequence number 2: Human Glypican-1 protein sequence (P35052) SEQ ID NO: 3: Anti-GPC-1 antibody 1-4 SEQ ID NO: 4: Anti-GPC-1 antibody 1-5 SEQ ID NO: 5: Anti-GPC-1 antibody 1-10 SEQ ID NO: 6: Anti-GPC-1 antibody 1 12 SEQ ID NO: 7: Anti-GPC-1 antibody 1-18 SEQ ID NO: 8: Anti-GPC-1 antibody 1-27 SEQ ID NO: 9: Anti-GPC-1 antibody 1-28 SEQ ID NO: 10: Anti-GPC-1 antibody 1-30 sequence SEQ ID NO: 11: anti-GPC-1 antibody 1-50 sequence SEQ ID NO: 12: anti-GPC-1 antibody 1-57 sequence SEQ ID NO: 13: anti-GPC-1 antibody 1-66 sequence SEQ ID NO: 14: anti-GPC- 1 antibody 1-77 SEQ ID NO: 15: anti-GPC-1 antibody 1-91 SEQ ID NO: 16: anti-GPC-1
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Abstract
Description
からなる群から選ばれる抗体、またはそのフラグメントもしくは機能的等価物を提供する。これらの抗体は、配列番号2の33~61位、または、339~358位および/もしくは388~421位、または430~530位をエピトープとして有していてもよい。これらの抗体は、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(single chain(Fv)2)、およびscFv-Fcから選択される抗体であってもよい。
(a)配列番号1に記載の塩基配列またはそのフラグメント配列を有するポリヌクレオチド;
(b)配列番号2に記載のアミノ酸配列からなるポリペプチドまたはそのフラグメントをコードするポリヌクレオチド;
(c)配列番号2に記載のアミノ酸配列において、1以上のアミノ酸が、置換、付加および欠失からなる群より選択される1つの変異を有する改変体ポリペプチドまたはそのフラグメントであって、生物学的活性を有する改変体ポリペプチドをコードする、ポリヌクレオチド;
(d)配列番号1に記載の塩基配列のスプライス変異体もしくは対立遺伝子変異体またはそのフラグメントである、ポリヌクレオチド;
(e)配列番号2に記載のアミノ酸配列からなるポリペプチドの種相同体またはそのフラグメントをコードする、ポリヌクレオチド;
(f)(a)~(e)のいずれか1つのポリヌクレオチドにストリンジェント条件下でハイブリダイズし、かつ生物学的活性を有するポリペプチドをコードするポリヌクレオチド;または
(g)(a)~(e)のいずれか1つのポリヌクレオチドまたはその相補配列に対する同一性が少なくとも70%、少なくとも80%、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%である塩基配列からなり、かつ、生物学的活性を有するポリペプチドをコードするポリヌクレオチドであり得る。ここで、生物学的活性とは、代表的に、Glypican-1の有する活性またはマーカーとして同じ生物内に存在する他のタンパク質から識別し得ることをいう。
Glypican-1のアミノ酸配列としては、
(a)配列番号2に記載のアミノ酸配列またはそのフラグメントからなる、ポリペプチド;
(b)配列番号2に記載のアミノ酸配列において、1以上のアミノ酸が置換、付加および欠失からなる群より選択される1つの変異を有し、かつ、生物学的活性を有する、ポリペプチド;
(c)配列番号1に記載の塩基配列のスプライス変異体または対立遺伝子変異体によってコードされる、ポリペプチド;
(d)配列番号2に記載のアミノ酸配列の種相同体である、ポリペプチド;または
(e)(a)~(d)のいずれか1つのポリペプチドに対する同一性が少なくとも70%、少なくとも80%、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%であるアミノ酸配列を有し、かつ、生物学的活性を有する、ポリペプチド、であり得る。ここで、生物学的活性とは、代表的に、Glypican-1の有する活性またはマーカーとして同じ生物内に存在する他のタンパク質から識別し得ること(例えば、抗原として用いられる場合特異的エピトープとして機能し得る領域を含むこと)をいう。
以下に本発明の好ましい実施形態を説明する。以下に提供される実施形態は、本発明のよりよい理解のために提供されるものであり、本発明の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本発明の範囲内で適宜改変を行うことができることは明らかである。また、本発明の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。
1つの局面において、本発明は、Glypican-1またはその発現産物あるいはそのフラグメントまたは誘導体を含む、食道がんを識別するためのマーカーを提供する。Glypican-1は生体内に存在するものであり、健常人またはその由来試料では、発現が少なく、食道がんにおいて発現が顕著に高いことから、食道がんの有効な指標マーカーとして用いることができることが本発明において見出された。
からなる群から選ばれる抗体、またはそのフラグメントもしくは機能的等価物を提供する。これらの抗体は、配列番号2の33~61位、または、339~358位および/もしくは388~421位、または430~530位をエピトープとして有していてもよい。これらの抗体は、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(single chain(Fv)2)、およびscFv-Fcから選択される抗体であってもよい。これらの抗体は、本明細書に記載される任意の用途で用いられ得る。
1つの局面において、本発明によれば、本発明による検出、検査および/または診断のための方法を実施するための検出、検査および/または診断のためのキットが提供される。このキットは、本発明の検出剤、検査剤および/または診断剤を含む。その実施形態としては、本明細書において記載された任意の実施形態を単独または組み合わせ用いることができる。
1つの局面では、本発明は、Glypican-1の抑制剤を含む、食道がんを予防または治療するための組成物または医薬(治療薬または予防薬)を提供する。この治療または予防薬を用いれば、食道がんを治療または予防することができる。またこの治療または予防薬は、抗体を使用するため、安全性の観点から優れている。
本明細書において用いられる分子生物学的手法、生化学的手法、微生物学的手法は、当該分野において周知であり慣用されるものであり、例えば、Sambrook J. et al.(1989). Molecular Cloning: A Laboratory Manual,Cold Spring Harborおよびその3rd Ed.(2001); Ausubel, F.M.(1987).Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel,F.M.(1989). Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience;Innis,M.A.(1990).PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel,F.M.(1992).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates;Ausubel,F.M. (1995).Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates;Innis,M.A. et al.(1995).PCR Strategies, Academic Press; Ausubel,F.M.(1999).Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology,Wiley, and annual updates; Sninsky, J.J.et al.(1999). PCR Applications: Protocols for Functional Genomics, Academic Press、別冊実験医学「遺伝子導入&発現解析実験法」羊土社、1997などに記載されており、これらは本明細書において関連する部分(全部であり得る)が参考として援用される。
食道がん手術組織は大阪大学医学部附属病院にてインフォームドコンセントについて同意を得た患者より提供していただいた。
本実施例では、ウェスタンブロットによるGlypican-1の各種細胞における発現を調べた。
正常食道上皮細胞HEEpic, Het1A、食道扁平上皮癌細胞株TE1,TE5, TE6, TE8, TE9, TE10, TE11, TE14, TE15について、氷冷PBS(-)で洗浄後、cell scraperではがし、遠心分離により細胞を回収した。細胞はLysisbuffer(20mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 x protease inhibitorcocktail (ナカライテスク), 1 x phosphatase inhibitor cocktail (ナカライテスク)) により溶解し、遠心分離(13,200rpm,4℃、15min)により上清をタンパク質抽出液として回収した。タンパク質濃度はタンパク質定量キット(DCProtein Assay kit (Bio-Rad Laboratories社))を用いてウシ血清アルブミン(BSA)
をスタンダードとして定量した。
その結果を図1に示す。Glypican-1について、特異的な抗体を用いてWestern blot法にて解析を行った。その結果、Glypican-1がHEEpicとHet1Aで発現しておらず、食道扁平上皮癌細胞株で発現していることを確認した。
本実施例では、Glypican-1(Accession No. P35052)の正常細胞および各種食道がん細胞株における相対発現量を調べた。
正常食道上皮細胞HEEpic,Het1Aに対して食道扁平上皮癌細胞株TE1, TE6, TE8, TE9, TE10, TE14におけるGlypican-1の発現量を相対的に評価した。
その結果を図2に示す。図2に示すように、正常株では、1.2台であったのに対して、食道がん細胞株では、TE6、TE9、TE10およびTE14では2倍以上、TE1およびTE8でも1.6~1.7と高い相対比率を示し、6種類の食道扁平上皮癌細胞株の内、4種類の癌細胞株にて2倍以上に高発現することが分かった。したがって、Glypican-1は食道がんのマーカーとして有用であることが示された。
次に、本実施例では、Glypican-1は食道がん細胞の細胞表面に発現することをFACSにて確認した。
細胞はPBS(Nacalai Tesque)で2回洗浄し、0.02% EDTA solution (Nacalai Tesque)でdishよりはがした。細胞をFACSstaining buffer (PBS supplemented with 1% FBS and 0.1% sodium azide)で2回洗浄し、5倍希釈したgoat anti-human Glypican-1 antibody (R&D Systems, Minneapolis, MN) で染色し、続いて50倍希釈したPE-labeled anti-goat IgG antibodyで染色した。染色した細胞はFACSCanto II (Becton Dickinson, Mountain View, CA, USA)で測定し、FlowJo software (TreeStar, Stanford, CA, USA)を用いてデータ解析した。
結果を図3に示す。示されるように、正常細胞では、Glypican-1の発現はバックグラウンド程度であったのに対して、いずれの食道がん細胞株においてもGlypican-1は有意に発現が増大していることが示された。
本実施例では、食道扁平上皮癌症例の組織(原発およびリンパ節転移)におけるGlypican-1を免疫組織化学染色により確認した。
パラフィン包埋組織の薄切は脱パラフィン処理、アルコールによる脱水を行った。Glypican-1に対する免疫組織化学染色はABC法により抗GPC-1抗体(Atlasantibodies:HPA030571)を用いて行った。
図4Aにその結果を示す。また、さらに別のシリーズで行った結果を図4Bに示す。いずれのシリーズにおいても、Glypican-1について、手術組織に対し免疫組織化学染色法を行うことで、細胞株でなく、癌組織における、癌抗原候補分子の発現について評価した。示されるように、免疫組織化学染色によっても、Glypican-1の食道がん細胞株における有意な発現増強が観察された。この実験の結果、Glypican-1が、食道扁平上皮癌組織の細胞膜において高発現することが判明した。
ヒトの各種正常組織由来RNAはHuman Total RNA Master Panel II (Clontech, Palo Alto, CA, USA)を用いた。食道扁平上皮癌細胞株TE11について、RNeasyminikit(QIAGEN)によりRNAを精製した。QuantiTect Reverse Transcription Kit(Qiagen)を用いてトータルRNAをcDNAへ逆転写した。
・GPC-1, forward primer 5’- GCCAGATCTACGGAGCCAAG-3’(配列番号27;NM_002046.3)
・GPC-1,reverse primer 5’- AGGTTCTCCTCCATCTCGCT-3’ (配列番号28;NM_002046.3)
・GAPDH, forward primer ’-AGCAATGCCTCCTGCACCACCAAC-3’(配列番号29;NM_002081.2)
・GAPDH, reverse primer 5’-CCGGAGGGGCCATCCACAGTCT-3’ (配列番号30;NM_002081.2)
・β-actin,forward primer ’-AGCCTCGCCTTTGCCGA-3’(配列番号31;NM_001101.3)
・β-actin, reverse primer 5’-CTGGTGCCTGGGGCG-3’ (配列番号32;NM_001101.3)。
結果を図5Aに示す。図に示すように、市販の各種ヒト正常組織由来RNAを用いて、リアルタイムPCR解析による各種正常組織におけるglypican-1の発現解析を行い、GAPDH発現に対する相対値としてglypican-1の発現量を調べたところ、正常組織での発現は低いことが確認された。いずれの組織においても、Glypican-1の有意な発現は見られなかったのに対して、食道癌細胞株では、顕著な発現増加がみられた。(図5A)。食道癌組織、非癌部位、TE14を加えた場合でも同様の傾向が見られた。図5BリアルタイムPCR解析による同一患者の食道癌組織および非癌部位におけるGlypican-1の発現解析を行った結果を示す。β-actin発現に対する相対値としてGlypican-1の発現量を調べたところ、食道癌組織では非癌部位と比較して有意な発現レベルの上昇が認められた。したがって、これらの結果からも、本発明のglypican-1は食道がんのマーカーとして有用であることが理解される。
健常人血清(N=5)、及び、食道がん患者血清(N=9)をPBSで2倍希釈し、USCNLife ScienceINC社のhuman ELISAキットで定量を行った。その結果、食道がん患者9例中6例において、健常人血清よりも血清中Glypican-1の濃度が高値を示すことが判明した(図6~図7)。
本実施例では、Glypican-1が食道扁平上皮癌細胞株の増殖と関係しているかどうかについて明らかにするため、Glypican-1に対するsiRNAを用いたGlypican-1の発現抑制を用いて、invitroでの抗腫瘍効果を検討した。
食道がん細胞は96ウェルプレートに2,000cells/wellまき、lipofectamine2000を用いたsiRNAトランスフェクション後72時間後にWST-8アッセイ法によって細胞増殖アッセイを行った。Glypican-1に対するsiRNAおよび、negative control siRNAはQIAGEN社より入手した。
lipofectamine2000を用いてTE6, TE8, TE14 細胞にsiRNAトランスフェクションした72時間後に、PBSで細胞を洗浄し、caspase-3fluorometric assay キット(R&D systems)を用いて測定した。
TE6,TE8, TE14について、氷冷PBS(-)で洗浄後、cell scraperではがし、遠心分離により細胞を回収した。細胞はRIPA buffer(10 mMTris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1%SDS, phosphatase ,1 x protease inhibitor (ナカライテスク)cocktail (ナカライテスク)) により溶解し、遠心分離(13,200rpm,4℃、15min)により上清をタンパク質抽出液として回収した。タンパク質濃度はタンパク質定量キット(DC Protein Assay kit (Bio-RadLaboratories社))を用いてウシ血清アルブミン(BSA)をスタンダードとして定量した。
その結果を図8、9、10A、10Bおよび10Cに示す。Glypican-1に対するsiRNA はGlypican-1陽性食道扁平上皮癌細胞(TE5、TE6、TE8、TE14およびTE15)に対して抗腫瘍効果を示した。このとき、Glypican-1のsiRNAによるGlypican-1の発現抑制により、TE6,TE8, TE14においてcaspase-3活性の上昇が認められたことから、アポトーシスが誘導されていることが明らかになった。また、TE6、TE8およびTE14においてGlypican-1の発現抑制はAKTのリン酸化レベルの低下を引き起こすことが明らかとなった。TE8細胞において、Glypican-1のsiRNAによるGlypican-1の発現抑制により、アポトーシス促進性のタンパク質(pro-apoptotic protein)であるPuma, Bik, Bimの発現増加と抗アポトーシス性のタンパク質(Anti-apoptotic protein)であるBcl-wの発現減少が認められ、さらにEGFRのリン酸化レベルの減少も認められた。
本実施例では、GPC-1ニワトリ抗体を作製し、特徴づけを行った。以下にプロトコールを示す。
ヒトGlypican-1のcDNAをほ乳動物発現ベクター(pcDNA3.1-V5/His-TOPO)にライゲーションし、C末端にV5/Hisタグ融合タンパク質となるようにクローニングした。次に、発現ベクターをニワトリリンパ芽球様細胞株にエレクトロポレーション法でトランスフェクトした後、2mg/mlのG418を添加して発現細胞の選択をおこなった。得られたLSR発現細胞株をニワトリに過免疫した。抗体価の測定はcell-ELISAにて実施した。細胞株はGlypican-1発現ニワトリリンパ芽球様細胞株を4×105cells/wellで使用した。
免疫をおこなったニワトリから脾臓を摘出した後、リンパ球を分離した。得られたリンパ球からRNAを抽出してcDNAの合成を行いscFv ファージ抗体ライブラリーを作製した。ファージ抗体ライブラリーの作製に関しては、[nakamuraet al., J Vet Med Sci. 2004 Jul;66(7):807-14]に記載の一般的な手法に従った。
scFv ファージライブラリーを用いてcell panningを行いGlypican-1特異的なファージの濃縮を行った。Glypican-1非発現細胞株に添加して非特異ファージの吸収操作をおこなった後、ヒトGlypican-1発現細胞株と反応させた。細胞株はリンパ芽球様細胞株を用いた。有機溶媒で洗浄後、Glypican-1発現細胞株に特異的に結合したファージを回収し、大腸菌に感染させた。4回パニングをおこなった後、ライブラリーの反応性をGlypican-1発現細胞株を用いたcell-ELISA、及びFACS解析で確認した。反応性が上昇し始めていたライブラリーからファージのクローニングを行いcell-ELISA、及びFACS解析にて反応性を確認した。陽性クローンを選択した後、配列を決定した。Cell panningに関しては、[Giordano et al., Nat Med. 2001 Nov;7(11):1249-53.]に記載の方法に従った。
抗体を用いたFACS解析を行うにあたり、Glypican-1陰性細胞として、ニワトリTリンパ芽球様細胞株(CT01細胞)、Glypican-1陽性細胞としてヒトGlypican-1を強制発現させたCT01細胞(CT01-GPC-1#42)を用いた。開発した抗体の各種クローンを一次抗体として用い、2次抗体としてはFITC標識GoatAnti-Mouse IgG(H+L chain specific)(southern biothech社)を用い、FACS CantoII (BD社)を用いて測定し、測定データはFlowJo(Tree Star社)を用いて解析した。その結果、Glypican-1をFACSで染色出来る抗体として、20種類のクローンを開発することに成功した。
結果を図11に示す。示されるように、いずれの抗体も反応性の結果から、これらの抗体は、診断薬・治療薬として使用される可能性があることが理解される。
Scidマウス(6週齢、メス)皮下に食道がん細胞株TE-8を2x106cells/100μl (PBS:マトリゲル=1:1)で移植する。移植後14日目にマウスを2群に分け、抗Glypican-1抗体あるいはisotype control抗体(MouseIgG2a、M7769、Sigma)を10mg/kg週2回の頻度で計6回、腹腔内に投与した。TE-8移植マウスは抗体投与開始後25日目に解剖し、腫瘍重量も計測する。腫瘍体積=長径x短径x高さより計算する。これにより抗腫瘍効果を確認することができる。
抗Glypican-1抗体の親和性解析はBiacore3000を用いて測定した。CM5 sensor tipにMouse Antibody Capture Kitウサギ抗マウスIgGポリクローナル抗体を固相化し、各種抗Glypican1抗体クローンをキャプチャーし、リガンドとしてリコンビナントGlypican-1(R&D systems 4519-GP-050)を各種濃度で添加し、結合と解離をモニターした。得られたセンサーグラムはBIAevaluation 4.1 softwareを用いて解析し、KD値を算出した(図12)。その結果抗GPC1抗体(#1-12)はKD値が2.61nMと高親和性を示す事が明らかとなった。
293細胞にマウスGlypican-1発現ベクターあるいは空ベクターを、lipofectamine2000を用いてトランスフェクションし、抗Glypican-1抗体各種クローンとの反応性をFACSにて解析した。その結果、#18(2-63)以外のクローンにおいてマウスGlypican-1と交差反応を示すことが明らかとなった(図13)。
TE14細胞を96ウェルプレートに2000cells/wellまき、37℃のCO2インキュベーターにて一晩インキュベートした。96ウェルプレートの細胞上清を捨て、抗Glypican-1抗体の希釈液(0μg/ml、1μg/ml、10μg/ml、100μg/ml)を100μL/wellずつ加えた。72時間後にWST-8アッセイ法によって細胞増殖アッセイを行った。また、コントロールとして、非抗LSR抗体であるマウスIgG2(biolegend社、400224、MOPC-173)を使用した。結果を図14に示す。抗Glypican-1抗体#19(2-70)を接触させることによって、食道癌細胞(TE14)の増殖が抑制された。
各種抗Glypican-1抗体のエピトープ解析を実施した。ヒトGlypican-1の全長の発現ベクターより33-61番目のアミノ酸を欠失させたtruncate mutantを作成した。この発現ベクターを293細胞にトランスフェクションし、各種抗Glypican-1抗体との反応性をFACSにより解析した。その結果、#7(1-28)と#19(2-70)の2クローンにおいて、FACSでの反応性が消失していたことから、#7(1-28)と#19(2-70)のエピトープは33-61番目の領域に存在すると考えられた(図16A、図16B)。
SCIDマウス(6週齢、メス)皮下に食道扁平上皮癌細胞株E14を2x106cells/100μl(PBS:マトリゲル=1:1)で移植した。移植後14日目にマウスを2群に分け、抗GPC1抗体#4(1-12)、抗GPC1抗体#19(2-70)あるいはisotypecontrol抗体(MouseIgG2a、M7769、Sigma)を10mg/kg週2回の頻度で計6回、腹腔内に投与した(図18)。TE14移植マウスは抗体投与開始後24日目に解剖し、腫瘍重量も計測した。腫瘍体積=長径x短径x短径x0.5より計算した。
NOD/SCIDマウス(6週齢、メス)皮下に食道扁平上皮癌細胞株TE14を2x106cells/100μl (PBS:マトリゲル=1:1)で移植した。移植後14日目にマウスを2群に分け、抗GPC1抗体#4(1-12)、抗GPC1抗体#19(2-70)あるいはisotypecontrol抗体(MouseIgG2a、M7769、Sigma)を10mg/kg週2回の頻度で計6回、腹腔内に投与した。TE14移植マウスは抗体投与開始後24日目に解剖し、腫瘍重量も計測した。腫瘍体積=長径x短径x短径x0.5より計算した。
SCIDマウス(6週齢、メス)皮下にGPC1陰性の肺扁平上皮癌細胞株であるLK2に2×106cells/100μl(PBS:マトリゲル=1:1)で移植した。移植後14日目にマウスを2群に分け、抗GPC1抗体(#1-12)あるいはisotype control抗体(MouseIgG2a、M7769、Sigma)を1週2回の頻度で計6回、腹腔内に投与した(図23)。マウスは抗体投与開始後24目に解剖し、腫瘍重量も計測した。腫瘍体積=長径x短径x短径x0.5より計算した。
抗Glypican-1抗体#4(1-12)はマウスGlypican-1とも交差反応を示すため、マウスに投与したときの急性毒性試験を実施した。C57BL/6J(8w)マウスの雄、雌それぞれにマウスIgG2a(Sigma,M7769)あるいは抗Glypican-1抗体 #4(1-12)を1mg腹腔内投与し、7日目にマウスを解剖し、脳、心臓、腎臓、肝臓、肺、脾臓を摘出しHE染色による病理解析を実施した。また、採血を行い、自動血球計測装置(VetScanHMII)、動物用生化学血液分析器(VetScanVS2)を用いて解析した(図26)。その結果、血球数のデータにおいて両者に有意な変化は認められなかった(図27、図28)。同様に血液生化学データにおいても両者に有意な変化は認められなかった(図29、図30)。このことから抗LSR抗体#1-25は毒性が少なく安全性が高いことが示唆された。
配列番号2:ヒトGlypican-1タンパク質配列(P35052)
配列番号3:抗GPC-1抗体1-4配列
配列番号4:抗GPC-1抗体1-5配列
配列番号5:抗GPC-1抗体1-10配列
配列番号6:抗GPC-1抗体1-12配列
配列番号7:抗GPC-1抗体1-18配列
配列番号8:抗GPC-1抗体1-27配列
配列番号9:抗GPC-1抗体1-28配列
配列番号10:抗GPC-1抗体1-30配列
配列番号11:抗GPC-1抗体1-50配列
配列番号12:抗GPC-1抗体1-57配列
配列番号13:抗GPC-1抗体1-66配列
配列番号14:抗GPC-1抗体1-77配列
配列番号15:抗GPC-1抗体1-91配列
配列番号16:抗GPC-1抗体2-11配列
配列番号17:抗GPC-1抗体2-14配列
配列番号18:抗GPC-1抗体2-57配列
配列番号19:抗GPC-1抗体2-60配列
配列番号20:抗GPC-1抗体2-63配列
配列番号21:抗GPC-1抗体2-70配列
配列番号22:抗GPC-1抗体2-77配列
配列番号23:Glypican-1 siRNAコア配列(ガイド配列)のセンス鎖配列
配列番号24:Glypican-1 siRNAコア配列(ガイド配列)のアンチセンス鎖配列
配列番号25:Glypican-1 siRNAセンス鎖全長配列
配列番号26:Glypican-1 siRNAアンチセンス鎖全長配列
配列番号27:GPC-1フォワードプライマー配列
配列番号28:GPC-1リバースプライマー配列
配列番号29:GAPDHフォワードプライマー配列
配列番号30:GAPDHリバースプライマー配列
配列番号31:βアクチンフォワードプライマー配列
配列番号32:βアクチンリバースプライマー配列
Claims (51)
- Glypican-1またはその発現産物に結合する物質を含む、食道がんを識別するための検出剤。
- 前記検出剤は、抗体またはそのフラグメントもしくは機能的等価物、あるいは核酸である、請求項1に記載の検出剤。
- 前記検出剤は、標識されたものである、請求項1に記載の検出剤。
- 前記Glypican-1は、配列番号1(核酸配列)または配列番号2(アミノ酸配列)あるいはその等価物である、請求項1に記載の検出剤。
- 前記検出剤は、抗体またはそのフラグメントもしくは機能的等価物であり、該抗体は以下:
(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号3の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(b)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号4の31~35位、50~66位、99~110位、159~166位、183~187位、および222~231位で示されるアミノ酸配列を含む抗体、
(c)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号5の31~35位、50~66位、99~113位、162~170位、187~193位、および226~237位で示されるアミノ酸配列を含む抗体、
(d)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号6の31~35位、51~66位、99~112位、160~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(e)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号7の31~35位、50~66位、99~120位、169~176位、193~199位、および232~242位で示されるアミノ酸配列を含む抗体、
(f)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号8の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(g)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号9の31~35位、50~66位、99~120位、169~176位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(h)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号10の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(i)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号11の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(j)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号12の31~35位、50~66位、99~113位、162~169位、186~192位、および225~234位で示されるアミノ酸配列を含む抗体、
(k)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号13の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(l)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号14の31~35位、50~66位、99~113位、162~169位、186~192位、および225~235位で示されるアミノ酸配列を含む抗体、
(m)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号15の31~35位、50~66位、99~113位、162~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(n)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号16の31~35位、50~66位、99~110位、159~166位、183~189位、および222~237で示されるアミノ酸配列を含む抗体、
(o)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号17の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(p)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号18の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(q)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号19の31~35位、50~66位、99~115位、164~175位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(r)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号20の31~35位、50~66位、99~117位、166~177位、194~200位、および233~242位で示されるアミノ酸配列を含む抗体、
(s)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号21の32~36位、51~67位、100~114位、163~171位、188~194位、および227~236位で示されるアミノ酸配列を含む抗体、および
(t)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号22の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
からなる群から選ばれる1種以上の抗体、あるいは該抗体の変異体であって、該変異体において該抗体のフレームワークに1または数個の置換、付加もしくは欠失を含むが、該CDRには変異を含まない、変異体
を有することを特徴とする、請求項3に記載の検出剤。 - 前記検出剤は、抗体またはそのフラグメントもしくは機能的等価物であり、該抗体は、配列番号2の33~61位、または、339~358位および/もしくは388~421位、または430~530位をエピトープとして有する抗体またはそのフラグメントもしくは機能的等価物である、請求項3に記載の検出剤。
- 前記検出剤は、核酸であり、該核酸はプローブまたはプライマーである、請求項1に記載の検出剤。
- 前記プローブまたはプライマーは、配列番号1に記載の核酸配列またはその相補鎖あるいはその変異体の断片である、請求項7に記載の検出剤。
- 前記プローブまたはプライマーは、配列番号25および/または26を有することを特徴とする、請求項7に記載の検出剤。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項1に記載の検出剤。
- Glypican-1またはその発現産物に結合する物質を用いる、食道がんを識別する方法。
- 対象検体のGlypican-1の発現が正常検体のものに比べて上昇している場合、該対象検体は食道がんを有すると診断されることを特徴とする、請求項11に記載の方法。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項11に記載の方法。
- Glypican-1またはその発現産物あるいはそのフラグメントまたは誘導体を含む、食道がんを識別するためのマーカー。
- 前記食道がんは扁平上皮癌および腺癌を含む、請求項14に記載のマーカー。
- 前記食道がんは扁平上皮癌を含む、請求項14に記載のマーカー。
- 前記食道がんはヒトのものである、請求項14に記載のマーカー。
- 前記食道がんはリンパ節転移部位のものを含む、請求項14に記載のマーカー。
- 前記Glypican-1は、配列番号1(核酸配列)もしくはその相補鎖または配列番号2(アミノ酸配列)あるいはその等価物である、請求項14に記載のマーカー。
- Glypican-1またはその発現産物を、食道がんを識別する指標とする方法。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項20に記載の方法。
- 対象検体のGlypican-1の発現が正常検体のものに比べて上昇している場合、該対象検体は食道がんを有すると診断されることを特徴とする、請求項20に記載の方法。
- 前記識別は、請求項1~10のいずれか1項に記載の検出剤を用いて行われる、請求項20に記載の方法。
- Glypican-1の抑制剤を含む、食道がんの予防または治療薬。
- 前記食道がんは、Glypican-1陽性である請求項24に記載の予防または治療薬。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項24に記載の予防または治療薬。
- 前記食道がんは扁平上皮癌を含む、請求項24に記載の予防または治療薬。
- Glypican-1陽性食道がんを発症していると判断された患者に対して投与するための、請求項24または25に記載の予防または治療薬。
- 前記Glypican-1の抑制剤は、抗体またはそのフラグメントもしくは機能的等価物、あるいは核酸である、請求項24に記載の予防または治療薬。
- 前記Glypican-1の抑制剤は、核酸であり、該核酸は、siRNAである、請求項24に記載の予防または治療薬。
- 前記siRNAは、配列番号25および/または配列番号26を含む、請求項30に記載の予防または治療薬。
- 前記Glypican-1の抑制剤は、抗体またはそのフラグメントもしくは機能的等価物であり、該抗体は以下:
(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号3の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(b)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号4の31~35位、50~66位、99~110位、159~166位、183~187位、および222~231位で示されるアミノ酸配列を含む抗体、
(c)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号5の31~35位、50~66位、99~113位、162~170位、187~193位、および226~237位で示されるアミノ酸配列を含む抗体、
(d)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号6の31~35位、51~66位、99~112位、160~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(e)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号7の31~35位、50~66位、99~120位、169~176位、193~199位、および232~242位で示されるアミノ酸配列を含む抗体、
(f)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号8の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(g)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号9の31~35位、50~66位、99~120位、169~176位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(h)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号10の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(i)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号11の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(j)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号12の31~35位、50~66位、99~113位、162~169位、186~192位、および225~234位で示されるアミノ酸配列を含む抗体、
(k)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号13の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(l)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号14の31~35位、50~66位、99~113位、162~169位、186~192位、および225~235位で示されるアミノ酸配列を含む抗体、
(m)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号15の31~35位、50~66位、99~113位、162~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(n)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号16の31~35位、50~66位、99~110位、159~166位、183~189位、および222~237で示されるアミノ酸配列を含む抗体、
(o)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号17の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(p)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号18の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(q)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号19の31~35位、50~66位、99~115位、164~175位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(r)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号20の31~35位、50~66位、99~117位、166~177位、194~200位、および233~242位で示されるアミノ酸配列を含む抗体、
(s)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号21の32~36位、51~67位、100~114位、163~171位、188~194位、および227~236位で示されるアミノ酸配列を含む抗体、および
(t)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号22の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
からなる群から選ばれる1種以上の抗体、あるいは該抗体の変異体であって、該変異体に
おいて該抗体のフレームワークに1または数個の置換、付加もしくは欠失を含むが、該CDRには変異を含まない、変異体
を有することを特徴とする、請求項24に記載の予防または治療薬。 - 前記Glypican-1の抑制剤は、抗体またはそのフラグメントもしくは機能的等価物であり、該抗体またはそのフラグメントもしくは機能的等価物は、配列番号2の33~61位、または、339~358位および/もしくは388~421位、または430~530位をエピトープとして有する抗体またはそのフラグメントもしくは機能的等価物である、請求項24に記載の予防または治療薬。
- 前記抗体は、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(single chain (Fv)2)、およびscFv-Fcから選択される抗体である、請求項32または33に記載の予防または治療薬。
- 有効量のGlypican-1の抑制剤を必要とする被験者に投与することを含む、該被験者の食道がんを予防または治療するための方法。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項35に記載の方法。
- 前記被験者のサンプルがGlypican-1陽性であることを検査する工程を含む、該被験者の食道がんを予防または治療するための方法。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項37に記載の方法。
- Glypican-1の結合剤を含む、食道がんの予防または治療薬。
- さらに細胞殺傷性薬剤を含む、請求項39に記載の予防または治療薬。
- 前記食道がんはリンパ節転移部位のもの、扁平上皮癌および/または腺癌を含む、請求項39に記載の予防または治療薬。
- 前記Glypican-1の結合剤は、抗体またはそのフラグメントもしくは機能的等価物、あるいは核酸である、請求項39に記載の予防または治療薬。
- 前記Glypican-1の結合剤は、抗体またはそのフラグメントもしくは機能的等価物であり、細胞殺傷性薬剤がさらに結合されたものである、請求項39に記載の予防または治療薬。
- 前記食道がんは、Glypican-1陽性である請求項39に記載の予防または治療薬。
- 前記食道がんは扁平上皮癌を含む、請求項39に記載の予防または治療薬。
- 前記Glypican-1の結合剤は、抗体またはそのフラグメントもしくは機能的等価物であり、該抗体は以下:
(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号3の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(b)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号4の31~35位、50~66位、99~110位、159~166位、183~187位、および222~231位で示されるアミノ酸配列を含む抗体、
(c)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号5の31~35位、50~66位、99~113位、162~170位、187~193位、および226~237位で示されるアミノ酸配列を含む抗体、
(d)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号6の31~35位、51~66位、99~112位、160~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(e)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号7の31~35位、50~66位、99~120位、169~176位、193~199位、および232~242位で示されるアミノ酸配列を含む抗体、
(f)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号8の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(g)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号9の31~35位、50~66位、99~120位、169~176位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(h)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号10の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(i)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号11の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(j)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号12の31~35位、50~66位、99~113位、162~169位、186~192位、および225~234位で示されるアミノ酸配列を含む抗体、
(k)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号13の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(l)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号14の31~35位、50~66位、99~113位、162~169位、186~192位、および225~235位で示されるアミノ酸配列を含む抗体、
(m)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号15の31~35位、50~66位、99~113位、162~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(n)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号16の31~35位、50~66位、99~110位、159~166位、183~189位、および222~237で示されるアミノ酸配列を含む抗体、
(o)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号17の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(p)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号18の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(q)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号19の31~35位、50~66位、99~115位、164~175位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(r)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号20の31~35位、50~66位、99~117位、166~177位、194~200位、および233~242位で示されるアミノ酸配列を含む抗体、
(s)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号21の32~36位、51~67位、100~114位、163~171位、188~194位、および227~236位で示されるアミノ酸配列を含む抗体、および
(t)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号22の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
からなる群から選ばれる1種以上の抗体、あるいは該抗体の変異体であって、該変異体において該抗体のフレームワークに1または数個の置換、付加もしくは欠失を含むが、該CDRには変異を含まない、変異体を有することを特徴とする、請求項39に記載の予防または治療薬。 - 前記Glypican-1の抑制剤は、抗体またはそのフラグメントもしくは機能的等価物であり、該抗体またはそのフラグメントもしくは機能的等価物は、配列番号2の33~61位、または、339~358位および/もしくは388~421位、または430~530位をエピトープとして有する抗体またはそのフラグメントもしくは機能的等価物である、請求項39に記載の予防または治療薬。
- 前記抗体は、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(single chain (Fv)2)、およびscFv-Fcから選択される抗体である、請求項46または47に記載の予防または治療薬。
- 以下の抗体:
(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号3の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(b)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号4の31~35位、50~66位、99~110位、159~166位、183~187位、および222~231位で示されるアミノ酸配列を含む抗体、
(c)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号5の31~35位、50~66位、99~113位、162~170位、187~193位、および226~237位で示されるアミノ酸配列を含む抗体、
(d)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号6の31~35位、51~66位、99~112位、160~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(e)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号7の31~35位、50~66位、99~120位、169~176位、193~199位、および232~242位で示されるアミノ酸配列を含む抗体、
(f)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号8の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(g)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号9の31~35位、50~66位、99~120位、169~176位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(h)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号10の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(i)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号11の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、
(j)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号12の31~35位、50~66位、99~113位、162~169位、186~192位、および225~234位で示されるアミノ酸配列を含む抗体、
(k)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号13の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(l)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号14の31~35位、50~66位、99~113位、162~169位、186~192位、および225~235位で示されるアミノ酸配列を含む抗体、
(m)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号15の31~35位、50~66位、99~113位、162~170位、187~193位、および226~236位で示されるアミノ酸配列を含む抗体、
(n)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号16の31~35位、50~66位、99~110位、159~166位、183~189位、および222~237で示されるアミノ酸配列を含む抗体、
(o)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号17の31~35位、50~65位、98~113位、162~171位、188~194位、および227~237位で示されるアミノ酸配列を含む抗体、
(p)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号18の31~35位、50~66位、99~116位、165~174位、191~197位、および230~240位で示されるアミノ酸配列を含む抗体、
(q)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号19の31~35位、50~66位、99~115位、164~175位、193~199位、および232~241位で示されるアミノ酸配列を含む抗体、
(r)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号20の31~35位、50~66位、99~117位、166~177位、194~200位、および233~242位で示されるアミノ酸配列を含む抗体、
(s)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号21の32~36位、51~67位、100~114位、163~171位、188~194位、および227~236位で示されるアミノ酸配列を含む抗体、
(t)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号22の31~35位、50~65位、98~114位、163~170位、187~193位、および226~235位で示されるアミノ酸配列を含む抗体、および
(u)該(a)~(t)のいずれかの抗体において、該抗体のフレームワークに1または数個の置換、付加もしくは欠失を含むが、該CDRには変異を含まない、抗体
からなる群から選ばれる抗体、またはそのフラグメントもしくは機能的等価物。 - 配列番号2の33~61位、または、339~358位および/もしくは388~421位、または430~530位をエピトープとして有する抗体またはそのフラグメントもしくは機能的等価物。
- 前記抗体は、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(single chain (Fv)2)、およびscFv-Fcから選択される抗体である、請求項49または50に記載の抗体またはそのフラグメントもしくは機能的等価物。
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CN113784987A (zh) * | 2019-01-22 | 2021-12-10 | 美国政府(由卫生和人类服务部的部长所代表) | 靶向磷脂酰肌醇蛋白聚糖-1的高亲和力单克隆抗体和使用方法 |
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JP6606793B2 (ja) | 2019-11-20 |
JP6500933B2 (ja) | 2019-04-17 |
US10077316B2 (en) | 2018-09-18 |
US20210340275A1 (en) | 2021-11-04 |
US20180362658A1 (en) | 2018-12-20 |
JP2017186343A (ja) | 2017-10-12 |
JP2018123136A (ja) | 2018-08-09 |
US20170066836A1 (en) | 2017-03-09 |
JPWO2015098112A1 (ja) | 2017-03-23 |
JP6455935B2 (ja) | 2019-01-23 |
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