WO2015098113A1 - 悪性腫瘍の治療薬 - Google Patents
悪性腫瘍の治療薬 Download PDFInfo
- Publication number
- WO2015098113A1 WO2015098113A1 PCT/JP2014/006456 JP2014006456W WO2015098113A1 WO 2015098113 A1 WO2015098113 A1 WO 2015098113A1 JP 2014006456 W JP2014006456 W JP 2014006456W WO 2015098113 A1 WO2015098113 A1 WO 2015098113A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- lsr
- malignant tumor
- positions
- chain cdr1
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3023—Lung
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3046—Stomach, Intestines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/57449—Specifically defined cancers of ovaries
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Definitions
- the present invention relates to a therapeutic agent or diagnostic agent for malignant tumors.
- LSR Lipolysis-stimulated lipoprotein receptor
- Non-Patent Document 1 describes that LSR expression is decreased in the liver of a mouse model of obesity and type 2 diabetes.
- Non-Patent Document 2 describes that LSR is expressed in bladder cancer cells.
- Non-Patent Document 3 describes that LSR is expressed in colon cancer cells.
- Non-Patent Document 4 describes that LSR is expressed in breast cancer cells.
- Patent Document 1 describes that LSR is expressed in ovarian cancer cells and the like.
- Patent Document 1 it is suggested that LSRRNA mRNA was detected in several cancers, and in the claims, anti-LSR antibodies that induce apoptosis of cancer cells are described in terms of words. Has been. However, Patent Document 1 does not include pharmacological data that has actually been successfully treated for cancer. In addition, Patent Document 1 does not describe diagnosing the presence or absence of LSR positivity in malignant tumor patients before treatment. For this reason, the results of Patent Document 1 alone cannot be said to be effective for the treatment of malignant tumors.
- the present invention provides a therapeutic or prophylactic agent for malignant tumors, including an inhibitor of LSR (Lipolysis stimulated lipoprotein receptor).
- LSR Lipolysis stimulated lipoprotein receptor
- the inhibitor in the present invention may include an anti-LSR (Lipolysis stimulated lipoprotein receptor) antibody, an antigen-binding fragment thereof, a functional equivalent thereof, or a nucleic acid.
- LSR Lipolysis stimulated lipoprotein receptor
- the inhibitor may comprise an anti-LSR (Lipolysis stimulated lipoprotein receptor) antibody, an antigen-binding fragment thereof, or a functional equivalent thereof.
- LSR Lipolysis stimulated lipoprotein receptor
- the inhibitor may be an RNAi molecule against LSR or a polynucleotide encoding the RNAi molecule.
- the malignant tumor may be an LSR-positive malignant tumor.
- the present invention may be for administration to a patient determined to have developed an LSR positive malignancy.
- the present invention may be for administration to a malignant tumor patient who has been determined to be an LSR-positive malignant tumor.
- the anti-LSR antibody may be an anti-LSR antibody that specifically binds to an epitope of LSR. More specifically, the antibody may have an epitope at positions 116 to 134 and / or 216 to 230 of SEQ ID NO: 7.
- the anti-LSR antibody may be an antibody having an ability to inhibit enhancement by VLDL.
- the anti-LSR antibody comprises: (a) heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are the positions 31 to 35, 50 to 50 of SEQ ID NO: 1, respectively.
- An antibody comprising an amino acid sequence shown at positions 66, 99-104, 153-165, 182-188, and 221-230,
- Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-104, 153-165, 182-188, and 221 of SEQ ID NO: 3, respectively.
- An antibody comprising an amino acid sequence shown at position 229, (d) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, and 99 to 99 of SEQ ID NO: 4, respectively.
- An antibody comprising the amino acid sequence shown at positions 104, 153-165, 182-188, and 221-229, (e) Strands CDR1, 2, 3, light chain CDR1, 2, and 3 are SEQ ID NO: 5 at positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-2, respectively.
- An antibody comprising the amino acid sequence shown at position 229, and (f) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-99 of SEQ ID NO: 6, respectively.
- a variant may be a variant that contains one or several substitutions, additions or deletions in the framework of the antibody, but does not contain a mutation in the CDR.
- the anti-LSR antibody may be a monoclonal antibody.
- the antibody class of the anti-LSR antibody may be IgG.
- the anti-LSR antibody may be an antigen-binding fragment.
- the present invention provides a cell division inhibitor for malignant tumor cells, comprising an anti-LSR antibody.
- the present invention provides a companion diagnostic agent for treating malignant tumors targeting LSR, which comprises a detection agent for LSR.
- the LSR detection agent may comprise an anti-LSR antibody.
- the present invention provides a companion diagnostic method for malignant tumor treatment targeting LSR, comprising the step of testing that a malignant tumor sample of a malignant tumor patient is LSR positive.
- the present invention provides: (a) Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, and 182 to 188 of SEQ ID NO: 1, respectively. And an antibody comprising the amino acid sequence shown at positions 221-230, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31-35 and 50-66 of SEQ ID NO: 2, respectively.
- antibodies selected from the group consisting of antibodies, or variants of the antibodies, wherein the variants contain one or several substitutions, additions or deletions in the framework of the antibodies, A variant is provided in which the CDR does not contain the mutation.
- 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 composition for preventing or treating a malignant tumor, comprising an LSR binding agent.
- the malignant tumor may be an LSR positive malignant tumor.
- the present invention may further comprise a cell killing agent.
- the LSR binding agent in the present invention may be an antibody, a fragment or functional equivalent thereof, or a nucleic acid.
- the LSR binding agent of the present invention is an antibody or a fragment or functional equivalent thereof, and may be further bound with a cell-killing agent.
- the malignant tumor in the present invention may comprise ovarian cancer.
- the ovarian cancer in the present invention may be recurrent ovarian cancer.
- the malignant tumor may be a metastasis of ovarian cancer.
- the malignant tumor may include ovarian cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
- the malignant tumor may be early ovarian cancer.
- the ovarian cancer can be ovarian serous adenocarcinoma or ovarian clear cell adenocarcinoma.
- the LSR binding agent in the present invention is an antibody or a fragment or functional equivalent thereof, and the antibody comprises: (a) heavy chain CDR1, 2, 3, light chain CDR1, 2, and An antibody, wherein 3 comprises the amino acid sequence shown at positions 31 to 35, 50 to 66, 99 to 104, 153-165, 182 to 188, and 221 to 230 of SEQ ID NO: 1, respectively (b) Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-103, 152-165, 182-188, and 221 of SEQ ID NO: 2, respectively.
- 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.
- a therapeutic agent for malignant tumors comprising an anti-LSR antibody is provided.
- a therapeutic agent for malignant tumor comprising an LSR antagonist is provided.
- a cell division inhibitor for malignant tumor cells comprising an anti-LSR antibody is provided.
- a companion diagnostic agent for treating malignant tumors targeting LSR including an anti-LSR antibody.
- the malignant tumor is determined to be LSR-positive by the companion diagnostic method of the present invention, and the LSR binding agent is subsequently administered. Also good.
- a companion diagnostic method for malignant tumor treatment targeting LSR which comprises the step of examining whether a malignant tumor sample of a malignant tumor patient is LSR positive.
- the malignancy may be an LSR positive malignancy.
- the therapeutic agent and in one embodiment of the present invention, the therapeutic agent is a therapeutic agent for administration to a patient determined to have developed an LSR-positive malignant tumor. It may be.
- the therapeutic agent may be a therapeutic agent for administration to a patient whose tumor is determined to be an LSR-positive malignant tumor among tumor patients.
- the anti-LSR antibody may be an anti-LSR antibody that specifically binds to an epitope of LSR.
- the anti-LSR antibody is (a) Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, and 182 to 188 of SEQ ID NO: 1, respectively. And an antibody comprising the amino acid sequence shown at positions 221-230, (b) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are positions 31-35 and 50-66 of SEQ ID NO: 2, respectively.
- heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 contain the amino acid sequences shown at positions 31 to 35, 50 to 66, 99 to 104, 153-165, 182-188, and 221-229 of SEQ ID NO: 6, respectively.
- the CDR may be a mutant that does not contain a mutation.
- 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 antibody to be used is not limited, but an antibody having SEQ ID NO: 7 positions 116 to 134 and / or positions 216 to 230 as an epitope can be advantageously used. This is because in this specification, advantageous effects are shown, and safety and stability are also shown.
- the anti-LSR antibody may be a monoclonal antibody.
- the antibody class of the anti-LSR antibody may be IgG.
- the anti-LSR antibody may be an antigen-binding fragment.
- the LSR antagonist may be an RNAi molecule against or a polynucleotide encoding the RNAi molecule.
- the present invention provides a marker for poor prognosis of malignant tumor treatment, which comprises a binding agent of LSR (Lipolysis stimulated lipoprotein receptor).
- a binding agent of LSR Lipolysis stimulated lipoprotein receptor
- the binder used in this aspect can be any form of the binder of the invention described herein.
- the binding agent can be an antibody or fragment or functional equivalent thereof, or a nucleic acid, and can be labeled.
- the present invention provides a method of using the expression level of LSR (Lipolysis stimulated lipoprotein receptor) as an indicator of poor prognosis in the treatment of malignant tumors.
- LSR Lipolysis stimulated lipoprotein receptor
- the binder used in this aspect can be any form of the binder of the invention described herein.
- the binding agent can be an antibody or fragment or functional equivalent thereof, or a nucleic acid, and can be labeled.
- the present invention provides a diagnostic agent for poor prognosis in the treatment of malignant tumors, comprising a LSR (Lipolysis stimulated lipoprotein receptor) binder.
- a LSR Lipolysis stimulated lipoprotein receptor
- the binder used in this aspect can be any form of the binder of the invention described herein.
- the binding agent can be an antibody or fragment or functional equivalent thereof, or a nucleic acid, and can be labeled.
- the present invention provides a therapeutic method, preventive method, use, etc. using the pharmaceutical composition, therapeutic agent or prophylactic agent of the present invention.
- a novel therapeutic agent or diagnostic agent for malignant tumors can be obtained.
- FIG. 1 shows the results of RT-PCR performed on nucleic acids obtained from an ovarian serous adenocarcinoma cell line. Two on the left show normal cells (HOSE2 from the left), and three on the right show cancer cells (OVCAR3, OVSAHO, JHOS4 from the left). The top row shows LSR, and the bottom row shows the background ⁇ -actin.
- FIG. 2 shows the results of RT-PCR performed on nucleic acids obtained from ovarian clear cell line cancer cell lines. Two on the left show normal cells (HOSE2C from the left), and four on the right show cancer cells (from the left, OVTOKO, OVMANA, OVISE, RMG-1). The top row shows LSR, and the bottom row shows the background ⁇ -actin.
- FIG. 3 shows the results of RT-PCR performed on nucleic acids obtained from endometrial cancer cell lines.
- the left end shows normal cells (E6 / E7 / TERT), and the rest are cancer cells. From left, HEC1, HEC1A, HEC6, HEC88nu, HEC108, HEC116, HEC251, and SNG-M are shown.
- the top row shows LSR, and the bottom row shows the background ⁇ -actin.
- FIG. 4 shows the results of Western blotting for proteins obtained from ovarian serous adenocarcinoma cell lines. Two on the left indicate normal cells (HOSE2C from the left), and three on the right indicate cancer cells (OVCAR3, OVSAHO, JHOS4 from the left).
- FIG. 5 shows the results of Western blotting performed on proteins obtained from ovarian clear cell line cancer cell lines. Two on the left show normal cells (HOSE2C from the left), and four on the right show cancer cells (from the left, OVTOKO, OVMANA, OVISE, RMG-1).
- FIG. 6 shows the results of Western blotting performed on proteins obtained from endometrial cancer cell lines. The upper left corner shows normal cells (E6 / E7 / TERT), and the rest are cancer cells. HEC1, HEC1A, HEC6, and HEC88nu are shown from the upper left, and HEC108, HEC116, HEC251, and SNG-M are shown from the lower left. Both indicate LSR.
- FIG. 8 is a view showing the results of Western blotting performed on a protein obtained from an endometrial cancer surgical tissue.
- FIG. 9 shows the amino acid sequence of the anti-LSR antibody described in the examples.
- FIG. 10 is a diagram showing the results of evaluation of reactivity to OVSAHO, JHSO4, RMG-I, and OVISE from the left for antibody # 9-7. The vertical axis represents intensity, and the horizontal axis represents cell frequency.
- FIG. 11 is a diagram showing the results of evaluation of reactivity to OVSAHO, JHSO4, RMG-I, and OVISE from the left for antibody # 16-6.
- FIG. 12 is a diagram showing the results of evaluation of reactivity to OVSAHO, JHSO4, RMG-I, and OVISE from the left for antibody # 26-2.
- the vertical axis represents intensity, and the horizontal axis represents cell frequency.
- FIG. 13 is a diagram showing the results of evaluation of reactivity to OVSAHO, JHSO4, RMG-I, and OVISE from the left for antibody # 27-6.
- the vertical axis represents intensity, and the horizontal axis represents cell frequency.
- FIG. 14 is a diagram showing the results of evaluation of reactivity to OVSAHO, JHSO4, RMG-I, and OVISE from the left for antibody # 1-25.
- FIG. 15 shows the results of LSR expression analysis performed on ovarian serous adenocarcinoma tissue by immunohistochemical staining using monoclonal antibody # 1-25.
- the top row shows ovarian serous G2, oviduct serous G3, ovarian serous G3, and ovarian serous G3.
- the lower row shows ovarian clear cells.
- FIG. 16 shows the results of LSR expression analysis performed on ovarian serous adenocarcinoma tissue by immunohistochemical staining using monoclonal antibody # 1-25.
- FIG. 17 shows the results of LSR expression analysis performed on endometrial cancer tissue by immunohistochemical staining using monoclonal antibody # 9-7 (4.5 ⁇ g / ml).
- FIG. 18 is a graph showing the results of evaluating the growth inhibitory effect on RMG-I for antibody # 9-7.
- the vertical axis shows the relative growth relative to the treatment.
- the horizontal axis represents the dose of IgG.
- FIG. 19 is a diagram showing the results of evaluating the growth inhibitory effect on RMG-I for antibody # 1-25.
- the vertical axis shows the relative growth relative to the treatment.
- the horizontal axis represents the dose of IgG.
- FIG. 20 shows # 1-25 antibody (3 on the left, 1 ⁇ g / ml, 10 ⁇ g / ml, 100 ⁇ g / ml from the left) and # 26-2 antibody (3 on the left, 1 ⁇ g / ml, 10 ⁇ g / ml respectively from the left) , 100 ⁇ g / ml) is a graph showing the results of evaluating the growth inhibitory effect on A2780.
- the control IgG is shown on the right (3 on the right, 1 ⁇ g / ml, 10 ⁇ g / ml, and 100 ⁇ g / ml from the left). Both were effective, but # 1-25 showed stronger effect than # 26-2.
- FIG. 21 is a diagram showing the results of evaluating the growth inhibitory effect against OVSAHO for the LSRsiRNA described in the Examples. From the left, the first day, the second day, the third day, the fourth day, and the fifth day are shown. From the left, the four bars indicate the untreated average amount, the control average value, the LSR siRNA1 average value, The average value of LSR siRNA2 is shown.
- FIG. 22 is a view showing a result of enhanced lipid (cholesterol) uptake in LSR stably expressing cells described in Examples. The left shows the effect on total cholesterol, the middle graph shows the effect on triglycerides, and the effect on phospholipids.
- cholesterol enhanced lipid
- the vertical axis represents each uptake (mg / ml), and the horizontal axis represents EMP1 low density, EMP1 high density, L45 low density, and L45 high density, respectively.
- EMP represents a vector-introduced cell
- L represents an LSR forced expression cell.
- FIG. 23 is a graph showing the results of enhanced lipid (cholesterol) uptake in high-density culture in LSR stably expressing cells described in Examples.
- the vertical axis shows total cholesterol uptake (mg / ml).
- EMP1 represents an empty vector-introduced cell
- L45 represents an LSR forced expression cell.
- FIG. 24 is a diagram showing the results of LSR expression described in the Examples that promotes VLDL metabolism, but inhibition of VLDL-induced metabolism is inhibited by administration of LSR antibody.
- # 9-7 inhibition of metabolic enhancement by VLDL is markedly observed, and some inhibition is also observed with antibody # 1-25.
- the vertical axis represents OCR (pmol / min)%
- the horizontal axis represents elapsed time (min).
- Squares indicate PBS (background control)
- triangles indicate control IgG
- diamonds indicate anti-LSR antibodies
- the upper panel is an empty vector (E1)
- the lower is LSR forced expression cells (L45).
- FIG. 25 is a view showing conditions when the anti-LSR antibody described in the Examples is administered to a malignant tumor model mouse.
- FIG. 26 is a graph showing the results of evaluating the anti-tumor effect of anti-LSR antibodies described in Examples after administration of # 9-7 or # 1-25 to malignant tumor model mice.
- the vertical axis represents tumor volume (mm 3 ), and the horizontal axis represents the number of days elapsed. Squares indicate control IgG, triangles indicate anti-LSR antibody (# 9-7), and diamonds indicate anti-LSR antibody (# 1-25).
- FIG. 27 is a diagram showing the results of evaluating the anti-tumor effect of anti-LSR antibodies described in Examples after administration of # 9-7 or # 1-25 to malignant tumor model mice.
- FIG. 29 shows that recurrent ovarian cancer has no effective treatment.
- ovarian cancer is characterized by rapid invasion and rapid progression such as lymph node and peritoneal dissemination.
- ovarian cancer is characterized by rapid invasion and rapid progression such as lymph node and peritoneal dissemination.
- more than 40% of ovarian cancers are serous, 24% are clear cells, 17% are endometrioid, and 13% are mucinous adenocarcinoma.
- Cisplatin and taxol were used as the Ist line, and Avastin was used for recurrent ovarian cancer, but it was said that there was no improvement in survival rate.
- FIG. 30 is an immunostaining diagram showing that LSR is expressed in ovarian cancer tissue.
- the left shows ovarian clear cell adenocarcinoma and the right shows ovarian serous adenocarcinoma.
- the lower panel shows a Western blot of each cell.
- the left three columns represent normal ovaries, the left to fourth columns represent clear cell adenocarcinoma, and the left to sixth columns to the right end represent serous adenocarcinoma.
- LSR indicates the band of LSR, and GAPDH indicates the control.
- FIG. 31 is a diagram showing that LSR is also expressed at ovarian cancer metastasis sites.
- the left column shows lymph node metastasis and the right column shows omental metastasis.
- the upper panel shows 100 times magnification and the lower panel shows 400 times magnification.
- FIG. 32 is a diagram showing that LSR is also expressed at ovarian cancer metastasis sites.
- the left column shows lymph node metastasis and the right column shows omental metastasis.
- the upper panel shows 100 times magnification and the lower panel shows 400 times magnification.
- FIG. 33 is a diagram showing that LSR is expressed early in ovarian cancer.
- Upper left shows hematoxylin and eosin staining (HE)
- middle upper shows # 1-25A
- upper right shows # 1-45A
- lower left is # 9-7B
- middle lower is # 1-25B
- lower right is # 1 -45B is shown.
- the cells are ovarian clear cells of Stage Ic / IIc.
- FIG. 34 shows that LSR is also specifically expressed in gastric cancer.
- FIG. 35 is a diagram showing that LSR is strongly expressed in gastric cancer (signature ring cell carcinoma).
- the upper left panel shows an enlarged photograph of 5 times, the upper right is 10 times, the lower left is 20 times, and the lower right is 40 times.
- FIG. 36 is a diagram showing expression analysis of LSR by IHC using a normal frozen tissue array.
- FIG. 37A is a diagram showing an expression analysis of LSR by IHC using a normal frozen tissue array.
- FIG. 37B shows the results of calculating the dissociation constant (K D ) of anti-LSR by FACS.
- RMG-I cells were stained with various concentrations of antibodies and analyzed by FACS.
- FIG. 38 shows the results of examining the prognosis of ovarian serous adenocarcinoma patients and ovarian clear cell adenocarcinoma based on the level of LSR expression.
- FIG. 39 shows a comparison of the epitope region of the hLSR antibody of the antibody of the present invention and the amino acid sequences of hLSR and mLSR.
- FIG. 40 shows that anti-hLSR antibody cross reacts with mLSR. The original figure is shown in red and blue, and red shows mIgG2a.
- Blue indicates the staining pattern of various anti-LSR antibody clones. Blue has an arrow in this figure. Reactions with various antibodies were confirmed by FACS on COS7 cells in which pCMV5-mLSR-myc / DDK was introduced into COS7 cells and mSR was transiently expressed. The upper row shows a mixture of COS7 and mLSR, and the lower row shows only COS7 cells. Various antibodies are shown from the left, # 9-7. # 16-6, # 26-2, # 27-2, # 1-25, and # 1-43 are shown. FIG. 41 shows that anti-LSR induces cell cycle arrest at the G0 / G1 phase on RMG-I cells.
- the graph shows the percentage of cells in G0 / G1, S and G2 / M phases, with no treatment from the left, treatment with control IgG, and treatment with antibody # 1-25 in each cycle, respectively.
- the result of the case is shown.
- the experiment was performed at 15000 cells / well in a 6-well plate, and was performed under the conditions of RPMI 1640 medium + 1% FBS + 1% penicillin streptomycin (100 ⁇ g / ml antibody condition, 96 hours).
- Treatment with antibody # 1-25 was statistically significant (p ⁇ 0.0001) (one-way ANOVA and Dunnett test).
- FIG. 42 shows that the anti-LSR antibody suppresses cell proliferation by suppressing p27 expression enhancement, cyclin D1 expression suppression, Rb and MAPK activities.
- the left panel shows p27, cyclin D1, phosphorylated Rb (retinoblastoma protein; Ser780), phosphorylated Rb (Se807 / 811), Rb alone, LSR, and GAPDH as a control from the top.
- the right panel shows phosphorylated-MEK1 / 2, MEK1 / 2, phosphorylated p44 / 42MAPK, p44 / 42MAPK, GAPDH from the top.
- no treatment is performed from the left, and the results using mouse IgG2a and anti-LSR mAb # 1-25 are shown.
- FIG. 43 shows that anti-LSR antibodies have ADCC-independent anti-tumor effects in addition to ADCC-mediated anti-tumor effects.
- This is an experiment modeled on RMG-I (NOD / SCID).
- FIG. 44 is another graph showing that anti-LSR antibodies have ADCC-independent antitumor effects in addition to ADCC-mediated antitumor effects.
- the model used was RMG-I (NOD / SCID). It was statistically significant (p ⁇ 0.001, Student's t test).
- FIG. 45 shows that anti-LSR antibodies reduce growth phase tumor cells in vivo. Immunohistochemical staining with anti-Ki67 antibody was performed using RMG-I (NOD / SCID).
- the left column shows the control IgG administration group, and the right column shows the anti-LSR antibody (# 1-25) administration group. The upper row shows 100 times and the lower row shows 400 times.
- SKOV3-E1, SKOV3-L45, xenograft model ovarian cancer cell lines
- # 1-25 was used as an LSR antibody
- mouse IgG2a (Sigma M7769) was used as a control.
- An intraperitoneal administration of 10 mg / kg was performed.
- SKOV3-E1 was used as the empty vector-introduced strain
- SKOV3-L45 was used as the LSR stable expression strain.
- the upper side of the arrow indicates intraperitoneal administration (every 2 days, up to the 14th day), and the lower side, tumor volume measurement was performed (every 4 days until the 16th day, and the 18th day was also measured).
- FIG. 47 shows that anti-LSR monoclonal antibodies show anti-tumor effects against ovarian cancer cell line xenograft models expressing LSR.
- the left graph shows SKOV3-L45 (SCID) (LSR stable expression strain), and the right graph shows SKOV3-E1 (SCID) (empty vector).
- SCID SKOV3-L45
- SCID SKOV3-E1
- the number of days after treatment is shown on the horizontal axis, and the vertical axis shows tumor volume (mm 3 ).
- FIG. 48 shows that anti-LSR monoclonal antibodies show anti-tumor effects against ovarian cancer cell line xenograft models expressing LSR.
- the left graph shows SKOV3-L45 (SCID) (LSR stable expression strain), and the right graph shows SKOV3-E1 (SCID) (empty vector).
- N 5
- the vertical axis represents tumor weight (mg).
- FIG. 49 shows that LSR takes up VLDL and promotes lipid metabolism.
- the vector-introduced cells are shown from the left.
- the right shows LSR forced expression cells.
- FIG. 50 shows the examination results of intracellular uptake of LSR monoclonal antibody.
- the upper row shows SKOV3-LSR # 45, and the lower row shows the result of SKOV3Empty # 1 (empty vector).
- FIG. 51 shows the examination results of intracellular uptake of LSR monoclonal antibody. This is the same as FIG. The upper row shows SKOV3-LSR # 45, and the lower row shows the result of SKOV3Empty # 1 (empty vector). From the left, antibodies # 16-6, # 26-2, # 27-6 and # 1-43 are shown. Arrows indicate various clones of anti-LSR antibodies incorporated into cells.
- FIG. 52 shows the protocol of the anti-LSR antibody safety test using mice.
- Mouse IgG2a (Sigma M7769) and anti-LSR antibody # 1-26 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. 54 shows a comparison between control IgG and anti-LSR antibody (female). Each value is the same as in FIGS. FIG.
- the abbreviations described in FIG. FIG. 56 shows a comparison of control IgG versus anti-LSR antibody (female). Each value is the same as that shown in FIGS.
- LSR Low density lipoprotein receptor
- LSR Low density lipoprotein
- accession numbers of LSR described in NCBI are, for example, NP_991403 (amino acid) and /NM_205834.3 (mRNA).
- the amino acid sequence of LSR is, for example, SEQ ID NO: 7.
- the base sequence of LSR mRNA is, for example, SEQ ID NO: 8.
- the amino acid sequence of LSR is not limited as long as it has LSR activity.
- a protein or a nucleic acid encoding it having an amino acid sequence described in a specific SEQ ID NO or accession number, but also a functionally active analog thereof Or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. It is understood that it can be used in the present invention.
- a “derivative”, “analog” or “variant” is preferably, but not intended to be limited, to a region that is substantially homologous to a protein of interest (eg, LSR).
- such molecules are at least when compared to sequences aligned over amino acid sequences of the same size or aligned by computer homology programs known in the art. Nucleic acids that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical or that encode such molecules are subject to (highly) stringent conditions It can hybridize to a sequence encoding a component protein under moderately stringent or non-stringent conditions.
- a functional domain of LSR for example, a transmembrane domain (positions 260-280), a phosphorylation site (positions 309, 328, 406, 493, 528, 530, 535, 540, 551) 586, 615, and 646) are preferably stored.
- an LSR fragment is a polypeptide containing any region of LSR, and as long as it functions as an object of the present invention (for example, a marker or therapeutic target), it does not necessarily have a biological function of natural LSR. You don't have to.
- the representative nucleotide sequence of LSR is (A) a polynucleotide having the base sequence of SEQ ID NO: 7 or a fragment sequence thereof; (B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 8 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: 8, 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 described in SEQ ID NO: 7 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: 8 or a fragment thereof; (F) a polynucleot
- the biological activity typically means that the LSR has an activity or can be distinguished from other proteins present in the same organism as a marker.
- amino acid sequence of LSR (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 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: 8 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: 7; (D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 8; or (e) at least 70% identity, 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% and having
- a substance that binds to LSR is a molecule or substance that binds to LSR at least temporarily.
- an LSR binding agent 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 LSR include antibodies, antisense oligonucleotides, siRNA, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes, and peptidomimetics.
- a substance that binds to LSR or “an LSR-interacting molecule may be an inhibitor of LSR, eg a binding protein or binding peptide directed against LSR, in particular directed against the active site of LSR,
- nucleic acids directed against the LSR gene which refers to, for example, double-stranded or single-stranded DNA or RNA, or a modification or derivative thereof that inhibits LSR gene expression or LSR activity. And including, but not limited to, antisense nucleic acids, aptamers, siRNA (small interfering RNA) and ribozymes
- a “binding protein” or “binding peptide” for LSR is any binding to LSR. Refers to a protein or peptide and is directed against an LSR (eg, a po Clonal or monoclonal antibody), 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. As long as the object of the present invention is satisfied, it may be natural or non-natural.
- 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.
- 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 means a predetermined amino acid or nucleotide or moiety in a polypeptide or polynucleotide to be compared in a polypeptide molecule or polynucleotide molecule (eg, LSR).
- Amino acids or nucleotides that have or are expected to have a similar action especially in the case of enzyme molecules, amino acids that are present at similar positions in the active site and that contribute similarly to catalytic activity, In the case of a complex molecule, it refers to a corresponding part (for example, a transmembrane domain).
- 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 LSR can find a corresponding LSR in other animals (especially mammals).
- Such corresponding genes can be identified using techniques well known in the art.
- a corresponding gene in an animal is a gene that serves as a reference for the corresponding gene (for example, an LSR) by using a sequence such as SEQ ID NO: 7 or SEQ ID NO: 8 as a query sequence. It can be found by searching a database containing animal sequences.
- 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.
- the functions of LSR involved in inhibition of VLDL uptake and the like can be mentioned, but the invention is 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 LSR can be determined by any method.
- the expression level of LSR can be determined by evaluating the amount of mRNA of LSR, the amount of LSR protein, and the biological activity of LSR protein. Such measurements can be used in companion diagnostics.
- the amount of mRNA or protein of LSR can be determined by a method as described in detail elsewhere in this specification or by other methods known in the art.
- LSR refers to any object having the same target function but different structure from the target original entity. Therefore, “LSR” or a functional equivalent of the antibody is not LSR or the antibody itself, but is a variant or a variant (for example, an amino acid sequence variant, etc.) of LSR or the antibody, and possessed by LSR. Those having a biological effect, and those that can change to LSR or its antibody itself or a variant or variant of this LSR or its antibody at the time of action (eg, LSR or its antibody itself or LSR or It is understood that nucleic acids encoding the antibody variants or variants, and vectors, cells, etc. containing the nucleic acids) are encompassed.
- a functional equivalent of LSR or an antibody thereof can be used in the same manner as LSR 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. Sci., USA 85: 2444- 2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol.
- Biological 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 to. 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 LSR. 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 LSR inhibitor of the present invention is a factor that can temporarily or permanently reduce or eliminate the function of a target LSR or a cell that expresses LSR.
- 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”.
- antibody broadly refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof such as Fv fragments, Fab ′ fragments, F (ab ′) 2 and Fab fragments, and other recombinantly produced conjugates or functional equivalents (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chains Antibody, scFV, diabody, sc (Fv) 2 (singlechain (Fv) 2 ), scFv-Fc).
- antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
- the anti-LSR antibody used in the present invention may be bound to the LSR protein, and its origin, type, shape, etc. are not limited. Specifically, 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. In the present invention, monoclonal or polyclonal antibodies can be used as antibodies, but monoclonal antibodies are preferred.
- the binding of the antibody to the LSR protein is preferably specific binding.
- the antibody includes an antibody modified product or an antibody unmodified product. In the modified antibody, 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.
- the “anti-LSR antibody” includes an antibody having binding ability to LSR.
- the method for producing this anti-LSR antibody is not particularly limited, and for example, it may be produced by immunizing mammals or birds with LSR.
- an antibody against LSR anti-LSR antibody
- a fragment thereof is, for example, in the case of an antibody, an antibody itself having an LSR binding activity, and an inhibitory activity if necessary, and a fragment thereof.
- the anti-LSR antibody according to an embodiment of the present invention is preferably an anti-LSR antibody that specifically binds to a specific epitope of LSR from the viewpoint of particularly strongly suppressing the growth of malignant tumors.
- the anti-LSR 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 with respect to LSR efficiently compared with a polyclonal antibody. From the viewpoint of efficiently producing anti-LSR monoclonal antibodies, it is preferable to immunize LSR to chickens.
- the antibody class of the anti-LSR 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-LSR 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-LSR 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 an anti-LSR 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-LSR antibody according to this embodiment.
- the anti-LSR antibody according to one embodiment of the present invention may be, for example, an antibody obtained through a step of immunizing a living organism with purified LSR, LSR-expressing cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect on LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization.
- the anti-LSR antibody according to an embodiment of the present invention may be an antibody having a CDR set of antibodies obtained through a step of immunizing a living organism with purified LSR, LSR-expressing cell vesicles, or LSR-containing lipid membranes. From the viewpoint of enhancing the therapeutic effect on LSR-positive malignant tumors, it is preferable to use LSR-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 “LSR-expressing cell” may be obtained, for example, by expressing a LSR after introducing a polynucleotide encoding LSR into the cell.
- the LSR includes an LSR fragment.
- the “LSR-containing lipid membrane” may be obtained, for example, by mixing LSR and a lipid bilayer membrane.
- the LSR includes an LSR fragment.
- the anti-LSR antibody according to one embodiment of the present invention is an antibody obtained through a step of immunizing a chicken with an antigen, or an antibody having a CDR set of the antibody, from the viewpoint of enhancing the therapeutic effect on LSR-positive malignant tumors. preferable.
- the anti-LSR antibody according to one 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 or more. , 5.0 ⁇ 10 6 or more, there may be mentioned a 1.0 ⁇ 10 7 or more without being limited to, usually, K D values (kd / ka) is an at 1.0 ⁇ 10 -7 or less It may be 1.0 ⁇ 10 ⁇ 9 (M) or 1.0 ⁇ 10 ⁇ 10 (M) or less.
- the anti-LSR antibody according to one embodiment of the present invention may have ADCC or CDC activity.
- the anti-LSR antibody according to one embodiment of the present invention may be an antibody that binds to a wild type or mutant type of LSR. Variants include those resulting from differences in DNA sequences between individuals.
- the amino acid sequence of the wild-type or mutant LSR is preferably 80% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more of the amino acid sequence shown in SEQ ID NO: 8. It has sex.
- an “antibody” comprises a molecule or population thereof that can specifically bind to a specific epitope on an antigen.
- the antibody may also 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 antibody (eg, scFv), dsFv, multivalent specific antibody (eg, bivalent specific antibody), peptide or polypeptide having antigen binding property, chimeric antibody (eg, mouse-human chimeric antibody, chicken-human chimeric antibody, etc.) ), One or more forms selected from the group consisting of mouse antibodies, chicken antibodies, humanized antibodies, human antibodies, or their equivalents (or equivalents).
- 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-LSR antibody used in the present invention may be bound to the LSR 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 LSR 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 “Kohler G, Milstein C., Nature. 1975 Aug7; 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 may be "Clackson et 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. The antibody 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 H chain on the N-terminal side and the entire L chain of fragments obtained by treating an antibody comprising a Fab region and an 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-LSR 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 a Fab region and an Fc region with a protease pepsin.
- F (ab ′) 2 can be obtained, for example, by treating an anti-LSR 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 the VH and VL of the anti-LSR antibody according to the embodiment of the present invention, constructs a polynucleotide encoding the VH-peptide linker-VL, and incorporates the polynucleotide into a vector. Can be produced using cells for expression.
- 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. (Reiteret al., Protein Eng. 1994 May; 7 (5): 697-704.) it can.
- 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-LSR 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 combination of an antibody variable region and an antibody constant region between different organisms, and can be constructed by a gene recombination technique.
- a mouse-human chimeric antibody can be prepared, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA. 1994 Feb 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 Jan 1:13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1:93 (11): 3922-3930), Re-surfacing (Roguska et al., Proc Natl Acad Sci US A.
- FR shuffle (Damschroder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060. Epub 2007 Jan 22.).
- amino acid residues in the human FR region may be substituted with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24; 332 (6162): 323-327.).
- 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 protein. For example, it can be produced by the method described in “Vaughan et al., Nat Biotechnol. 1996 Mar; 14 (3): 309-314.”
- the antibody may be any antibody by CDR-grafting (Ozaki et al., Blood. 1999 Jun 1:93 (11): 3922-3930.) Or the heavy chain CDR or light chain of the anti-LSR antibody according to the embodiment of the present invention.
- the chain CDR may be prepared by grafting.
- the encoded DNA can be obtained by ligation to a vector according to a method known in the art, and then expression using a known cell.
- a method known in the art for example, a method in which amino acid residues of an antibody are randomly mutated to screen for highly reactive or phage
- the region excluding the heavy chain CDR or the light chain CDR may be optimized using a display method or the like.
- FR shuffle (Damschroder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060.
- the FR region may be optimized using JP-A-2006-241026 or Footeet al., J Mol Biol.1992 Mar 20; 224 (2): 487-499.
- 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
- VH-only molecule was produced in "Reiter et al., J Mol Biol. 1999 Jul 16; 290 (3): 685-98.”
- camel antibodies only heavy chain antibodies without light chains exist. Are listed.
- 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 definition Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)
- Chothia definition Chothiaet al., J. Mol. , 1987; 196: 901-917.
- 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. This refers to the target substance.
- markers can include genes, gene products, metabolites, enzymes, and the like.
- expression product refers to a protein or mRNA encoded by a gene.
- LSR gene products
- a “malignant tumor” includes, for example, a tumor that occurs when a normal cell is mutated. Malignant tumors can arise from any organ or tissue throughout the body. This malignant tumor is, for example, lung cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, ovarian cancer, uterine cancer, bladder cancer, prostate cancer, urine Duct cancer, renal pelvis cancer, ureteral cancer, penile cancer, testicular cancer, brain tumor, cancer of central nervous system, cancer of peripheral nervous system, head and neck cancer, glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer Including one or more selected from the group consisting of salivary gland cancer, malignant lymphoma, carcinoma, sarcoma, and hematological malignancy.
- ovarian cancer includes, for example, ovarian serous adenocarcinoma or ovarian clear cell line carcinoma.
- Uterine cancer includes, for example, endometrial cancer or cervical cancer.
- Head and neck cancer includes, for example, oral cancer, pharyngeal cancer, laryngeal cancer, nasal cavity cancer, sinus cancer, salivary gland cancer, or thyroid cancer.
- Lung cancer includes, for example, non-small cell lung cancer or small cell lung cancer.
- the malignant tumor may be LSR positive.
- the anti-LSR antibody according to the embodiment of the present invention can be a new therapeutic agent for serous adenocarcinoma and clear cell line cancer.
- the “LSR positive malignant tumor” includes a malignant tumor that significantly or excessively expresses LSR. Whether a malignant tumor is LSR-positive may be evaluated by, for example, RT-PCR, Western blot, or immunohistochemical staining. Alternatively, when the total protein of malignant tumor cells is provided for Western blotting and a band corresponding to LSR (for example, a band near 649aa) can be visually confirmed, it may be determined as LSR positive. Alternatively, when the LSR expression level of a patient-derived malignant tumor cell is significantly larger than that of a normal cell, it may be determined as LSR positive. From the viewpoint of achieving more optimal dosing by accurately diagnosing LSR positivity, it is preferable to test the expression of LSR using an anti-LSR antibody.
- 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 associated with a disease, disorder, condition (eg, malignant tumor), etc. in a subject and determines the current or future of such disease, disorder, or condition. That means.
- 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”.
- prognosis predicts the likelihood of death or progression due to cancer, such as recurrence of a neoplastic disease, such as a malignant tumor (eg, ovarian cancer), metastatic spread, and drug resistance. Means. Therefore, in the present specification, “good prognosis” means a state in which no recurrence cancer originating from the cancer is observed over a certain period (for example, 4 years) after cancer tissue resection. "Poor prognosis” or “poor prognosis” refers to a condition in which recurrent cancer originating from the cancer is observed over a certain period (for example, 4 years) after resection of the cancer tissue.
- Prognostic factors are variables related to the natural course of malignant tumors, which affect the recurrence rate and outcome of patients once they have developed a malignant tumor.
- Clinical indicators associated with worsening prognosis include, for example, lymph node metastasis and high-grade tumors.
- Prognostic factors are often used to classify patients into subgroups with different underlying risk of recurrence.
- the expression of the LSR of the present invention can be used as a prognostic factor.
- the term “prediction” means the likelihood that a patient will have a specific clinical outcome, whether good or bad, after removal of the primary tumor. Therefore, the LSR of the present invention can be used as a poor prognosis marker.
- the prediction method of the present invention can be used clinically to determine a therapy by selecting the optimal therapy for a particular patient.
- the prediction method of the present invention is a valuable tool in predicting if a patient is likely to respond well to epilepsy, such as a treatment plan, eg, a surgical intervention.
- the prediction can include prognostic factors.
- 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 state (for example, a disease such as a malignant tumor).
- treatment refers to prevention of worsening of a disease or disorder when the disease or disorder (eg, malignant tumor) becomes such a condition, Preferably, it refers to reduction, 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”.
- therapeutic agent refers to any agent that can treat a target condition (for example, a disease such as a malignant tumor) in a broad sense.
- 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 malignant tumor includes a drug (prophylactic agent) used for preventing malignant tumor, or a growth inhibitor of malignant tumor cells.
- prevention means that a certain disease or disorder (for example, malignant tumor) is prevented from becoming such a state before it becomes such a state. Diagnosis can be performed using the drug of the present invention, and malignant tumors, for example, 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, malignant tumor
- prophylactic agent refers to any agent that can prevent a target condition (for example, a disease such as a malignant tumor) 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, examination 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” describes a 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 instructions are so-called package inserts and are usually provided in a paper medium, but are not limited thereto, and are 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 novel therapeutic or prophylactic agent for malignant tumors.
- This therapeutic or prophylactic agent is a malignant tumor therapeutic or prophylactic agent containing an LSR inhibitor, for example, an anti-LSR antibody.
- an LSR inhibitor for example, an anti-LSR antibody.
- the therapeutic agent of the present invention is preferably administered to patients whose malignant tumor is determined to be an LSR-positive malignant tumor among the malignant tumor patients.
- the therapeutic agent of the present invention is preferably administered to patients whose malignant tumor is determined to be an LSR-positive malignant tumor among the malignant tumor patients.
- composition or medicament (such as a therapeutic or prophylactic agent) of the present invention is administered by administration to a patient determined to develop an LSR-positive malignant tumor. Formulated on the assumption.
- the LSR inhibitor used in the present invention is an antibody or a fragment or functional equivalent thereof, or a nucleic acid.
- the LSR inhibitor used in the present invention also has an ability to inhibit enhancement by VLDL.
- the present invention provides that the LSR antibody is an antibody that also has an ability to inhibit enhancement by VLDL.
- LSR acts on cancer growth by taking up VLDL and enhancing lipid metabolism. That is, it is considered that cancer cells expressing LSR are strengthened by suppressing the growth of cancer cells by suppressing the function of LSR.
- the antibody of the present invention can enhance the suppression of cancer cell proliferation by suppressing the incorporation of VLDL of LSR into the cancer cell.
- the cancer or cancer cell targeted by the present invention may be a cancer or cell related to VLDL (eg, cancer or cancer cell related to cancer cell shown in Examples, ovarian cancer, etc.).
- the LSR inhibitor used in the present invention is a nucleic acid, and the nucleic acid is an antisense nucleic acid, siRNA, or the like.
- the siRNA includes SEQ ID NOs: 9 to 14 or the like. May be included.
- the inhibitor of LSR is an antibody or a fragment or functional equivalent thereof.
- the antibody of the present invention may be a specific sequence described elsewhere herein.
- 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.
- embodiments described elsewhere in this specification and / or techniques known in the art can be used.
- such an antibody or a fragment or functional equivalent thereof preferably has inhibitory activity downstream of LSR or its signal transduction pathway.
- Such activity can be determined by examining the expression level of LSR or its activity, or by directly using a malignant tumor cell line such as ovarian specific fat cancer cell, cell proliferation inhibition, cell in antibody-dependent cytotoxicity (ADCC) It may be confirmed by observing the damage activity or observing tumor regression after transplantation into a model animal.
- ADCC antibody-dependent cytotoxicity
- the present invention provides a method for preventing or treating a malignant tumor in a subject, comprising administering to the subject in need of an effective amount of an LSR inhibitor. It will be understood that any form described elsewhere herein can be utilized as an LSR inhibitor used in the prevention or treatment method of the present invention.
- the present invention also provides a composition or medicament (therapeutic or preventive agent) for preventing or treating malignant tumors, which comprises a binding agent for LSR.
- a composition or medicament for preventing or treating malignant tumors, which comprises a binding agent for LSR.
- 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 LSR binding agent is an antibody or a fragment or functional equivalent thereof, or a nucleic acid. In a preferred embodiment, the LSR binding agent is an antibody or a fragment or functional equivalent thereof and further conjugated 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 drugs include Gail D. et al., Cancer Res 2008; 68: 9280-9290 .; Ian Krop andEric P.
- 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 LSR on the cell surface and the susceptibility of cancer cells to cell killing 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 LSR binding agent is an antibody or fragment or functional equivalent thereof, which can be a sequence specifically listed elsewhere herein.
- 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 ovarian region, it can be further administered by any suitable route, such as by direct injection into the affected area such as the ovary.
- 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.
- 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 an LSR positive malignant tumor. At this time, the determination or diagnosis is preferably performed by detecting the protein level of LSR.
- 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.
- heavy chain CDR1, 2, 3, and light chain CDR1, 2, and 3 are the 31-35, 50-66, 99-104, 153 of SEQ ID NO: 1, respectively.
- Antibodies comprising amino acid sequences shown at positions 165, 182-188, and 221-230 are each represented by SEQ ID NO: 31
- heavy chain CDR1, 2, 3, light Strands CDR1, 2, and 3 represent the amino acid sequences shown in SEQ ID NO: 3 at positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229, respectively.
- the antibody comprising, (d) heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-104, 153-165, 182 of SEQ ID NO: 4, respectively.
- (f) Heavy chain CDR1, 2, 3, light chain CDR1, 2, and 3 are 31-35, 50-66, 99-104, 153-165, 182-188, and 221 of SEQ ID NO: 6, respectively.
- the CDR may be an anti-LSR antibody that is a mutant that contains an addition or deletion, but that does not contain a mutation. If this anti-LSR antibody is used, the growth of LSR-positive malignant tumor cells can be particularly effectively suppressed. In addition, LSR positive malignant tumors can be diagnosed efficiently.
- Another embodiment of the invention is an anti-LSR antibody comprising at least one set of the amino acid sequence sets of heavy chain CDR1, 2, and 3 listed above.
- 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 anti-LSR antibody 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, At least one, preferably 2, 3, 4, 5, 6, 7, or all frameworks of light chains FR1, 2, 3, and 4 are any of SEQ ID NOs: 1-6 Can be the same or substantially the same or the same except for conservative substitutions.
- One or more antibodies may be used.
- Another embodiment of the invention is an anti-LSR antibody comprising at least one set of the amino acid sequence sets of heavy chains FR1, 2, 3, and 4 listed above.
- the anti-LSR 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 is located at positions 116 to 132 of SEQ ID NO: 1, and from 116 to 132 of SEQ ID NO: 2. Even if it has the amino acid sequence shown at positions 132, 116 to 132 of SEQ ID NO: 3, 116 to 132 of SEQ ID NO: 4, 116 to 132 of SEQ ID NO: 5, or 116 to 132 of SEQ ID NO: 6 Good.
- VH of # 9-7, # 16-6, No.26-2, No.27-6, No.1-25, No.1-43 described in Example 2 described later is the SEQ ID NO. 1 to 115 of 1, 1 to 115 of SEQ ID NO: 2, 1 to 115 of SEQ ID NO: 3, 1 to 115 of SEQ ID NO: 4, 1 to 115 of SEQ ID NO: 5, 1 to 115 of SEQ ID NO: 6 It is rank.
- VL of # 9-7, # 16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in Example 2 described later are arranged respectively. No.
- the amino acid sequence listed above is (i) an amino acid sequence in which one or several nucleotide sequences are deleted, substituted, inserted, or added in the above amino acid sequence. , (Ii) a polynucleotide having a nucleotide sequence complementary to the amino acid sequence having 90% or more homology to the above amino acid sequence, and (iii) a base sequence encoding the above 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 various conditions.
- a transformant can be prepared by introducing a polynucleotide or vector encoding an anti-LSR antibody according to an embodiment of the present invention into a cell.
- the anti-LSR 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.
- 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 a 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; Elbashir et al.
- 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.
- an LSR inhibitor of the invention comprises 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.
- an LSR inhibitor of the present invention comprises a nucleotide sequence that is complementary to the nucleotide sequence of a target gene.
- the LSR inhibitor of the present invention interacts with the nucleotide sequence of the target gene so as to inhibit the expression of the target gene.
- LSR inhibitors 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 bond or association comprising one or more nucleotides with a 2′-OH group Group, moiety or chain.
- an agent that inhibits LSR of the present invention may comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
- LSR inhibitors are nucleic acid molecules that can mediate sequence-specific RNAi, such as small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin 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 short hairpin RNA
- small interfering oligonucleotide small interfering nucleic acid
- small interfering modified oligonucleotide chemically modified siRNA
- 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 an LSR-positive malignant tumor comprising an RNAi molecule against LSR or a polynucleotide encoding the RNAi molecule. If this RNAi molecule or a polynucleotide encoding the RNAi molecule is used, the growth of LSR-positive malignant tumor 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 LSR-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 Berge et al., J 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. For solvates, see, 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 LSR 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 Example 3 described later includes the nucleotide sequence of SEQ ID NO: 9 or 10. These base sequences are base sequences complementary to a part of the LSR mRNA, and are considered to be a part that functions as a guide strand.
- One embodiment of the present invention includes such an RNAi molecule comprising the nucleotide sequence of SEQ ID NO: 9 or 10.
- the RNAi molecule may further include a base sequence complementary to the base sequence represented by SEQ ID NO: 9 or 10 (for example, SEQ ID NOs: 11 and 12, respectively).
- the “complementary base sequence” is a base sequence possessed by another highly complementary polynucleotide capable of hybridizing to one polynucleotide.
- the sense strand full length of siRNA used in Example 3 described later is the base sequence of SEQ ID NO: 13 or 14, and the antisense strand full length is the base sequence of SEQ ID NO: 15 or 16.
- the above listed base sequences are (i) amino acid sequences in which one or several base sequences are deleted, substituted, inserted or added in the above base acid sequences 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.
- the present invention is a therapeutic agent for LSR-positive malignant tumors, including an LSR antagonist.
- the LSR antagonist includes a substance that inhibits the expression or function of LSR. By using this LSR antagonist, the growth of LSR-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 LSR, 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 LSR.
- As the DNA strand a DNA strand encoding an RNAi molecule against LSR 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 reducing the amount of protein as a result, eg, 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, as a result of inhibiting gene expression, the production amount of mRNA or protein is reduced.
- 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 amount of expression may be the amount of mRNA or protein as an indicator.
- “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 malignant tumor treatment method.
- This therapeutic method is, for example, a therapeutic method including a step of administering an anti-LSR antibody to a patient. With this treatment method, LSR positive malignant tumors can be treated. This therapeutic method is superior from the viewpoint of safety because it uses antibodies.
- the malignant tumor treatment method preferably includes a step of diagnosing that the patient has developed an LSR-positive malignant tumor.
- this treatment method may include a step of examining that malignant tumor cells derived from a patient express LSR.
- the onset diagnosis of LSR-positive malignant tumor 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 dosage by accurately diagnosing LSR positive.
- the protein expression diagnosis may be performed using, for example, an anti-LSR antibody.
- the protein obtained from the test malignant tumor cells derived from the patient was subjected to Western blot, and when a band corresponding to LSR was confirmed visually, it was judged that an LSR-positive malignant tumor had developed. Also good.
- the LSR expression level of a patient-derived malignant tumor cell is significantly larger than that of a normal cell or LSR-negative malignant tumor cell, it may be determined that an LSR-positive malignant tumor has developed.
- the derived malignant tumor cell may be determined to have developed an LSR-positive malignant tumor when the band intensity corresponding to LSR is significantly stronger.
- serum or plasma obtained from a malignant tumor patient and serum or plasma obtained from a healthy person or an LSR-negative malignant tumor patient are subjected to an ELISA method using an anti-LSR antibody.
- serum or plasma derived from a malignant tumor patient has a significantly stronger LSR expression level, it may be determined that an LSR-positive malignant tumor has developed.
- Serum and plasma samples themselves may be quantified, or exosomes may be isolated from serum and plasma, and LSR in exosomes may be subjected to an ELISA method for analysis.
- RT-PCR may be used instead of Western blot.
- the method for treating a malignant tumor may include a step of administering an LSR antagonist to a patient.
- the method may include the step of administering to the patient an RNAi molecule against LSR, or a polynucleotide encoding the RNAi molecule.
- One embodiment of the present invention is a novel diagnostic agent for malignant tumors, including an anti-LSR antibody.
- This diagnostic agent may be, for example, a companion diagnostic agent for treating malignant tumors targeting LSR, including anti-LSR antibodies. Since there are LSR positive and non-LSR positive patients among malignant tumor patients, if this malignant tumor is tested for LSR positive in advance using this companion diagnostic agent, LSR will be targeted It is possible to diagnose the therapeutic efficacy of malignant tumor treatment. In this diagnosis, if an LSR positive result is obtained, it can be judged that malignant tumor treatment targeting LSR is effective.
- “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.
- LSR-positive patients For clinical application of antibody drugs with anti-LSR antibodies, selective treatment of patients with malignant tumors that express LSR is thought to lead to personalized medicine. Therefore, a method for selecting LSR-positive patients is necessary.
- a method for selecting LSR-positive patients a method of examining LSR in a biopsy tissue of cancer by immunohistochemical staining is considered to be highly useful. However, since obtaining a biopsy tissue is highly invasive, a method with low invasiveness is preferable.
- certain malignant tumors such as ovarian cancer have a problem that it is difficult to obtain biopsy tissue due to the problem of the site of cancer.
- LSR or its extracellular domain expressed in cancer may be present in the blood.
- LSR in malignant tumor patient blood can be quantified, it is suggested that patients with high blood LSR concentration may have high LSR expression levels in ovarian cancer tissues.
- Blood samples have the advantage of being less invasive than biopsy.
- the LSR concentration in the blood is quantified by ELISA, and it is highly possible that LSR is highly expressed in patient tissues where the blood LSR concentration is higher than that in healthy individuals, and the blood LSR concentration is a companion diagnostic agent. This measurement is considered to be highly useful.
- the diagnostic agent for a malignant tumor may be a diagnostic agent containing an anti-LSR antibody for diagnosing the therapeutic efficacy of an anti-LSR antibody or an LSR antagonist against a malignant tumor. Since there are LSR positive and non-LSR positive patients among malignant tumor patients, if this malignant tumor is tested for LSR positive in advance using this diagnostic agent, anti-LSR against the patient The therapeutic efficacy of the antibody or LSR antagonist can be diagnosed.
- One embodiment of the present invention is a companion diagnostic method for malignant tumor treatment targeting LSR, which comprises the step of examining whether a malignant tumor sample of a malignant tumor patient is LSR positive. Since there are LSR positive and non-LSR positive patients among malignant tumor patients, if this malignant tumor is tested for LSR positive in advance using this companion diagnostic method, LSR will be targeted It is possible to diagnose the therapeutic efficacy of malignant tumor treatment.
- the diagnostic method may further comprise the step of isolating or extracting a malignant tumor sample from a malignant tumor patient.
- a “malignant tumor sample” may be a malignant tumor tissue or cell obtained from a malignant tumor patient.
- One embodiment of the present invention is a method for diagnosing the prognosis of a malignant tumor using the expression level of LSR as an index. It has been demonstrated that high LSR expression is a poor prognostic marker (FIG. 38). From this, it can be said that high LSR expression is a poor prognostic marker.
- the cancer to be prognosed is ovarian serous adenocarcinoma, but is not limited thereto, and can also be applied to ovarian clear cell adenocarcinoma, etc. It is understood that it can be applied.
- the method of using such a prognostic (bad) marker may include, for example, a step of examining that a patient-derived malignant tumor cell expresses LSR.
- the prognosis (diagnosis) of an LSR-positive malignant tumor may be performed by, for example, mRNA expression diagnosis or detection, or protein expression diagnosis or detection.
- This diagnosis or detection is preferably a protein expression diagnosis from the viewpoint of realizing a more optimal dosage by accurately diagnosing LSR positive.
- the protein expression diagnosis may be performed using, for example, an anti-LSR antibody. In this onset diagnosis, if the protein obtained from the test malignant tumor cell derived from the patient is subjected to Western blot and the enhancement of the band corresponding to LSR can be confirmed visually, it is judged that the prognosis of the malignant tumor is poor. May be.
- LSR expression level of the patient-derived malignant tumor cells is significantly greater than that of normal cells or LSR-negative malignant tumor cells (eg, LSR-negative cell lines such as OVTOKO), It may be determined that the prognosis is poor.
- the total protein obtained from patient-derived malignant tumor cells and the total protein obtained from normal cells or LSR-negative malignant tumor cells are subjected to Western blot, and normal cells
- LSR-negative cell lines for example, LSR-negative cell lines such as OVTOKO
- the malignant tumor It may be determined that the prognosis is poor.
- serum or plasma obtained from a patient with malignant tumor and serum or plasma obtained from a healthy subject or a patient with LSR-negative malignant tumor are treated with anti-LSR.
- Serum or plasma derived from patients with malignant tumors is significantly more prognostic when the LSR expression level is significantly higher than that of healthy subjects or patients with LSR-negative malignant tumors. You may judge that it is bad.
- Serum and plasma samples themselves may be quantified, or exosomes may be isolated from serum and plasma, and LSR in exosomes may be subjected to an ELISA method for analysis.
- RT-PCR may be used instead of Western blot.
- One embodiment of the present invention is a method for examining the therapeutic efficacy of an anti-LSR antibody or LSR antagonist against malignant tumors.
- This method is, for example, a test method including a step of testing that a malignant tumor sample of a malignant tumor patient is LSR positive.
- This test method may include the step of detecting the presence of LSR in a malignant tumor sample.
- a step of detecting a large size may be included.
- For detection of LSR for example, RT-PCR, Western blot, or immunohistochemical staining may be used.
- the evaluation criteria for the presence or absence of LSR may be the same as in the case of the onset diagnosis of the above-mentioned LSR positive malignant tumor.
- Methods for testing therapeutic effectiveness include methods for testing efficacy for treatment.
- One embodiment of the present invention is a malignant tumor cell growth inhibitor comprising an anti-LSR antibody.
- it is a method for inhibiting the growth of malignant tumor cells, comprising a step of bringing an anti-LSR antibody into contact with a malignant tumor cell.
- it is a malignant tumor cell growth inhibitor containing an LSR antagonist.
- it is a method for inhibiting the growth of malignant tumor cells, comprising a step of bringing an LSR antagonist into contact with a malignant tumor cell.
- the growth inhibitory agent or therapeutic agent for malignant tumor cells has a growth rate, growth amount, or volume of a malignant tumor that is 10 compared to when no growth inhibitor is added or no therapeutic agent is added, It may be an agent that reduces 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 a cell division inhibitor for malignant tumor cells, comprising an anti-LSR antibody.
- the method is a method for inhibiting the suppression of cell division of malignant tumor cells, comprising a step of bringing an anti-LSR antibody into contact with a malignant tumor cell.
- it is a cell division inhibitor for malignant tumor cells, including an LSR antagonist.
- it is a method for inhibiting cell division of malignant tumor cells, comprising a step of bringing an LSR antagonist into contact with malignant tumor cells.
- the agent for inhibiting cell division of malignant tumor cells according to an embodiment of the present invention is a drug that reduces the division rate of malignant tumor cells by 10, 20, 30, or 50% or more compared to when no agent for inhibiting cell division is added. It may be. 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 LSR-dependent malignant tumors, including an anti-LSR antibody. With this therapeutic agent, LSR-dependent malignant tumors can be treated.
- One embodiment of the present invention is the use of anti-LSR antibodies or LSR antagonists for the production of therapeutic agents for malignant tumors. Yet another embodiment is the use of an anti-LSR antibody for the manufacture of a companion diagnostic for the treatment of malignant tumors targeting LSR.
- a step of introducing a polynucleotide encoding LSR into a cell, a step of expressing the LSR in the cell, and an immunization of a chicken with an antigen containing the cell expressing the LSR A method for producing an anti-LSR antibody. According to this production method, an excellent anti-LSR antibody can be efficiently produced for the treatment or diagnosis of LSR-positive malignant tumors.
- the “bond” may be a covalent bond or a non-covalent bond, and may be, for example, an ionic bond, a hydrogen bond, a hydrophobic interaction, or a hydrophilic interaction.
- 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.
- LSR expression analysis 1.1 Quantitative analysis of cell surface membrane proteins by iTRAQ method Higher expression in ovarian serous adenocarcinoma cell lines (OVCAR3, OVSAHO, JHOS4) compared to normal ovarian epithelial cell lines (HOSE2C, E7 / TERT)
- HOSE2C normal ovarian epithelial cell lines
- HOSE2C normal ovarian epithelial cell lines
- a cell surface membrane protein was labeled with biotin with sulfo-NHS-SS-biotin on a cell line 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 sample was mixed into one and roughly fractionated into 24 fractions by ion exchange HPLC, and each fraction was desalted and then measured by mass spectrometer (nanoLC-MS / MS) analysis.
- the obtained data was subjected to database search with proteome discoverer ver1.1 to identify and quantify proteins.
- the ovarian cancer surgical tissue used in the Examples was provided by a patient who had obtained informed consent from Osaka University Hospital.
- LSR was specifically highly expressed in the ovarian serous adenocarcinoma cell line as described below.
- HOSE2C ovarian epithelial cell line
- OVCAR3, OVSAHO, JHOS4 ovarian clear cell line cancer cell line
- E6 / E7 normal endometrial cell line
- E6 / E7 normal endometrial cell line
- E6 / E7 normal endometrial cell line
- E6 / E7 normal endometrial cell line
- E6 / E7 endometrial cancer
- HEC1, HEC1A, HEC6, HEC88nu, HEC108, HEC116, HEC251, SNGM endometrial cancer
- RNAs were purified with RNeasymini kit (QIAGEN), respectively.
- reverse transcription was performed using a QuantiTect Reverse Transcription Kit (Qiagen).
- RT-PCR was performed using TaKaRaEx Taq DNA polymerase (Takara Bio, Shiga, Japan).
- primers having the following sequences were used.
- LSR forward primer5'-GGGAGGACCTCAGGGGTGGC-3 '(SEQ ID NO: 17), and reverse primer5'-TGGTGGGGGTGGGGTCTTGG-3 '(SEQ ID NO: 18)
- ⁇ -actin forward primer5'-AGCCTCGCCTTTGCCGA-3 '(SEQ ID NO: 19
- LSR mRNA in ovarian serous adenocarcinoma cell lines OVCAR3, OVSAHO, JHOS4, ovarian clear cell line cancer cell lines OVMANA, OVISE, RGMI, and endometrial cancer cell lines HEC1, HEC1A, HEC6, HEC88nu, HEC108, HEC116, HEC251, SNGM A band corresponding to was detected. In normal ovarian epithelial cell lines, it was not detected by HOSE2C.
- electrophoresis was carried out at 40 mA for 50 min, and transferred to a PVDF membrane at 120 mA for 1 hour.
- the cells were blocked with 1% BSA / TBST (TBS + 0.1% Tween20) for 1 hour at room temperature and incubated with an anti-LSR antibody (Santa Cruz Biotechnology) for 1 hour at room temperature.
- an anti-LSR antibody Santa Cruz Biotechnology
- the PVDF membrane was incubated at room temperature for 1 hour using an HRP-labeled anti-rabbit antibody (GE healhcare) diluted 5,000 times with TBST.
- the PVDF membrane was washed with TBST for 3 minutes for 10 minutes, and then the reacted protein was detected with a fluorescence reaction system (PerkinElmer).
- anti-LSR antibody (Santa Cruz Biotechnology) against proteins obtained from normal ovarian tissue, ovarian serous adenocarcinoma surgical tissue, ovarian clear cell carcinoma surgical tissue, normal endometrial tissue, and endometrial cancer surgical tissue Western blotting was performed using An anti-GAPDH antibody (SantaCruz Biotechnology) was used as a loading control.
- the tissues used for Western blot were obtained from healthy individuals or patients suffering from each cancer.
- Example 2 Preparation and evaluation 2.1 Preparation of human LSR-expressing chicken cell line and immunization to chicken
- the human LSR cDNA (SEQ ID NO: 7) was cloned into a mammalian expression vector (pcDNA3.1-V5 / His-TOPO).
- An LSR expression vector was prepared. This LSR expression vector encodes a fusion protein in which a V5 / His tag is fused to the C-terminus of human LSR.
- the LSR 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 flow cytometry (FACS) analysis. For FACS analysis, the protocol of FACSCalibur (BD, USA) was followed.
- scFv phage antibody library from immunized 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 an scFv phage antibody library.
- scFv phage antibody library the procedure described in “Nakamura et al., J Vet Med Sci. 2004 Jul; 66 (7): 807-14” was followed.
- the scFv phage antibody library was added to an LSR non-expressing cell line to absorb non-specific phage, and then reacted with the LSR expressing cell line.
- Lot1 was cell panned using a mammalian cell line, and Lot2 was a chicken lymphoblastoid cell line used for immunization. After washing with an organic solvent, phages specifically bound to the LSR-expressing cell line were recovered and infected with E. coli. After panning four times, the reactivity of the library was confirmed by FACS analysis using an LSR-expressing cell line.
- Phages were cloned from the library with the highest reactivity, and positive clones were selected, and then the sequences of 6 clones were determined (SEQ ID NOs: 1 to 6, FIG. 9).
- the method described in “Giordano et al., Nat Med. 2001 Nov; 7 (11): 1249-53.” was followed.
- Immunohistochemical staining LSR expression was analyzed by immunohistochemical staining in tissues of ovarian cancer (84) cases.
- the primary antibody used was cloud clone (PAD744Hu01) and was stained using Dako ChemMate ENVISION Kit / HRP (DAB) -universal kit (K5007).
- the results of immunohistochemical staining were scored. Scoring is 0+ (no holding cells), 1+ (pale holding in any proposal of cells), 2+ (darkly staining cells ( ⁇ 25% of area)), 3+ (darkly staining cells (25-49% of area) ) And 4+ (dark staining (> 50% area)), score 0, 1 and 2 were LSR low expression group, score 3 and 4 were LSR high expression group, LSR low expression group and LSR Divided into high expression groups, a survival curve was created using the Kaplan-Meier method, and a log rank test was performed.
- ovarian cancer As shown in FIG. 29, there has been no effective treatment for recurrent ovarian cancer.
- ovarian cancer As an epidemiology and characteristics of ovarian cancer, ovarian cancer is characterized by rapid invasion and rapid progression such as lymph node and peritoneal dissemination.
- more than 40% of ovarian cancers are serous, 24% are clear cells, 17% are endometrioid, and 13% are mucinous adenocarcinoma.
- Cisplatin and taxol were used as the Ist line, and Avastin was used for recurrent ovarian cancer, but it was said that there was no improvement in survival rate.
- Antibody drugs approved as cancer therapeutics are listed in the table of FIG. 29 (Carter PJ Nat. Rev. Immunol.
- LSR LSR-specific recombinant protein
- the protocol is as follows.
- the immunostaining of LSR used the same method as described above in this example.
- the expression of LSR was analyzed by immunohistochemical staining.
- the primary antibody used was cloud clone (PAD744Hu01) and was stained using Dako ChemMate ENVISION Kit / HRP (DAB) -universal kit (K5007).
- LSR expression was examined by Western blotting using proteins extracted from ovarian cancer surgical tissues. As a result, it was revealed that LSR was highly expressed in ovarian clear cell adenocarcinoma and ovarian serous adenocarcinoma compared with normal ovarian tissue. GAPDH indicates a control group.
- Results are shown in FIG. As shown in FIG. 30, it was confirmed that LSR was expressed at the primary site of ovarian cancer.
- LSR LSR-specific kinase-like protein
- the protocol is shown below.
- the immunostaining of LSR used the same method as described above in this example. That is, the expression of LSR was analyzed by immunohistochemical staining.
- the primary antibody used was cloud clone (PAD744Hu01) and was stained using Dako ChemMate ENVISION Kit / HRP (DAB) -universal kit (K5007).
- LSR LSR protein kinase kinase kinase
- FIGS. 33 to 35 show that LSR was also expressed in other cells. These include early ovarian clear cell adenocarcinoma, gastric cancer other than ovarian cancer, gastric cancer, and gastric cancer signet ring cell carcinoma.
- Immunohistochemical staining of LSR was performed in the same manner as described above. Specifically, the expression of LSR was analyzed by immunohistochemical staining. The primary antibody used was cloud clone (PAD744Hu01) and was stained using Dako ChemMate ENVISION Kit / HRP (DAB) -universal kit (K5007). .
- LSR was also expressed in early ovarian clear cell adenocarcinoma.
- LSR was shown to be expressed in gastric cancer as an adenocarcinoma other than ovarian cancer.
- FIG. 35 it was shown that LSR is also expressed in signet ring cell carcinoma of gastric cancer. From these, it is understood that ovarian cancer can be used for treatment even at an early stage, and that other adenocarcinomas such as gastric cancer can be treated.
- PepStar TM peptide microarrays were prepared on glass slides obtained from JPT Peptide Technologies (GmbH). A 15-mer overlapping peptide that overlapped 10 amino acids in the extracellular domain region of LSR was synthesized and immobilized on a glass slide. Binding of the purified recombinant antibody to the peptide was performed according to the instructions, with some changes (www.jpt.com). The primary antibody was reacted at a concentration of 1.0 ⁇ g / mL, and the glass slide was washed with TBST (50 mM TBS-buffer including 0.1% Tween 20, pH 7.2).
- TBST 50 mM TBS-buffer including 0.1% Tween 20, pH 7.2
- the reaction was performed using Cy5-labeled goat anti-chicken IgY (Jackson Immuno Research), and the glass slide was washed 5 times with TBST and 5 times with ddH2O. The glass slide was dried by gently blowing argon gas. The fluorescence signal was detected with a GenePix 4200AL scanner (Molecular Devices) with a resolution of 10 ⁇ m.
- amino acids 116-135 corresponding to antibody # 9-7, 1-25, 16-6, 26-2, 1-43
- amino acids 216-230 amino acids 216-230
- Immunohistochemical staining using normal frozen tissue array Immunohistochemical staining was performed on FDA standard human tissue microarray (T6234701-2, Biochain) using anti-LSR antibody # 1-25. Among various normal tissues, LSR expression was observed in the liver and testis (FIGS. 36 and 37).
- K D 2.52nM
- RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodiumdeoxycholate, 0.1% SDS, 1 ⁇ phosphatase inhibitorcocktail (Nacalai Tesque) and 1 ⁇ protease inhibitorcocktail (Nacalai Tesque )) was used for protein extraction, and protein expression differences were analyzed by Western blotting. The following primary antibodies were used.
- Anti-LSR antibody (sc-133765), anti-GAPDH antibody (sc-25778) (Santa Cruz Biotechnology (Santa Cruz, CA)), anti-cyclin D1 antibody (# 2926), anti-p27 antibody (# 3686), anti-phospho-Rb (Ser780) antibody (# 9307), anti-phospho-Rb antibody (Ser807 / 811) (# 9308), anti-Rb antibody (# 9313), anti-phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) antibody (# 4370), anti-p44 / 42 MAPK (Erk1 / 2) antibody (# 4695), anti-phospho-MEK1 / 2 (Ser217 / 221) antibody (# 9154), anti-MEK1 / 2 antibody (# 9126) (Cell Signaling Technology) ⁇ Example 3> Inhibition of growth of LSR-positive malignant tumors 3.1 Growth inhibition assay using anti-LSR antibody Ovarian clear cell line
- Ovarian clear cell line cancer (A2780) was seeded in a 96-well plate at 1000 cells / well and incubated overnight in a 37 ° C. CO 2 incubator. The cell supernatant of the 96-well plate was discarded, and a diluted solution (1 ⁇ g / ml, 10 ⁇ g / ml, 100 ⁇ g / ml) of anti-LSR antibody (# 9-7 or # 26-2) was added at 100 ⁇ L / well. Cell proliferation assay was performed 72 hours later by WST-8 assay. As a control, mouse IgG2 (biolegend, 400224, MOPC-173), which is a non-anti-LSR antibody, was used. The results are shown in FIG. Proliferation of ovarian cancer cells (A2780) was suppressed by contacting with an anti-LSR antibody.
- Ovarian clear cell carcinoma (RMG-I) was seeded in a 6-well plate at 15,000 cells / well and incubated overnight in a 37 ° C. CO 2 incubator. The cell supernatant of the 6-well plate was removed, and 2 ml / well of anti-LSR antibody (# 1-25) diluted to a concentration of 100 ⁇ g / ml with RPMI 1640 medium (containing 1% FBS and 1% penicillin-streptomycin) was added. .
- mouse IgG2 biolegend, 400224, MOPC-173
- intracellular DNA was stained using Cycle Test Plus DNA Reagent kits (BD Biosciences), and cell cycle analysis was performed using a FACSCanto flow cytometer.
- results are shown in FIG.
- the cell cycle of ovarian cancer cells (RMG-I) was significantly decreased in S and G2 / M phases and significantly increased in G0 / G1 phase compared to the control antibody treatment group Admitted.
- Proteins were extracted 72 hours after the addition of the antibody, and the changes in the expression of proteins related to the cell cycle were detected by Western blotting (anti-cyclin D1 (# 2926), anti-p27 (# 3686), anti-phospho- Rb (Ser780) (# 9307), anti-phospho-Rb (Ser807 / 811) (# 9308), anti-Rb (# 9313), anti-phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (# 4370), anti-p44 / 42 MAPK (Erk1 / 2) (# 4695), anti-phospho-MEK1 / 2 (Ser217 / 221) (# 9154), anti-MEK1 / 2 (# 9126) (Cell Signaling Technology).
- the anti-LSR antibody obtained in 2.4 above was specifically identified by analyzing the epitope as described above.
- the anti-LSR antibody whose epitope has been identified is examined for the growth inhibitory effect of LSR-positive malignant tumors.
- an anti-LSR antibody that recognizes a specific epitope significantly suppresses the growth of LSR-positive malignant tumors compared to an anti-LSR antibody that recognizes another epitope.
- LSR siRNA and negative control siRNA were obtained from QIAGEN.
- LSR siRNA has an RNA sequence complementary to LSR mRNA (LSR siRNA 1: SEQ ID NO: 9, LSR siRNA 2: SEQ ID NO: 10). The results are shown in FIG. Contact with LSR siRNA suppressed the growth of ovarian cancer cells (OVSAHO).
- Lipid quantification was performed using SKOV3 empty vector strain (EMP1) and LSR forced expression strain (L45). Low concentration was 5 ⁇ 10 5 per 10 cm dish, and high concentration was 5 ⁇ 10 5 cells and cultured for 48 hours. The medium was not changed. A methanol + chloroform mixture was added from cells suspended in PBS, and the lower organic layer was recovered by centrifugation, and lipid extraction was performed.
- EMP1 SKOV3 empty vector strain
- LSR forced expression strain L45
- Lipid quantification is performed using Lab Assay TM triglyceride (GPO / DAOS method, Wako Pure Chemical Industries, Ltd.), Lab Assay TM cholesterol (cholesterol oxidase / DAOS method, Wako Pure Chemical Industries, Ltd.), Phospholipid C-Test Wako (choline oxidase / DAOS). Method, Wako Pure Chemical Industries, Ltd.). Metabolic enhancement by VLDL was measured using an extracellular flux analyzer XFe24 (Primetech Inc.). Glucose was removed from the buffer, Glutamine was added, and the antibody amount was increased from 10 ug / ml to 100 ug / ml for assay.
- lipid (cholesterol) uptake was enhanced in the LSR stably expressing cells described in the Examples.
- the uptake of lipid (cholesterol) was enhanced in the high-density culture in the LSR stably expressing cells described in Examples.
- LSR expression described in the Examples promotes VLDL metabolism, but the administration of LSR antibody (# 9-7) inhibited the enhancement of metabolism by VLDL. Even though # 1-25 showed some inhibition of hypermetabolism, the degree was less than that of # 9-7. Without wishing to be bound by theory, it is believed that this difference is due to differences in the epitope recognition sites by clones.
- anti-LSR antibody # 1-25
- isotype control antibody MouseIgG2a, M7769, Sigma
- Tumor volume calculated from major axis x minor axis x height.
- the anti-LSR antibody administration group showed a significant inhibitory effect on tumor growth in vivo compared to the control IgG administration group (FIGS. 26-28). There was also a significant difference in tumor weight.
- anti-LSR antibody # 1-25
- isotype control antibody MouseIgG2a, M7769, Sigma
- Tumor volume calculated from major axis x minor axis x height.
- the anti-LSR antibody-administered group showed a significant inhibitory effect on tumor growth in vivo compared to the control IgG-administered group (FIG. 44). There was also a significant difference in tumor weight.
- a significant decrease in the number of Ki-67 positive cells was observed in the anti-LSR antibody administration group compared to the control IgG administration group. It was revealed that anti-LSR antibody showed an activity to induce cell cycle arrest in vivo. (FIG. 45).
- FIG. 50 shows that an antibody-conjugated drug can be applied as an antibody-drug conjugate (ADC).
- ADC antibody-drug conjugate
- ADC antibody-drug conjugate
- Example 7 Safety test
- mice 1 mg of mouse IgG2a (sigma, M7769) or anti-LSR antibody # 1-25 was intraperitoneally administered to males and females of C57BL / 6J (8w) mice. The mice were dissected on the 7th day, and the brain, heart, kidney, The liver, lung and spleen were removed and pathological analysis was performed by HE staining.
- Examples 1-4 above show that (i) when an anti-LSR antibody is brought into contact with a malignant tumor cell, the proliferation of the malignant tumor cell is suppressed, and (ii) when an LSR antagonist is allowed to act on the malignant tumor cell, Tumor cell growth is suppressed; (iii) malignant tumor patients can be treated with anti-LSR antibodies; (vi) malignant tumor patients have LSR positive patients, but LSR negative It shows that there are a certain number of patients, and (v) in the treatment of malignant tumors targeting LSR, it is important to diagnose the presence or absence of LSR positivity in malignant tumor patients before treatment. .
- a malignant tumor marker and a malignant tumor control technique are provided, and a technique that can be used in industries (reagents, pharmaceuticals, etc.) related to a technique related to diagnosis, treatment and prevention of a malignant tumor is provided.
- SEQ ID NO: 1 anti-LSR antibody 9-7
- SEQ ID NO: 2 anti-LSR antibody 16-6
- SEQ ID NO: 3 anti-LSR antibody 26-2
- SEQ ID NO: 4 anti-LSR antibody 27-6
- SEQ ID NO: 5 anti-LSR Antibody 1-25
- SEQ ID NO: 6 Anti-LSR antibody 1-43
- SEQ ID NO: 7 Human LSR protein sequence (NP_991403.1)
- SEQ ID NO: 10 LSR siRNA 2 core sequence (guide sequence)
- SEQ ID NO: 12 antisense sequence of LSR siRNA 2 core sequence (guide sequence)
- SEQ ID NO: 13 sense full-length sequence of LSR siRNA 1
- SEQ ID NO: 14 LSR siRNA 2 sense full
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Reproductive Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hospice & Palliative Care (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pregnancy & Childbirth (AREA)
- Gynecology & Obstetrics (AREA)
Abstract
Description
(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230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2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230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3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、および(f)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、50~66位、99~104位、153~165位、182~188位、および221~229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、変異体を提供する。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)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230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2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230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3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、および(f)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、50~66位、99~104位、153~165位、182~188位、および221~229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、変異体であってもよい。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。使用される抗体としては、限定されるものではないが、抗体は、配列番号7の116~134位および/または216~230位をエピトープとして有するものが有利に用いられ得る。本明細書において、有利な効果が示されており、安全性および安定性も示されているからである。
(a)配列番号7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8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8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7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8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、LSR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。
LSRのアミノ酸配列としては、
(a)配列番号8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8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7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8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、LSR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。
抗体が好ましい。
ができる。
が挙げられるが、これらに限定されない。本発明のマーカーまたはマーカーの検出剤、検査剤、診断剤(プライマーまたはプローブ等であり得る)にはこのようなタグを結合させてもよい。
成され、その監督官庁により承認を受けた旨が明記される。指示書は、いわゆる添付文書(package insert)であり、通常は紙媒体で提供されるが、それに限定されず、例えば、電子媒体(例えば、インターネットで提供されるホームページ、電子メール)のような形態でも提供され得る。
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、新規の悪性腫瘍の治療または予防薬を提供する。この治療または予防薬は、LSRの抑制剤、例えば、抗LSR抗体を含む、悪性腫瘍の治療または予防薬である。この治療または予防薬を用いれば、LSR陽性悪性腫瘍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 in Molecular 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: ACompendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel,F.M. (1995).Short Protocols in Molecular Biology: 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 from Current 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。
1.1 iTRAQ法による細胞表面膜タンパク質の定量解析
正常卵巣上皮細胞株(HOSE2C、E7/TERT)に比べて卵巣漿液性腺癌細胞株(OVCAR3、OVSAHO、JHOS4)で高発現する細胞表面膜タンパク質を探索することによって、卵巣癌特異的な癌抗原タンパク質の同定を試みた。まず、150mmシャーレで培養した細胞株に対して、sulfo-NHS-SS-biotinで細胞表面膜タンパク質をビオチン標識した。抽出したタンパク質をNeurto-avidinビーズにて精製した。このとき、サンプル間での誤差を補正するため、sulfo-NHS-SS-biotinで標識したウシ血清アルブミンを内部標準として等量ずつ加え、質量分析計による定量結果の補正に用いた。精製したタンパク質をトリプシンで消化し、iTRAQ試薬で標識した。サンプルを1つに混合し、イオン交換HPLCにて24個のフラクションに粗分画し、それぞれの分画を脱塩後、質量分析計(nanoLC-MS/MS)解析にて測定した。得られたデータをproteome discoverer ver1.1にてデータベースサーチすることで、タンパク質の同定と定量を行った。なお、実施例で使用した卵巣癌手術組織は、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。
正常卵巣上皮細胞株(HOSE2C)、卵巣漿液性腺癌細胞株(OVCAR3、OVSAHO、JHOS4)、卵巣明細胞線癌細胞株(OVTOKO、OVMANA、OVISE、RGMI)、正常子宮内膜細胞株(E6/E7/TERT)および子宮内膜癌(HEC1、HEC1A、HEC6、HEC88nu、HEC108、HEC116、HEC251、SNGM)のRNAを、それぞれRNeasymini kit (QIAGEN)で精製した。さらに、QuantiTect Reverse Transcription Kit (Qiagen)を用いてcDNAへ逆転写した。RT-PCRを、TaKaRaEx Taq DNA polymerase(Takara Bio, Shiga, Japan)を用いて行った。RT-PCRには、以下の配列のプライマーを用いた。
LSR:
forward primer5’-GGGAGGACCTCAGGGGTGGC-3’ (配列番号17)、および
reverse primer5’-TGGTGGGGGTGGGGTCTTGG-3’ (配列番号18)
β-actin:
forward primer5’-AGCCTCGCCTTTGCCGA-3’ (配列番号19)、および
reverse primer5’-CTGGTGCCTGGGGCG-3’ (配列番号20)
以上の結果を図1~3に示す。卵巣漿液性腺癌細胞株OVCAR3、OVSAHO、JHOS4、卵巣明細胞線癌細胞株OVMANA、OVISE、RGMI、および子宮内膜癌細胞株HEC1、HEC1A、HEC6、HEC88nu、HEC108、HEC116、HEC251、SNGMにおいて、LSRmRNAに相当するバンドが検出された。正常卵巣上皮細胞株では、HOSE2Cでは検出されなかった。
正常卵巣上皮細胞株(HOSE2C)、卵巣漿液性腺癌細胞株(OVCAR3、OVSAHO、SKOV3、JHOS2、JHOS4)、卵巣明細胞線癌細胞株(OVTOKO、OVMANA、OVISE、RGMI)、正常子宮内膜細胞株(E6/E7/TERT)および子宮内膜癌(HEC1、HEC1A、HEC6、HEC88nu、HEC108、HEC116、HEC251、SNGM)から得られた10μgのタンパク質を、それぞれSDS-PAGE(5-20%グラジェントゲル(和光純薬))にアプライした。次に、40mAで50min泳動し、PVDF膜に120mA、1時間転写した。転写後、1%BSA/TBST (TBS+ 0.1% Tween20)にて室温で1時間ブロッキングし、抗LSR抗体(Santa Cruz Biotechnology)で、室温で1時間インキュベートした。TBSTで10分間、3回ずつ洗浄した後、TBSTで5,000倍希釈したHRP標識抗ウサギ抗体(GE healhcare)を用いてPVDF膜を室温で1時間インキュベートした。PVDF膜をTBSTで10分間、3回ずつ洗浄した後、蛍光反応システム(PerkinElmer社)により、反応したタンパク質を検出した。
2.1 ヒトLSR発現ニワトリ細胞株の作製とニワトリへの免疫
ヒトLSRのcDNA(配列番号7)をほ乳動物発現ベクター(pcDNA3.1-V5/His-TOPO)にクローニングし、LSR発現ベクターを作成した。このLSR発現ベクターは、ヒトLSRのC末端にV5/Hisタグが融合した融合タンパク質をコードする。次に、LSR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。抗体価の測定はフローサイトメトリ(FACS)解析にて実施した。FACS解析に関してはFACSCalibur(BD、USA)のプロトコ ルに従った。
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。scFvファージ抗体ライブラリーの作製に関しては、"Nakamura et al., J Vet Med Sci. 2004 Jul;66(7):807-14"に記載の手法に従った。
scFvファージ抗体ライブラリーをLSR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、LSR発現細胞株と反応させた。Lot1はほ乳類細胞株を用い、Lot2は免疫に使用したニワトリリンパ芽球様細胞株を用いてセルパニングを行った。有機溶媒で洗浄後、LSR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LSR発現細胞株を用いたFACS解析で確認した。反応性が最も上昇し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、陽性クローンを選択した後、6種のクローンについて配列を決定した(配列番号1~6、図9)。セルパニングに関しては、"Giordano et al., Nat Med. 2001 Nov;7(11):1249-53."に記載の方法に従った。
scFvファージ抗体をコードするDNA鎖を鋳型にして、ニワトリ由来抗体遺伝子のVH、VLのPCR増幅を行った後、マウス/ニワトリキメラ(IgG2a)発現ベクター(H鎖:pcDNA3.1、L鎖:pcDNA4(Invitrogen))へクローニングした。作製したH鎖、L鎖のコンストラクトをほ乳類培養細胞にトランスフェクトした後、発現した抗体(抗LSRマウス/ニワトリキメラモノクローナル抗体)の精製をProteinG Sepharose(GE)を用いておこなった。以上により、6種の抗LSR抗体のクローンを得た(#9-7、#16-6、No.26-2、No.27-6、No.1-25、No.1-43)。組換えに関しては、"Tateishi et al., J Vet Med Sci. 2008 Apr;70(4):397-400."に記載の手法に従った。
上記2.4で得られた抗LSR抗体から5種(#1-25、#9-7、#16-6、No.26-2、No.27-6)を用いて、各種卵巣癌細胞株への反応性を、FACS解析により調査した。その結果を図10~14に示す。卵巣漿液性腺癌細胞株(OVSAHO、JHOS2)、および卵巣明細胞線癌細胞株(RGM-I、OVISE)において、抗LSR抗体の有無によって、有意なシフト差が見られた。
卵巣癌(84)症例の組織に対して、LSRの発現を免疫組織化学染色により解析した。1次抗体はcloud clone社(PAD744Hu01)を使用し、Dako ChemMate ENVISION Kit/HRP (DAB)-universal kit (K5007)を用いて染色した。
PepStarTMペプチドマイクロアレイはJPT Peptide Technologies (GmbH)より入手したガラススライド上に作成した。LSRの細胞外ドメインの領域に対して、10アミノ酸ずつオーバーラップした15-merのオーバーラッピングペプチドを合成し、ガラススライドに固相化した。精製リコンビナント抗体のペプチドへの結合は説明書に従って実施したが一部変更点を含めた(www.jpt.com)。一次抗体は1.0 μg/mLの濃度で反応させ、TBST(50 mM TBS-buffer including 0.1% Tween20, pH 7.2)でガラススライドを洗浄した。次に、Cy5標識ヤギ抗ニワトリIgY (Jackson Immuno Research)を用いて反応し、TBSTで5回、ガラススライドをddH2Oで5回洗浄した。ガラススライドはアルゴンガスを穏やかに吹き付けることで乾燥させた。蛍光シグナルはGenePix 4200AL scanner (Molecular Devices)を用いて、10 μmの解像度で検出した。
抗LSR抗体がマウスLSRと交差反応することを調べるため、COS7細胞にマウスLSR発現ベクターあるいはコントロールベクターをトランスフェクションし、本実施例で調製される抗LSR抗体各種クローンとの反応性をFACSにて解析した。結果を図40に示す。その結果、いずれのクローンもマウスLSRと交差反応を示すことが明らかとなった。このように、抗LSR抗体がマウスLSRと交差することは、安全性試験の動物にマウスを用いることが出来る事になる。実際の急性毒性試験は図52以降で実施しているのでこれらを参照されたい。
卵巣漿液性腺癌組織、および子宮内膜癌組織のパラフィン包埋組織の薄切に対して、脱パラフィン処理、アルコールによる脱水を行った。次に、抗LSR抗体(#1-25、または#9-7)を用いて、ABC法で、LSRに対する免疫組織化学染色を行った。その結果を図15~17に示す。卵巣漿液性腺癌組織、および子宮内膜癌組織においてLSRが腫瘍部位に高発現していた。
FDA standard human tissue microarray(T6234701-2,Biochain)を抗LSR抗体#1-25を用いて免疫組織化学染色を実施した。各種正常組織の内、肝臓と精巣にてLSRの発現が見られた(図36,37)。
RMG-I細胞に対して、各種抗LSR抗体を様々な濃度で反応させ、ヤギ抗マウスIgG-FITC (Southern Biotech, Birmingham, AL, USA)を用いて染色し、FACSCanto II cytometer (Becton Dickinson)を用いて解析した。FITCの蛍光強度はGraphPadPrism Software Version 6.0 for Windows (GraphPad Software Inc., San Diego, CA, USA)を用いてKD値を解析した。結果を図37Bに示す。作製したLSR抗体の結合能をFACSで解析したところ、結合能の高い抗体が得られた。この中で最も結合能が高いクローン2種類を用いて以後の解析に用いた。(図37B)。#9-7はKD=2.52nM、#1-25はKD=2.03nM、#16-6はKD=2.33nM、#26-2はKD=4.04nM、#27-6はKD=4.29nM、#1-43はKD=24.62nMであった。
卵巣明細胞線癌(RMG-I)を6ウェルプレートに15,000cells/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。培養上清を捨て、RPMI 1640 medium (1% FBS, 1% penicillin-streptomycin含有)で希釈した100μg/mlの抗LSR抗体あるいはマウスIgG2aを2mL/wellずつ加えた。96時間後にCycle Test Plus DNA Reagent kits (BD Biosciences)を用いて細胞周期解析を行った。
卵巣明細胞線癌(RMG-I)を6ウェルプレートに15,000cells/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。培養上清を捨て、RPMI 1640 medium (1% FBS, 1% penicillin-streptomycin含有)で希釈した100μg/mlの抗LSR抗体あるいはマウスIgG2aを2mL/wellずつ加えた。96時間後にRIPA buffer (10 mM Tris-HCl,pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodiumdeoxycholate, 0.1% SDS, 1× phosphatase inhibitorcocktail (Nacalai Tesque) and 1× protease inhibitorcocktail (Nacalai Tesque))を用いてタンパク質抽出を行い、ウェスタンブロット法によりタンパク質発現差の解析を行った。一次抗体は以下のものを用いた。抗LSR抗体(sc-133765)、抗GAPDH抗体(sc-25778) (Santa Cruz Biotechnology (Santa Cruz, CA))、抗cyclin D1抗体(#2926), 抗p27抗体(#3686), 抗phospho-Rb(Ser780)抗体(#9307), 抗phospho-Rb抗体(Ser807/811) (#9308)、抗Rb抗体(#9313), 抗phospho-p44/42 MAPK(Erk1/2) (Thr202/Tyr204)抗体(#4370), 抗p44/42 MAPK (Erk1/2)抗体(#4695), 抗phospho-MEK1/2 (Ser217/221) 抗体(#9154)、抗MEK1/2抗体(#9126) (Cell Signaling Technology)
<実施例3>LSR陽性悪性腫瘍の増殖抑制
3.1 抗LSR抗体による増殖阻害アッセイ
卵巣明細胞線癌(RMG-I)を96ウェルプレートに1000cells/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LSR抗体(#9-7、または#1-25)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18、19に示す。抗LSR抗体を接触させることによって、卵巣癌細胞(RMG-I)の増殖が抑制された。
卵巣明細胞線癌(RMG-I)を6ウェルプレートに15,000cells/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。6ウェルプレートの細胞上清を除き、RPMI 1640 培地(1% FBSおよび1% penicillin-streptomycin含有)で100μg/mlの濃度に希釈した抗LSR抗体(#1-25)を2mL/wellずつ加えた。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。抗体添加後、96時間後にCycle Test Plus DNA Reagent kits (BD Biosciences)を用いて細胞内のDNAを染色し、FACSCanto flow cytometerを用いて細胞周期解析を実施した。
卵巣明細胞線癌(RMG-I)を6ウェルプレートに15,000cells/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。6ウェルプレートの細胞上清を除き、RPMI 1640 培地(1% FBSおよび1% penicillin-streptomycin含有)で100μg/mlの濃度に希釈した抗LSR抗体(#1-25)を2mL/wellずつ加えた。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。抗体添加後、72時間後にタンパク質を抽出し、ウェスタンブロット法により細胞周期に関連するタンパク質の発現変動を各種抗体(anti-cyclin D1 (#2926), anti-p27 (#3686), anti-phospho-Rb(Ser780) (#9307), anti-phospho-Rb (Ser807/811) (#9308), anti-Rb (#9313), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370), anti-p44/42 MAPK (Erk1/2) (#4695), anti-phospho-MEK1/2 (Ser217/221)(#9154),anti-MEK1/2 (#9126) (Cell Signaling Technology)を用いて解析した。
上記2.4で得られた抗LSR抗体について、上述のようにエピトープを解析して具体的に同定した。また、エピトープを特定した抗LSR抗体について、LSR陽性悪性腫瘍の増殖抑制効果を調べる。その結果、特定のエピトープを認識する抗LSR抗体は、他のエピトープを認識する抗LSR抗体に比べて、顕著にLSR陽性悪性腫瘍の増殖を抑制すると理解される。
卵巣漿液性腺癌細胞(OVSAHO)を96ウェルプレートに1000cells/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、lipofectamine2000でsiRNAをトランスフェクションした。120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。LSR siRNA、およびnegative control siRNAはQIAGEN社より入手した。LSR siRNAは、LSR mRNAに相補的なRNA配列(LSR siRNA 1:配列番号9、LSRsiRNA 2:配列番号10)を有している。結果を図21に示す。LSR siRNAを接触させることによって、卵巣癌細胞(OVSAHO)の増殖が抑制された。
実施例に記載のLSR安定発現細胞では脂質(コレステロール)の取り込みが亢進するかどうかを確認し、VLDL代謝を亢進させるかどうかを確認したのち、LSR抗体投与下により、VLDLによる代謝の亢進が阻害されるかどうかを確認した。
Scidマウス(6週齢、メス)皮下に卵巣明細胞腺癌細胞株RMG-Iを1x106cells/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LSR抗体(#1-25)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(図25)。RMG-I移植マウスは抗体投与開始後25日目に解剖し、腫瘍重量も計測した。腫瘍体積=長径x短径x高さより計算した。
NOD/Scidマウス(6週齢、メス)皮下に卵巣明細胞腺癌細胞株RMG-Iを1x106cells/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LSR抗体(#1-25)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(図43)。RMG-I移植マウスは抗体投与開始後25日目に解剖し、腫瘍重量も計測した。腫瘍体積=長径x短径x高さより計算した。
また、腫瘍組織を抗Ki-67抗体で免疫組織化学染色を行った結果、control IgG投与群と比較して抗LSR抗体投与群ではKi-67陽性細胞数の有意な減少が認められたことから、抗LSR抗体はin vivoにおいて細胞周期の停止を誘導する活性を示していることが明らかとなった。(図45)。
Scidマウス(6週齢、メス)皮下にLSR陰性の卵巣漿液性腺癌細胞株であるSKOV3にLSRを安定発現させたSKOV3-L45あるいは空ベクターを遺伝子導入したSKOV3-E1を5x105cells/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LSR抗体(#1-25)あるいはisotypecontrol抗体(MouseIgG2a、M7769、Sigma)を10mg/kg一日おきの頻度で計8回、腹腔内に投与した(図46)。マウスは抗体投与開始後18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高さより計算した。
次に、本発明の抗体について安全性試験を行った。抗LSR抗体 #1-25はマウスLSR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LSR抗体 #1-25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(VetScan HMII)、動物用生化学血液分析器(VetScanVS2)を用いて解析した(図52)。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53、54)。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55、56)。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理解される。
配列番号2:抗LSR抗体16-6配列
配列番号3:抗LSR抗体26-2配列
配列番号4:抗LSR抗体27-6配列
配列番号5:抗LSR抗体1-25配列
配列番号6:抗LSR抗体1-43配列
配列番号7:ヒトLSRタンパク質配列(NP_991403.1)
配列番号8:ヒトLSR核酸配列(NM_205834.3)
配列番号9:LSR siRNA 1のコア配列(ガイド配列)
配列番号10:LSR siRNA 2コア配列(ガイド配列)
配列番号11:LSR siRNA 1のコア配列(ガイド配列)のアンチセンス配列
配列番号12:LSR siRNA 2コア配列(ガイド配列)のアンチセンス配列
配列番号13:LSR siRNA 1のセンス全長配列
配列番号14:LSR siRNA 2のセンス全長配列
配列番号15:LSR siRNA 1のアンチセンス全長配列
配列番号16:LSR siRNA 2のアンチセンス全長配列
配列番号17:LSRフォワードプライマー配列
配列番号18:LSRリバースプライマー配列
配列番号19:β-アクチンフォワードプライマー配列
配列番号20:β-アクチンリバースプライマー配列
Claims (46)
- LSR(Lipolysis stimulated lipoprotein receptor)の抑制剤を含む、悪性腫瘍の治療または予防薬。
- 前記抑制剤は、抗LSR(Lipolysis stimulated lipoprotein receptor)抗体またはその抗原結合性断片あるいはその機能的等価物、あるいは核酸を含む、請求項1に記載の悪性腫瘍の治療または予防薬。
- 前記抑制剤は、抗LSR(Lipolysisstimulated lipoprotein receptor)抗体またはその抗原結合性断片あるいはその機能的等価物を含む、請求項1に記載の悪性腫瘍の治療または予防薬。
- 前記抑制剤は、LSRに対するRNAi分子、またはそのRNAi分子をコードするポリヌクレオチドである、請求項1に記載の悪性腫瘍の治療または予防薬。
- 前記悪性腫瘍は、LSR陽性悪性腫瘍である請求項1に記載の治療または予防薬。
- LSR陽性悪性腫瘍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1に記載の治療薬。
- 悪性腫瘍患者の中で、前記悪性腫瘍がLSR陽性悪性腫瘍と判断された患者に対して投与するための、請求項1に記載の治療薬。
- 前記抗LSR抗体は、LSRのエピトープに特異的に結合する抗LSR抗体である、請求項3に記載の悪性腫瘍の治療または予防薬。
- 前記抗LSR抗体は、VLDLによる亢進阻害能をも有する抗体である、請求項3に記載の治療または予防薬。
- 前記抗LSR抗体は、
(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230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2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230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3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、および
(f)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、50~66位、99~104位、153~165位、182~188位、および221~229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、変異体である、請求項3に記載の治療または予防薬。 - 前記抗LSR抗体は、モノクローナル抗体である、請求項3に記載の治療または予防薬。
- 前記抗LSR抗体の抗体クラスは、IgGである、請求項3に記載の治療または予防薬。
- 前記抗LSR抗体は、抗原結合性断片である、請求項3に記載の治療または予防薬。
- 前記抗LSR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10に記載の予防または治療薬。
- 前記抗LSR抗体は、配列番号7の116~134位および/または216~230位をエピトープとして有する、請求項3に記載の予防または治療薬。
- 前記悪性腫瘍は卵巣癌を含む、請求項1に記載の予防または治療薬。
- 前記卵巣癌は、再発性卵巣癌である、請求項16に記載の予防または治療薬。
- 前記悪性腫瘍は、卵巣癌が転移したものである、請求項1に記載の予防または治療薬。
- 前記悪性腫瘍は、卵巣癌、膵臓癌、肺癌、胃癌または大腸癌を含む、請求項1に記載の予防または治療薬。
- 前記悪性腫瘍は早期卵巣癌である、請求項1に記載の予防または治療薬。
- 前記卵巣癌は、卵巣漿液性腺癌または卵巣明細胞腺癌である、請求項16に記載の予防または治療薬。
- 抗LSR抗体を含む、悪性腫瘍細胞の細胞分裂抑制薬。
- LSRの検出剤を含む、LSRを標的とした悪性腫瘍治療のためのコンパニオン診断薬。
- 前記LSRの検出剤は抗LSR抗体を含む、請求項23に記載のコンパニオン診断薬。
- 悪性腫瘍患者の悪性腫瘍サンプルがLSR陽性であることを検査する工程を含む、LSRを標的とした悪性腫瘍治療のコンパニオン診断法。
- (a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230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2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230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3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、および
(f)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、50~66位、99~104位、153~165位、182~188位、および221~229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。 - 前記抗LSR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26に記載の抗体または該抗体の変異体。
- 前記抗LSR抗体は、配列番号7の116~134位および/または216~230位をエピトープとして有する、請求項26に記載の予防または治療薬。
- LSRの結合剤を含む、悪性腫瘍を予防または治療するための組成物。
- 前記悪性腫瘍は、LSR陽性悪性腫瘍である請求項29に記載の治療または予防薬。
- さらに細胞殺傷性薬剤を含む、請求項29に記載の組成物。
- 前記LSR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29に記載の組成物。
- 前記LSR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29に記載の組成物。
- 前記悪性腫瘍は卵巣癌を含む、請求項29に記載の組成物。
- 前記卵巣癌は、再発性卵巣癌である、請求項34に記載の組成物。
- 前記悪性腫瘍は、卵巣癌が転移したものである、請求項29に記載の組成物。
- 前記悪性腫瘍は、卵巣癌、膵臓癌、肺癌、胃癌または大腸癌を含む、請求項29に記載の組成物。
- 前記悪性腫瘍は早期卵巣癌である、請求項29に記載の組成物。
- 前記卵巣癌は、卵巣漿液性腺癌または卵巣明細胞腺癌である、請求項34に記載の組成物。
- 前記悪性腫瘍は、請求項24のコンパニオン診断法によりLSR陽性であると判断されるものであり、前記LSRの結合剤がその後に投与されることを特徴とする、請求項29に記載の組成物。
- 前記LSR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1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230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2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230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3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229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5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、および
(f)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、50~66位、99~104位、153~165位、182~188位、および221~229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29に記載の組成物。 - 前記抗LSR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41に記載の組成物。
- 前記LSR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、前記抗LSR抗体は、配列番号7の116~134位および/または216~230位をエピトープとして有する、請求項29に記載の組成物。
- LSR(Lipolysis stimulated lipoprotein receptor)の結合剤を含む、悪性腫瘍の治療の予後不良のマーカー。
- LSR(Lipolysis stimulated lipoprotein receptor)の発現レベルを悪性腫瘍の治療の予後不良の指標とする方法。
- LSR(Lipolysis stimulated lipoprotein receptor)の結合剤を含む、悪性腫瘍の治療の予後不良の診断剤。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015554577A JP6372930B2 (ja) | 2013-12-27 | 2014-12-25 | 悪性腫瘍の治療薬 |
US15/108,242 US10174111B2 (en) | 2013-12-27 | 2014-12-25 | Therapeutic drug for malignant tumors |
EP14873694.5A EP3088001B1 (en) | 2013-12-27 | 2014-12-25 | Anti-lipolysis stimulated liporotein receptor antibodies for treatment of ovarian cancer |
US16/151,232 US20190023781A1 (en) | 2013-12-27 | 2018-10-03 | Therapeutic drug for malignant tumors |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013272084 | 2013-12-27 | ||
JP2013-272084 | 2013-12-27 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/108,242 A-371-Of-International US10174111B2 (en) | 2013-12-27 | 2014-12-25 | Therapeutic drug for malignant tumors |
US16/151,232 Division US20190023781A1 (en) | 2013-12-27 | 2018-10-03 | Therapeutic drug for malignant tumors |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2015098113A1 true WO2015098113A1 (ja) | 2015-07-02 |
Family
ID=53478009
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2014/006456 WO2015098113A1 (ja) | 2013-12-27 | 2014-12-25 | 悪性腫瘍の治療薬 |
Country Status (4)
Country | Link |
---|---|
US (2) | US10174111B2 (ja) |
EP (1) | EP3088001B1 (ja) |
JP (2) | JP6372930B2 (ja) |
WO (1) | WO2015098113A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11788067B2 (en) | 2016-05-25 | 2023-10-17 | University Of Cape Town | Production of a horseradish peroxidase-igg fusion protein |
WO2024101382A1 (ja) * | 2022-11-09 | 2024-05-16 | 学校法人 岩手医科大学 | 免疫チェックポイントの調節に用いるための組成物 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101842679B1 (ko) * | 2015-10-15 | 2018-03-28 | 한국과학기술원 | Rna 올리고뉴클레오티드 및 이를 포함하는 면역 활성제 |
WO2017065369A1 (ko) * | 2015-10-15 | 2017-04-20 | 한국과학기술원 | Rna 올리고뉴클레오티드 및 이를 포함하는 면역 활성제 |
CN109758579A (zh) * | 2019-03-19 | 2019-05-17 | 深圳大学 | 一种脂代谢激活脂蛋白受体在促进yap蛋白磷酸化增加中的应用 |
CN109884319B (zh) * | 2019-03-21 | 2022-05-10 | 福建师范大学 | 一种卵巢癌肿瘤标记物的双模式免疫分析方法 |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
WO1999007409A1 (fr) | 1997-08-04 | 1999-02-18 | Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) | Produit comprenant au moins un arn double brin en association avec au moins un agent anti-viral |
WO1999032619A1 (en) | 1997-12-23 | 1999-07-01 | The Carnegie Institution Of Washington | Genetic inhibition by double-stranded rna |
WO2000001846A2 (en) | 1998-07-03 | 2000-01-13 | Devgen N.V. | Characterisation of gene function using double stranded rna inhibition |
WO2000044895A1 (de) | 1999-01-30 | 2000-08-03 | Roland Kreutzer | Verfahren und medikament zur hemmung der expression eines vorgegebenen gens |
WO2000044914A1 (en) | 1999-01-28 | 2000-08-03 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna |
WO2001029058A1 (en) | 1999-10-15 | 2001-04-26 | University Of Massachusetts | Rna interference pathway genes as tools for targeted genetic interference |
WO2001036646A1 (en) | 1999-11-19 | 2001-05-25 | Cancer Research Ventures Limited | Inhibiting gene expression with dsrna |
JP2006241026A (ja) | 2005-03-01 | 2006-09-14 | Hiroshima Univ | 抗体およびその利用 |
WO2012140627A1 (en) | 2011-04-15 | 2012-10-18 | Compugen Ltd. | Polypeptides and polynucleotides, and uses thereof for treatment of immune related disorders and cancer |
WO2013190555A1 (en) * | 2012-06-21 | 2013-12-27 | Compugen Ltd. | Lsr antibodies, and uses thereof for treatment of cancer |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2767135B1 (fr) | 1997-08-06 | 2002-07-12 | Genset Sa | Recepteur complexe lsr, activite, clonage, et application au diagnostic, a la prevention et/ou au traitement de d'obesite et des risques ou complications associes |
AU4082100A (en) | 1999-06-18 | 2001-01-09 | Immunex Corporation | Lsr, a cdna of the ig superfamily expressed in human leukocytes |
-
2014
- 2014-12-25 JP JP2015554577A patent/JP6372930B2/ja active Active
- 2014-12-25 US US15/108,242 patent/US10174111B2/en active Active
- 2014-12-25 EP EP14873694.5A patent/EP3088001B1/en active Active
- 2014-12-25 WO PCT/JP2014/006456 patent/WO2015098113A1/ja active Application Filing
-
2018
- 2018-06-12 JP JP2018112077A patent/JP2018199676A/ja active Pending
- 2018-10-03 US US16/151,232 patent/US20190023781A1/en not_active Abandoned
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
WO1999007409A1 (fr) | 1997-08-04 | 1999-02-18 | Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) | Produit comprenant au moins un arn double brin en association avec au moins un agent anti-viral |
WO1999032619A1 (en) | 1997-12-23 | 1999-07-01 | The Carnegie Institution Of Washington | Genetic inhibition by double-stranded rna |
WO2000001846A2 (en) | 1998-07-03 | 2000-01-13 | Devgen N.V. | Characterisation of gene function using double stranded rna inhibition |
WO2000044914A1 (en) | 1999-01-28 | 2000-08-03 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna |
WO2000044895A1 (de) | 1999-01-30 | 2000-08-03 | Roland Kreutzer | Verfahren und medikament zur hemmung der expression eines vorgegebenen gens |
WO2001029058A1 (en) | 1999-10-15 | 2001-04-26 | University Of Massachusetts | Rna interference pathway genes as tools for targeted genetic interference |
WO2001036646A1 (en) | 1999-11-19 | 2001-05-25 | Cancer Research Ventures Limited | Inhibiting gene expression with dsrna |
JP2006241026A (ja) | 2005-03-01 | 2006-09-14 | Hiroshima Univ | 抗体およびその利用 |
WO2012140627A1 (en) | 2011-04-15 | 2012-10-18 | Compugen Ltd. | Polypeptides and polynucleotides, and uses thereof for treatment of immune related disorders and cancer |
WO2013190555A1 (en) * | 2012-06-21 | 2013-12-27 | Compugen Ltd. | Lsr antibodies, and uses thereof for treatment of cancer |
Non-Patent Citations (98)
Title |
---|
"Japanese Society of Obstetrics and Gynecology, Fuj inka Shuyo Iinkai Hokoku", GYNECOLOGY TUMOR COMMITTEE REPORT, vol. 64, no. 6, 2012 |
"KOD-Plus-Mutagenesis Kit", TOYOBO CO. , LTD. |
"Molecular Cloning, 2nd ed.", vol. SUPPL. 1, 1995, OXFORD UNIVERSITY PRESS, article "Current Protocols in Molecular Biology" |
"Revised 4th edition Shin Idenshikogaku Handobukku [New Genetic Engineering Handbook]", 2003, YODOSHA, pages: 152 - 179 |
"Sequences of Proteins of Immunological Interest, 5th ed.", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH |
"Tanpakushitsu Jikken Handobukku [Protein experiment handbook]", 2003, YODOSHA, pages: 128 - 142 |
"Tanpakushitsu Jikken Handobukku [Protein experiment handbook]", 2003, YODOSHA, pages: 86 - 91 |
"Tanpakushitsu Jikken Handobukku [Protein experiment handbook]", 2003, YODOSHA, pages: 92 - 96 |
ADAMS, R.L. ET AL.: "The Biochemistry of the Nucleic Acids", 1992, CHAPMAN&HALL |
ALLSHIRE, SCIENCE, vol. 297, 2002, pages 1818 - 1819 |
ALMAGRO ET AL., FRONT BIOSCI., vol. 13, 1 January 2008 (2008-01-01), pages 1619 - 1633 |
ALTSCHUL ET AL., J.MOL.BIOL., vol. 215, 1990, pages 403 - 410 |
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1995, WILEY INTERSCIENCE PUBLISHERS |
AUSUBEL, F. M.: "Current Protocols in Molecular Biology", 1987, GREENE PUB. ASSOCIATES AND WILEY-INTERSCIENCE |
AUSUBEL, F. M.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", 1992, GREENE PUB. ASSOCIATES |
AUSUBEL, F. M.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", 1995, GREENE PUB. ASSOCIATES |
AUSUBEL, F. M.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", 1999, WILEY |
AUSUBEL, F.M.: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", 1989, GREENE PUB . ASSOCIATES AND WILEY-INTERSCIENCE |
BASS, NATURE, vol. 411, 2001, pages 428 - 429 |
BATZER ET AL., NUCLEIC AC ID RES., vol. 19, 1991, pages 5081 |
BERGE ET AL., J. PHARM. SCI., vol. 66, 1977, pages 1 - 19 |
BLACKBURN, G. M. ET AL.: "Nucleic Acids in Chemistry and Biology", 1996, OXFORD UNIVERSITY PRESS |
CARTER P, JNAT. REV. IMMUNOL., no. 5, 6 May 2006 (2006-05-06), pages 343 - 357 |
CARTER PJ, NAT. REV. IMMUNOL., vol. 6, no. 5, pages 343 - 357 |
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917 |
CLACKSON ET AL., NATURE, vol. 352, no. 6336, 15 August 1991 (1991-08-15), pages 624 - 628 |
DAMSCHRODER ET AL., MOL IMMUNOL., vol. 44, no. 11, 2007, pages 3049 - 3060 |
DAMSCHRODER ET AL., MOL IMMUNOL., vol. 44, no. 11, 22 January 2007 (2007-01-22), pages 3049 - 3060 |
E. W. MARTIN: "Remington's Pharmaceutical Sciences", MARK PUBLISHING COMPANY |
ECKSTEIN, F.: "Oligonucleotides and Analogues : A Practical Approach", 1991, IRL PRESS |
ELBASHIR ET AL., NATURE, vol. 411, 2001, pages 494 - 498 |
FOOTE ET AL., J MOL BIOL., vol. 224, no. 2, 20 March 1992 (1992-03-20), pages 487 - 499 |
GAIL D. ET AL., CANCER RES, vol. 68, 2008, pages 9280 - 9290 |
GAIT, M.J.: "Oligonucleotide Synthesis: A Practical Approach", 1985, IRL PRESS |
GAIT, M.J.: "Oligonucleotide Synthesis: A Practical Approach", 1990, IRL PRESS |
GARCIA ET AL.: "Prognostic value of LISCH7 mRNA in plasma and tumor of colon cancer patients", CLIN CANCER RES, vol. 13, no. 21, 1 November 2007 (2007-11-01), pages 6351 - 8 |
GIORDANO ET AL., NAT MED., vol. 7, no. 11, November 2001 (2001-11-01), pages 1249 - 53 |
GYNECOLOGY TUMOR COMMITTEE REPORT, vol. 64, no. 6, 2012 |
HALL ET AL., SCIENCE, vol. 297, 2002, pages 2232 - 2237 |
HERBSLEB ET AL.: "Increased cell motility and invasion upon knockdown of lipolysis stimulated lipoprotein receptor (LSR) in SW780 bladder cancer cells", BMC MED GENOMICS, vol. 1, 22 July 2008 (2008-07-22), pages 31 |
HERMANSON, G.T.: "Bioconjugate Techniques", 1996, ACADEMIC PRESS |
HUTVAGNER; ZAMORE, SCIENCE, vol. 297, 2002, pages 2056 - 60 |
IAN KROP; ERIC P. WINER, CLIN CANCER RES, vol. 20, no. 1, pages 1 - 6 |
INNIS, M. A. ET AL.: "PCR Strategies", 1995, ACADEMIC PRESS |
INNIS, M. A.: "PCR Protocols : A Guide to Methods and Applications", 1990, ACADEMIC PRESS |
J.HONIG ET AL., THE VAN NOSTRAND CHEMIST'S DICTIONARY, 1953, pages 650 |
JENUWEIN, SCIENCE, vol. 297, 2002, pages 2215 - 2218 |
K NAITO ET AL., LEUKEMIA, vol. 14, 2000, pages 1436 - 144 |
KABAT ET AL.: "Sequence of Proteins of Immunological Interest", 1983, US DEPT. HEALTH AND HUMAN SERVICES |
KOHLER G; MILSTEIN C., NATURE, vol. 256, no. 5517, 7 August 1975 (1975-08-07), pages 495 - 497 |
LETH-LARSEN ET AL.: "Functional heterogeneity within the CD44 high human breast cancer stem cell-like compartment reveals a gene signature predictive of distant metastasis", MOL MED., vol. 18, 25 September 2012 (2012-09-25), pages 1109 - 21 |
LONBERG ET AL., INT REV IMMUNOL., vol. 13, no. 1, 1995, pages 65 - 93 |
MARK ET AL., PROC NATL ACAD SCI USA., vol. 81, no. 18, September 1984 (1984-09-01), pages 5662 - 5666 |
MARKS ET AL., JMOLBIOL., vol. 222, no. 3, 5 December 1991 (1991-12-05), pages 581 - 597 |
MARTINEZ, CELL, vol. 110, 2002, pages 563 - 574 |
MCMANUS ET AL., RNA, vol. 8, 2002, pages 842 - 850 |
MURRAY, JAH: "Antisense RNA and DNA", 1992, WILEY-LISS INC |
NAITO ET AL., BMC BIOINF ORMATICS, vol. 10, 30 November 2009 (2009-11-30), pages 392 |
NAKAMURA ET AL., J VET MED SCI., vol. 66, no. 7, July 2004 (2004-07-01), pages 807 - 14 |
NARVEKAR ET AL.: "Liver-specific loss of lipolysis-stimulated lipoprotein receptor triggers systemic hyperlipidemia in mice", DIABETES., vol. 58, no. 5, May 2009 (2009-05-01), pages 1040 - 9 |
NAT GENET., vol. 32, December 2002 (2002-12-01), pages 526 - 32 |
NEEDLEMAN; WUNSCH, J.MOL.BIOL., vol. 48, 1970, pages 443 - 453 |
OHTSUKA ET AL., J.BIOL.CHEM., vol. 260, 1985, pages 2605 - 2608 |
OZAKI ET AL., BLOOD, vol. 93, no. 11, 1 June 1999 (1999-06-01), pages 3922 - 3930 |
PEARSON; LIPMAN, PROC.NATL.ACAD.SCI., USA, vol. 85, 1988, pages 2444 - 2448 |
PROC.NATL.ACAD.SCI.USA, vol. 86, 1989, pages 9268 - 9272 |
REINHART ET AL., GENE & DEV., vol. 16, 2002, pages 1616 - 1626 |
REINHART; BARTEL, SCIENCE, vol. 297, 2002, pages 1831 |
REITER ET AL., J MOL BIOL., vol. 290, no. 3, 16 July 1999 (1999-07-16), pages 685 - 98 |
REITER ET AL., PROTEIN ENG., vol. 7, no. 5, May 1994 (1994-05-01), pages 697 - 704 |
RIECHMANN ET AL., NATURE, vol. 332, no. 6162, 24 March 1988 (1988-03-24), pages 323 - 327 |
ROGUSKA ET AL., PROC NATL ACAD SCI U S A., vol. 91, no. 3, 1 February 1994 (1994-02-01), pages 969 - 973 |
ROGUSKA ET AL., PROC NATL ACAD SCI USA., vol. 91, no. 3, 1 February 1994 (1994-02-01), pages 969 - 973 |
ROSSOLINI ET AL., MOL. CELL. PROBES, vol. 8, 1994, pages 91 - 98 |
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual, 3rd ed.", vol. 1, 2001, COLD SPRING HARBOR LABORATORY PRESS, pages: 7.42 - 7.45 |
SAMBROOK J. ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR |
SAMBROOK J. ET AL.: "MOLECULAR CLONING: A LABORATORY MANUAL, 3rd ed.", 2001, COLD SPRING HARBOR |
SARIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 7448 - 7451 |
SCHWARZ ET AL., MOLECULAR CELL, vol. 10, 2002, pages 537 - 568 |
See also references of EP3088001A4 * |
SHABAROVA, Z. ET AL.: "Advanced Organic Chemistry of Nucleic Acids", 1994, WEINHEIM |
SHUJUNSHA: "Cellular engineering, Extra issue", article "DNA Maikuroarei to Saishin PCR ho (DNA Microarrays and Latest PCR Methods)" |
SMITH; WATERMAN, J.MOL.BIOL., vol. 147, 1981, pages 195 - 197 |
SNINSKY, J. J. ET AL.: "PCR Applications: Protocols for Functional Genomics", 1999, ACADEMIC PRESS |
STEIN ET AL., NUCL. ACIDS RES., vol. 16, 1988, pages 3209 |
TATEISHI ET AL., J VET MED SCI., vol. 70, no. 4, April 2008 (2008-04-01), pages 397 - 400 |
VAUGHAN ET AL., NAT BIOTECHNOL., vol. 14, no. 3, March 1996 (1996-03-01), pages 309 - 314 |
VAUGHNANDMARTIENSSEN, SCIENCE, vol. 309, 2005, pages 1525 - 1526 |
VOLPE ET AL., SCIENCE, vol. 297, 2002, pages 1833 - 1837 |
WANG ET AL., SCIENCE, vol. 224, no. 4656, 29 June 1984 (1984-06-29), pages 1431 - 1433 |
WILLY ET AL., BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 356, no. 1, 27 April 2007 (2007-04-27), pages 124 - 128 |
WOLFSON W, CHEM BIOL., vol. 13, no. 12, December 2006 (2006-12-01), pages 1243 - 1244 |
YODOSHA: "Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume)", 1997, YODOSHA, article "Idenshi Donyu Oyobi Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis & Expression Analysis]" |
YODOSHA: "Tanpakushitsu Jikken Handobukku [Protein experiment handbook]", 2003, YODOSHA, pages: 27 - 52 |
YUSUKE NAKAMURA: "Genomu Kaiseki Jikkenho Nakamura Yusuke Labo Manyuaru [ Genome analysis experimental method Yusuke Nakamura Lab Manual]", 2002, YODOSHA |
ZAMORE ET AL., CELL, vol. 101, 2000, pages 25 - 33 |
ZAMORE; HALEY, SCIENCE, vol. 309, 2005, pages 1519 - 1524 |
ZOLLER ET AL., NUCLEIC ACIDS RES., vol. 10, no. 20, 25 October 1982 (1982-10-25), pages 6487 - 6500 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11788067B2 (en) | 2016-05-25 | 2023-10-17 | University Of Cape Town | Production of a horseradish peroxidase-igg fusion protein |
WO2024101382A1 (ja) * | 2022-11-09 | 2024-05-16 | 学校法人 岩手医科大学 | 免疫チェックポイントの調節に用いるための組成物 |
Also Published As
Publication number | Publication date |
---|---|
US20170051056A1 (en) | 2017-02-23 |
JPWO2015098113A1 (ja) | 2017-03-23 |
JP6372930B2 (ja) | 2018-08-15 |
JP2018199676A (ja) | 2018-12-20 |
EP3088001B1 (en) | 2020-02-12 |
US20190023781A1 (en) | 2019-01-24 |
EP3088001A1 (en) | 2016-11-02 |
US10174111B2 (en) | 2019-01-08 |
EP3088001A4 (en) | 2017-09-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6606793B2 (ja) | 食道がんのマーカーおよびその利用 | |
JP7138735B2 (ja) | Ror1癌の治療および転移の阻害に使用するための抗体およびワクチン | |
JP6372930B2 (ja) | 悪性腫瘍の治療薬 | |
US7959923B2 (en) | Method for treating cancer using anti-Wnt2 monoclonal antibodies and siRNA | |
WO2018199318A1 (ja) | 抗gpc-1抗体 | |
Cui et al. | CD147 receptor is essential for TFF3-mediated signaling regulating colorectal cancer progression | |
ES2717908T3 (es) | Anticuerpos S100A4 y usos terapéuticos de los mismos | |
JP6649941B2 (ja) | Fstl1を利用した抗がん剤・転移抑制剤およびその併用剤 | |
JP5756014B2 (ja) | がんの診断および治療のためのvhz | |
US20150216891A1 (en) | Nucleic acids targeting tctp for use in the treatment of chemo-or hormone- resistant cancers | |
JP2007517763A (ja) | Her2アンタゴニストを用いた治療への感受性を回復させる組成物および方法 | |
WO2021251459A1 (ja) | ヒト化抗gpc-1抗体 | |
US20110027297A1 (en) | Methods Involving MS4A12 and Agents Targeting MS4A12 for Therapy, Diagnosis and Testing | |
US20210155709A1 (en) | Antibodies anti tumor associated antigens and method for obtaining them | |
WO2024101382A1 (ja) | 免疫チェックポイントの調節に用いるための組成物 | |
JP2024068984A (ja) | 悪性腫瘍治療薬としてのlsrを認識する抗体薬物複合体 | |
WO2024036192A2 (en) | Compositions and methods for assessing the severity of and treating covid-19 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14873694 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2015554577 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15108242 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2014873694 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2014873694 Country of ref document: EP |