WO2010024293A1 - Ensemble d'anticorps monoclonaux anti-vasohibines - Google Patents

Ensemble d'anticorps monoclonaux anti-vasohibines Download PDF

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WO2010024293A1
WO2010024293A1 PCT/JP2009/064862 JP2009064862W WO2010024293A1 WO 2010024293 A1 WO2010024293 A1 WO 2010024293A1 JP 2009064862 W JP2009064862 W JP 2009064862W WO 2010024293 A1 WO2010024293 A1 WO 2010024293A1
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antibody
vasohibin
monoclonal antibody
monoclonal
amino acid
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PCT/JP2009/064862
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Japanese (ja)
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光 園田
英樹 太田
ちひろ 山内
靖史 佐藤
丘 近藤
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塩野義製薬株式会社
国立大学法人東北大学
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Priority to JP2010526744A priority Critical patent/JP5430573B2/ja
Publication of WO2010024293A1 publication Critical patent/WO2010024293A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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

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  • the present invention relates to a set of anti-vasohibin monoclonal antibodies. More specifically, a set of anti-vasohibin monoclonal antibodies comprising a combination of specific monoclonal antibodies, a monoclonal antibody constituting the set, a method for measuring a complex of the set and a biological sample derived from a subject, A method for determining the onset of diseases in which angiogenesis is enhanced, such as cancer and intraocular neovascular diseases, a kit for carrying out the method, a method for evaluating the efficacy of anti-angiogenic drugs using the set, and the method It is related with the kit for doing.
  • Angiogenesis is controlled by both angiogenesis-promoting factors and inhibitory factors, not only in the developmental phase but also in the somatic phase, and maintaining the balance between the stimulating factor and the inhibitory factor maintains vascular homeostasis. Is important to.
  • angiogenesis is promoted by overexpression of angiogenesis-promoting factor, resulting in further increase in tumor tissue. Therefore, anticancer treatment includes a method of administering an angiogenesis inhibitory factor, a method of administering an inhibitor of angiogenesis promoting factor, and the like.
  • Non-Patent Document 1 reports that the survival rate of rectal cancer patients is improved by administering a monoclonal antibody (Avastin) against VEGF, which is an angiogenesis-promoting factor, together with chemotherapy.
  • VEGF monoclonal antibody
  • endogenous inhibitors such as endostatin and angiostatin are also administered (see Non-Patent Document 2).
  • Vasohibin is a polypeptide found by the present inventors, and is expressed in vascular endothelial cells by stimulation of angiogenesis promoting factors (VEGF, FGF-2, etc.) secreted from tumor cells, stromal cells, macrophages and the like ( (See Patent Document 1), which acts on the endothelial cells themselves in an autocrine manner and suppresses angiogenesis.
  • VEGF angiogenesis promoting factors
  • FGF-2 FGF-2, etc.
  • Vasohibin is characterized by being secreted into the blood from vascular endothelial cells undergoing angiogenesis, so by measuring the amount of vasohibin in the blood and urine, the degree of angiogenesis occurring in the body can be determined. I can know.
  • HPLC high performance liquid chromatography
  • GC gas chromatography
  • a mass spectrometer or the like can be used for detection of in vivo vasohibin. It can also be measured by a Western method using a commercially available anti-vasohibin monoclonal antibody.
  • an anti-Vasohibin-1 monoclonal antibody (clone 411208) manufactured by R & D®systems can be used, but it is not yet sufficient in terms of sensitivity and specificity, and it can be quickly and easily In addition, further technology development is necessary for accurate and accurate detection.
  • An object of the present invention is to provide a set of anti-vasohibin monoclonal antibodies capable of detecting in vivo vasohibin with good sensitivity and specificity, a monoclonal antibody constituting the set, and the set and a biological sample derived from a subject.
  • a method for measuring a complex a method for determining the onset of a disease in which angiogenesis is enhanced, such as cancer or intraocular neovascular disease using the set, and a cancer or intraocular blood vessel containing the set for performing the method
  • a kit for determining the onset of a disease in which angiogenesis is promoted such as a neoplastic disease, a method for evaluating the efficacy of an anti-angiogenic agent using the set, and the efficacy of an anti-angiogenic agent containing the set for performing the method It is to provide an evaluation kit.
  • a set (set A) which is a monoclonal antibody (antibody A-1) that recognizes amino acid numbers 132 to 145 and a monoclonal antibody (antibody A-2) that recognizes amino acid numbers 351 to 365
  • a set (set C) which is a monoclonal antibody (antibody C-1) recognizing amino acid numbers 1 to 76 and a monoclonal antibody (antibody C-2) recognizing amino acid numbers 132 to 145
  • a set (set D) which is a monoclonal antibody (antibody D-1) that recognize
  • a monoclonal antibody that recognizes amino acids 132 to 145, a monoclonal antibody that recognizes amino acids 77 to 318, a monoclonal antibody that recognizes amino acids 319 to 365, and a monoclonal antibody that recognizes amino acids 3 to 15 A monoclonal antibody that is at least one selected from the group consisting of: [3] A method of measuring the abundance of the complex by contacting a biological sample derived from a subject and the monoclonal antibody constituting the set of [1] to form a complex, [4] Step (I): A biological sample derived from a subject is brought into contact with the monoclonal antibody constituting the set described in [1] to form a complex, and the abundance of the complex is measured.
  • Step (II) a step of comparing the abundance measured in the step (I) with an abundance in the control
  • step (III) abundance in the subject in the comparison performed in the step (II)
  • a method of determining the onset of a disease with increased angiogenesis comprising determining that the subject is more likely to have developed a disease with increased angiogenesis when the amount of [5]
  • a kit for determining the onset of a disease that promotes angiogenesis comprising the set of monoclonal antibodies according to [1], [6]
  • Step (B) for measuring the abundance of the body the step of comparing the abundance measured in the step (A) with the abundance before administration
  • the step (C) performed in the step (B)
  • the method includes a step of determining that the anti-angiogenic drug is likely to have
  • the set of anti-vasohibin monoclonal antibodies of the present invention has an excellent effect of being able to detect in vivo vasohibin with good sensitivity and specificity.
  • FIG. 1 is a diagram showing that a monoclonal antibody obtained by immunization with a partial peptide of Vasohibin-1 recognizes each peptide as an epitope.
  • FIG. 2 is a diagram showing the result of mapping whether the monoclonal antibody obtained by immunizing the full-length Vasohibin-1 protein recognizes the amino terminus, the central part, or the carboxyl terminus of the Vasohibin protein as an epitope.
  • FIG. 3 is a diagram showing the results of detecting Vasohibin-1 protein in the blood of healthy subjects and lung cancer patients by the Western method.
  • FIG. 4 is a diagram showing the results of detection of Vasohibin-1 protein by Western method using antibody VC1-4E12 and a commercially available antibody (clone 411208).
  • FIG. 5 is a diagram showing the results of examining a solid phase antibody when the antibody FcC-3E6 is used as a labeled antibody.
  • A is the result of absorbance when measuring Vasohibin-1 protein using each solid phase antibody
  • B is the result of creating a standard curve based on the absorbance
  • C is the result of an addition recovery test.
  • FIG. 6 is a diagram showing the results of examining a solid phase antibody when the antibody VhW-2B4 is used as a labeled antibody.
  • FIG. 7 is a diagram showing the results of examining a labeled antibody when the antibody FcC-9H5 is used as a solid phase antibody.
  • A is the result of absorbance when Vasohibin-1 protein is measured using each labeled antibody
  • B is the result of creating a standard curve based on the absorbance
  • C is the result of an addition recovery test.
  • FIG. 8 shows the results of a standard curve and addition recovery test in ELISA B ′.
  • FIG. 9 shows the results of a standard curve and addition recovery test in ELISAELF ′.
  • (A) shows a standard curve, and (B) shows the results of an addition recovery test.
  • FIG. 10 shows the standard curve and the result of the addition recovery test in ELISA F ”.
  • (A) shows the standard curve and (B) shows the result of the addition recovery test.
  • FIG. 11 is a diagram showing the results of detecting the amount of Vasohibin-1 protein in blood before and after surgery for cancer patients by ELISA. The left of each specimen is the preoperative value and the right is the postoperative value.
  • the set of anti-vasohibin monoclonal antibodies of the present invention is constituted by combining two types of monoclonal antibodies against a specific antigen, and has a great feature in that the combination is specific.
  • vasohibin examples include vasohibin-1 and vasohibin-2.
  • the anti-vasohibin monoclonal antibody means a monoclonal antibody against vasohibin-1.
  • Vasohibin 1 and Vasohibin 2 are different genes existing on different chromosomes, but the amino acid sequence of the protein encoded by these genes is 58%. Have homology.
  • Vasohibin 1 is a protein encoded by the KIAA1036 polynucleotide consisting of the base sequence represented by the 386th A to 1480th C of SEQ ID NO: 1, and the KIAA1036 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
  • peptides having a cysteine residue added to the amino terminus or carboxyl terminus are synthesized according to a known method. These peptide sequences correspond to the Vasohibin-1 polypeptides Gly3-Ala15, Leu132-Lys145, Ala217-Lys229, Gly286-Arg299, and Glu351-Val365, respectively, and are VN1, VN3, VR1, VC1, and VC, respectively. .
  • a hapten antigen is prepared through SH group of the peptide using maleimide KLH (Keyhole Limpet Hemocyanin, Imject (registered trademark) Maleimide Activated mcKLH, manufactured by Pierce).
  • KLH Keyhole Limpet Hemocyanin, Imject (registered trademark) Maleimide Activated mcKLH, manufactured by Pierce.
  • Vasohibin-1-KLH fusion protein or a rabbit IgG Fc region and Vasohibin-1 fusion protein is also used as an antigen.
  • These fusion proteins can be prepared according to known methods.
  • Vasohibin-1-KLH fusion protein introduces SH group into purified Vasohibin-1 protein with 10-fold molar amount of SPDP reagent, and 20 mM dithiothreitol After reduction, it can be fused with the maleimidated KLH to make an antigen.
  • the Fc fusion Vasohibin-1 protein is transiently introduced by introducing a plasmid vector into which a polynucleotide sequence having Fc added to the amino terminus or carboxyl terminus of Vasohibin-1 is introduced, for example, into Freestyle 293-F Cells (Invitrogen). And then purified using a protein A column.
  • the expression and purification of Vasohibin-1 protein is not particularly limited and can be performed according to a known method.
  • the anti-vasohibin monoclonal antibody in the present invention (hereinafter also referred to as the monoclonal antibody of the present invention) is not particularly limited, and can be prepared according to a known method.
  • a mammal is immunized with the antigen obtained as described above.
  • a mammal Generally, a mouse
  • the age of the mammal varies depending on the animal species used and is not particularly limited. In the case of a mouse or rat, it is usually about 4 to 12 weeks old, preferably about 5 to 10 weeks old. These mammals can be selected in consideration of compatibility with plasma cells to be cell-fused for the production of the monoclonal antibody of the present invention.
  • the antigen is used as an immunogen mixed with an adjuvant to enhance the immune response.
  • an adjuvant there is no limitation and a well-known thing can be used.
  • the adjuvant and the antigen can be mixed according to a method known in the art for the adjuvant to be used.
  • Mammal immunization is performed according to methods known in the art.
  • the immunogen is administered by injection into a mammal subcutaneously, intradermally, intravenously or intraperitoneally.
  • administration of the immunogen is repeated several times after the first immunization, and the administration interval can be adjusted as appropriate. Since the immune response varies depending on the type and strain of the mammal to be immunized, the immunization schedule and the dose of the immunogen can be appropriately set according to the animal to be used.
  • desired antibody-producing cells can be prepared in the immunized mammal.
  • Such antibody-producing cells are preferably splenocytes isolated 3 to 5 days after the final administration of the immunogen.
  • boost additional injection of immunogen
  • the amount of immunogen administered in boost is preferably about 4 to 5 times the amount of immunogen initially administered, but can be appropriately increased or decreased based on this.
  • the obtained antibody-producing cells are fused with myeloma-derived cells (myeloma cells) to prepare hybridomas.
  • the myeloma cells are preferably compatible with the mammal from which the antibody-producing cells to be fused are derived. Examples thereof include myeloma cells such as mouse myeloma P3U1, X63-Ag8.653.
  • the cell fusion method a method known in the art can be used, and examples include a method using polyethylene glycol (PEG), a method using Sendai virus, and a method using an electrofusion device.
  • PEG polyethylene glycol
  • Sendai virus a method using Sendai virus
  • electrofusion device a method using an electrofusion device.
  • the obtained hybridoma can be separated by culturing in a selective medium according to a known method.
  • the culture supernatant can be collected and an antibody titer assay can be performed based on a known method, for example, the DELFIA method.
  • hybridoma producing the desired monoclonal antibody can be obtained.
  • the hybridoma can be subcultured in a normal medium and can be stored semipermanently in liquid nitrogen.
  • Desired monoclonal antibodies can be prepared in large quantities by in vivo and in vitro culture methods.
  • the in vitro culture method can be performed by culturing the hybridoma in an appropriate serum medium or serum-free medium, and the desired monoclonal antibody is produced in the medium. According to this culture method, a desired antibody having a relatively high purity can be obtained as a culture supernatant.
  • the in vivo culture method can be carried out by inoculating the hybridoma with a hybridoma, for example, an intraperitoneal cavity of a mammal that is compatible with the hybridoma, such as a mouse, and proliferating, and the desired antibody can be recovered in large amounts as mouse ascites. .
  • the obtained culture supernatant and ascites fluid such as mouse can be used as a crude antibody solution as they are.
  • These are purified according to conventional methods, for example, by appropriately combining DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, etc. to obtain purified antibodies. be able to.
  • monoclonal antibodies can be obtained, and the monoclonal antibodies used in the present invention are shown in Table 1.
  • FcN is amino terminal
  • FcC is carboxyl terminal
  • Fso is Vasohibin-1 protein
  • VhW is full length Vasohibin-1-KLH fusion protein.
  • each monoclonal antibody is antigen protein and clone.
  • it can be expressed as antibody VN1-2H11.
  • a monoclonal antibody obtained using VR1 as an antigen that is, the antibody VR1-12E7 is H.HSonoda et al., Multiple processing forms and their biological activities of a novel angiogenesis inhibitor vasohibin.Biochem Biophys Res Commun.
  • the monoclonal antibodies shown in Table 1 excluding the monoclonal antibody constitute the set of monoclonal antibodies of the present invention. Provided as a monoclonal antibody.
  • the above monoclonal antibody may be prepared by using the above-mentioned method to recognize a specific antigen.
  • a hybridoma that produces the monoclonal antibody the National Institute of Advanced Industrial Science and Technology, The cells deposited under the following deposit number or receipt number at the deposit center (1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture) can also be suitably used.
  • FERM P-21585 (monoclonal antibody produced is VN3-11E11, indicated VN3-11E11, date of commissioning June 5, 2008)
  • FERM ABP-11169 (Monoclonal antibody produced is VC-12F6, indicated VC-12F6, domestic consignment date: June 5, 2008, transfer application pending)
  • FERM ABP-11168 (Monoclonal antibody produced is VR1-12E7, labeled VR1-12E7, domestic contract date was accepted on June 5, 2008, and application for transfer)
  • FERM P-21589 (monoclonal antibody produced is FcC-3E6, designated FcC-3E6, date of trust June 5, 2008)
  • FERM P-21590 (monoclonal antibody produced is FcC-9H5, designated FcC-9H5, date of trust June 5, 2008)
  • FERM P-21588 (monoclonal antibody produced is FcN-6A5, indicated FcN-6A5, date of trust June 5, 2008)
  • FERM P-21590
  • ⁇ Immunoassay method using the set of monoclonal antibodies of the present invention sandwich-enzyme immunometric assay (ELISA) was performed using VC1-4E12 as a labeled antibody and VR1-12E7 as a solid phase antibody, and the purified Vasohibin-1 protein in the buffer was measured. It has been reported that this is possible (H. Sonoda et al., Multiple processing forms and their biological activities of a novel angiogenesis inhibitor vasohibin. Biochem Biophys Res Commun. 342 (2006), 640-646.). However, when an attempt was made to measure Vasohibin-1 protein in blood using the above antibody, it was not possible to measure. Therefore, for the antibodies shown in Table 1, sandwich ELISA antibodies that enable measurement of blood Vasohibin-1 protein, that is, combinations of solid phase antibodies and labeled antibodies are identified.
  • the preparation of the solid phase antibody and the labeled antibody that is, the solid phase immobilization and labeling of the monoclonal antibody obtained above is not particularly limited and can be performed according to a known method.
  • the subclass of the antibody is IgG2b, which makes it difficult to modify Fab ′ necessary for high sensitivity. Since it is expected, it is excluded from the subject of examination as a labeled antibody in advance.
  • the obtained combination of solid phase antibody and labeled antibody is examined for the quantitative and specific binding properties of Vasohibin-1 protein according to a known ELISA method. Quantitative evaluation and specific binding evaluation are performed according to the following method to identify a set of anti-vasohibin monoclonal antibodies of the present invention.
  • an increase in signal intensity is observed depending on Vasohibin-1 concentration in a concentration range of at least 2 pmol / mL, preferably 0 to 5 pmol / mL.
  • a combination that provides a standard curve with good linearity is determined as a good combination.
  • the linearity can be judged from the slope and intercept of the standard curve.
  • the slope is preferably 0.5 to 1.5, more preferably 0.7 to 1.5, and the Y-axis intercept is preferably 0.15 or less, more preferably. Is 0.10 or less, it is determined that the combination has good linearity.
  • the specific binding property is obtained when measuring Vasohibin-1 in a blood sample to which a known amount of Vasohibin-1 is added or in a blood sample to which no known amount is added by the sandwich EILSA method described in Example 1 described later. It can be evaluated by calculating the addition recovery rate from the signal intensity.
  • a combination in which the recovery rate of the specimen containing Vasohibin-1 is preferably 75 to 125%, more preferably 80 to 125%, and still more preferably 85 to 115% is determined as a good combination.
  • a set of anti-vasohibin monoclonal antibodies of the present invention that is, an anti-vasohibin monoclonal antibody capable of measuring blood vasohibin for combinations determined to be good in all of the above quantitative and specific binding properties Identified as a set of Accordingly, the present invention also provides the use of said anti-vasohibin monoclonal antibody set for the measurement of blood vasohibin.
  • blood vasohibin means vasohibin present in at least one biological sample selected from the group consisting of blood, serum and plasma.
  • Immunoassay An immunoassay is performed using the set of antibodies identified above. Immunoassays include enzyme immunoassay (EIA), enzyme immunometric assay (ELISA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay, immunoblot, Western blot
  • EIA enzyme immunoassay
  • ELISA enzyme immunometric assay
  • FFA fluorescence immunoassay
  • RIA radioimmunoassay
  • luminescence immunoassay immunoblot
  • Western blot The ELISA method is preferable because the antibody can be easily detected with high sensitivity.
  • the ELISA method includes a general competitive method, a sandwich method, and the like. Since the set of the anti-vasohibin monoclonal antibody of the present invention can be used as a solid phase antibody and a labeled antibody in the sandwich method, the sandwich method is used. Is preferred.
  • the sandwich ELISA method provides a method for measuring the abundance of the complex by contacting a biological sample derived from a subject with the monoclonal antibody constituting the set of the present invention to form a complex.
  • the present invention also provides a method for determining the onset of a disease in which angiogenesis is enhanced.
  • the set of anti-vasohibin monoclonal antibodies of the present invention can detect in vivo vasohibin with good sensitivity and specificity.
  • Vasohibin is expressed in vascular endothelial cells by angiogenesis-promoting factors (VEGF, FGF-2, etc.) secreted from tumor cells, stromal cells, macrophages and the like. A large amount of vasohibin is considered to exist.
  • VEGF angiogenesis-promoting factors
  • the method is a method for measuring the onset factor of a disease in which angiogenesis is enhanced Or as a determination method.
  • diseases in which angiogenesis is enhanced include cancer, intraocular angiogenic diseases, rheumatoid arthritis and the like, but cancer and intraocular angiogenic diseases are preferable from the viewpoint of enhancing angiogenesis by onset.
  • intraocular neovascular diseases include age-related macular degeneration, diabetic retinopathy, and the like.
  • Step (I) A step of contacting a biological sample derived from a subject and a monoclonal antibody constituting the set of the present invention to form a complex, and measuring the abundance of the complex
  • Step (II) a step of comparing the abundance measured in the step (I) with an abundance in the control
  • step (III) abundance in the subject in the comparison performed in the step (II) If the subject is found to be more than the control, the subject is determined to have a high possibility of developing a disease in which angiogenesis is enhanced.
  • the above “determination method” is not only a test for establishing a treatment strategy for a subject exhibiting a symptom of a disease, but also a prevention performed to determine whether the subject is susceptible to the disease. Also included is a test for or whether or not already affected. Therefore, “the possibility of developing a disease in which angiogenesis is increased is high” means that the degree of onset of a disease in which angiogenesis is increased is serious, a disease in which angiogenesis is increased, or It means determining the onset of a disease in which angiogenesis is enhanced.
  • step (I) of the determination method For the measurement of the abundance of the complex in step (I) of the determination method, a method well known to those skilled in the art can be used as long as it is a method using the set of monoclonal antibodies of the present invention. preferable.
  • step (II) the abundance obtained as described above is compared by performing a statistical analysis based on the abundance in the control person.
  • a statistical analysis method There is no limitation in particular as an analysis method, A well-known method can be used.
  • the determination in the subsequent step (III) is, for example, determined that there is a high possibility of developing a disease in which angiogenesis is enhanced when the abundance in the subject is larger than the abundance in the control Is done.
  • a control person refers to a patient diagnosed as not having developed a disease that promotes angiogenesis.
  • cancer treatment for a subject whose onset of cancer has been determined as a disease in which angiogenesis is enhanced, cancer treatment, for example, surgical treatment, treatment by radiation therapy, treatment by chemotherapy, etc.
  • angiogenesis is reduced compared to before treatment, and thus the amount of vasohibin in the living body is considered to be reduced.
  • the healing state of the cancer can also be determined.
  • control person in this case means the subject before treatment of the subject whose onset of cancer is determined, and the amount of vasohibin present is higher than that before treatment by comparison in step (II) In some cases, it is highly likely that the cancer has developed, that is, the cancer is still developing.
  • kits for diseases that promote angiogenesis In another aspect of the present invention, a kit for determining the onset of a disease that promotes angiogenesis is provided.
  • kits of the present invention include those containing the set of monoclonal antibodies of the present invention. If the detection method uses the set of monoclonal antibodies of the present invention when detecting vasohibin in a sample, the kit is used. be able to. Since the set of monoclonal antibodies of the present invention can detect in vivo vasohibin with good sensitivity and specificity, the use of the kit may greatly contribute to the determination of the onset of a disease that promotes angiogenesis. it can.
  • VEGF is an angiogenic factor that is secreted from cancer cells and plays a central role in tumor angiogenesis in solid cancer tissues.
  • VEGF receptor antagonists have been actively developed as anticancer agents.
  • Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. Clin. Invest. 114 (2004), 898-907, Vasohibin-1 expression is induced by stimulation of VEGF.
  • VEGF produced by vascular endothelial cells themselves is required for homeostasis of vascular endothelial cells (S.
  • VEGF signals in systemic vascular endothelial cells are blocked, resulting in a decrease in Vasohibin-1 production and a change in blood Vasohibin-1 levels.
  • vascular endothelial cell responsiveness to VEGF receptor antagonists in each patient is expected to vary from individual to individual, that responsiveness is reflected in the anti-angiogenic effects of those drugs. Therefore, by measuring the fluctuation amount of blood Vasohibin-1 before and after the administration of these drugs, the reactivity to the drug in each patient is predicted, or the efficacy after the actual administration is evaluated. It will be possible.
  • the drug efficacy evaluation method is as follows: Step (A): A biological sample derived from a subject after administration of an anti-angiogenic agent and a monoclonal antibody constituting the set of the present invention are contacted to form a complex, and the abundance of the complex is determined. Step (B) of measuring: A step of comparing the abundance measured in the step (A) with an abundance before administration, and a step (C): in the comparison performed in the step (B), A step of determining that the anti-angiogenic drug is likely to have a medicinal effect when it is recognized that the amount of the complex is small after administration compared to before administration of the new drug.
  • step (A) of the above-mentioned drug efficacy evaluation method a method well known to those skilled in the art can be used as long as it uses the set of monoclonal antibodies of the present invention. Is preferred.
  • step (B) the abundance obtained as described above is compared by performing a statistical analysis based on the abundance before administration of the anti-angiogenic drug.
  • a statistical analysis method There is no limitation in particular as an analysis method, A well-known method can be used.
  • step (C) for example, when the amount of the anti-angiogenic agent in the sample after administration is smaller than the amount in the sample before administration, the anti-angiogenic agent causes angiogenesis. It is judged that there is a high possibility that the effect of suppressing is shown.
  • kits for anti-angiogenic drugs In another aspect of the present invention, a kit for evaluating the efficacy of an anti-angiogenic drug is provided.
  • kits of the present invention include those containing the set of monoclonal antibodies of the present invention. If the detection method uses the set of monoclonal antibodies of the present invention when detecting vasohibin in a sample, the kit is used. be able to. Since the set of monoclonal antibodies of the present invention can detect in vivo vasohibin with good sensitivity and specificity, the use of the kit can greatly contribute to the evaluation of the efficacy of anti-angiogenic drugs.
  • Vasohibin-1 partial peptides that is, Vasohibin-1 polypeptide Gly3-Ala15, Leu132-Lys145, Ala217-Lys229, Gly286-Arg299, Glu351-Val365C amino terminus or carboxyl terminus added with cysteine residue
  • Vasohibin-1 polypeptide Gly3-Ala15, Leu132-Lys145, Ala217-Lys229, Gly286-Arg299, Glu351-Val365C amino terminus or carboxyl terminus added with cysteine residue These peptides were synthesized by Sigma-Aldrich and designated as VN1, VN3, VR1, VC1, and VC, respectively.
  • the hapten antigen was prepared through SH group of the peptide using maleimide KLH [Keyhole Limpet Hemocyanin, Imject (registered trademark) Maleimide Activated mcKLH, manufactured by Pierce Co., Ltd.].
  • Vasohibin-1-KLH fusion protein or a fusion protein of Fc region of rabbit IgG and Vasohibin-1 was prepared as an antigen. That is, the Vasohibin-1-KLH fusion protein was introduced into the purified Vasohibin-1 protein with a 10-fold molar amount of SPDP reagent, reduced with 20 mM dithiothreitol, and then fused with the maleimidated KLH to obtain an antigen.
  • the Fc-fused Vasohibin-1 protein is transiently introduced by introducing a plasmid vector into which a polynucleotide sequence having Fc added to the amino terminus or carboxyl terminus of Vasohibin-1 is introduced into Freestyle 293-F Cells (Invitrogen). After expression, it was prepared by purification using a protein A column (MAPS II kit, Bio-Rad Laboratories Inc.). Vasohibin-1 protein is expressed in baculovirus expression system as described in K. ⁇ Watanabe et al., Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. Clin. Invest. 114 (2004), 898-907. Expression and purification.
  • Vasohibin-1 hapten antigen, Vasohibin-1-KLH fusion protein, or Fc fusion Vasohibin-1 protein can be used as an immunogen by mixing equal amounts of complete adjuvant for the first immunization and incomplete adjuvant for the second and subsequent immunizations. An emulsion was prepared.
  • Monoclonal antibodies were produced by the method described in K. Watanabe et al., Vasohibin asan anendothelium-derived negative feedback regulator, of angiogenesis, J. Clin. Invest. 114 (2004), 898-907. That is, a 5-week-old female A / J mouse was subcutaneously administered with 50 ⁇ g hapten antigen or 10 ⁇ g protein antigen (Vasohibin-1-KLH fusion protein, Fc fusion Vasohibin-1 protein) per administration per administration. The dose was divided into equal doses intraperitoneally. Thereafter, after the fourth or fifth immunization, the spleen was extracted from the mouse subjected to the final booster (4 days before cell fusion) to prepare spleen cells.
  • the antibody titer of the antiserum and the hybridoma supernatant was evaluated by the DELFIA method described below. That is, antiserum or hybridoma supernatant is added to a 96-well microplate on which goat anti-mouse IgG antibody is solid-phased, and biotinylated Vasohibin-1 protein and europium-labeled streptavidin are mixed and stirred, and then at room temperature for 2 hours or 4 hours. The reaction was allowed to proceed overnight at 0 ° C.
  • the plate was washed three times with a washing solution (saline containing 0.01% Tween 20 and 0.1% ProClin 150), 100 mL DELFIA enhancing reagent was added, and the mixture was shaken and mixed for 5 minutes.
  • Time-resolved fluorescence at 615 nm was measured with ARVO MX (Perkin Elmer), and a case showing a signal intensity exceeding three times the signal obtained when no antiserum or hybridoma was added was judged as positive.
  • Each monoclonal antibody was purified from a serum-free medium of antibody-producing hybridoma or ascites obtained by administering the hybridoma to mice using a protein A column (MAPS II kit, Bio-Rad Laboratories Inc.). As described above, all the monoclonal antibodies shown in Table 1 were prepared.
  • Test Example 1 [Confirmation of epitope of monoclonal antibody obtained by immunization with partial peptide of Vasohibin-1] According to the above DELFIA method, it was examined whether the monoclonal antibody obtained by the immune system of Vasohibin-1 partial peptide surely recognizes and reacts with the partial peptide as an epitope.
  • antibody solutions were prepared by diluting various monoclonal antibodies VN1-2H11, VN3-11E11, VR1-12E7, VC1-4E12, and VC-12F6 obtained using hapten antigen as an immunogen to various concentrations.
  • the DELFIA method was performed using this antibody solution, and the amount of antibody showing a certain signal (100,000 counts) was calculated.
  • various concentrations of the peptide used for immunization were added, and it was examined whether a competitive reaction occurred depending on the peptide concentration. If a competitive reaction occurs, the antibody will be reactive with the peptide.
  • a peptide having a sequence unrelated to Vasohibin-1 was used as a control. The results are shown in FIG.
  • Test Example 2 [Epitope mapping of monoclonal antibody obtained by immunization with full-length Vasohibin-1] As shown in Table 1, monoclonal antibodies (FcN-5A3, FcN-6A5, FcC-3E6, FcC-6E5, FcC-9H5) obtained by immunization with full-length Vasohibin-1 protein (FcN, FcC, VhW) VhW-1F7, VhW-2B4) were investigated by epitope mapping to determine which part of Vasohibin-1 protein was recognized.
  • anti-mouse IgG is solid-phased on a plate, each anti-Vashibin monoclonal antibody is bound, biotinylated full-length Vasohibin-1 protein is further bound, and biotinylated full-length Vasohibin-1 is detected by europium-labeled streptavidin. .
  • Unlabeled full-length Vasohibin-1 a polypeptide in which the amino terminal 76 amino acid residues are deleted (Vh (77-365)), or a polypeptide in which the carboxyl terminal 47 is further deleted (Vh (77-318)) is added, and it is detected whether or not the biotinylated full-length Vasohibin-1 is replaced by recognition of the added protein by each monoclonal antibody.
  • the antibody When replaced by full length Vasohibin-1 alone, the antibody recognizes the amino terminal 76 amino acid residues of Vasohibin-1, and when replaced by full length Vasohibin-1 and Vh (77-365), the antibody is of Vasohibin-1 If it recognizes the 47 amino acid residues at the carboxyl terminal and is replaced by full length Vasohibin-1, Vh (77-365), or Vh (77-318), the antibody is an intermediate corresponding to the 77th to 318th amino acids. You will recognize the part. As a result, as shown in FIG.
  • FcN-5A3, FcN-6A5 and FcC-3E6 have amino acid terminal 76 amino acid residues
  • FcC-6E5 and FcC-9H5 have intermediate parts corresponding to amino acids 77 to 318. It was revealed that VhW-1F7 and VhW-2B4 recognize the carboxyl terminal 47 amino acid residues, respectively.
  • Test Example 3 Detection of blood Vasohibin-1 protein by Western method
  • Vasohibin-1 in blood can serve as a marker for angiogenesis of cancer. A test was conducted.
  • EDTA plasma Blood was collected from healthy subjects and lung cancer patients using EDTA blood collection tubes to prepare EDTA plasma. From 30 ⁇ L of this EDTA plasma, 500 ⁇ L of albumin and globulin removed using anti-albumin / globulin gel of Albumin and IgG Removal kit (GE Healthcare Biosciences), 2 mL of acetone was added and stirred, It left still at 20 degreeC for 2 hours or more. Thereafter, the precipitate obtained by centrifugation was dried and dissolved in 60 ⁇ L of Tris-buffered physiological saline. 15 ⁇ L of this solution was added to a 10% SDS-PAGE gel by the method described in K.
  • FIG. 3 shows the results of detection using ECL (advance (GE Healthcare Bioscience).
  • FIG. 3 a band corresponding to Vasohibin-1 indicated by an arrow was obtained from a lung cancer patient specimen. Therefore, it was shown that Vasohibin-1 protein in blood can be measured using the prepared monoclonal antibody, and that Vasohibin-1 can be used as an angiogenesis marker for cancer patients.
  • Test Example 4 Evaluation of Sensitivity and Specificity of Anti-Vasohibin-1 Monoclonal Antibody.
  • the Western method was used for comparative evaluation of the performance of the anti-Vasohibin-1 monoclonal antibody prepared above and the anti-Vasohibin-1 monoclonal antibody (clone 411208) commercially available from R & D systems.
  • the antibody VC1-4E12 was used as the anti-Vasohibin-1 monoclonal antibody prepared above.
  • Vasohibin-1 protein which was transiently transfected with Vasohibin-1 expression vector to express Vasohibin-1 protein, or Vasohibin-1 protein produced and purified in baculovirus expression system, 10% SDS- Electrophoresis was performed on a PAGE gel, and the protein in the electrophoresis gel was transferred to a nitrocellulose membrane (Invitrogen). After blocking with 5% skim milk, each anti-Vasohibin-1 antibody (1 ⁇ g / mL) was blotted overnight at 4 ° C. with HRP-labeled anti-mouse IgG antibody (KPL) for 2 hours at room temperature. 1 was detected. The result is shown in FIG.
  • Vasohibin-1 protein in 1 ⁇ g of COS cell extract could be detected.
  • a commercially available antibody (clone 411208) manufactured by R & D®systems was used, Vasohibin-1 could not be confirmed in any volume of COS cell extract of 1 to 20 ⁇ g.
  • Example 1 Combination of anti-Vasohibin-1 monoclonal antibody
  • sandwich ELISA antibodies To search for combinations of sandwich ELISA antibodies that enable measurement of blood Vasohibin-1 protein, blood Vasohibin-1 protein measurement was performed for all combinations of antibodies shown in Table 1 that can be prepared as solid phase or labeled antibodies.
  • a sandwich ELISA was performed. It should be noted that the antibody FcC-6E5 and the antibody FcC-9H5 are excluded from the subject of examination as labeled antibodies because preparation of labeled antibodies is difficult.
  • a 96-well microplate on which all monoclonal antibodies shown in Table 1 were solid-phased was prepared. That is, 100 ⁇ L / well of each antibody solution prepared to 10 ⁇ g / mL with phosphate buffered saline (PBS) was added to a 96-well microplate and allowed to react overnight. Thereafter, the reaction solution is removed, and after washing 3 times with a washing solution (saline containing 0.01% Tween 20 and 0.1% ProClin 150), 2% Block Ace (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) is added at 200 ⁇ L / well. Blocking was performed by allowing to stand overnight at 0 ° C.
  • PBS phosphate buffered saline
  • Vasohibin standard solution (Vasohibin-1 final concentration 0 to 3.2 pmol / mL)
  • 50 ⁇ L of a solution containing labeled streptavidin was added, followed by stirring at 4 ° C. overnight. Thereafter, the reaction solution was removed, and after washing 3 times with the above washing solution, 100 ⁇ L of TMB solution (Colorburst Blue, manufactured by ALerCHEK) was added and reacted at room temperature for 15 minutes.
  • 50 ⁇ L of 0.5N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm of each well was measured.
  • FIG. 5 shows the results when the antibody FcC-3E6 is a labeled antibody
  • FIG. 6 shows the results when the antibody VhW-2B4 is a labeled antibody
  • FIG. 7 shows the antibody FcC-9H5. The results for a solid phase antibody are shown.
  • the absorbance results when measuring serially diluted Vasohibin-1 protein in a concentration range of 0 to 2.0 pmol / mL using each solid phase antibody (A), and the results of creating a standard curve by absorbance (B)
  • the absorbance was 1.5 or more with 2.0 pmol / mL Vasohibin-1 protein in the above result (A) and showed good results
  • the result (C) of conducting an addition recovery test on 9H5 is shown.
  • the recovery rate of antibody VC1-4E12 was less than half of the added amount (37.8%), whereas the recovery rates of antibodies FcC-6E5 and FcC-9H5 were 82.1% and 81.0%. A sufficient recovery rate was obtained. From these results, it was found that when antibody VhW-2B4 was used as a labeled antibody, excellent sensitivity and specificity were exhibited when solid phase antibodies were used as antibodies FcC-6E5 and FcC-9H5. The antibody FcC-6E5 and the antibody FcC-9H5 are considered to recognize a common epitope, and since almost no difference was found in the performance of these two antibodies, the antibody VhW-2B4 was selected as a representative of both antibodies. The antibody FcC-9H5 was used as the solid phase antibody in the case of the labeled antibody.
  • FIG. 7 shows the result of examining the labeled antibody when the antibody FcC-9H5, which was determined to be a preferable solid phase antibody when the antibody VhW-2B4 was used as the labeled antibody, was used as the solid phase antibody.
  • recovery test about -12E7, FcN-6A5, FcC-3E6, and VhW-2B4 is shown.
  • VhW-2B4 was determined to be a suitable combination in FIG. 6, and the recovery rate was as good as 91.7%.
  • antibodies VR1-12E7 and FcN-6A5 showed good results with recovery rates of 112.9% and 79.2%.
  • antibodies VN3-11E11 and FcC-3E6 were unsuitable as a combination because the recovery rates were 184.1% and 9.0% which were too high or too low.
  • Table 2 shows the results of all the studies conducted on the antibody combinations shown in Table 1.
  • the antibody combinations that are quantitative and show good results in the additive recovery test are marked with “ ⁇ ”, the other combinations are marked with “x”, and the combinations that were not examined are marked with “-”. .
  • the antibody FcC-6E5 and the antibody FcC-9H5 are considered to recognize a common epitope, and almost no difference was found in the performance of these two antibodies. Since the combination using VhW-2B4 was considered to have the same performance, the following tests were conducted for combinations other than the combination of solid phase antibody FcC-6E5 and labeled antibody VhW-2B41.
  • Example 2 [Construction of ELISA method using combination of anti-Vasohibin-1 monoclonal antibody]
  • an ELISA method was performed using a labeled antibody prepared by directly labeling the antibody.
  • Fab ′ was prepared by reduction with 10 mM 2-mercaptoethylamine and the above column.
  • HRP or alkaline phosphatase (ALP) was maleimidized with Sulfo-HMCS and purified with a PD-10 column (GE Healthcare).
  • An equimolar Fab ′ and maleimidated enzyme were mixed and reacted overnight at 4 ° C., and then purified by the above TSKgel G2000SWXL column of HPLC to prepare a labeled antibody.
  • the obtained labeled antibodies are shown in Table 3.
  • VC-to which was added in advance an assay buffer (1 mM magnesium chloride, 0.1 mM calcium chloride, 0.5% BSA, 0.01% Tween 20, 50 ⁇ M tris-HCl buffered saline pH 7.4 containing 10 ⁇ g / mL mouse ⁇ -globulin).
  • an assay buffer 1 mM magnesium chloride, 0.1 mM calcium chloride, 0.5% BSA, 0.01% Tween 20, 50 ⁇ M tris-HCl buffered saline pH 7.4 containing 10 ⁇ g / mL mouse ⁇ -globulin.
  • reaction solution was removed, and after washing 4 times with the above washing solution, 100 ⁇ L of a chemiluminescent substrate solution (Lumigen APS-5) was added, and after stirring, the amount of chemiluminescence in each well was measured.
  • a chemiluminescent substrate solution Liigen APS-5
  • FIG. 11 shows that the average decrease rate in the measured values before and after the operation was 0.69 times. From this result, the amount of Vasohibin-1 protein in the blood decreased by removing the cancer tissue, so a part of Vasohibin-1 protein detected in the blood of cancer patients was secreted from the vascular endothelial cells of the cancer tissue. It was speculated that the tumor angiogenesis could be monitored by the set of anti-vasohibin monoclonal antibodies of the present invention.
  • Example 3 Evaluation of efficacy of anti-angiogenic drugs by measuring Vasohibin-1
  • anti-angiogenic drugs such as anti-VEGF drugs such as VEGF-neutralizing antibodies such as Avastin and VEGF receptor inhibitors such as Sorafenib
  • the efficacy of the drug is measured in blood Vasohibin-1 before and after administration. Judgment can be made by changes in level.
  • an anti-angiogenic drug such as an anti-VEGF drug
  • the drug for cancer patients whose blood Vasohibin-1 level could be measured in advance by ELISA using any of the monoclonal antibody combinations shown in Table 3
  • the blood Vasohibin-1 level is measured after administration, and it is considered that anti-angiogenic drugs such as anti-VEGF drugs show a medicinal effect in patients with a large change in the measured value.
  • the degree can be shown by the ratio of the measured value after administration to the measured value before administration.
  • the set of anti-vasohibin monoclonal antibodies of the present invention can detect in vivo vasohibin with good sensitivity and specificity, it is preferably used for monitoring tumor angiogenesis.
  • Sequence number 1 of a sequence table is KIAA1036 polynucleotide and polypeptide encoded therein. It is. Sequence number 2 of a sequence table is KIAA1036 polypeptide.

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Abstract

La présente invention porte sur un ensemble d'anticorps monoclonaux anti-vasohibine, qui est au moins un élément choisi dans le groupe constitué par un ensemble comprenant un anticorps monoclonal (MA) capable de reconnaître la séquence se situant entre l'acide aminé (aa)-132 et aa-145 dans la séquence d'acides aminés représentée dans SEQ ID NO : 2, et un MA capable de reconnaître la séquence se situant entre aa-351 et aa-365 dans la séquence d'acides aminés, un ensemble comportant un MA capable de reconnaître la séquence se situant entre aa-217 et aa-229 dans la séquence d'acides aminés et un MA capable de reconnaître la séquence se situant entre aa-351 et aa-365 dans la séquence d'acides aminés, un ensemble comportant un MA capable de reconnaître la séquence se situant entre aa-1 et aa-76 dans la séquence d'acides aminés et un MA capable de reconnaître la séquence se situant entre aa-132 et aa-145 dans la séquence d'acides aminés, un ensemble comportant un MA capable de reconnaître la séquence se situant entre aa-77 et aa-318 dans la séquence d'acides aminés et un MA capable de reconnaître la séquence se situant entre aa-1 et aa-76 dans la séquence d'acides aminés, un ensemble comportant un MA capable de reconnaître la séquence se situant entre aa-77 et aa-318 dans la séquence d'acides aminés et un MA capable de reconnaître la séquence se situant entre aa-319 et aa-365 dans la séquence d'acides aminés, et un ensemble comportant un MA capable de reconnaître la séquence se situant entre aa-3 et aa-15 dans la séquence d'acides aminés et un MA capable de reconnaître la séquence se situant entre aa-351 et aa-365 dans la séquence d'acides aminés. L'ensemble d'anticorps monoclonaux anti-vasohibine peut détecter la vasohibine in vivo avec une bonne sensibilité et une bonne spécificité, et par conséquent peut être utilisé de façon appropriée pour la surveillance de l'angiogenèse de tumeur ou similaire.
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WO2014087810A1 (fr) * 2012-12-03 2014-06-12 国立大学法人東北大学 Anticorps anti-vasohibine-2
CN110028550A (zh) * 2016-07-07 2019-07-19 华东理工大学 降血压肽和降血压蛋白质及其应用

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WO2002090546A1 (fr) * 2001-05-07 2002-11-14 Shionogi & Co., Ltd. Polypeptide servant de marqueur angiogenique et son adn
WO2007037245A1 (fr) * 2005-09-29 2007-04-05 Shionogi & Co., Ltd. Polypeptide présentant une activité anti-angiogénique
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WO2007037245A1 (fr) * 2005-09-29 2007-04-05 Shionogi & Co., Ltd. Polypeptide présentant une activité anti-angiogénique
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2014087810A1 (fr) * 2012-12-03 2014-06-12 国立大学法人東北大学 Anticorps anti-vasohibine-2
JPWO2014087810A1 (ja) * 2012-12-03 2017-01-05 国立大学法人東北大学 抗バソヒビン２抗体
US9701744B2 (en) 2012-12-03 2017-07-11 Tohoku University Anti-Vasohibin 2 antibody
CN110028550A (zh) * 2016-07-07 2019-07-19 华东理工大学 降血压肽和降血压蛋白质及其应用
CN110028550B (zh) * 2016-07-07 2022-08-05 华东理工大学 降血压肽和降血压蛋白质及其应用

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