WO2006063735A1 - Endogline utilisee comme cible/marqueur de la resistance insulinique - Google Patents

Endogline utilisee comme cible/marqueur de la resistance insulinique Download PDF

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Publication number
WO2006063735A1
WO2006063735A1 PCT/EP2005/013198 EP2005013198W WO2006063735A1 WO 2006063735 A1 WO2006063735 A1 WO 2006063735A1 EP 2005013198 W EP2005013198 W EP 2005013198W WO 2006063735 A1 WO2006063735 A1 WO 2006063735A1
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Prior art keywords
endoglin
insulin resistance
compound
protein
diabetes
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PCT/EP2005/013198
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English (en)
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Peter Berndt
Stefan Evers
Stefan Foser
Michael Fountoulakis
Mitchell Lee Martin
Elena Sebokova
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F. Hoffmann-La Roche Ag
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Publication of WO2006063735A1 publication Critical patent/WO2006063735A1/fr

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Endoglin as target/marker for insulin resistance
  • Type 2 diabetes is a disease of fast growing worldwide importance and can be described as a failure of the pancreatic beta-cell (beta-cell failure) to compensate, with enhanced insulin secretion of the beta-cells, for peripheral insulin resistance. Insulin resistance can be considered the first step in the development of Type 2
  • EHC euglycemic-hyperinsulinemic clamp
  • the aim of the present invention is to identify and provide a novel target to screen for compounds that prevent, attenuate, or inhibit Insulin Resistance, and for a marker that allows for monitoring and/or diagnosis of Insulin Resistance at an earlier stage of type II diabetes and more reliably than can presently be done.
  • Endoglin also known as CD105, is a 180 kDa homodimeric co-receptor for members of the TGF-beta superfamily. This large type I integral membrane glycoprotein may have roles in hematopoiesis, cardiovascular development and angiogenesis. Although the function of Endoglin remains elusive, levels of a circulating soluble form of Endoglin are elevated in patients with atherosclerosis (Blann et al., 1996, Atherosclerosis 120, 221-226) and certain cancers including breast cancer (Li et al, 2000, Int. J. Cancer 89, 122-126), colorectal cancer (Takahashi et al., 2001, Clin. Cancer Res.
  • Endoglin binds several members of the TGF-beta superfamily including TGF- betal, TGF- beta3, BMP-2, BMP-7, and Activin A (Barbara et al., 1999, J. Biol. Chem. 274, 584-594). Endoglin does not bind TGF-beta ligands by itself, but does so by associating with both ligand and a corresponding receptor (Barbara et al., 1999, J. Biol. Chem. 274, 584-594).
  • Endoglin binds TGF-beta 1 and TGF-beta 3 by associating with TGF-beta type II receptor (TGF-beta RII), or interacts with Activin A and BMP-7 via either the Activin type II or type HB receptors.
  • TGF-beta RII TGF-beta type II receptor
  • Endoglin binds BMP-2 in vitro via either BMPR-IA (ALK-3) or BMPR-IB (ALK-6).
  • ALK-3 BMPR-IB
  • Endoglin does not bind TGF-beta by itself, it can bind the receptor in the absence of ligand.
  • TGF-beta RI and TGF-beta RII each associate with both the intracellular and extracellular domains of Endoglin (Guerrero-Esteo et al., 2002, J. Biol. Chem. 277, 29197-29209). Binding leads to differential regulation of the phosphorylation state of both TGF-beta RI and TGF-beta RII. Endoglin appears to have both positive and negative effects as a modulator of TGF- beta signaling. In vitro, Endoglin enhances phosphorylation of the downstream TGF-beta effector, Smad2 (Guerrero-Esteo et al., 2002, J. Biol. Chem.
  • TGF-beta 1 effects on proliferation and migration are markedly enhanced (Li et al., 1999, FASEB J. 14, 55-64).
  • over-expression of Endoglin in myelomonocytic cells suppresses TGF-beta 1 activities (Lastres et al., 1996, J. Cell Biol. 133, 1109 ff).
  • Interaction assays may also be performed with recombinant proteins in an assay format similar to the assay described in Examples 4 or 5, which can easily be adapted by the skilled person to fulfill this purpose.
  • the present invention provides a target for the treatment and/or prevention of Insulin Resistance, and a novel marker for the early diagnosis of Insulin Resistance in diabetes.
  • Endoglin is a type I transmembrane protein implies that in order for a secreted for to appear in plasma, fragments of the sequence of Seq ID No. 1 have to be generated.
  • the target used for the methods of the present invention, or the markers detectable by the methods of the present invention also includes soluble fragments of Seq ID No. 1.
  • Such soluble fragments of Endoglin have been shown to be present in atherosclerosis or in cancers (Li et al., 2000, Int. J. Cancer 89, 122-126; Takahashi et al., 2001, Clin. Cancer Res. 7, 524 ff; Calabro et al., 2002, J. Cell. Physiol. 194, 171-175).
  • Endoglin and “protein Endoglin”, as used herein, are understood to include soluble fragments of Seq ID No. 1 as well as the protein of Seq ID No. 1 or mutants thereof which are at least 90 % homologous to Seq ID No. 1.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid molecule).
  • the amino acid residues or nucleotides at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position.
  • amino acid "homology" is equivalent to amino acid
  • identity The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology equals the number of identical positions/total number of positions times 100).
  • the marker Endoglin consists of any fragment or mutant or native form of Seq ID No. 1 which can be detected by any one of the ELISAs described in Examples 4 and 5.
  • the novel target and/or marker Endoglin maybe used for diagnostic, monitoring as well as for screening purposes.
  • the diagnostic method according to the present invention may help to assess efficacy of treatment and recurrence of Insulin Resistance in the follow-up of patients. Therefore, the present invention provides the use of protein Endoglin, for monitoring the efficacy of treatment of diabetes.
  • the diagnostic method according to the present invention is used for patient screening purposes. I.e., it is used to assess subjects without a prior diagnosis of diabetes by measuring the level of Endoglin and correlating the level of Endoglin to the presence or absence of Insulin Resistance.
  • the methods of the present invention are useful for monitoring progression of the disease through the different stages leading to diabetes, namely Insulin Resistance, Impaired Glucose Tolerance and Diabetes.
  • the present invention thus provides a method for monitoring the progression of diabetes, comprising the steps of (a) providing a liquid sample obtained from an individual, (b) contacting said sample with a specific binding agent for Endoglin under conditions appropriate for formation of a complex between said binding agent and Endoglin, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in Insulin Resistance.
  • the present invention also provides a method for monitoring the efficacy of treatment of diabetes, comprising the steps of (a) providing a liquid sample obtained from a patient treated against diabetes, (b) contacting said sample with a specific binding agent for Endoglin under conditions appropriate for formation of a complex between said binding agent and Endoglin, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in the absence of treatment.
  • the present invention provides a method of screening for a compound which interacts with Endoglin, comprising the steps of a) contacting protein Endoglin with a compound or a plurality of compounds under compositions which allow interaction of said compound or a plurality of compounds with Endoglin; and b) detecting the interaction between said compound or plurality of compounds with said polypeptide.
  • the present invention provides a method of screening for a compound that prevents and/ or inhibits and/ or attenuates Insulin Resistance, comprising the steps of a) contacting a compound with protein Endoglin; and b) measuring the activity of protein Endoglin; wherein a compound which inhibits or stimulates the activity of protein Endoglin is a compound that may prevent and/ or inhibit and/or attenuate Insulin Resistance.
  • said method additionally comprises the step of immobilizing protein Endoglin prior to step a) or between steps a) and b).
  • the term “bul activity” as used herein relates e.g. to the ability of Endoglin to interact with a TGF-beta superfamily member and a corresponding receptor and to modulate their functions.
  • Such assays are known in the art (e.g. from Guerrero-Esteo et al., 2002, J. Biol. Chem. 277, 29197-29209; Barbara et al., 1999, J. Biol. Chem. 274, 584-594; Li et al., 1999, FASEB J. 14, 55-64).
  • the present invention also includes cell-free assays.
  • Such assays involve contacting a form of Endoglin (e.g., full-length polypeptide, a biologically active fragment of said polypeptide, or a fusion protein comprising all or a portion of said polypeptide) with a test compound and determining the ability of the test compound to bind to said polypeptide. Binding of the test compound to said polypeptide can be determined either directly or indirectly as described above.
  • the assay includes contacting the said polypeptide with a known compound which binds said polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with said polypeptide, wherein determining the ability of the test compound to interact with said polypeptide comprises determining the ability of the test compound to preferentially bind to the said polypeptide as compared to the known compound.
  • determining the ability of the test compound to interact with said polypeptide comprises determining the ability of the test compound to preferentially bind to the said polypeptide as compared to the known compound.
  • the cell-free assays of the present invention are amenable to use of either a membrane-bound form of a polypeptide or a soluble fragment thereof.
  • a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • solubilizing agents include non- ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X- 114, Thesit, Isotridecypoly( ethylene glycol ether)n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimemylamminio] ⁇ 2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl-N, N-dimethyl-3-ammonio-l -propane sulfonate.
  • non- ionic detergents such as n-oct
  • binding of a test compound to a polypeptide, or interaction of a polypeptide with a binding molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed binding protein or polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of a polypeptide hereinbefore described can be determined using standard techniques.
  • a polypeptide hereinbefore described or its binding molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with a polypeptide or binding molecules can be derivatized to the wells of the plate. Unbound binding protein or polypeptide of the invention are trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with a polypeptide hereinbefore described or binding molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with a polypeptide or binding molecule.
  • the present invention also provides a method of screening for a compound that prevents and/ or inhibits and/ or delays Insulin Resistance, comprising the step of detecting soluble Endoglin secreted from a host in the presence or absence of said compound, wherein a compound that prevents and/ or inhibits and/ or delays Insulin Resistance is a compound with which the level of Endoglin secreted from a host is changed.
  • a host maybe a model cell representing beta-cells in culture, or an animal which can be used as a model for Insulin Resistance.
  • the present invention also provides for a use of protein Endoglin as a target and/or as a marker for screening for a compound that prevents and/or inhibits Insulin Resistance.
  • the diagnostic, monitoring or patient screening methods according to the present invention are based on a liquid sample which is derived from an individual. Unlike to methods known from the art Endoglin is specifically measured from this liquid sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for Endoglin or an antibody to Endoglin.
  • a receptor for Endoglin or an antibody to Endoglin.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for Endoglin. A level of less than 5% cross-reactivity is considered not significant.
  • a specific binding agent preferably is an antibody reactive with Endoglin.
  • the term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.
  • Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 ( 1990) the whole book, especially pages 43-78; Elsevier, Amsterdam).
  • polyclonal antibodies raised in rabbits have been used.
  • polyclonal antibodies from different species e.g. rats or guinea pigs, as well as monoclonal antibodies can be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to Endoglin in a method according to the present invention is yet another preferred embodiment.
  • Endoglin has been identified as a marker which is useful in the diagnosis of Insulin Resistance
  • alternative ways may be used to reach a result comparable to the achievements of the present invention.
  • alternative strategies to generate antibodies maybe used.
  • Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of Endoglin for immunization.
  • DNA immunization also known as DNA vaccination maybe used.
  • the liquid sample obtained from an individual is contacted with the specific binding agent for Endoglin under conditions appropriate for formation of a binding agent Endoglin- complex.
  • Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of Insulin Resistance or to a respective control, as hereinbefore described.
  • the skilled artisan will appreciate there are numerous methods to measure the amount of the specific binding agent Endoglin-complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al o eds. (1996) Immunoassay, Academic Press, Boston).
  • Endoglin is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture Endoglin on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable, is used on the other side.
  • Endoglin can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample are required to apply the marker Endoglin in the diagnosis of Insulin Resistance.
  • the method according to the present invention is practiced with serum as liquid sample material.
  • the method according to the present invention is practiced with plasma as liquid sample material.
  • the method according to the present invention is practiced with whole blood as liquid sample material.
  • Antibodies to Endoglin with great advantage can be used in established procedures, e.g., to Insulin Resistance in situ, in biopsies, or in immunohistological procedures.
  • an antibody to Endoglin is used in a qualitative (Endoglin present or absent) or quantitative (Endoglin amount is determined) immunoassay.
  • the present invention relates to use of protein Endoglin as a marker molecule in the diagnosis of Insulin Resistance from a liquid sample obtained from an individual.
  • marker molecule is used to indicate that changes in the level of the analyte Endoglin as measured from a bodily fluid of an individual marks the presence of Insulin Resistance.
  • novel marker Endoglin in the early diagnosis of type II diabetes. It is especially preferred to use the novel marker Endoglin in the early diagnosis of glucose intolerance.
  • the use of protein Endoglin itself represents a significant progress to the challenging field of Insulin Resistance diagnosis. Combining measurements of Endoglin with other known markers for diabetes, like insulin, or with other markers of Insulin Resistance yet to be discovered, leads to further improvements. Therefore in a further preferred embodiment the present invention relates to the use of Endoglin as a marker molecule for diabetes, preferably for Insulin Resistance, in combination with another marker molecule for diabetes, preferably for Insulin Resistance, in the diagnosis of diabetes, preferably of Insulin Resistance from a liquid sample obtained from an individual.
  • Preferred selected other diabetes markers with which the measurement of Insulin Resistance may be combined are insulin, pre-insulin, and/ or C-peptide.
  • Diagnostic reagents in the field of specific binding assays like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for Endoglin and auxiliary reagents for measurement of Endoglin.
  • One way of assessing clinical utility of the novel marker Endoglin is by measuring its levels in 17 patients that were diagnosed as being insulin resistant by measuring the glucose disposal rate with the EHC method and comparing the levels with those measured in 17 patients with demonstrated normal glucose disposal rate as determined by the same methodology. For statistical analysis, standard Student's t-test evaluation is performed with values ⁇ 0.05 being taken as significant.
  • ROC receiver-operating characteristics
  • the ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision threshhold over the entire range of data observed.
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify" subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true- positive test results) (number of true-positive + number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • false-positive fraction or 1 - specificity [defined as (number of false-positive results)/(number of true-negative + number of false-positive results)] . It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • VECs Human Vascular Endothelial Cells
  • the HUVECs was cultured in pi medium for 48h. After 48h the cells were harvested by scraping and the total cellular RNA was extracted with RNA-BeeTM. From each sample 10 ⁇ g of total cellular RNA were reverse transcribed (Invitrogen, U.S.), labelled (Ambion, U.S.) and processed by using commercial kits according to the supplier's instructions. The methods of the alkaline heat fragmentation and the following hybridization of the cDNA with the U133 A and B GeneChip arrays were standard procedure provided by the manufacturer of the microchips (Affymetrix, U.S.).
  • the cell intensity values of the arrays were recorded with a confocal laser scanner (Hewlett Packard, U.S.) and data were analyzed using GeneChip v3.1 software (Affymetrix, U.S.).
  • the expression level for each gene was calculated as normalized average difference of fluorescence intensity as compared to hybridization to mismatched oligonucleotides, expressed as average difference (A.D.). This experiment was performed in triplicate in order to account for biological variation.
  • HMMs Hidden Markov Models
  • the "signal" and “anchor” scores that any input sequence is assigned are fed into a Support Vector Machine (SVM) in a second analysis step (Cristianini N, Shawe-Taylor J. An Introduction to Support Vector Machines and other Kernel-based Learning Methods. Cambridge University Press, Cambridge, England, 2000).
  • SVM Support Vector Machine
  • the SVM was trained on a set of bonafi.de examples for both classes. On this training set, the SVM obtained the following results on three training sets (signal - anchor - neither).
  • the proteins predicted as extracellular (“signal" or “anchor”) were further evaluated for organ specificity.
  • a search for public domain expressed sequence tags encoding the candidate proteins was carried out and grouped according to tissue source. Only those protein were retained that were expressed in blood vessels and that did not show a strong expression in other secretory organs (e.g. liver, pancreas).
  • Polyclonal antibody to the Insulin Resistance marker Endoglin is generated for further use of the antibody in the measurement of serum and plasma and blood levels of Endoglin by immunodetection assays, e.g. Western Blotting and ELISA.
  • recombinant expression of the protein is performed for obtaining immunogens.
  • the expression is done applying a combination of the RTS 100 expression system and E.coli.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY" system. This is a commercial web based service (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No. 3186237) system to generate linear PCR templates from the cDNA and for in-vitro transcription and expression of the nucleotide sequence coding for the Endoglin protein is used.
  • the expressed protein contains a His-tag.
  • the best expressing variant is identified. All steps from PCR to expression and detection are carried out according to the instructions of the manufacturer.
  • the respective PCR product, containing all necessary T7 regulatory regions (promoter, ribosomal binding site and T7 terminator) is cloned into the pBAD TOPO ® vector (Invitrogen, Düsseldorf,
  • the construct is transformed into E. coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89) and the transformed bacteria are cultivated in a 1 1 batch for protein expression. Purification of His-Endoglin fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-Endoglin fusion protein is pelleted by centrifugation.
  • the cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidinium chloride, imidazole and thioglycerole, followed by homogenization using an Ultra- Turrax" .
  • Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acidic conditions.
  • Endoglin This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g Endoglin adsorbed to aluminum hydroxide and 10 9 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g Endoglin in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46.
  • ca. l*10 8 spleen cells of the immunized mouse are mixed with 2xlO 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300 g and 4°C). The cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 g in a 50 ml conical tube.
  • FCS fetal calf serum
  • the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and sown in hypoxanthine-azaserine selection medium (100 mmol/1 hypoxanthine, 1 ⁇ g/ml azaserine in RPMI 1640 + 10% FCS).
  • Interleukin 6 at 100 U/ml is added to the medium as a growth factor.
  • the primary cultures are tested for specific antibody.
  • Endoglin-positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of IxIO 5 cells per ml in RPMI
  • 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050).
  • concentrations of 100 ⁇ g monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Brack, C, et al., Methods in Enzymology 121 (1986) 587-695).
  • Endoglin Endoglin
  • complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti-Endoglin serum used for further experiments as described in examples 3 and 4.
  • IgG immunoglobulin G
  • One volume of rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4 0 C), the pellet discarded and the supernatant collected. The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered (0.2 ⁇ m). The immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8000 x g, 15 min, 4°C). The supernatant is discarded. The pellet is dissolved in 10 mM NaH 2 P(VNaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed. The dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, pH 7.5,
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim,
  • Protein samples enriched and isolated from the medium by Heparin columns are solved in sample buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl 5 0.05 % Tween 20, 1 % SDS, and centrifuged at 12,000 g for 10 min at 4°C.
  • sample buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl 5 0.05 % Tween 20, 1 % SDS, and centrifuged at 12,000 g for 10 min at 4°C.
  • the protein concentration of the supernatant is measured by Bradford using a standard curve constructed from a range of known bovine serum albumin standards.
  • sample buffer 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8
  • sample buffer 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8
  • sample buffer 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8
  • samples are separated by 12.5% homogenous ExcelGel SDS gels (Amersham Bioscience) and electro transferred onto Nitrocellulose membranes.
  • blocking solution 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% non-fat dry milk
  • membranes are incubated with rabbit anti-rat antibody for 2 hrs at room temperature, respectively.
  • membranes are incubated with a horseradish peroxidase conjugated anti-rabbit IgG (H+L), anti-mouse IgGi and anti-mouse IgG2a (Southern Biotechnology Associates, Inc., Birmingham, AL), respectively, for 1 hr at room temperature.
  • H+L horseradish peroxidase conjugated anti-rabbit IgG
  • anti-mouse IgGi and anti-mouse IgG2a Southern Biotechnology Associates, Inc., Birmingham, AL
  • Membranes are washed 3 times for 10 min and antigen-antibody complexes are visualized by an enhanced chemiluminescence's reagent (Western Lightning TM, PerkinElmer Life Sciences, Inc., Boston, MA) on an X-ray film according to the manufacturer's protocol.
  • a sandwich ELISA For detection of Endoglin in human serum or plasma, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-Endoglin polyclonal antibody (see Example 2) are conjugated with biotin and digoxigenin, respectively.
  • Streptavidin-coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-Endoglin polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween 20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted plasma samples from patients. After binding of Endoglin, plates are washed three times with 0.9% NaCl , 0.1% Tween 20.
  • wells are incubated with 100 ⁇ l of digoxigenylated anti- Endoglin polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. Thereafter, plates are washed three times to remove unbound antibody.
  • wells are incubated with 20 mU/ml anti-digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20.
  • Plates are subsequently washed three times with the same buffer.
  • wells are incubated with 100 ⁇ l ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an ELISA reader.
  • Endoglin was detected in plasma of insulin-resistant and insulin-sensitive individuals using the Quantikine Human Endoglin/ CD 105 Immunoassay kit from R&D Systems.
  • the ELISA was perfomed according to the manufacturer's instructions. Briefly, this assay employed the quantitative sandwich enzyme immunoassay technique.
  • a monoclonal antibody specific for Endoglin had been pre-coated onto a microplate. Standards and samples were pipetted into the wells and any Endoglin present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for Endoglin was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of Endoglin bound in the initial step. The color development was stopped and the intensity of the color is measured.
  • the assay was performed as follows:
  • Endoglin Microplate Part 892530
  • 96 well polystyrene microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against human Endoglin.
  • Endoglin Conjugate Part 892531
  • Endoglin Standard Part 892532
  • Assay Diluent RDlS (Part 895137) - 11 mL of a buffered protein base with preservatives.
  • Calibrator Diluent RD5K (Part 895119) - 21 mL of a buffered protein base with preservatives.
  • Wash Buffer Concentrate (Part 895003) - 21 mL of a 25-fold concentrated solution of buffered surfactant with preservatives.
  • Color Reagent A (Part 895000) - 12.5 mL of stabilized hydrogen peroxide.
  • Color Reagent B (Part 895001) - 12.5 mL of stabilized chromogen (tetramethylbenzidine) .
  • Stop Solution Part 895032 - 6 mL of 2 N sulfuric acid. Plate Covers - 4 adhesive strips.
  • wash Buffer 400 ⁇ L
  • squirt bottle a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step was essential to good performance. After the last wash, any remaining Wash Buffer was removed by aspirating or decanting. The plates were inverted and blotted against clean paper towels.
  • the optical density of each well was determined within 30 minutes, using a microplate reader set to 450 nm and a correction set to 540 nm or 570 nm.
  • the mean concentration of Endoglin measured in plasma of 17 insulin resistant individuals was 4.2235 ng/ml +/- 0.8828 ng/ml, and in 17 insulin sensitive individuals 3.5235 ng/ml +/- 0.7259 ng/ml.
  • Clinical utility of the novel marker Endoglin is further assessed by measuring its levels in 60 samples from patients with varying degrees of insulin resistance and 20 healthy control subjects. Statistical analysis is performed by standard Student's t-test evaluation with values ⁇ 0.05 taken as significant.

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Abstract

La présente invention concerne la suivi de la progression de la maladie et le diagnostic de la résistance insulinique du diabète par la mesure des niveaux d'endogline dans un prélèvement de liquide, et le classement des nouveaux composés de prévention et/ou de traitement du diabète.
PCT/EP2005/013198 2004-12-14 2005-12-09 Endogline utilisee comme cible/marqueur de la resistance insulinique WO2006063735A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3739041A1 (fr) * 2014-03-27 2020-11-18 The Salk Institute for Biological Studies Compositions et procédés pour le traitement du diabète de type 1 et 2 et de troubles apparentés
US11685901B2 (en) 2016-05-25 2023-06-27 Salk Institute For Biological Studies Compositions and methods for organoid generation and disease modeling
US11981931B2 (en) 2015-02-27 2024-05-14 Salk Institute For Biological Studies Reprogramming progenitor compositions and methods of use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040053329A1 (en) * 2000-09-04 2004-03-18 Roland Kontermann Endoglin-specific polypeptide, production and use thereof

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Publication number Priority date Publication date Assignee Title
US20040053329A1 (en) * 2000-09-04 2004-03-18 Roland Kontermann Endoglin-specific polypeptide, production and use thereof

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AZIZ W ET AL: "LEVELS OF SOLUBLE TGF-BETA RECEPTOR-CD105 AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN THE PLASMA AND VITREOUS OF DIABETIC PATIENTS WITH PROLIFERATIVE RETINOPATHY", DIABETOLOGIA, BERLIN, DE, no. SUPPL 1, August 1998 (1998-08-01), pages A18, XP008061824, ISSN: 0012-186X *
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TAKAHASHI N ET AL: "Association of serum endoglin with metastasis in patients with colorectal, breast, and other solid tumors, and suppressive effect of chemotherapy on the serum endoglin.", CLINICAL CANCER RESEARCH : AN OFFICIAL JOURNAL OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH. MAR 2001, vol. 7, no. 3, March 2001 (2001-03-01), pages 524 - 532, XP002373974, ISSN: 1078-0432 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3739041A1 (fr) * 2014-03-27 2020-11-18 The Salk Institute for Biological Studies Compositions et procédés pour le traitement du diabète de type 1 et 2 et de troubles apparentés
US10912800B2 (en) 2014-03-27 2021-02-09 Salk Institute For Biological Studies Compositions and methods for treating type 1 and type 2 diabetes and related disorders
US11981931B2 (en) 2015-02-27 2024-05-14 Salk Institute For Biological Studies Reprogramming progenitor compositions and methods of use thereof
US11685901B2 (en) 2016-05-25 2023-06-27 Salk Institute For Biological Studies Compositions and methods for organoid generation and disease modeling
US11760977B2 (en) 2016-05-25 2023-09-19 Salk Institute For Biological Studies Compositions and methods for organoid generation and disease modeling

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