EP0711341A1 - Procedes de determination de la fonction d'un recepteur - Google Patents

Procedes de determination de la fonction d'un recepteur

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Publication number
EP0711341A1
EP0711341A1 EP94924067A EP94924067A EP0711341A1 EP 0711341 A1 EP0711341 A1 EP 0711341A1 EP 94924067 A EP94924067 A EP 94924067A EP 94924067 A EP94924067 A EP 94924067A EP 0711341 A1 EP0711341 A1 EP 0711341A1
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EP
European Patent Office
Prior art keywords
receptor
cells
antibody
activated
test
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94924067A
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German (de)
English (en)
Other versions
EP0711341A4 (fr
Inventor
Neill A. Giese
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
COR Therapeutics Inc
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COR Therapeutics Inc
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Publication date
Application filed by COR Therapeutics Inc filed Critical COR Therapeutics Inc
Publication of EP0711341A1 publication Critical patent/EP0711341A1/fr
Publication of EP0711341A4 publication Critical patent/EP0711341A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • Signal-transducing receptors mediate fundamental aspects of cell physiology, growth, differentiation, endocrine, paracrine and exocrine signalling, and other properties of cells. Because of the importance of these receptors (and their cognate ligands) in human health, the identification of compounds that antagonize or agonize receptor activity will have profound consequences in medicine. The development of efficient, high capacity assays for such compounds is important for the development of therapeutic agents to treat human diseases.
  • Receptor-mediated signal transduction occurs when the receptor binds its ligand and is activated, initiating a variety of cellular responses.
  • the particular responses to ligand binding depend on the receptor and cell type, the repertoire of early responses characteristic of receptor activation includes dimerization or oligomerization of multi-subunit receptors, receptor autophosphorylation, substrate phosphorylation, and receptor association with specific cellular proteins.
  • receptor activation by ligand subsequent changes in the cell physiology occur (involving, e.g., mitogenesis, specific gene activation, phosphatidylinositol turnover, phospholipase A2 activation, and changes in cell shape, cellular calcium concentration, and intracellular pH) .
  • signal transduction and the resulting changes in cell physiology can be blocked by interference with an early response characteristic of receptor activation.
  • inhibition of the tyrosine kinase activity by phosphotyrosine-containing peptides can prevent signal transduction (Pazin & Williams, 1992) .
  • FGF-R wild-type fibroblast growth factor receptor
  • PDGF-R platelet-derived growth factor receptor
  • mutant receptors subsequent signal transduction is blocked (Ueno et al.. Science 252:844-848) .
  • growth factor receptors One class of signal-transducing receptors, the growth factor receptors, is of particular interest, due in part to the implication of some growth factor receptors in a wide range of human diseases (e.g., cancer, cardiovascular disease, and inflammatory diseases) .
  • Many growth factor receptors belong to the tyrosine kinase family of receptors, which includes platelet-derived growth factor receptors (PDGF-R) , fibroblast growth factor receptors (FGF-R) , and others.
  • PDGF-R platelet-derived growth factor receptors
  • FGF-R fibroblast growth factor receptors
  • Tyrosine-kinase activity of such growth factor receptors is induced by ligand binding to the receptor extracellular domain, and results in both autophosphorylation and substrate phosphorylation; additionally, transphosphorylation (the phosphorylation of one receptor subunit by another, associated, subunit) may occur.
  • Activation of some growth factor receptors is also characterized by dimerization or oligomerization of receptor subunits. Activation is also characterized, for some receptors, by receptor association with specific cytoplasmic proteins. Because of the fundamental role of signal transduction in biology and medicine, it would be very useful to have efficient methods for assaying receptor function.
  • an assay that measures traits characteristic of receptor activation e.g., early responses such as receptor dimerization, receptor-protein association, and tyrosine kinase activity
  • traits characteristic of receptor activation e.g., early responses such as receptor dimerization, receptor-protein association, and tyrosine kinase activity
  • therapeutics targeted to receptors e.g., growth factor receptors
  • receptor dimerization has been measured by chemical cross-linking or i munoprecipitation followed by Western blot analysis or autoradiography.
  • the primary means for identifying tyrosine phosphorylation of a specific protein within the cell is by immunoprecipitation of that protein followed by Western blot analysis using an antiphosphotyrosine antibody.
  • Reed-Gitomer (U.S. Patent Application No. 07/341,949) describes an ELISA-based assay used to distinguish between various dimeric forms of platelet-derived growth factor (PDGF) .
  • PDGF platelet-derived growth factor
  • Keating et al. J. Biol. Chem. 263:12805, 1988
  • Nelson et al. J. Clin. I muno.
  • the invention provides methods for assaying the function of signal-transducing receptors are that are useful for screening for agonists or antagonists of receptor activation. These methods utilize whole cells expressing the receptor of interest and comprise the steps of (i) treating the cells with agonists, antagonists, and/or test compounds, (ii) preparing a cell lysate from the treated cells, and (i ⁇ ) assaying for receptor function using a two-antibody sandwich assay.
  • The- two-antibody sandwich assay employs an immobilized first antibody (abl) that specifically binds and captures the receptor of interest along with any tightly associated proteins or polypeptides and a second antibody (ab2) directed against (i) an epitope of the receptor that is characteristic of the activated but not the unactivated state, (ii) an epitope of a protein or molecule characteristically bound to activated receptor but not to unactivated receptor, or (iii) an epitope of the receptor that is characteristic of the unactivated but not the activated state.
  • An important characteristic of the first antibody (abl) is that it captures (specifically binds) the receptor of interest whether activated or"not activated by the treatment.
  • the level of binding of the second antibody is determined and correlated with the presence of activated (or unactivated) receptor, and thus with activation (or lack of activation) in response to the treatment received by the cells.
  • the quantity of receptors that are activated in cells can be determined.
  • additional antibodies are used to quantitate the total amount of receptor present (allowing calculation of the fraction of total receptor in the activated state) and for detection of other antibodies.
  • the agonizing or antagonizing activity of a compound can be determined.
  • the agonizing or antagonizing activity of a compound can be determined.
  • These methods may be easily adapted to high-throughput formats (e.g., by using microtiter plates and colori etric detection) making them ideally suited for the discovery of compounds that influence receptor activation.
  • the methods provided by the invention will find particular use for detecting activation of tyrosine kinase growth factor receptors as assessed by receptor and substrate phosphorylation, receptor dimerization and oligomerization, and receptor association with specific cytoplasmic proteins.
  • Fig. 1 shows assay results showing induction of PDGF-R dimerization by PDGF.
  • Fig. 2 shows assay results showing induction of
  • PDGF-R autophosphorylation by PDGF.
  • Fig. 3 shows assay results showing association of PDGF-R and phospholipase c ⁇ in the presence of PDGF.
  • the receptors of interest will be growth factor receptors, but function of any receptor can be assayed by this method provided that the receptor has at least one epitope that is characteristic of the activated state but not the unactivated state or at least one epitope that is characteristic of the unactivated state but not the activated state, and at least one epitope that is characteristic of both the activated and unactivated (i.e., not activated) states.
  • the receptor will have at least two epitopes characteristic of both the activated and unactivated state.
  • Total receptor activated receptor + unactivated receptor.
  • a receptor is in an activated state when (i) it has bound a ligand (i.e., a natural ligand or other agonist that binds the receptor) and is transducing a signal (e.g., association with specific cytoplasmic proteins) in the manner characteristic of the binding of the natural ligand or (ii) the receptor is transducing a signal in the manner characteristic of the binding of the natural ligand even though no ligand is bound to the extracellular ligand binding site.
  • a ligand i.e., a natural ligand or other agonist that binds the receptor
  • a signal e.g., association with specific cytoplasmic proteins
  • a tyrosine kinase growth factor receptor is in an activated state (a) if the tyrosine kinase activity of the receptor is elevated, (b) if receptor polypeptides are associated in dimers or oligomers, when such association is elevated when the receptor is bound by a natural ligand of the receptor, compared to unoccupied receptor, or, (c) if the receptor is tightly associated with the intracellular or membrane proteins whose association with the receptor is elevated when the receptor is bound to its natural ligand, compared to unoccupied receptor.
  • a ligand as used herein, is a molecule or macromolecule that binds an extracellular region of a receptor.
  • a "natural" ligand for a receptor is a ligand found in nature functionally associated with the receptor.
  • PDGF in the forms found in nature, is a natural ligand of the PDGF receptors; insulin is a natural ligand of the insulin receptors, etc.
  • An epitope is the site on an antigen (e.g., a receptor) that is recognized by an antibody or antibody fragment. For the practice of this invention it is not necessary that the epitope bound be characterized in molecular terms.
  • the epitope is characterized in functional terms (e.g., the association of the epitope with activated receptor) .
  • An essential aspect of the invention is the use of cells that express the receptor of interest (hereafter referred to as "receptor-expressing cells”) .
  • the level of receptor expression will typically be at least 0.5xl0 4 receptor polypeptides/cell, with at least 1-0 5 receptor polypeptides/cell more preferred, and at least 10 6 receptor polypeptides/cell most preferred.
  • receptor-expressing cells are produced by transforming eukaryotic cells with a recombinant polynucleotide encoding the receptor polypeptide of interest (e.g., a receptor cDNA) to produce cells expressing or overexpressing the receptor polypeptides.
  • a recombinant polynucleotide encoding the receptor polypeptide of interest e.g., a receptor cDNA
  • mammalian cells are used. Methods for recombinant expression of polypeptides are well known in the art (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide. Vols 1-3, Cold Spring Harbor Press, which is incorporated herein by reference) .
  • a cell line that endogenously expresses a receptor can be identified by selection and screening. When selecting receptor-expressing cells, cells that express high levels of receptor will generally be preferred over cells expressing lower levels of receptor.
  • Methods for assessing receptor expression levels are well known in the art and include immunological, binding competition, and functional assays (see, e.g.. Methods in Enzymology. Vol. 198, 1991, Barnes, D. , Mathers, J.P., Sato, G.H. (Eds.) Academic Press, Inc. , which is incorporated herein by reference) .
  • the assays of the instant invention are suited to a wide variety of receptors, but will be especially useful for assaying function of growth factor receptors, especially tyrosine kinase growth factor receptors.
  • tyrosine kinase growth factor receptors include platelet-derived growth factor receptors (PDGF-R) , fibroblast growth factor receptors (FGF-R) , insulin receptor (In-R) , epidermal growth factor receptor (EGF-R) , erythropoietin receptor (EPO-R) , colony stimulating factor receptor (CSF-R) , stem cell factor receptor (SCF-R) , vascular endothelial growth factor receptor (VEGF-R) , FLK-1-receptor, FLT-1-receptor, ERB-2-receptor and ERB-3- receptor.
  • PDGF-R platelet-derived growth factor receptors
  • FGF-R fibroblast growth factor receptors
  • In-R insulin receptor
  • receptor- expressing cells will be grown under conditions appropriate for growth and proliferation and for expression of the receptor of interest.
  • General tissue culture techniques are well known in the art and are described in, e.g., Methods in Enzymology, Vol. 58, 1979, Jakoby, W. B. and Paston, I. (Eds.), Academic Press, Inc. , which is incorporated herein by reference.
  • Media appropriate for eukaryotic cells are well known and readily available.
  • the cell medium will be supplemented with serum (e.g., fetal bovine serum, FBS) , which provides growth factors and essential nutrients.
  • serum e.g., fetal bovine serum, FBS
  • cells will be grown in ultiwell cell culture plates (sometimes referred to as microtiter plates) suitable for cell cultures, because of the ease of screening large numbers of samples using these plates.
  • ultiwell cell culture plates sometimes referred to as microtiter plates
  • 96-well cell culture plates will be used (e.g.. Falcon 96-well cell culture plates; Costar 96-well cell culture plates, cat. # 3596). Cells will be seeded according to usual tissue culture practices at an appropriate cell density.
  • This density will depend on cell-type, but typically will be about 10 4 cells/well for adherent cells in 96-well microtiter plates. If possible, cells should be seeded so that they reach confluency in 1-2 days. The cells will be grown to about confluency. For cells grown in the presence of serum, it will sometimes be desirable to incubate the confluent cells for about 24 hours (at least 6 hours and typically not more than 48 hours) in a serum-free medium in order to remove growth factors and/or compounds that interact with the receptor of interest.
  • the assay can also be carried out using nonadherent cells, by making modifications that will be apparent to one of ordinary skill in the art.
  • non-adherent cells can be grown and incubated with test compounds in suspension, and can be centrifuged and resuspended according to usual practice to exchange media, collect for cell lysis, etc (see, e.g.. Methods in Enzymology Vol. 58, supra) .
  • test compounds which may be, or may contain, agonists or antagonists.
  • An agonist, antagonist, or test compound may be a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues.
  • Test compounds are evaluated for potential activity as agonists or antagonists of receptor activation by inclusion in screening assays described herein.
  • An "agonist” enhances the activity of a receptor; an “antagonist” diminishes the activity of a receptor.
  • the terms "agonist” and "antagonist”, as used herein, do not imply a particular mechanism of function.
  • receptor-expressing cells when assaying for agonist activity, receptor-expressing cells will be incubated with a known agonist, a test compound, or a combination of the two.
  • the cells are incubated with the agonist or test compound for about 1 hour at about 4°C.
  • the time and temperature of incubation may range from about 1 minute to about 5 hours, and from about 4°C to about 37°C, but will usually be at least enough time for receptor activation by the natural ligand, if such time known.
  • Lower temperatures e.g., 4°C are generally preferred to reduce the possibility of proteolysis, dephosphorylation, dissociation of associated proteins or other processes that could interfere with the assay.
  • the receptor will be activated if there is a suitable receptor agonist present (e.g., in the test compound) and no antagonist present.
  • receptor- expressing cells When assaying for antagonist activity, receptor- expressing cells will be incubated with a known antagonist, a test compound, or a combination of the two, and, in addition, will be incubated with an agonist. According to the assay, the presence of the agonist will cause receptor activation unless antagonist is present.
  • the order of addition of antagonist (or test compound) and agonist can vary.
  • the agonist and antagonist may be added to the receptor-expressing cells simultaneously, or, alternatively, the antagonist (or test compound) can be added before addition of the agonist
  • the conditions (time and temperature) for incubations when testing for antagonist activity are essentially the same as those described to agonists, supra. However, when they are added in sequence total time will not usually be longer than five hours.
  • receptor-expressing cells that express a receptor variant that is in a constitutively activated state (i.e., activated in the absence of ligand; see, e.g., Roussel et al. (1990) Oncogene 1:25-30).
  • an assay for a compound with receptor antagonist activity will not necessarily require incubation with a known agonist.
  • the amount or concentration of agonist/antagonist added will, when known, vary depending on the compound, but will generally range from about lOpM to lOO ⁇ M. Typically, a variety of concentrations will be used. In the case of uncharacterized test compounds it may not be possible, and it is not necessary, to determine the concentration of agonist/antagonist. It will also be desirable to include various experimental controls in the assay. Examples of appropriate controls include negative controls and positive controls. In testing for agonist activity, negative controls can include incubation of cells with inert compounds (i.e., compounds known not to have agonist activity) or in the absence of added compounds. Positive controls can include incubation of cells with compounds known to have agonist activity (e.g., the natural ligand) .
  • cells are homogenized in the presence of buffers (e.g., Tris-HCl, HEPES) , nonionic detergents (e.g., Tween-20; Triton X-100) , and protease inhibitors (e.g., PMSF, aprotinin, leupeptin) .
  • buffers e.g., Tris-HCl, HEPES
  • nonionic detergents e.g., Tween-20; Triton X-100
  • protease inhibitors e.g., PMSF, aprotinin, leupeptin
  • Other inhibitors such as phosphatase inhibitors (e.g., orthovanadate) , may also be desirable.
  • conditions are chosen that are nondenaturing to the cellular proteins of interest.
  • a preferred method for preparation of a cell lysate is by addition of lysis buffer (20mM Tris-HCl, pH 7.3, 150 mM NaCl, 1% Triton X-100, lmM PMSF, lmM sodium orthovanadate, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin) followed by cell disruption (e.g., by shaking or scraping).
  • lysis buffer 20mM Tris-HCl, pH 7.3, 150 mM NaCl, 1% Triton X-100, lmM PMSF, lmM sodium orthovanadate, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin
  • cell disruption e.g., by shaking or scraping.
  • the insoluble matter in the cell lysate is removed by centrifugation (e.g., 10,000 xg for 15 min.) and the clarified supernatant is recovered.
  • the resulting cell lysate is transferred to a well of a microtiter plate to which an antibody (abl) against the receptor of interest is immobilized.
  • an antibody (abl) against the receptor of interest is immobilized.
  • Methods for immobilizing antibodies are known in the art and are described in Harlow, E. and D. Lane, Antibodies: A Laboratory Manual. (1988) , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (hereafter referred to as "Harlow”) , which is incorporated herein by reference.
  • the antibody may be either polyclonal or monoclonal.
  • the antibody (abl) will be selected as able to specifically bind ("capture") the receptor in both activated and unactivated states. Typically the antibody will bind the receptor with an affinity of at least about 10 "7 M "1 .
  • a cell membrane fraction especially a plasma membrane fraction can be purified from the cells treated with a test compound or, e.g., a known agonist or antagonist, using standard methods (see, e.g.. Methods in Enzymology Vol. 198 f supra.) and used in the assay.
  • a membrane preparation will be that the purified membranes can be frozen and stored for a period of time, which may, in some cases, increase the convenience of the assay and allow use of a cell membrane preparation to be used for several assays over a period of time.
  • it will sometimes be useful to further disrupt the membrane fraction before adding combining the membranes and antibodies.
  • Antibodies can be generated to receptor polypeptides or peptides in a variety of ways known to those skilled in the art including injection of antigen into animals, by hybridoma fusion, and by recombinant methods involving bacteria or phage systems (see, e.g., Marks et al. (1992) Jj. Biol. Chem. 267:16007; Marks et al. (1992) Biotechnology .1():779; Lowman et al. (1991) Biochem. 30:10832; Lerner et al. (1992) Science 258:1313; each of which is herein incorporated by reference) .
  • Antibodies may be produced by immunizing an appropriate vertebrate host, e.g. , mouse, with the antigen (e.g.,- a receptor polypeptide, peptide, or extracellular domain) , itself or in conjunction with an adjuvant. Usually two or more immunizations will be involved, and the blood or spleen will be harvested a few days after the last injection. For polyclonal antisera, the i munoglobulins may be precipitated, isolated and purified, including affinity purification. For monoclonal antibodies, the splenocytes normally will be fused with an immortalized lymphocyte, e.g., a myeloid line, under selective conditions for hybridomas. The hybridomas may then be cloned under limiting dilution conditions and their supernatants screened for antibodies having the desired specificity.
  • the antigen e.g.,- a receptor polypeptide, peptide, or extracellular domain
  • an adjuvant e.g.,
  • a variety of screening strategies are suitable including antibody capture, antigen capture and functional assays (see, e.g., Harlow) .
  • One preferred method for screening monoclonals and polyclonal antibodies that bind receptor is antibody capture. Techniques for producing antibodies are well known in the literature and are exemplified by U.S. Patent Nos. 4,381,292, 4,451,570, and 4,618,577, and are described in Harlow, supra.
  • an antibody directed against a receptor will have a binding affinity of at least lxlO 7 M "1 .
  • the first antibody (abl) is directed against the extracellular domain of the receptor of interest; this is advantageous because the possibility that the epitope bound by the first antibody (abl) would interfere with the binding of a candidate second antibody (ab2) directed to an intracellular epitope of the receptor (e.g., a phosphotyrosine) or a cytoplasmic region of receptor association with other proteins is reduced.
  • a candidate second antibody (ab2) directed to an intracellular epitope of the receptor e.g., a phosphotyrosine
  • a cytoplasmic region of receptor association with other proteins is reduced.
  • the cell lysate and the first antibody (abl) will be incubated together for a time and at a temperature sufficient to allow binding of the receptor and antibody. Typically, this will be about 30 minutes to about 12 hours at a temperature of 4°C to 37°C.
  • a second antibody (ab2) that is specific for an epitope characteristic of the activated form of the receptor, or for an epitope of a protein that is tightly associated with the activated, but not unactivated receptor, is added to the well and allowed to bind under suitable conditions for antibody binding, as described for abl-receptor binding, supra.
  • epitopes characteristic of an activated receptor can include sites of tyrosine phosphorylation.
  • an antibody against phosphotyrosine would be a suitable second antibody (ab2) .
  • Such tyrosine phosphorylation can be on the receptor polypeptide bound by the first antibody (abl; e.g., autophosphorylation) or on a tightly associated protein.
  • the identity- of tightly associated proteins will vary according to the receptor being studied but can include a second receptor polypeptide (e.g., in homodimers and heterodimers and oligomers) or other proteins (e.g., phosphoinositide-3-kinase, GTPase activating protein, phospholipase C ⁇ [PLC ⁇ ] and nonreceptor tyrosine kinases such as those of the src family [e.g., pp60 c-src , p59 fyn , and pp62 c" yes ]).
  • a second receptor polypeptide e.g., in homodimers and heterodimers and oligomers
  • proteins e.g., phosphoinositide-3-kinase, GTPase activating protein, phospholipase C ⁇ [PLC ⁇ ] and nonreceptor tyrosine kinases such as those of the src family [e.g
  • a protein is considered to be tightly associated with a receptor if the receptor and protein remain associated in the presence of a nonionic detergent (e.g., Tween-20 nonionic detergent, Triton X-100 nonionic detergent) .
  • a tightly associated protein remains associated with a receptor in the presence of a nonionic detergent at a concentration of 0.1% (v/v) , more usually at a concentration of about 0.25%, still more usually about 0.5%, even more usually at about 0.75%, and most usually at a concentration of about 1% (v/v) .
  • Such tight association will typically be determined under conditions of near physiological salt (e.g., 0.1 - 0.2 M NaCl) and pH (e.g., 7.2 - 7.6).
  • any polypeptide or biomolecule that is associated with the activated, but not unactivated receptor, and which remains associated with the receptor when captured by the first antibody (abl) will be useful in the assay of the invention and will be considered tightly associated.
  • the second antibody (ab2) can be directed against other protein modifications characteristic of an activated receptor (e.g., phosphorylation, cleavage).
  • the second antibody (ab2) can be directed against an epitope of a protein or other biomolecule (e.g., a lipid, polysaccharide, glycoprotein, glycolipid, peptide, polypeptide, nucleic acids and other biological macromolecules) tightly associated with an activated receptor.
  • the unbound material will be removed by gently washing the wells.
  • the second antibody (ab2) will be labeled- so that bound antibody can be detected and, if desired, quantitated.
  • an enzyme e.g., alkaline phosphatase, horseradish peroxidase / S-galactosidase
  • the second antibody (ab2) can be used to detect and/or binding by the second antibody (ab2) (see, e.g., Harlow, supra) .
  • the second antibody (ab2) is conjugated with peroxidase which is detected, e.g., by monitoring product formation at 650 nm following the addition of ABTS (2,2'- az-inobis (3-ethyl-benzthiazoline-6-sulfonic acid) .
  • An alternative method for detection of the second antibody (ab2) involves using a third antibody (ab3) directed against the second antibody (ab2) .
  • the second antibody (ab2) is not itself labeled but is dictated by binding of the third antibody (ab3) .
  • the third antibody (ab3) can be labeled as described for the second antibody (ab2) , supra.
  • This alternative method for detection will be particularly preferred when the first (abl) and second (ab2) antibodies have different xenotypes (e.g., mouse and goat) , and may allow increased sensitivity of the assay.
  • the quantity of activated receptors can be determined.
  • Quantity of activated receptors can refer to a measurement of the actual number of activated receptors per cell (or the equivalent) or to a qualitative comparison between two (or among several) measurements. For example, in making a qualitative comparison it will be possible to determine that there are more activated receptors in one well than there are in a second well, without measuring the actual number of receptors in either well.
  • a fourth antibody (ab4) is used in the assay to quantitate the total amount of receptor captured.
  • the fourth antibody (ab4) is directed against an epitope characteristic of both activated and unactivated receptor.
  • the fourth antibody must be able to bind receptor that is bound (i.e., captured) by the first antibody (abl) and bound- by the second antibody (ab2).
  • the specificity of the first (abl) and fourth (ab4) antibodies is similar, if used in combination they must be able to bind the receptor polypeptide at the same time.
  • an index of activation- can be determined.
  • the index of activation (IA) is defined as follows:
  • IA activated receptor bound/total receptor bound - moles ab2 bound/ moles ab4 bound
  • receptor activation of a tyrosine kinase growth factor receptor is measured.
  • the second antibody (ab2) is directed against phosphotyrosine.
  • the first antibody (abl) recognizes a first monomer of a homo- or hetero- dimer or oligomer
  • the second antibody (ab2) recognizes a second monomer of a homo- or hetero- dimer or oligomer, and the first and second monomers are tightly associated only or primarily when the receptor is in an activated state (i.e., when such association is elevated when- the receptor is bound by its natural ligands, compared to unoccupied receptor) .
  • the second antibody is directed against a protein (other than a second receptor subunit or polypeptide) that is tightly associated with the receptor only or primarily when the receptor is in an activated state.
  • a protein other than a second receptor subunit or polypeptide
  • the first antibody (abl) will be immobilized on solid phase substrates other than a microtiter plate (e.g., cell culture beads).
  • the order of addition of the first (abl) and second (ab2) antibodies will be reversed.
  • a second antibody (ab2) is added to a cell lysate prepared as described above, and allowed to bind an epitope characteristic of activated receptor (e.g., phosphotyrosine) .
  • the lysate-antibody mixture including ab2-phosphorylated protein complex if present, is then combined with the immobilized first antibody (abl) which captures the receptor along with any second antibody (ab2) molecules bound to the receptor or to a molecule tightly associated with the receptor (e.g., a second monomer of a receptor dimer).
  • the second antibody (ab2) is then quantitated as a measure of activated receptor.
  • Monoclonal antibody against the PDGF receptor (anti- jS-PDGF-R 1C7D5) was prepared according to standard methods (see, e.g., Harlow) using recombinant extracellular domain protein as previously described (Ramakrishnan et al. (1993) Growth Factors 8 . :253-265) .
  • Polyclonal antibodies against PDGF (anti- ⁇ -PDGF-R-3980) and ⁇ (anti-3-PDGF-R-3982) receptor extracellular domains were made in rabbits using recombinant extracellular domain protein (Fretto et al.. supra) according to standard procedures (see, e.g., Harlow, supra) .
  • Anti-phosphotyrosine antibodies were obtained from commercial sources (International Biotechnologies, Inc.).
  • Anti phospholipase C- ⁇ -1 was obtained from Upstate Biotechnology Inc. (UBI) .
  • Conjugated antibodies directed against mouse monoclonal antibodies or rabbit polyclonal antibodies used for colorimetric detection were obtained commercially.
  • Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Boehriger Mannheim) and HRP-conjugated monoclonal antibody PY20 were obtained commercially.
  • a collection of human tumor cell lines obtained from the American Type Culture Collection were screened for receptor levels.
  • the cell lines screened included (ATCC numbers in parentheses): SKLMS1 (HTB88) , A204 (HTBA2) , HS68 (CRL1635) , MG63 (CRL1427) , HS27 (CRL1634) , WS1 (CRL1502) , HT1080 (CRL121) and G402 (CRL1440) .
  • PDGF receptor levels were measured using a two-antibody sandwich assay (see, Harlow, supra) .
  • MG 63 cells were seeded at 300,000 cells/well in 6-well dishes (Falcon tissue culture plates) and incubated at 37°C in 2 ml/well of Dulbecco's minimum essential media (DMEM) with 10% fetal bovine serum for 3 days at which time ceils were confluent.- The media was removed from the wells and replaced with serum-free DMEM and incubation was continued at 37°C for 18 hours.
  • DMEM Dulbecco's minimum essential media
  • PDGF AA, AB or BB was added at 2-200 ng/ml to each well containing 700 ⁇ l DMEM and cells were incubated for 2 hours at 4°C.
  • the media was drained and 60 ⁇ l of freshly prepared lysis buffer (20 mM Tris at pH 7.3, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM sodium orthovanadate, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin) was added to each well and cells were scraped from the wells to prepare the cell lysate.
  • freshly prepared lysis buffer (20 mM Tris at pH 7.3, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM sodium orthovanadate, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin
  • monoclonal antibody 1C7D5 which reacts specifically with beta PDGF receptor was immobilized by incubating 0.7 ⁇ g of antibody per well at 4°C for >18 hours (5 days) in 23 mM Tris at pH 8.0, 68 mM NaCl, 14 mM ammonium bicarbonate and .01% sodium azide. After antibody immobilization, the wells were blocked for 2 hours at room temperature with 25 mM HEPES pH 7.6, 100 mM NaCl, and 0.2% Tween 20 just prior to the addition of cell lysate.
  • alpha PDGF receptor is in a complex with beta PDGF receptor following ligand stimulation, both receptor subtypes will be captured by the immobilized antibody.
  • polyclonal anti-alpha-PDGF-R 3980 was added to the washed wells (0.29 ⁇ g/well in 100 ⁇ l of wash buffer) and incubated for 105 minutes at 4°C. After washing the wells 3 times with 150 ⁇ l of wash buffer, 100 ⁇ l of horseradish peroxidase conjugated anti-rabbit IgG (Boehringer Mannheim cat. # 1238850) was added to each well at a 1 to 1000 dilution.
  • PDGF AB and BB responses were observed at concentrations as low as 0.17 nM and were maximal at 4 nM. This is the concentration range at which PDGF mitogenic responses occur in vivo, demonstrating that the assay is functioning at physiologically relevant concentrations.
  • HR5 cells were seeded at 10,000 cells/well in microtiter plates (Falcon 96 well plates) and incubated at 37°C in RPMI with 10% fetal bovine serum for 3 days at which time confluency was reached. The media was removed from the wells and replaced with 100 ⁇ l of serum free RPMI and incubation was continued at 37°C for 18 hours.
  • PDGF AA or BB Retto et al. (1993) J. Biol.
  • Chem 268:3625-363) was added to each well containing 100 ⁇ l RPMI and the cells are incubated for 90 minutes at 90 minutes at 4°C.
  • the media was drained and 50 ⁇ l of freshly prepared lysis buffer (20 mM Tris at pH 7.3, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, l mM sodium orthovanadate, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin) was added to each well and the plate was shaken vigorously to prepare the cell lysate.
  • monoclonal antibody 1C7D5 directed against the beta PDGF receptor was immobilized by incubating 1.0 ⁇ g of antibody per well at 4°C for 18 hours in 23 mM Tris at pH 8.0, 68 mM NaCl, 14 mM ammonium bicarbonate and .01% sodium azide. After antibody immobilization, the wells were blocked with 25 mM HEPES pH- 7.6, 100 mM NaCl, and 0.2% Tween 20 just prior to the addition of cell lysate. The cell lysate was incubated with immobilized antibody against beta PDGF receptor for 2-3 hours at 4°C and wells are washed 3 times with 200 ⁇ l of wash buffer (0.3% gelatin, 25 mM HEPES,
  • beta PDGF receptor phosphorylation 100 mM NaCl and .01% Tween 20 at pH 7.4.
  • horseradish peroxidase conjugated monoclonal antibody PY20 International Biotechnologies, Inc.
  • PY20 horseradish peroxidase conjugated monoclonal antibody directed against phosphotyrosine was added at 0.12 ⁇ g/well in 100 wash buffer and incubated for 1 hour at 37°C.
  • the wells were drained and washed 3 times with 200 ⁇ l of wash buffer, peroxidase substrate (ABTS) in 100 ⁇ l of phosphate buffered saline was added and product formation is monitored as described, supra.
  • ABTS peroxidase substrate
  • beta PDGF-R phosphorylation is detected (Fig.
  • HR5 cells were seeded at 5 million cells per 100 mm tissue culture dish (Falcon) in RPMI 1640 containing 10% fetal bovine serum and grown at 37°C for 3 days at which time the cells had reached confluency. The media was replaced with serum-free RPMI 1640 and incubation was continued for 18 hours at 37°C. The media was removed and 3 ml of RPMI 1640 or RPMI 1640 containing PDGF BB (50 ng/ml) was added and the incubation continued for 10 minutes at 37°C.
  • the media was drained and 250 ul of freshly prepared lysis buffer (20 mM Tris at pH 7.3, 150 mM NaCl, 1% Triton X-100, l mM PMSF, 1 mM sodium orthovanadate, 100 ⁇ g/ml aprotinin and 10 ⁇ g/ml leupeptin) was added to each 100 mm plate and cells were scraped from the plates and collected into a microfuge tube. The microfuge tube was vortexed and centrifuged in a microfuge for 10 minutes at 4°C to remove insoluble material. In a microtiter plate (Immulon II, Dynatech cat.
  • rabbit polyclonal IgG directed against the beta PDGF receptor was immobilized by incubating 0.5 ug of antibody per well in 100 ul of 23 mM Tris at pH 8.0, 68 mM NaCl, 14 mM ammonium bicarbonate and .01% sodium azide at 4°C for 18 hours. After antibody immobilization, the wells were washed once with 150 ul of 25 mM HEPES pH 7.6, 100 mM NaCl, and 0.2% Tween 20 and then 200 ⁇ l/well of this buffer was added and incubated at room temperature for 1 hour to block the wells.
  • the wells were washed 2 times with 150 ⁇ l/well of incubation buffer (0.3% gelatin, 25 mM HEPES, 100 mM NaCl and 0.01% Tween 20 at pH 7.6) and 50 ⁇ l of cell lysate plus 50 ⁇ l of incubation buffer were added to each well and the plates were incubated for 2.5 hours at room temperature.
  • the plates were washed 3 times with 150 ul/well of incubation buffer and 0.05 ug of a mixed preparation of mouse monoclonal antibodies directed against PLC gamma (Upstate Biotechnology, Inc.) was added to each well in 100 ⁇ l of- incubation buffer and the plates were incubated at 37°C for 1 hour.
  • the wells were washed 3 times with 150 ⁇ l of incubation buffer and 100 ⁇ l/well of a 1:1000 dilution of horseradish peroxidase coupled anti-mouse antibody (Sigma) was added and incubated at 37°C for 1 hour.
  • the wells were washed 3 times with 150 ⁇ l of incubation buffer and 1 time with 150 ⁇ l of phosphate buffered saline.
  • 100 ⁇ l/well of peroxidase substrate (ABTS, supra) was added and product formation was monitored at 650 nM (Molecular Devices) .

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Abstract

L'invention concerne des procédés de détermination de la fonction (par exemple, l'activation) de récepteurs transducteurs de signaux sur des cellules. Les procédés sont efficaces pour l'identification de composés agonistes ou antagonistes de la fonction du récepteur. Ils mettent en application une méthode de détermination sandwich à deux anticorps utilisant un premier anticorps immobilisé qui se fixe au récepteur et le capture avec les polypeptides et protéines étroitement associés et un deuxième anticorps dirigé contre (i) un déterminant antigénique du récepteur caractéristique de l'état activé mais non de l'état inactivé, (ii) un déterminant antigénique d'une protéine ou d'une molécule fixée caractéristiquement au récepteur activé mais pas au récepteur inactivé ou (iii) un déterminant antigénique du récepteur caractéristique de l'état inactivé mais non de l'état activé. Dans un mode de réalisation, le niveau de fixation du deuxième anticorps est déterminé et mis en corrélation avec la présence du récepteur activé et, de ce fait, avec l'activation en réponse au traitement reçu par les cellules. Les procédés décrits par l'invention sont particulièrement efficaces pour détecter l'activation des récepteurs du facteur de croissance de tyrosine kinase.
EP94924067A 1993-07-29 1994-07-29 Procedes de determination de la fonction d'un recepteur Withdrawn EP0711341A4 (fr)

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DE19741716A1 (de) * 1997-09-22 1999-03-25 Hoechst Ag Adressierbares modulares Erkennungssystem, seine Herstellung und Verwendung
US6406869B1 (en) 1999-10-22 2002-06-18 Pharmacopeia, Inc. Fluorescent capture assay for kinase activity employing anti-phosphotyrosine antibodies as capture and detection agents
DE10323081A1 (de) * 2003-05-22 2004-12-16 Aventis Pharma Deutschland Gmbh Verwendung eines Polypeptides
JP2008544285A (ja) * 2005-06-24 2008-12-04 ベックマン コールター， インコーポレイテッド リン酸化タンパク質のイムノアッセイ
CA2704048A1 (fr) 2007-10-29 2009-05-07 Eisai R & D Management Co., Ltd. Procedes de pronostic de l'aptitude a traiter le cancer d'un compose analogue de la zearalenone
JP5819307B2 (ja) * 2009-10-20 2015-11-24 ネステク ソシエテ アノニム 発癌性融合タンパク質を検出するための近接媒介性アッセイ

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WO1992013870A1 (fr) * 1991-01-31 1992-08-20 The Regents Of The University Of California Recepteurs du facteur de croissance d'origine plaquettaire humain
WO1995014930A1 (fr) * 1993-11-23 1995-06-01 Genentech, Inc. Methode d'activation du recepteur de kinase
EP1000617A2 (fr) * 1994-01-07 2000-05-17 Sugen, Inc. Utilisation d'inhibiteurs du récepteur de facteur mitogénique plaquettaire dans un médicament pour le traitement du cancer

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US4376110A (en) * 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
US5045455A (en) * 1984-01-12 1991-09-03 Chiron Corporation Factor VIII:C cDNA cloning and expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013870A1 (fr) * 1991-01-31 1992-08-20 The Regents Of The University Of California Recepteurs du facteur de croissance d'origine plaquettaire humain
WO1995014930A1 (fr) * 1993-11-23 1995-06-01 Genentech, Inc. Methode d'activation du recepteur de kinase
EP1000617A2 (fr) * 1994-01-07 2000-05-17 Sugen, Inc. Utilisation d'inhibiteurs du récepteur de facteur mitogénique plaquettaire dans un médicament pour le traitement du cancer

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Title
See also references of WO9504136A1 *

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