WO2002095400A2 - Essai de fixation de recepteurs modifies - Google Patents

Essai de fixation de recepteurs modifies Download PDF

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Publication number
WO2002095400A2
WO2002095400A2 PCT/IB2002/002990 IB0202990W WO02095400A2 WO 2002095400 A2 WO2002095400 A2 WO 2002095400A2 IB 0202990 W IB0202990 W IB 0202990W WO 02095400 A2 WO02095400 A2 WO 02095400A2
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receptor
viral
ligand
binding
cell
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PCT/IB2002/002990
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WO2002095400A3 (fr
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Mark P. Chadwick
Francois-Lois Cosset
Dimitri Lavillette
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Biofocus Discovery Limited
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Priority to AU2002319858A priority Critical patent/AU2002319858A1/en
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Publication of WO2002095400A3 publication Critical patent/WO2002095400A3/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • 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
    • G01N33/5011—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 for testing antineoplastic activity
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/05—Epstein-Barr virus
    • 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
    • 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
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
    • 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
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • 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
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • This invention relates to methods of identifying receptor modulators.
  • Cells use their surface receptors to monitor and respond to subtle changes in the composition of their immediate environment. Substances which block or activate specific receptors, or increase or decrease cell surface receptor number or affinity, can therefore be used to modify cellular functions for medicinal purposes.
  • Retroviral envelope glycoproteins mediate specific viral attachment to cell surface receptors and subsequently trigger fusion between the viral envelope and the target cell membrane.
  • Retroviral envelope glycoproteins consist of an external glycoprotein moiety (SU) noncovalenffy attached, via its C-terminus, to a smaller transmembrane polypeptide moiety (TM).
  • SU external glycoprotein moiety
  • TM transmembrane polypeptide moiety
  • Each surface projection (or spike), visible by electron microscopy on the viral surface is a trimer of identical envelope glycoprotein subunits.
  • SU comprises two domains connected by a proline-rich hinge, the N-terminal domain conferring receptor specificity and exhibiting a high degree of conservation between murine leukemia viruses (MLNs) with different host ranges (Battini et al, 1992, J. Virol., 66, p. 1468-1475).
  • MSNs murine leukemia viruses
  • a general method has been disclosed that allows the display of a polypeptide ligand (which may be glycosylated) on the surface of a retroviral vector as a genetically encoded extension of the viral envelope protein (WO 94/06920).
  • the engineered retroviral envelope vector then adopts the binding specificity of the displayed ligand.
  • MN murine leukemia virus
  • WO 94/06920 single chain antibodies, cellular growth factors and immunoglobulin binding domains
  • this technology should allow the display of many different structural classes of binding domains on retroviral vectors, including glycopolypeptides and glycoproteins.
  • a novel biological phenomenon called ligand-dependent, receptor-mediated viral sequestration has recently been described.
  • a polypeptide is fused (by genetic engineering) to the envelope protein of an MLN (murine leukemia virus)-based retroviral vector such that the envelope protein to which it has been grafted remains substantially intact and capable of binding to its natural receptor, and the fused non-viral polypeptide ligand is displayed on the viral surface.
  • the virus displaying the fused non-viral polypeptide ligand is then capable of multivalent attachment to the natural virus receptor and to the cognate receptor for the non-viral ligand; attachment to the natural virus receptor leads to infection of the target cell, whereas attachment to the cellular receptor for the displayed non-viral ligand may not lead to infection of the target cell.
  • the two binding reactions envelope protein to natural receptor and non- viral ligand to its cognate receptor
  • the two binding reactions proceed in competition and the infectivity of the virus for the target cells is reduced in proportion to the efficiency with which the second binding reaction competes virus away from the natural virus receptor.
  • EGF epidermal growth factor
  • amphotrophic retroviral vector the engineered vector bound preferentially to EGF receptors present on EGF receptor-positive human cells and gene transfer was significantly impaired.
  • EGF receptor- negative cells were fully susceptible to the engineered retroviral vector but showed reduced susceptibility when they were genetically modified to express EGF receptors. The reduction in susceptibility was in proportion to the level of EGF receptor expression.
  • soluble EGF was added to competitively inhibit virus capture by the EGF receptors, gene transfer was restored (Cosset, et al., 1995, J. Virol, 69(10), 6314-22).
  • the degree to which gene transfer is inhibited depends, at least in part, on the relative affinities of the two binding reactions (envelope protein to natural receptor and non- viral ligand to its cognate receptor), the relative densities of the two receptors on the target cell surface, and the relative densities of the non- viral ligand and the intact envelope protein on the viral surface. Inhibition of gene transfer is additionally influenced by intrinsic properties of the receptor for the non-viral ligand, such as the distance it projects from the target cell membrane, its mobility within the target cell membrane and its half life on the cell surface after engagement of ligand.
  • a novel drug discovery assay based on the concept of ligand-dependent, receptor- mediated viral sequestration has been developed (WO 97/03357 and U.S.
  • This assay platform does not require knowledge or measurement of the downstream signaling cascade of a receptor to test for substances which are receptor agonists, antagonists, or down-modulators.
  • the assay platform described in WO 97/03357 is dependent upon the re-targeting of a virus particle, containing a transferable label and a first member of a specific binding pair, to a receptor of interest comprising a second member of a specific binding pair, wherein the receptor comprises wild-type, unmodified, endogenously or exogenously expressed receptor.
  • the assay described in WO 97/03357 must be performed with a cell expressing the receptor of interest. If the virus particle fuses with the membrane via the natural viral receptor of the cell the label is transferred. Transfer of label is inhibited by the formation of a complex between the first and second members of the specific binding pair.
  • virus particles has been achieved by using cells with wild-type, unmodified, endogenously or exogenously expressed receptor (as in WO 97/03357) and viral re- targeting also has been achieved with particles which display a peptide ligand or the receptor binding portion of a ligand at the N-terminus of the viral envelope surface protein (U.S. Patent 5,723,287 and WO 94/06920).
  • virus re-targeting can occur wherein re-targeting is characterized by the nature of the molecular target (receptor or protease), the nature of the targeting element (soluble adapter molecule or genetically modified viral envelope glycoprotein) or the nature of host-range modification (by restriction or extension (Russell and Cossett, 1999, J. Gene Med, 1(5), 300-11).
  • retargeting of a retrovirus can be achieved without modification to a receptor of interest.
  • a linear hemagglutinin (HA) epitope tag was incorporated into the adenovirus envelope fiber protein.
  • the modified molecular target was an anti-HA single chain antibody (scFv) anchored to the cell surface by a transmembrane domain derived from platelet derived growth factor (Einfeld et al., supra).
  • scFv anti-HA single chain antibody
  • Retroviral envelope proteins that are not bound to vector particles, or free virus-receptor- binding domains have been shown to exert a "receptor blockade effect" wherein cell entry of viral particles displaying the corresponding wild-type viral envelope protein is blocked
  • the "receptor blockade effect” and the “viral activation effect” provide the basis for a new assay method, ideally suited to the screening of compounds which modulate the level of cell surface receptor expression.
  • the invention relates to a method of identifying a receptor upmodulator or downmodulator that is based upon regulating the targeting of a virus particle to a receptor of interest by using a fusion protein comprising a viral envelope protein or receptor binding domain (RBD) of a viral envelope protein or a receptor binding moiety fused to a polypeptide that can be sequestered by another cell surface receptor.
  • a fusion protein comprising a viral envelope protein or receptor binding domain (RBD) of a viral envelope protein or a receptor binding moiety fused to a polypeptide that can be sequestered by another cell surface receptor.
  • a method of identifying a receptor modulator that requires that a specific ligand or binding domain for a receptor is displayed can be problematic for the following reasons.
  • the display of some ligands or binding domains on virus particles may result in low levels of viral incorporation.
  • the ligand or binding domain may be oligomeric such that the display of the monomer may result in the formation of an oligomeric cap that inhibits normal envelope biology (Morling et al, (1997) Virology. 234:51-61).
  • the ligand maybe a chemical rather than a peptide.
  • the ligand may be unknown, for example, orphan G protein-coupled receptors. In the first two scenarios, the resulting reagent would be inappropriate for drug discovery using prior technologies.
  • the invention provides a method of screening for a receptor upmodulator that increases the number or affinity of cell surface receptors that bind specifically to a ligand, comprising the steps of: (a) mixing a candidate upmodulator, a virus particle comprising a transferable label, a target cell comprising a viral receptor, wherein the target cell does not express a cell surface receptor for the ligand, and a fusion protein comprising a viral receptor binding moiety and a ligand, under conditions that permit binding of the virus particle to the viral receptor or binding of the viral receptor binding moiety to the viral receptor; and (b) monitoring formation of a complex between the virus particle and the viral receptor, wherein the monitoring comprises measuring the amount of label transferred from the virus particle to the target cell, and wherein transfer of label is activated by binding of the virus particle and the viral receptor.
  • the invention also provides a method of screening for a receptor downmodulator that decreases the number or affinity of cell surface receptors that bind specifically to a ligand, comprising the steps of: (a) mixing a candidate downmodulator, a virus particle comprising a mutant envelope protein and further comprising a transferable label, a target cell comprising a viral receptor, wherein the target cell expresses a high level of a cell surface receptor for the ligand, and a fusion protein comprising a viral receptor binding moiety and a ligand, under conditions that permit binding of the virus particle to the viral receptor or binding of the viral receptor binding moiety to the viral receptor; and (b) monitoring formation of a complex between the virus particle and the viral receptor, wherein the monitoring comprises measuring the amount of label transferred from the virus particle to the target cell, and wherein transfer of label is activated by binding of the virus particle and the viral receptor.
  • the invention also provides for a downmodulator identified by this method.
  • a "mutant envelope protein” refers to an envelope protein that can bind to a virus receptor with a Kd of lOO ⁇ M or lower, but is impaired in its ability to mediate entry into a cell.
  • a viral particle comprising a "mutant envelope protein” encompasses a virus particle that can bind to a virus receptor but exhibits impaired entry into a target cell.
  • “impaired entry” refers to less than 100% (i.e., 90%, 50%, 10%, 1%, 0.1%, 0.01% etc... or less, including no detectable infection) of the amount of entry observed with a viral particle expressing a corresponding wild-type envelope protein.
  • a “mutant envelope protein” according to the invention also encompasses an envelope protein that exhibits an impaired ability to trigger virus- cell fusion.
  • an "impaired ability to trigger virus-cell fusion refers to less than 100% (i.e., 90%, 50%, 10%, 1%, 0.1%, 0.01% etc... or less, including no detectable infection) of the amount of virus-cell fusion observed with a viral particle expressing a corresponding wild- type envelope protein. Entry of a virus particle into a cell or virus cell fusion is determined by incubating a virus particle comprising a transferable label with an appropriate target cell expressing a viral receptor, as described in any of Examples 1-5 presented herein. "Mutant envelope proteins" according to the invention are disclosed in Lavaillette et al, 2000, supra.
  • the target cell expresses a "low level" of a receptor.
  • low level refers to less than 10 2 , less than 10 3 , less than 10 4 or less than 10 5 of the number of cell surface copies of the receptor on a cell, in the absence of a substance that increases the cell surface density of the receptor.
  • the invention also provides for an upmodulator identified according to this method.
  • the virus particle of the invention expresses a wild-type or a mutant viral envelope protein.
  • a "viral envelope protein” comprises any polypeptide displayed on a lipid-enveloped particle, typically of viral origin (i.e., substantially similar to one naturally encoded by a viral gene).
  • wild-type refers to the normal, naturally occurring form.
  • a viral envelope protein according to the invention also comprises a chimeric protein comprising a region derived from a first viral envelope protein and at least a second region derived from a second viral envelope protein.
  • a chimeric envelope protein useful according to the invention can bind to a virus receptor with a Kd of lOO ⁇ M or less and preferably lO ⁇ M or less, i.e., l ⁇ M, 500nM, 100 nM, etc.).
  • a "viral envelope protein” according to the invention also encompasses a modified viral envelope protein that can bind to a virus receptor with a Kd of lOO ⁇ M or less.
  • modified includes a viral envelope protein comprising an amino acid deletion of one or more (i.e., 2, 3, 4, 5, 10, 20, etc..) contiguous or scattered amino acids, or comprising at least one amino acid difference as compared to the wild-type protein, or encompassing a chimeric molecule, as discussed above and hereinbelow.
  • modified refers to a viral envelope protein that has a variation in the nucleotide or amino acid sequence as compared to the wild-type envelope protein.
  • the variation in the nucleotide sequence may result from the deletion, insertion or substitution of more than one nucleotide (e.g., 2, 3, 4, or more nucleotides) or a single nucleotide change such as a deletion, insertion or substitution.
  • a “viral envelope protein” includes a mutant viral envelope protein that binds to a virus receptor with a Kd of lOO ⁇ M or less.
  • a mutant viral envelope protein exhibits an impaired ability to trigger virus-cell fusion, as defined below.
  • a "mutation" refers to a variation in the nucleotide or amino acid sequence as compared to the wild-type envelope protein. The variation in the nucleotide sequence may result from the deletion, insertion or substitution of more than one nucleotide (e.g., 2, 3, 4, or more nucleotides) or a single nucleotide change such as a deletion, insertion or substitution.
  • a viral envelope protein according to the invention also includes any mutant form of an MLV viral envelope protein known in the art or any mutant form of an MLN viral envelope protein that binds to a virus receptor with a Kd of 1 OO ⁇ M or less.
  • a viral envelope protein is a mutant MLN viral envelope protein comprising a deletion of at least one amino acid residue from the ⁇ -terminus and capable of binding to a virus receptor with a Kd of lOO ⁇ M or less.
  • a "viral envelope protein” includes a viral envelope protein derived from any virus including but not limited to MLN, influenza, measles, HIN, adenovirus, etc..
  • a "viral envelope protein” according to the invention also includes an envelope protein comprising a deletion of one or more contiguous or scattered amino acids of the PHQ motif of the viral envelope protein.
  • PHQ motif refers to a conserved peptide motif centering on a histidine residue and known in the art to be critical for postbinding events that lead to membrane fusion.
  • the PHQ motif is present near the amino terminus of the SU of the 4070A and Moloney virus envelope proteins as well as on proteins that are homologous to the 4070A and Moloney virus envelope proteins.
  • homologous refers to any protein that is significantly similar (i.e., at least 29%, preferably 50%, more preferably 60-75% and most preferably 75-100%) in sequence to an MLN envelope protein. Sequence similarity is defined by aligning the target sequence with the MLN sequence using standard methods (e.g., BLAST, ClustalW) and demonstrating that the probability that the alignment arose by chance is less than 5% (PO.05).
  • the target cell expresses a low level of a cell surface receptor.
  • a "low level of a cell surface receptor” refers to less than 100% (i.e., 99%, 90%, 80%), 70%, 60%, 50%, etc%) of the level of a cell surface receptor on a cell, in the absence of a substance that increases the cell surface density of the receptor.
  • the transfer of label is activated by binding of the virus particle and the viral receptor in the presence of a fusion protein.
  • the invention provides for a mutant envelope protein that binds to a virus receptor and exhibits an impaired ability to trigger virus-cell fusion.
  • "free" viral receptor binding moiety stimulates virus-cell fusion of a virus expressing such a mutant envelope protein.
  • free refers to a fusion protein comprising a receptor binding domain or a receptor binding moiety and a ligand, that is not bound to or complexed to a target cell via with the ligand receptor. In certain embodiments, “free” refers to a fusion protein comprising a receptor binding domain or a receptor binding moiety and a ligand that is not bound to or complexed to a target cell via the virus receptor.
  • a "receptor upmodulator” refers to a compound that is capable of increasing the number of cell surface receptors present on the surface of a cell by at least 2-fold, preferably 5-fold, more preferably 10-fold and most preferably, 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold, etc%), as compared to the number of cell surface receptors present on the surface of a cell in the absence of a receptor modulator according to the invention.
  • a receptor upmodulator also refers to a compound that is capable of increasing the affinity of a receptor for its ligand by at least 10%, preferably 15-25%, more preferably 25- 50% and most preferably 50-100, as compared to the affinity of the receptor for its ligand in the absence of a compound.
  • a receptor upmodulator also refers to a compound that is capable of increasing the affinity of a receptor for its ligand by at least 2-fold, preferably 5 -fold, more preferably 10-fold and most preferably, 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500- fold, 1000-fold, 10,000-fold, etc...), as compared to the affinity of the receptor for its ligand in the absence of a compound.
  • a "receptor upmodulator” includes an agonist, an antagonist, an inverse agonist, a protease inhibitor, a protease activator, or any compound that increases the number or affinity of a cell surface receptor.
  • the invention also provides for a "receptor upmodulator” that stimulates expression of a detectable level of a cell surface receptor on a cell that does not express a detectable level of cell surface receptor in the absence of a "receptor upmodulator”.
  • the invention also provides for a "receptor upmodulator” that stimulates expression of a detectable level of a cell surface receptor on a cell that expresses a "low level of cell surface receptor in the absence of a "receptor upmodulator”.
  • a “detectable level” refers to an amount of cell surface receptor that can be detected by standard binding assays well known in the art, or by immunological methods (e.g., immunofluorescence, FACS analysis, immunoprecipitation or western blot analysis).
  • a "virus particle” refers to a virus capsid or core particle that comprises a viral envelope protein that binds to its cognate receptor, and the viral nucleic acid, and is capable of fusing to and infecting a target cell (e.g. a eukaryotic cell) and being released into the cytoplasm of the target cell.
  • a target cell e.g. a eukaryotic cell
  • a “virus particle” also refers to a virus capsid or core particle comprising viral nucleic acid and a mutant viral envelope protein, according to the invention, wherein the mutant viral envelope protein only mediates fusion with, and infection of a target cell (e.g., a eukaryotic cell) and release of the virus into the cytoplasm of the target cell in the presence of a free receptor binding domain or receptor binding moiety.
  • a target cell e.g., a eukaryotic cell
  • a "viral envelope protein” useful according to the invention includes a wild-type viral envelope protein, a mutant viral envelope protein wherein the PHQ motif is altered or deleted or a chimeric viral envelope protein comprising a region derived from a first viral envelope protein and at least a second region derived from a second viral envelope protein.
  • ligand or “second-binding moiety” refers to a moiety that is capable of associating or binding to a receptor.
  • a ligand and a receptor have a binding constant that is sufficiently strong to allow detection of binding by an assay method that is appropriate for detection of a ligand binding to a receptor (e.g. a binding assay to measure protein-ligand binding or an immunoassay to measure antibody-antigen interactions).
  • a ligand according to the invention includes the actual molecule that binds a receptor (e.g. EGF is the ligand for the EGF receptor) or a ligand may be an antibody, an antigen an enzyme or any polypeptide capable of binding to the receptor.
  • a ligand according to the invention also includes a tag or a moiety that binds to a tag according to the invention.
  • Additional ligands useful according to the invention include but are not limited to IGF, EGF, heregulin, PDGF, NEGF, ligands that bind to G-protein coupled receptors, ion channel ligands, tags, as defined herein, and tag-binding proteins, (e.g., calmodulin).
  • IGF IGF
  • EGF EGF
  • PDGF PDGF
  • NEGF ligands that bind to G-protein coupled receptors
  • tags as defined herein
  • tag-binding proteins e.g., calmodulin
  • a ligand and receptor specifically bind to each other (e.g. via covalent or hydrogen bonding or via an interaction between, for example, a protein and a ligand, an antibody and an antigen or protein subunits).
  • a “transferable label” refers to a label that can be transferred from a viral particle to an infected target cell following fusion of the viral particle with the target cell membrane.
  • a “transferable label” that is useful according to the invention can be any label whose presence can be detected in the target cell upon fusion of the viral particle and the target cell membrane (e.g., a gene encoding a selectable marker or a reporter gene, a protein that can be detected using immunochemical techniques employing antibodies that specifically bind to the protein, or an enzyme that catalyzes a reaction wherein a colored product is produced from a chromogenic substance or a nucleic acid molecule encoding a such an enzyme.
  • a chromogenic polypeptide is any polypeptide that can be detected by the absorption of light or by fluorescence or that can act upon a substrate to generate a detectable color change, or a change in fluorescence or luminescence.
  • a transferable label can also be a radioactive or an otherwise-modified lipid or non-lipid component of a viral particle, a cytotoxic agent such as perform, ribonucleases, and exotoxins or an enzyme.
  • the label may not be, strictly speaking, "transferred" to the cell because the particle may cease to have an independent existence.
  • the label will be acquired by the cell, and such is understood as being within the meaning of the term "transferable” label.
  • the presence or absence of a transferable label in a target cell can be detected by any means known in the art which is sensitive to the particular label employed.
  • a “cell surface receptor” refers to a protein that binds a specific extracellular signaling molecule or ligand and initiates a response in the cell, wherein the protein is located in the plasma membrane such that the ligand-binding site is exposed to the external medium.
  • Cell surface receptors that are useful according to the invention include but are not limited to, G-protein coupled receptors, tyrosine kinase receptors, serine-threonine kinase receptors, ion channels, immune recognition receptors and transporters (e.g., Glut4 glucose transporter).
  • Receptors useful according to the invention also include IGFR, EGFR (erbBl), erbB2, erbB3, erbB4 and NEGF receptors.
  • a receptor according to the invention comprises a tag.
  • a cell surface receptor according to the invention also includes a constitutively active mutant.
  • constitutively active refers to a receptor that is stabilized in an active state by means other than binding of the receptor with its endogenous ligand or an equivalent thereof.
  • constitutively active describes a state of the receptor in which a receptor can signal in the absence of an agonist.
  • a receptor may be stabilized by an agonist or an inverse agonist.
  • agonists and inverse agonists change the conformation of the receptors such that they are stabilized at the cell surface rather than being retained in or recycled into intracellular compartments.
  • does not express refers to a target cell that expresses less than 5%, and preferably less than 3% of the amount of a cell surface receptor for a ligand on a cell that expresses a cell surface receptor for a ligand, as determined in the absence of an upmodulator. "Does not express” also refers to a target cell that expresses less than 10 or less than 1 copy of a cell surface receptor in the absence of a receptor upmodulator.
  • does not express refers to a target cell that does not express a detectable level of a cell surface receptor for a ligand wherein receptor number is measured by standard binding assays known in the art, or wherein a cell surface receptor is detected by immunological methods (e.g., immunofluorescence, FACS analysis, immunoprecipitation or western blot analysis).
  • immunological methods e.g., immunofluorescence, FACS analysis, immunoprecipitation or western blot analysis.
  • fusion protein comprising a viral receptor binding moiety and a ligand or a “fusion partner” refers to a protein encoded by a nucleic acid comprising a sequence encoding a viral envelope glycoprotein that is fused without disruption of the translational reading frame to a sequence encoding a ligand, as defined herein.
  • the resulting hybrid gene codes for a chimeric glycoprotein in which the virus envelope glycoprotein comprises a domain capable of binding to a virus receptor and the ligand is capable of binding to the corresponding receptor.
  • a “viral receptor binding moiety” or an “RBD” comprises a viral envelope binding protein or a portion of a viral envelope protein that binds to a virus receptor with a Kd of lOO ⁇ M or lower.
  • a “viral receptor binding moiety” according to the invention includes a viral envelope protein from any virus including, but not limited to MLN, influenza, measles, HIV, adenovirus, etc...
  • a “viral receptor binding moiety according to the invention encompasses a complete viral envelope protein (i.e., 100% of the viral envelope protein) or any portion (i.e., less than 100%) of the complete viral envelope protein that binds to a virus receptor with a Kd of lOO ⁇ M or less.
  • a viral receptor binding moiety also includes any of the mutant forms of the MLV envelope proteins known in the art as well as any mutant MLV envelope protein comprising at least one amino acid residue deletion and capable of binding to a virus receptor with a Kd of lOO ⁇ M or lower.
  • a viral receptor binding moiety useful according to the invention can include the 'intact' SU portion of the viral envelope protein, just the RBD, any portion of the SU that includes the RBD or any mutant of the above, wherein the PHQ motif is mutated or deleted.
  • a fusion protein comprising a viral receptor binding moiety can comprise a tag to facilitate purification (e.g., a histidine, myc, FLAG, influenza HA calmodulin-binding or any other peptide or protein that has a known binding partner.
  • a tag to facilitate purification e.g., a histidine, myc, FLAG, influenza HA calmodulin-binding or any other peptide or protein that has a known binding partner.
  • the invention also encompasses a receptor binding moiety that is a portion of a viral envelope protein or an RBD.
  • a "portion of a viral envelope protein" or an RBD refers to the smallest portion of the viral envelope protein that can fold into a 3 -dimensional structure and bind to its cognate virus receptor with a Kd of lOO ⁇ M or lower (i.e., 50 ⁇ M, l ⁇ M, .5 ⁇ M, etc .).
  • a “fusion protein comprising a viral receptor binding moiety and a ligand”, according to the invention also refers to a hybrid gene coding for a chimeric glycoprotein in which the virus envelope glycoprotein comprises a domain capable of binding to a virus receptor and is a chimera comprising a region derived from a first envelope protein and a region derived from at least a second envelope protein.
  • region refers to less than 100% (i.e., 99%, 90%, 80%, 70%, 60%, 50% etc%) of the virus envelope glycoprotein.
  • a "fusion protein comprising a viral receptor binding moiety and a ligand” wherein the viral receptor binding moiety is a hybrid molecule comprising a region derived from the amphotropic (4070A) viral envelope protein and a region derived from the ecotropic (Moloney) viral envelope protein is useful according to the invention.
  • a "fusion protein comprising a viral receptor binding moiety and a ligand” in which the virus envelope glycoprotein comprises a domain capable of binding to a virus receptor and is a chimera comprising a region derived from a first envelope protein and a region derived from at least a second envelope protein, useful according to the invention, are provided in Lavillette et al., 2001, J. Virology, (in press).
  • a "fusion protein comprising a viral receptor binding moiety and a ligand” comprises a viral binding moiety that binds to the same receptor to which the viral particle binds such that the viral binding moiety and the viral particle can both bind to the same viral receptor.
  • a "fusion protein comprising a viral receptor binding moiety and a ligand” comprises a viral binding moiety that does not bind to the same receptor to which the viral particle binds.
  • a fusion protein comprising a viral receptor binding moiety and a ligand can be produced in a cell (i.e., a target cell according to the invention or a producer cell).
  • a fusion protein comprising a viral receptor binding moiety and a ligand can also be produced in a baculovirus system.
  • the invention also provides for a fusion protein comprising a viral receptor binding moiety and a ligand that is produced from a recombinant nucleic acid construct in prokaryotic or eukaryotic cells and is purified according to methods of protein purification well-known in the art.
  • the "fusion protein" is generated by cross-linking the ligand to the viral receptor binding moiety or RBD.
  • a “tag” refers to a sequence of amino acids that is covalently bound to an extracellular portion of a cell surface receptor and encodes a protein or epitope that mediates specific binding to the first member of a specific binding pair.
  • a “tag” also includes a fusion of short peptides or protein domains.
  • a “tag” that is useful according to the invention includes but is not limited to histidine, hemagglutinin, glutathione-S-transferase, myc, lacZ, trpE, thioredoxin, FLAG tag and calmodulin-binding tag.
  • inhibittion of transfer of label refers to a decrease in the transfer of label that is at least 10%, preferably 10-30%, more preferably 30-50% and most preferably 50- 100% of the amount of label transferred in the absence of a receptor modulator.
  • activation of transfer of label refers to an increase in the transfer of label that is at least 10%, preferably 10-30%, more preferably 30-50% and most preferably 50- 100% of the amount of label transferred in the absence of a receptor modulator. "Activation of transfer of label” also refers to an increase in the transfer of label that is 10%, 50%, 100%, or 2- fold, 5-fold, preferably 10-fold, more preferably 50-fold, and most preferably 100-fold or more
  • a "receptor downmodulator” refers to a compound that is capable of decreasing the number of cell surface receptors present on the surface of a cell by at least 2-fold, preferably 5-fold, more preferably 10-fold and most preferably, 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold, etc .), as compared to the number of cell surface receptors present on the surface of a cell in the absence of a receptor downmodulator according to the invention.
  • a receptor downmodulator also refers to a compound that is capable of decreasing the affinity of a receptor for its ligand by 10%, preferably 15-25%, more preferably 25-50% and most preferably 50-100%, as compared to the affinity of the receptor for its ligand in the absence of a compound.
  • a receptor downmodulator also refers to a compound that is capable of decreasing the affinity of a receptor for its ligand by at least 2-fold, preferably 5-fold, more preferably 10-fold and most preferably, 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500- fold, 1000-fold, 10,000-fold, etc...), as compared to the affinity of the receptor for its ligand in the absence of a compound.
  • a "receptor downmodulator” includes an agonist, an antagonist, an inverse agonist, a protease inhibitor, a protease activator or any compound that decreases the number or affinity of a cell surface receptor.
  • a "high level of cell surface receptor for a ligand” is preferably more than 10 receptors per cell, more preferably more than 10 5 receptors per cell and most preferably more than 10 6 receptors per cell.
  • a "high level of a cell surface receptor for a ligand” also refers to a target cell that expresses 2-fold more, preferably 5-fold more, more preferably 10- fold more and most preferably, greater than 100-fold more than the number of cell surface receptors present on the surface of a cell in the presence of a downmodulator.
  • the level of a cell surface receptor for a ligand is measured by standard binding assays known in the art. Alternatively, a cell surface receptor for a ligand is detected by immunological methods known in the art.
  • the invention also provides a method of simultaneously screening for an upmodulator or a downmodulator of a cell surface receptor that binds specifically to a ligand.
  • This method comprises the steps of: mixing a candidate upmodulator or downmodulator, a virus particle comprising a first transferable label, a second virus particle comprising a mutant viral envelope protein and a second transferable label, a target cell comprising a plurality of viral receptors, wherein the target cell expresses a cell surface receptor for the ligand, and a plurality of fusion proteins comprising a viral receptor binding moiety and a ligand, under conditions that permit binding of the virus particle to the virus receptor or binding of the viral receptor binding moiety to the virus receptor; and monitoring formation of a complex between at least one of the virus particle(s) and the virus receptor(s), wherein the monitoring comprises measuring the amount of the first or second label transferred from the virus particle to the target cell.
  • transfer of label is activated by binding of the virus particle and the virus receptor.
  • the invention also provides for an upmodulator or a downmodulator identified according to this method.
  • the target cell expresses a low level of the cell surface receptor for a ligand.
  • a low level of a cell surface receptor for a ligand refers to less than 10 , preferably less than 10 3 , more preferably less than 10 4 and most preferably less than 10 5 cell surface receptors per cell.
  • a "low level of a cell surface receptor for a ligand” also refers to 2-fold less, preferably 5-fold less, more preferably 10-fold less and most .preferably 100- fold or more, less than the number of cell surface receptors present on the surface of a cell in the presence of an upmodulator.
  • a "low level of a cell surface receptor for a ligand” also refers to 5-10%, preferably 10-20% and most preferably 20-25% of the level of a cell surface receptor for a ligand as compared to the level of expression of a cell surface receptor on a cell that expresses the cell surface receptor in the presence of an upmodulator according to the invention.
  • the viral receptor binding moiety comprises a viral envelope glycoprotein.
  • viral envelope glycoprotein refers to a glycoprotein encoded by a virus in its natural state.
  • the viral glycoprotein is typically a spike glycoprotein, i.e. a protein which in its natural state:
  • oligomeric having 2 to 6 subunits which maybe identical or non identical, i.e. homo or heterodimers;
  • a viral glycoprotein is glycosylated.
  • the viral envelope glycoprotein is modified.
  • modified refers to containing at least one amino acid substitution that does not alter the receptor binding properties.
  • the viral receptor binding moiety comprises at least apart of a viral envelope glycoprotein.
  • at least a part of a viral envelope glycoprotein refers to a domain of a glycoprotein that is of sufficient size (i.e., from 20 amino acids to 500 amino acids or more) to allow binding to a viral receptor.
  • the non-mutated RBD from the 4070A virus is 208 amino acids.
  • the invention also provides for "at least a part of a viral envelope glycoprotein” that binds to a viral receptor with a Kd that is less than 1 OO ⁇ M and preferably less than lO ⁇ M.
  • At least a part of a viral envelope glycoprotein is modified.
  • the receptor is tagged.
  • tagged refers to being covalently or non-covalently associated with a tag, wherein a tag includes but is not limited to a his, hemagglutinin, GST, FLAG, myc moiety, or calmodulin binding tags, and wherein the tag can be bound by a fusion protein comprising a domain (e.g., an antibody) capable of specifically binding to the tag.
  • the receptor is substantially intact.
  • a "substantially intact" receptor according to the invention has normal or near normal pharmacology (e.g. receptor activity, the response of a receptor to an activator or an inhibitor, or ligand binding by the receptor are at least 90% of the level of activity, response or binding exhibited by a wild-type receptor).
  • substantially intact refers to a receptor comprising at least part of a wild-type receptor wherein the post-translational and functional activities of a "substantially intact” receptor are identical to a wild-type receptor.
  • the receptor is selected from the group consisting of G protein- coupled receptors, tyrosine kinase receptors, serine-threonine kinase receptors, ion channels, transporters and immune recognition receptors.
  • the receptor is a constitutively active mutant.
  • the receptor is a constitutively active mutant of a G-protein coupled receptor.
  • the ligand is selected from the group consisting of growth factors, antibodies, cytokines or enzymes.
  • the ligand is a small molecule (e.g., of a molecular weight less than 1000).
  • the fusion protein further comprises a linker separating the viral receptor binding moiety and the ligand.
  • a “linker” refers to a short stretch of amino acids, from 0 to 20 amino acids or more (i.e., 21, 25, 30, 40, 50, 100 etc%) in length that separates the virus RBD or viral receptor binding moiety, and the 'ligand' of a fusion protein, such that the two domains fold independently and both have sufficient conformational flexibility to bind to their respective targets.
  • the virus RBD and the ligand do not bind simultaneously to their respective targets.
  • a “linker” according to the invention may also include a protease cleavage site to allow separation of the two domains of the fusion protein.
  • the fusion protein further comprises a protease cleavage site separating the viral receptor binding moiety and the ligand.
  • the protease cleavage site is located in a linker, according to the invention.
  • a protease cleavage site refers to a specific site on a protein that is susceptible to cleavage by one or more protease enzymes.
  • a protease cleaves a cell surface receptor to activate or inhibit the action of the receptor, i.e., by cleaving off a specific component.
  • a protease inhibitor refers to a compound that prevents a proteolytic enzyme or protease from cleaving aprotein at a protease cleavage site.
  • Protease activity i.e. cleavage of a protein at a protease cleavage site
  • a protease inhibitor inhibits a protease on the eukaryotic export pathway, wherein the protease is present on the plasma membrane or is secreted.
  • the receptor upmodulator is an agonist.
  • an "agonist" refers to a ligand which activates an intracellular response when it binds to a receptor.
  • An agonist may alternatively enhance GTP binding to membranes when it binds to a receptor.
  • An agonist, according to the invention may increase intemalization of a cell surface receptor such that the cell surface expression of a receptor is decreased by at least 2-fold, preferably 5-fold, more preferably 10-fold and most preferably, 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold etc.), as compared to the number of cell surface receptors present on the surface of a cell in the absence of an agonist.
  • an agonist stabilizes a cell surface receptor and increases the cell surface expression of a receptor by at least 2-fold, preferably 5-fold, more preferably 10- fold and most preferably, 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000- fold, 10,000-fold etc...), as compared to the number of cell surface receptors present on the surface of a cell in the absence of agonist.
  • the receptor upmodulator is an antagonist.
  • an "antagonist” is a ligand which competitively binds to the receptor at the same site as an agonist, but does not activate an intracellular response initiated by an active form of a receptor, and thereby inhibits the intracellular response induced by an agonist by at least 10%, preferably 15-25%, more preferably 25-50% and most preferably, 50-100%, as compared to the intracellular response in the presence of an agonist and in the absence of an antagonist.
  • An antagonist does not diminish the base line intracellular response that occurs in the absence of an agonist.
  • the receptor downmodulator is an agonist.
  • the receptor downmodulator is an antagonist.
  • the receptor upmodulator is an inverse agonist that increases the cell surface expression of the cell surface receptor.
  • an "inverse agonist” or “negative antagonist” is a ligand that binds to either the inactive form of a receptor or to a constitutively activated form of a receptor.
  • An inverse agonist inhibits the baseline, spontaneous activity of a receptor below the normal baseline level of activity which is observed in the absence of agonists or partial agonists.
  • An inverse agonist according to the invention can also decrease the GTP binding of G protein coupled receptors.
  • the baseline intracellular response is inhibited in the presence of an inverse agonist by at least 30%, more preferably by at least 50%, and most preferably by at least 75%, as compared with the baseline response in the absence of the inverse agonist.
  • a neutral antagonist is a ligand that has the same affinity for the inactive and the active form of a receptor and binds competitively with both agonists and inverse agonists.
  • the receptor downmodulator increases intemalization of the cell surface receptor.
  • the receptor downmodulator is an agonist.
  • intemalization occurs by homologous desensitization.
  • homologous desensitization refers to loss of cell surface receptor following a direct interaction between the receptor downmodulator and the receptor.
  • intemalization occurs by heterologous desensitization.
  • heterologous desensitization refers to loss of cell surface receptor due to an interaction between the receptor downmodulator and an entity that is not the cell surface receptor.
  • the transferable label is selected from the group consisting of a nucleic acid encoding a selectable marker or a reporter molecule, a fluorescent moiety, a radioactive moiety, a cytotoxic agent or an enzyme.
  • Figure 1 is a schematic diagram demonstrating a method of assaying for receptor modulators that increase the cell surface number or availability of EGF receptors.
  • Figures 2A and 2B demonstrate the sequestration of EGF-amphotropic receptor binding domain fusion protein onto EGF receptors.
  • Figure 3 shows the effect of an agonist on a wild-type G protein-coupled receptor.
  • Figure 4 shows the effect of stabilization on a constitutively active mutant G protein- coupled receptor.
  • Figure 5 shows the effect of stabilization on a constitutively active mutant G protein- coupled receptor.
  • Figure 6 is a graph demonstrating stabilization of CAM adrenergic receptor with inverse agonists.
  • Figure 7 demonstrates an assay for test substances that decrease the cell surface number/availability of EGF receptors.
  • the invention provides novel drag discovery assays wherein a virus particle competes for binding to a viral receptor with a fusion protein comprising a viral receptor binding moiety and a ligand.
  • a virus particle competes for binding to a viral receptor with a fusion protein comprising a viral receptor binding moiety and a ligand.
  • targeting of a fusion protein is achieved in the presence of a compound that increases or decreases the number or affinity of cell surface receptors that bind specifically to a ligand.
  • the target cells express virus receptor (e.g. amphofropic virus receptor), and may or may not express the ligand receptor (e.g. EGFR).
  • virus receptor e.g. amphofropic virus receptor
  • a wild-type viral particle e.g., an amphofropic viral particle
  • a fusion protein is also present, consisting of a virus receptor-binding moiety (e.g., amphofropic) fused to a ligand (e.g. EGF).
  • the fusion protein binds to the viral receptor, reducing gene delivery by the amphofropic viral particle.
  • ligand receptor may be expressed on the cell surface (or otherwise increase the availability/affinity of free ligand-binding sites).
  • the fusion protein is sequestered onto the ligand receptor and away from the virus receptor. The virus receptor is then free, and the viral particle delivers the reporter gene.
  • the invention provides for identifying a receptor upmodulator or downmodulator in a 'high throughput screening' format, by assaying for the level of reporter gene present in the target cell.
  • the virus particle comprising a transferable label of the invention and a wild type viral envelope protein fuses with the membrane of a target cell of the invention by binding to a natural viral receptor present on the target cell, the virus particle will infect the cell and transfer of label will occur.
  • a fusion protein comprising a viral receptor binding moiety and a ligand, as well as cells that do not express, or express low levels of, a cell surface receptor for the ligand, the viral particle will not bind to the cell and there will be no transfer of label, or a reduction in the amount of transferred label.
  • the fusion protein will bind to the ligand receptors rather than to the viral receptor and the virus particle fuses with the membrane of the target cell by binding to an unoccupied viral receptor of the target cell.
  • the invention provides an assay comprising such virus particles, fusion proteins comprising a viral receptor binding moiety and a ligand, and target cells lacking or expressing low levels of, a cell surface receptor for the ligand wherein receptor upmodulators are identified by measuring label transfer from the viral particle to the cell.
  • the invention also provides an assay comprising virus particles that express a mutant viral envelope protein according to the invention, a fusion protein comprising a viral receptor binding moiety and a ligand, and target cells expressing a cell surface receptor for a ligand wherein receptor downmodulators are identified by measuring label transfer from the viral particle to the cell.
  • mutant envelope proteins are any envelope proteins that can still bind receptor but do not trigger virus-cell fusion or trigger virus-cell fusion less efficiently than wild-type viruses.
  • "less efficiently" as it refers to virus-cell fusion refers to less than 100% (i.e., 99%, 90%, 80%, 70%, 60%, 50% etc%) of the amount of virus-cell fusion that occurs with a viras that expresses a corresponding wild-type envelope protein.
  • the invention provides for a viral particle expressing wild type envelope proteins, as well as viral particles that express mutant viral envelope proteins.
  • binding of a viral particle comprising a transferable label is determined by measuring transfer of the label from the viral particle to a target cell expressing a viral receptor.
  • Binding of a viral particle to a viral receptor is determined by exposure of a target cell expressing a viral receptor for 1-48 hours or more to a virus containing supernatant containing the viral particles comprising a transferable label (e.g. an antibiotic resistance gene or a reporter gene (e.g., ⁇ -galactosidase, luciferase etc...)) in the presence of polybrene.
  • a transferable label e.g. an antibiotic resistance gene or a reporter gene (e.g., ⁇ -galactosidase, luciferase etc...)
  • Target cells are selected (e.g.
  • a viral particle capable of binding to a viral receptor can transfer an amount of label to a cell wherein the amount is sufficient to be detected in an assay that is appropriately selected for the label being utilized.
  • transfer of a label comprising an antibiotic resistance gene is detected by performing a binding assay (as described above) and detecting cells that are capable of growing in the presence of antibiotic.
  • Transfer of a label comprising a reporter gene is detected by performing an assay for the protein product of the reporter gene and detecting a signal (e.g., light).
  • the invention provides for a downmodulator assay wherein the particle associated viral envelope protein and the RBD of the fusion protein bind to different receptors on the target cell.
  • the invention also provides for an upmodulator that simultaneously assays for upmodulation of more than one target receptor on a target cell.
  • a viral particle expressing a mutant envelope protein according to the invention is mixed with a plurality of fusion proteins, each comprising a different ligand.
  • the invention also provides for an up- or downmodulator assay that simultaneously probes the cell surface levels of target receptors by mixing more than one set of viral particle (comprising a transferable labe ⁇ )-fusion protein pairs, wherein each viral particle interacts with a different viral receptor on the target cell (as compared to every other viral particle) and delivers its transferable label to the target cell after interacting with a specific viral receptor.
  • Each viral particle and fusion protein of a particular pair interacts with the same viral receptor on the target cell.
  • the invention also provides for simultaneously performing a downmodulator and an upmodulator assay of the invention using a mixture of the combination of 1. a first viral particle that displays a wild-type envelope protein (i.e., an amphofropic envelope protein) and comprises a first transferable label, 2. a second viral particle that displays a mutant envelope protein (i.e., a mutant amphofropic envelope protein) and comprises a second transferable label, 3. a target cell that expresses a viras receptor (X) for the wild type and mutant viral envelope protein and a decoy receptor (Y) for a ligand according to the invention and 4.
  • a first viral particle that displays a wild-type envelope protein i.e., an amphofropic envelope protein
  • a mutant envelope protein i.e., a mutant amphofropic envelope protein
  • a target cell that expresses a viras receptor (X) for the wild type and mutant viral envelope protein and a decoy receptor (Y) for a ligand according to
  • a fusion protein comprising a viral receptor binding moiety or RBD that binds receptor X and a ligand that binds decoy receptor (Y).
  • downmodulation of receptor Y causes an increase in the delivery of the second transferable label and a decrease in the delivery of the first transferable label.
  • Upmodulation of receptor Y causes an increase in the delivery of the first transferable label and a decrease in the delivery of the second transferable label.
  • the invention also provides for viral particles that display viral envelope proteins that bind to a viral receptor that is not recognized by the viral binding moiety of the fusion protein. These viral particles comprise a different transferable label from a viral particle that displays viral envelope proteins that bind to the same viral receptor as the viral binding moiety of the fusion protein.
  • the ability of a candidate upmodulator to increase or decrease the cell surface availability of a target cell surface receptor is determined by comparing the amount of delivery of the two different transferable labels.
  • the invention also provides for viral particles expressing an envelope glycoprotein that exhibits impaired ability (e.g. at least 1% less than, preferably 5-25% less than, and more preferably 25-100% less than a viral particle expressing a wild-type envelope glycoprotein, as determined in a binding assay as described above) to effect virus-cell fusion.
  • an envelope glycoprotein that exhibits impaired ability e.g. at least 1% less than, preferably 5-25% less than, and more preferably 25-100% less than a viral particle expressing a wild-type envelope glycoprotein, as determined in a binding assay as described above
  • Binding of a fusion protein to the virus receptor according to the invention i.e., expressed on a target cell, will enhance gene delivery from a virus particle expressing a glycoprotein that exhibits an impaired ability to trigger subsequent fusion.
  • a viral particle of the invention also includes a viral particle expressing a viral envelope protein that is a chimeric protein comprising a region derived from a first envelope glycoprotein and at least a second region derived from a second viral envelope protein (for example as disclosed in Lavillette et al., 2001, supra)
  • Viral particles of the invention comprise a transferable label.
  • Retro viruses and refroviral vectors can efficiently transfer genes into eukaryotic cells. Gene delivery is initiated by retroviral envelope glycoproteins (Env), which mediate attachment to cell-surface viral receptors and subsequent fusion between viral and cellular membranes. Amphofropic retroviruses bind to a receptor which is present on most mammalian cells and are therefore used for gene transfer to human target cells. Ecotropic retroviruses bind to a receptor which is present on murine and some other rodent cells, but is absent from human cells.
  • Env retroviral envelope glycoproteins
  • Env proteins are homooligomers containing two to four heterodimeric subunits (Doms et al, 1993, Virology, 193(2), 545-62). In the case of murine leukemia viras (MLV), the envelope proteins, are homotrimers (Kamps et al, (1991) Virology. 184:687-94).
  • Each Env subunit comprises gp70, a surface glycoprotein (SU) which mediates attachment to a cellular receptor and gpl5E, a transmembrane protein (TM) which is responsible for fusion between viral and cellular membranes.
  • SU surface glycoprotein
  • TM transmembrane protein
  • the human immunodeficiency virus HIV envelope glycoproteins g l20 and gp41
  • Fusion of HIV with the cellular membrane depends on the sequential interaction of gpl20 and gp41 with the cellular receptor CD4 and co-receptors from the GPCR chemokine receptor family.
  • 4070A (amphofropic) MLV Env interacts with Pit-2 cellular receptor (Miller et al, 1994, Proc. Natl. Acad. Sci. USA, 91(1)78-82); Van Zeijl et al, (1994) Proc. Natl. Acad. Sci. U S A. 91 : 1168-72) which is present on mouse and human cells.
  • Moloney (ecotropic) MLV Env interacts with CAT-1 cellular receptor (Albritton et al, 1989, Cell, 57(4), 659-666), which is present on mouse cells but not human cells. Both Pit-2 and CAT-1 receptors have multiple transmembrane regions.
  • ALV-A and ALV-B viras have been well characterized.
  • the cellular receptors of ALV-A and ALV-B viras are TVA and TVB, respectively. Both TVA and TVB have a single transmembrane region and a discrete extracellular domain (Young et al., 1993, J. Virol., 67(4), 1811-6).
  • the extracellular domain of TVA has been shown to support viral infection when placed at various distances from the cell surface membrane (Zingler et al., 1995, J. Virol., 69(7), 4261-6); Rong & Bates, (1995) J. Virol. 69:4847-53; Zingler et al, (1995) J. Virol. 69:4261-6.
  • envelope proteins of adenovirus, togavirus, rhabdoviras, and refroviras families, as well as envelope proteins from enveloped viruses such as paramyxoviras and orthomyxoviras are useful envelope proteins according to the invention.
  • Murine leukemia viras envelope proteins, such as the 4070A and Moloney MLV envelope proteins, are particularly useful.
  • viruses whose envelope proteins conform to this pattern (examples include other retroviruses, orthomyxoviruses and paramyxovirases) should therefore be useful according to the invention. Possible advantages offered by other viruses include higher virus titers, higher mutation or recombination rates, ability to use different cell surface molecules as surrogate receptors, ease and speed of viras production, higher density and absolute number of non- viral polypeptides incorporated per virion.
  • the invention provides for fusion proteins comprising a viral receptor binding moiety and a ligand.
  • a viral receptor binding moiety can be any viral envelope protein, including but not limited to all of the viral envelope proteins described in the section entitled, "Envelope proteins of virus particles", that can bind specifically to the appropriate viral receptor.
  • a receptor binding moiety is also a portion of a viral envelope protein or an RBD that can bind specifically to the corresponding viral receptor.
  • a "portion of a viral envelope protein" or an RBD (receptor binding domain) refers to the smallest portion of the viral envelope protein that can fold into a 3 -dimensional structure and bind to its cognate virus receptor with a Kd of 1 OO ⁇ M or lower (i.e., 50 ⁇ M, 1 ⁇ M, .5 ⁇ M, etc .).
  • a viral envelope protein or a portion of a viral envelope protein can contain one or more amino acid substitutions that do not prevent binding to the cognate viral receptor.
  • a ligand includes any moiety that is capable of associating with or binding to a receptor.
  • a ligand can be any molecule that binds to a receptor (e.g. EGF, PDGF etc...) or a ligand may be an antigen or antibody, an enzyme or any polypeptide that can bind specifically to a receptor.
  • a fusion protein may comprise a linker that separates the viral receptor binding moiety and the ligand.
  • a linker is a peptide that comprises from 0-20 or more (i.e., 21, 25, 30, 40, 50, 100 etc...) amino acids in length.
  • the linker may optionally include a specific subsfrate sequence for a protease such that the binding domains of the fusion protein may be physically separated upon cleavage by a protease.
  • a fusion protein is provided as a purified protein that is prepared and purified (according to methods well known in the art) prior to incubation with a viral particle and a target cell.
  • the fusion protein can be added to the target cells at the same time as the viras particle.
  • the fusion protein is provided by a cell line transfected with a fusion protein expression plasmid and added to a mixture of viral particles and cells.
  • the fusion protein can be provided by a target cell that has been transfected with the fusion protein expression plasmid.
  • the invention provides for a fusion protein comprising a single chain antibody or wherein at least a portion of the fusion protein is a single-chain antibody.
  • a viral particle according to the invention comprises a transferable label to facilitate identification and selection of target cells that have been infected by a viral particle.
  • a transferable label can be any label whose presence can be detected in the target cell upon fusion of the viral particle and the target cell membrane.
  • the fransferable label is a gene encoding a selectable marker or a reporter gene.
  • Transferable labels also include proteins that can be detected using immunochemical techniques employing antibodies that specifically bind to the protein.
  • the fransferable label can be an enzyme that catalyzes a reaction wherein a colored product or luminescence is produced from a chromogenic substance or a nucleic acid molecule encoding a such an enzyme.
  • a chromogenic substrate produces a colored product or a product that can be detected by the absorption of light or by fluorescence.
  • the invention also provides for a transferable label comprising a fluorescent polypeptide (e.g., green fluorescent protein (GFP)) or a nucleic acid molecule encoding a fluorescent polypeptide.
  • a transferable label comprising a fluorescent polypeptide (e.g., green fluorescent protein (GFP)) or a nucleic acid molecule encoding a fluorescent polypeptide.
  • the transferable label may also be radioactive or an otherwise-modified lipid or non-lipid component of a viral particle, or a cytotoxic agent such as perform, ribonucleases, and exotoxins.
  • Genes encoding selectable markers are preferably antibiotic-resistance genes, such as a neomycin, puromycin, or phleomycin resistance genes.
  • Reporter genes encode a detectable product, such as galactosidase, luciferase, glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), or chloramphenicol acetylfransferase (CAT). Many such genes are known in the art.
  • Polynucleotides encoding selectable markers or reporter gene products can be fransfected into target cells according to methods well known in the art (See Sambrook et al., supra, Ausubel et al., supra) for subsequent packaging into viral display packages. Methods of preparing a viral display package comprising a gene encoding a selectable marker or reporter gene are described in U.S. Patent 5,723,287 and WO 97/03357.
  • Viral display packages comprising other fransferable labels useful according to the invention are prepared using an alternative packaging cell line.
  • TELCeB6 cells package ⁇ - galactosidase, as they stably express the ⁇ -gal gene that had been previously inserted in a refroviral packaging plasmid (essentially flanked by long terminal repeats and with a refroviral packaging signal upstream of the gene), hi order to make a new packaging cell line, an appropriate cell line (e.g., TE-FLY cells), are fransfected with a retroviral packaging plasmid containing the reporter gene of interest, and stable clones are selected.
  • TE-FLY cells e.g., TE-FLY cells
  • the presence or absence of a fransferable label in a target cell can be detected by any means known in the art which is sensitive to the particular label employed.
  • the label is a fluorescent molecule, or a nucleic acid encoding a fluorescent molecule such as a fluorescent polypeptide
  • a suitable method of detection would be by fluorescence specfroscopy of a cell suspension, fluorescence microscopy, or use of a fluorescence-activated cell sorter (FACS; see e.g., WO 97/04311; Herzenberg (1978) in W. Knapp et al, eds., Immunofluorescence and related staining techniques, Elsevier, Amsterdam; and D.R. Parks & L.A.
  • Methods of detecting labels useful according to the invention are disclosed in Sambrook et al., supra and Ausubel et. al., supra.
  • the invention provides methods for identifying up or downmodulators of a cell surface receptor that specifically binds to a ligand.
  • Receptors of the invention include, but are not limited to, G protein-coupled receptors, tyrosine kinase receptors, serine-threonine kinase receptors, ion channels, transporters and immune recognition receptors.
  • the receptors can be constitutively active or wild-type (i.e., non- constitutively active).
  • constitutively active mutant (CAM) receptors include a CAM of a G-protein coupled receptor.
  • Receptors useful according to the invention include M5 muscarinic receptor, Bl bradykinin receptor, PTH/PTHrP receptor, CXCR2 receptor and 5- HT2A receptor are presented in the following publications, herein incorporated by reference:
  • a receptor acts to receive a specific extracellular signal and transmits its presence across the cell membrane to activate an infracellular biochemical response.
  • the signal molecule is believed to stabilize the receptor in a different three-dimensional shape (conformation), which in turn causes that infracellular part of the receptor to interact with a transducing protein.
  • the transducing protein participates in further intracellular biochemical activity.
  • This further activity is designated a "second messenger” system and typically includes second messengers such as 3'5'-cyclic AMP (cAMP), 3'5'-cyclic GMP (cGMP), 1,2-diacylglycerol, inositol 1,4,5,- triphosphate, and Ca 2+ , or a signalling cascade of phosphorylation in the case of kinase receptors.
  • the second messengers in turn activate one or more enzymatic systems.
  • An intracellular response can be measured using assays for the second messengers; these assays are well known in the art.
  • a receptor according to the invention preferably is substantially intact and has normal or near normal pharmacology.
  • a receptor according to the invention can be tagged.
  • tagged refers to a receptor that is covalently or non-covalently attached to a tag or detectable moiety including but not limited to his, hemagglutinin, myc, FLAG, GST or calmodulin binding peptides.
  • a tagged receptor can be used in an assay comprising a fusion protein, wherein the fusion protein comprises a viral receptor binding moiety and a ligand (for example an antibody) that is capable of binding specifically to the tag.
  • ligand for example an antibody
  • the receptor is expressed in a preferred cell line resulting in standardization between assay screens performed in identical cell lines.
  • a receptor upmodulator or downmodulator according to the invention can be an agonist that increases or decreases cell surface expression of a receptor, an antagonist, an inverse agonist, a compound that is not an agonist or inverse agonist, and increases or decreases cell surface expression of a receptor, a protease inhibitor or a protease activator.
  • the candidate receptor modulator may be a synthetic compound, or a mixture of compounds, or may be a natural product (e.g. a plant extract or culture supernatant).
  • a candidate receptor modulator according to the invention includes a small molecule that can be synthesized, a natural extract, peptides, proteins, carbohydrates, lipids etc...
  • Candidate modulator compounds from large libraries of synthetic or natural compounds can be screened. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich
  • Useful compounds may be found within numerous chemical classes. Useful compounds maybe organic compounds, or small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, preferably less than about 750, more preferably less than about 350 daltons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents.
  • peptide agents may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • an unnatural amino acid such as a D-amino acid, particularly D-alanine
  • a useful concentration of a candidate modulator according to the invention is from about l ⁇ M to about 60 ⁇ M or more (i.e., lOO ⁇ M, lmM, lOmM, lOOmM, 1M etc).
  • the primary screening concentration will be used as an upper limit, along with nine additional concentrations, wherein the additional concentrations are determined by reducing the primary screening concentration at half-log intervals (e.g. for 9 more concentrations) for secondary screens or for generating concentration curves.
  • the cell used in an assay of the invention may be a eukaryotic cell such as a yeast, mammalian, or insect cell; however a prokaryotic cell, such as a bacterial cell may also be used. Most preferably the cell will be a mammalian cell line.
  • an upmodulator or a downmodulator is detected when a virus particle infects a target cell expressing the cognate viral receptor and label is transferred from the viral particle to the target cell.
  • a receptor upmodulator or downmodulator which increases or decreases receptor number or affinity and, consequently increases viral particle binding to a target cell and label transfer from the viral particle to the target cell can be detected by comparing the amount of transferable label in a cell in the presence or absence of the modulator.
  • the method of contacting a viral particle, a cell comprising a viral receptor and a fusion protein comprising a viral receptor binding moiety and a ligand will typically involve incubating the cell in a medium containing a sufficient concentration of the viral particle and the fusion protein, and for a sufficiently long incubation period, so that at least about 0.1, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99 percent of the cells will be infected and will take up the transferable label (See U.S. Patent 5,723,287 and WO 97/03357).
  • the invention provides for an incubation period that is 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 12 hours, 24 hours, or 48 hours or more long.
  • the incubation period of the compound with the cells and the viras with the cells can be similar.
  • the compound is incubated with the cells, and the fusion protein is added (if it is not already produced by the target cells). Virus is added last.
  • the invention provides for assays wherein the concentration of fusion protein is lfM, lOfM, lOOfM, lpM, lOpM, lOOpM, InM, lOnM, lOOnM, l ⁇ M, lO ⁇ M, lOO ⁇ M, lmM, lOmM, lOOmM or more, i.e., lmM, lOOmM, 1M.
  • the label After the transferable label has been taken up by the cells the label can be detected and compounds capable of increasing or decreasing receptor levels or affinity are identified.
  • the method of label detection should be appropriately chosen based on the particular label. Some labels will be detectable immediately after or shortly after contacting the cell with the viral display package. Other labels, such as nucleic acid molecules encoding a detectable product, will require a period of time, usually minutes to hours, before sufficient product accumulates in the host cell to enable detection.
  • the duration of contact between the virus particle, the cell and the fusion protein, the temperature and the biochemical composition (e.g., pH or salt concentration) of the medium in which the contact takes place can be varied and optimized by routine trial and error. After sufficient time has elapsed for transfer of the label from the viras particle to the cell and, where the label is a gene, for its expression in the cell, the presence of the label in the cell is detected qualitatively or, preferably, quantitatively.
  • a washing step can optionally be performed prior to the step of detecting, in order to remove any label that was not taken up by the cells.
  • the label is a fluorescent protein
  • the cells can be washed in a physiological saline solution or cell culture medium and then observed by fluorescence microscopy. Comparison of the fluorescence and visible light images of a field of cells will reveal cells that display the receptor as those with little or no fluorescence.
  • Receptor regulation mechanisms include such mechanisms as intemalization of receptors and stabilization of constitutively active mutant receptors. Intemalization of receptors, including wild- type and mutant receptors may occur by, for example, direct agonist activation (homologous desensitization) or heterologous desensitization. See Figure 3. Stabilization of receptors, including constitutively active receptors, such as G protein-coupled receptors is another receptor regulatory mechanism that can be monitored by the assays of the invention. For example, where receptors are unstable at the cell surface and consequently expressed at low levels, inverse agonists and agonists stabilize the receptor and result in an elevation of the cell surface receptor number. See Figures 4 and 5. For example, a constitutively active mutant adrenergic receptor is stabilized by several inverse agonists. See Figure 6.
  • the invention provides methods of screening for a receptor modulator that functions via any of the above-recited receptor regulation mechanisms.
  • Assays of the invention can be used to screen for candidate compounds or substances that have agonist, antagonist, or inverse agonist activity.
  • the assays of the invention can also be used to screen for candidate protease inhibitors.
  • an assay for identifying a receptor upmodulator comprises incubating a viral particle comprising a fransferable label and capable of binding to a cognate viral receptor, a fusion protein comprising a viral receptor binding moiety and a ligand, wherein the viral receptor binding moiety competes with the viral particle for binding to the viral receptor, and a target cell that expresses a viral receptor and does not express, or expresses low levels of, a cell surface receptor capable of binding to the ligand component of the fusion protein.
  • a viral particle is mixed with a cell and a fusion protein according to the invention. It may be sufficient, for example, to pre-incubate the candidate receptor modulator with a viral particle or a cell or both. If a pre-incubation step is used, a particle or a cell or both will optionally be washed to remove unbound candidate receptor modulator prior to the performance of the assay.
  • the invention provides an assay to detect an increase or decrease in the numbers/affinity of a cell surface receptor.
  • the invention also provides an assay to detect a compound that increases or decreases the number/affinity of a cell surface receptor.
  • the assays of the invention can be used as follows:
  • Detection of substances that upregulate receptors to alleviate a disease state e.g. a compound that upregulates TNF receptors in certain tumor types or a compound that upregulates GLUT4 in adipocytes and/or muscle cells for treatment of diabetes.
  • kits to assay for up or downregulation of certain receptors e.g. as a diagnostic tool to profile the cell-surface receptors on tumor cells from different patients.
  • This example describes an assay for detecting compounds that increase the cell-surface numbers of tumor necrosis factor receptor II.
  • the assay requires:
  • Amphofropic Env SU portion-single chain anti-TNFRIJ fusion protein hereafter designated ASU-anti-TNFR ⁇
  • Viras supernatant is collected from TELCeB6 cells that have been transfected with an amphofropic MLV envelope expression plasmid (i.e., as described in Cosset et al., 1995, J. Virol., 69(12), 7430-6). Supernatant is passed through a 0.45 ⁇ m filter prior to use. The resultant viral particles display wild-type amphofropic envelope proteins and encapsulate ⁇ -galactosidase reporter genes under the control of Moloney MLV promoters.
  • TE671 cells (ATCC CRL-8805) are fransfected with an ASU-anti-TNFRfl expression vector under the control of a CMV promoter in the vector pRcCMV2 (Invifrogen).
  • the ASU-anti-TNFRII sequence is generated by PCR assembly of cDNA fragments coding for 4070A SU and anti-TNFRII single chain antibody, then inserted into the multiple cloning site of pRc/CMV2.
  • Stable chimera-expressing cells are then pooled following neomycin selection at 500 ⁇ g/ml for 2 weeks. Clones of cells with high levels of expression are selected by FACS sorting and are expanded. Supernatant is harvested from these cells as in (i), above.
  • the target cell line is rat C6 glioma (ATCC CCL-107).
  • Candidate upmodulators are derived from a chemical library. Rat C6 glioma cells are dispensed into 96-well tissue culture plates at 20,000 cells per well. One candidate upmodulator is then added per well (final concentration: 20 ⁇ M) and the cells are incubated overnight at 37°C/5% CO 2 . On the following day, virus supernatant (i) and ASU-anti-TNFRII supernatant (ii) are added to the cells at a ratio of 1:10 (v/v). The cells are incubated for an additional 4 hours. The cells are then washed with PBS and fresh medium is dispensed into each well, prior to overnight incubation at 37°C/5% CO 2 .
  • the cells are washed and lysis/development buffer is added containing ⁇ -galactosidase substrate (CPRG). After a final incubation of 90 minutes, the plates are read in a specfrophotometer at 578 nm. Those samples producing an elevated optical density reading, as compared to untreated cells, correspond to cells that have been treated with an upmodulator that increases the cell surface level of TNFRH.
  • CPRG ⁇ -galactosidase substrate
  • This example describes an assay for detecting an upmodulator that is an 'inverse agonist' that increases the stability (and hence cell surface availability) of 'tagged' constitutively active mutant para-thyroid hormone (PTH)/parathyroid hormone related peptide (PTHrP) receptors.
  • PTH para-thyroid hormone
  • PTHrP parathyroid hormone related peptide
  • the assay requires:
  • ARBD-anti-FLAG the nucleotide sequence coding for amphofropic (4070A) receptor-binding domain (corresponding to the first 208 amino acids) is fused to the sequence coding for a single-chain antibody that binds the FLAG peptide (DYKDDDDK), via a sequence coding for a flexible linker (GGGGS) in a pPICZ vector.
  • the vector is then used to fransform Pichia pastoris and fusion protein is purified according to manufacturers instructions (Invitrogen); (iii) Target TE671 cells expressing constitutively active PTH/PTHrP receptors tagged at the N-terminus with the 'FLAG' peptide (DYKDDDDK) (the cDNA coding for constitutively active PTH PTHrP receptors is inserted into the pIRESneo2 expression vector (Clontech). The sequence coding for the FLAG tag is then inserted at the 5' end of the receptor gene (e.g. by insertion of annealed oligos following appropriate plasmid digestion).
  • the resultant plasmid is then fransfected into TE671 cells and neomycin selection is performed for 2 weeks at 500 ⁇ g/ml. Following selection, single-cell clones are seeded into 96-well plates by FACS. The clones are grown up and characterized for expression of the tagged receptors by Western blot with an anti- FLAG antibody. The clone with the highest level of expression is subsequently used in the assay) ; and
  • TE671 cells are fransfected with an ARBD-anti-FLAG expression vector under the control of a CMV promoter. Clones of cells with high levels of expression are detected by FACS sorting and expanded. Supernatant is harvested from these cells as in Example 1.
  • TE671 cells expressing FLAG-tagged constitutively active PTH/PTHrP receptors are dispensed into 96-well tissue culture plates at 20,000 cells per well. The cells are then incubated overnight at 37°C/5% CO 2 . On the following day, one candidate upmodulator is added per well to a final concentration of lO ⁇ M and the cells incubated for 2 hours at 37°C/5% CO 2 .
  • Virus supernatant (i) and purified ARBD-anti-FLAG fusion protein (at a final concentration of l ⁇ M) (ii) are then added to the cells at a ratio of 1 : 10 (v/v). The cells are incubated for an additional 4 hours.
  • This example describes an assay for detecting compounds that increase the cell surface availability and/or affinity of EGF (epidermal growth factor) receptors.
  • the assay requires:
  • Target cells expressing low levels of EGFRs at the cell surface e.g., 3T3 cells
  • TE671 cells are fransfected with an EXA1-ST expression vector under the control of a Moloney viral promoter.
  • the expression vector is constructed by replacing the 4070A envelope protein sequence in pFB MoSALF with a construct encoding 1) EGF, 2) a factor Xa cleavage site (IEGR) and 3) the SU portion of 4070A envelope protein. Pools of stable producers are selected following freatment with phleomycin at 50 ⁇ g/ml for 2 weeks. Clones of cells with high levels of EXA1-ST secretion are selected for their capacity to express EXA1-ST that can be detected in a binding assay that uses A431 cells and an antibody against the fusion protein. Supernatant is harvested from these cells as described above.
  • 3T3 cells expressing low levels of EGF receptors are dispensed into 96-well tissue culture plates at 20,000 cells per well. The cells are then incubated overnight at 37°C/5% CO 2 . On the following day, one test compound is added per well to a final concentration of lO ⁇ M and the cells are incubated for 10 hours at 37°C/5% CO 2 . EXA1-ST supernatant and then viras supernatant are then added to the cells at a ratio of 1 : 10 (v/v). The cells are incubated for a further 4 hours. The cells are then washed with PBS and fresh medium is dispensed into each well, prior to overnight incubation at 37°C/5% CO 2 .
  • the cells are washed and lysis/development buffer is added containing ⁇ -galactosidase chemiluminescent 1,2-dio ⁇ ethane subsfrate (Galacton-StarTM). After a final incubation of 10 minutes, the plates are read in a luminometer. Those samples producing an elevated light emission reading, as compared to untreated cells, correspond to cells that have been treated with an upmodulator of the EGF receptor.
  • Figure 2 shows the difference in infectivity of amphofropic lacZ retroviral particles for cells expressing low versus high levels of EGF receptors, after pre-incubation in the absence of RBD fragment (Control), in the presence of wild-type amphofropic RBD (A2-ST), or in the presence of EGF fused to amphofropic RBD via a factor Xa cleavable linker (EXAl -ST).
  • PiT-2 amphofropic receptor-positive cells that express a low level (NTH-3T3 mouse cells) or a high level (A431 human cells) of EGF receptors were used.
  • This example describes the use of an assay to detect compounds that decrease the cell surface availability and/or affinity of EGF (epidermal growth factor) receptors.
  • the assay requires:
  • Amphofropic MLV viral particles containing a reporter gene and incorporating envelope proteins in which Histidine residue 6 has been deleted envelope proteins comprising a deletion of histidine residue 6 are generated in the envelope expression plasmid by methods known in the art, for example PCR mutagenesis
  • Target cells expressing high levels of EGFRs at the cell surface eg, HT-1080 cells
  • HT-1080 cells Target cells expressing high levels of EGFRs at the cell surface
  • Virus supernatant is collected from TELCeB6 cells that have been transfected with an amphofropic MLV mutant envelope [delHis ⁇ ] expression plasmid. Supernatant is passed through a 0.45 ⁇ m filter prior to use. The viral particles produced display wild-type amphofropic envelope proteins and encapsulate ⁇ -galactosidase reporter genes under the control of Moloney MLV promoters .
  • TE671 cells are fransfected with an EXAl -ST expression vector under the control of a CMV promoter. Clones of cells with high levels of EXAl -ST secretion are selected for their capacity to express EXAl -ST that can be detected by a binding assay that uses A431 cells and an antibody against the fusion protein. Supernatant is harvested from these cells as described above.
  • HT-1080 cells expressing high levels of EGF receptors are dispensed into 384- well tissue culture plates at 5,000 cells per well. The cells are then incubated overnight at 37°C/5% CO 2 . On the following day, one candidate downmodulator is added per well to a final concentration of lO ⁇ M and the cells incubated for 4 hours at 37°C/5% CO 2 . EXAl -ST supernatant and then virus supernatant are then added to the cells at a ratio of 1:10 (v/v). The cells are incubated for an additional 4 hours. The cells are then washed with PBS and fresh medium is dispensed into each well, prior to overnight incubation at 37°C/5% CO 2 .
  • the cells are washed and lysis/development buffer is added containing ⁇ -galactosidase chemiluminescent 1,2-dioxethane substrate (Galacton-StarTM). After a final incubation of 10 minutes, the plates are read in a luminometer. Those samples producing an elevated light emission reading, as compared to untreated cells, correspond to cells treated with a downmodulator of the EGF receptor.
  • This example describes an assay for detecting a substance that increases the cell surface levels of Glut4 glucose transporters.
  • This example describes a fusion protein, according to the invention, produced in a baculoviras system, that comprises a mutant virus RBD that lacks the first six residues of the RBD.
  • the invention also describes a target cell receptor that is tagged in an exofacial loop (i.e., not on the N or C-terminus).
  • the envelope protein that is expressed on the surface of the virus particle is a chimera comprising the SU portion of the 4070 (amphofropic) envelope protein and the TM portion of the Moloney (ecofropic) envelope protein, designated TMMO (as described in Lavilette et al. 2001, supra).
  • the assay requires :
  • [dell-6] amphofropic receptor binding domain-cahnodulin fusion protein (hereafter designated [dell-6]ARBD-calmodulin), produced as follows:
  • the nucleotide sequence coding for del[l-6] amphofropic (4070A) receptor-binding domain (corresponding to amino acids 7 to 208) is fused to the sequence coding for calmodulin via a sequence coding for a flexible linker (GGGGS) in a pMelBac vector.
  • the vector is then used to produce a fusion protein from insect SF9 cells that is subsequently purified according to manufacturers instructions (Invifrogen).
  • Target HIR 3T3/Glut4 cpep cells Mouse NTH 3T3 cells that express high levels of human insulin receptor, Ram-1 (4070A/amphofropic receptor) and C-peptide- tagged Glut 4 receptor:
  • H R 3T3 cells (See Lammers et al., 1989, EMBO Journal 8(5), 1369-1375) are fransfected with the vector pcDNA 3.1/ Glut4 cpep (pcDNA 3.1 vector (Invifrogen) with chimaeric Glut 4 sequence inserted under the control of a CMV promoter:
  • the sequence coding for a calmodulin-binding peptide (AAARWKKAFIAVSAANRFKKIS:
  • Stable clones are selected by growing the cells in the presence of neomycin for 2 weeks at 500 ⁇ g/ml. The clone with the highest level of expression is subsequently used (determined by Western blotting) and
  • IR/3T3 cells expressing Glut4 cpep are dispensed into 96-well tissue culture plates at 20,000 cells per well. The cells are then incubated overnight at 37°C/5% CO 2 . On the following day, one candidate upmodulator is added per well to a final concentration of lO ⁇ M and the cells incubated for 2 hours at 37°C/5% CO 2 . Recombinant [dell-6]ARBD-cahnodulin fusion protein (ii) is then added to each well to give a final concentration of l ⁇ M and the cells incubated for 30 minutes at 37°C. 50 ⁇ l virus supernatant (i) is subsequently added to the cells. The cells are incubated for an additional 4 hours.
  • the cells are then washed with PBS and fresh medium is dispensed into each well, prior to overnight incubation at 37°C/5% CO 2 .
  • the cells are washed and lysis/development buffer is added containing ⁇ -galactosidase chemiluminescent 1,2-dioxethane substrate (Galacton-StarTM).
  • lysis/development buffer is added containing ⁇ -galactosidase chemiluminescent 1,2-dioxethane substrate (Galacton-StarTM).
  • the plates are read in a luminometer. Those samples producing an elevated light output, as compared to untreated cells, correspond to cells that have been treated with an upmodulator that increases the cell surface levels of Glut4 glucose transporter.

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Abstract

L'invention porte sur un système des bio-essais servant à identifier un modulateur de récepteur consistant: (i) à mettre en contact un marqueur transférable avec un candidat modulateur de récepteur, une protéine hybride et un ligand, et une cellule cible exprimant un récepteur de ligand de surface de cellule, et (ii), en variante, à mettre en contact une particule virale, comportant une protéine mutante d'enveloppe, pouvant se fixer à un récepteur de virus mais s'avérant incapable de pénétrer dans une cellule, et comportant en outre un marqueur transférable, avec une protéine hybride comprenant un fragment se fixant à un récepteur de virus, et un ligand, et une cellule cible exprimant un récepteur de surface de cellule pouvant se fixer au ligand de la protéine hybride. La fixation de la protéine hybride à un récepteur de virus de la cellule cible inhibe ou active le transfert du marqueur transférable de la particule de virus à la cellule.
PCT/IB2002/002990 2001-05-18 2002-05-16 Essai de fixation de recepteurs modifies WO2002095400A2 (fr)

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US9777044B2 (en) 2003-05-02 2017-10-03 Centre National De La Recherche Scientifique (Cnrs) GLUT-1 as a receptor for HTLV envelopes and its uses

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US9777044B2 (en) 2003-05-02 2017-10-03 Centre National De La Recherche Scientifique (Cnrs) GLUT-1 as a receptor for HTLV envelopes and its uses
US8349571B2 (en) 2004-09-24 2013-01-08 Cellaura Technologies Limited High content screening
WO2010079208A1 (fr) * 2009-01-09 2010-07-15 Centre National De La Recherche Scientifique Nouveaux ligands de liaison de récepteur, et leur utilisation dans la détection de cellules ayant un intérêt biologique
US20130203080A1 (en) * 2009-01-09 2013-08-08 Universite Montpellier 2 New receptor binding ligands, their use in the detection of cells with biological interest
US9791435B2 (en) 2009-01-09 2017-10-17 Centre National De La Recherche Scientifique Receptor binding ligands, their use in the detection of cells with biological interest

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