WO2001062971A1 - Detection de recepteurs de ligands orphelins par presentation retrovirale - Google Patents

Detection de recepteurs de ligands orphelins par presentation retrovirale Download PDF

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Publication number
WO2001062971A1
WO2001062971A1 PCT/US2001/005388 US0105388W WO0162971A1 WO 2001062971 A1 WO2001062971 A1 WO 2001062971A1 US 0105388 W US0105388 W US 0105388W WO 0162971 A1 WO0162971 A1 WO 0162971A1
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receptor
ligand
cells
cell
oφhan
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PCT/US2001/005388
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English (en)
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Mark P. Chadwick
Gerard J. Mcgarrity
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Cambridge Drug Discovery, Ltd.
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Priority to AU2001238533A priority Critical patent/AU2001238533A1/en
Publication of WO2001062971A1 publication Critical patent/WO2001062971A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the invention is related to the area of cell surface receptors and their ligands. In particular it is related to methods of identifying, enriching for, and cloning cells which display a particular receptor, such as a receptor for an orphan ligand.
  • Ligand-receptor interactions are central to most regulatory phenomena in biology and medicine. Many drugs function by modulating ligand-receptor interactions or by interacting with receptors for endogenous ligands. A large number of ligands and their cognate receptors have been identified and characterized. However, many ligands or candidate ligands exist for which no receptor is known. These "orphan ligands" can serve as the basis for the development of new drugs, whose utility depends on knowledge of the corresponding receptor. The sequencing of the human genome has revealed new orphan ligands. These include peptide hormones, neurotransmitters, and chemokines which are known to bind to seven-transmembrane- domain receptors. Hinuma et al. (1999) J. Mol.
  • G protein-coupled receptors which are important targets of drug development. Many G protein-coupled receptors are orphan receptors that have been discovered from new genomic data, but whose cognate ligand and physiological role is unknown. Wilson et al. (1998) Br. J. Pharmacol. 125: 1387-1392. In many cases, knowledge of the function and utility of an orphan ligand as well as the discovery of new drugs based on it cannot go forward without identification of a corresponding receptor, which may be an orphan receptor or a known receptor.
  • One embodiment of the invention provides a method of identifying a cell that displays a receptor.
  • a cell is contacted with a viral display package comprising a fusion protein and a transferable label.
  • the fusion protein comprises a viral envelope protein and an o ⁇ han ligand that binds to its receptor, when it is present on the cell surface.
  • the cell comprises receptors for the viral envelope protein and may, or may not comprise receptors for the o ⁇ han ligand. Binding of the displayed o ⁇ han ligand to its receptor inhibits transfer of label to the cell via interaction of the viral envelope protein with the virus receptor. Transferable label is detected in the cell, whereby the absence of transferable label in the cell indicates that the cell displays the receptor for the o ⁇ han ligand.
  • the transferable label is a chromogenic polypeptide, a gene encoding a chromogenic peptide, a cytotoxic agent, a gene encoding a cytotoxic agent, or a gene encoding antibiotic resistance.
  • a preferred chromogenic peptide is green fluorescent protein.
  • Preferred cytotoxic agents are perforin, ribonucicases, and cxotoxins.
  • a preferred viral display package is a retrovirus, which preferably is a lentivirus. It is also preferred that the cell is not proliferating. Furthermore, the cell preferably expresses a hetcrologous receptor, where the heterologous receptor is a virus receptor.
  • Another embodiment of the invention provides another method of identifying a cell that displays a receptor.
  • a cell is first contacted with a viral display package comprising a fusion protein and a transferable label.
  • the fusion protein comprises a viral envelope protein and an o ⁇ han ligand that binds to the receptor. Binding of the fusion protein to the receptor inhibits transfer of label to the cell. Transferable label is detected in the cell, whereby the absence of transferable label in the cell indicates that the cell displays the receptor for the o ⁇ han ligand.
  • the cell is contacted with the viral display package in the presence of a free form of the o ⁇ han ligand and transferable label is detected in the cell.
  • the cell is identified as displaying the receptor for the o ⁇ han ligand if transferable label is detected in the presence of the free form of the o ⁇ han ligand but less transferable label is detected in the absence of the free form of the o ⁇ han ligand.
  • Still another embodiment of the invention provides another method of identifying a cell that displays a receptor for an o ⁇ han ligand.
  • a cell is contacted with a viral display package in the presence of a free form of an o ⁇ han ligand that binds to the receptor.
  • the viral display package comprises a fusion protein and a transferable label.
  • the fusion protein comprises the o ⁇ han ligand and a viral envelope protein. Binding of the fusion protein to the receptor inhibits transfer of label to the cell.
  • the free form of the o ⁇ han ligand inhibits binding of the fusion protein to the receptor.
  • Transferable label is detected in the cell.
  • the cell is identified as displaying the receptor if transferable label is detected in the presence of the free form of the oiphan ligand but less transferable label is detected in the absence of the free form of the O ⁇ han ligand.
  • Another embodiment of the invention provides still another method of identifying a cell that displays a receptor.
  • a cell is contacted with a viral display package comprising a fusion protein and a transferable label.
  • the fusion protein comprises a viral envelope protein and an o ⁇ han ligand that binds to its receptor.
  • a protease cleavage site is located between the o ⁇ han ligand and the viral envelope protein. Binding of the fusion protein to the receptor for the o ⁇ han ligand inhibits transfer of label to the cell. Transferable label is detected in the cell.
  • the viral display package is treated with a protease that cleaves the o ⁇ han ligand from the viral envelope protein.
  • the cell is contacted with the protease-treated viral display package, and transferable label is detected in the cell.
  • the cell is identified as displaying the receptor if transferable label is detected when the protease-treated viral display package is used but less transferable label is detected when the untreated viral display package is used.
  • Yet another embodiment of the invention is a method of providing a clone of cells that display a receptor.
  • a plurality of cells is contacted with a viral display package, which comprises a fusion protein and a transferable label.
  • the plurality of cells has been previously transfected with a cDNA expression library comprising a gene for the receptor.
  • the fusion protein comprises a viral envelope protein and an o ⁇ han ligand that binds to its receptor. Binding of the fusion protein to the receptor for the o ⁇ han ligand inhibits transfer of label to the cell. Transferable label is detected in the cells, whereby the absence of transferable label in a cell indicates that the cell displays the receptor.
  • the transferable label is a cytotoxic agent or a gene encoding a cytotoxic agent, and the cytotoxic agent kills cells not displaying the receptor.
  • Preferred cytotoxic agents are perforin. ribonucleases, or exotoxins.
  • the transferable label results in a change in cell fluorescence, and the step of detecting is performed using a fluorescence-activated cell sorter which separates two or more distinct populations of cells based on the intensity of cell fluorescence. In some embodiments, two or more populations of cells are separated based on the density of the receptor.
  • the transferable label results in a change in cell luminescence.
  • a preferred viral display package is a retrovirus, which preferably is a lentivirus. It is also preferred that the cell is not proliferating. Furthermore, the cell preferably expresses a heterologous receptor, where the heterologous receptor is a virus receptor.
  • a further embodiment of the invention is a method of providing a clone of cells that are deficient in a receptor.
  • a plurality of cells is contacted with a viral display package comprising a fusion protein and a transferable label.
  • the fusion protein comprises a viral envelope protein and an O ⁇ han ligand that binds to its receptor. Binding of the fusion protein to the receptor for the o ⁇ han ligand inhibits transfer of label to the cell via interaction of the viral envelope protein with the virus receptor. Transferable label is detected in the cells, whereby the presence of transferable label in a cell indicates that the cell is deficient in the receptor.
  • One or more cells that are deficient in the receptor are cultured to form a clone of cells that are deficient in the receptor.
  • the transferable label is a gene encoding a selectable marker. In other embodiment the transferable label is a chromogenic polypeptide, a gene encoding a chromogenic peptide, or a gene encoding antibiotic resistance.
  • Still another embodiment of the invention is a method of enriching a population of cells with cells that display a ligand receptor.
  • a population of cells is contacted with a viral display package comprising a fusion protein and a transferable label.
  • the fusion protein comprises a viral envelope protein and an o ⁇ han ligand that binds to their respective receptors receptor. Binding of the displayed ligand to the ligand receptor inhibits transfer of label to the cells via interaction of the viral envelope protein with the virus receptor. Transferable label is detected in the cells, whereby the absence of transferable label in a cell indicates that the cell displays the ligand receptor.
  • the population of cells is cultured and becomes enriched with cells that display the ligand receptor.
  • the transferable label is a cytotoxic agent or a gene encoding a cytotoxic agent, and the cytotoxic agent kills the cells not displaying the receptor.
  • a preferred viral display package is a retrovirus, which preferably is a lentivirus. It is also preferred that the cell is not proliferating. Furthermore, the cell preferably expresses a heterologous receptor, where the heterologous receptor is a virus receptor.
  • the invention thus provides the art with novel methods of identifying, screening, and cloning cells that display or do not display a receptor for an o ⁇ han ligand. as well as cells that possess a desired density of such a receptor.
  • An o ⁇ han ligand is a ligand for which no specific receptor has been identified as the target for that ligand.
  • An o ⁇ han ligand may bind to either a previously known receptor or an o ⁇ han receptor.
  • An o ⁇ han receptor is a receptor for which no specific ligand has been identified as binding to that receptor.
  • a "receptor” is a cell surface protein or glycoprotein that has a specific binding site for a ligand present in the extracellular medium.
  • a receptor for an o ⁇ han ligand may be either a previously known receptor or an o ⁇ han receptor.
  • a receptor for an o ⁇ han ligand can be present naturally on the target cell membrane, or it can be introduced into the target cell using standard molecular biological techniques such that it is expressed as a heterologous receptor on the cell surface.
  • a receptor can be part of a family of genetically related receptors whose gene sequences share some degree of homology and whose function may or may not be closely related.
  • Viral display packages are well known in the art (see, e.g., U.S. Patent 5,723,287). Viral display packages of the invention display either recombinant viral envelope proteins or chimeric viral envelope proteins on their surface. Production of viral display packages is taught, for example, in U.S. Patent 5,723,287, and in Chadwick et al., ( 1999) J. Mol. Biol. 285:485-494. Viral packaging cells such as Psi 2, TELCeB.6, or PA317, are conveniently used to produce viral display packages. The packaging cells comprise either a nucleic acid molecule that encodes a chimeric envelope protein or a nucleic acid molecule that encodes a recombinant envelope protein.
  • the cells producing the vector potentially could themselves be susceptible to the cytotoxic genes that the vector inco ⁇ orates. It is therefore preferred that the cytotoxic gene product is either inactive in the producer cells or is not made in the producer cells.
  • One potential means of addressing this issue is to put the cytotoxic gene under the control of a promoter that is induciblc by addition of an exogenous substance, such as the insect hormone ecdysone.
  • a cytotoxic agent could be used that required a component in the target cell (e.g. a receptor) that is not present in the producer cells.
  • Yet another alternative is to use the thymidine kinase gene as the cytotoxic gene. In this case, gancyclovir is added to the target cells as a means of selection, whereas gancyclovir is not added to the producer cells.
  • a cell "displays" a receptor if that receptor is expressed by the cell in its surface membrane and if a ligand binding site on the receptor is accessible from the extracellular medium.
  • a fusion protein is a protein comprising within its linear amino acid sequence one or more portions of another polypeptide, or portions of other polypeptides, and expressed from a nucleic acid construct created by the linkage of coding sequences from two or more genes to form a combined cistron.
  • the invention uses a fusion protein, also called a "chimeric envelope protein", as part of a viral display package.
  • That fusion protein is formed by linking the coding sequence for an o ⁇ han ligand or a known ligand to the coding sequence for a viral envelope protein, such that the C-te ⁇ uinus of the o ⁇ han ligand is expressed in covalent attachment to the N-terminus of the viral envelope protein.
  • An additional sequence of linking amino acids can, if desired, be included between the C-terminal amino acid of the o ⁇ han ligand and the N-terminal amino acid of the viral envelope protein.
  • Nucleic acid molecules encoding fusion proteins can be produced using recombinant DNA technology or can be synthesized using standard nucleic acid synthesis techniques.
  • a viral display package can also comprise a "transferable label" to facilitate identification and selection of target cells that have been infected by a viral display package.
  • a transferable label can be any label whose presence can be detected in the target cell upon fusion of the viral display package and the target cell membrane.
  • the transferable 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. Envelope proteins of adenovirus, togavirus, rhabdovirus, and retrovirus families, as well as from enveloped viruses such as paramyxovirus and orthomyxovirus, are useful in the chimeric envelope protein.
  • Murine leukemia virus envelope proteins such as the 4070A and Moloney MLV envelope proteins, are particularly useful for this pu ⁇ ose. It is important that the viral envelope protein is substantially intact, i.e. retains all its domains, to conserve post-translational processing, oligomerization, viral inco ⁇ oration, and fusogenic activities. However, certain alterations, such as mutations, deletions, or additions, can be made to the viral envelope protein which do not significantly affect these functions, and viral envelope proteins with such modifications are considered substantially intact. It is not necessary that an entire viral envelope protein be used. However, if only a portion of a viral envelope protein is included in the chimeric envelope protein, that portion must be able to mediate fusion between the viral display package and the surface membrane of the target cell.
  • 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.
  • the transferable label can be a chromogenic polypeptide or a nucleic acid molecule encoding a chromogenic polypeptide.
  • a chromogenic polypeptide is any polypeptide that can be detected by the abso ⁇ tion of light or by fluorescence.
  • cytotoxic agents include perforin, ribonucleases, and exotoxins.
  • a cytotoxic agent is any agent delivered to a cell that can result in death of the cell within an appropriate time period, e.g., within 1, 2, 5, 10, 24, 48, or 72 hours.
  • a cytotoxic agent delivered to a cell as part of a viral display package i.e., one which serves as a transferable label, can produce its toxic effect either directly or indirectly, for example by expression of a toxic protein or enzyme within the target cell.
  • Suitable cytotoxic agents include a gene encoding perforin (Liu et al. ( 1995) Immunol. Rev. 146: 145-75), a ribonuclease (Youle et al. ( 1993) Crit. Rev. Ther.
  • Drug Carrier Syst.10 1 -28
  • a bacterial toxin such as Pseudomonas exotoxin (Fitzgerald et al. ( 1993) Ann. N.Y. Acad. Sci. 685:740-745).
  • Genes encoding selectable markers are preferably antibiotic-resistance genes, such as a neomycin, puromycin, or phleomycin resistance gene.
  • Reporter genes encode a detectable product, such as galactosidase, luciferase, glucuronidase, green fluorescent protein (GFP), autofluorcscent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), or chloramphenicol acetyltransferase (CAT). Many such genes are known in the art.
  • Polynucleotides encoding selectable markers or reporter gene products can be transfected into target cells as described above, for subsequent packaging into viral display packages.
  • an antibiotic resistance gene or reporter gene can be included in a candidate polynucleotide, either upstream or downstream from the open reading frame of the candidate polynucleotide.
  • a free form of an o ⁇ han ligand is any form of the ligand which is soluble in the extracellular fluid.
  • the free form may have the o ⁇ han ligand as one of its domains or it may be an o ⁇ han ligand which is not covalently bound to or part of a macromolecule such as a fusion protein.
  • a free form of an o ⁇ han ligand can be used as a control to demonstrate the reversibility of the effect of a fusion protein containing the o ⁇ han ligand.
  • Such a free form of an o ⁇ han ligand will possess adequate binding affinity and specificity for the receptor of the o ⁇ han ligand to displace or inhibit the binding of the fusion protein at the concentration of free o ⁇ han ligand employed in the control experiment.
  • a “protease” according to the invention is an enzyme which hydrolyzes a peptide bond between a pair of amino acids located in a polypeptide chain; this is also called an “endoprotease.”
  • Proteases are typically defined by reference to the nucleophile in the catalytic center of the enzyme. The most common nucleophiles arise from the side chains of serine, aspartic acid, or cysteine, resulting in families of proteases, such as serine proteases (Paetzel et al.. Trends Biochem. Sci. 22, 28-31 , 1997), aspartyl proteases (Spinelli et al.. Biochemie 73. 1391 -96, 1991 ), and cysteine proteases (Altschuh et al., Prot. Eng. 7, 769-75. 1994).
  • Metalloproteases usually contain a zinc catalytic metal ion at the catalytic site (Klimpel et al., 1994, Mol. Microbiol.13, 1093- 100).
  • the protease cleavage site can be a variation of a cleavage site of a known protease.
  • a variation of the known cleavage site is formed by modifying at least one amino acid of the known protease recognition site.
  • the protease cleavage site in the fusion protein of the viral display package is inco ⁇ orated between the N-terminus of the viral envelope protein and the C-terminus of the o ⁇ han ligand.
  • Figure 1 depicts a method of selective gene delivery to cells based on the presence or absence of a displayed receptor.
  • Figure 1(a) shows that no gene delivery occurs if a cell displays a receptor for the ligand displayed on a viral vector that inco ⁇ orates the gene.
  • vectors displaying ligands on their envelope proteins are sequestered onto the ligand receptor, preventing gene delivery via the virus receptor.
  • Figure 1(b) shows that the gene is delivered to a cell that does not display a receptor for the ligand displayed on the viral vector. If the target cell lacks the ligand receptor, the vector is free to initiate gene delivery by binding to virus receptor.
  • Figure 2 illustrates a method of selecting for cells that display a ligand receptor by selectively delivering a cytotoxic gene to cells that do not display the receptor.
  • a viral vector is constructed which contains a cytotoxic gene and displays a ligand on a chimeric envelope protein. If the vector is contacted with a mixture of cells, some of which express the ligand receptor and others of which do not.
  • Figure 2(a) shows that cells which express the ligand receptor sequester vectors onto the receptor for the displayed ligand, thereby preventing delivery of the cytotoxic gene (labeled "cyt" to signify any cytotoxic gene).
  • Figure 2(b) shows that the vector is able to deliver the cytotoxic gene to cells which do not express the receptor for the displayed ligand by binding to the virus receptor. Therefore, cells which express the displayed ligand receptor will survive, but cells which do not express and display the receptor on their surface will be destroyed by the product of the cyt gene.
  • Figure 3 demonstrates a method of cloning cells expressing receptors for o ⁇ han ligands.
  • a cDNA library is generated from a cell line known to express or suspected of expressing the receptor for an o ⁇ han ligand.
  • the library is inserted into an expression vector and transfected into cells that are o ⁇ han ligand receptor negative.
  • the letters "a” through “g” represent distinct transgenes from the library.
  • a vector is added which delivers a cytotoxic gene (cyt) only if the target cell lacks the receptor for the o ⁇ han ligand displayed on the surface of the vector.
  • O ⁇ han ligand receptor-negative cells then die, leaving cells that express the o ⁇ han ligand receptor to grow ("a" represents the gene for the o ⁇ han ligand receptor). After multiple rounds of such selection, genomic DNA can be extracted from the cells and the o ⁇ han ligand receptor gene can be sequenced and identified.
  • the inventors have developed novel methods for rapidly identifying receptors of o ⁇ han ligands and selecting for cells based on display of such receptors.
  • the methods use viral vectors comprising chimeric envelope proteins (viral display packages) to selectively deliver a transferable label to cells that do not display a receptor for an o ⁇ han ligand.
  • the chimeric envelope proteins are generated by fusing a nucleotide sequence encoding a viral envelope protein with a nucleotide sequence encoding an o ⁇ han ligand.
  • the viral envelope protein serves as the key to infecting host cells which bear a receptor for the envelope protein (unrelated to the receptor for the o ⁇ han ligand).
  • a host cell displays on its surface a receptor for the o ⁇ han ligand, then formation of the o ⁇ han ligand-receptor complex will trap the viral vector and prevent it from infecting the cell. If a host cell does not display a receptor for the o ⁇ han ligand, then the viral vector will infect the cell and deliver the transferable label to the cell. Transferable labels can conveniently identify or modify cells which do not express a receptor for the o ⁇ han ligand.
  • Viral display packages of the invention are viral vectors suitabie for transfecting animal cells.
  • Each viral display package displays an o ⁇ han ligand on its surface.
  • the o ⁇ han ligand is part of a chimeric viral envelope protein.
  • the viral envelope protein functions to infect host cells by interacting with a virus receptor on the host cell surface membrane.
  • the chimeric envelope protein is a fusion protein comprising an o ⁇ han ligand whose C-terminal amino acid is attached to the N-terminal amino acid of a viral envelope protein, optionally through a linker peptide of one or more amino acids.
  • a viral display package also comprises a transferable label.
  • the transferable label Upon infection of a host cell, the transferable label is taken up into the cytoplasm of the host cell, where it can be directly detected or where it becomes activated to produce a product that can be detected.
  • the transferable label can be a nucleic acid sequence that encodes a detectable product and is enclosed within an envelope of a viral display package.
  • the viral display package is a retrovirus.
  • Retroviral display of functional polypeptides is disclosed in WO 94/06920. The sequence coding for a heterologous polypeptide is fused to the substantially intact envelope protein as an N-terminal extension. Retroviral N-terminal display has been described for the display of functional growth factors, including epidermal growth factor (Cosset et al. ( 1995) J. Virol. 69:6314-6322), stem cell factor (Fielding et al. ( 1998) Blood 91 : 1802- 1809), and insulin-like growth factor I (Chadwick et al. ( 1999) J. Mol. Biol. 285:485-494), among others.
  • functional growth factors including epidermal growth factor (Cosset et al. ( 1995) J. Virol. 69:6314-6322), stem cell factor (Fielding et al. ( 1998) Blood 91 : 1802- 1809), and insulin-like growth factor I (Chadwick et al.
  • the target cell In order for the viral display packages of the invention to effectively deliver the transferable label to a target cell, the target cell must display both a first receptor for the o ⁇ han ligand and a second receptor through which the viral vector binds and infects the cell. Therefore, the target cell and viral envelope protein should be selected so that infection of the target cell will occur in the absence of the binding of the o ⁇ han ligand to its receptor.
  • MMV amphotropic murine leukemia virus
  • Env envelope protein binds to Pit-2 receptors on the target cell. Van Zeijl et al. (1994) Proc. Natl. Acad. Sci. U.S.A.
  • the presence of an o ⁇ han ligand receptor can be detected as a reduction in viral titer caused by the display of the o ⁇ han receptor, whose binding to the o ⁇ han ligand receptor competes with the viral Env binding to its receptor.
  • comparison of viral titers obtained 8 hours after infection with vectors made with and without the o ⁇ han ligand might show a decrease of five-fold or more when the ligand is included in the vector.
  • the number of receptors varies in different cell types. Pit-2 expression in cardiac muscle is high, but is lower in lymphocytes. Alternate approaches include use of an alternate viral envelope protein such as that of GALV, or transfection of the target cells with an appropriate virus receptor expression plasmid.
  • the cell can be contacted with a viral display package comprising a chimeric envelope protein that displays the o ⁇ han ligand. If the o ⁇ han ligand receptor is not present on the surface of a cell, then the viral display package is capable of infecting the cell and transferring the label to the cell.
  • the method of contacting will typically involve incubating the cell in a medium containing a sufficient concentration of the viral display package, and for a sufficiently long incubation period, so that at least about 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99 percent of the cells that do not display a receptor for the o ⁇ han ligand will be infected and will take up the transferable label.
  • the label can be detected so that the cells displaying the receptor are identified.
  • the method of 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.
  • 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. Cells that have taken up the label either do not display the o ⁇ han ligand receptor or display a low level of the receptor. Comparison of the fluorescence and visible light images of a field of cells will reveal cells that display the o ⁇ han ligand receptor as those with little or no fluorescence.
  • the invention can be used to automatically select for cells expressing a receptor for an o ⁇ han ligand or cells not expressing such a receptor.
  • the transferable label is cytotoxic.
  • the transferable label can be either a cytotoxic protein, such as a bacterial toxin, or a nucleic acid molecule encoding a toxin. If the transferable label is toxic, the viral display package will infect and kill those cells which do not display a receptor for the o ⁇ han ligand, leaving a population of cells enriched in such receptors.
  • the transferable label is only mildly toxic, or otherwise limits the growth or proliferation of the infected host cells, then the method outlined above can be employed to select for cells which have a higher density of the receptor, as those cells will have a growth advantage.
  • the transferable label is a gene encoding a selectable marker.
  • the selectable marker can be, for example, an enzyme conferring antibiotic resistance. This will allow cells not displaying the o ⁇ han ligand receptor to grow in the presence of an antibiotic, while cells displaying the receptor will be selectively killed by the antibiotic.
  • a fluorescence-activated cell sorter can be adapted to select either for cells displaying a receptor or for cells not displaying a receptor.
  • the transferable label should be a fluorescent molecule or should encode a fluorescent molecule.
  • the FACS can be used to collect different fractions of cells based on their individual fluorescence intensity corresponding to the amount of a given label transferred to or expressed in the cell. For example, if the label appears only in cells which do not display a particular receptor, then collecting a fraction of cells with higher than a specified background level of fluorescence intensity will result in a population of cells that do not display the receptor.
  • a suitable background level can be determined, for example, as the fluorescence at the respective excitation and emission wavelengths of cells that have not been contacted with a viral display package or a transferable label.
  • collecting a fraction of cells with lower than a threshold level of fluorescence intensity will result in a population of cells that display the receptor.
  • the threshold level of fluorescence intensity should be somewhat higher than the background fluorescence of untreated cells. For example, in order to capture nearly all of the treated cells with only background fluorescence, i.e., those cells which display the receptor, then the threshold might be set at 2 above the mean background fluorescence of untreated cells.
  • Any method used to select for cells which display or do not display a receptor can be applied cither in an all-or-nothing manner or in a graduated manner. That is, if the selection criteria are sufficiently stringent, then all or nearly all of the cells selected will either display or not display the receptor. However, if the selection criteria are less stringent, then the method will merely select for cells which display a higher or lower density of the receptor.
  • the threshold of fluorescence intensity used to discriminate between the resulting fractions can be set far from the distribution of fluorescence values for labeled cells so as to completely eliminate cells that do not display a receptor, or it can be set closer to or within the range of fluorescence levels for labeled cells, so that cells with a lower density of receptor will be included in the selected fraction.
  • the selection could also be 'punctuated', in which the vector containing a cytotoxic gene is added in order to kill approximately 80% of the cells. After a period during which cells recover, the same amount of vector is added again. The process is then repeated until the majority of the cells contain the receptor (or receptor density) required.
  • thymidine kinase and gancyclovir Another means of selection is application of thymidine kinase and gancyclovir.
  • virus is first added cells, and then the cells are plated at mid-density. The cells are grown for 24 hours, then gancyclovir is added for selection. After an appropriate recovery period, the process is repeated until the majority of cells contain the receptor (or receptor density) required.
  • the methods outlined above result in a negative observation to identify cells displaying an o ⁇ han receptor; cells displaying the receptor are the ones which do not become labeled.
  • a control experiment can be additionally performed using a free form of the o ⁇ han ligand of interest. If a cell that displays an o ⁇ han ligand receptor is contacted with the viral display package in the presence of the free form of the same o ⁇ han ligand contained in the viral display package, then interaction of the viral display package with the o ⁇ han ligand receptor will be competitively inhibited. This will result in cells displaying the o ⁇ han ligand receptor, whose infection was previously blocked, now becoming infected and acquiring the transferable label.
  • the hallmark of a cell that displays a receptor for an o ⁇ han ligand is that the cell does not become labeled by contact with a viral display package bearing the o ⁇ han ligand, but does become labeled when a free form of the same o ⁇ han ligand is added during the period of contact with the viral display package.
  • the amount of the free ligand used must be sufficient to competitively inhibit at least about 50, 60, 70, 80, 90, 95, 99, or 100 percent of the binding of the corresponding viral display package to the o ⁇ han ligand receptor.
  • the concentration of free ligand added to the incubation can be at least 3, 5, 10, 20, 50, 100, or 1000 times the dissociation constant, K D , for the binding of the free ligand to the receptor.
  • One possible form of free ligand is represented by the amino acid sequence identical to that integrated into the chimeric envelope protein of the viral display package. However, it is not required that the free ligand be identical to the o ⁇ han ligand used in the viral display package.
  • the free form of the ligand should have sufficiently high affinity for the receptor so that, at the concentration employed, it effectively inhibits binding of the chimeric envelope protein to the o ⁇ han ligand receptor.
  • Another means to control for the ability of a viral display package to transfect a host cell and deliver transferable label can be performed by proteolysis of the o ⁇ han ligand from the chimeric envelope protein.
  • a chosen protease recognition and/or cleavage site can be inco ⁇ orated by design into the chimeric envelope protein as a linker or part of a linker between the N-terminal amino acid of the viral envelope protein and the C-terminal amino acid of the o ⁇ han ligand.
  • a factor Xa protease cleavage site can be introduced between the Env protein and the o ⁇ han ligand. Cosset et al. (1995).
  • a viral display package inco ⁇ orating such a chimeric envelope protein is pre-treated, prior to contacting the host cell, with the protease that recognizes the linker, then the o ⁇ han ligand will be cleaved away from the viral display package. The viral display package will then infect a host cell bearing the o ⁇ han ligand receptor. Similar to the above case where free o ⁇ han ligand is used as a control, a cell which displays a receptor for the o ⁇ han ligand is characterized by no detectable transferable label, or a low level of label, following contact with the untreated viral display package and by the detection of a higher level of transferable label when the protease-treated viral display package is used.
  • the invention enables the generation of a clone of cells that display a receptor for a selected orphan ligand.
  • any of the procedures outlined above is carried out to identify cells that display a receptor for a selected o ⁇ han ligand. Then, one or more cells that are so identified are cultured to produce a clone of cells, essentially all of which display the receptor. Once those cells displaying a particular o ⁇ han ligand receptor are identified, a small number of cells is picked and transferred under sterile conditions to an empty culture dish, and allowed to grow to form a clone. Any suitable technique known in the art can be used to pick cells identified as expressing and displaying the receptor. For example, about 1 , 2.
  • 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 cells can be drawn under gentle suction into a small capillary tube and sterilely transferred under a laminar flow hood to a new culture dish with fresh culture medium suitable for growing the cell type involved.
  • the culture dish is placed in an incubator providing the standard growth conditions for the cell type, e.g., 37°C, 95% air, 5% CO 2 , and the culture medium is replaced with fresh medium every few days.
  • a culture of cells is obtained which all derive from one or more of the picked cells; therefore, all of the cells in the clone will express and display the o ⁇ han ligand receptor.
  • the homogeneity of the clone of cells derives from the small number of receptor-positive cells selected for cloning. Therefore, the smallest possible number of cells should be selected for cloning, preferably 1, 2, 3, 4, or 5 cells.
  • a recombinant DNA library is used to generate a corresponding library of transfected cells, each expressing a different fragment of DNA from the library which might express a receptor for an o ⁇ han ligand.
  • the transferable label is cytotoxic
  • the surviving cells, or most of the surviving cells will each express a distinct o ⁇ han ligand receptor.
  • the surviving cells can be individually cultured, each yielding a clone of cells expressing a single receptor for an o ⁇ han ligand.
  • the surviving cells could express the same o ⁇ han ligand receptor, in which case individually cultured cells will clones expressing the same receptor for an o ⁇ han ligand.
  • the invention can be used to rapidly identify and analyze a large number of gene sequences whose function can be related to a particular ligand encoded in a recombinant library.
  • the viral display packages of the invention can be used to enrich or deplete a population of cells based on the display of o ⁇ han ligand receptors if the appropriate transferable label is chosen. If the transferable label is an agent which confers any disadvantage to the growth or replication of a recipient cell, and the label is transferred to cells that do not display a receptor for the orphan ligand displayed on the viral display package, then a population of cells treated with the display package will in time become enriched in cells that display one or more receptors for the orphan ligand. Examples of agents which interfere with the growth or replication of a recipient cell include cytotoxic agents or genes encoding cytotoxic agents.
  • the cytotoxic agent can be perforin, a ribonuclcase, an exotoxin, thymidine kinase (derived from He ⁇ es Simplex Virus) or other cytotoxic agent.
  • the transferable label is an agent which confers any advantage to the growth or replication of a recipient cell, and the label is transferred to cells that do not display a receptor for the o ⁇ han ligand displayed on the viral display package, then a population of cells treated with the display package will in time become depleted in cells that display one or more receptors for the o ⁇ han ligand.
  • agents which selectively enhance the growth or replication of a recipient cell include selectable markers such antibiotic resistance genes.
  • Selective depletion or enrichment of a cell population based on the display of an o ⁇ han ligand receptor requires that similar steps are carried out as for the identification or cloning of such cells.
  • the population of cells is contacted with the appropriately designed viral display package and a sufficient incubation time is allowed to permit the transferable label to have the desired effect on the target cell population. If desired, the effects of the transferable label can be monitored, for example by monitoring the transfer, expression, function, or resulting effects f the label on the target cell population.
  • the viral display package can be removed, e.g., by centrifugation. filtration, affinity chromatography, or other suitable method, after the desired degree of enrichment or depletion has been attained.
  • An o ⁇ han ligand is identified from analysis of genomic DNA based on its homology to an existing family of ligands.
  • the sequence of the o ⁇ han ligand is inserted into a 4070A MLV vector, where it is expressed as an N-terminal extension of the Env protein.
  • the fusion protein is displayed on the surface of the vector.
  • the RNA genome of the vector has been engineered to include the gene for GFP as a transferable label; cells infected by the retrovirus express detectable amounts of GFP.
  • a population of human cells is transfected with an expression vector containing fragments of a cDNA library that contains a gene coding for a receptor which binds the o ⁇ han ligand whose sequence is displayed on the MLV vector.
  • the transfected cells express a variety of proteins from the cDNA library, and one or more cells express and display on their surface membrane the receptor for the o ⁇ han ligand. About 8 hours following transfection, most of the cells are infected with the retrovirus, and the transfected cells begin to show the fluorescence of GFP, which they are also expressing. An individual cell which does not reveal any GFP fluorescence is picked with a capillary and transferred to a sterile culture dish containing fresh sterile culture medium. Over a period of days, the cell proliferates to form a clone of cells which all express the receptor for the o ⁇ han ligand and display the receptor on their surfaces.
  • An o ⁇ han ligand is identified from analysis of genomic DNA based on its homology to an existing family of ligands.
  • the sequence of the o ⁇ han ligand is inserted into a 4070A MLV vector, where it is expressed as an N-terminal extension of the Env protein.
  • the fusion protein is displayed on the surface of the vector.
  • the RNA genome of the vector has been engineered to include the gene for perforin, which is expressed in cells infected by the vector, leading to the death of the infected cells within a few hours following transfection.
  • a population of human cells is transfected with an expression vector containing fragments of a cDNA library that contains a gene coding for a receptor which binds the o ⁇ han ligand whose sequence is displayed on the MLV vector.
  • the transfected cells express a variety of proteins from the cDNA library, and one or more cells express and display on their surface membrane the receptor for the o ⁇ han ligand.
  • About 8 hours following transfection most of the cells are infected with the retrovirus and have been killed as a result of the expression of perforin.
  • the cells are then allowed to recover by overnight incubation at 37°C/5%CO 2 .
  • the process of vector addition and cell killing is then repeated each day for five days. At the end of this period, the cells are grown up to confluence and genomic DNA is extracted.
  • the sequence of the cDNA coding for the o ⁇ han ligand receptor is then determined using standard sequencing techniques.

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Abstract

L'invention concerne des méthodes d'identification de cellules présentant des récepteurs pour des ligands orphelins. Les méthodes utilisent un ensemble de présentation virale comprenant un ligand orphelin fusionné à une protéine d'enveloppe virale et une étiquette transférable. La fixation du ligand orphelin à un récepteur sur la cellule cible empêche l'infection de la cellule cible par l'ensemble de présentation virale. L'ensemble de présentation virale infecte et transfère l'étiquette vers des cellules ne présentant pas de récepteur du ligand orphelin. Des cellules présentant un récepteur pour le ligand orphelin sont identifiées par absence de l'étiquette. Des étiquettes cytotoxiques peuvent être utilisées pour enrichir sélectivement une population de cellules pour celles exprimant et présentant un récepteur pour un ligand orphelin.
PCT/US2001/005388 2000-02-25 2001-02-20 Detection de recepteurs de ligands orphelins par presentation retrovirale WO2001062971A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1171638A1 (fr) * 2000-02-25 2002-01-16 Cambridge Drug Discovery Limited Methodes d'identification d'un inhibiteur de protease
US20100261180A1 (en) * 2004-03-01 2010-10-14 Peter Maccallum Cancer Institute Recombinant perforin, expression and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686592A (en) * 1990-06-11 1997-11-11 Nexstar Pharmaceuticals, Inc. High-affinity oligonucleotide ligands to immunoglobulin E (IgE)
US5766923A (en) * 1994-07-22 1998-06-16 President & Fellows Of Harvard College Isolated nucleic acid encoding ligands for FGFR
US5945279A (en) * 1991-05-02 1999-08-31 Baylor College Of Medicine Screening system for identifying compounds that regulate steroid and orphan receptors mediation of DNA transcription

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686592A (en) * 1990-06-11 1997-11-11 Nexstar Pharmaceuticals, Inc. High-affinity oligonucleotide ligands to immunoglobulin E (IgE)
US5945279A (en) * 1991-05-02 1999-08-31 Baylor College Of Medicine Screening system for identifying compounds that regulate steroid and orphan receptors mediation of DNA transcription
US5766923A (en) * 1994-07-22 1998-06-16 President & Fellows Of Harvard College Isolated nucleic acid encoding ligands for FGFR

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1171638A1 (fr) * 2000-02-25 2002-01-16 Cambridge Drug Discovery Limited Methodes d'identification d'un inhibiteur de protease
EP1171638A4 (fr) * 2000-02-25 2005-01-19 Biofocus Discovery Ltd Methodes d'identification d'un inhibiteur de protease
US20100261180A1 (en) * 2004-03-01 2010-10-14 Peter Maccallum Cancer Institute Recombinant perforin, expression and uses thereof

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