WO1998033819A1 - Recepteurs cellulaires des adenovirus du sous-groupe c et des virus coxsackie du groupe b - Google Patents

Recepteurs cellulaires des adenovirus du sous-groupe c et des virus coxsackie du groupe b Download PDF

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WO1998033819A1
WO1998033819A1 PCT/US1998/001724 US9801724W WO9833819A1 WO 1998033819 A1 WO1998033819 A1 WO 1998033819A1 US 9801724 W US9801724 W US 9801724W WO 9833819 A1 WO9833819 A1 WO 9833819A1
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hcar
protein
mcar
glycoprotein
cell
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PCT/US1998/001724
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WO1998033819A9 (fr
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Richard P. Tomko
Lennart Philipson
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New York University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the invention in the field of virology, molecular genetics and medicine relates to the HCAR and MCAR protein molecules, human and mouse proteins (respectively) that serve as common cellular receptors for adeno viruses of the serotypes 2 and 5 (subgroup C) and for the group B coxsackieviruses.
  • the invention provides peptide fragments of these proteins which correspond to the extracellular domains and more discrete virus-binding peptides, as well as other functional derivatives, DNA molecules encoding these proteins, peptides and functional derivatives, and antibodies to the proteins.
  • the DNA molecules are used for recombinant production of the proteins and peptides.
  • the isolated proteins or fragments or variants thereof are used to prevent or treat virus infections.
  • the invention also concerns expressing the DNA encoding these virus receptors in cells which lack them, rendering the cells susceptible to transformation by adenoviral vectors carrying genes for gene therapy.
  • HIV-1 human immunodeficiency virus
  • Epstein-Barr virus binds to the complement receptor protein CR2
  • human rhinoviruses bind to the cell adhesion molecule ICAM-1
  • rabies virus binds to the acetylcholine receptor
  • reo viruses bind to ⁇ -adrenergic receptors
  • herpes simplex virus appears to use fibroblast growth factor receptor as a binding site.
  • virus binding proteins or receptors are a strong determinant of susceptibility to virus infection. For example, binding is required for virus uptake by the target cell, an event that may occur at the cell surface or within an acidified endosome after receptor-mediated endocytosis (White et al, Quant. Rev. Biophys. 16: 151-195 (1983)). After internalization, the virion nucleic acid enters the cytoplasm and the viral replication process is initiated.
  • Adeno viruses are DNA viruses, some members of which are human pathogens. In general, viruses of different families do not compete for binding to a common receptor. However, two serotypes of adenovims, serotype 2 (Ad2) and serotype 5 (Ad5), as well as the RNA vims group known as group B coxsackievimses (CVB) are human pathogens that do share a common receptor (Lonberg-Holm, K. et al, Nature 259:679-681 (1976)). Adenovimses attach to a cellular receptor with the "knob" portion of their capsid fiber (Xia, D. et al, Struct. 2:1259-1270 (1994)). In contrast, the CVB are presumed to attach to cells through insertion of the receptor into the canyons on the virion surface
  • adenovimses unlike CVB, do not have a broad host range and therefore encounter other restrictions in their life cycle following receptor attachment ((Shenk, T., In: VIROLOGY, Fields B.N. et al, eds., Lippincott-Raven, Philadelphia, 1996, pp. 2111-2148).
  • the expression of mRNA encoding HCAR, this common receptor, in several tissues indicates that adenovims-mediated gene deliver to a variety of tissues and organs will be possible.
  • the present inventors have isolated human cDNA and mouse cDNA that encode, respectively, the .uman CVB andA l and Ad5 receptor (HCAR), included in and the mouse C B Ad2 and Ad5 receptor (MCAR).
  • the cDNA clone which includes the HCAR coding sequence is SEQ ID NO: 1
  • the cDNA clone which includes the MCAR encoding sequence is SEQ ID NO: 3.
  • the amino acid sequence of HCAR is SEQ ID NO:2 and that of MCAR is SEQ ID NO:4. Both proteins encoded by these new DNA molecules have not previously been isolated in totally pure form.
  • a human receptor for these vimses has not been characterized beyond recognition of a relationship between receptor type (biological activity with appropriate vimses) ) and molecular weight or isoelectric pH. It is only with the making of the present invention that it is possibly to characterize these receptor proteins as membrane-spanning glycoproteins that contain two extracellular immunoglobulin- (Ig-) like domains and, therefore, are new members of the Ig superfamily. The two protein molecules described herein are expected to have other biological functions than serving as vims receptor sites, although these have not yet been identified.
  • a different human receptor for CVB has been shown to be decay accelerating factor (DAF), a complement receptor protein (Mohanty et al. ,supra: Bergelson et al, supra.).
  • HCAR genetic locus localizes to human chromosome 21 and not to chromosome 19 as had been proposed by others (Couillin, P. et al, Path. Biol 24:195-203 (1976).
  • the present invention provides an isolated DNA molecule which encodes a coxsackievims B and Ad2 and Ad5 receptor protein or glycoprotein molecule, termed HCAR, or a protein or glycoprotein termed MCAR, or encodes a functional derivative thereof, which protein, glycoprotein or functional derivative binds group C adenovimses or coxsackievims B. Also intended is an allelic variant of such a DNA molecule as understood in the art.
  • SEQ ID NO:l and SEQ ID NO:3 were deposited with Genbank under accession number U90716 and were put on line on 22 April 1997.
  • SEQ ID NO:2 and SEQ ID NO:4 were deposited with Genbank under accession number U90715 and were put on line on 22 April 1997.
  • the DNA molecule has the sequence SEQ ID NO:l, more preferably the coding sequence thereof. In another embodiment, the DNA molecule has the sequence SEQ ID NO:3, more preferably, the coding sequence thereof.
  • DNA molecule as above wherein the protein has the amino acid sequence SEQ ID NO:2 or SEQ ID NO:4.
  • the DNA molecule is one which hybridizes under stringent conditions with the coding sequence of SEQ ID NO:l, SEQ ID NO:3 or with any stretch of twenty contiguous nucleotides of the coding sequence of SEQ ID NO: l or SEQ ID NO:3
  • the derivative is preferably a peptide of an extracellular immunoglobulin domain of HCAR.
  • the peptide is selected from the group consisting of residues 35-130 of SEQ ID NO:2, and residues 155-220 of SEQ ID NO:2.
  • Other preferred DNA molecules encode a Ad2, Ad5 or CVB binding peptides comprising one or more repeats of the sequence LSPEDQGP, PEDQG, LDIEW, QVIIL, QMIIL, ILYSGD, DKII, NDLKS, NDVKS or VKKAPG.
  • the above DNA molecule may be a cDNA molecule or a genomic DNA molecule.
  • DNA molecule as above which is an expression vector, such as a plasmid.
  • the present invention is directed to a host cell, prokaryotic or eukaryotic, most preferably mammalian, transformed or transfected with a DNA molecule as above.
  • this invention provides a process for preparing HCAR or MCAR protein, glycoprotein or functional derivative molecule which, if naturally occurring, is substantially free of other proteins or glycoproteins with which it is natively associated, comprising culturing a host cell transformed or transfected as above under culturing conditions to express the DNA molecule and recovering the protein, glycoprotein or functional derivative molecule produced by the host cell from the culture.
  • the present invention is directed to HCAR or MCAR protein, glycoprotein or derivative made according to the above method.
  • HCAR or MCAR protein, glycoprotein or functional derivative molecule which, if naturally occurring, is substantially free of other proteins or glycoproteins with which it is natively associated, which protein, glycoprotein or functional derivative is encoded by a DNA molecule as described above.
  • the functional derivative is a peptide of an extracellular immunoglobulin domain of HCAR, preferably a peptide selected from the group consisting of (a) residues 35-130 of SEQ ID NO:2 and (b) residues 155-220 of SEQ ID NO:2.
  • Preferred peptides comprise one or more repeats or combinations of the sequence LSPEDQGP, PEDQG, LDIEW, QVIIL, QMIIL, ILYSGD, DKII, NDLKS, NDVKS, VKKAPG.
  • the present invention is also directed to a method for detecting the presence of nucleic acid encoding a normal or mutant HCAR or MCAR protein in a nucleic acid-containing sample comprising (a) contacting the sample with an oligonucleotide probe encoding at least a portion of the normal or mutant HCAR or MCAR under hybridizing conditions; and (b) measuring the hybridization of the probe to the nucleic acid of the cell, thereby detecting the presence of the nucleic acid.
  • This method may additional comprise, before step (a), the step of selectively amplifying the DNA encoding the HCAR or MCAR protein.
  • kits useful for foregoing detection method are adapted to receive therein one or more containers, and comprises a first container containing the oligonucleotide probe and a second container or plurality of containers containing a reagent or reagents capable of detecting the binding of the oligonucleotide to the sample nucleic acid.
  • a method for detecting the presence or measuring the quantity of HCAR or MCAR protein, glycoprotein or functional derivative in a biological sample comprising contacting the biological sample that is suspected of containing the protein, glycoprotein or derivative with a binding partner capable of binding to the HCAR or MCAR protein; and detecting the binding of the binding partner to a substance in the sample or measuring the quantity of the binding partner bound, thereby determining the presence or measuring the quantity of the HCAR or MCAR protein, glycoprotein or derivative.
  • the binding partner may be an antibody or an antigen-binding fragment thereof.
  • the binding partner is a viral protein or peptide which binds to the HCAR or MCAR protein or is an HCAR or MCAR- binding functional derivative of the viral protein or peptide.
  • a method for identifying in a sample an analyte capable of binding to a HCAR or MCAR protein, glycoprotein or functional derivative as above comprises: (a) incubating the sample which is suspected of containing the analyte in the presence of an HCAR or MCAR protein or functional derivative, or a ligand-binding portion thereof, such that the HCAR or MCAR protein, glycoprotein, derivative or portion binds to the analyte; and (b) detecting the analyte which is bound to the protein, glycoprotein, derivative or potion.
  • the HCAR or MCAR protein, glycoprotein, derivative or portion is immobilized to a solid support.
  • the analyte may be an HCAR or MCAR-binding vims, viral protein or peptide, or an HCAR or MCAR-specific antibody.
  • the present invention also provides a composition
  • a composition comprising a solid support to which is immobilized an isolated HCAR or MCAR protein, glycoprotein or functional derivative as described.
  • the invention is directed to a method for isolating from a complex mixture a composition capable of binding to HCAR or MCAR protein, glycoprotein or functional derivative as described, comprising: (a) immobilizing the HCAR or MCAR protein, glycoprotein or derivative, or a ligand-binding portion thereof, to a solid support; (b) contacting the complex mixture with the solid support of step (a) so that any of the composition binds to the immobilized HCAR or MCAR protein, glycoprotein or derivative; (c) washing away any unbound material from the mixture; and (d) eluting the bound composition, thereby isolating the composition.
  • compositions useful for preventing or treating an infection by a vims which utilizes HCAR or MCAR as its cellular receptor comprising a HCAR or MCAR protein, glycoprotein or functional derivative as described above; and a pharmaceutically acceptable carrier or excipient.
  • This invention is directed to a method for preventing or treating in a subject an infection with a vims which utilizes HCAR or MCAR as its cellular receptor, comprising administering to the subject an effective amount of the above pharmaceutical composition.
  • the vims infection is typically with adenovims serotype 2 or serotype 5, or a group B coxsackievims.
  • a method for inhibiting the infectivity of adenovims serotype 2 or serotype 5, or of a group B coxsackievims comprising contacting the vims, in vitro or in vivo with an effective amount of the HCAR or MCAR protein molecule or derivative as above, and allowing the molecule to prevent the vims from attaching to a cell, thereby inhibiting the infectivity
  • Another embodiment includes a method for rendering a cell which is normally not susceptible to infection with an adenovims susceptible to infection by, and vims-mediated gene transfer by, an adenovims vector, comprising the steps of (a) transforming a cell of the non-susceptible species or cell type with an expression vector as described above; (b) expressing the HCAR or MCAR protein, glycoprotein or derivative on the surface of the cell, thereby rendering the cell susceptible to infection by the adenoviral vector.
  • This invention is also directed to a transgenic non-human mammal essentially all of whose germ cells and somatic cells contain a DNA sequence encoding HCAR or MCAR as described above.
  • the DNA molecule has been introduced into the mammal or an ancestor of the mammal at an embryonic stage.
  • Figure 1 is an electropherogram showing expression of unique RNA's found in receptor-expressing TCMK-1 cells.
  • L cells was probed with a 32 P labeled fragment of RTMCR-4 to reveal differentially expressed 6 kb (large arrow) and 1.4 kb (small arrow) RNA's.
  • Figures 2 A and 2B show an amino acid alignment (Fig. 2 A) of HCAR and MCAR and a domain model (Fig. 2B) for a CAR protein.
  • the deduced amino acid sequence (SEQ ID NO:2) of cloned HCAR cDNA and the deduced amino acid sequence (SEQ ID NO:4) of cloned MCAR cDNAs were aligned using the Gene Works software program.
  • ) represent amino acid identities, dashes (-) are gaps in the alignment. The positions of the potential signal peptide (thinner underscore at the N-terminus), transmembrane region (thicker underscore), and N-linked glycosylation sites (*) are shown.
  • FIG. 2B IG1 and IG2 represent immunoglobulin domains.
  • SS represents the signal sequence and TM represents the transmembrane-spanning region.
  • Figure 3 is a set of four photomicrographs showing that pRTHR and pRTMR confer susceptibility to Ad2 entry. Only TCMK-1 positive controls (panel A) or NIH3T3 cells transfected with either pRTHR (panel B) or pRTMR (panel C) stained for ⁇ -galactosidase expression following incubation with a CMV- ⁇ gal recombinant Ad5 vector. pBK-CMV controls (panel D) showed no reactivity following incubation under identical conditions.
  • Figures 4A and 4B show western blots of HCAR and MCAR as detected by CVB receptor-specific antibodies. 46 kDa proteins are visible in lysates from
  • HCAR and MCAR transfected cells that are absent in pBK-CMV transfected cells. Similar sized proteins (about 46kDa) are also detectable in two positive control cell lines: HeLa and TCMK-1. Immunodetection was performed using a mAb specific for HCAR (RmcB) (Fig. 4A, lanes 1-3) or the anti-p46 antiserum (Fig. 4B, lanes 4-6).
  • Figure 5 shows detection of hybridizing 6 kb and 1.4 kb mRNAs in poly A northern blots of TCMK-1 cells but not in mouse L cells.
  • six species of mRNA ranging in size from 6kb to 1.2kb were detected in HeLa cells that that were not found in receptor-negative rhabdomyosarcoma (Rd) cells.
  • the sizes of the RNAs that correlate with the size of the isolated HCAR (large arrow) and MCAR (small arrow) cDNAs are indicated.
  • Figure 6 shows that HCAR mRNA is expressed in a variety of tissues.
  • Human multiple tissue Northern blots were purchased from Clontech and hybridized with a 32 P-labeled fragment co ⁇ esponding to the open reading frame of HCAR. Hybridizing sequences are similar in size to those detected in HeLa cells ( Figure 4A-4B).
  • Figure 7 shows MCAR mRNA is expressed in multiple mouse tissues.
  • Mouse multiple tissue Northern blots (Clontech) were hybridized with a
  • Hybridizing sequences are similar in size to those detected in TCMK-1 cells ( Figure 5).
  • Figures 8-11 are a series of views of a 3 dimensional (3D) model of the HCAR/MCAR receptor.
  • Fig. 8 shows a 3D model of the complete HCAR/MCAR receptor.
  • IG1 and IG2 domains were built by homology with an immunoglobulin and the CD4 protein.
  • the C-terminal cytoplasmic Ser-Pro-Thr-rich domain was assigned an arbitrary conformation.
  • Tyr 269 is a potential kinase phosphorylation site.
  • Fig. 9 shows a model of a fiber knob interacting with three IG1 receptor domains.
  • Fig. 10 shows a top view of the HCAR/MCAR receptor docked into the canyon of CVB3.
  • Figure 12 shows the amino acid sequences or HCAR (SEQ ID NO: 2) and MCAR (SEQ ID NO:4) with demarcation of the residues involved in binding to Ad2, Ad5 and/or CVB.
  • the present invention is directed to a DNA molecule discovered by the inventors which encodes a human vims receptor protein termed HCAR and a cDNA molecule which encodes the murine homologue termed MCAR. Collectively, these proteins are termed CAR proteins (Coxsackie and Adenovims Receptor) .
  • CAR proteins Coxsackie and Adenovims Receptor
  • the present inventors have conceived of a method of using a CAR protein, or a functional derivative thereof, preferably a soluble form of the protein, to bind Ad2, Ad5 or CVB in a manner that prevents entry of these vimses into susceptible cells.
  • the methods of the present invention which identify normal or mutant CAR genes or measure the presence or amount of CAR protein associated with a cell or tissue can serve as methods for identifying susceptibility to infection by these vimses.
  • the invention is directed to a naturally occurring HCAR or MCAR protein substantially free from impurities of human or murine origin (respectively) with which it is natively associated. In another embodiment, the invention is directed to a recombinant HCAR or MCAR protein.
  • substantially free of other proteins indicates that the protein has been purified away from at least about 90%(on a weight basis), and preferably, from at least about 99%, if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them.
  • substantially pure is meant any protein or peptide of the present invention (or any DNA encoding any such protein or peptide), which is essentially free of other proteins (or polynucleotides) or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature. Such purity can be achieved by subjecting lysates or extracts of cells, tissue or fluids containing the HCAR or MCAR protein to protein purification techniques.
  • HCAR or MCAR protein of the present invention can be purified biochemically or physicochemically from a variety of cell or tissue sources.
  • human pancreas or prostate tissue and cells are preferred.
  • polypeptides of desired sequence may be synthesized using well-known methods employing solid phase supports.
  • the HCAR or MCAR gene (genomic DNA or cDNA) can be isolated or all or part of the coding sequence can be synthesized
  • the HCAR or MCAR polypeptide, or a functional derivative thereof can be prepared substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired.
  • a preferred use of this invention is the production by chemical synthesis or recombinant DNA technology of a fragment of the HCAR or MCAR molecule, preferably as small as possible, while still retaining sufficient specificity and affinity of binding to CVB, Ad2 or Ad5 to act as an inhibitor of infection.
  • Preferred fragments of HCAR or MCAR include extracellular domains IGI and IG2 (see Figures 2A and 2B) or shorter fragments of these domains which may be derived from IGI, IG2 or both. Examples of preferred peptides are shown in Table 1, below, and are indicated in the sequences shown in Figure 12. These oligopeptides of 4-8 amino acids may be used singly or in combination as inhibitors of viral infection.
  • peptides comprising these sequences are included in this invention up to and including the full length HCAR or MCAR protein molecule.
  • synthetic (or recombinant) peptides which comprise one or more of these sequences.
  • vims binding peptides may include between one and about 20 repeats of a single peptide sequence or various of these oligopeptides. The ordering of the peptides may be the same or different from the order in the HCAR or MCAR protein.
  • spacer groups of between one and ten amino acids may be placed between the vims-binding oligopeptides described herein to permit proper folding.
  • Shorter peptides are expected to have two advantages over the larger proteins: (1) greater stability and diffusibility, and (2) less immunogenicity.
  • the identification of the HCAR or MCAR as a potential receptor or site of entry of a Ad2, Ad5 or CVB vims into target cells establishes a critical mechanism to explain how the adenovims or CVB enters the cell. Based on the inventors' recognition of these molecules as cellular receptors, this invention provides specific HCAR or MCAR receptor "mimics" or “decoys” that can prevent vimses that use these receptors (Ad2, Ad5 or CVB ) from binding to and being taken up into target cells. This permits a method for limiting initial infection and for controlling spread of an established infection by Ad2, Ad5 or CVB which would then prevent or treat vims-induced disease.
  • vims infections for which the present invention is useful include Ad2, Ad5, CVB and any other vims now known or yet to be discovered which bind to
  • HCAR or MCAR as their cellular receptor or which enter the cell via an HC AR- or MCAR-dependent mechanism.
  • the invention provides an approach to systematic identification of critical sites in HCAR or MCAR which must be present for susceptibility of a cell to infection by Ad2, Ad5 or CVB. This knowledge allows development of rationally designed small molecule therapeutic agents for dismpting viral entry into target cells.
  • the viral receptor of the present invention can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic or non-mammalian eukaryotic cell.
  • the HCAR or MCAR protein molecule is produced by recombinant means and expressed in mammalian cells, most preferably in human cells.
  • the receptor molecule of the present invention endows human cells, murine cells, or cells of other normally resistant species which are not normally capable of being infected by Ad2, Ad5 or CVB and which express the receptor with the ability to be infected by these vimses. This is particularly useful for targeted gene therapy using adenovims vectors.
  • HCAR or MCAR one skilled in the art, using known binding and inhibition assays, will be able, without undue experimentation, to identify the single or multiple amino acid substitutions which are responsible for binding of adenovims vectors with sufficiently high affinity to permit infection and genetic transformation of a cell expressing HCAR or MCAR. Substitution of between about 1 and 5 residues is preferred. Substitution of as few as one amino acid may alter the vims specificity of the altered receptor protein. Another means for modifying the vims binding specificity of HCAR or MCAR is by deletion of one or more of the extracellular amino acid residues in HCAR or MCAR. Preferred deletions of between about 1 and 5 residues from HCAR or MCAR at positions 35 to 130 of the protein are preferred.
  • a major advantage of transfecting human cells with HCAR or murine cells with MCAR if these cells are to be used in cellular therapy or cell transfer to achieve gene therapy is the decrease in immunogenicity resulting from use of the receptor molecule native to the species.
  • human cells normally resistant, but which are to be infected with a Ad2 or Ad5 are made to express HCAR or a functional derivative having as few amino acid residues of HCAR as necessary to confer infectibility by the vims. If cells bearing this receptor are to be introduced on multiple occasions into the same human subject, the fact that the receptor is largely of human origin decreases the chances of an undesirable immune response which would otherwise be directed to sequences of foreign origin.
  • the HCAR or MCAR protein can be expressed on the cell surface as a transmembrane protein in a number of cell types, particularly cells of pancreas and liver lineages, consistent with in vitro tropism of Ad2, Ad5 or CVB.
  • the protein will permit cells of these lineages in the human, which are normally resistant to Ad2, Ad5 or CVB vims infection, to be infected with an adenovims vector.
  • HCAR or MCAR functional derivatives thereof can be used in gene therapy.
  • an abnormal HCAR or MCAR molecule which results in enhanced susceptibility to disease may be replaced by infusion of cells of the desired lineage (such as hemopoietic cells, for example) transfected with a modified HCAR or MCAR protein, under conditions where the infused cells will preferentially replace the endogenous cell population.
  • Adenovimses have been recognized as useful vectors for transferring genes efficiently into human cells, for example to correct enzyme deficiencies.
  • vimses have varying host ranges and recognize some but not all human cells.
  • an adenoviral vector used for gene therapy be capable of infecting only desired cells and not cause generalized infection of cells throughout the body of the individual being treated. In the past, this has generally been accomplished by using defective vims preparations, or mutants lacking the El and E3 regions of the viral genome. Even in murine model work, it is important that the mouse be protected from generalized infection.
  • the present invention provides an additional and improved measure of safety compared to the prior art approaches in that it permits use of murine cells or transgenic mice with a selected susceptibility to the vims.
  • a human cell which is not infectable by a Ad2, Ad5 or CVB vims or is infectable only at very low efficiency due to lack of sufficient receptor protein on its surface is transfected with the HCAR gene or a functional derivative, and the HCAR protein (or functional derivative) is expressed, resulting in Ad2, Ad5 or CVB vims receptor appearing on the cell surface.
  • Ad2, Ad5 or CVB vims receptor appearing on the cell surface.
  • This approach can be used both in cells expressing no CAR or less CAR than is desirable for optimal gene transfer by the viral vector.
  • Such a transfected cell can then be infected with an Ad2, Ad5 or CVB vector carrying a gene of interest, in order to transfer the gene of interest permanently (or transiently, if desired) into the cell.
  • adenovims vectors for human gene therapy include the fact that no human malignancies are known to be associated with such vimses, the adenovims genome is double stranded DNA which can be manipulated to accept foreign genes of up to 7.5 kb in size, and live adenovims is a safe human vaccine entity.
  • the present invention is intended to encompass Ad2, Ad5 or CVB vimses, or any other mammalian vims with similar receptor specificity, which attaches to the HCAR or MCAR molecule of the present invention as its cellular receptor or enters the cell via an HCAR-or MCAR-dependent mechanism.
  • the invention is directed to the general concept of generating a viral receptor which will allow selected cells or cells of a selected animal species to be infected with a Ad2, Ad5 or CVB vims which normally does not infect cells of that type or species.
  • Ad2, Ad5 or CVB vims which normally does not infect cells of that type or species.
  • Examples of cells which are relatively resistant to CVB infection include WI-38 cells and other fibroblast- type cell lines such as various strains of NIH-3T3 mouse fibroblasts, mouse L cells, various strains of Chinese hamster ovary (CHO) )saroma origin and RD cells originating from human rhabdomyosarcoma. .
  • HCAR or MCAR HCAR or MCAR.
  • a vims it is possible to modify the receptor attachment site of a vims so that it will not bind to its natural receptor.
  • changes in the DNA sequence of Ad2 or Ad5 will render these vimses non-infective for HCAR-bearing cells.
  • Corresponding changes may be introduced into HCAR so that this mutant adenovims will bind to it.
  • a safer adenovims preparation can be generated which binds only to select cells bearing the appropriate mutant/variant receptor, but not to the normal targets of adenovims.
  • a human cell can be infected with "normal” or a corresponding mutant adenovims in vitro in a transient fashion, and can be manipulated by the judicious use of cytokine growth or differentiation factors.
  • Such cells can be introduced into a recipient.
  • a second vims which binds to a second genetically engineered receptor can be introduced into the individual to infect stably alter only those introduced cells bearing the second viral receptor.
  • the preferred animal subject of the present invention is a mammal.
  • the invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well.
  • the present invention includes soluble forms of HCAR or MCAR (or chimeric receptors), as well as functional derivatives thereof having similar bioactivity for all the uses described herein. Also intended are all active forms of HCAR or MCAR derived from the HCAR or MCAR transcript, and all muteins with HCAR or MCAR activity. Methods for production of soluble forms of receptors which are normally transmembrane proteins are well known in the art (see, for example, Smith, D.H. et al, Science 235:1704-1707
  • Such methods are generally based on truncation of the DNA encoding the receptor protein to exclude the transmembrane portion, leaving intact the extracellular domain (or domains) capable of interacting with specific ligands, such as an intact Ad2, Ad5 or CVB vims or a adenoviral protein or glycoprotein.
  • specific ligands such as an intact Ad2, Ad5 or CVB vims or a adenoviral protein or glycoprotein.
  • HCAR or MCAR or a functional derivative of HCAR or MCAR, comprise the elements of the binding site of the HCAR or MCAR that permits binding to a Ad2, Ad5 or CVB vims.
  • Ad2, Ad5 or CVB vims Of the many amino acid residues of HCAR or MCAR, only a few are critically involved in vims recognition and binding.
  • the HCAR or MCAR proteins or peptides of the present invention may be further modified for purposes of drug design, such as, for example, to reduce immunogenicity, to promote solubility or enhance delivery, or to prevent clearance or degradation.
  • “Functional derivative” of the HCAR or MCAR protein is defined as a "fragment,” “variant,” “analogue,” or “chemical derivative” of the HCAR or MCAR protein.
  • a functional derivative retains at least a portion of the function of the HCAR or MCAR protein which permits its utility in accordance with the present invention, preferably the capacity to bind to Ad2, Ad5, CVB or to a viral protein or glycoprotein which is responsible for vims binding to the receptor.
  • a "fragment" of the HCAR or MCAR protein is any subset of the molecule, that is, a shorter peptide.
  • variant of the HCAR or MCAR refers to a molecule substantially similar to either the entire protein or fragment thereof.
  • Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well- known in the art.
  • amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide.
  • variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final constmct possesses the desired activity.
  • the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA stmcture (see European Patent Publication No. EP 75,444).
  • these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al, DNA 2:183 (1983)) of nucleotides in the DNA encoding the HCAR or MCAR molecule or peptide , thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • Preferred variants exhibit the same qualitative biological activity as the nonvariant peptide.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al, Proc. Natl. Acad. Sci. (USA) 75:5765 (1978).
  • This primer is then annealed with the single-stranded protein-sequence-containing vector, and subjected to DNA- polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutant strand.
  • a mutated sequence in the second strand bears the desired mutation.
  • This heteroduplex vector is used to transform appropriate cells, and clones are selected that include recombinant vectors bearing the mutant sequence.
  • the mutant sequence encoding the mutant protein region may be removed and placed in an appropriate vector for protein expression in appropriate host cells.
  • variants involves a terminal insertion such that a signal sequence, whether heterologous or homologous to the host cell is fused to the N- terminus of the peptide molecule to facilitate the secretion of mature peptide molecule from recombinant hosts.
  • the natural signal sequences of HCAR or MCAR, as shown in Figure 2A may be used for this purpose.
  • substitution variants comprises a protein or peptide in which at least one amino acid residue, and preferably, only one, has been removed and a different residue inserted in its place.
  • substitutions are defined herein as exchanges within one of the following five groups:
  • Gly is the only residue lacking any side chain and imparts flexibility to the chain.
  • Pro because of its unusual geometry, tightly constrains the chain.
  • Cys participates in disulfide bond formation which is important for protein folding.
  • Tyr because of its hydrogen bonding potential, has some kinship with Ser, Thr, etc.
  • substitutions that are less conservative, such as between, rather than within, the above five groups, which will differ more significantly in their effect on maintaining (a) the stmcture of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions are (a) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (b) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, He, Phe, Val or Ala; (c) substitution of a Cys residue for (or by) any other residue;
  • substitution of a residue having an electropositive side chain e.g., Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g., Glu or Asp; or
  • deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect is evaluated by routine screening assays, either immunoassays or bioassays.
  • a variant typically is made by site-specific mutagenesis of the peptide-encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification of the protein or peptide from the cell culture supernatant or cell extracts, for example, by immunoaffinity chromatography using an immobilized antibody specific for the or the viral ligand for the receptor on a column.
  • the activity of a cell lysate containing HCAR or MCAR or a functional derivative thereof, or a purified preparation of HCAR or MCAR can be screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the protein molecule expressed as altered binding to a given antibody, is measured by a competitive type immunoassay (see below). Biological activity is screened in an appropriate bioassay, such as vims binding or infectivity, as described herein.
  • Modifications of peptide properties such as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
  • an “analogue” of the HCAR or MCAR protein refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a “chemical derivative” of the HCAR or MCAR protein contains additional chemical moieties not normally a part of the protein. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo- beta-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK ⁇ of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as l-cyclohexyl-3-(2- morpholinyl-(4-ethyl) carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • carbodiimides R'-N-C-N-R'
  • carbodiimides Rosinyl or glutamyl
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers.
  • Commonly used cross-linking agents include, e.g., l,l-bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patents 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • the recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques.
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
  • cDNA is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a "cDNA clone” means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • a "cDNA library” is a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire expressible genome of an organism. Such a cDNA library may be prepared by methods known to those of skill, and described, for example, in Sambrook et al, supra.
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene. Preferred for the purposes of the present invention are mammalian cell lines.
  • Oligonucleotides representing a portion of the HCAR or MCAR sequence are useful for screening for the presence of homologous genes and for the cloning of such genes. Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu, R., et al, Prog. Nucl. Acid. Res. Molec. Biol 27:101-141
  • the oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding an HCAR or MCAR fragment is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences.
  • An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the HCAR or MCAR gene or a yet unidentified homologous gene (Sambrook et al, supra).
  • a suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the HCAR or MCAR gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified, synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the HCAR or MCAR gene or putative homologue.
  • Single stranded oligonucleotide molecules complementary to the "most probable" HCAR or MCAR peptide coding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art (Belagaje, R., et al, J. Biol. Chem. 254:5765- 5780 (1979); Maniatis, T., et al, In: Molecular Mechanisms in the Control of Gene Expression, Nierlich, D.P., et al, Eds., Acad. Press, NY (1976); Wu, R., et al, Prog. Nucl. Acid Res. Molec. Biol.
  • DNA synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al. ⁇ supra), and by Haymes, B.D., et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)).
  • the HCAR or MCAR gene is expression cloning, wherein a library of expression vectors is prepared by cloning DNA, preferably, cDNA (from a cell capable of expressing HCAR or MCAR) into an expression vector.
  • the library is screened for members capable of expressing a protein which binds, for example, to an anti-HCAR or anti-MCAR antibody or a viral protein ligand, and which therefore has a nucleotide sequence capable of encoding polypeptides that have the same sequence as HCAR or MCAR proteins or peptides, or fragments thereof.
  • genomic DNA or cDNA is fragmented (by shearing, endonuclease digestion, etc.) to produce a pool of DNA fragments which are cloned into an expression vector in order to produce a genomic library (or cDNA library) of expression vectors whose members each contain a unique cloned DNA fragment.
  • Procedures for preparing cDNA and for producing a genomic library are disclosed by Sambrook et al. ⁇ supra).
  • a “vector” is a DNA molecule, derived from a plasmid or bacteriophage or animal vims, into which selected fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replication or integration into the DNA of its host such that the cloned sequence is reproducible.
  • An "expression vector” is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing the encoded peptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell.
  • the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.
  • the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences.
  • nucleic acid “functional derivative” which is defined as a polynucleotide or oligonucleotide which encodes a "fragment” "variant” or “analogue” of the native CAR protein.
  • a functional derivative may be “substantially similar” in nucleotide sequence to the CAR- encoding sequence and thus encode a protein possessing similar activity to the native protein.
  • a “functional derivative” of a polynucleotide can also be a chemical derivative which retains its functions. Such a chemical derivative is useful as a molecular probe to detect CAR-encoding sequences in nucleic acid hybridization assays.
  • a molecule is said to be "substantially similar” to another molecule if the sequence of amino acids or nucleotides in both molecules is substantially the same.
  • Substantially similar protein or peptide molecules will possess a similar biological activity.
  • two molecules possess a similar activity they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.
  • a DNA sequence encoding the CAR protein or functional derivative may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Sambrook, J. et al, supra, and are well known in the art.
  • a nucleic acid molecule such as DNA is "capable of expressing" a polypeptide if (a) it contains a nucleotide sequence which includes transcriptional and translational regulatory information and (b) such sequences are “operably linked” to the coding sequence.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • the precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region (which in prokaryotes contains both the promoter as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis).
  • Such regions will normally include those 5'- non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the coding sequence may be obtained by the above- described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3 '-region naturally contiguous to the DNA coding sequence , the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a different 3' region functional in the host cell may be substituted.
  • Two sequences of a nucleic acid molecule are said to be "operably linked” when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence.
  • two sequences such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence.
  • two sequences such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence.
  • the promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Suitable promoters are repressible, or, more preferably, constitutive. Strong promoters are preferred.
  • the present invention encompasses the expression of the HCAR or MCAR protein (or a functional derivative thereof) or a chimeric HCAR or MCAR protein in either prokaryotic or eukaryotic cells, although preferred expression is in eukaryotic cells, most preferably in human cells.
  • a prokaryotic cell such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.
  • a functional prokaryotic promoter examples of which are well-known in the art.
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325, etc.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage 1 (P L and P R ), the trp, recA, lacZ, lad, and gal promoters of E. coli (reviewed in Glick, B.R., J. Ind. Microbiol 7:277-282 (1987); Cenatiempo, Y. Biochimie 68:505-516 (1986); Gottesman, S. ⁇ Ann. Rev. Genet. 75:415-442 (1984).
  • Eukaryotic hosts include yeast, insects, fungi, and mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites.
  • Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO, or cells of lymphoid origin, such as the hybridoma SP2/O-Agl4 or the murine myeloma P3-X63Ag8, and their derivatives.
  • Prefe ⁇ ed mammalian cells are cells which are intended to replace the function of the genetically "deficient" cells in vivo.
  • Prefe ⁇ ed eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al, J. Mol. Appl. Gen. 7:273-288 (1982)); the TK promoter of Herpes vims (McKnight, S., Cell 37:355-365 (1982)); the SV40 early promoter (Benoist, C, et al, Nature (London) 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston, S.A., et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al, Proc. Natl.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovims, bovine papilloma vims, Simian vims, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, etc., may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.
  • any of a series of yeast gene expression systems can be utilized which incorporate promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in glucose-rich medium.
  • Known glycolytic genes can also provide very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.
  • Production of HCAR or MCAR molecules in insects can be achieved, for example, by infecting the insect host with a baculovims engineered to express HCAR or MCAR by methods known to those of skill (Jasny, Science 238: 1653 (1987).
  • the HCAR or MCAR coding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non- replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the HCAR or MCAR protein may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g. , antibiotics, or heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol. Cell. Biol. 3:280 (1983).
  • the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose.
  • Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, D., et al, Miami Winter. Symp. 79:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.
  • Prefe ⁇ ed vectors for transient expression of the HCAR or MCAR in CHO cells is the pSG5 or pCDM8 expression vector.
  • the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment (Johnston et al, Science 240:1538 (1988)), etc.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of HCAR or MCAR or the chimeric HCAR or MCAR protein and its expression on the cell surface.
  • the expressed HCAR or MCAR protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • HCAR or MCAR onto the transmembrane and intracytoplasmic portions of another molecule, resulting in yet another type of chimeric molecule.
  • the present invention is also directed to a transgenic non-human eukaryotic animal (preferably a rodent, such as a mouse) the germ cells and somatic cells of which contain genomic DNA according to the present invention which codes for the HCAR or MCAR protein or a functional derivative thereof capable as serving as a human Ad2, Ad5 or CVB vims receptor.
  • the HCAR or MCAR DNA is introduced into the animal to be made transgenic, or an ancestor of the animal, at an embryonic stage, preferably the one-cell, or fertilized oocyte, stage, and generally not later than about the 8-cell stage.
  • transgene means a gene which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of protein in the transgenic animal.
  • a gene can be introduced into the genome of the animal embryo so as to be chromosomally incorporated and expressed.
  • One method is to transfect the embryo with the gene as it occurs naturally, and select transgenic animals in which the gene has integrated into the chromosome at a locus which results in expression.
  • Other methods for ensuring expression involve modifying the gene or its control sequences prior to introduction into the embryo.
  • One such method is to transfect the embryo with a vector (see above) containing an already modified gene.
  • transgene is present in all of the germ cells and somatic cells of the transgenic animal and has the potential to be expressed in all such cells.
  • the presence of the transgene in the germ cells of the transgenic "founder" animal in turn means that all its progeny will carry the transgene in all of their germ cells and somatic cells.
  • Introduction of the transgene at a later embryonic stage in a founder animal may result in limited presence of the transgene in some somatic cell lineages of the founder; however, all the progeny of this founder animal that inherit the transgene conventionally, from the founder's germ cells, will cany the transgene in all of their germ cells and somatic cells.
  • Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the HCAR or MCAR DNA of the present invention, such as animals produced when fewer than all of the cells of the momla are transfected in the process of producing the transgenic mammal, are also intended to be within the scope of the present invention.
  • the techniques described in Leder, U.S. Patent 4,736,866 (hereby incorporated by reference) for producing transgenic non-human mammals may be used for the production of the transgenic non-human mammal of the present invention.
  • the animals carrying the HCAR or MCAR gene can be used to test compounds or other treatment modalities which may prevent, suppress or cure a human Ad2, Ad5 or CVB vims infection or a disease resulting from such infection for those Ad2, Ad5 or CVB vimses which infect the cells using the HCAR or MCAR molecule as a receptor.
  • These tests can be extremely sensitive because of the ability to adjust the vims dose given to the transgenic animals of this invention.
  • Such animals will also serve as a model for testing of diagnostic methods for the same human Ad2, Ad5 or CVB vims diseases.
  • Transgenic animals according to the present invention can also be used as a source of cells for cell culture.
  • This invention is also directed to an antibody specific for an epitope of HCAR or MCAR protein.
  • this antibody is used to prevent or treat Ad2, Ad5 or CVB vims infection, to detect the presence of, or measure the quantity or concentration of, HCAR or MCAR protein in or on the surface of a cell, or in a cell or tissue extract, or a biological fluid.
  • antibody is meant to include polyclonal antibodies and monoclonal antibodies (mAbs) (Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent 4,376,110); Hartlow, E. et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988); Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, NY (1980)); Campbell, A., In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R., et al, eds.), Elsevier, Amsterdam (1984).
  • mAbs monoclonal antibodies
  • a mAb may be of any immunoglobulin class including IgG, IgM, IgE, IgA and any subclass thereof.
  • the hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently prefe ⁇ ed method of production, and mAbs of isotype IgM or IgG may be purified from ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • This invention also includes humanized antibodies or chimeric antibodies (Morrison et al, Proc. Natl. Acad. Sci. USA 5/:6851-6855 (1984); Neuberger et al, Nature 314:268-270 (1985); Sun et al, Proc. Natl. Acad. Sci. USA 54:214-218 (1987); Better et al, Science 240:1041- 1043 (1988); Better, M.D. WO 91/07494).
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325
  • Fab and F(ab') 2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of HCAR or MCAR protein according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • Antibody molecules or fragments may be purified by known techniques, e.g., immunoabsorption or immunoaffinity chromatography (Hartlow et al, supra), chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof, etc.
  • the antibodies, or fragments of antibodies, of the present invention may be used to quantitatively or qualitatively detect the presence of cells which express HCAR or MCAR protein (or a chimeric receptor having an HCAR or MCAR- derived epitope) on their surface or intracellularly. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • the antibodies of the present invention may be employed histo logically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of HCAR or MCAR protein.
  • In situ detection may be accomplished by removing a histo logical (cell or tissue) specimen from a subject and providing the a labeled antibody of the present invention to such a specimen.
  • a histo logical (cell or tissue) specimen from a subject and providing the a labeled antibody of the present invention to such a specimen.
  • histological methods such as staining procedures
  • the antibody of the present invention can be used to detect the presence of soluble HCAR or MCAR molecules in a biological sample. Used in this manner, the antibody can serve as a means to monitor the presence and quantity of HCAR or MCAR proteins or derivatives used therapeutically in a subject to prevent or treat human Ad2, Ad5 or CVB vims infection.
  • Immunoassays for HCAR or MCAR protein typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leucocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying HCAR or MCAR protein, and detecting the antibody by any of a number of techniques well-known in the art.
  • the biological sample may be contacted with a solid phase support or carrier (which terms are used interchangeably herein) such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled CAR-specific antibody.
  • the solid phase support may then be washed with the buffer to remove unbound antibody.
  • the amount of bound label on the solid support may then be detected by conventional means.
  • solid phase support or “carrier” is intended any support capable of binding an antigen or an antibody.
  • supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible stmctural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • suitable carriers for binding antibody or antigen or will be able to ascertain the same by use of routine experimentation.
  • the binding activity of a given lot of anti-HCAR or MCAR antibody may be determined according to weir known methods.
  • Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.
  • a prefe ⁇ ed type of immunoassay to detect an antibody specific for a CAR protein is an enzyme-linked immunosorbent assay (ELISA) or more generally termed an enzyme immunoassay (El A).
  • ELISA enzyme-linked immunosorbent assay
  • El A enzyme immunoassay
  • a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the reagents useful in the present invention include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, ⁇ -galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, ⁇ -5- steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detectable label may be a radiolabel, and the assay thus used termed a radioimmunoassay (RIA), which is well known in the art. See, for example, Yalow, R. et al, Nature 184:1648 (1959); Work, T.S., et al, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, 1978, incorporated by reference herein.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes which are particularly useful for the purpose of the present invention are 125 1, 131 1, 35 S, 3 H and 14 C.
  • fluorophore it is also possible to label the antigen or antibody reagents with a fluorophore.
  • fluorescently labeled antibody When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence of the fluorophore.
  • fluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, fluorescamine or fluorescence-emitting metals such as 152 Eu or other lanthanides. These metals are attached to antibodies using metal chelators.
  • the antigen or antibody reagents useful in the present invention also can be detectably labeled by coupling to a chemiluminescent compound.
  • a chemiluminescent-tagged antibody or antigen is then determined by detecting the luminescence that arises during the course of a chemical reaction.
  • useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound such as a bioluminescent protein may be used to label the antigen or antibody reagent useful in the present invention. Binding is measured by detecting the luminescence.
  • Useful bioluminescent compounds include luciferin, luciferase and aequorin.
  • Detection of the detectably labeled reagent according to the present invention may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorophore.
  • the detection can be accomplished by colorimetry which employs a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • the immunoassay of this invention may be a "two-site” or “sandwich” assay.
  • a first antibody specific for an epitope of the CAR protein is adsorbed to a solid support.
  • a quantity of detectably labeled soluble second antibody is added to permit detection and/or quantitation of the ternary complex formed between solid- phase antibody, antigen, and labeled antibody.
  • Sandwich assays are generally described by Wide, Radioimmune Assay Method, Kirkham et al, Eds., E. & S. Livingstone, Edinburgh, 1970, pp. 199-206.
  • HCAR or MCAR cellular receptor protein
  • the protein or functional derivative may be adsorbed to an insoluble support or carrier, as in an immunoassay.
  • the biological sample e.g., semm, suspected of containing Ad2, Ad5 or CVB vims is then contacted with the HCAR or MC AR-containing support and the vims allowed to bind to its receptor material.
  • the presence of the bound vims is then revealed in any of a number of ways well known in the art, for example, by addition of a detectably-labeled antibody specific for the vims.
  • the same assay can be used to detect the presence in a biological sample of a viral component such as a viral protein or glycoprotein which has affinity for the HCAR or MCAR protein.
  • the vims or viral protein may be labeled and binding measured in a competitive assay using an antibody specific for the vims-binding portion of the HCAR or MCAR molecule.
  • prevention of infection involves administration of the HCAR or MCAR protein, peptide derivative, or antibody (see above) prior to the clinical onset of the disease.
  • successful administration of a composition prior to initial contact with a Ad2, Ad5 or CVB vims results in "prevention" of the disease.
  • Administration may be after initial contact with the vims, but prior to actual development of the disease.
  • Treatment involves administration of the protective composition after the clinical onset of the disease.
  • successful administration of a HCAR or MCAR protein or peptide or anti-HCAR or MCAR antibody according to the invention after development of a Ad2, Ad5 or CVB vims infection in order to delay or suppress further vims spread comprises "treatment" of the disease.
  • the HCAR or MCAR protein, peptides or antibody of the present invention may be administered by any means that achieve their intended purpose, for example, to treat local infection or to treat systemic infection in a subject who has, or is susceptible to, such infection.
  • an immunosuppressed individual is particularly susceptible to viral infection and disease.
  • administration may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, intracranial, transdermal, or buccal routes.
  • administration may be by the oral route.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • compositions of the present invention are by topical application, primarily for disinfectant uses to minimize the risk of entry of Ad2, Ad5 or CVB vimses via the skin.
  • the proteins, peptides and pharmaceutical compositions of the present invention may be incorporated into topically applied vehicles such as salves or ointments as a means for administering the active ingredient directly to the affected area.
  • the carrier for the active ingredient may be either in sprayable or nonsprayable form.
  • Non- sprayable forms can be semi-solid or solid forms comprising a carrier conducive to topical application and having a dynamic viscosity preferably greater than that of water.
  • Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like.
  • Prefe ⁇ ed vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol- 1000 (PEG- 1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material.
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the proteins or peptides of the present invention.
  • the active principles in accordance with the present invention may be packaged in a squeeze bottle, or in a pressurized container with an appropriate system of valves and actuators.
  • metered valves are used with the valve chamber being recharged between actuation or dose, all as is well known in the art.
  • a compound according to the present invention for topical applications, it is prefe ⁇ ed to administer an effective amount of a compound according to the present invention to an infected area, e.g., skin surfaces, mucous membranes, etc. This amount will generally range from about
  • a prefe ⁇ ed topical preparation is an ointment wherein about 0.01 to about 50 mg of active ingredient is used per cc of ointment base, the latter being preferably PEG- 1000.
  • a typical regimen for preventing, suppressing, or treating Ad2, Ad5 or CVB vims infection comprises administration of an effective amount of the HCAR or MCAR protein or functional derivative thereof, administered over a period of one or several days, up to and including between one week and about six months.
  • the dosage administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concu ⁇ ent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the ranges of effective doses provided below are not intended to be limiting and represent prefe ⁇ ed dose ranges. However, the most prefe ⁇ ed dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the protein, functional derivative thereof or antibody may be administered alone or in conjunction with other therapeutics directed to the viral infection, or directed to other symptoms of the viral disease.
  • Effective amounts of the HCAR or MCAR protein, functional derivative thereof, or antibody thereto are from about 0.01 ⁇ g to about 100 mg/kg body weight, and preferably from about 10 ⁇ g to about 50 mg/kg body weight.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art.
  • Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods.
  • Pharmaceutical compositions comprising the proteins, peptides or antibodies of the invention include all compositions wherein the protein, peptide or antibody is contained in an amount effective to achieve its intended pu ⁇ ose.
  • the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • compositions include suitable solutions for administration by injection or orally, and contain from about 0.01 to 99%, preferably from about 20 to 75% of active component ⁇ i.e., the HCAR or MCAR protein, peptide or antibody) together with an excipient.
  • Pharmaceutical compositions for oral administration include tablets and capsules.
  • Compositions which can be administered rectally include suppositories.
  • the present invention provides methods for evaluating the presence and the level of normal or mutant HCAR or MCAR protein or mRNA in a subject. Absence, or more typically, low expression of the HCAR or MCAR gene or presence of a mutant HCAR or MCAR in an individual may serve as a predictor of resistance to Ad2, Ad5 or CVB vims infection. Alternatively, over-expression of HCAR or MCAR, may serve as a predictor of enhanced susceptibility to Ad2, Ad5 or CVB vims infection.
  • Oligonucleotide probes encoding any of a number of segments of the HCAR or MCAR DNA sequence are used to test cells from a subject for the presence HCAR or MCAR DNA or mRNA.
  • a prefe ⁇ ed probe would be one directed to the nucleic acid sequence encoding at least 12 and preferably at least 15 nucleotides of the HCAR or MCAR sequence.
  • Qualitative or quantitative assays can be performed using such probes. For example, Northern analysis (see below) is used to measure expression of an HCAR or MCAR mRNA in a cell or tissue preparation.
  • Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques.
  • Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments are well-known and typically involve introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment (U.S. Patent 4,237,224; Sambrook et al. ⁇ supra))
  • a method based on polymerase chain reaction is used to selectively increase the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.
  • the method can be used to amplify either single- or double-stranded DNA.
  • the oligonucleotide sequences of the probes of the PCR method are selected such that they contain sequences identical to, or complementary to, sequences which flank the particular nucleic acid sequence whose amplification is desired.
  • the oligonucleotide sequences of the "first" probe is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3' to the desired sequence
  • the oligonucleotide sequence of the "second” probe is selected such that it contains an oligonucleotide sequence identical to one present 5' to the desired region.
  • Both probes possess 3' hydroxy groups which permits primer extension.
  • the PCR reaction is capable of exponential amplification of specific nucleic acid sequences because the extension product of the "first" probe, of necessity, contains a sequence which is complementary to a sequence of the "second” probe, and thus can serve as a template for the production of an extension product of the "second” probe.
  • the extension product of the "second" probe contains a sequence which is complementary to a sequence of the "first" probe, and thus can serve as a template for the production of an extension product of the "first” probe.
  • a geometric increase in the concentration of the desired nucleic acid molecule can be achieved. Reviews of the PCR are provided by Mullis, K.B. ⁇ Cold Spring Harbor Symp. Quant. Biol. 57:263-273 (1986)); Saiki, R.K., et al. ⁇ Bio/Technology 3:1008-1012 (1985)); and Mullis, K.B., et al. ⁇ Meth. Enzymol.
  • Oligonucleotide probes co ⁇ esponding to a portion of the HCAR or MCAR DNA sequence are useful in the new method of reverse transcriptase-PCR ("RT- PCR"). Isolated mRNA is reverse-transcribed into DNA, and then the DNA fragments co ⁇ esponding to HCAR or MCAR are specifically amplified by PCR.
  • RT-PCR permits comparison the patterns of expression of HCAR or MCAR with another gene.
  • RT-PCR for the detection of circulating tumor cells is as a potential technique for detecting or staging cancer involving HCAR or MCAR-expressing cells.
  • TCMK-1 RNA selected twice with oligo was used to constmct a cDNA library in the ⁇ ZAP Express® vector using the ZAP Express cDNA synthesis kit (Stratagene).
  • a library of 4x10 5 primary clones was amplified once in the XLl-Blue MRF' strain of E. coli.
  • the library was plated at a density of 5x10 4 PFU on 150mm diameter NZY agar plates and overlaid with nitrocellulose filters that had been wet in sterile dH 2 0 containing 10 mM IPTG (isopropylthio- ⁇ -D-galactoside) after incubation for 2.5 hours at 42°C.
  • the plates were transfe ⁇ ed to 37°C for 8 hours before the filters were removed and washed once in Tris buffered saline to remove agarose and bacteria. Thereafter, filters were assayed for the presence of the expressed protein product with anti-p46 antiserum (Xu, R. et al, supra) using the BCIP/NBT detection system.
  • 3xl0 5 NIH3T3 cells were seeded into 35mm plates 24 hours prior to transfection. Forty- five minutes prior to transfection, a solution was prepared by mixing 2 ⁇ g of supercoiled plasmid diluted in lOO ⁇ l of DMEM (Gibco) and 7 ⁇ l of lipofectamine (Life Technologies, GIBCO) diluted in lOO ⁇ l of DMEM. Following incubation at room temperature, 800 ⁇ l of DMEM was added and the solution overlaid onto plates that were washed twice with DMEM. The plates were incubated for 5 hours at 37°C in 5% CO 2 before the solution was removed and 2 ml of DMEM containing 10% fetal calf serum (FCS) was added. The plates were incubated for an additional 18 hours at 37°C. Cells were used 18-24 hours post-transfection for experiments.
  • FCS fetal calf serum
  • Vims Assays Twenty hours following transfection, 35mm plates containing 5xl0 5 cells were washed twice, and 200 ⁇ l DMEM containing lxl 0 7 pfii of vims were added. Plates were incubated for 90 minutes at room temperature before being washed 4 times with 2ml of DMEM. Plates were overlaid with 5ml DMEM/ 10% FCS and twenty- four hours later alternately frozen and thawed three times. Cellular debris was removed by centrifugation, and dilutions of the resulting supernatants evaluated for vims content by plaque assay (Crowell, R.L. et al, J. Exp. Med. 773:419-435 (1961)).
  • Immunofluorescence lxl 0 6 transfectant cells were incubated in 100 ⁇ l of IX PBS containing 2 ⁇ g of RmcB for 1 hour at 4°C. Cells were washed three times with IX PBS and incubated for 30 minutes at 4°C in 100 ⁇ l of IX PBS containing 0.35 ⁇ g of a goat anti-mouse FITC conjugated antibody. Fluorescence was monitored using a Zeiss Axioplan fluorescent microscope.
  • the present inventors employed the classical approach of screening a ⁇ phage expression cDNA library with an antiserum containing antibodies specific for a mouse CVB binding protein.
  • the protein is termed p46 and the antibodies, anti-p46 (Xu, R. et al, Vir. Res. 35:323-340 (1995)).
  • This antibody has several properties that indicate its specificity for the viral receptors sought. First, it protects mouse cells from infection with CVB. Second, the antibody specifically identified a 46 kDa protein from vims-susceptible mouse cells (Xu, R. et al, Vir. Res. 35:323-340 (1995)).
  • RTMCAR-4 a 1 kb cDNA clone (RTMCAR-4) was isolated that hybridized in Northern blots with (6kb and 1.4kb RNA's from TCMK-1 cells that were not found in receptor-negative mouse L cells (Hsu K-H. L. et al, J. Virol. 63:3105-8 (1989)) ( Figure 1).
  • RTMC AR-4 revealed two IMAGE consortium clones from newborn human melanocyte (265680) and pancreas islet cell (328668) cDNA libraries that were significantly homologous to RTMCAR-4. These findings suggested the human clones may encode a homologue of RTMCAR-4.
  • clone 265680 was purchased from the American Type Culture Collection and the nucleotide and deduced amino acid sequence compared to that ofRTMCAR-4. Based upon best fit alignments, both 265680 and RTMCAR-4 contained single open reading frames that exhibited 67% amino acid identity. Clone 265680 was slightly larger, containing an additional 34 amino acids N-terminal of the protein encoded by RTMCAR-4.
  • Vims Receptor cDNA size (approx) Number of amino Molecular mass of acid residues encoded protein (approx.)
  • HCAR and MCAR are Functional Viral Receptors
  • HCAR and MCAR cDNA's encode functional receptors, they were inserted into pBK-CMV vectors, transfected into NIH3T3 cells.
  • the plasmid pRTHR contains a 1. lkb HCAR cDNA that lacks the 3 'UTR.
  • the plasmid pRTMR contains the complete 1.4kb MCAR cDNA. Transfectants were assayed for vims production or reporter gene expression..
  • the transfected cells upon viral infection, yielded 10 2 - to 10 3 -fold higher titers of infectious Coxsackie vims, CVB3 and CVB4, than did cells transfected with control plasmid (pBK-CMV) alone.
  • the infectability was specific for CVB, since no increase in titer was seen when another picornavirus family member, poliovims Tl, was used (Table 3).
  • HCAR and MCAR are specific adenovims binding proteins, since 35 S-Ad3 (a group B adenovims), which does not compete with group C adenovimses for the same receptor (Defer, C. et al, J. Virol. 64:3661-3673 (1990)), exhibits 5-fold less binding to transfectants than does 35 S-Ad2.
  • the binding of labeled vims could be competed with cold vims, as previously described (Lonberg-Holm et al, supra).
  • these results confirm that HCAR and MCAR are the receptors for Ad2, A5, and the CVB.
  • HCAR-specific mAb did not react with the MCAR product, and the anti-MCAR serum did not react with the HCAR product, as previously reported (Crowell, R.L. et al, In: Cellular Receptors for Animal Viruses, Wimmer, E., ed., Cold Spring Harbor Press, New York, 1994, pp. 75-79 .
  • RNA's of about 6kb and about 1.4 kb from TCMK-1 cells identified RNA's of about 6kb and about 1.4 kb from TCMK-1 cells, and a range of RNA's from about 6kb to about 1.3 kb from HeLa cells (Figure 5).
  • RNA's of the respective receptor-negative cells of human (rhabdomyosarcoma) or murine (L cell) origin Neither fragment hybridized to RNA's of the respective receptor-negative cells of human (rhabdomyosarcoma) or murine (L cell) origin.
  • the larger mRNA's may be incompletely processed transcripts or RNA's that are related to the receptors by sequence homology. Nonetheless, the sizes of the identified cDNA's co ⁇ elate with sizes of RNA's detected by these hybridizations ( Figure 5), indicating they are tmly representative of the parental RNA species.
  • RNA's from multiple human tissues or mouse tissues were probed with HCAR probes or MCAR probes, respectively, in Northem blots.
  • the pancreas, brain, heart, small intestine, testis, and prostate had the highest amount of HCAR message (Figure 6).
  • the liver and lung had small amounts of HCAR mRNA. No signal was detected in kidney, placenta, peripheral blood leukocytes, thymus, and spleen. In contrast, in mouse tissue, the highest level of MCAR RNA was observed in liver ( Figure 7).
  • Mouse kidney, heart, lung and brain tissue also expressed detectable levels of MCAR RNA.
  • Isolation of the genomic sequences for both HCAR and MCAR will provide insight into the regulation of their expression which is useful information for identifying possible targets for adenovims gene therapy or devising strategies to inhibit adenovims and CVB infectivity.
  • the hydrophobicity profile for the HCAR and MCAR sequences revealed two strongly hydrophobic stretches: fourteen N-terminal residues, a hypothetical signal peptide, and residues 239-259, a possible transmembrane helix.
  • a sensitive sequence search (Abagyan, R.A., et al. Submitted to J. Mol. Biol. (1996)) of fragment 15-235 through the Swissprot database and the NCBI non-redundant database revealed a number of similarities with membrane-bound proteins, most of them containing Ig-like domains.
  • IGI Ig-like domain
  • the sequence of IGI and IG2 domains was threaded onto ltet and 3cd4; the initial sequence alignment was adjusted to make it consistent with the immunoglobulin topology.
  • the side-chains were placed by global energy minimization (Abagyan, R.A., et al. J.Comp.Chem., 75:488-506 (1994)).
  • the cytoplasmic domain was found similar to a number of so called Ser-Pro-Thr-rich domains, which are found in a TFG gene related to the papillary thyroid carcinoma and p68 TRK-T3 oncoprotein. No related proteins with known function or 3D stmcture were found.
  • RKKRREEKY fragments 265-273 in SEQ ID NO:2 and SEQ ID NO:4
  • Tyrosine 269 may be involved in signal transduction via tyrosine phosphorylation and dephosphorylation.
  • Three other tyrosines do not have the required upstream sequence pattern, and are less likely, although still possible, candidates for signal transduction.
  • To build a 3D model of the IGI domain we used homology to the heavy chain of an antibody (ltet) with known 3D stmcture. The rest of the extracellular domain was built by homology with CD4 (3cd4).
  • CD4 3cd4
  • ICAM-1 (Olson, N.H., et al. Proc. Natl. Acad. Sci. USA, 90:507-511 (1993)), showed that the monomeric N-terminal Ig-like domain DI of ICAM-1 contains the primary binding site which interacts with the canyon of HRV16.
  • Two Ig-like amino-terminal domains of CD4 receptor of the HIV-1 for which the stmcture has been determined at atomic resolution Wang, J.H., et al. Nature, 345:411-418
  • Receptor interaction with the adenovims fiber knob Xia et al. (Xia, D., et al. Structure, 2:1259-1270 (1994)) solved the stmcture of the carboxy-terminal knob domain of the Ad5 fiber at 1.7 A resolution and suggested that both the central surface depression surrounding the three-fold axis ( Figure 9) and the side valleys are conserved and are candidates for binding sites of the cellular receptor.
  • MCAR transcripts were analyzed by reverse transcriptase PCR (RT-PCR).
  • RT-PCR reverse transcriptase PCR
  • Total RNA extracted from various tissues of a 3 week old female Balb/C mouse was reverse transcribed, and the resulting cDNA PCR was amplified using a 3'- anchor primer and a 5 '-primer specific for MCAR.
  • Several amplified products were obtained and subjected to DNA sequencing. Three distinct sequences were identified that give rise to alternate receptor carboxyl termini. Two of the sequences appear to be present in all tissues analyzed. The third has been isolated only from heart.
  • the amino acid sequence of these alternate forms is likely to be conserved from residues 1- 339 of SEQ ID NO:2 or SEQ ID NO:4. Following lysine residue 339, the sequences deviate among the alternate forms, presumably through alternate splicing.
  • CAGTCTCCCC CAAATTAGTA CAGAAATATC CATGACAAAA TTACTTACGT ATGTTTGTAC 1585
  • GGC TCC CTT CCA CTA CAG TTT GAA TGG CAG AAA CTG TCG GAC TCC CAG 702 Gly Ser Leu Pro Leu Gin Phe Glu Trp Gin Lys Leu Ser Asp Ser Gin 170 175 180

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Abstract

Les molécules protéiques HCAR et MCAR sont, respectivement, des protéines humaines et des protéines de souris servant de récepteurs cellulaires pour les adénovirus des sérotypes 2 et 5 (sous-groupe C) et pour les virus Coxsackie du groupe B. L'invention se rapporte à des molécules d'ADN codant ces protéines ou codant des dérivés fonctionnels de ces dernières, à des cellules hôtes transformées par les molécules d'ADN et à des procédés permettant de produire les protéines recombinées et leurs dérivés. L'invention concerne aussi ces protéines, les fragments peptidiques de ces protéines qui correspondent à leurs domaines extracellulaires, ainsi que d'autres dérivés fonctionnels, en particulier les oligopeptides qui se lient au virus. Les protéines HCAR ou MCAR isolées ou des fragments ou des variants de ces dernières sont utilisées pour prévenir ou traiter les infections virales. L'expression de l'ADN précité dans des cellules qui ne possèdent pas ces récepteurs viraux rendent les cellules susceptibles d'être transformées par des vecteurs d'adénovirus portant des gènes de thérapie génique.
PCT/US1998/001724 1997-01-30 1998-01-30 Recepteurs cellulaires des adenovirus du sous-groupe c et des virus coxsackie du groupe b WO1998033819A1 (fr)

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EP1017716A1 (fr) * 1997-09-19 2000-07-12 The General Hospital Corporation Recepteurs car, molecules et procedes apparentes
EP1132396A4 (fr) * 1998-11-13 2002-05-08 Kyowa Hakko Kogyo Kk Peptides actifs physiologiquement
EP1132396A1 (fr) * 1998-11-13 2001-09-12 Kyowa Hakko Kogyo Co., Ltd. Peptides actifs physiologiquement
US6794186B2 (en) * 2000-07-07 2004-09-21 Novartis Ag pCAR and its uses
WO2002019813A3 (fr) * 2000-09-07 2002-09-26 Index Pharmaceuticals Ab Procede permettant d'obtenir un mammifere non humain susceptible de fournir un gene a mediation par adenovirus, procede permettant de fournir un tel gene, et mammifere non humain susceptible de fournir un tel gene
WO2002019813A2 (fr) * 2000-09-07 2002-03-14 Index Pharmaceuticals Ab Procede permettant d'obtenir un mammifere non humain susceptible de fournir un gene a mediation par adenovirus, procede permettant de fournir un tel gene, et mammifere non humain susceptible de fournir un tel gene
WO2002070744A2 (fr) * 2001-03-07 2002-09-12 Galapagos Genomics B.V. Test de criblage de banque adénovirale destiné à des procédés et à des gènes régulateurs e2f, et compositions destinées au criblage de composés
WO2002070744A3 (fr) * 2001-03-07 2003-10-09 Galapagos Genomics B V Test de criblage de banque adénovirale destiné à des procédés et à des gènes régulateurs e2f, et compositions destinées au criblage de composés
US7365160B2 (en) * 2002-02-19 2008-04-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Mutated constitutively active nuclear orphan receptor
US20110294986A1 (en) * 2004-04-28 2011-12-01 Eisai R&D Management Co., Ltd. Methods For Detecting Th1 Cells
US20130231466A1 (en) * 2004-04-28 2013-09-05 Eisai R&D Management Co., Ltd. Methods for detecting th1 cells
US9175073B2 (en) 2004-04-28 2015-11-03 Eisai R&D Management Co., Ltd. Methods for detecting Th1 cells
US9200066B2 (en) 2004-04-28 2015-12-01 Eisai R&D Management Co., Ltd. Methods for detecting Th1 cells
US9453070B2 (en) 2004-04-28 2016-09-27 Eisai R&D Management Co., Ltd. Methods for detecting Th1 cells
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