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WO1993025682A9 - Polypeptides recepteurs mutants, proteines h13 humaines et leurs utilisations - Google Patents

Polypeptides recepteurs mutants, proteines h13 humaines et leurs utilisations

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Publication number
WO1993025682A9
WO1993025682A9 PCT/US1993/005569 US9305569W WO9325682A9 WO 1993025682 A9 WO1993025682 A9 WO 1993025682A9 US 9305569 W US9305569 W US 9305569W WO 9325682 A9 WO9325682 A9 WO 9325682A9
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WIPO (PCT)
Prior art keywords
chimeric receptor
cell
leu
receptor polypeptide
ala
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PCT/US1993/005569
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English (en)
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WO1993025682A3 (fr
WO1993025682A2 (fr
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Priority to EP93915287A priority Critical patent/EP0644936A1/fr
Priority to JP50172794A priority patent/JP3623230B2/ja
Priority to AU45322/93A priority patent/AU4532293A/en
Priority to US08/132,990 priority patent/US5834589A/en
Publication of WO1993025682A2 publication Critical patent/WO1993025682A2/fr
Publication of WO1993025682A9 publication Critical patent/WO1993025682A9/fr
Publication of WO1993025682A3 publication Critical patent/WO1993025682A3/fr
Priority to US08/912,332 priority patent/US7071301B1/en

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  • the present invention in the field of virology and molecular genetics, relates to nucleic acid, methods and proteins involving mammalian mutant viral receptor polypeptides, which bind viruses and/or envelope protein binding domains thereof, as well as to methods of making and using thereof, including diagnostic and therapeutic applications, such as gene therapy.
  • Viruses infect a cell by first attaching to a cell at a viral receptor.
  • a number of virus-specific cellular receptors have been identified, and most of these receptor molecules have other known cellular functions.
  • the expression of these virus binding proteins or receptors is a strong determinant of susceptibility to virus infection. Binding is required for fusion of the virus envelope protein (env) to the target cell, an event that may occur at the cell surface or within an acidified endosome after receptor-mediated endocytosis (White et al., Quaint . Rev. Biophys . 16: 151-195 (1983)). After fusion, the virion core enters the cytoplasm and the viral replication process is initiated. For example, in the case of HIV, recent studies suggested that cell surface molecules other than CD4 may also be important for virus entry into human cells.
  • Susceptibility of cells to infection with ecotropic (species specific) or amphotropic (species non-specific) murine leukemia virus (E-MuLV) may also be determined by binding of the virus envelope to a membrane receptor.
  • MuLV receptors Based on viral interference assays, four types of specific MuLV receptors have been postulated: (a) receptors for E-MuLV; (b) receptors for wild-type amphotropic MuLV; (c) receptors for recombinant viruses derived from E-MuLV, such as the "mink cell focus-inducing" or MCF virus; and (d) receptors for a recombinant virus derived from an amphotropic MuLV (Rein, A. et al.. Virology 136:144-152 (1984)). Recently, a cDNA clone (termed Wl) encoding the murine ecotropic retroviral receptor (ERR) (SEQ ID NO:4) was identified (Albritton, L.W. et al., Cell 57:659-666 (1989)). This study demonstrated that susceptibility to E-MuLV infection was acquired by the expression of a single mouse gene in human EJ cells.
  • ERR murine ecotropic retroviral receptor
  • HIV is an example of a virus exhibiting receptor- mediated tissue restriction, apparently based on its use of the CD4 protein as its primary receptor.
  • cell- specific receptors are unlikely to be the sole determinant of tissue specificity.
  • the tissue tropism of retroviruses is likely to result from a complex series of factors, such as the tissue specificity of long terminal repeats, variations in viral env proteins, cellular factors, and the expression of appropriate cell surface receptors (Kabat, Curr. Top. Microbiol . Immunol . 148:1-31 (1989)) .
  • the tea (T cell early activation, SEQ ID NO:5) gene as exemplified by clone 20.5 (MacLeod et al., 1990, J. Biol . Chem. 1:271-279) , is the first example of a cloned gene or cDNA that has the potential to encode a multiple transme brane-spanning protein which is induced during T cell activation (Crabtree, Science 243:355-361 (1989)).
  • the func ⁇ tion of the tea gene is not yet known.
  • the sequence of 20.5 cDNA (SEQ ID NO:5) was found to be strikingly homologous to the murine ERR cDNA clone (SEQ ID NO: 3) discussed above (the Rec-l gene) .
  • amphotropic retrovirus- mediated gene transfer (Gilboa, E., Bio-Essays 5:252-258 (1987); Williams, D.A. et al., Nature 310:476-480 (1984); Weiss, R.A. et al., .RNA Tumor Viruses, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1985) .
  • Recombinant amphotropic retroviruses have been studied with the possibility of being used as vector for the transfer of genes into human cells (Cone, R.D. et al., Proc. Natl . Acad. Sci . USA 81:6349-6353 (1984); Danos, 0. et al.
  • amphotropic viruses such as murine amphotropic E-MuLV can infect human cells.
  • amphotropic retroviruses that have been rendered replication-defective are sometimes capable of generating wild-type variants through recombinational events which provide replication ability. Such an alteration could lead to the widespread retroviral infection in cells and tissues which were not intended to be genetically modified.
  • the present invention is intended to overcome one or more deficiencies of the related background art.
  • the present invention is also intended to provide new and/or superior methods for gene therapy, wherein nucleic acid having a therapeutic and/or diagnostic effect can be delivered to target cells with improved safety and/or selectivity, with reduced nonspecific cell incorporation or association of the nucleic acid.
  • Such selective gene therapy may be provided according to the present invention by using vectors having viral binding domains which may bind chimeric receptor polypeptides of the present invention; which may be associated with selected target cells.
  • the target cell is made selective for a viral binding domain of a species other than the species of the target cell by association with a chimeric receptor polypeptide.
  • the mutant receptor polypeptide comprises a viral receptor binding site of the same species as the target cell, as a first species, which binding is modified to also bind a non-first species virus.
  • the present invention is also intended to provide chimeric receptor polypeptides, corresponding to at least the binding portion of a first species-specific virus receptor, which is modified to allow binding of, and/or infection by, a non-first species-specific virus or at least the bindings of the envelope protein (env) binding domain thereof.
  • the amino acid sequence of the chimeric receptor polypeptide including the modification in the viral receptor binding site of the chimeric receptor polypeptide is termed the chimeric receptor binding site.
  • the use of the sequence regardless of its origin, is embodied in the concept of a chimeric receptor.
  • a chimeric receptor polypeptide may also contain a binding site sequence of a first species viral receptor which is modified to provide at least one amino acid substitution, deletion or addition, corresponding to at least one amino acid of a viral receptor binding site of a non-first species specific virus.
  • the substitution, deletion or addition confers the ability of the chimeric receptor polypeptide to bind an env binding domain of the non-first species specific virus.
  • the first species is selected from a human, primate or rodent species.
  • a chimeric receptor polypeptide is intended to be provided as an isolated, purified, recombinant and/or organically synthesized polypeptide.
  • the resulting chimeric receptor polypeptide, or a chimeric receptor cell expressing, or associated at its surface with, the chimeric receptor polypeptide is thus capable of binding, and/or being infected by, a non-first species specific virus, or a binding domain thereof, in vivo, in situ, or in vitro.
  • the present invention is therefore intended to include all compounds, compositions, and methods of making and using such chimeric receptor polypeptides, without undue experimentation, based on the teachings and guidance presented herein.
  • a chimeric receptor polypeptide of the present invention is further intended to be used to selectively target cells expressing, or associated at their surface with, a chimeric receptor binding site of the chimeric receptor polypeptide for diagnostic, research or therapeutic applications, such as human gene therapy or cancer treatment or diagnosis, as non-limiting examples.
  • a chimeric receptor polypeptide may be used to provide selective binding of non-first species specific viruses, or an env binding domain thereof, to target cells or tissues expressing or associated with a chimeric receptor polypeptide.
  • a non- first species specific virus, or a conjugate containing an env binding domain thereof may be used as a target cell specific delivery vector to deliver at least one diagnostic or therapeutic agent to the target cell or tissue.
  • the invention also is intended to provide nucleic acid coding for, and/or cells or tissues expressing, such a chimeric receptor polypeptide.
  • the present invention is thus intended to provide delivery vectors, containing one or more therapeutic and/or diagnostic agents, including vectors suitable for gene therapy, having an improved measure of safety compared to related, art approaches.
  • the present invention permits the use of cell or tissue specific delivery vectors for therapeutic and/or diagnostic agents which substantially bind only target cells as chimeric receptor cells or tissues expressing cell surface chimeric receptor polypeptides, wherein the delivery vectors may have substantially reduced or substantially no binding to and/or infecting of non-target cells or tissues.
  • the present invention is also intended to provide gene therapy to selected cells or tissues of a eukaryote, such as a mammal, bird, insect or yeast, in vitro, in situ, or in vivo, through the use of transient or constitutive expression of a chimeric receptor polypeptide associated with, or on the surface of, resulting chimeric receptor cells of a first species.
  • a eukaryote such as a mammal, bird, insect or yeast
  • Such chimeric receptor cells or tissues are thus rendered susceptible to exclusive infection or binding by non- first species specific viral vectors, or env binding domains thereof.
  • the infection of the non- uman specific viral vector or binding domain may stably integrate a gene, such as a therapeutic gene, into the selected cells or tissues in vivo, in vi tro, or in situ, such that the selected cells or tissues express the gene product having the desired effect on the target cell or tissues.
  • a gene such as a therapeutic gene
  • the present invention is also intended to provide gene therapy to specific target cells or tissues, with substantially no or substantially reduced non-target cell viral vector infection and/or binding.
  • the present invention is further intended to provide gene therapy having substantially no, or substantially reduced reversion or recombination to replication competence or viral packaging of viral vectors used for delivering the therapeutic agent of the gene therapy.
  • the target cells may express or be associated with a chimeric receptor polypeptide of the present invention.
  • the present invention is additionally intended to provide a chimeric receptor polypeptide or retroviral polypeptide which can be used to inhibit retroviral infection of a human cell or tissue, in vivo or in vitro or in situ . Methods of using receptor polypeptides of the present invention are also provided to prevent or treat a viral infection, such as a retroviral infection (e.g. HIV) , as a non-limiting example.
  • a retroviral infection e.g. HIV
  • a recombinant nucleic acid (SEQ ID NO:8) is also provided, comprising a nucleotide sequence which encodes the H13 molecule, or a sequence substantially corresponding to, or consisting essentially of, the H13 amino acid sequence or mutant thereof, which nucleic acid consists of DNA or RNA.
  • Figure 2 shows a schematic diagram of the alignment of one strand of the H13 and ERR cDNA sequence (SEQ ID NOS:7 and 3, respectively). The sequences were analyzed using the Genetics computer group sequence analysis software package (Devereux, J. et al., Nucl . Acids Res . 12:387-395 (1984)).
  • Figure 3 shows the alignment of H13, ERR and TEA deduced amino acid sequences. Vertical lines indicate sequence homology. Dots indicate lack of homology, with double dots representing conservative amino acid changes.
  • the sequences were analyzed as in Figure 2. Shown in brackets are the sequences of H13 corresponding to Extracellular Domain 3 (residues 210-249) and Extracellular Domain 4 (residues 310- 337) .
  • Figure 4 shows an autoradiogram of the hybridization pattern of EcoRl-digested nucleic acid of human (CCL120,
  • Figure 5 shows an autoradiogram of a Southern blot analysis nucleic acid from various species with H13 cDNA (SEQ ID N0:1) .
  • Nucleic acid hybridized was EcoRl-digested nucleic acid of human (CCL120, CCL 119, SupTl, H9, M0LT4) , hamster (CH0-K1) and mouse thymocytes (Balb/c or BI0T6R) origin.
  • Figure 6 shows an autoradiogram of H13 gene expression. RNA from the indicated human cell lines was hybridized with the H13 cDNA (SEQ ID NO:l) .
  • Figure 7 shows an autoradiogram of the hybridization pattern of RNA of human (CEM, H9, M0LT4, SupTl, CCL120, CCL119) , hamster (CHO Kl) and mouse (RL12) origin, probed with the KpnI-Kpnl fragment (390 bp) of murine ERR cDNA.
  • Figure 8 shows a Northern Blot demonstrating the acquisition of susceptibility to infection with murine ecotropic retrovirus by transfection of a resistant cell with ERR cDNA. After transfection of ERR cDNA into hamster CHO Kl cells, the transfectants expressing the murine retroviral receptor gene were infected with murine radiation leukemia virus (RadLV) . Two weeks later, Northern blot analysis was performed using a viral probe, and reverse transcriptase (RT) activity of the cell supernatants was measured.
  • Figure 9 shows hydropathy plots for H13, ERR and TEA predicted proteins.
  • FIG. 10 shows a graph indicating the antigenicity of H13 predicted protein, analyzed using the PEPTIDESTRUCTURETM program.
  • One of the highly antigenic peptides was prepared using an AccI-EcoRI fragment as shown in Figure 14.
  • Figure 11 shows a SDS-PAGE autoradiogram depicting the synthesis of a fusion protein including the H13 protein with glutathione-S-transferase (GST) .
  • the fusion protein was prepared by ligating the 180 bp AccI-EcoRI fragment of H13 cDNA to the plasmid pGEX-2T, which expresses antigens as fusion proteins, was induced by addition of isopropyl-beta- thiogalacto-pyranoside (IPTG) , and was purified using glutathione-Sepharose chromatography.
  • IPTG isopropyl-beta- thiogalacto-pyranoside
  • Figure 12A-B shows the genetic mapping of the H13 gene to human chromosome 13.
  • the autoradiogram shows the hybridization pattern of EcoRI-digested nucleic acid from human-hamster somatic cell hybrids probed with H13 cDNA (SEQ ID N0:1) .
  • Lane 1 and 11 contain nucleic acid from human and hamster, respectively.
  • Lanes 2-10 contain nucleic acid which is derived from the chromosomes as designated in the table in Figure 12B.
  • Figure 13 is a schematic diagram of the genetic structure of the H13 and ERR genes, and four chimeric constructs there between. The infectivity of E-MuLV on human cells transfected with the various constructs is also indicated.
  • Figure 14 shows a comparison of sequences (nucleotide and amino acid) of the region of H13 and ERR termed Extracellular Domain 3 (as also depicted as part of SEQ ID NO:7, SEQ ID NO:8 and Figure 1).
  • This region of the receptor protein is the most diverse between the human and mouse sequences.
  • the sequences were aligned using Genetics computer group sequence analysis software package (See, e.g., Devereux, J. et al. , Nucl . Acids Res . 12:387-395 (1984)).
  • Figure 15 shows a schematic illustration of several cDNA clones from which the H13 sequence was derived, and their general structural relationship to the murine ERR homolog.
  • Clone 7-2 (H-13.7-2) represents a part of the complete H13 nucleic acid sequence; this was the first H13 clone sequenced, yielding SEQ ID N0:1 and SEQ ID NO:2.
  • Clones 1-1 (H13.1-1) and 3-2 (H13.3-2) each contain parts of the H13 sequence. The combined sequencing of these three clones resulted in the full H13 nucleic acid and amino acid sequences (SEQ ID NO:7 and SEQ ID NO:8, respectively).
  • Figure 16 shows a schematic illustration of extracellular domains 3 and 4, wherein (*) marks positions which contain critical amino acids for infection by MuLV-E.
  • Mouse-human chimeric receptor molecules (Chimera I-III) were prepared by substitution using common restriction sites in murine ERR and human H13, and their abilities to function as a receptor for MuLV-E was determined using the recombinant MuLV- E, CRE/BAG virions (se, e.g., Price et al. Proc. Natl . Acad. Sci . USA 84: 156-160 (1987); Danos et al. Proc. Natl . Acad. Sci . USA 15: 6460-6464 (1988)). Black boxes on top of the figure indicate extracellular domains of ERR and H13 ( See, e.g., Albritton et al.
  • Figure 17 shows a comparison of nucleotide and amino acid sequences of extracellular domains 3 and 4 in murine ERR and human H13.
  • the alignment was made using the Genetics computer group sequence analysis software package (See, e.g., Devereux et al Nucleic Acids Res. 12:387-395 (1984)). To pinpoint the critical amino acid residues, oligonucleotide- directed mutagenesis was carried out and 13 individual mutant ERR molecules were created which contain one or two amino acids substitutions or insertions as marked by boxes.
  • Figure 18 shows results demonstrating the acquirement of ability of H13 to function as a receptor for MuLV-E by mutation.
  • Figure 19 shows the alignment of gp70 amino acid sequences associated with receptor specificity with leukemia virus sequences.
  • Non-ecotropic retrovirus sequences Xenotropic MLV NZB.1U.6(2); polytropic MCF 247 (2); amphotropic MLV 4070A(2) , (+) - amino acid identity; (- ) - gap in sequence; Caps - conserved amino acid substitutions lower case - non-conserved amino acid substitutions. Number indicate the ⁇ position of amino acids in the gp70 sequence.
  • Figure 20 shows an amino acid and nucleic acid comparison of the N-terminal env region of the ecotropic AKv and amphotropic MLV 4070A which contains a 30 amino acid gap within the amphotropic sequence. Nucleotide sequence and corresponding amino acids are shown.
  • AKv sequence (9) Genbank Accession number V01164 Amphotorpic MLB 4070 (2) Genbank
  • the present invention relates to new methods of gene therapy and to chimeric receptor polypeptides which are discovered to provide target cell specific binding sites for specific viruses, viral vectors and delivery vectors of therapeutic and/or diagnostic agents.
  • Such chimeric receptor polypeptides of the present invention may be used for diagnostic, research and therapeutic applications, including gene therapy, wherein target cells having such chimeric receptor polypeptides on their surface may be exclusively and/or substantially infected by non-human specific delivery vectors.
  • the present invention thus overcomes one or more problems associated with the use of known viral vectors which are susceptible to non-specific infection of non-target cells, as well as reversion or mutation to replication competence of such viral vectors.
  • cells or tissues of humans, or other mammals may be treated to be associated with, or to transiently or constitutively express a chimeric receptor polypeptide, as a chimeric receptor cell or tissue, in vivo, in situ or in vitro.
  • Animal or human subjects having such chimeric receptor cells or tissues can thus be treated or diagnosed according to methods of the present invention with therapeutic and/or diagnostic agents further comprising an env binding domain polypeptide which binds a cell surface chimeric receptor polypeptide of the present invention.
  • a "chimeric receptor binding site” or “chimeric receptor” refers to a modified first species viral receptor binding site, which modification is comprised of a chimeric receptor polypeptide wherein at least two amino acids are substituted, deleted or added from a non-first species specific viral receptor binding site.
  • the at least two amino acid deletion, substitution or addition confers binding capability on the chimeric receptor for an env binding domain of a surface protein of a non-first species specific virus.
  • the substitution or addition corresponds to domain 3 of human H13 substituted non human specific virus receptor, such as ERR, , MuLV or GALV.
  • the substituted, deleted, or added amino acid residues correspond to residues 200-280 or 220-260, 230-245 or any range or value therein, of H13 (SEQ ID NO: 8), e.g., similarly to the substitutions presented in Table 1 below.
  • chimeric receptor cell or "chimeric receptor tissue” refers to a cell or tissue of a first species which has on, or associated with, its cell surface a chimeric receptor polypeptide, such that a non-first species specific virus or env binding domain specific for the chimeric receptor polypeptide may bind the chimeric cell or tissue, in vivo, in si tu, or in vitro .
  • the chimeric receptor polypeptide may preferably be associated with the chimeric receptor cell via a cell specific receptor associated, with a chimeric receptor polypeptide which combination preferably binds to a first species specific cell or tissue type to provide a chimeric receptor cell of the present invention. In another preferred embodiment, such association is provided by recombinant expression of the specific cell or tissue type of at least one chimeric receptor polypeptide.
  • association in the context of the present invention refers to any type of covalent or non-covalent binding or association such as, but not limited to, a covalent bond, hydrophobic/hydrophilic interaction, Van der Wahls forces, ion pairs, ligand-receptor interaction, epitope- antibody binding site interaction, enzyme-substrate interaction, liposome-hydrophobic interaction, nucleotide base pairing, membrane-hydrophobic interaction, and the like.
  • chimeric receptor polypeptide refers to a polypeptide of at least 10 amino acids corresponding to a human viral receptor protein or consensus sequence thereof, wherein the chimeric receptor polypeptide contains a chimeric receptor binding site capable of binding a an env binding domain of a non-human specific virus such as 10-700, 10-100, 10-50, 10-30, 20-30, 20-40, 40-60 or any range or value therein.
  • chimeric receptor polypeptides of the present invention may be defined as amino acid sequences of at least 10 residues having at least 80% (such as 81-99%, or any value or range therein, such as 83-85, 87-90, 93-95, 97-98, or 99% or any range or value therein.) homology with the corresponding amino acid sequence of a first species viral receptor, as any ecotropic or amphotropic first species viral receptor, have been modified to include amino acids from a non-first species viral receptor, and which have the biological activity of binding a binding domain of an env polypeptide of a non-first species virus.
  • the first species is human and the second species is rodent, or vice versa.
  • a non-limiting example of such a corresponding human viral receptor sequence is SEQ ID NO: 8, such as corresponding to 10 to 629 amino acids of SEQ ID NO: 8 or any value or range therein.
  • a chimeric receptor polypeptide of the present invention may further comprise a hydrophobic amino acid sequence corresponding to at least one to 20 transmembrane domains of a first species viral receptor protein which is at least 80% (such as 80-100%, or any range or value therein) homologous to the corresponding first species viral receptor.
  • transmembrane domains may be analogous to those described, e.g., a human H13 sequence (SEQ ID NO:8) or a murine ERR (SEQ ID NO:4) having 14 potential transmembrane domains (See, e.g., Eisenberg et al. , J. Mol. Biol. 179:125-142 (1984)), and which can be determined using hydrophobicity plots according to known method steps, e.g., as referenced therein or herein.
  • a non-limiting example of a first species viral receptor, as human viral receptor, is the human H13 protein (SEQ ID NO:8), which can be provided as an H13 polypeptide according to the present invention in a non-naturally occurring form, such as purified or chemically synthesized, or recombinantly produced, in either case according to known method steps, e.g., as referenced herein.
  • Such a non-limiting example of a chimeric receptor polypeptide of the present invention may be, e.g., a modified human H13 amino acid sequence (e.g., such as SEQ ID NO: 8) of at least 10 amino acids which is modified to provide binding capability to a non-human specific virus, such as the non- limiting examples of E-MuLV, gp-70 or ERR receptor protein (SEQ ID NO: 4) .
  • a modified human H13 amino acid sequence e.g., such as SEQ ID NO: 8
  • SEQ ID NO: 4 the non-human specific virus
  • Such modifications may preferably include substitution at a receptor binding site of a human viral receptor sequence by at least one non-human viral receptor amino acid, such as a murine ERR amino acid in the corresponding site in H13, to permit infection of a human or non-murine target cell having the chimeric receptor polypeptide, preferably a human cell, with a virus or retrovirus, such as E-MuLV.
  • a non-human viral receptor amino acid such as a murine ERR amino acid in the corresponding site in H13
  • Domain 3 comprises residues between positions 210 and 250 (SEQ ID N0:7). Preferred substitution is with 1-10 amino acid residues, or any number or range therein, from the corresponding domain of ERR, between amino acid residues 210 and 242 (SEQ ID N0:4) , preferably amino acids 238, 239 and 242, with more preferably at least 242 being substituted. Substitution of between 1 and 4 residues is preferred. For example, residues and positions which differ in extracellular domain 3 of H13 and ERR are listed below in Table 1.
  • At least Pro242 of H13 is replaced by Tyr, and at least one of Gly240 and Val244 (SEQ XD NO:8) is replaced by Val and Glu, respectively, and as presented in Figure 18.
  • at least one of H13 amino acid 239 may be preferably replaced by the corresponding ERR amino acid 233.
  • Non-limiting examples of chimeric receptor polypeptides according to the present invention may include Tyr242, Phe242 and/or Trp242 and at least one of Val240, Met240, Leu240, Ile240, Glu244, Gln244, Asp244, or Asn244; and Asn239, Asp239, Glu239 or Gln239 (SEQ ID NO:3), wherein at least Pro242 of H13 (SEQ ID NO:8) is replaced by Tyr, and at least one of Gly240 and Val244 (SEQ ID NO:8) is replaced by Val and Glu, respectively.
  • Another means for modifying virus binding specificity of H13 is by deletion of one or more of the "extra" amino acid residues in H13 that do not correspond to residues of ERR.
  • Preferred deletions are of between one and six residues from H13 positions 326 to 331 (SEQ ID N0:1), most preferably, deletion of all six of these residues.
  • an H13 chimeric receptor polypeptide may be provided which confers the binding ability of a human cell, to bind an env binding domain of a non-human virus, wherein 1 to 30 amino acids of H13 are substituted, deleted or modified by corresponding amino acids from ERR, in-order to confer such binding ability.
  • such a chimeric receptor polypeptide may preferably comprises a peptide wherein at least Pro242 of the H13 polypeptide (SEQ ID NO:8) is replaced by Tyr, and at least one of Gly240 and Val244 (SEQ ID NO:8) is replaced by Val and Glu, respectively, or fragments having amino acid sequences substantially corresponding to the amino acid sequence of H13, such that the resulting chimeric receptor is selectively bound by a murine ecotropic retrovirus, which cannot infect other human or non-murine cell or tissue types.
  • a homologous chimeric receptor polypeptide of an amphotropic first species viral receptor corresponding to H13 may be similarly modified to allow binding of a non-first species specific amphotropic virus, such as a chimeric receptor cell or tissue.
  • a non-first species specific amphotropic virus such as a chimeric receptor cell or tissue.
  • the first species is human or rodent.
  • Also intended are all active forms of chimeric receptors or H13 polypeptide derived from the chimeric receptor or H13 transcript, respectively, and all mutants with H13-like activity.
  • Such methods are generally based on truncation of the nucleic acid 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 retrovirus or a retroviral protein or glycoprotein.
  • specific ligands such as an intact retrovirus or a retroviral protein or glycoprotein.
  • it is important that the soluble chimeric receptor polypeptide or H13 polypeptide comprise elements of the binding site of the chimeric receptor or H13 that permits binding to a virus.
  • a chimeric receptor polypeptide or H13 polypeptide has many amino acid residues, only one or two or more, such as 2-15, or any value or range therein, such as 3-5, 6-9 or 10-15, which are critically involved in virus recognition and binding.
  • H13 proteins or chimeric receptor polypeptides 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, according to known method steps.
  • the invention provides muteins of a chimeric receptor polypeptide of the present invention.
  • mutein is meant a "fragment,” “variant, " or “chemical derivative” of an H13 protein or a chimeric receptor polypeptide.
  • a mutein retains at least a portion of the function of the H13 protein which permits its utility in accordance with the present invention.
  • a "fragment" of the H13 protein or chimeric receptor polypeptide is any subset of the molecule, that is, a shorter peptide.
  • a "mutein" of the H13 chimeric receptor polypeptide refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Muteins may be conveniently prepared by direct chemical synthesis or recombinant production, including mutagenesis, of the mutein, using methods well-known in the art. See, e.g., Sambrook, supra, Ausubel, supra, Coligan, supra. Alternatively, muteins of an H13 or chimeric receptor polypeptide can be prepared by mutations in the nucleic acid which encodes the synthesized peptide. Such mutants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence as presented herein.
  • any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • the mutations that will be made in the nucleic acid encoding the mutein peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see, e.g., European Patent Publication No. EP 75,444).
  • these muteins ordinarily are prepared by site-directed mutagenesis (as exemplified by
  • muteins typically exhibit the same qualitative biological activity as a chimeric peptide.
  • H13 polypeptide having critical amino acid residues derived from other non-human or non-animal specific viruses, such as ERR, may be produced using site-directed mutagenesis.
  • muteins are those in which at least one amino acid residue in the protein molecule, and preferably, only one, has been removed and a different residue inserted in its place.
  • protein chemistry and structure see Schulz, G.E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T.E., Proteins; Structure and Molecular
  • Substantial changes in functional or immunological properties are made by selecting 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 structure 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, ile, phe, val or ala; (c) substitution of a cys residue for (or by) any other residue; (d) 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 (e) substitution of a residue having a bulky side chain, e.g., phe, for (or by) a residue not having such a side chain, e.g., gly.
  • a hydrophilic residue e.g., ser or thr
  • a hydrophobic residue e.g., leu
  • 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 will be evaluated by routine screening assays, either immunoassays or bioassays.
  • a mutein typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid ⁇ in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using a specific antibody on a column (to absorb the mutein by binding to at least one epitope) .
  • the activity of the cell lysate containing H13 or a chimeric receptor polypeptide, or of a purified preparation of H13 or chimeric receptor can be screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the protein molecule, such as binding to a given antibody, is measured by a competitive type immunoassay (as described herein) .
  • Bioactivity is screened in an appropriate bioassay, such as virus infectivity, as described herein.
  • modified amino acids or chemical derivatives of amino acids of a chimeric receptor polypeptide according to the present invention may be provided, which polypeptides contain additional chemical moieties or modified amino acids not normally a part of the protein. Covalent modifications of the peptide are thus included within the scope of the present invention. Such modifications may be introduced into a chimeric receptor polypeptide by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers, according to known method steps.
  • cross-linking agents include, e.g., 1,1-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-1,8-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. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 (which are herein entirely incorporated by reference) , may be employed for protein immobilization.
  • a chimeric receptor polypeptide of the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins : Structure and Molecule Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups, according to known method steps.
  • Such derivatized moieties may improve the solubility, absorption, biological half life, and the like.
  • Such moieties or modifications of a chimeric receptor polypeptide may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington ' s Pharmaceutical Sciences, 16th ed. , Mack Publishing Co., Easton, PA (1980). Such chemical derivatives of a chimeric receptor polypeptide also may provide attachment to solid supports, such as for purification, generation of antibodies or cloning; or to provide altered physical properties, such as resistance to enzymatic degradation or increased binding affinity or modulation for a chimeric receptor polypeptide, which is desired for therapeutic compositions comprising a chimeric receptor polypeptide, antibodies thereto or fragments thereof. Such peptide .derivatives are known in the art, as well as method steps for making such derivatives.
  • H13 polypeptide refers to a polypeptide having an amino acid sequence substantially corresponding to, or consisting essentially of, at least a 10 amino acid portion of the amino acid sequence of SEQ ID NO:8 and which binds a retrovirus, such as the HIV env binding domain.
  • substantially corresponding to an amino acid sequence of a chimeric receptor polypeptide or H13 protein, refers to those amino acid sequences in which at least one amino acid residue in a soluble portion of the protein molecule has been removed and a different residue inserted in its place, the number of substitutions being relatively small and well characterized or conservative, as described herein.
  • a preferred use of this invention is the production by chemical synthesis or recombinant nucleic acid technology a chimeric receptor polypeptide or chimeric receptor polypeptide or fragments thereof, where the fragments are small as possible, while still retaining sufficiently high affinity in binding to a non-human specific retrovirus or HIV.
  • Preferred fragments of H13 include extracellular domain 3, as presented herein.
  • an extracellular fragment of the H13 or chimeric receptor polypeptide of the present invention may bind to a human or non-human specific virus, respectively.
  • a human or non-human specific virus By production of smaller fragments of this peptide, one skilled in the art, using known binding and inhibition assays, will readily be able to identify the minimal peptide capable of binding a binding domain of a virus or retrovirus with sufficiently high affinity to inhibit infectivity, without undue experimentation based on the teaching and guidance presented herein. Shorter peptides are expected to have two advantages over the larger proteins: (1) greater stability and diffusibility, and (2) less immunogenicity.
  • a mutant chimeric receptor polypeptide of the present invention endows human cells or other non-murine cells expressing the receptor with the ability to be infected by a non-human specific virus, such as murine E-MuLV or HuLV, which may be modified to reduce the possibility of reversion to replication competent forms.
  • a non-human specific virus such as murine E-MuLV or HuLV
  • chimeric receptor polypeptides of the present invention may be provided without undue experimentation, based on the teaching and guidance presented herein.
  • the present invention is also intended to encompass binding of a chimeric receptor polypeptide to any non-human ecotropic or amphotropic virus with similar receptor specificity, including retroviruses, such as MuLV or HuLV, and adenoviruses.
  • nucleic acid encoding a viral receptor suitable for use in providing a chimeric receptor polypeptide of the present invention from any species or cell type, without undue experimentation, based on the teaching and guidance presented herein.
  • a cDNA library of the species or cell type of interest for example, a human T cell cDNA library
  • a probe based on the sequence of a human viral receptor, such as H13 is a probe based on the sequence of a human viral receptor, such as H13.
  • clone and sequence the hybridizing nucleic acid to obtain the sequence of the "new" retroviral receptor.
  • a sequence having one or more amino acid substitutions such that a chimeric receptor between the new receptor and a known receptor is created.
  • the chimeric receptor can then be expressed in a. cell of choice and its function can easily be tested using conventional virus binding assays or virus infectivity assays.
  • the receptor attachment site of a virus so that it will not bind to its natural receptor, or bind to a different receptor, based on knowledge of receptor choice determinants in envelope glycoproteins of viruses, such as murine leukemia viruses. See, e.g., Battini et al J. Virology 66(3) :1468-1475 (1992) .
  • a human cell can be infected with one virus strain in vi tro 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 virus which binds to a second expressed chimeric receptor polypeptide can be introduced into the individual to infect stably and alter only those introduced cells bearing the second retroviral receptor.
  • Oligonucleotides representing a portion of the H13 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 . 21:101-141 (1978)), Ausubel et al., infra; Sambrook, infra . (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.
  • DNA synthesis may be achieved through the use of automated synthesizers. Techniques of DNA hybridization are disclosed by Sambrook et al. supra, and by Haymes, B.D., et al. (In: DNA
  • a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing H13, a receptor mutant or homolog) into an expression vector.
  • the library is then screened for members capable of expressing a protein which binds to anti- H13, anti-mutant or anti-homolog antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as H13, receptor mutant or homolog proteins or peptides, or fragments thereof.
  • a nucleic acid sequence encoding the H13, receptor mutant or homolog, polypeptide or protein of the present invention, or a functional derivative thereof, 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 and expressed in an appropriate host cell. Techniques for such manipulations are disclosed by Sambrook, J. et al., supra, and are well known in the art.
  • ribosome binding sites are disclosed, for example, by Gold, L., et al. Ann. Rev. Microbiol . 35:365-404 (1981)).
  • 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 SP2/0-Agl4 or the murine myeloma P3-X63Ag8, and their derivatives.
  • Preferred mammalian cells are cells which are intended to replace the function of the genetically deficient cells in vivo. Bone marrow stem cells are preferred for gene therapy of disorders of the hemopoietic or immune system.
  • many possible vector systems are available for the expression of a chimeric receptor polypeptide or chimeric receptor polypeptide.
  • 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 adenovirus, bovine papilloma virus, Simian virus, 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, protein production.
  • silk moth caterpillars and baculoviral vectors are presently preferred hosts for large scale a chimeric receptor polypeptide or chimeric receptor polypeptide production according to the invention. See, e.g., Ausubel, infra, Sambrook, supra.)
  • the expressed a chimeric receptor polypeptide or chimeric receptor polypeptide 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.
  • the chimeric receptor proteins may be isolated by the use of specific antibodies, such as an anti-receptor polypeptide antibody that still reacts with the protein containing ERR-derived amino acid substitutions. Such antibodies may be obtained by well-known methods.
  • manipulation of the genetic constructs of the present invention allow the grafting of a particular virus-binding domain onto the transmembrane and intracytoplasmic portions of a chimeric receptor polypeptide or chimeric receptor polypeptide, or grafting the retrovirus receptor binding domain of a chimeric receptor polypeptide or chimeric receptor polypeptide onto the transmembrane and intracytoplasmic portions of another molecule, resulting in yet another type of chimeric molecule.
  • the present invention also relates to a method for rendering a human or other eukaryotic cell or tissue which is susceptible to binding by an env binding domain of a non-human virus is provided such as retroviral infection.
  • the method may optionally first comprise transforming, in vi tro, in vivo or in si tu, a eukaryotic cell or tissue with an expressible nucleic acid encoding a chimeric receptor polypeptide to produce a recombinant chimeric receptor cell or tissue which is capable of expressing a chimeric receptor polypeptide capable of binding an extracellular viral env binding domain of a non-specific virus.
  • Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al. , Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al., Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, B.M. , Genes II, John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W. , et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, publisher, Berkeley, CA (1981); Sambrook, J. et al.
  • the in vivo transforming is carried out by one selected from: injection of the mutant nucleic acid into the tissue or cell; retroviral infection using a recombinant retrovirus comprising the mutant nucleic acid under control of at least one tissue specific regulatory sequence specific for the tissue or cell; liposome delivery of the mutant nucleic acid to the tissue or cell; antibody delivery of the mutant nucleic acid to the tissue or cell; or contacting a cell or tissue specific antibody conjugated to the mutant nucleic acid to the tissue or cell, according to known method steps.
  • the in si tu or in vitro transforming be carried out by one selected from: injection of the mutant nucleic acid into the tissue or cell; retroviral infection using a recombinant retrovirus comprising the mutant nucleic acid in expressible form; liposome delivery of the mutant nucleic acid; antibody delivery of the mutant nucleic acid; transfection of the tissue or cell with the mutant nucleic acid; or contacting the cell or tissue specific antibody conjugated to the mutant nucleic acid to the tissue or cell, according to known methods steps.
  • the virus is a recombinant murine ecotropic or hamster amphotropic retrovirus and the chimeric receptor polypeptide is a chimeric receptor polypeptide as presented herein.
  • a chimeric receptor polypeptide of the present invention can be expressed on the cell surface as an integral membrane protein in a number of cell types, particularly cells of the T lymphocyte and monocyte/macrophage lineages, consistent with in vitro tropism of known human retroviruses such as HIV-l and HTLV-l.
  • a chimeric receptor polypeptide of the present invention will permit cells of these lineages in the human, which are normally resistant to non-human specific viral infection, to be infected with such viruses.
  • the present invention includes a modified chimeric receptor cell or tissue produced by a herein-described method, wherein the eukaryotic cell or tissue is selected from, but not limited to mammalian, insect, bird or yeast origin. It is preferred that the mammalian cell or tissue is of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used.
  • 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 a chimeric receptor polypeptide or chimeric receptor polypeptide capable as serving as a human retrovirus receptor.
  • the a chimeric receptor polypeptide or chimeric receptor polypeptide nucleic acid 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 as used herein, 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.
  • Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain the a chimeric receptor polypeptide or chimeric receptor polypeptide nucleic acid of the present invention, such as animals produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic mammal, are also intended to be within the scope of the present invention.
  • Chimeric non-human mammals having human cells or tissue engrafted therein are also encompassed by the present invention, which may be used for testing expression of chimeric receptor polypeptides in human tissue and/or for testing the effectiveness of therapeutic and/or diagnostic agents associated with delivery vectors which preferentially bind to a chimeric receptor polypeptide of the present invention.
  • Methods for providing chimeric non-human mammals are provided, e.g, in U.S. serial Nos.
  • the animals carrying a chimeric receptor polypeptide or chimeric receptor polypeptide gene can be used to test compounds or ⁇ other treatment modalities which may prevent, suppress or cure a human retrovirus infection or a disease resulting from such infection for those retroviruses which infect the cells using the a chimeric receptor polypeptide or chimeric receptor polypeptide as a receptor.
  • These tests can be extremely sensitive because of the ability to adjust the virus 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 retrovirus diseases.
  • diseases include, but are not limited to AIDS, HTLV- induced leukemia, and the like.
  • Transgenic animals according to the present invention can also be used as a source of cells for cell culture.
  • the transgenic animal model of the present invention has numerous economic advantages over the "SCID mouse” model (McCune, J.M et al., Science 241:1632-1639 (1988)) wherein it is necessary to repopulate each individual mouse with the appropriate cells of the human immune system requiring a significantly greater amount of time and experimentation.
  • Chimeric receptor or H13 Specific Antibodies and Methods This invention is also directed to an antibody specific for an epitope of a chimeric receptor polypeptide or chimeric receptor polypeptide.
  • the antibody of the present invention is used to prevent or treat retrovirus infection, to detect the presence of, or measure the quantity or concentration of, a chimeric receptor polypeptide or chimeric receptor polypeptide in a cell, or in a cell or tissue extract, or a biological fluid. See, generally, Coligan, supra, and harlow, infra .
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies (mAbs) , chimeric antibodies, anti-idiotypic (anti-Id) antibodies, and fragments thereof, provided by any known method steps, such as by hybridomas, recombinant techniques or chemical synthesis.
  • an antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope 1 is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • An "antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen may have one, or more than one epitope.
  • polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110, see, e.g., Ausubel et al. eds. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., (1987, 1992) ; and Harlow and Lane, Antibodies : A Laboratory Manual , Cold Spring Harbor Laboratory (1988) ; Coligan et al, eds, Current Protocols in Immunology, Greene Publishing Associates and Wiley Wiley Interscience, N.Y. (1992, 1993) .
  • Such antibodies 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 vi tro or in vivo . Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs..
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (see, for example, Morrison et al., Proc. Natl . Acad. Sci . USA 81:6851- 6855 (1984); Neuberger et al., Nature 314:268-270 (1985); Sun et al., Proc. Natl . Acad. Sci . USA 84:214-218 (1987); Better et al., Science 240:1041- 1043 (1988); Better, M.D. International Patent Publication WO 9107494, which references are hereby entirely incorporated by reference) .
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody) .
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may bear structural similarity to the original mAb which induced the anti-Id.
  • antibodies to the idiotypic determinants of a mAb it is possible to identify other clones expressing antibodies of identical specificity.
  • mAbs generated against a chimeric receptor polypeptide or chimeric receptor polypeptide of the present invention may be used to induce anti-Id antibodies in suitable animals, such as Balb/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional Balb/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an a chimeric receptor polypeptide or chimeric receptor polypeptide epitope.
  • KLH keyhole limpet hemocyanin
  • the anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as an epitope of a chimeric receptor polypeptide or chimeric receptor polypeptide.
  • the term "antibody”, as presented above, 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 (1983) ) .
  • Antibody Diagnostic Assays are 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 F
  • 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 a chimeric receptor polypeptide or chimeric receptor polypeptide 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).
  • the antibodies, or fragments of antibodies, of the present invention may be used to quantitatively or qualita- tively detect the presence of cells which express a chimeric receptor polypeptide or chimeric receptor polypeptide on their surface or intracellularly. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light micros: :opic, flow cytometric, or fluorometric detection.
  • the antibodies of the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in si tu detection of a chimeric receptor polypeptide or chimeric receptor polypeptide. Through the use of such a procedure, it is possible to determine not only the presence of a chimeric receptor polypeptide or chimeric receptor polypeptide but also its distribution on the examined tissue. Additionally, the antibody of the present invention can be used to detect the presence of soluble a chimeric receptor polypeptide or chimeric receptor polypeptides in a biological sample, such as a means to monitor the presence and quantity of a chimeric receptor polypeptide or chimeric receptor polypeptide used therapeutically.
  • Such immunoassays for a chimeric receptor polypeptide or chimeric receptor polypeptide typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying H13 protein, and detecting the antibody by any of a number of techniques well-known in the art.
  • the biological sample may be treated 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 a chimeric receptor polypeptide- or chimeric receptor polypeptide-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support may then be detected by conventional means.
  • solid phase support or “carrier” is intended any support capable of binding antigen or antibodies.
  • Well-known supports, or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the . binding activity of a given lot of anti- chimeric receptor polypeptide or anti-chimeric receptor polypeptide antibody may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • One of the ways in which the chimeric receptor polypeptide- or chimeric receptor polypeptide-specific anti ⁇ body can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) according to known method steps.
  • Detection may be accomplished using any of a variety of other immunoassays, such as (Laboratory Techniques and Biochemistry in Molecular Biology, Work, et al., North Holland Publishing Company, New York (1978) with particular reference to the chapter entitled “An Introduction to Radioimmune Assay and Related Techniques” by T. Chard, incorporated entirely herein by reference.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds examples include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the antibody molecules of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to "extract" the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule”) . After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
  • tissue or cells having at least one expressible chimeric receptor polypeptide encoding nucleic acid are subjected to infection by a recombinant non-human specific retroviral vector that recognizes only cells expressing, or bound on their surface, a chimeric receptor polypeptide, and the retroviral vector cannot infect other human cells, due to the non-human specificity of the vector and the relative lack of ability to revert, mutate or recombine to provide replication competence.
  • a chimeric receptor polypeptide can be selectively associated with a target cell or expressed on the target cell by allowing infection by a recombinant retrovirus having nucleic acid encoding the chimeric receptor polypeptide.
  • This temporary and/or permanent association allows target cells, such as pathologic cells having a particular receptor, to selectively expose or express a chimeric receptor polypeptide, as well as progeny of such pathologic cells in the case of constitutively expressed chimeric receptor polypeptide, making possible specific delivery of therapeutic agents to such target cells and/or their progeny, according to the present invention.
  • a procedure for marking specific types of target cells (and, opotionally, with recombinant, chromosomal expression of a chimeric receptor polypeptide, their progeny) is provided, by temporarily or permanently associating a chimeric receptor polypeptide with a target cell specific cell surface molecule, such as cell surface receptor, and then administering a recombinant retrovius which binds the chimeric receptor polypeptide and infects the target cell.
  • the infecting virus vector may carry any of a variety of therapeutic nucleic acids or therapeutic genes conferring one or more functions.
  • a chimeric receptor polypeptide is specifically delivered to a target cell of a first species by association with a target cell specific vector, such as an antibody, liposome or target cell specific receptor ligand, such that the chimeric receptor polypeptide is associated with the target cell for a sufficient time to allow treatment or diagnosis using a therapeutic agent or diagnostic agent associated with the receptor binding or env domain of a non-first species specific virus which is capable of binding the chimeric receptor polypeptide.
  • a therapeutic or diagnostic agent such as a therapeutic or diagnostic nucleic acid
  • the target cell e.g., where the nucleic acid is incorporated into the chromosome of the target cell, to the exclusion of the non-target cells.
  • the virus vector could carry into the cell a modified retrovirus receptor gene.
  • the target cell Once the target cell is infected, the target cell has incorporated the chimeric receptor polypeptide encoding DNA, such that subsequently the target and cell and all progeny will express the chimeric receptor polypeptide on the target cell surface.
  • a chimeric receptor polypeptide By marking such target cells and their progeny with a chimeric receptor polypeptide according to the present invention, an animal subject suffering from the pathology can be treated using gene therapy, wherein the gene therapy vector specifically binds the chimeric receptor polypeptide and delivers a therapeutic or diagnostic agent to the target cell.
  • specific pathologies may be treated with or without substantially reduced risk of non-specific retroviral vector infection and gene insertion in to the chromosome.
  • a chimeric receptor polypeptide encoding nucleic acid may be combined with a coding sequence for a polypeptide that specifically binds a receptor specific for a particular type of target cell.
  • the expression of such a nucleic acid in a recombinant host provides a fusion protein in recoverable amounts, suitable for ⁇ therapeutic administration.
  • the pharmaceutically acceptable fusion protein may then be administered to a subject having a pathology such that the fusion protein will specifically bind target cells and will act as an env binding domain for a non-human specific virus according to the present invention.
  • the subsequent administration of a non-human specific virus which has nucleic acid encoding any of a large variety of genes, may confer one or more functions on the infected cell and could have positive or negative therapeutic effects on that cell.
  • human tumors may be treated using a chimeric receptor polypeptide as a fusion protein to an antibody fragment of an antibody specific for a human tumor cell surface receptor.
  • a fusion protein in pharmaceutically acceptable form may be administered to an animal model or human subject to mark tumor cells for infection by a non-human specific retrovirus which has DNA encoding a chimeric receptor polypeptide.
  • a second step may consist of administration of the retrovirus and infection of the tumor cell results in expression of the chimeric receptor polypeptide by subsall of the infected tumor cells, as well as their progeny.
  • step 2 can be dispensed with altogether as determined by one skilled in the art.
  • B3 antibody fragments specific for human tumor cells may be used to provide a fusion protein and gene therapy, wherein a specific pathologic cell specific antibody or binding protein is first expressed as fusion protein, shown to: (a) bind the pathologic cells and allow infection by a non-human specific virus having nucleic acid encoding a chimeric receptor polypeptide in vitro and in vivo, such that the chimeric receptor polypeptide is expressed on the surface of the virus infected pathologic cell, (b) in vitro and in vivo killing of the virus infected target cell by at least one therapeutic agent associated with an env binding domain as the delivery vector that binds the chimeric receptor polypeptide.
  • Animal model systems may be preferably used before clinical treatment of humans, according to known methods steps.
  • a chimeric receptor polypeptide encoding nucleic acid into animal or human cells or tissues, which are to be infected by the recombinant non- human specific virus, such as an ecotropic or amphotropic virus, may be accomplished according to known method steps.
  • Non-limiting examples include in vitro transfection of human cells or tissue, such as bone marrow cells (as stem cells or stromal cells) , white blood cells, and differentiated or undifferentiated granulocytes, monocytes, macrophages, lymphocytes, erythrocytes, megakaryocytes, cells of the central nervous system, and tissue cells, such as nerve tissue, liver cells, kidney cells, muscle cells, heart cells or myocardial cells, atrial or venus cells or tissue, eye cells, connective tissue or cells, lung tissue or cells, spleen cells or tissue, endocrine tissue or cells, CSF, or cells of the central nervous system, with nucleic acid encoding a chimeric receptor polypeptide, followed by reintroduction into the human subject; or by direct injection of a nucleic acid encoding the chimeric receptor polypeptides into the tissue in vivo or in si tu, such as muscle, heart, liver, kidney, brain, nerve, spleen, pancreas
  • a method for transferring at least one therapeutic agent or diagnostic agent to a chimeric receptor bearing cell or tissue.
  • the method comprises providing a chimeric receptor cell or tissue according to the present invention and contacting the chimeric receptor cell or tissue, in vitro, in vivo or in situ, with a delivery vector comprising an env binding domain of a non-human virus and at least one therapeutic pr diagnostic agent, such that the delivery vector binds the chimeric modified cell and the therapeutic or diagnostic agent has a therapeutic or diagnostic effect on the chimeric receptor cell.
  • a therapeutic agent or diagnostic agent is selectively delivered according to a method of the present invention to a target cell as a chimeric receptor cell or tissue by a delivery vector which comprises an env binding domain of a virus envelope protein which is specific for the chimeric receptor of the chimeric receptor polypeptide expressed extracellularly on the chimeric receptor cell.
  • Delivery Vectors which comprise an env binding domain of a virus envelope protein which is specific for the chimeric receptor of the chimeric receptor polypeptide expressed extracellularly on the chimeric receptor cell.
  • the delivery vector may be, but is not limited to, a viral vector, a liposome, or a conjugate of the env binding domain associated with diagnostic or therapeutic agent.
  • the delivery vector may further comprise any diagnostic or therapeutic agent which has a therapeutic or diagnostic effect on the chimeric receptor cell as the target cell for the delivery vector. Diagnostic or Therapeutic Agent
  • the diagnostic or therapeutic agent may be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which may be detectably labeled as for labeling antibodies, as described herein.
  • labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.
  • any other known diagnostic or therapeutic agent can be used in a method of the present invention.
  • a therapeutic agent used in the present invention may have a therapeutic effect on the target cell as a chimeric receptor cell, the effect selected from, but not limited to: correcting a defective gene or protein, a drug action, a toxic effect, a growth stimulating effect, a growth inhibiting effect, a metabolic effect, a catabolic affect, an anabolic effect, an antiviral effect, an antibacterial effect, a hormonal effect, a neurohumoral effect, a cell differentiation stimulatory effect, a cell differentiation inhibitory effect, a neuromodulatory effect, an antineoplastic effect, an anti- tumor effect, an insulin stimulating or inhibiting effect, a bone marrow stimulating effect, a pluripotent stem cell stimulating effect, an immune system stimulating effect, and any other known therapeutic effects that may be provided by a therapeutic agent delivered to a chimeric receptor cell via a delivery vector according to the present invention.
  • a therapeutic nucleic acid as a therapeutic agent may have, but is not limited to, at least one of the following therapeutic effects on a chimeric receptor cell: inhibiting transcription of a DNA sequence; inhibiting translation of an RNA sequence; inhibiting reverse transcription of an RNA or DNA sequence; inhibiting a post-translational modification of a protein; inducing transcription of a DNA sequence; inducing translation of an RNA sequence; inducing reverse transcription of an RNA or DNA sequence; inducing a post-translational modification of a protein; transcription of the nucleic acid as an RNA; translation of the nucleic acid as a protein or enzyme; and incorporating the nucleic acid into a chromosome of a chimeric receptor cell for constitutive or transient expression of the therapeutic nucleic acid.
  • Therapeutic effects may include, but are not limited to: turning off a defective gene or processing the expression thereof, such as antisense RNA or DNA; inhibiting viral replication or synthesis; gene therapy as expressing a heterologous nucleic acid encoding a therapeutic protein or correcting a defective protein; modifying a defective or underexpression of an RNA such as an hnRNA, an mRNA, a tRNA, or an rRNA; encoding a toxin in pathological cells; encoding a drug or prodrug, or an enzyme that generates a compound as a drug or prodrug in pathological or normal cells expressing the chimeric receptor; encoding a thymidine kinase varicella- zoster virus thymidine kinase (VZV TK) (see, e.g., Huber et al Proc.
  • VZV TK varicella- zoster virus thymidine kinase
  • a therapeutic nucleic acid of the present invention which encodes, or provides the therapeutic effect any known toxin, prodrug or drug gene for delivery to pathogenic cells may also include genes under the control of a tissue specific transcriptional regulatory sequence (TRSs) specific for pathogenic cells, such as neoplastic cells, including ⁇ - fetoprotein TRS or liver-associated albumin TRS (see, e.g., Dynan and Tjian Nature (London) 316:774-778 (1985)).
  • TRSs tissue specific transcriptional regulatory sequence
  • Such TRSs would further limit the expression of the cell killing toxin, drug or prodrug in the target cell as a cancer cell expressing a chimeric receptor polypeptide of the present invention.
  • a brain tumor could be injected, transfected or viral vector transformed in vivo with a chimeric receptor polypeptide encoding nucleic acid of the present invention, followed by therapeutic treatment of the chimeric receptor brain tumor cells with a recombinant ecotropic retrovirus encoding a thymidine kinase, such that the brain tumor cells would be selectively killed by expression of the thymidine kinase.
  • a brain tumor could be injected, transfected or viral vector transformed in vivo with a chimeric receptor polypeptide encoding nucleic acid of the present invention, followed by therapeutic treatment of the chimeric receptor brain tumor cells with a recombinant ecotropic retrovirus encoding a thymidine kinase, such that the brain tumor cells would be selectively killed by expression of the thymidine kinase.
  • an abnormal H13 molecule which results in enhanced susceptibility to disease may be replaced by infusion of cells of the desired lineage (such as hemopoietic stem cells, for example) transfected with a chimeric receptor polypeptide, such as a mutant H13 protein, under conditions such that the infused cells will preferentially replace the endogenous cell population.
  • cells of the desired lineage such as hemopoietic stem cells, for example
  • a chimeric receptor polypeptide such as a mutant H13 protein
  • the delivery vector is a recombinant, non-human specific virus, such that binding of the non-human specific virus to the chimeric receptor cell or tissue results in infection of the modified receptor cell and the therapeutic effect of the therapeutic nucleic acid in the chimeric receptor cell.
  • the delivery vector includes a complex fusion protein, or nucleic acid encoding therefor, comprising a non-human specific env binding domain bound by a linker to a therapeutic or diagnostic agent, such that the binding or contacting results in a desired therapeutic or diagnostic effect.
  • the delivery vector may further comprise a liposome, the liposome containing the env binding domain and the therapeutic agent, such that the env binding domain is capable of binding the chimeric receptor binding site of a chimeric receptor cell.
  • the contacting of the delivery vector to the chimeric receptor cell or tissue may result in the chimeric receptor cell or tissue expressing a therapeutically effective amount of the expression product of a therapeutic nucleic acid.
  • the therapeutic nucleic acid encodes a toxin which acts to selectively kill the chimeric receptor containing cell or tissue.
  • the pathologic cell may be a cancer cell.
  • the therapeutic nucleic acid may further encode a growth factor selected from epidermal growth factor, interleukin-2, interleukin-4, interleukin-6, tissue growth factor- ⁇ ;, insulin growth factor-1 or fibroblast growth factor.
  • Th toxin may be a purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin, Pseudomonas exotoxin, diphtheria toxin, endotoxin, a venom toxin, and the like.
  • the therapeutic nucleic acid may encode at least one member selected from a single chain ribosome inhibitory protein acting to block expression of an abnormal protein in the chimeric receptor cell or tissue; a cytokine; or a growth factor.
  • a cytotoxic or a chemotherapeutic agent may be attached directly to a delivery vector having an env binding domain or to an antibody or fragment, or a growth factor, that preferentially binds pathologic cells as target chimeric receptor cells.
  • the targets for this type of therapy can also be growth factor receptors, differentiation antigens, or other less characterized cell surface antigens specifically associated with other pathologic cells. It is now established that many cancers overproduce growth factor receptors which can function as oncogenes or in an autocrine way to promote the growth of the cancer cells (Pastan and Fitzgerald, 1991; Velu et al, 1987; Kawano et al, 1988; Hellstrom & Hellstrom, 1989) .
  • the epidermal growth factor receptor is present in large amounts (up to 3 x 10 6 receptors per cell) in many squamous cell and epidermoid carcinomas, glioblastomas, and some metastatic ovarian and bladder cancer (Hender et al, 1984; Jones et al, 1990; Lau et al, 1988) .
  • normal cells can contain a magnitude less receptors per cell (Dunn et al, 1986) .
  • the interleukin-2 (IL- 2) receptor is present in substantially higher numbers on the cells of patients with adult T cell leukemia (ATL; 3 x 10 4 receptors per cell) than in normal T cells.
  • B lymphocytes are often also present on tumor cell such as B cell lymphomas. Because such antigens are not present on the stem cells that produce B cells, any mature B cells that are killed by targeted therapy will be replaced from the stem cell population from the stem cell population, whereas the cancer cells will not be replaced (Ghetie et al, 1988) . Finally, there are antigens preferentially expressed on cancer cells whose functions are not yet understood. Some of these, such as carcinoembryonic antigens (CEA) (Muraro et al, 1985), are fetal antigens, which are either not present or only present in small amounts on normal adult tissues.
  • CEA carcinoembryonic antigens
  • This group also contains antigens of unknown origin that are only defined by their reactivity with a monoclonal antibody (Fraenkel et al, 1985; Varki et al, 1984; Willingham et al, 1987) .
  • Single-chain antigen-binding proteins which may be used as components of therapeutic or diagnostic delivery vectors of the present invention have numerous advantages in clinical applications because of their small size. These proteins are cleared from serum faster than monoclonal antibodies or Fab fragments. Because they lack the Fc portion of an antibody, which is recognized by cell receptors, they have a lower background for use in imaging applications and they are less immunogenic. They are also expected to penetrate the microcirculation surrounding solid tumors better than monoclonal antibodies.
  • the therapeutic agent is a toxin or toxin fragment or domain; such as a purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin selected from at least one of ricin, Pseudomonas exotoxin, diphtheria toxin, thymidine kinase, and the like.
  • recombinant toxins e.g., for cancer treatment is known in the art (see, e.g., Pastan and Fitzgerald Science, November 22, 1991, pages 1173-1177, and the articles cited therein, which references are herein entirely incorporated by reference) .
  • ⁇ Obtaining a safer vector for in vivo gene therapy.
  • the use of three different tools can be used to increase the margin of safety in vivo gene therapy treatments is provided according to the present invention. These tools are: (a) the use of different packaging cell lines; (b) the use of ecotropic, rather than amphotropic-based vectors, and (c) providing means to target retroviral vectors to the desired cell population in vivo.
  • a Chinese hamster cell expressing -HaLV; and stably expressing the murine ecotropic virus receptor (cell line A) is used to provide helper virus, and co-cultivated with a second Chinese hamster cell expressing - ecotropic MuLV (cell line B) , but not expressing the murine ecotropic virus receptor.
  • virus propagated in cell line A is able to infect cell line B through the HaLV receptor.
  • Virus replicated in this cell now has the murine ecotropic gp70, and is able to infect cell line A. This process is expected to continue until a theoretical maximum number of particle production is achieved (about 10 9 to 10 10 PFU) (Bodine et al. Proc. Natl . Acad. Sci .
  • a Chinese hamster cell line containing multiple copies of ecotropic virus sequences integrated into genome, and producing at least about 10 7 -10 10 , preferably about 10 9 to 10 10 , with no particles, recombinant or otherwise, capable of infecting human cells, unless their virus receptors are modified according to the present invention.
  • appropriate viral infectivity assays are performed with a variety of human and murine cells lines, normally used to detect viral infectivity. This approach is expected to yield a far safer packaging line than previously available.
  • HaLV is cloned and a deletion in its packaging signal is produced.
  • the removal of the packaging signal is obtainable by known method steps.
  • transfection and stable expression of the murine ecotropic virus receptor into a CHO hamster cell is provided according to known method steps, e.g., as described in Yoshimoto et al. (1993).
  • a cloned helper virus is introduced into these cells as split genomes for added safety.
  • plasmids pgragr-polgpt and penv derived from the 3P0 plasmid representing the ecotropic Moloney murine leukemia virus with a 134 base pair deletion of the M packaging sequences, are readily obtained from commercial sources or published investigators as presented herein. Such plasmids have been used successfully used to generate PCLs with somewhat greater safety than those using unsplit helper virus genomes.
  • clone HaLV known procedures are used, e.g., the procedure of Anderson et al. (1991), similar to cloning defective retrovirus particles from a recombinant Chinese Hamster ovary cell line.
  • clones include the pCHOC.MLlO sequence identified by Anderson or endogenous, polytropic murine leukemia virus (MuLV) isolate, MX27 (Stoye and Coffin, 1987).
  • the later probe consists of a 12.3 kb mouse genomic fragment encompassing a complete 9.3 kb provirus genome.
  • Hamster C-type related sequences are isolated from a randomly primed cDNA library of particle RNA in lgtlO.
  • Extracellular particles are prepared from culture fluid recombinant CHO cell subclone, 3- 3000-44 (Lasky et al., 1986). This subclone was derived from a dihydrofolate reductase (dhrf) -deficient CHO-DuxBll cells (Simonsen and Levinson, 1983; Urlab and Chasin, 1980) following transfection with an expression vector containing the genes for murine dhfr and recombinant envelope glycoprotein (gpl20) of human immunodeficiency virus type 1
  • dhrf dihydrofolate reductase
  • gpl20 envelope glycoprotein
  • the CH0-K1 cell line (progenitor of CH0-DUXB11 line) was originally derived from an ovarian biopsy of an adult Chinese Hamster (Puck et al. , 1958) and is readily available from commercial sources such as the American Type Culture Collection (ATCC) . Alternatively, repair of the hamster sequence with equivalent portion of the murine ecotropic virus is used. As observed by Anderson et al., virus particles are expected to be produced.
  • the endonuclease region can be replaced with that of a homologous retrovirus genome, such as from ecotropic MuLV.
  • the HaLV surface envelope proteins is appropriately expressed so that the particles can infect other hamster cells; all other HaLV genes are not essential (LTRs possibly excluded) , and replaceable by homologous MuLV sequences.
  • a modified ecotropic retroviral receptor (MERR) for gene delivery should lower the potential incidence of cancer and related diseases during gene therapy.
  • any recombinant viruses that my arise will not be able to infect human cells, as murine ecotropic viruses cannot replicate in these cells.
  • the gene therapist will be able to limit with extraordinarily specificity the infection only to those target cells desired to be infected. Potential random insertions of viruses all over the human genome in various types of organs will not be expected or shown to occur, as would be expected whenever human infectable amphotropic-retroviruses-based vectors are used.
  • methods of the present invention use a human viral receptor protein which is minimally modified, the possibility of rejection of the infected human cells by the immune system is substantially reduced or eliminated.
  • a preferred aspect of the present invention is the method of treating an animal using a delivery vector which permits specific targeting of cells to be infected by recombinant, non-human specific ecotropic viruses which provide a therapeutic effect on a target cell.
  • a delivery vector which permits specific targeting of cells to be infected by recombinant, non-human specific ecotropic viruses which provide a therapeutic effect on a target cell.
  • Non-limiting examples of such an agent include a fusion protein encompassing the V H and V L regions of a specific antibody to a cell surface molecule (such as an MHC Class 1 antigen) joined with an appropriate linker peptide and the mouse ecotropic virus receptor or the modified human ecotropic receptor.
  • a ligand for a membrane receptor such as the epidermal growth factor receptor
  • compositions comprising the proteins, peptides or antibodies of the present invention, such as at least one chimeric receptor polypeptide or antibody include all compositions wherein at least one therapeutic agent is contained in an amount effective to achieve its intended purpose.
  • pharmaceutical compositions containing at least one therapeutic agent 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 0.001 to 99 percent, preferably from about 20 to 75 percent of active component (i.e., the therapeutic together with the excipient.
  • Pharmaceutical compositions for oral administration include tablets and capsules.
  • Compositions which can be administered rectally include suppositories.
  • 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. If desired, these may be sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • Preferred 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.
  • 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 therapeutic agents 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 therapeutic agent of the present invention may be administered by any means that achieve its 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 retroviral 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.
  • a therapeutic agent of the present invention may be incorporated into topically applied vehicles such as salves or ointments, which have both a soothing effect on the skin as well as a means for administering the active ingredient directly to the affected area.
  • topically applied vehicles such as salves or ointments, which have both a soothing effect on the skin as well as a means for administering the active ingredient directly to the affected area.
  • a preferred topical preparation is an ointment wherein about 0.001 to about 50 mg of active ingredient is used per cc of ointment base, the latter being preferably PEG-1000.
  • a typical regimen for treatment or prophylaxis comprises administration of an effective amount over a period of one or several days, up to and including between one week and about six months.
  • the dosage of a therapeutic agent of the present invention administered in vivo or in vitro will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent 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 preferred dose ranges. However, the most preferred 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 therapeutic agent 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 a therapeutic agent of the present invention are from about 0.001 ⁇ g to about 100 mg/kg body weight, and preferably from about 1 ⁇ g to about 50 mg/kg body weight.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous 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.
  • the present invention provides methods for evaluating the presence and the level of normal or chimeric receptor or H13 protein or mRNA in a subject. Absence, or more typically, low expression of the H13 gene or presence of • a mutant H13 in an individual may serve as an important predictor of resistance to retrovirus infection and thus to the development of AIDS or certain types of leukemia or other retrovirus-mediated diseases. Alternatively, over-expression of H13, may serve as an important predictor of enhanced susceptibility to retrovirus infection.
  • the present invention provides a means for detecting a human retrovirus-infected or retrovirus-transformed cell in a subject.
  • Oligonucleotide probes • encoding various portions of a chimeric receptor polypeptide or chimeric receptor polypeptide encoding nucleic acid sequence are used to test cells from a subject for the presence a chimeric receptor polypeptide or chimeric receptor polypeptide DNA or mRNA.
  • a preferred probe would be one directed to the nucleic acid sequence encoding at least 12 and preferably at least 15 nucleotide of a chimeric receptor polypeptide or chimeric receptor polypeptide sequence.
  • Qualitative or quantitative assays can be performed using such probes.
  • Northern analysis is used to measure expression of a chimeric receptor polypeptide or chimeric receptor polypeptide mRNA in a cell or tissue preparation.
  • Such methods can be used even with very small amounts of nucleic acid obtained from an individual, for knowing uses of selective amplification techniques.
  • Recombinant nucleic acid methodologies capable of amplifying purified nucleic acid fragments have long been recognized.
  • Such methodologies involve the 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. Examples of such methodologies are provided by Cohen et al. (U.S. Patent 4,237,224), Sambrook et al., supra, etc.
  • the polymerase chain reaction provides a method for selectively increasing the concentration of a particular DNA 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 essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired DNA molecule.
  • CCL120 ATCC# CCL120
  • CCL119 CEM, ATCC# CCL119
  • SupTl a human non-Hodgkin's T lymphoma cell line
  • H9 a single cell clone derived from HUT78, a huma ⁇ cutaneous T cell lymphoma cell line
  • M0LT4 ATCC# CRL1582
  • HOS ATCC# CRL1543
  • HLa ATCC# CCL2
  • CH0-K1 ATCC #61
  • EcoRI-EcoRI or EcoRI-Hindlll fragments in the cosmids were subcloned into pBluescript or pSport 1 (GIBCO BRL, Gaithersburg, MD) .
  • the exons and exon-intron junctions were sequenced using synthetic oligonucleotides as primers. Sequences were compiled and analyzed using the Genetics computer group sequence analysis software package (Devereux, J. et al., Nucl . Acids Res . 12:387-395 (1984)).
  • the complete nucleotide sequence of H13 (SEQ ID NO:7) including non-coding sequences at the 5' and 3' end of the coding sequence are shown in Figure 1.
  • This sequence includes the partial sequence originally obtained from clone 7-2 (SEQ ID N0:1); nucleotide 1-6-and 1099-1102 of SEQ ID N0:1 were originally incorrectly determined.
  • Figure 1 also shows the complete amino acid sequence predicted from the nucleotide sequence (SEQ ID NO:8).
  • This sequence includes the originally described partial amino acid sequence (SEQ ID NO:2) with the exception of the N-terminal Pro-Gly and the C-terminal Pro, which were originally incorrectly predicted from the nucleotide sequence.
  • ERR is shown in Figure 2 and the amino acid sequence comparison of H13, ERR and TEA is shown in Figure 3.
  • the homology between the compared sequences is very high, for example 87.6% homology between H13 and ERR DNA, and 52.3% homology between H13 and TEA amino acids.
  • Murine retroviral receptor (ERR) cDNA was cotransfected into hamster CHO cells, which can not be infected by murine ecotropic retroviruses, with the selectable marker plasmid DNAP, pSV 2 Neo, using calcium phosphate (Wigler, M. et al., Cell 14: 725-731 (1978)).
  • the transfectant expressing the receptor gene was, then, infected by murine radiation leukemia virus (RadLV) .
  • RadLV murine radiation leukemia virus
  • RT reverse transcriptase activity of the supernatant was measured (Stephenson, J.R. et al., Virology 48: 749-756 (1972)), and Northern Blot analysis was performed using a viral probe after preparing its RNA.
  • the RT activity detected in untransfected CHO cells which do not express the receptor gene was indistinguishable from the activity of tissue culture medium (background) . This indicates that the cells were not infected by MuLV.
  • the MuLV viral probe detected transcripts in RNA prepared from the transfectant, but not in RNA prepared from untransfected CHO cells. The results indicate that the cells transfected with the ERR cDNA can acquire the susceptibility to ecotropic murine leukemia virus.
  • the DNA sequence encoding a highly antigenic portion (SEQ ID NO:2, amino acid residues 309-367) was prepared by cutting with the restriction enzymes AccI and EcoRI yielding a 180 bp AccI-EcoRI fragment.
  • This fragment of H13 cDNA was ligated to the cloning sites of pGEX-2T plasmid vector (Pharmacia LKB Biotechnology) , which can express antigens as fusion proteins with glutathione-S-transferase (GST) , in the orientation that permit [s] the expression of the open reading frames (Smith, D.B. et al. , Gene 67: 31-40 (1988)).
  • the fusion protein was induced by addition of isopropyl-beta-thiogalactopyranoside (IPTG) to cultures, and was purified using glutathione Sepharose 4B chromatography (Pharmacia LKB Biotechnology) (see Figure 11) .
  • IPTG isopropyl-beta-thiogalactopyranoside
  • the purified fusion protein injected intramuscularly and subcutaneously into rabbits with Freund's complete adjuvant to obtain antisera.
  • the antisera are shown to bind specifically to the
  • H-13 protein and epitopic fragments thereof are epitopic fragments thereof.
  • Membrane proteins from human cells are prepared according to standard techniques and are separated by polyacrylamide gel electrophoresis, an blotted onto nitrocellulose for Western Blot analysis.
  • the H-13 specific antibodies are shown to bind to proteins on these blots.
  • H13 Chromosomal location of the H13 gene was determined using Chromosome Blots (Bios Corp. , New Haven, Connecticut) containing DNA from a panel of human-hamster somatic cell hybrids (Kouri, R. E. et al., Cytogenet. Cell Genet. 51:1025 (1989)). By comparison of which human chromosomes remained in the human-hamster hybrid cell and the expression of H13 cDNA, the H13 gene was mapped to human chromosome 13 (see Figure 12) .
  • Human genes (or diseases caused by mutations therein ) linked to chromosome 13 include: retinoblastoma, osteosarcoma, Wilson's disease, Letterer-Siwe disease, Dubin-Johnson syndrome, clotting factor Vii and X, collagen IV ⁇ l and CU2 chains, X-ray sensitivity, lymphocyte cytosolic protein-1, carotid body tumor-1, propionyl CoA carboxylase ( ⁇ subunit) , etc.
  • Extracellular Domain 3 is the region of the receptor protein which is most diverse between the human and mouse sequences, as shown in Figure 14.
  • the sequences in Figure 14 (derived from the sequences shown in Figure 1-3) were aligned using Genetics computer group sequence analysis software package (Devereux, J. et al, Nucl . Acids Res . 12:387-395 (1984)).
  • oligonucleotide-directed mutagenesis was employed to produce chimeric molecules containing individual amino acid substitutions within extracellular domain 3. These were transfected as above and the transfectant cells are tested for susceptibility to infection by E-MuLV as shown above.
  • Human H13 amino acid residues were substituted by murine ERR residues, as described in Figure 18.
  • Mouse-human chimeric receptor molecules were made by substitution using common restriction sites which clarified that the extracellular domains 3 and/or 4 contain the critical amino acid residues. Oligonucleotide-directed mutagenesis was then used to create 13 individual mutant ERR molecules containing one or two amino acids substitutions or insertions within these two extracellular domains.
  • H13 molecule without modification by substitution or deletion of amino acids lacks the ability to function as a receptor for MuLV-E.
  • modifications, as presented in Figure 18, for MuLV-E infection H13 modified proteins were prepared by substitution using the common and single restriction site, Kpnl , and determined their abilities to function as a receptor for MuLV-E (Fig. 16) .
  • Figure 17 shows the comparison of sequences of extracellular domains 3 and 4 in murine ERR and human H13, which are aligned using the Genetics computer group sequence analysis software package (Devereaux et al Nucleic Acid Res . 12:387-395 (1984)).
  • Extracellular domain 3 is the most diverse region between murine ERR molecules (Mutants 1-11) containing one or two amino acid substitutions or insertions within these two domains (Fig. 17 and Table 2) . For each substitution, amino acid residues of ERR were replaced with those found in equivalent position of H13 sequence. For each insertion amino acid residues of H13 were added into equivalent position of ERR sequence which aligned as shown in Fig. 17.
  • Mutant 7 has two amino acid substitutions, Tyr (235 amino acid residue in ERR) to Pro (corresponding to amino acid residue 242 in H13) and Glu (corresponding to amino acid residue 244 in ERR) to Val (244 amino acid residue in H13) .
  • Mutants 7A and B which contain just one amino acid substitution (Mutant 7A: Tyr to Pro and Mutant 7B: Glu to Val) , were prepared and tested for their abilities to function as the receptor. Although Mutant 7B has almost the same ability to function as the receptor as the intact ERR, Mutant 7A was found to almost completely abrogate the ability (Table 2) . These results suggest that the Tyr located at 235 amino acid residue in ERR sequence is very important to function as a receptor protein for MuLV-E and substitution of this amino acid residue leads ERR to lose its ability to function as the receptor.
  • H13 To determine whether the H13 molecule would acquire the ability to function as the receptor if certain amino acid residues in the H13 are substituted by the corresponding amino acid residues in ERR, eight mutants of H13 were made as shown in Fig. 18, and their abilities to function as the receptor were determined.
  • the H13 mutants were created by the method of altered site-directed mutagenesis using a phagemid vector, pSELECT-l (Promega) , e.g., as presented in Lewis and Thompson, Nucleic Acids Res . 18:3439-3443 (1990) .
  • the mutagenesis is based on the use of single stranded DNA and two primers, one mutagenic and a second correction primer which corrects a defect in the vector to ampicillin resistance.
  • H13 mutants 1-3 and 5 were prepared using the pSELECT-l antisense H13 as a template and oligonucleotides: AAAGAAGGGAAGTACGGRGRRGGRGG (SEQ ID NO:9) (H13 Mutant 1) ;
  • H13 Mutant 4 was prepared using pSELECT-l antisense H13 Mutant 8 and oligonucleotide TGAAGTACGGTGTTGGTGGATTCATG (SEQ ID NO:13) .
  • H13 Mutants 6-8 were prepared using pSELECT-l antisense H13 Mutant 5 and oligonucleotides ACACAAAAGAAGTGAAGTACGGTGA (SEQ ID NO:14) (H13 Mutant 6) ; AATGACACAAAAAACGTGAAGTACGGTGA (SEQ ID NO: 15) (H13 Mutant 7) ; and AACAATGACACAAACGTGAAGTACGGTGAGGGTGGATTCATG (SEQ ID NO: 16) (H13 Mutant 8) .
  • the ERR mutants were also created by the method of altered site-directed mutagenesis using a phagemid vector, pSELECT-l (Promega) , e.g., as presented in Lewis and Thompson, Nucleic Acids Res. 18:3439-3443 (1990).
  • the mutagenesis is based on the use of single stranded DNA and two primers, one mutagenic and a second correction primer which corrects a defect in the vector to ampicillin resistance.
  • pSG5ERR The insert of pSG5ERR was partially digested with BamHI and EcoRI, and subcloned to the BamHI and EcoRI sites in pSELECT-l to obtain pSELECT-l sense and anti-sense ERR.
  • Single stranded DNA was prepared from pSELECT-l sense (for preparation of Mutants 2 and 6) and antisense (for preparation of the other mutants) ERR and mutagenesis was carried out according to the manufacturer's directions (Promega).
  • the correctly mutated clones were selected by directly sequencing using Sequenase (USB) and two ERR specific antisense oligonucleotides GGTGGCGATGCAGTCAA (SEQ ID NO: 17) for mutants 1-7 and TCAGCCATGGCATAGATA (SEQ ID NO:18) for Mutants 8-11) as primers.
  • Mutated inserts of the phagemids prepared by mini- preps were excised with EcoRI and subcloned into the EcoRI site of pSG5. The presence of mutations was confirmed by sequencing each plasmid using the same primers as used above. Each mutant was then transiently transfected into CH0-K1 cells by the method using Lipofectin reagent and their susceptibilities to infection by MuLV-E were determined as above for results shown in Figure 16.
  • mutant H13 polypeptides according to the present invention containing Tyr242 and at least one of Val 240 and Glu244 provide a mutant H13 receptor binding region that is functionally ecognized by ecotropic murine retroviruses, such as MuLV-E, such that expression of such a chimeric receptor polypeptide on the extracellular surface of a human cells allows binding and infection by a murine ecotropic retrovirus.
  • ecotropic murine retroviruses such as MuLV-E
  • Such a method can thus be used according to the present invention as a method for gene therapy in vitro, in vitro or in situ.
  • such a method of the present invention can be used to introduce heterologous or exogenous genes into human cells or tissues using a murine ecotropic retroviral vector.
  • an H13 mutant according to the present invention provides a much safer means for gene therapy than the use of amphotropic retroviral vectors, which overcomes the problems of unintended infection of non-target cells by amphotropic retroviruses, as well as immunogenicity reduction for the use of relatively lower dosages of recombinant ecotropic virus.
  • the modified gene for the ecotropic retroviral receptor will be delivered to target cells and transiently expressed.
  • the murine ecotropic-virus- vector would then be used to infect, stably integrate, direct the expression of the desired therapeutic gene. Zero to some time may elapse between the first step of the infection.
  • the ecotropic virus vector to be used will carry deletions of the structural genes and be propagated in "safe" packaging cell lines for added safety.
  • the construction of novel packaging lines producing virus titers in the 10 8 to 10 10 particles/ml range is expected and devoid of recombinant viruses capable of infecting human cells.
  • modification of the viral envelope glycoprotein will eliminate any determinants which would interfere with virus infectivity in vivo or diminish virus titers.
  • the human ecotropic virus receptor homolog also is studied to determine its normal gene function and gain sufficient understanding of the protein to eliminate the likelihood that gene therapy protocols would affect its normal function in a deleterious manner.
  • the amphotropic receptor is cloned for development of gene therapy vectors.
  • the receptor for amphotropic-MLV are cloned by a similar strategy to that used to clone the receptors for Gibbon ape leukemia virus and mouse ecotropic virus (E-MLV) according to known method steps (e.g., Brown et al, 1990; Anderson et al, 1991) .
  • E-MLV mouse ecotropic virus
  • the strategy relies on the fact that human cells can be infected by A-MLV but hamster cells cannot..
  • the inability to infect hamster cells result from their lack of a suitable receptor, making possible the transfection the human gene into hamster cells, rendering them infectable by A-MLV.
  • the receptor gene is accessible to cloning by virtue of its association with human repetitive DNA.
  • amphotropic receptor gene is cloned, its similarities and dissimilarities to the ecotropic virus receptor is studied in a variety of assays and by a variety of techniques (e.g., Yoshimoto et al, 1993) .
  • CHO cells are plated on the morning of transfection. Sheared human genomic DNA (50 ⁇ g) and pSV 2gpt DNA (1 ⁇ g) are coprecipitated with calcium phosphate by the method of Wigler et al. (1978) and applied to CHO cells. The next day, transfected cells are passaged at 2 x 10 5 cells per plate in gpt selection medium (DMEM/10% FCS, hypoxanthine 15 ⁇ g, xanthine ⁇ g/ml, thymidine 10 ⁇ g/ml, glycine 10 ⁇ g/ml, methotrexate 0.1 ⁇ M, and mycophenolic acid 25 ⁇ g/ml). After 21 days under selection, colonies are dispersed by brief exposure to trypsin/EDTA and replaced prior to exposure to viruses, allowing for enrichment of cells that have acquired human DNA.
  • DMEM/10% FCS hypoxanthine 15 ⁇ g, xanthine ⁇ g/ml, thy
  • PA317/LNL6 amphotropic retrovirus producer fibroblasts are grown to confluence and refed with fresh medium. Twelve to twenty hours later, the culture medium is filtered (0.45 ⁇ m, Nalge) , brought to 8 ⁇ g/ml of polybrene, and incubated with the transfected CHO cells. After 4-12 hours fresh medium is added, and the infection protocol repeated the next day. Three days later, these cells are replated at 2 x 10 5 cells per 82 mm plate in DMEM/10% FCS containing 1 mg/ml of G418, and selection medium is replaced every 3 days for 15 days. It is expected that out of 20,000 transfectants, only a few (10-20) will develop into g418 resistant colonies.
  • amphotropic virus ⁇ -2-AM-ZIP-DHFR
  • ⁇ -2-AM-ZIP-DHFR amphotropic virus
  • cells To authenticate that clones are infectable by this amphotropic virus indeed express the amphotropic virus receptor, they are also infected by a second amphotropic virus ( ⁇ -2-AM-ZIP-DHFR) . It is possible for cells to acquire the virus through a low efficiency pathway not involving the virus receptor. G418-resistant colonies are isolated with cell cloning cylinders and each is exposed to - 2-AM-ZIP-DHFR virus as described above for the neomycin virus. Following exposure to the virus, cells are selected in DMEM/10% dialyzed FCS containing methotrexate (150 nM) . After 14 days, plates are stained for the presence of methotrexate- resistant colonies with 1% crystal violet.
  • DNA is then prepared from this primary transfected cell line (1°TF) and used in a second cycle of the transfection/infection protocol.
  • To identify the receptor gene is the secondary transfectant cell lines, Southern blots using a panel of human repetitive sequences as probes are made. Because of the low efficiency of DNA transfection (0.1% genome/cycle) , cycles of transfection/selection are adequate to segregate the receptor gene away from the remainder of the murine genome (Murray et al., 1981).
  • a lambda phage library is prepared from secondary transfectants DNA and hybridized with the radiolabeled repetitive probe that seems most appropriate from the Southern blot screening.
  • RNA transcript present in the 2°TFs growing the amphotropic viruses are identified.
  • the specific transfer of the gene in question is expected to transfer susceptibility to amphotropic virus infection in a consistent manner, and a large panel of cells infectable by these viruses express this molecule is provided.
  • a complementary DNA (cDNA) from the antibody B3 (e.g., Brinkmann et al, 1991) is used to construct an Fv fragment that is fused to a chimeric receptor polypeptide of the present invention.
  • This single-chain recombinant receptor then is used to allow retrovirus infection of targeted human cells.
  • a single-chain Fv and two different (B3 (Fv) immunotoxins, B3(Fv)-PE40 and B3 (Fv)PE38KDEL vectors are used via standard recombinant DNA technologies to insert into the chimeric receptor polypeptide encoding nucleic acid, or substitute its recombinant toxin with the virus binding domain of the gene encoding the modified region of the human ecotropic virus receptor.
  • the resulting plasmid (B3 (Fv) -mH13) as well as the immunotoxin vector B3 (Fv)PE38KDEL are expressed in a host, such as E. coli , and the single chain immunoreceptor and single-chain immunotoxin are purified to homogeneity as known in the art.
  • the antitumor activity of the B3 (Fv) -mH13 is determined first in vitro.
  • the B3 antibody reacts uniformly with the surface of many mucinous carcinomas of the colon, stomach, and ovary and with normal tissues, such as glands of the stomach, epithelia of the trachea and bladder, differentiated epithelium of the esophagus, and small bowel mucin. (Pastan et al. ( Cancer Res . 51:3781-3787, 1991)).
  • the B3 antibody also reacts uniformly with many human tumor cell lines, including MCF7, MDA-MB-468, and HTB20 (breast), A431 (epidermoid) , TH29 (colon) , HTB33 (cervical) , and DU145 (prostrate) . Infection in some or all of these cells is expected, such as A431, of a non-human specific recombinant retroviral vector, such as a murine ecotropic retrovirus vector carrying the neomycin resistant gene, after first delivering to those cells the modified receptor peptide by use of the fusion protein derived from B3(Fv)-mH13.
  • a non-human specific recombinant retroviral vector such as a murine ecotropic retrovirus vector carrying the neomycin resistant gene
  • the viral vector having an env binding domain which binds the chimeric receptor polypeptides and a therapeutic agent, such as a murine ecotropic retrovirus vector carrying the thymidine kinase gene, is used preliminarily to cause cell death of cultured tumor cells, such as A431 cells, by the addition of ganciclovir to the cell cultures.
  • murine ecotropic virus-based vectors of the present invention are expected to be incapable of infecting these cells, unless a chimeric receptor polypeptide or the corresponding region of the murine ecotropic virus receptor is expressed on the selected animal model cell surface via the delivery vector presented herein.
  • a viral vector carrying the thymidine kinase gene is used to infect the animal models expressing the chimeric receptor polypeptide on the selected target cells. This expression is followed by the administration of ganciclovir to the animals. This protocol is expected to achieve tumor reduction as the ganciclovir is phosphorylated within tumor cells to its toxic form and in conjunction with the associated "bystander effect".
  • the expression plasmid pULl contains the gene for the immunotoxin B3(Fv)-PE40, which is a fusion protein including an antibody fragment to an antibody to B3 specific of carcinoma cells, conjugated to the toxin
  • the pULl expression plasmid is modified to replace the PE40 toxin encoding portion with a chimeric receptor polypeptide of the present invention or the corresponding region of the murine ecotropic virus receptor to provide a delivery vector that transforms carcinoma cells in vivo, in situ or in vi tro, to express a chimeric receptor polypeptide.
  • B3 (Fv) is a single chain antigen-binding protein derived from a monoclonal antibody to B3.
  • the B3 antibody fragment recognizes a carbohydrate antigen which is found on the surface of many mucinous carcinomas. However, the antibody fragment reacts with only a limited number of normal tissues, such that the antibody fragment will preferentially bind carcinoma cells in vivo .
  • PE40 is a truncated derivative of Pseudomonas exotoxin.
  • the PE40 coding region has a Hind III restriction site at the 5' end, the point of connection to the DNA encoding B3 (Fv) , and an EcoRI site just beyond 3' end.
  • This Hind III-EcoRI fragment encoding PE40 is removed from pULl and both termini of the linearized pULl are partially filled-in with dATP, yielding cohesive ends -AA.
  • the -TTCGA at 3' end of B3 (Fv) coding region and AATTC- at the other terminus of the linearized plasmid are similarly modified to complement the chimeric receptor polypeptide encoding DNA, as follows.
  • Nru 1-Pst 1 fragment of a chimeric receptor polypeptide the present invention as a modified H13 cDNA, which contains whole coding region of modified H13, is digested with Tfi I and the 850 bp fragment, which contains the region encoding the third extracellular domain of the modified H13, is purified on a 1.5% agarose gel.
  • the purified 850 bp Tfi 1 fragment is then digested with BsrI and 85 bp Bsr 1-Tfi I fragment, which encodes the whole third extracellular domain of the modified H13 designated Ex3mH13.
  • the resulting restriction fragment EX3ml3 is purified on 2.0% agarose gel.
  • the purified 85 bp Bsr 1-Tfi 1 fragment has cohesive ends AGC- at 5' end and -GG at '3 end.
  • This 3' end is partially filled-in with dATP, making the 3' end
  • the 85 bp Bsr 1-Tfi fragment is be ligated to the partially filled-in Hind III-
  • GATTAATCTT SEQ ID NO:21 which are specially designed to prevent any frame shift.
  • the resulting plasmid is designated pBH30.
  • the gene encoding B3(Fv) has a Ndel site at 5' end.
  • the resulting plasmid pBH30 is then digested with Ndel and the termini are filled in with dATP and dTTP to become blunt ends.
  • the purified fragment is integrated into an expression vector pTrcHisB at the Bglll-EcoRI site, which is positioned downstream of the series of a Tre promoter, an ATG initiation codon, a polyhistidine coding region and an enterokinase-cleavable site coding region, using an adaptor GATCCCCGGG (SEQ ID NO:22)
  • the resulting plasmid is designated pBH3.
  • the expression plasmid pBH3 allows B3 (Fv) -Ex3MH13 to be expressed as a fusion protein composed of a polyhistidine metal binding domain, an enterokinase-cleavable site and
  • E. coli HB101 is then transformed with pBH3.
  • the expression is induced with isopropyl
  • the suspension is sonicated and the supernatant is loaded on a Ni 2 + metal affinity resin column.
  • the protein bound to the resin is eluted by competition with glycine.
  • the eluted protein is then treated with enterokinase for the polyhistidine sequence to be removed.
  • the resulting fusion protein is expected to specifically bind the pathologic cells described and is suitable for .providing a chimeric cell as a target for therapy as described herein.
  • the present invention provides for the dissection of viral envelope elements required for binding to both receptors' as well as the characterization of the degree of modification that these proteins will tolerate without abrogating their capacity to bind the receptors, as well as to examine how the limited differences between the human and mouse ecotropic receptors responsible for allowing binding of the virus.
  • a second aim is to eliminate any potential complement binding region on the viral envelope which might lead to virus lysis in humans, a problem which has been argued by some investigators to probably limit the infectious potential in vivo of therapeutic vectors based on murine retroviruses.
  • the finding of nonspecific inactivation and lysis of murine, feline, and simian C-type viruses was originally published by.Welsh et al. (1975, 1976) .
  • the lysis is due to antibody-independent binding of the human Clq complement component to gp70, leading to the activation of the classic complement pathway (Cooper et.al., 1976).
  • Murine leukemia virus (MuLV) surface glycoprotein (gp70 su ) is to determine which regions of gp70 su participate in the specific interactions with cell surface receptors.
  • MuLV Murine leukemia virus
  • a variety of studies have suggested that the determinants for receptor specificity lie in the N-terminal two thirds of gp70 su (Ott and Rein J. Virol 66:4632-4638, 1992 -- INSERT REFS 13, 16).
  • Heard and Danos (1991) have recently shown than an Env fragment containing most of this region of Friend MULV gp70 su can bind to the ecotropic receptor in NIH 3T3 cells.
  • MuLVE-gp70 which is critical for binding to its receptor.
  • the MuLVE- gp70 is modified by in-frame deletions within the amino- terminal domain, and by oligonucleotide directed mutagenesis.
  • a comparison of envelope sequences shows that MLVgp70s differ in two limited regions in their amino-terminal domains. These are amino acids 50 to 116 and 170 to 183. They also differ in the proline-rich segment, amino acids 244 to 283.
  • one or more of these three hypervariable regions are expected to include receptor binding cells.
  • Defective retroviral vector transducing a modified E. coli lacZ gene is used to infect cells expressing wild-type or modified MuLVE-gp70s. Susceptibilities to infection are determined by counting X-Gal-positive foci.
  • the ecotropic envelope N-terminal domain vectors is transiently transfected into simian Cos-7 cells that express the exogenous murine ecotropic retroviral receptor gene (Albritton et al, 1989) .
  • the Cos cells (expressing to T antigen gene) permits a high level of env gene expression (Gluzman, 1981) .
  • Transfected cells are infected with the ecotropic pseudotype CRE/BAG (ATCC CRL 1850) virions (Price et al, 1987) and the number of jS-galactosidase (gal) foci are assayed. Modified env fragments which decrease the number of j ⁇ -gal foci are further examined for their ability to interact with the ERR..
  • Cells are fixed with 0.5% glutaraldehyde in physiological buffered saline and stained with a histochemical solution containing 1 mg/ml X-gal (Sanes et al, 1986) . Those cells having the highest number of j3-gal foci are used for interference assays.
  • Recombinant env gene constructs are transiently transfected into Cos-7 cells containing the murine ERR. This system is expected to eliminate any potential problems caused by endogenous env transcripts that might be present in murine cells.
  • Cos cells are expected to permit high expression of the SV40 promoter-containing expression plasmids (Gluzman, 1981) .
  • the cells are transfected by the DEAE dextran method according to Stratagene protocols. Approximately 48 hrs after env gene transfection, 2xl0 5 cells are plated onto a six-well culture dish and infected with 10 2 -10 3 CRE/BAG virions in the presence of 8ug polybrene/ml according to the method of Heard and Danos (1991) .
  • the cells When the cells have grown to confluence, they are fixed in 0.5 % gluteraldelhyde in PBS and stained with a histochemical solution containing lmg/ml X-gal (Sanes et al, 1986) .
  • the number of ⁇ -gal foci are scored relative to cells which were transfected with the pSG5 vector alone.
  • a decreased number of ⁇ -gal foci are expected to be scored relative to cells which were transfected with the pSG5 vector alone.
  • a decreased number of 0-gal foci determines that the transfected env construct is likely to be able to bind the ERR and block virion interaction.
  • env RNA transcripts is determined by extracting total RNA from the transfected cells followed by northern blot analysis using an AKv env sequence probe.
  • Fragments which are able to be detected by immunoprecipitation and which block CRE/BAG virion infection are utilized in further examples and methods of the present invention.
  • a 163bp Smal fragment is present (404bp, 567bp) within the variable region of the Akv env N-terminal sequence (Lenz et al, 1992) .
  • This fragment includes the 30 amino acids which are deleted from the amphotropic sequence ( Figures 19 and 20) . Simple removal of this sequence will create an incorrect reading frame since one Smal site (404bp) lacks 1 nucleotide of an amino acid triplet, b) Creation of Insertions.
  • PCR polymerase chain reaction
  • the insertion of the 30 amino acid region of the ecotropic virus into the amphotropic virus sequence is accomplished by the following steps.
  • the product is run on a 2% agarose gel, the 90bp fragment is excised, purified and ligated to the amphotropic env sequence at the Rsal site. Recombinant clones are sequenced to determine those having the correct orientation of the PCR fragment.
  • sucrose banded and purified, cloned Radiation leukemia virus is diluted with PBS to a standardized concentration, so that at least 100,000 cpm are detectable in our standard reverse transcriptase assay (Brown et al, 1990) .
  • Human serum at various dilutions are added to the virus preparation and incubated for 30 minutes under normal laboratory lighting conditions.
  • Control samples are treated with .5% Triton X-100 (Sigma). All samples are then analyzed for RT activity as previously described (Meruelo et al, 1988) .
  • a putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.
  • CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature, 312:763-767, 1984.
  • Gluzman, Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell, 23:175-182, 1981.
  • RNA tumor viruses molecular biology of tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984.
  • CA A (SEQ ID No: 30)
  • each oligonucleotide sequence are those in the original ERR sequence. - means the absence of corresponding nucleotide sequence in ERR sequence according to the alignment (Fig.
  • AAA AAC ATG GAG CCT TCC AAA ATC TCT GGG CTA ATT GTG AAC CCG G 1102 Lys Asn Met Glu Pro Ser Lys Ile Ser Gly Leu Ile Val Asn Pro 355 360 365
  • GAA AAT GCT GGC CCT GCC ATC GTC ATC TCC TTC TTG ATT GCT GCT CTC 423 Glu Asn Ala Gly Pro Ala Ile Val Ile Ser Phe Leu Ile Ala Ala Leu 60 65 70 75
  • GGT ACT TCA AGC GTG GCA AGA GCC TGG AGT GCG ACT TTT GAC GAG CTG 615 Gly Thr Ser Ser Val Ala Arg Ala Trp Ser Ala Thr Phe Asp Glu Leu 125 130 135
  • GAG AAA GAA ACT CTG GAA TCA TGT ACC AAT GCG ACT TTG AAG AGC
  • GAG 1183 Glu Lys Glu Thr Leu Glu Ser Cys Thr Asn Ala Thr Leu Lys Ser Glu 245 250 255
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:13:
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA

Abstract

L'invention concerne des polypeptides récepteurs chimères contenant des récepteurs viraux chimères appartenant à une première espèce qui lie des virus spécifiques n'appartenant pas à la première espèce et/ou les domaines de liaison de leur enveloppe protéinique env, des acides nucléiques de codage de ceux-ci, des cellules ou des tissus d'expression du polypeptide récepteur chimère, ainsi que des procédés de préparation et d'utilisation de ceux-ci. L'invention concerne également des procédés de sensibilisation sélective de cellules ou de tissus eucaryotes à la liaison et/ou à l'infection par des vecteurs viraux recombinés ou à des vecteurs de transfert qui se lient à un récepteur chimère d'un polypeptide récepteur chimère exprimé dans des cellules ou des tissus cibles. On peut ainsi utiliser des procédés de thérapie génétique pour traiter des cellules ou des tissus sélectionnés d'un mammifère, d'un oiseau, d'un insecte ou d'une levure en utilisant l'expression transitoire ou constitutive ou l'association vectorielle d'un polypeptide récepteur chimère ayant un site de liaison du réceptor chimère. Les cellules ou tissus à récepteurs chimères deviennent ainsi susceptibles de s'infecter ou de se lier à des vecteurs viraux ou à des domaines de liaison de l'enveloppe protéinique de celui-ci qui se lient spécifiquement au récepteur chimère. Les cellules à récepteur chimère peuvent ainsi être infectées par et/ou se lier à des agents thérapeutiques et/ou diagnostiques comprenant un domaine de liaison de l'enveloppe protéinique d'un virus spécifique n'appartenant à la première espèce et capable de se lier au polypeptide récepteur chimère décrit.
PCT/US1993/005569 1990-12-14 1993-06-11 Polypeptides recepteurs mutants, proteines h13 humaines et leurs utilisations WO1993025682A2 (fr)

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AU45322/93A AU4532293A (en) 1992-06-11 1993-06-11 Chimeric receptor polypeptides, human h13 proteins and uses thereof
US08/132,990 US5834589A (en) 1990-12-14 1993-10-07 Chimeric viral receptor polypeptides
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