EP1100944A1 - Alphavirus cible et vecteurs alphaviraux - Google Patents

Alphavirus cible et vecteurs alphaviraux

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Publication number
EP1100944A1
EP1100944A1 EP99937686A EP99937686A EP1100944A1 EP 1100944 A1 EP1100944 A1 EP 1100944A1 EP 99937686 A EP99937686 A EP 99937686A EP 99937686 A EP99937686 A EP 99937686A EP 1100944 A1 EP1100944 A1 EP 1100944A1
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Prior art keywords
cell
alphavirus
cells
virus
cancer
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EP99937686A
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German (de)
English (en)
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Boro Dropulic
Lesia Dropulic
J. Marie Hardwick
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Johns Hopkins University
School of Medicine of Johns Hopkins University
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Johns Hopkins University
School of Medicine of Johns Hopkins University
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Publication of EP1100944A1 publication Critical patent/EP1100944A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/855—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants

Definitions

  • the present invention relates to recombinant alphaviral vectors comprising at least one nonalphaviral sequence wherein the nonalphaviral sequence results in targeting the vector to a specific target cell.
  • the invention also concerns methods to treat cancer using the targeted vectors of the invention.
  • Alphavirus is a member of the togavirus family.
  • Alphaviruses are positive strand RNA viruses that are replicated and packaged in the cytoplasm of infected cells. There are many known strains which are trophic for humans. These include eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus (including variants Ockelbo and Babanki viruses), chikungunya virus, o'nyong-nyong virus, Igbo Ora virus, Ross River virus, Mayaro virus, and Barmah Forest virus.
  • alphaviruses that are normally trophic for nonhuman animals include eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus and Getah virus.
  • alphaviruses are described by the International Committee of Taxonomy of Viruses in their published reports entitled "The Classification and Nomenclature of Viruses," the sixth report of which was published in 1995.
  • the large number of viruses that constitute the alphavirus genus are closely related in terms of molecular characteristics and structure.
  • Sindbis virus is a typical prototype alphavirus and has been studied extensively. Sindbis virus is distributed over vast areas of Europe, Africa, Asia and Australia and infects humans, but usually without recognized clinical disease. In certain defined regions of the world, Sindbis virus is a significant cause of fever, arthritis and rash. Because symptoms of infection with Sindbis virus are mild and transient, medical attention is usually not sought. Given that Sindbis virus used in the context of the present invention will be targeted, it will be attenuated and, therefore, not likely to cause disease.
  • Sindbis virus contains a nucleocapsid enclosed within a lipoprotein envelope having T-4 icosahedral symmetry.
  • Sindbis comprises a single-stranded positive strand RNA genome (11 ,703 bases) of positive polarity that is associated with 240 copies of the 29 kd capsid protein (C).
  • C 29 kd capsid protein
  • the lipid bilayer of the lipoprotein envelope is derived from the plasma membrane of a host cell during budding. The general structure of the genome is shown in Figure 1.
  • the complete genomic sequence of the Sindbis virus has been disclosed by Straus, E.G. et al. Virology (1984) 133:92-110, incorporated herein by reference.
  • the genome is capped at the 5' end; approximately two-thirds of the genome encodes nonstructural proteins which are sufficient to effect replication of the positive strand to a negative complement.
  • the 3' one-third of the genome comprises a subgenomic promoter which controls the transcription of the portion encoding structural genes which comprise the capsid and envelope proteins as described above.
  • Immediately upstream of the promoter is a viral junction region required for replication.
  • a polyA chain At the 3' end is a polyA chain.
  • the portion of the minus strand corresponds to the region encoding the structural proteins is transcribed and capped as a subgenomic plus strand RNA and translated into the structural proteins which then package the genomic RNA to complete the virions.
  • Alphavirus in particular Sindbis, has been used for a number of years as a vector for expression of heterologous genes.
  • U.S. Patent No. 5,091,309 is directed to a Sindbis virus Dl RNA vector, in particular a vector comprising nts 1-241 and 1928- complementary to a restriction endonuclease site for further insertion of a heterologous sequence.
  • the vector also comprises a polyA tail adjacent to nt 2314.
  • the vectors are described as useful for expression of a heterologous sequence in a cell.
  • 5,217,879 is directed to infectious Sindbis viral vectors comprising (i) a heterologous coding sequence upstream from the viral structural coding region and at least five nts downstream from the start of the viral subgenomic RNA and (ii) a viral junction region located upstream from the viral structural coding region.
  • the vectors are described as useful for expression of the heterologous sequence in a cell. Schlesinger, TIBTECH (January 1993) 11 :18-22, in addition to disclosing recombinant Sindbis virus that expresses a heterologous gene, discloses alphaviral vectors that express a heterologous epitope that can function as an immunogen.
  • alphaviruses as carriers for heterologous polypeptides by incorporation of heterologous sequences into permissive sites in the virion envelope proteins is discussed by Bredenbeck et al. Seminars in Virology (1992) 3:297-310. Random insertional mutagenesis was used to identify insertion sites in the envelope proteins of Sindbis virus, e.g., El and E2, that allow recovery of chimeric viruses with growth properties similar to parental Sindbis virus. It has been suggested that such chimeric viruses could be used to develop live-attenuated viral vaccines and, by insertion of peptide ligands or larger binding domains into viral surface proteins, may ultimately allow delivery of recombinant RNA expression constructs to specific cell types.
  • Alphavirus-based vaccines are also described in PCT application WO99/11808 and alphavirus vectors with reduced cytopathic effects are described in PCT application WO99/18226.
  • Specific expression of an alphaviral vector in bone marrow cells is described in WO98/36779 and U.S. Patent No. 5,811,407.
  • Frolov, et al. Proc NatlAcadSci USA (1996) 93:11371-11377 provide a review of the use of alphavirus as a recombinant vector as of that date. This article, which is incorporated herein by reference, provides a helpful description of the alphavirus itself as well as its life cycle. It is also suggested that it would be helpful to target alphavirus to specific cell types in a genetic therapy context.
  • Vectors other than alphaviral vectors have been modified to target them to specific cell targets in the context of gene therapy. Miller et al. FASEB J(1995) 9:190-199. Retro viral vectors, adenoviral vectors, liposomal vectors and vectors comprising molecular conjugates have been described as useful in the context of cell- surface targeted gene therapy as has been transcription targeting. A retrovirus that displays a single-chain antibody against a cell-surface protein expressed on various human carcinoma cell lines has been disclosed (Chu et al. J Virol 69(4):2659-2663 (1995)).
  • the envelope protein of avian retrovirus has been modified to comprise an RGD-containing peptide that binds integrins (Valsesia-Wittmann et al. J Virol 68(7):4609-4619 (1994)). Similar modifications have been suggested for other retroviruses, such as murine leukemia viruses.
  • Moloney murine leukemia virus MoMLV
  • MoMLV Moloney murine leukemia virus
  • erythropoietin sequence as an insertion into its viral envelope, thereby enabling targeting to the erythropoietin receptor (Kasahara et al. Science 266:1373-1376 (1994)).
  • the fiber of adenovirus has been modified to comprise a ligand that is specific for a cell-surface receptor (U.S. Pat. No. 5,543,328).
  • An MoMLV vector comprising a fusion protein of an antibody fragment and the viral envelope protein Pr80 env has been disclosed to target binding to mouse fibroblasts (Russell et al. Nucleic Acids Research (1993) 21(5):1081-1085).
  • MoMLV vector targeted to human low density lipoprotein via a chimeric envelope protein comprising a single-chain variable fragment derived from an mAb also has been disclosed (Somia et al. Proc NatlAcadSci USA (1995) 92:7570-7574). Han et al.
  • Proc Natl Acad Sci USA (1995) 92:9747-9751 discloses the insertion of sequences encoding human heregulin into the envelope of MoMLV in order to target the virus to human breast cancer cells.
  • Adenovirus expressing a single chain antibody also has been described (WO 94/10323).
  • WO 94/10323 discloses modification of influenza and vaccinia viruses and virus-like particles so that they bind to a target cell. Modifications listed include mAbs, ScFvs, dAbs and minimal recognition units.
  • Retroviral vectors for example, are disadvantageous due to low transduction and infection efficiencies.
  • retroviral vectors pose a risk of insertional mutagenesis upon integration of the pro viral DNA into the host genome and a risk of generation of replication- competent virus.
  • retroviral vectors cannot infect nonproliferating target cells because they are unable to integrate their proviral DNA into the genomes of nonproliferating cells.
  • Targeted retroviral vectors also require the presence of wild- type envelope glycoprotein and modified envelope glycoprotein in the mature virion in order to establish an infection.
  • adenoviral vectors can be produced in high titers and efficiently transfer genes to replicating and nonreplicating cells, they are disadvantageous because, due to their complexity, they are difficult to modify in the receptor-binding domain.
  • infection with adenoviral vectors results in nonspecific inflammation and antivector cellular immunity and, consequently, only transient gene expression.
  • the use of alphavirus as a vector specifically targeted to a cell to be treated has advantages over previously constructed targeted vectors.
  • the targeted cell is a cancer cell and the alphavirus effects a negative effect either through infection per se or by virtue of an additional heterologous sequence.
  • the vectors of the invention provide advantages over those available in the prior art.
  • alphaviruses In vitro, alphaviruses, due to their simple genomic structure, render construction of recombinant alphavirus easy, and enable production of high titer recombinant viral stocks from cells transfected with RNA, without the need for tedious amplification or selection procedures. In vivo, alphaviruses offer the advantages of no risk of insertional mutagenesis, the ability to infect proliferating and resting cells, no requirement for wild-type envelope glycoprotein for the production of infectious mature virions, and infection of targeted cells that is as good as or better than infection by wild-type, i.e., unmodified, alphavirus. In addition, high levels of heterologous gene expression can be realized because the viral RNA replication and translation signals are used.
  • RNA transcripts does not involve DNA intermediates and replication is cytoplasmic, which, therefore, does not involve integration of the viral genome into the host genome.
  • alphaviruses have the capacity to kill a targeted cell by apoptosis or by eliciting a host immune response to the viral structural proteins expressed on the surface of a targeted cancerous cell. The immune response against a cell infected with an alphavirus aids in the clearance of the cells killed by apoptosis.
  • the present invention provides a recombinant alphavirus comprising an epiviral nonalphaviral sequence designed to target the virus to a specific cell type.
  • the epiviral nonalphaviral sequence is present in the El, E2 and or E3 glycoprotein as an insertion or as a replacement of one or more amino acid residues.
  • the epiviral sequence is directed to a receptor on a cancer cell.
  • the at least one epiviral nonalphaviral sequence may be, for example, a single-chain antibody (scAb) for a specific cell-surface receptor or a ligand for a specific cell-surface receptor, or a binding domain for a specific cell-surface receptor.
  • scAb single-chain antibody
  • the binding domain for the specific, cell-surface receptor can contain a single epitope or two or more epitopes. Additional epiviral nonalphaviral sequences which reinforce the ability of the vectors to target the desired cells may also be included. Thus, an additional amino acid sequence which also binds receptors characteristic of the desired cells may be included.
  • Preferred target cells are cancer cells, or, more generally, cells characteristic of cancer progression. Cells characteristic of cancer progression are the cancer cells themselves or may be cells which aid in metastasis or cancer growth, such as cells of the neovasculature required for the cancer to thrive.
  • the epiviral nonalphaviral sequence binds erbB-2.
  • the epiviral nonalphaviral sequence bind erbB-2 when it is heterodimerized with erbB-3 or erbB-4.
  • a preferred sequence that binds heterodimerized erbB-2 is the EGF domain of ⁇ -heregulin.
  • the recombinant alphavirus can further comprise and express a heterologous sequence preferably under the control of the viral subgenomic promoter .
  • the heterologous sequence may be a full-length or partial coding sequence for a protein or factor that induces cell death directly or indirectly.
  • the heterologous sequence is a full-length or partial coding sequence of Bak or Bax or encodes a ribozyme that binds, cleaves and destroys Bcl-2 mRNA.
  • the heterologous sequence induces cell death in the presence of a cytotoxic drug, such as when the heterologous sequence encodes thymidine kinase and the cytotoxic drug is ganciclovir.
  • the heterologous sequence may encode any desired protein including a marker for infected cells.
  • the transcription of the genome of the recombinant alphavirus can be under the control of a nonalphaviral promoter. If so, the promoter preferably is a cancer- specific promoter or an inducible promoter. More specifically, the present invention provides a recombinant Sindbis virus as described above.
  • the present invention further provides a recombinant DNA vector from which can be transcribed the RNA genome of an above-described recombinant alphavirus.
  • a substantially pure RNA genome of an above-described recombinant alphavirus also is provided.
  • the present invention provides methods of treating cancer in a mammal.
  • the cancer to be treated is a cancer of epithelial cell origin, such as breast cancer or ovarian cancer.
  • One method comprises administering to the mammal a cancer treatment-effective amount of an above-described recombinant alphavirus.
  • the recombinant alphavirus treats the cancer by binding to and subsequently infecting and lysing the cancerous cell.
  • Another method comprises administering to the mammal a cancer treatment-effective amount of an above- described recombinant vector or RNA genome.
  • the recombinant vector or RNA genome treats the cancer by entering the cancerous cell, wherein it produces an RNA genome, which is then packaged into a viral particle, which lyses the cancerous cell and selectively binds to, infects and lyses another cancerous cell.
  • a method of inserting a heterologous sequence into the E2 glycoprotein encoding region of the Sindbis viral genome is also provided by the present invention.
  • a method of replacing a portion of the E2 glycoprotein encoding region of the Sindbis viral genome with a heterologous sequence is provided.
  • the method comprises contacting a cell with a recombinant alphavirus, which is desirably noncytolytic and replication-defective and which comprises a marker gene, such as green fluorescent protein, and detecting the expression of the marker gene, wherein detection of the expression of the marker gene indicates the presence of cancerous cells.
  • a recombinant alphavirus which is desirably noncytolytic and replication-defective and which comprises a marker gene, such as green fluorescent protein
  • Another method provided by the present invention is a method of determining a new cancer-specific, cell-surface molecule or receptor.
  • the method comprises introducing a sequence from a library of binding domains or a random sequence as an insertion or replacement of one or more amino acid residues of the glycoprotein El, E2 or E3 of a recombinant alphavirus, contacting the recombinant alphavirus with a cancerous cell, selecting the recombinant alphavirus that binds to the cancerous cell, and using the recombinant alphavirus that binds to the cancerous cell to identify the cancer-specific, cell-surface molecule or receptor to which the recombinant alphavirus bound.
  • Fig. 1 is a depiction of the Sindbis RNA genome.
  • Fig. 2 is a map of the alphaviral expression vectors TotoCat5 double subgenomic vector (top) and single subgenomic vector (bottom).
  • Fig. 3 is a schematic diagram of part of the E2 glycoprotein showing two major neutralization epitopes/receptor-binding domains and the location of three introduced restriction enzyme sites.
  • Alphaviral infection of humans can range from asymptomatic seroconversion to disease, although most alphaviruses cause subclinical infections or result in transient, temporarily incapacitating febrile illness (e.g., Semliki Forest virus and Venezuelan equine encephalitis virus (VEE)). Some alphaviruses, in a small percentage of infections, will enter the central nervous system and cause encephalitis (e.g., Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) and Highlands J virus). Inactivated whole virus vaccines are available as investigational new drugs for EEE, WEE and VEE infection in humans.
  • encephalitis e.g., Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) and Highlands J virus.
  • EEE Eastern equine encephalitis virus
  • WEE Western equine encephalitis virus
  • Highlands J virus Highlands J virus
  • a live attenuated VEE vaccine has been used in laboratory workers without adverse events and was used to contain the equine epizootic spread of VEE in Texas.
  • Other alphaviruses can cause acute arthropathy with permanent arthritic sequellae (e.g., Chikungunya, Mayaro, O'nyong-nyong, Igbo Ora, Ross River, Sindbis and
  • the alphavirus of the invention in a preferred embodiment, contains the epiviral nonalphaviral amino acid sequence responsible for targeting a desired cell embedded in the envelope.
  • the targeting sequence is provided as a fusion protein with the envelope proteins normally found therein, and responsible for the trophism of the virus in its natural state.
  • a description of the nature of the envelope is relevant.
  • Two viral glycoproteins, El and E2 are embedded in the lipoprotein envelope.
  • El and E2 are distributed as 80 trimers on the outer surface of the viral envelope.
  • Each trimer consists of 3 E1-E2 heterodimers, which flare out in a triangular shape from the membrane.
  • the 3 heterodimers twist around one another to form the stalk of the spike and then separate to form a tripartite head.
  • a one-to-one interaction exists between a glycoprotein heterodimer and a capsid protein subunit.
  • the C-terminal region of E2 is involved in binding to the capsid.
  • Cross-linking studies suggest that the trimers are maintained by noncovalent El-El interactions.
  • the E1-E2 heterodimers Upon binding of the viral envelope with a cell membrane during infection, the E1-E2 heterodimers disassemble and form E2 monomers and El homotrimers. El has been demonstrated to play a role in fusion of the viral envelope with the cell membrane.
  • Each of El and E2 has a molecular mass of about 50 kilodaltons and is anchored in the viral envelope by membrane-spanning anchors in the C-terminal regions.
  • the Sindbis virus E2 is 432 amino acids long and has a 33 residue cytoplasmic domain.
  • the Sindbis virus El is 439 amino acids long and has a cytoplasmic domain of 2 residues.
  • Both El and E2 are glycosylated with E2 bearing 2 or 3 carbohydrate chains and El bearing 2 carbohydrate chains. The number and positions of the carbohydrate chains are not absolutely conserved among alphaviruses.
  • the El and E2 of Sindbis and Semliki Forest viruses are known to have palmitic acid residues attached in or near the membrane-spanning anchors.
  • the major neutralization epitope present in the region of E2 amino acids 170-220 varies in sequence between strains of Sindbis virus, is poorly structured as determined by the Chou-Fasman and Robson-Garnier computer analyses of predicted secondary structure, is highly charged (i.e., hydrophilic), and contains a glycosylation site, which indicates that it is epiviral on the surface of the virion.
  • the viral structural proteins are translated from a 26S subgenomic mRNA as a polyprotein, H NC-E3-E2-6K-El-COOH.
  • the capsid (C) possesses serine protease activity and cleaves itself from the polypeptide chain as it is being synthesized.
  • E2 is derived from the precursor PE2, which is cleaved into E2 and a small glycoprotein, E3, in the trans-Golgi network. E3 does not remain associated with the mature
  • Sindbis virion but remains associated with the Semliki Forest virion.
  • 6K is a small hydrophobic peptide that serves as a linker between El and E2 in the polyprotein and has been found to be associated with the mature virion in submolar quantities.
  • an N-terminal signal sequence leads to the insertion of PE2 into the endoplasmic reticulum with subsequent cleavage of the polyprotein into individual proteins by signalases in the endoplasmic reticulum.
  • Carbohydrate chains are added as the proteins transit through the secretory pathway.
  • PE2 is cleaved to E2 in the trans-Golgi network by calcium-dependent serine proteases.
  • the envelope glycoproteins bear domains involved in neutralization, hemagglutination, initial events in cell infection, and virulence.
  • Three neutralization sites have been identified on E2 and one neutralization site has been identified on El .
  • One of the neutralization sites on E2, namely Ec is a conformational epitope at amino acids 62, 96 and 152.
  • the remaining two neutralization sites on E2, namely Ea and Eb, are at amino acids 190-216 and, together, constitute the major neutralization domain of Sindbis. There are analogous regions on other alphaviruses.
  • the major neutralization domain in Venezuelan equine encephalitis virus is at E2 amino acids 180-210 and includes sequences at the extreme N-terminus
  • the major neutralization domain in Ross River virus includes E2 amino acids 216, 234 and 246-251.
  • E2a and E2b are in close physical proximity because, while they are independently mutable, they compete strongly for E2a and E2b mAbs in ELISA.
  • Transitional El and E2 epitopes become epiviral during the initial stages of infection due to conformational alterations.
  • These and other cryptic epitopes become epiviral during treatment with high temperature, reducing agents or low pH. Some of these changes may involve reduction of critical disulfide bonds in the glycoproteins, which may disrupt protein-protein associations, such as in disassembly.
  • Antibodies directed against the E2 protein primarily neutralize viral infectivity. Accordingly, the E2 protein is believed to be important in binding the virus to the cell surface. Ionic interactions between Sindbis virus and the host cell surface are also important for attachment and initiation of infection.
  • the genomic RNA serves as mRNA for translation of a polyprotein, which is co- and post-translationally cleaved to give the nonstructural proteins nsPl, nsP2, nsP3 and nsP4.
  • the genomic RNA also serves as a template for synthesis of the complementary negative strand, which, in turn, provides the template for the synthesis of the positive strand genomic RNA and the transcription of the 26S subgenomic RNA, which is translated into the capsid, PE2, 6K and El proteins.
  • the recombinant alphavirus can be replication-defective, preferably it is replication-competent and comprises an epiviral nonalphaviral sequence.
  • the epiviral nonalphaviral sequence is preferably present in the El and/or E2 or E2 and/or E3 glycoprotein as an insertion or as a replacement of one or more amino acid residues and directly and selectively binds to at least one region on a target-cell- specific surface receptor.
  • nonalphaviral is meant that the sequence does not occur naturally in any alphavirus.
  • epiviral is meant that the sequence is part of any part of the viral envelope or viral protein coat, which contains El, E2 and, possibly, E3, if E3 is part of the mature virion, other than on the internal surface of the viral envelope or viral protein coat.
  • the epiviral sequence is on an external surface of the viral protein coat or, as a result of conformational changes that occur during the initial stages of infection (as indicated by the exposure of transitional El and E2 epitopes in infecting virions at the plasma membrane within the first 30 min. after establishment of virus-cell complexes), is an external surface of the viral protein coat.
  • the epiviral sequences are designed to target the virus to a selected cell population having characteristic cell surface receptors.
  • cell surface receptors any molecule present on the cell surface that can be recognized by a peptide of any kind.
  • the "receptor” may or may not have any transduction capability when bound to a suitable ligand or other binding agent. All that is required of the "receptor” is that it be present in sufficient quantity on the desired target cell to interact successfully with the epiviral sequences in the recombinant alphavirus so as to mediate infection by the virus and that it be sufficiently characteristic of and selective for the desired host cell in the environment where the host cell is to be targeted to cause selective infection of the desired target cell in comparison to cells in the treated environment.
  • the reason for selecting a particular cell population to be targeted can be varied. For example, selective gene therapy which will be suitable for particular types of differentiated cells or of undifferentiated cells would require such selectivity.
  • a particularly preferred embodiment discussed hereinbelow is the targeting of cancer cells or other undesired cells whose destruction is the ultimate goal of infection.
  • the vectors are not limited to this specific purpose.
  • the nature of the targeted cells is dependent on the purpose of infection.
  • a cell surface receptor characteristic of a target cell can be recognized by a single-chain antibody which is directed to an antigen-specific interaction with said receptor.
  • Particular receptors, however, which may exist normally on specific types of cells may have ligands specific for them which themselves can then be used as the epiviral amino acid sequence. If the ligand is a nonpeptide, a peptide which mimics the conformation and charge pattern of the actual ligand may be used.
  • the invention herein is exemplified using the prototypic representative of the alphavirus family, Sindbis virus.
  • the appropriate position for insertion or replacement of a sequence by an epiviral sequence can be described in terms of positions in the Sindbis virus genome and its encoded proteins. It will be understood that other members of the alphavirus family, if substituted for the Sindbis virus, have permissible positions for insertion or replacement that are analogous to those of Sindbis. Thus, the positions described in the following paragraph and elsewhere in this application which are specific to the Sindbis virus, can be extrapolated to other alphavirus family members by selecting corresponding positions in these strains.
  • the epiviral nonalphaviral sequence is present in any region of the E2 glycoprotein. More preferably, it is present in the region of E2 amino acids from about 60 to about 243 of Sindbis. Most preferably, it is present in the region of E2 amino acids from about 60 to about 114, from about 114 to about 175, from about 60 to about 175, or from about 175 to about 243. Alternatively, it is present in the N- terminal half of E2, preferably from about E2 amino acid 1 to about E2 amino acid 172, most preferably from about E2 amino acid 62 to about E2 amino acid 172.
  • the envelope glycoprotein domain replaced preferably is poorly conserved among strains of a given alphavirus, poorly structured so as to avoid altering or losing important secondary structure, and easily accessible (i.e., hydrophilic).
  • the nonalphaviral sequence is present as an insertion, preferably the insertion comprises from about 3 to about 423 amino acids, more preferably from about 6 to about 200 amino acids, and most preferably from about 9 to about 68 amino acids. If the nonalphaviral sequence is present as a replacement, preferably the replacement comprises from about 3 to about 423 amino acids, more preferably from about 6 to about 200 amino acids, and most preferably from about 9 to about 68 amino acids.
  • Such recombinant alphavirus can further comprise at least one more epiviral nonalphaviral sequence as described above, which, together with or separate from the epiviral nonalphaviral sequence, is present in the E2 glycoprotein as an insertion at an E2 amino acid residue from about 60 to about 243 of Sindbis or from about 1 to about 172 or as a replacement of one or more amino acid residues of the E2 glycoprotein from about E2 amino acid residue 60 to about E2 amino acid residue 243 or from about E2 amino acid residue 1 to about E2 amino acid residue 172.
  • the present invention thus also provides a method of inserting a heterologous sequence into the E2 glycoprotein encoding region of the alphaviral genome.
  • the method comprises introducing into a vector from which can be transcribed the RNA genome of alphavirus a restriction enzyme site at a nucleotide site corresponding to the E2 amino acid residue 60, 114 or 175 of Sindbis virus and inserting into the restriction enzyme site a heterologous sequence.
  • the present invention also provides a method of replacing a portion of the E2 glycoprotein encoding region of the Sindbis viral genome with a heterologous sequence.
  • the method comprises introducing into a vector from which can be transcribed the RNA genome a restriction enzyme site at a nucleotide site corresponding to the E2 amino acid residue 60, 114 or 175 of Sindbis.
  • the restriction enzyme site is then used in combination with either of another restriction enzyme site at one of the other two remaining amino acid residues or the naturally occurring Bel I restriction enzyme site at the nucleotide site corresponding to the E2 amino acid residue 243 of Sindbis to delete the nucleotides corresponding to E2 amino acids 60-114, 60-175, 114-175, 114-243 or 175-243.
  • the deleted nucleotides are replaced with a nucleic acid sequence encoding the heterologous sequence.
  • Whether or not a given site in a viral structural protein is permissive (i.e., replacement of or insertion into the site results in synthesis of structural proteins, production of virus and the ability to establish a productive infection in a cancerous or a noncancerous cell line) for insertion or replacement can be determined in accordance with any one of a number of methods known in the art.
  • anti- Sindbis virus antibodies can be used in immunoprecipitation or a Western blot to look for synthesis of viral proteins in cells and to look for the presence of viral proteins in supernatant fluids or in purified virions.
  • the presence of the one or more nonalphaviral sequences also can be determined in accordance with methods known in the art.
  • antibodies against the nonalphaviral sequence(s) can be used to look for the presence of the nonalphaviral sequence(s) in purified virions by immunoprecipitation or Western blot.
  • the recombinant alphavirus can be assayed for its ability to kill a cancerous or noncancerous-derived cell line.
  • Whether or not a given recombinant alphavirus has sufficient activity for use in the present inventive method can be determined in accordance with methods known in the art.
  • the degree of cell killing of a cancer cell line after infection with a targeted alphavirus can be determined by trypan blue exclusion or propidium iodide assay followed by FACS analysis.
  • the cancer cell-killing capacity of a recombinant alphavirus can be determined using an animal model, such as the nude mouse model.
  • the recombinant alphavirus kills from about 20% to about 50% cancer cells, more preferably from about 50% to about 80% cancer cells, most preferably from about 80% to about 100% cancer cells.
  • a recombinant alphavirus can be determined to have sufficient activity if it spreads throughout a tumor without causing death of tumor cells because the expression of viral structural proteins of the tumor cells will serve as immunotherapy and result in the destruction of tumor cells by the host's immune system.
  • the E2 glycoprotein has two N-linked glycosylation sites, one of which is located in the neutralization epitope E2ab at amino acid residue 196. Replacement of this region with a ligand or an scAb, for example, may result in misfolding of the glycoprotein, since carbohydrate chains are required for proper folding.
  • the neutralization epitope E2c contains regions that have been found to be important for viral penetration. Replacement of this region with a nonalphaviral sequence could interfere with this function.
  • the fusion function of alphaviruses resides in the El glycoprotein.
  • the El and E2 glycoproteins heterodimerize and, therefore, their proper folding and, hence, function are dependent on each other. Care also must be exercised to ensure that modification of a receptor binding domain by insertion is not one that can be easily removed from the virus by RNA recombination, thereby generating wild-type alphavirus, which is no longer targeted.
  • the only risk attendant generation of most wild-type alphaviruses is an asymptomatic infection, leading to polyarthritis and a rash, which is mild and resolves as the virus is cleared by the immune system as described above.
  • RNA transfection of cells If virus is not produced after RNA transfection of cells, pulse-chase analysis and immunoprecipitation of metabolically labeled infected cell lysates can be performed to determine whether the modified glycoprotein is misfolded and not transported to the cell surface. If the glycoprotein is misfolded, for example, a shorter, less critical portion of the glycoprotein should be replaced with the nonalphaviral sequence, a shorter nonalphaviral sequence should be used, or the nonalphaviral sequence should be introduced into the alphavirus as an insertion, instead of a replacement.
  • a chimeric glycoprotein can be generated in a recombinant alphavirus by using specific oligonucleotide primers containing the nonalphaviral sequence of choice (the "insert") and homologous alphaviral sequences, which include a unique restriction endonuclease site, to amplify the insert for insertion into or replacement of all or part of an alphaviral structural protein.
  • specific oligonucleotide primers containing the nonalphaviral sequence of choice the "insert”
  • homologous alphaviral sequences which include a unique restriction endonuclease site
  • the recombinant alphavirus can further comprise at least one more epiviral nonalphaviral sequence.
  • the binding of additional sequence(s) can improve the specificity of targeting of the recombinant alphavirus.
  • the at least one more epiviral nonalphaviral sequence, together with or separate from the above-described epiviral nonalphaviral sequence, preferably also is present in the El, E2 or E3 glycoprotein as an insertion or as a replacement of one or more amino acid residues and can be a scAb, a ligand or a binding domain that directly and selectively binds a target- specific, cell-surface receptor, or is an immunogen.
  • the at least one more epiviral nonalphaviral sequence binds to the same receptor as the first epiviral nonalphaviral peptide, it preferably binds to a different region from that which is bound by the first epiviral nonalphaviral peptide.
  • a bi-targeted virus can be created with nonalphaviral sequences in E2 and E3 or a nonalphaviral sequence can replace a region that includes the cleavage site between E2 and E3, which is not part of the mature virion.
  • incorporation of PE2 into mature virions results in a noninfectious virus. This "lethality" can be suppressed by second site mutations in E3 or E2.
  • the immunogen may elicit an immune response against a cell to which the recombinant alphavirus directly and selectively binds and/or may promote cytotoxicity to the cell or may generate, a cytokine that promotes immunity.
  • a surface antigen is the B.7 histocompatibility antigen; an example of a cytokine is GM-CSF.
  • Further improvement of the recombinant alphavirus that specifically targets cancer cells can be achieved through selection of optimized recombinant alphavirus by passaging the recombinant alphavirus through multiple rounds of infection in the target cancer cell. This allows the engineered targeting epitope to drift and make a best fit epitope. This selection process also can be used to determine novel epitopes that are on the surface of specific types of cancer cells.
  • epiviral sequence refers to an amino acid sequence that is fused into the envelope protein or proteins of the virus. It can also refer to the nucleotide sequence encoding the amino acid sequence displayed on the viral surface. Thus, “sequence” refers either to the peptide or nucleotide sequence level and which is intended will be clear from the context.
  • the epiviral sequence preferably is a scAb, ligand, or, generally, binding domain for a cancer-specific, cell-surface.
  • the binding domain for a cancer-specific, cell-surface receptor can contain a single epitope or two or more epitopes.
  • nonalphaviral sequence to be used for cancer treatment is dependent upon a number of factors, including the type of cancer to be treated, the ability to achieve direct and selective binding between the recombinant alphavirus and a given cancer-specific, cell-surface receptor, and the ability to achieve specific infectivity of a targeted cancer cell.
  • the ability to achieve direct and selective binding between the recombinant alphavirus and a given cancer-specific, cell-surface receptor is, in turn, dependent upon a number of other factors, including the capacity to incorporate a nonalphaviral sequence into the El or E2 glycoprotein of the alphavirus such that the nonalphaviral sequence is epiviral and packaging of the virus is not adversely affected.
  • the ability to achieve specific infectivity of a targeted cancer cell is, in turn, dependent upon a number of other factors, including whether or not binding to a cancer-specific, cell-surface receptor effects internalization of the RNA genome of the alphavirus, viral replication and packaging, and, ultimately, cell lysis.
  • cancer-specific, cell-surface receptors include placental alkaline phosphatase (testicular and ovarian cancer), pan carcinoma (small cell lung cancer), polymorphic epithelial mucin (ovarian cancer), prostate-specific membrane antigen, ⁇ -fetoprotein, B-lymphocyte surface antigen (B-cell lymphoma), truncated EGFR (gliomas), idiotypes (B-cell lymphoma), gp95/gp97 (melanoma), N-CAM (small cell lung carcinoma), cluster w4 (small cell lung carcinoma), cluster 5A (small cell carcinoma), cluster 6 (small cell lung carcinoma), PLAP (seminomas, ovarian cancer, and nonsmall cell lung cancer), CA-125 (lung and ovarian cancers), ESA (carcinoma), CD19, 22 or 37 (B-cell lymphoma), 250 kD proteoglycan (melanoma), P55 (breast cancer), TCR-IgH fusion (childhood T
  • cancer-specific, cell-surface receptors include erbB-2, erbB-3, erbB-4, IL-2 (lymphoma and leukemia), IL-4 (lymphoma and leukemia), IL-6 (lymphoma and leukemia), MSH (melanoma), transferrin (gliomas), tumor vasculature integrins, and the like.
  • Preferred cancer-specific, cell-surface receptors include erbB-2 and tumor vasculature integrins, such as CD1 la, CD1 lb, CD1 lc, CD 18, CD29, CD51 , CD61 , CD66d, CD66e, CD 106, and CDwl 45.
  • a ligand can be identified by using a radio-receptor assay to screen conditioned media from a number of cells. Then, the ligand can be isolated from the cells using known techniques. For example, protein purification techniques, such as extraction, precipitation, ion exchange chromatography, affinity chromatography, gel filtration and the like can be used. Any of a wide variety of procedures then can be used to clone the gene encoding the ligand. For example, a shuttle vector library of DNA inserts derived from the cells that express the ligand can be analyzed for the presence of the gene.
  • the amino acid sequence of the ligand is partially determined using, for example, an automated sequencer or fragmentation with cyanogen bromide or a protease, such as papain, chymotrypsin or trypsin. Then, an oligonucleotide having a complementary nucleotide sequence to the oligonucleotide sequence capable of encoding the sequenced amino acid fragment is identified, synthesized and hybridized to a cDNA library, which has been generated from RNA extracted from cells cultured under conditions that induce ligand synthesis.
  • cyanogen bromide or a protease such as papain, chymotrypsin or trypsin.
  • a library of expression vectors can be prepared by cloning DNA or cDNA from ligand-expressing cells into expression vectors.
  • the expression vector library can be screened with an antiligand molecule.
  • the DNA molecule comprising the ligand gene or a portion thereof can be further amplified by PCR, for example, and then introduced into a DNA vector encoding an alphavirus to generate a recombinant alphavirus comprising an epiviral ligand for binding to a cancer-specific, cell-surface receptor.
  • binding domains Nucleic acids encoding many of the binding domains are known and can readily be found by reference to publicly accessible nucleotide sequence databases, such as EMBL and GenBank. Polymerase chain reaction, for example, can then be used to amplify the DNA, which can be incorporated into a DNA vector encoding an alphavirus to generate a recombinant alphavirus comprising an epiviral binding domain for binding to a cancer-specific, cell-surface molecule or receptor as demonstrated in the Examples.
  • binding domains include the EGF domain of ⁇ -heregulin, ⁇ - integrin domain, tumor vasculature peptide motifs, and those described in the http://ampere.doe-mbi.ucla. edu:8801/dat/dip.dat and http://bones.biochem.ualberta.ca/pedro/rt-l .html, databases.
  • the epiviral nonalphaviral sequence can be one of a library of binding domains or a random sequence. Such sequences can be used to find specific epitopes on the surface of cancerous cells.
  • the resultant recombinant alphavirus is selected by passage, with the "correct" type being amplified during the selection process.
  • the resultant recombinant alphavirus can be sequenced in the region of the epiviral nonalphaviral sequence to determine the specific sequence that binds to the target cell population. This information then can be used to engineer more specific targeted alphaviruses.
  • the erbB-2 receptor has been found in breast, ovarian, gastric, salivary gland and adeno-carcinomas and in nonsmall cell carcinomas of the lung. Over-expression of the erbB-2 receptor on such cancers has been found to correlate with poor prognosis. In vitro studies strongly suggest that over-expression of erbB-2 may play an important role in tumor progression.
  • scAb An example of a scAb is that which binds c-erbB-2 (WO 93/16185). See, also, WO 93/21232 and http://www.antibody resource.com for antibody sequences that can be used to construct scAbs.
  • An example of a ligand which binds to erbB-2 is ⁇ -heregulin. ⁇ -Regulin is bound to erbB-2 when it is heterodimerized with erbB-3 or erbB-4 (WO 92/12174).
  • a preferred binding domain is the EGF domain of ⁇ -heregulin.
  • Alpha-heregulin is a ligand with affinity for breast cancer cells expressing the human epidermal growth factor receptors erbB-2, erbB-3 and erbB-4. Heregulin interacts indirectly with erbB- 2 via heterodimerization with erbB-3 or erbB-4.
  • Specificity of binding can be determined by viral binding assays, using radiolabeled virus in the presence and absence of a competitive monoclonal antibody (mAb), for example.
  • the degree of specific infectivity can be determined by measuring infectivity in the presence or absence of an mAb and assaying for activity of a reporter gene, such as chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP), which is present in the recombinant alphavirus, determining the number of cells infected by indirect immunofluorescence, and assaying for cell death by trypan blue exclusion.
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • a vector with greater specificity of binding can be selected by serial passaging of the recombinant alphavirus in cultures of cells expressing the targeted receptor using positive and negative selection procedures.
  • a modified alphavirus can be incubated with baby hamster kidney (BHK) cells for 1 hr at 4°C to remove viruses in the population that are able to bind BHK cells. After this negative selection, the supernatant fluids can be used to infect breast cancer cells over- expressing erbB-2 or erbB-2 and erbB-4, i.e., a positive selection.
  • BHK baby hamster kidney
  • the supernatant fluids can be used to infect breast cancer cells over- expressing erbB-2 or erbB-2 and erbB-4, i.e., a positive selection.
  • Other suitable methods are known in the art.
  • the recombinant alphavirus can further comprise and express a heterologous sequence under the control of the viral subgenomic promoter.
  • a heterologous sequence will be dependent on the nature of the target cell and the purpose to be achieved. For example, if the cell is targeted for genetic therapy, the heterologous sequence will be that encoding the desired protein. If the selected cell population is one to be labeled, the heterologous sequence will encode a reporter gene such as that encoding green fluorescent proteins (GFP). If the target cell is to be destroyed, the heterologous sequence will be designed to produce a product which aids in this destruction.
  • GFP green fluorescent proteins
  • the heterologous sequence is a full-length or partial coding sequence for a protein or factor that induces cell death directly, or indirectly and potentiates apoptosis induced by the recombinant alphavirus.
  • preferred coding sequences for a protein or factor that induces cell death include Bak, Bax, Bid, Bad, Bik, Bif-2, a cell death-inducing coding sequence of Bcl- 2 (e.g., comprises an N-terminal deletion), a cell death-inducing coding sequence of BC1-X L (e.g., comprises an N-terminal deletion), IAP-1, IAP-2, a caspase, and a kinase, such as protein kinase C ⁇ , protein kinase C ⁇ , Akt/PI(3)-kinase, DNA-PK, PITSLRE, DAP kinase, RIP, JNK/SAPK, Daxx, Raf-1, Pim-1, NIK
  • the heterologous sequence is a full-length or partial coding sequence of Bak or Bax.
  • a cell death-inducing sequence such as a full-length or partial coding sequence of Bak or Bax, is desirably used in a recombinant alphavirus targeted to erbB-2 on cancerous cells that do not express erbB-3 and/or erbB-4, which, as pointed out above, heterodimerize with erbB-2 in indirectly interacting with heregulin, for example.
  • the heterologous sequence comprises or encode a ribozyme or an antisense molecule against a gene with antiapoptotic activity so as to induce cell death to express a coding sequence with antiapoptotic activity to create persistently infected cells so as to facilitate spread of the recombinant alphavirus.
  • genes include Bcl-2, Bcl-x L , Bcl-w, Bag-1, Mcl-1, the clap family of genes, viral IAp genes, CrmA, P35, and dominant negative forms of death-inducing genes such as FADD, ASK, JUN/SAPK and PKC.
  • the heterologous sequence comprises or encodes a ribozyme that binds, cleaves and destroys Bcl-2 mRNA.
  • the recombinant alphavirus can further comprise and express a coding sequence that promotes cell death in the presence of a cytotoxic drug as the heterologous sequence.
  • a coding sequence is that of the thymidine kinase gene (e.g., from Herpes virus). Expression of the thymidine kinase gene in cells that are treated with ganciclovir results in their death. Other such prodrug strategies can be used.
  • the recombinant alphavirus comprises and expresses a heterologous sequence, one of two types of vectors can be used (see, for example, Huang et al.
  • One type of vector contains two copies of the viral subgenomic promoter.
  • One promoter controls the synthesis of the viral structural proteins and the other controls the expression of a heterologous sequence.
  • the vector is self-replicating, produces infectious viral particles and can spread from cell to cell.
  • the other type of vector is one in which the heterologous sequence replaces one or more of the structural protein genes.
  • This type of vector requires helper RNA for packaging of recombinant RNA into viral particles. The vector does not produce infectious viral particles and, therefore, cannot spread from cell to cell.
  • the recombinant alphavirus can be further modified such that the transcription of the genome is under the control of a nonalphaviral promoter, such as a cancer-specific promoter or an inducible promoter.
  • a nonalphaviral promoter such as a cancer-specific promoter or an inducible promoter.
  • the cancer-specific promoter is one that is only activated in a cell of the cancer that is directly and selectively bound by the recombinant alphavirus.
  • the use of a cancer-specific promoter provides another layer of protection to ensure that the alphavirus replicates only in a cancer cell.
  • An example of a cancer-specific promoter is CEA.
  • promoters can be found on the Internet in the eukaryotic promoter database at http://www.genome.ad.jp/dbget-bin/www_bFind7epdtable.
  • the promoter can be a cell-specific promoter, from which the target cancerous cell or target supporting cell actively transcribes mRNA.
  • the present invention also provides a recombinant
  • RNA genome of the above-described recombinant alphavirus including the recombinant Sindbis virus.
  • substantially pure is meant free from recombinant DNA vectors and cellular components, if the RNA genome is generated from a recombinant DNA vector, or free from viral coat proteins and other viral components, if the RNA genome is purified and isolated from alphaviral particles, such as Sindbis viral particles.
  • cancer includes cancers that are characterized by abnormal cellular proliferation and the absence of contact inhibition, which can be evidence by tumor formation.
  • the term encompasses cancer localized in tumors, as well as cancer not localized in tumors, such as, for instance, cancer that expands from a tumor locally by invasion, or systemically by metastasis.
  • any type of cancer can be targeted for treatment according to the invention.
  • the cancer is of epithelial origin, such as breast cancer or ovarian cancer.
  • One method involves the administration to a mammal in need of cancer treatment a cancer treatment effective amount of an above-described recombinant alphavirus, such as a recombinant Sindbis virus, wherein, upon binding to a cell of the cancer to be treated, the recombinant alphavirus infects and lyses the cell, thereby treating the cancer.
  • Treatment of the cancer can be assessed, for example, by monitoring the attenuation of tumor growth and/or tumor regression, wherein "tumor growth” includes an increase in tumor size and/or the number of tumors and "tumor regression" includes a reduction in tumor mass.
  • the recombinant alphavirus comprises at least one more epiviral nonalphaviral sequence and that sequence is an immunogen, preferably the immunogen elicits an immune response against the cancer to be treated.
  • the cancer is a cancer of epithelial origin, such as breast cancer or ovarian cancer.
  • Another method of treating cancer in a mammal involves the administration to a mammal in need of cancer treatment a cancer treatment effective amount of an above-described recombinant DNA vector or RNA genome.
  • the recombinant DNA vector or RNA genome Upon entry of the recombinant vector or RNA genome into a cancerous cell, the recombinant DNA vector or RNA genome produces an RNA genome, which is then packaged into a viral particle, which lyses the cancerous cell and selectively binds to and infects another cancerous cell, which is consequently lysed, thereby treating the cancer.
  • the recombinant DNA vector or RNA genome encodes at least one more epiviral nonalphaviral sequence and that sequence is an immunogen, preferably the immunogen elicits an immune response against the cancer to be treated.
  • the cancer is a cancer of epithelial origin, such as breast cancer or ovarian cancer.
  • a method of treating cancer in accordance with the present invention can further comprise targeting noncancerous cells, such as latently virus- infected, precancerous cells or cells that support tumor growth, such as tumor angiogenic endothelial cells.
  • Targeting a tumor angiogenic endothelial cell can involve targeting an integrin, for example, with a recombinant alphavirus comprising an RGD peptide as an epiviral, nonalphaviral sequence.
  • the present inventive methods of treating cancer in a mammal can be used alone or in combination with radiation, chemotherapy and/or surgery.
  • combinatorial treatment can be used in the early or late stages of the progression of cancer, including the metastatic stage.
  • Recombinant alphavirus in accordance with the present invention can be introduced into a mastectomy or ovarectomy site, for example, to infect residual tumor cells following surgery.
  • Recombinant alphavirus also can be introduced into the mammary gland by ductal cannulation.
  • a recombinant alphavirus preferably a recombinant Sindbis virus, a recombinant DNA vector from which can be transcribed an RNA genome of a recombinant alphavirus, or an RNA genome of a recombinant alphavirus is administered to a mammal in need thereof.
  • the means of administration can be by any suitable means, which, in part, is determined by whether a recombinant alphavirus or a recombinant DNA vector or RNA genome is being administered.
  • Suitable routes of administration include peritumoral, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, oral, rectal, intraocular, intranasal, and the like.
  • Peritumoral and intratumoral routes of administration are preferred. Administration by lipofection, direct DNA injection, microprojectile bombardment, liposomes, molecular conjugates (with respect to recombinant DNA vectors and RNA genomes, for example), and the like, also can be effected.
  • cells can be removed, contacted with recombinant alphavirus, recombinant DNA vector or RNA genome and returned to the mammal in an ex vivo technique.
  • the method is not dependent on any particular means of administration and is not to be so construed. Means of administration are well-known to those skilled in the art, and also are exemplified herein.
  • the targeted alphavirus can be delivered as a virus after packaging in an appropriate packaging cell line. Since the TA can only enter the targeted cell (e.g., breast cancer cell), the packaging cell line must be made from a cell line of the same type as that of the target cell line. So, for a TA that targets breast cancer, the packaging cell line must be the same type of breast cancer cell to which the TA is to be targeted.
  • the erb-B2 targeted TA is packaged in an in vitro cultured erb-B2-overexpressing breast cancer or ovarian cancer cell line. The virus then can be concentrated (e.g., by filtration or ultracentrifugation) and can be administered directly to the tumor site, or the site of tumor resection (as an adjunct to surgical therapy).
  • the targeted alphaviral vector can be delivered directly to the tumor site or to a peripheral site, where the TAV can produce TA that can spread through the tumor.
  • the TAV can be initially delivered to a packaging cell line or to the patient's own irradiated tumor cells, which act as packaging cells and then the cells can be delivered back to the tumor site.
  • the number of cells that can be introduced back into the patient is about 10 3 to about 10 14 cells per inoculation. Multiple inoculations can be required at the same or different sites.
  • the TAV is delivered as a nucleic acid, it can be delivered either as RNA or DNA. If it is delivered as RNA, it would first have to be transcribed from a DNA template to make RNA.
  • the RNA can be made by using standard in vitro transcription techniques that are known in the art.
  • the R A then can be complexed with a liposomal or nonliposomal (collectively termed 'lipoid') delivery system in order to transfect cells efficiently with the TAV RNA. Once the RNA enters the cytoplasm of the cell (which can be the target cell or a cell that is not the target cell), it can amplify itself to produce TA particles.
  • TA can be produced that then specifically infects and spreads through a population of target cancerous cells.
  • the cells that produce first round virus will die from productive viral infection, but this is not of consequence since the number of cells transfected is relatively small in comparison to subsequent replication and amplification of the TA through the target cell population, e.g., the tumor.
  • the TAV can be further targeted to tumors by incorporating in the liposome or nonliposome delivery system molecules that target the lipoid-RNA and complexes more specifically to tumor vasculature.
  • Tumor vasculature has specific surface molecules, i.e., integrins, and by incorporating the appropriate ligand that targets these integrins into the lipoid-RNA complexes, first round TA can be produced from the tumor vasculature, which then can seed TA particles into the tumor. The TA can then amplify and spread through the tumor.
  • the incorporation of the ligand in the lipoid-DNA complexes additionally serves to facilitate targeting of the integrin-tropic TAV to tumor vasculature.
  • the TAV lipoid-RNA can more specifically infect tumor vasculature endothelial cells and TA particles can then spread through neighboring tumor endothelial cells and kill them, cutting off the tumor's blood supply.
  • the TAV also can be delivered as a DNA-lipoid complex.
  • a promoter must be placed proximally to the virus to ensure first round expression of the TAV, since alphaviruses are RNA viruses with no DNA intermediate.
  • alphaviruses are RNA viruses with no DNA intermediate.
  • One way to do this is to transfect the plasmid DNA TAV directly into cells without a eukaryotic promoter system.
  • virus can be produced from cells that are transfected with eukaryotic promoter-deficient plasmid DNA that encodes the alphavirus. This first round virus then can be amplified by passage in an appropriate packaging cell line or in vivo.
  • Another way of producing first round virus from DNA-lipoid TAV is to express simultaneously a bacteriophage polymerase gene in cells that contain the TAV plasmid DNA, which contains the promoter sequences for binding of the polymerase.
  • An example of this is simultaneous expression of the bacteriophage SP6 polymerase in cells that contain the TAV with an SP6 polymerase promoter sequence.
  • the SP6 polymerase can then bind to the SP6 promoter sequence and transcribe first round TAV RNA.
  • the TAV RNA can then amplify and infect and spread through the target cell population tumor cells.
  • the targeting of the TAV can be made more specific by linking the SP6 polymerase gene to a promoter that is only expressed in target cells.
  • the SP6 polymerase can be linked to a tumor-specific promoter so that first round of virus is only produced from tumor cells and not other cell types.
  • Such multi-layered approaches aid targeting of the TAV and decrease their toxicity (infection of noncancerous/nontumorous cell types).
  • first round virus from DNA-lipoid TAV is by linking the TAV plasmid DNA sequence directly with a eukaryotic promoter.
  • the TCV can be made more specific for tumor cells by using tumor-specific promoters.
  • Concepts that promote gradual increase in specificity using multi-layered strategies are especially important for clinical applications.
  • a preferred way of producing first round virus is by placing the TAV sequences inside a carrier vector (e.g., an adenoviral vector or preferably a crHIV vector (Dropulic et al. WO 97/20060)) driven by an appropriate promoter for the expression of TAV RNA inside cells.
  • First round delivery can be accomplished by delivery of the carrier vector to the target site, which then produces first round TAV targeted alphaviral particles, which can then specifically infect the target cell population.
  • a more preferred method of delivery of the TAV is inside a crHIV vector. Since the size of the crHIV genome is smaller than the alphaviral genome, TAV segregates into two components that contain sequences to permit their recombination. Introduction of two heterologous crHIV TAV components into a packaging cell system permits co-packaging of the two heterologous TAV component RNAs into one virion (since two crHIV RNAs are co-packaged into one virion) and upon infection of the target cell with the crHIV vector, reverse transcription of the crHIV RNA results in their recombination and production of a whole infectious targeted alphaviral provirus.
  • the targeted alphaviral provirus can be driven by a eukaryotic promoter and expressed in cells to produce TA particles.
  • two targeted alphaviral proviruses express their component RNAs, which then produce a whole TA RNA genome for its amplification and production of targeted alphaviral particles.
  • a most preferred method of delivery for TAV is to layer the two-component crHIV-TCA/AV inside a vector-helper crHIV direct delivery system.
  • crHIV vector-helper DNA-lipoid complexes Upon the infection of cells with crHIV vector-helper DNA-lipoid complexes, first-round, two- component crHIV-TA particles are produced for infection of cells.
  • Cells infected with two-component, crHIV-TA produces cells containing targeted alphaviral proviruses, which produce targeted alphaviral particles for specific infection and killing of target cell populations.
  • An array of promoter/vector permutations can be used to produce TA from specific cell types. This would aid toward the development of highly specific TA.
  • Recombinant alphavirus for use in the methods of the present invention can be preserved in either crude or purified form.
  • virus-producing cells can be cultivated in a bioreactor and viral particles are released from the cells into the culture medium.
  • the virus can then be preserved in crude form by adding a sufficient amount of a formulation buffer to the culture medium containing the recombinant alphavirus to form an aqueous suspension.
  • the virus can be preserved in purified form by clarifying the crude recombinant virus by passing it through a filter and then concentrating the virus, such as by cross flow concentration.
  • DNase can be added to digest exogenous DNA and the digest diafiltrated to remove excess media components and establish the virus in a more desirable buffered solution.
  • the diafiltrate is then passed over a column and the purified recombinant alphavirus eluted.
  • a sufficient amount of formulation buffer can then be added to the eluate to reach a desired final concentration of the substituents and to dilute recombinant virus minimally.
  • the aqueous suspension can then be stored (e.g., at -70°C) or immediately dried.
  • Crude recombinant alphavirus also can be purified by ion exchange column chromatography.
  • Aqueous suspensions can be dried by lyophilization or evaporated at ambient temperature. In this regard, such aqueous suspensions comprise components to preserve the activity of the recombinant alphavirus upon freezing and lyophilization or drying through evaporation.
  • the suspension can comprise a buffer, a high molecular weight structural additive, a saccharide and water (see, for example, WO 96/17072). Lyophilized or dehydrated recombinant alphavirus can then be reconstituted using water or dilute salt solution, for example.
  • Other additives include components that enhance the transduction efficiency of the reconstituted virus.
  • the recombinant alphavirus is administered to the mammal in the form of a pharmaceutically acceptable composition.
  • the composition must be such that it does not compromise the ability of the recombinant alphavirus to bind directly and specifically to a cancer-specific, cell-surface molecule or a cancer-specific, cell- surface receptor on the cancer to be treated.
  • the recombinant DNA vector or RNA genome is administered by means of cationic lipids, e.g., liposomes.
  • cationic lipids e.g., liposomes.
  • liposomes are commercially available (e.g., Lipofectin , Lipofectamine , and the like, supplied by Life Technologies, Gibco BRL, Gaithersburg, MD).
  • liposomes having increased transfer capacity and/or reduced toxicity in vivo e.g., as reviewed in PCT patent application no. WO 95/21259) can be employed in the present invention.
  • the recommendations identified in WO 93/23569 can be followed.
  • other delivery vehicles include hydrogels and controlled-release polymers.
  • liposomal formulations and the like can be targeted to cancer cells by causing the liposomes to display a mAb, scAb, ligand or binding domain, for example, for a cancer-specific, cell-surface molecule or receptor (see, for example, Miller et al. (1995), supra).
  • a recombinant DNA vector can be targeted to a cell by coupling the DNA to a mAb, scAb, ligand or binding domain, for example, by covalently linking a polycation, such as polylysine, to the ligand.
  • the polycation then binds to and condenses plasmid DNA via electrostatic interactions, leaving the ligand epiviral (see, for example, Miller et al. (1995), supra).
  • a recombinant DNA vector or RNA genome of the present invention can be formulated into various compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and can be formulated to be appropriate for either human or veterinary applications.
  • a composition for use in the method of the present invention can comprise one or more of the aforementioned recombinant alphaviruses, recombinant DNA vectors or RNA genomes, preferably in combination with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well-known to those skilled in the art, as are suitable methods of administration.
  • the choice of carrier will be determined, in part, by whether a recombinant alphavirus or a recombinant DNA vector or RNA genome is to be administered, as well as by the particular method used to administer the composition.
  • a recombinant alphavirus or a recombinant DNA vector or RNA genome is to be administered, as well as by the particular method used to administer the composition.
  • routes of administering a composition are available, and, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, there are a wide variety of suitable formulations of compositions that can be used in the present inventive methods.
  • a recombinant alphavirus, a recombinant DNA vector, an RNA genome or a composition comprising such an alphavirus, a recombinant DNA vector or an RNA genome, alone or in further combination with one or more other active agents can be made into a formulation suitable for parenteral administration, preferably intraperitoneal administration.
  • a formulation can include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneously injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets, as described herein.
  • a formulation suitable for oral administration can consist of liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or fruit juice; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solid or granules; solutions or suspensions in an aqueous liquid; and oil-in-water emulsions or water-in-oil emulsions.
  • diluents such as water, saline, or fruit juice
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as solid or granules
  • solutions or suspensions in an aqueous liquid and oil-in-water emulsions or water-in-oil emulsions.
  • Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.
  • a formulation suitable for oral administration can include lozenge forms, which can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
  • An aerosol formulation suitable for administration via inhalation also can be made. The aerosol formulation can be placed into a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • a formulation suitable for topical application can be in the form of creams, ointments, or lotions.
  • a formulation for rectal administration can be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • a formulation suitable for vaginal administration can be presented as a pessary, tampon, cream, gel, paste, foam, or spray formula containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the infected individual over a reasonable time frame.
  • the dose will be determined by the potency of the particular recombinant alphavirus, recombinant DNA vector or RNA genome employed for treatment, the severity of the cancer, as well as the body weight and age of the infected individual.
  • the size of the dose also will be determined by the existence of any adverse side effects that may accompany the use of the particular recombinant alphavirus, recombinant DNA vector or RNA genome employed. It is always desirable, whenever possible, to keep adverse side effects to a minimum.
  • the dosage can be in unit dosage form, such as a tablet or capsule.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a vector, alone or in combination with other anticancer agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
  • the specifications for the unit dosage forms of the present invention depend on the particular embodiment employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.
  • the dose administered should be a "cancer treatment effective amount” or an amount necessary to achieve an "effective level" in the individual patient.
  • the effective level is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on interindividual differences in pharmacokinetics, drug distribution, and metabolism.
  • the "effective level” can be defined, for example, as the blood or tissue level desired in the patient that corresponds to a concentration of one or more recombinant alphaviruses or recombinant DNA vectors according to the invention, which lyses targeted cancerous cells in an assay predictive for clinical anticancer activity.
  • the "effective level” for a recombinant alphavirus, a recombinant DNA vector, or RNA genome of the present invention also can vary when the compositions of the present invention are used in combination with other known anticancer agents.
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective level" in the individual patient.
  • One skilled in the art also can readily determine and use an appropriate indicator of the "effective level" of the compounds of the present invention by a direct (e.g., tumor biopsy or radio-imaging of the tumor) or indirect (e.g., PSA levels in the blood) analysis of appropriate patient samples (e.g., blood and/or tissues).
  • suitable animal models are available and have been widely implemented for evaluating the in vivo efficacy against cancer of recombinant DNA protocols (see, e.g., PCR). These models include nude mice and SCID mice. Such models also can be used to evaluate the in vivo efficacy of an RNA genome.
  • an amount of recombinant alphavirus, recombinant DNA vector or RNA genome sufficient to achieve a tissue concentration of about 10 2 to about 10 12 particles per ml is preferred, especially of about 10 6 to about 10 10 particles per ml.
  • multiple daily doses are preferred.
  • the number of doses will vary depending on the means of delivery and the particular recombinant alphavirus, recombinant DNA vector or RNA genome administered.
  • the pharmaceutical composition can contain other pharmaceuticals, in conjunction with a recombinant alphavirus, a recombinant DNA vector or an RNA genome according to the invention, when used to treat cancer therapeutically.
  • an anticancer agent be employed, such as, preferably, a recombinant virus, a nucleic acid/liposomal formulation (or other nucleic acid delivery formulation), or another vector system (e.g., retrovirus or adenovirus), either as a viral particle or as a nucleic acid/liposomal formulation.
  • chemotherapeutic agents include, but are not limited to, angiostatin, endostatin, anti-HER-2/neu antibody, and tamoxifen.
  • Immunomodulators and immunostimulants include, but are not limited to, various interleukins, cytokines, antibody preparations, and interferons.
  • the method of treating cancer in a mammal can be adapted for use in vitro to determine, for example, cancer-specific expression of a protein.
  • a nonalphaviral sequence can be introduced into a recombinant alphavirus or, more specifically, a recombinant Sindbis virus, and cancer-specific expression of a protein to which the nonalphaviral sequence can bind can be assessed by lysis of cancerous cells by the recombinant alphavirus.
  • the present invention provides a method of detecting cancerous cells.
  • the method comprises contacting a cell with a present inventive recombinant alphavirus, which is desirably noncytolytic and replication-defective and which comprises a marker gene, such as green fluorescent protein, and detecting the expression of the marker gene, wherein detection of the expression of the marker gene indicates the presence of cancerous cells.
  • a present inventive recombinant alphavirus which is desirably noncytolytic and replication-defective and which comprises a marker gene, such as green fluorescent protein, and detecting the expression of the marker gene, wherein detection of the expression of the marker gene indicates the presence of cancerous cells.
  • Another method provided by the present invention is a method of determining a new cancer cell-specific, cell-surface molecule or receptor.
  • the method comprises introducing a sequence from a library of binding domains or a random sequence as an insertion or replacement of one or more amino acid residues of the glycoprotein El, E2 or E3 of a recombinant alphavirus, contacting the recombinant alphavirus with a cancerous cell selecting the recombinant alphavirus that binds to the cancerous cell to identify the cancer-specific, cell-surface molecule or receptor to which the recombinant alphavirus bound.
  • Example 1 Introduction of unique restriction endonuclease sites into the E2 glycoprotein region of Sindbis virus
  • Each of three unique restriction endonuclease sites namely Mlu I, Sph I and Bst EII, were introduced into the E2 glycoprotein of Sindbis virus clone TE (available from Dr. Diane Griffith, The Johns Hopkins University School of Hygiene and Public Health) by site-directed mutagenesis using PCR in two separate reactions. In one reaction, a 5' flanking primer and a 3' mutagenic primer were used and, in another reaction, a 5' mutagenic primer and a 3' flanking primer were used.
  • the 5' flanking primer (5'SV-Stu I) and the 3' mutagenic primer for introduction of the Mlu I site at E2 amino acid residue 175 were 5'-CT CGA CAT CCT TGA AGA G-3' (SEQ ID NO:l) and 5'-ATG TAT ACG CGT GCG GTC TCG GC-3' (SEQ ID NO:2), respectively, whereas the 5' mutagenic primer and the 3' flanking primer (3'SV-Bcl I) for introduction of the Mlu I site were 5'-CGC ACG CGT ATA CAT CCT ACC TG- 3' (SEQ ID NO:3) and 5'-ATT TCC CTT GGG CCG TGT-3' (SEQ ID NO:4), respectively.
  • the 5' flanking primer (5'SV-Stu I) and the 3' mutagenic primer for introduction of the Sph I site at E2 amino acid residue 60 were 5'-CT CGA CAT CCT TGA AGA G-3' (SEQ ID NO:5) and 5'-TTT GCG CAT GCT GCT CCG CT-3' (SEQ ID NO:6), respectively, whereas the 5' mutagenic primer and the 3' flanking primer (3'SV-Mlu I) were 5'-AGC AGC ATG CGC AAA CAA GTA CC-3* (SEQ ID NO:7) and 5'-ATG TAT ACG CGT GCG GTC TCG GC-3' (SEQ ID NO:8), respectively.
  • the 5' flanking primer (5'SV-Sph I) and the 3' mutagenic primer for introduction of the Bst EII site at E2 amino acid residue 114 were 5'-AGC AGC ATG CGC AAA CAA GTA CC-3' (SEQ ID NO:9) and 5'-CA CTA TGG TAA CCG TTA CGC TOTS' (SEQ ID NO: 10), respectively.
  • the 5' mutagenic primer and the 3' flanking primer (3'SV-Mlu I) for introduction of the Bst EII site were 5'-ACG GTT ACC ATA GTG AGT AGC AA-3' (SEQ ID NO:l 1) and 5'-ATG TAT ACG CGT GCG GTC TCG GC-3' (SEQ ID NO:12), respectively.
  • the final PCR product was gel-purified, digested with enzymes immediately upstream or downstream of the flanking primers and run on an agarose gel to confirm that the fragment is of the correct size. Then, a 1200 bp Stu I-BSSH II fragment from the Sindbis virus clone TE (the "parent virus"), which contains the three introduced restriction enzyme sites in the E2 glycoprotein, was subcloned into the Stu I-BSSH II- digested double subgenomic vector TotoCat5, thereby replacing the corresponding TotoCat5 E2 sequence, as shown in Fig. 1, which is a map of the alphaviral expression vectors TotoCat5 double subgenomic vector (top) and single subgenomic vector (bottom).
  • Fig. 1 is a map of the alphaviral expression vectors TotoCat5 double subgenomic vector (top) and single subgenomic vector (bottom).
  • ⁇ -heregulin is obtained from p-GEX-HRG a177"244 (Wallasch et al. EMBO J (1995) 17:4267-4275), which is available from Axel Ullrich (Department of Molecular Biology, Max Planck Institut fur Biochemie, Martinsried,
  • the ⁇ -heregulin sequence (EGF domain) cloned into the SV vector is 200 bp in length.
  • the sequence is amplified using PCR and the primers set forth in Table
  • a control vector comprising an irrelevant scAb, such as anti-gpl20 scAb, or an irrelevant ligand, such as the CD-4 binding ligand from gpl20, also is generated.
  • an irrelevant scAb such as anti-gpl20 scAb
  • an irrelevant ligand such as the CD-4 binding ligand from gpl20
  • Example 3 Construction of a recombinant Sindbis virus clone TE comprising the nonalphaviral sequence ⁇ -heregulin as a replacement of E2 amino acids 175-243 (TEMluBcl)
  • the 200 bp sequence for ⁇ -heregulin was obtained from p-GEX-HRG a177"244 and amplified using PCR and primers encoding restriction endonuclease sites Mlu I and/or Bel I at their 5' ends as shown in Table 1.
  • the PCR-amplified heregulin was subcloned into the E2 glycoprotein sequence of the recombinant Sindbis virus clone TE by replacing E2 amino acids 175-243 to generate TEMluBcl.
  • Example 4 Construction of a recombinant Sindbis virus clone TE comprising the nonalphaviral sequence ⁇ -heregulin as an insertion into the Bel I site at E2 amino acid 243 (TEBcl)
  • the 200 bp sequence for ⁇ -heregulin was obtained from p-GEX-HRG a177"244 and amplified using PCR and primers encoding the restriction endonuclease site Bel I at their 5' ends as shown in Table 1 (5'BclI and 3'BclI).
  • the PCR-amplified heregulin was subcloned into the E2 glycoprotein sequence of the recombinant Sindbis virus clone TE by inserting the 200bp sequence into the Bel I site at E2 amino acid 243 to generate TEBcl. Correct construction of the modified virus was confirmed by restriction enzyme analysis and DNA sequencing.
  • the erbB-2 sequence is 711 bp in length.
  • the sequence is amplified using PCR and is subcloned into
  • TotoCat5-E2ab or -E2c or the Sindbis virus clone TE For cloning of the anti-erbB-2 scA into the E2ab site of TotoCat5, the 5' Mlu I primer and the 3' Bel I primer of the anti-erbB-2 scA into the E2ab site of TotoCat5, the 5' Mlu I primer and the 3' Bel I primer of the anti-erbB-2 scA into the E2ab site of TotoCat5, the 5' Mlu I primer and the 3' Bel I primer of
  • Table 2 (in which underlining indicates restriction enzyme sites) are used.
  • the 5' Sph I primer and the 3' Mlu I primer of Table 2 are used.
  • a control vector comprising anti-gpl20 scAb also is generated.
  • Sindbis virus clone TE comprising the nonalphaviral sequence NGR binding domain specific for binding to angiogenic endothelial cells, as a replacement at one of two different sites in the E2 glycoprotein or at a site spanning the E2 and E3 glycoproteins
  • the NGR binding domain was obtained from Arap et al. Science (1998)
  • the PCR-amplified NGR domain was subcloned into the E2 or E2/E3 glycoprotein sequence of the recombinant Sindbis virus clone TE by replacing E2 amino acids 61-
  • Example 7 Production of recombinant alphaviral stocks
  • Infectious recombinant alphaviral RNA is transfected into BHK cells and the supernatant fluids are harvested at 24 hrs after transfection.
  • the post-transfection supernatant fluids contain recombinant virus.
  • the amount of virus present is determined by titering on BHK cells to determine the number of plaque forming units (pfu) per ml.
  • the amount of infectious units of virus per ml of supernatant fluid also can be determined by growing the virus in the presence of actinomycin-D, which inhibits cellular RNA polymerase, and H-uridine, which results in the incorporation of H- uridine into viral RNA.
  • Supernatant fluids containing H-uridine-labeled viral RNA are TCA precipitated, counted in a liquid scintillation counter and compared to a labeled wild-type viral culture with known titer in order to determine the number of infectious units/ml.
  • the viral stock supernatant fluids can be immunoblotted to determine the relative amount of viral proteins present in supernatant fluids.
  • the wild-type and heregulin-containing viral stocks are expected to contain
  • plaque titration on BHK cells does not work because the tropism of the virus has changed and the virus cannot bind and spread virus can be quantified using a stably transfected BHK cell line that expresses the wild-type E2 glyco-protein.
  • the wild-type protein can complement the nonalphaviral sequence containing E2 to allow spread of the virus and plaque titration.
  • Recombinant alphaviral stocks also can be produced by (i) cotransfection with helper RNA construct, (ii) transfection of RNA into packaging cell line, (iii) transfection of DNA, and (iv) cotransfection of DNA with SP6.
  • Synthesis of recombinant viral structural proteins by recombinant alphavirus Vector DNA (2-5 ⁇ g) is linearized with Sst I and transcribed in vitro using an SP6 DNA-dependent RNA polymerase (RiboMAX; Promega, Madison, Wisconsin) with 5 mM each of rATP, rCTP and rUTP, 0.6 mM rGTP, 3 mM cap analog (7 m G5'ppp5'G). The reaction is incubated for 2 hr at 37 °C. The RNA is phenol/chloroform extracted, precipitated with isopropanol, and resuspended in RNase-free Tris-Cl-EDTA.
  • RNA is transfected into BHK 21 (ATCC) cells using Lipofectin (GibcoBRL, Gaithersburg, Maryland) or electroporation.
  • Virus-containing supernatant fluids are harvested 24 hrs after transfection, clarified to remove cellular debris and stored at -80 °C.
  • proteins from the virus-containing supernatant fluids are radioimmunoprecipitated or immunoblotted using anti-Sindbis virus rabbit serum that recognizes an epitope in the E2 glycoprotein that was not deleted from the recombinant virus.
  • Example 9 Production of recombinant viral structural proteins by the recombinant alphaviruses generated from the plasmids TEMluBcl and TEBcl
  • the plasmids of Examples 4 and 5 were linearized and recombinant Sindbis viral RNA was transcribed in vitro using SP6 DNA-dependent RNA polymerase (RiboMAX) with 5 mM each of rATP, rCTP and rUTP, 0.6 mM rGTP, 3 mM cap analog (7 m G5'ppp5'G).
  • proteins from the virus-containing supernatant fluids 50 ⁇ l from infected cell lysates and 100 ⁇ l from stock supernatants
  • proteins from the virus-containing supernatant fluids 50 ⁇ l from infected cell lysates and 100 ⁇ l from stock supernatants
  • anti-Sindbis virus rabbit serum that recognizes an epitope in the E2 glycoprotein that was not deleted from the recombinant virus.
  • protein immunoblots using anti-scA antibodies and antiheregulin antibodies are performed. El, E2 and capsid proteins were detected in the cell lysates infected with TE,
  • the cell lysates derived from TEBcl-infected cells revealed a band on Western blot equal in size to wild-type E2. This indicated that heregulin, which was inserted at the Bel I site, may have been deleted from the virus by RNA recombination.
  • the slowest migrating band in the TEBcl-infected lane may represent the heregulin-containing E2 glycoprotein, which would be expected to be approximately 7300 daltons larger than the wild-type E2, or this band may represent PE2, the precursor of E2.
  • the cell lysates were immunoprecipitated with anti-Sindbis virus rabbit serum (1:80 dilution) and the immune complexes were precipitated using protein A sepharose (Pharmacia). After washing the immune complexes, the samples were boiled and the proteins were separated by electrophoresis through an SDS- 15% polyacrylamide (PAGE) gel.
  • PAGE polyacrylamide
  • the RIP assay of the transfected cell lysates revealed similar findings as the protein immunoblot of the stock virus infected cell lysates.
  • E2 the size of wild-type protein, was detected in TEMluBcl-transfected cells. A prominent band just below 68 kd along with an E2 band the size of wild-type was detected in the TEBcl-transfected cells.
  • the wild-type E2 band was what is expected for the TEMluBcl-transfected cells because the heregulin sequence is nearly the same size as the Sindbis virus sequence that was removed.
  • the PE2 in the TEMluBcl-transfected cells appeared to be completely processed.
  • a band corresponding to the wild-type E2 was seen in the TEBcl-transfected cells, suggesting the presence of a mixed infection with wild-type virus or the removal of the heregulin sequence by RNA recombination.
  • the precursor protein, pi 10 composed of PE2 and El in wild-type infected cells, was of a larger size in the TEBcl-transfected cells. This was consistent with the presence of heregulin in this larger precursor protein.
  • the PE2 in TEBcl-transfected cells did not appear to be completely processed. This was most likely the result of the heregulin insert.
  • alphavirus can be made, despite the removal of a 68 amino acid domain and replacement with a heterologous sequence.
  • the data also indicate that alphavirus with an insertion of a larger heterologous sequence (approximately 200 bp) in the E2 glycoprotein also can be made.
  • Example 10 Infection of BHK cells and MDA-MB-231 and breast cancer cells with wild-type Sindbis virus or a heregulin-containing Sindbis virus BHK cells and MDA-MB-231 cells (which express low or undetectable levels of erbB-2 and erbB-4 receptors) were infected with wild-type Sindbis virus or a heregulin-containing Sindbis virus, namely TEMluBcl (heregulin replaces E2 amino acid residues 175-243) or TEBcl (heregulin sequence at amino acid position 243), at a multiplicity of infection of 5. At various times after infection, the amount of cell death as a result of viral infection was determined by trypan blue exclusion.
  • Example 11 Transfection of BHK cells and SKBR3 breast cancer cells with Sindbis wild-type and modified viral RNA genomes Infectious RNA was transcribed from Clone 12 (TE Sph I Bst EII heregulin) and Clone 15 (TE Bst EII Mlu I heregulin) in vitro and transfected into BHK cells and SKBR3 breast cancer cells (available from American Type Culture Collection (ATCC), Rockville, Maryland; over-expresses erbB-2) using Lipofectin. The number of dead cells was determined 48 hrs after transfection using the trypan blue exclusion assay (Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, Inc., 1996, Suppl. 18, Unit 11.5.1, Cell Viability Test by Trypan Blue Exclusion).
  • the parental virus which was derived from cDNA of the virus TE and which contains the three unique restriction enzyme sites Sph I at E2 amino acid residue 60, Bst EII at E2 amino acid residue 114, and Mlu I at E2 amino acid residue 175 as generated in a two-step PCR reaction was able to replicate in both cell lines, albeit less efficiently in SKBR3 cells.
  • 80% of cells in the BHK cultures were dead versus 20% of cells in the SKBR3 cultures.
  • the heregulin-containing viruses Clone 12, with heregulin replacing E2 amino acid residues 60-114, and Clone 15, with heregulin replacing E2 amino acid residues 114- 175, were able to kill only SKBR3 cells (at approximately the same level as TE) and not BHK cells, when compared to mock transfected cells (lipofectin only) and to cells transfected with a Sindbis virus replicon (an expression vector containing no structural proteins) expressing the ⁇ -galactosidase gene.
  • the heregulin-containing viruses exhibited cytopathic effect only in the SKBR3 cells.
  • Example 12 Evaluation of intracellular synthesis of alphaviral structural proteins by Clone 12, TE Bst EII Mlu I heregulin (Clone 15) andLKD 102.10 (derived from parental virus TE) Clone 12, Clone 15 and the parental virus were pulse-labeled and the transfected cell lysates were immunoprecipitated. The presence of the capsid protein was readily detected in all samples but was diminished compared to the wild-type virus TE. A band of slightly larger molecular weight than wild-type PE2 was detected for cells transfected with Clone 12. This band most likely represented PE2, which is slightly larger than wild-type PE2 because of the presence of heregulin. The presumed PE2 for Clone 15 was barely detectable.
  • the post-transfection supernatant fluids were harvested 32 hours after electroporation, clarified to remove cell debris, and 200 ⁇ l were placed onto BHK cell monolayers in 35mm wells for 1 hour at 37°C. After infection, 1.5 ml of media was added to the culture and the cells were incubated for 26 hours at 37°C. At 26 hours after infection, when approximately 60-70% of the cells exhibited a cytopathic effect (CPE), the supernatant fluids were harvested and clarified to remove cell debris. The cells were lysed with RIPA buffer. BHK or SKBR3 cells were infected with 200 ⁇ l of these supernatant fluids for 1 hour at 37°C.
  • CPE cytopathic effect
  • RNAs from Sindbis viruses encoding CAT and comprising anti-erbB-2 scA, ⁇ -heregulin, no modification, an irrelevant scA or an irrelevant ligand are separately transfected into BHK-21 cells using Lipofectin and the progeny virions are labeled in media containing S-methionme and S-cysteine
  • the radiolabeled progeny virions are precipitated from the supernatant fluids 24 hrs post-infection using 10% polyethylene glycol 8000 in 0.5 M NaCl and purified by passage over a 15-40% potassium tartrate gradient.
  • the purified radiolabeled virus is diluted in binding medium (RPMI without NaHCO 3 , 0.2% BSA, 20 mM HEPES, pH 7.4) and stored at -80°C.
  • the number of infectious units/ml is determined by growing the virus in a permissive breast cancer cell line in the presence of actinomycin-D (at a dose sufficient to inhibit cellular RNA polymerase) and H-uridine, which results in incorporation of the H-uridine into viral RNA.
  • Supernatant fluids containing H-uridine-labeled viral RNA are TCA precipitated, counted in a liquid scintillation counter, and the proportion of 3 H-uridine incorporated into viral RNA is calculated and compared to a labeled wild-type viral culture with known titer.
  • Specific binding of the viruses is determined by separately adding a given number of infectious units of test and control vectors to cell monolayers in the absence or presence of increasing dilutions of anti-erbB-2 or a GST ⁇ -heregulin fusion protein as appropriate and incubating the cell monolayers at 4°C, which allows the virus to bind to the cells but not enter the cells.
  • IB4 an EBV-transformed B- lymphocyte cell line
  • Supernatant fluids are collected at 30 min intervals after addition of virus, virus and mAb, or virus and ⁇ -heregulin as appropriate, and counted in a liquid scintillation counter to determine the amount of unbound virus. After washing, the cells are lysed with 1% SDS and counted in a liquid scintillation counter to determine the amount of bound virus.
  • Example 15 Specific infectivity of the targeted recombinant Sindbis viruses Specific infectivity of SKBR3, MDA-MD-453, MDA-MD-361 (ATCC), BT 474 (ATCC), MCF-7, MDA-MB-231 , BHK-21 , N 18 and IB4 cells by Sindbis viruses encoding CAT or green fluorescent protein (GFP, Clonetech, Palo Alto, California) and comprising anti-erbB-2 scA, ⁇ -heregulin, no modification, an irrelevant scA or an irrelevant ligand in the presence or absence of anti-erbB-2 or GST ⁇ -heregulin fusion protein as appropriate and at the antibody dilution that maximally inhibited viral binding in the binding assay is determined by assaying for CAT activity.
  • GFP green fluorescent protein
  • Cells are incubated with vector and mAb for 1 hr at 37°C, the vector or combination of vector and antibody is/are removed, the cells are washed twice with phosphate-buffered saline, and fresh growth medium is added. At 6 hrs post-infection, the cells are harvested and assayed for CAT activity using standard techniques (If GFP is used, fluorescence microscopy is used to detect its expression). The cells are lysed by three successive freeze-thaw cycles. The lysate is centrifuged and the supernatant assayed for CAT activity. Because of the high level of CAT expression achieved with TotoCat5, cell extracts usually need to be diluted 1,000 times or more to be within the linear range of the assay.
  • TLC sheet is then air-dried.
  • the amount of monoacetylated chloramphenicol is quantified using a phosphoimager (Molecular Dynamics).
  • specific infectivity is measured by indirect immunofluorescence assay.
  • Test and control cells are grown on glass coverslips to 70% confluency and counted with a hemacytometer to determine the number of cells plated.
  • the cells are then infected with a given number of infectious units of targeted or untargeted vector for 1 hr at 37°C.
  • the coverslips are washed twice with PBS (calcium and magnesium free) and then fixed in -20°C methanol for 5 min. The methanol is removed and the cells are washed three times with PBS.
  • the cells are then incubated in blocking buffer (0.5% w/v gelatin/ 0.2% w/v BSA in PBS) for 30 min at room temperature.
  • the blocking buffer is removed and primary antibody, i.e., anti-Sindbis virus rabbit serum, anti-E2 mAb or anti-CAT rabbit serum (5'3', Inc., Boulder, Colorado), is added for 30 min at room temperature.
  • primary antibody i.e., anti-Sindbis virus rabbit serum, anti-E2 mAb or anti-CAT rabbit serum (5'3', Inc., Boulder, Colorado
  • secondary antibody i.e., fluorescein-conjugated antimouse IgG or antirabbit IgG, is added for 30 min at room temperature.
  • the coverslips are washed three times with PBS, air-dried, and mounted on glass slides using Moviol 4-88 (Calbiochem) solution containing 2.5% DABCO (l,4-diazobicyclo-[2.2.2]-octane, Sigma Chem. Co., St. Louis, MO). Immunofluorescent staining is performed and the cells expressing CAT or Sindbis antigens are counted. The percentage of cells infected is calculated.
  • erbB-2-expressing breast cancer cells and control cells also are infected with a given number of infectious units of test and control vectors. At 12 hrs post-infection, the cells are harvested by incubation for 1-2 min in trypsin. After washing with PBS, an aliquot of the cells is diluted five-fold in 0.2% trypan blue. The number of dead, i.e., blue, and living cells is counted using a hemacytometer and the percentage of dead cells is calculated.
  • the vascular cell line SLK is a human Kaposi's sarcoma cell line that expresses the NGR receptor which is characteristic of cells in the vasculature.
  • Arap, W. et al. Science (1998) 279:377-380 have suggested targeting tumor vasculature with "NGR" peptides.
  • NGR NGR peptides.
  • a number of these peptides are disclosed in this publication, which is incorporated herein by reference.
  • the exemplified peptide described below is for illustrative purposes only, and other related peptides could, of course, also be used.
  • a nucleotide sequence 5'-TGC AAC GGT CGC TGT GTA TCG GGA TGT GCC GGT CGA TGT-3' encodes the peptide sequence C-N-G-R-C-V-S-G-C-A-G-R- C.
  • the nucleotide sequence was ligated into the portion of the Sindbis virus genome either between amino acid 58 of E3 and amino acid 6 of E2 (NGR1) or between amino acid 61 and amino acid 73 of E2 (NGR2). Sindbis virus was produced having these modifications in the envelope protein.
  • the resulting virus was then tested for cytopathic activity against either the SLK cell line that expresses at its surface an NGR receptor or against the BHK cell line which is normally infected with wild-type Sindbis virus.
  • the NGR1 and NGR2 strains are specifically targeted to the SLK cell line and are ineffective against BHK.
  • the wild-type Sindbis virus infects only BHK.
  • Example 17 Targeting of the targeted recombinant Sindbis viruses in vivo
  • SKBR3 cells (1 x 10 ) are infected in vitro at a multiplicity of infection of 5 with targeted alphaviral vectors or control viral vectors at 37 °C for 1 hr. After infection, the cells are injected into the mammary fat pads of six- week-old Swiss athymic nude mice (Jackson Laboratories, Bar Harbor, Maine). For 4-6 wks, the tumors are observed for a change in rate and extent of growth as compared to tumors formed from control, uninfected SKBR3 cells.
  • TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling
  • Tissue sections are deparaffinized, rehydrated, permeabilized by incubation in 10 ⁇ g of proteinase K per ml (Boehringer Mannheim, Indianapolis, Indiana) for 15 min at room temperature and washed twice for 5 min in PBS.
  • the tissue sections are covered with 40 ⁇ l of labeling mix (25 mM Tris-HCl, pH 7.2, 0.2 M potassium cacodylate, 5 mM cobalt chloride, 30 U of terminal deoxynucleotidyltransferase, 0.6 nmol of digoxigenin-11-dUTP, 0.15 mM dATP) and incubated in a humid chamber at 37°C for 60 min.
  • labeling mix 25 mM Tris-HCl, pH 7.2, 0.2 M potassium cacodylate, 5 mM cobalt chloride, 30 U of terminal deoxynucleotidyltransferase, 0.6 nmol of digoxigenin-11-dUTP, 0.
  • the reaction is terminated by incubation in 2 x SSC for 15 min at room temperature.
  • the probe is detected using alkaline phosphatase-conjugated antidigoxigenin Fab fragments with the Genius 3 nonradioactive nucleic acid detection kit from Boehringer Mannheim.
  • Blocking buffer buffer 1 (Boehringer Mannheim) plus 0.1% Triton X-100 and 2% normal lamb serum (NLS, GibcoBRL, Gaithersburg, Maryland) is added to the tissue sections and the sections are incubated for 30 min.
  • TUNEL assay controls include omission of terminal transferase or labeled nucleotide.
  • Immunoperoxidase staining using standard techniques also is performed to detect the presence of and to determine the distribution of viral antigens in resected tumors.
  • Tissue sections are stained immunocytochemically with antialphaviral rabbit serum using the avidin-biotin-peroxidase complex method. Sections are first blocked with 2% normal rabbit serum in PBS for 30 min and then incubated with antialphaviral rabbit serum at a 1/100 stock dilution for approximately 45 min. The slides are rinsed in PBS and then incubated with biotinylated secondary antibody (1 :100 dilution in 2% normal rabbit serum) for 30 min. The slides are then washed and treated with 1% hydrogen peroxide in methanol to block endogenous peroxidase.
  • Example 18 Methods of delivering a targeted alphavirus (TA) or an alphaviral vector (TAV) to a mammal in accordance with the present inventive methods
  • the TAV can be delivered as a virus after packaging in an appropriate packaging cell line.
  • An example of a packaging cell line for production of TAV to be used in the treatment of a breast cancer is a breast cancer cell line that expresses the target protein or target factor for TAV infection.
  • any cell line that can produce high titers of virus can be used.
  • the TA is then concentrated (e.g., by filtration, ultracentrifugation or another method) and is administered, for example, directly to the tumor site, or the site of tumor resection (as an adjunct to surgical therapy).
  • TAV construct DNA or in vitro transcribed RNA transfection into a packaging cell line is TA particle production.
  • Standard transfection reagents are used, e.g., Lipofectin (for RNA), lipofectamine, lipofectamine plus, effectene, and clonfectin and the like (for DNA).
  • the packaging cell line contains the target protein or factor for TA infection, the TA particles produced from transfected cells will infect neighboring cells, resulting in amplification of TA particles.
  • the virus is harvested 6-96 hrs after transfection, more preferably at 12-72 hrs after transfection (this will vary depending upon the packaging cell line used), after which the supernatant is clarified of cellular debris by centrifugation.
  • the TA particles are then concentrated, e.g., preferably by filtration or ultracentrifugation. If filtration is used, a Centricon 100-500 concentrator is used to concentrate the virus away from nonviral proteins and factors present in the supernatant. Use of Centricon concentrators is known by users in the art and can be found in any Centricon users manual. If ultracentrifugation is used, the virus can be centrifuged at a speed of 39,000 rpm for 1/2 hr using a SW-41 rotor (Beckman). The concentrated virus is then titered on an indicator cell line.
  • an analogous erb-B2 TAV expressing the green fluorescent protein (GFP) can be titered on an erb- B2-overexpressing breast cancer cell line and the virus titer can be determined by counting GFP-positive cells by fluorescent microscopy.
  • GFP green fluorescent protein
  • Plaquing the TA on breast cancer cells is another method that can be employed to determine virus titer. Limiting dilutions of TCA are infected on breast cancer cell monolayers and the titer is determined by examining the monolayers for viral plaques 48-72 hrs later.
  • the titer of the virus desirably is at about 10 2 - 10 13 TA particles/ml for clinical application.
  • concentrated TA particles can be resuspended in a suitable solution (e.g., saline or tris-buffered saline) and then applied or injected at or into the tumor site.
  • a suitable solution e.g., saline or tris-buffered saline
  • TA particles can be applied to surgical tumor resection sites as an adjunct to surgical therapy. TA particles would not only infect and kill any residual cells in the surgical site, but would also be taken up by regional lymph nodes and infect any residual tumor cells present in the lymph node that escaped surgical therapy.
  • the TA suspension can be directly injected into the tumor mass without the need for surgery. Multiple injections would be performed into the tumor, at periodic intervals, preferably weekly or monthly, to decrease the tumor mass and prevent metastases.
  • the TA suspension could be injected intravenously at concentrations, such as concentrations greater than about 10 10 TA particles per ml.
  • concentrations such as concentrations greater than about 10 10 TA particles per ml.
  • the intravenous virus would then bind to angiogenic endothelial cells, replicate in them and kill them.
  • the virus produced from the first round of infection of the endothelial cells would then spread to neighboring tumor angiogenic endothelial cells and kill them also, thus killing the cells that line the blood vessels that supply the tumor with nutrients. This would then result in tumor necrosis.
  • the format for delivery of TAV can be one of two types - (a) direct nucleic acid delivery to the tumor site or a peripheral site, where the nucleic acid can produce TA that would spread through the tumor, or (b) delivery of the nucleic acid to a packaging cell line (or the patient's own irradiated tumor cells - to act as a packaging system) followed by delivery of the cells containing TA in the patient.
  • the number of cells that would be introduced into the patient desirably would be from about 10 3 to about 10 14 cells per inoculation. Multiple inoculations can be required and can be administered at the same or different sites.
  • the TAV is delivered as a nucleic acid, it can be delivered either as RNA or DNA. If it is delivered as RNA, it would first have to be transcribed from a DNA template to make RNA. Such in vitro transcription of RNA is known in the art.
  • the plasmid construct of the TAV is linearized with an appropriate restriction enzyme prior to in vitro transcription in reaction buffer (e.g., 0.5 ⁇ g DNA, 20 U RNAsin, 2.5mM dNTPs, 100mm DTT, 40mM Tris-HCl, pH 7.5, 25mM MgCl 2 , 2mM Spermidine, lOmM NaCl ), containing a polymerase to drive transcription (e.g., SP6 polymerase).
  • reaction buffer e.g., 0.5 ⁇ g DNA, 20 U RNAsin, 2.5mM dNTPs, 100mm DTT, 40mM Tris-HCl, pH 7.5, 25mM MgCl 2 , 2mM
  • RNA is then complexed with a liposomal or nonliposomal (termed here collectively as "lipoid”) delivery system in order to transfect cells efficiently with the TAV RNA.
  • lipoid delivery systems can be, for example, lipofectin (Life technologies), clonfectin (Qiagen), superfect and effectene (Qiagen), which could be complexed with other compounds (such as polybrene or polyamines) or other proteins and the like to enhance the efficiency and targeting of TAV transfection into cells.
  • the TAV RNA enters the cytoplasm of the cell, which can be the target cell or a cell that is not the target cell.
  • a target cell is an erb-B2 overexpressing breast cancer cell that is to be killed by the TA/TAV.
  • An example of a nontarget cell is a packaging cell line, a cell in a tissue around a resected tumor, a lymph node cell, or an endothelial cell.
  • TAN R ⁇ A can die from virus production. Death occurs when a lytic production of virus kills that particular cell type or when the cell is destroyed by the host's immune system as a result of viral proteins being expressed on the cell's surface.
  • virus can be produced chronically so that TA/TAV production occurs for longer periods of time (Griffin and Hardwick, Annual Review of Microbiology (1997) 51 :565-92 and Dryga et al. Virology (1997) 228(1 ):74-83).
  • TA/TAV can be modified to infect certain cell types, such as the nontarget cells, to persistently produce virus from nontarget cells and at the same time produce a cytolytic infection in target cancer cells.
  • TA/TAV can be modified to infect cancer cells persistently and not induce a lytic cytopathology as a result of viral replication. Cell death then can be induced by the expression of a viral or a heterologous protein or genetic element.
  • This protein can be a cell death protein or a ribozyme that destroys antiapoptotic protein mRNAs.
  • the heterlogous protein can be expressed on the surface of the cancer cell, which then can become a target for destruction by the host's immune system.
  • the heterologous protein can be encoded by a cell death inducing or preventing gene, its cognate antisense or ribozyme molecule, or an immunogen (defined broadly as an antigen, cytokine or an antibody or part thereof) that promotes target tumor cell destruction by the immune system, such as a nonhost antigen, a cytokine or a scAb.
  • a nonhost antigen are the 4- IBB ligand (Melero et al. European Journal of Immunology (1998) 28(3): 1116-21).
  • An example of a cytokine is GM-CSF (Dunussi-Joannopoulos et al. Blood (1998) 91(l):222-30).
  • the heterologous gene can be inserted into an alphaviral vector, such as a Sindbis viral vector, which contains a second subgenomic promoter for the expression of a heterologous gene.
  • an alphaviral vector such as a Sindbis viral vector
  • the heterologous gene can function to inhibit tumor growth and/or increase TA TAV production.
  • the vector kills tumor cells in the host.
  • Nontarget cells that produce first round virus can die from productive virus infection, but this is not of consequence since the number of nontarget cells transfected is relatively small (1-20%), preferably (5-10%), in comparison to subsequent replication and amplification of the TA/TAV through the target cell population, e.g., the tumor (approaching 100%).
  • the TA/TAV can be further targeted to tumors by incorporating in the liposome or nonliposome delivery system molecules that target the lipoid-RNA complexes more specifically to tumor vasculature.
  • peptides that express the RGD domains that target tumor vasculature can be mixed with the lipoid-RNA complexes to target the TAV RNA to cells of the tumor vasculature integrin molecules, specifically the tumor angiogenic endothelial cells (Ruoslahti, Annual Review of Cell & Developmental Biology (1996) 12:697-715.
  • the TA/TAV infected tumor vasculature then produces first round virus, which can seed TA particles from the nonluminal endothelial surface into the cellular mass of tumor cells.
  • the tumor-specific TA would then spread from tumor cell to tumor cell and, thus, destroy the tumor.
  • the necrosis that arises from destruction of tumor cells close to the blood supply can produce a tumor necrosis that prevents other tumor cells from receiving the necessary nutrients from the blood for survival.
  • the TAV RNA can be targeted to tumor vasculature by incorporating peptide RGD ligands into the lipoid-RNA complex, where the TAV itself, is targeted to RGD domains of tumor vasculature integrin molecules. This can be done by adding RGD peptide ligands at a concentration from about lpM to about lOOmM, preferably from about InM to about 1 ⁇ m, to the lipoid TAV RNA complex.
  • this then becomes a two-layered system, where ligands in the lipoid RNA complex first promote one- level of targeting to the tumor vasculature and then the specific RGD domains on the virus promote a second level of targeting through spread and amplification of the virus through tumor vascular endothelial cells. Further layers of specific targeting can be added to promote tumor specificity and low toxicity.
  • the TAV can be delivered as a DNA-lipoid complex rather than as a RNA-lipoid complex.
  • a promoter must be placed proximally to the virus to ensure first round expression of the TA, since alphaviruses are RNA viruses with no DNA intermediate.
  • One way is to transfect directly the plasmid DNA into cells without a eukaryotic promoter system. Even though the plasmid DNA does not contain a eukaryotic promoter (but does contain cryptic sequences for eukaryotic transcription of the vector, such as an SP6 bacteriophage promoter), virus can be produced from cells that are transfected with eukaryotic promoter-deficient plasmid DNA that encodes the alphavirus.
  • the SP6 promoter TAV DNA can be transfected into cells using the above-mentioned lipoid transfection reagents at concentrations that are known in the art.
  • TAV Tranfectin
  • 5 ⁇ g of TAV can be mixed with 20 ⁇ l of Clonfectin (Qiagen) for transfection into packaging cells or directly into a breast cancer surgical site.
  • Transfected cells can produce first round virus, which then amplifies and spreads to other tumor cells in a cell-specific manner.
  • One preferred method of producing first round virus from DNA-lipoid complexes is to express simultaneously a SP6 bacteriophage polymerase gene in cells that contain the TAV plasmid DNA that contains the SP6 promoter sequences proximally to the alphaviral genome.
  • the SP6 polymerase is expressed from a helper plasmid.
  • the SP6 gene can be cut out from the pSR3 plasmid (Moss et al. Biotechniques (1993) 14:222-223) and cloned into the pREPIO plasmid (Invitrogen, CA) or expressed from a recombinant Vaccinia virus.
  • the SP6 plasmid can then express in eukaryotic cells where the SP6 polymerase can bind to the SP6 promoter sequences located on the TAV. Expression of the TAV RNA would result in the translation of the alphaviral NS RNA dependent RNA polymerase genes. The polymerase can then bind to TAV RNA promoter sequences and amplify TAV genomes within cells and produce TA particles. These particles would then infect and spread through the target cell population ⁇ the tumor cells or the cells that support their growth.
  • This system can be further improved by driving SP6 expression from a tumor- specific promoter, or a tumor vascular capillary endothelial cell promoter.
  • the latter promoter is preferred because it would prevent expression in hematogenously exposed cells other than endothelial cells.
  • a promoter that is more specifically expressed in endothelial cells is the Factor VIII promoter.
  • Another promoter is the gamma- glutamyl transpeptidase promoter (Dropulic et al. In Vitro Cellular & Developmental Biology (1987) 23(11):775-81).
  • TAV DNA-lipoid
  • a eukaryotic promoter Dubensky et al. Journal of Virology (1996) 70(1):508-19.
  • the alphaviral genome is cut from its plasmid vector and cloned into the pcDNA3.1 Amp expression vector (Invitrogen), where the alphaviral genome is located distal to the cytomegalovirus promoter (CMV).
  • CMV cytomegalovirus promoter
  • Initial TAV RNA is transcribed from the CMV- driven plasmid DNA, after which the TAV RNA-dependent RNA polymerase binds to the transcription initiation site on TAV RNA to amplify the amount of TAV RNA in the cell.
  • a ribozyme can be inserted into the 3' region of the viral genome to cleave the 5' leader sequence between the CMV transcription initiation site and the true 5' terminus of the TAV RNA. This ribozyme is constructed so that it binds to the 5' terminus and cleaves right at the first nucleotide of the TAV RNA 5' terminus.
  • the TAV can be made more specific for tumor cells by using tumor-specific or vascular-specific promoters.
  • a preferred way of producing first round virus is by placing the TAV sequences inside a carrier vector (e.g., a vaccinia vector, an adenoviral vector or, preferably, a crHIV vector as described in Dropulic et al. WO 97/20060) driven by an appropriate promoter for the expression of TAV RNA inside cells.
  • First round delivery can be accomplished by delivery of the carrier vector to the target site, which can then produce first round targeted alphaviral particles, which can then specifically infect the target cell population.
  • a preferred method for delivery of the TCA/AV is inside a crHIV vector. Since the size of the crHIV genome is smaller than the alphaviral genome, the TCA/AV can be segregated into two components that contain sequences to permit their recombination.
  • the Non-Structural (NS) region, the J (junction) region and a limited number of downstream nucleotides (to permit efficient recombination between the Non-Structural and the Structural (S) TAV RNAs, if required) of the TAV genome can be amplified by PCR, using appropriate primers containing restriction enzyme sites for cloning into the crHIV vector.
  • the PCR product is amplified using Vent polymerase (New England Biolabs) to prevent mutations being inserted into the PCR product.
  • the PCR product is then cut with restriction enzymes, run on a gel and the appropriate band is purified from the gel using a Qiagen gel extraction kit (Qiagen).
  • Qiagen Qiagen gel extraction kit
  • the Structural region containing the modified envelope proteins that are targeted to cancer cells, the J region and a limited number of upstream sequences (to permit efficient recombination between the Non-Structural and Structural TAV RNAs, if required) of the TAV genome can be similarly amplified by PCR and cloned into the crHIV vector.
  • the TAV sequences in the two crHIV vectors can be linked to a eukaryotic promoter (eg., CMV, a tumor-specific promoter, or a vascular-specific promoter) or can be directly expressed from the crHIV Long Terminal Repeat (LTR), which acts as a promoter in this vector system.
  • a eukaryotic promoter eg., CMV, a tumor-specific promoter, or a vascular-specific promoter
  • LTR crHIV Long Terminal Repeat
  • the two crHIV-TAV (NS and S) constructs can be co-transfected with a helper construct into a packaging cell system to permit the production of recombinant crHIV-TAV. Since HIV encapsidates two genomic RNAs into its viral particles, some of the particles will contain both crHIV-TAV NS and S genomes, which can then infect cells. During reverse transcription of crHIV-TAV RNAs, recombination can occur between NS and S crHIV-TAV genomes, resulting in a whole TAV genome being incorporated into the genome of the host cell.
  • the TAV then can be expressed off the eukaryotic promoter to produce initial TAV RNA, which would then amplify using the Sindbis virus nonstructural RNA-dependent RNA polymerase.
  • the targeted alphaviral particles would then specifically infect tumor cells and spread by the receptor-specific mechanism.
  • a most preferred method for delivery of TAV is to layer the two component crHIV-TAV inside a vector-helper crHIV direct delivery system.
  • two-component crHIV-TAV particles can be produced for infection of cells.
  • Cells infected with the two-component crHIV-TAV can produce cells containing targeted alphaviral proviruses, which would go on to produce targeted alphaviral particles for specific infection and killing of target cell populations.
  • An array of promoter/vector permutations can be used to produce TAV specifically from specific cell types. This can aid toward the development of highly specific TA/TAV.

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Abstract

L'invention concerne un alphavirus recombinant contenant au moins une séquence non alphavirale épivirale qui (i) est présente dans la glycoprotéine E1, E2 ou E3 comme insertion ou en remplacement d'un ou plusieurs résidus d'acide aminé et qui (ii) se lie directement et sélectivement à une région sur un récepteur de surface cible qui est spécifique de cellule. L'invention concerne également les acides nucléiques liés à cet alphavirus et des méthodes de traitement du cancer chez un mammifère au moyen de cet alphavirus et des acides nucléiques correspondants.
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