WO2003087329A2 - Virionoides cytocides inhibiteurs d'angionenese - Google Patents

Virionoides cytocides inhibiteurs d'angionenese Download PDF

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WO2003087329A2
WO2003087329A2 PCT/US2003/011142 US0311142W WO03087329A2 WO 2003087329 A2 WO2003087329 A2 WO 2003087329A2 US 0311142 W US0311142 W US 0311142W WO 03087329 A2 WO03087329 A2 WO 03087329A2
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Frederick L. Hall
Erlinda M. Gordon
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University Of Southern California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—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
    • C12N2810/50—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
    • C12N2810/851—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from growth factors; from growth regulators

Definitions

  • the present invention relates to the medical arts, and in particular, to viral medicines.
  • the molecular modification of the env protein prevents the obligatory conformational change that occurs in response to receptor binding which is required for viral fusion and core entry.
  • pathology-targeted vector which, when injected intravenously, seeks and accumulates in diseased or cancerous lesions within the body.
  • pathology-targeted vector which, when injected intravenously, seeks and accumulates in diseased or cancerous lesions within the body.
  • escort proteins has been introduced to improve cell-specific targeting and gene delivery to activated endothelial cells.
  • TVTMs tumor vasculature targeting motifs
  • Incorporation of tumor vasculature targeting motifs (TVTMs) into MLV env escort proteins enhances retroviral binding and transduction of human endothelial cells, J Virol, 74:5320-5328; Masood, R, Gordon, EM, Whitley, MD, Wu, BW, Cannon, P, Evans, L, Anderson, WF, Gill, P, Hall, FL (2001) Retroviral vectors bearing IgG-binding motifs for antibody-mediated targeting of vascular endothelial growth factor receptors, Int'l J Mol Med 8:335-343).
  • Escort proteins are defined as non- infectious retroviral env proteins that accompany the infectious wild type env to provide a gain-in- function-phenotype (i.e., targeting) to the composite vector.
  • escort proteins display specific ligands or targeting peptides, which essentially replace the deleted receptor binding domains of a modified ecotropic env construct.
  • a wild-type CAE env encoding amphotropic 4070 gp70 envelope protein
  • this modified env construct bearing a specific targeting motif
  • the vectors arrayed in the CAE plus "escort" env protein configuration exhibited the desired gain-of-function phenotype (e.g.
  • vascular endothelial growth factor also known as vascular permeability factor
  • NEGF vascular endothelial growth factor
  • vascular permeability factor is a secreted selective mitogen and prominent regulator of angiogeneisis and vascular permeability in vivo.
  • Vascular permeability factor an endothelial cell mitogen related to PDGF, Science 246: 1309- 1312; Connolly., D.T., D.M. Heuvelman, R. Nelson, J.V. Olander, B.L. Eppley, J. J. Delfino, N.R. Siegel, R. M. Leimgruber, and J. Feder, (1989) Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis, J. Clin. Invest.
  • Vascular endothelial growth factor a potent and selective angiogenic agent, J. Biol. Chem. 271: 602- 606; Senger, D.R., S. J. Galli, A. M. Dvorak, CA. Perruzzi, V.S. Harvey, and H. F. Dvorak (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of as cites fluid, Science 219: 983-985; Senger, et al (1986) A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines, Cancer Res. 46: 5629-5632).
  • VEGF isoforms differ in the efficiency of secretion and the potency of mitogenic activities, which are specific for vascular endothelial cells.
  • VEGF gene expression is normally quite low in the absence of overt angiogenesis.
  • Vascular permeability factor vascular endothelial growth factor
  • VEGF gene expression is found to be up-regulated both by oncogenic gene mutations and hypoxia present in ischemic tissues and solid tumors.
  • Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis, Nature 359:843-845; Liu, Y, Cox SR, Morita T, Kourembanas S. (1995) Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5" enhancer, Circ Res 77:638-643; Mazure, NM, Chen, EY, Yeh, P, Laderoute, KR, Giaccia, AJ, (1996) Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression, Cancer Res. 56: 3436-3440).
  • the homodimeric VEGF polypeptides bind to one of two tyrosine kinase receptors: (1) a high affinity receptor (25 pM) receptor designated Flt-1 (de Vries, C, Escobedo JA, Ueno H, Houck KA, Ferrara N, Williams LT, (1992) The fins-like tyrosine kinase, a receptor for vascular endothelial growth factor, Science 255:989-990; Shibuya, M, Yamaguchi S, Yamane A, Ikada T, Tsushime H, Sato M, (1990) Nucleotide sequence and expression of human receptor-type tyrosine kinase (fit) closely related to the fins family, Oncogene 8:519-527), and (2) a lower affinity receptor (125 pM) designated KDR/flk-1 (Terman, Bl, Carrion ME, Kovacs E, Rasmussen BA, Shows TB.
  • VEGF is an important factor in driving the growth, metastasis, and angiogenesis of solid tumors.
  • Vascular endothelial growth factor and glioma angiogenesis coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms, Int. J. Cancer 59: 520-529; Claffey, K.P., L.F. Brown, L.F. del Aguila, K. Tognazzi, K.T. Yeo, E.J. Manseau, and H.F (1996) Dvorak.
  • vascular permeability factor/vascular endothelial growth factor increases tumor growth, angiogenesis, and experimental metastasis, Cancer Res. 56: 172-181).
  • VEGF vascular endothelial growth factor
  • its cognate receptor(s) on tumor vascular endothelium.
  • the VEGF/receptor complex is a highly specific marker of tumor endothelium.
  • Dvorak et al. (1991) H.F., J.A. Nagy, A.M. Dvorak, (1991) Structure of solid tumors and their vasculature: implications for therapy with monoclonal antibodies, Cancer Cells 3: 77-85; Ke-Lin et al., (1996) Vascular targeting of solid and ascites tumours with antibodies to vascular endothelial growth factor, Eur. J. Cancer 32A: 2467-2473).
  • the VEGF/receptor complex can be utilized for the targeting and/or imaging of tumor vasculature.
  • VEGF receptor complex is among the most specific markers of human tumor-associated vasculature, as Flt-1 and KDR are expressed almost exclusively on endothelial cells.
  • Flt-1 and KDR Endothelial receptor tyrosine kinases involved in angiogenesis, J Cell Biol 129:895-898.
  • up-regulation of both the ligand and the cognate receptor(s) of VEGF are observed within solid tumors, when compared with the endothelium of normal tissues.
  • VEGF expression serves, not only as a specific marker of tumor endothelium (Brekken,R.A., X. Huang, S. W. King, and P.E. Thorpe (1998) Vascular endothelial growth factor as a marker of tumor endothelium, Cancer Res. 58: 1952-1959), but as a potential target for therapeutic intervention in the treatment of neoplastic disease.
  • the present invention is based on an unexpected discovery made while studying a series of constructs comprising VEGF congeners that displayed within the context of Moloney murine leukemia virus (MLN) envelope "escort proteins".
  • MLV Moloney murine leukemia virus
  • escort proteins a series of constructs comprising VEGF congeners that displayed within the context of Moloney murine leukemia virus (MLN) envelope "escort proteins.
  • MLV-based vectors bearing a ⁇ -galactosidase marker gene.
  • the performance of these chimeric retroviral vectors were then evaluated in vitro in terms of targeting and transduction of activated human endothelial cells.
  • vascular endothelial cell growth factor/vascular permeability factor (VEGFNPF) receptors selectively expressed on the surface of tumor-activated endothelial cells (EC) provide an advantageous locus for targeting vectors to angiogenic tissues and/or tumor vasculature.
  • VEGFNPF vascular endothelial cell growth factor/vascular permeability factor
  • VEGF-targeted escort proteins were incorporated without a wild-type amphotropic env partner into virions, which thereby bound to endothelial cells without triggering fusion and entry of the retroviral core.
  • the observed cytotoxicity was abolished when a wild-type amphotropic envelope protein was co-expressed with the VEGF- targeted escort construct.
  • cytocidal virionoids identify a new class of therapeutic viral particles, which we have designated “targeted cytocidal virionoids.”
  • inventive artificial predatory virionoids lacking a fusogenic envelope, seek (by selective VEGF receptor-mediated targeting) and destroy target cells by contact cytotoxicity in the absence of gene delivery, thereby establishing the prototype of a new breed of engineered viral medicines that are distinguishable from vaccines and classical gene therapy vectors.
  • the inventive targeted cytocidal virionoids have an advantage over retroviral vectors that have been used in the art to target angiogensis, in that they do not deliver exogenous genetic material into the target cell.
  • a "virionoid” is a non-infectious cytotoxic or cytocidal viral particle that has its envelope artificially modified so that it displays to a specific ligand or targeting peptide that binds to a cell surface receptor target and induces cytotoxicity and/or death without gene delivery to the target cell.
  • the native receptor binding moiety or domain of the viral particle is deleted and replaced with the specific targeting ligand.
  • a most preferred embodiment is a virionoid constructed from a retroviral vector, such as, but not limited to, a lentiviral vector.
  • inventive virionoids are constructed from adenoviral vectors, adeno-associated virus vectors, herpes virus vectors (e.g., herpes simplex virus-derived vectors, and pseudotyped viruses.
  • the inventive virionoid can have a core containing a complete native viral genome or an artificially modified genome (e.g., a recombinant nucleic acid construct).
  • inventive retroviral particles can be constructed employing known techniques, such that they need not even contain nucleic acid (i.e., empty capsids; e.g., Seung SY et al, The 17 nucleotides downstream from the env gene stop codon are important for murine leukemia virus packaging, J Virol 74:8775- 8780 [2000]).
  • nucleic acid i.e., empty capsids; e.g., Seung SY et al, The 17 nucleotides downstream from the env gene stop codon are important for murine leukemia virus packaging, J Virol 74:8775- 8780 [2000]).
  • VEGF peptide is an isoform, congener, or fragment of VEGF polypeptide that is at least about 110 contiguous amino acid residues long, or up to the length of a full length native VEGF polypeptide of interest.
  • a useful full-length human VEGF is 165 contiguous amino acid residues long.
  • the useful VEGF peptide is one that will bind the VEGF receptor of interest on the target cell.
  • a preferred embodiment is a human VEGF peptide including a VEGF receptor binding region, useful for binding human vascular endothelial cells, such as those comprised in the neovasculature of human malignant tumors.
  • a VEGF peptide can be preselected that binds the VEGF receptor of the mammalian species of interest, such as, but not limited to, a rodent, lagomorph, canine, feline, or non-human primate species.
  • a useful VEGF peptide can also contain one or more amino acid substitutions or deletions, as long as the VEGF peptide still binds specifically to a VEGF receptor of interest, which binding capacity can be determined by routine in vitro screening using cultured vascular endothelial cells originating from a mammalian species of interest.
  • the present invention also provides a producer cell for producing the inventive VEGF peptide- bearing non-infectious retroviral particle.
  • the present invention also provides a method of inhibiting angiogenesis in a mammal, including in a human, which is especially useful for targeting the vascular endothelial cells comprised in neovasculature of, for example, malignant tumors, or in proliferative vascularization which develops in retinopathies, such as diabetic retinopathy.
  • the virionoid is administered to a mammalian subject of a preselected (i.e., predetermined) mammalian species of interest, such as a human, the subject being in need of treatment for a tumor, retinopathy, or other lesion involving abnormal proliferative vascularization.
  • a useful virionoid employed in the method thus, comprises a VEGF peptide on its surface that specifically binds to a VEGF receptor of the preselected mammalian species.
  • a human VEGF peptide is incorporated into the inventive virionoid intended for administration to a human subject; a rabbit or rat VEGF peptide is incorporated into the virionoid intended for administration to a rabbit subject or rat subject, respectively.
  • virionoids comprising a human VEGF peptide can also be used in treating rodents (e.g., mice, rats, guinea pigs, and hamsters) and non-human primates, because their VEGF receptors also specifically bind human VEGF.
  • a particular embodiment of the inventive virionoids can be used in the method for a particular mammalian species, e.g., a virionoid comprising a human VEGF peptide for administering to an animal of any particular other mammalian species, can be determined by prior in vitro screening using appropriate vascular endothelial cells corresponding to the mammalian species of interest.
  • the virionoid is administered by any suitable means, for example by injection. Injection can be intrarterial, intravenous, intrathecal, intraocular, intramuscular, intraperitoneal, or by direct (e.g., stereotactic) injection into a tumor or other lesion.
  • the virionoid targets and specifically binds to vascular endothelial cells bearing the VEGF receptor.
  • an effective amount of the non-infectious viral particle inhibits the proliferation of new vasculature in a tumor or lesion in the subject compared to the rate of proliferation before treatment. This is determined by routine clinical means, and a clinical outcome such as inhibition of tumor growth can be used as a proxy in determining the effective amount.
  • the effective amount is about 1 x 10 7 to about 1 x 10 9 virionoid particles per kg body mass, more preferably about 4 x 10 to about 2 x 10 virionoid particles per kg body mass.
  • Figure 1A Human sequences of various lengths, including VEGF 1 10 ([SEQ ID NO:4], encoding human VEGF peptide having the first 110 amino acid residues [SEQ ID NO:5]), VEGF 121 ([SEQ ID NO:6], encoding human VEGF peptide having the first 121 amino acid residues [SEQ ID NO:7]), and VEGF165 ([SEQ ID NO:8], encoding complete human VEGF peptide [SEQ ID NO:9]), were generated by RT-PCR from a cDNA template, which also added the respective linkers and cloning sites as descriibed in Liu L et al., Incorporation of tumor vasculature targeting motifs (TVTMs) into ML V env escort proteins enhances retroviral binding and transduction of human endothelial cells, J Virol, 74:5320-5328 (2000]) (see also; Wu, BW et al, Characterization of the pro
  • Figure IB The PCR products were cloned in-frame into a strategically modified MLV-based envelope construct designated CEEC-BA as described in Liu et al. (2000) and Wu et al. (1998), to generate envelope escort proteins which were devoid of the ecotropic receptor binding domain.
  • Figure 1C VEGF165 was also cloned in-frame as N-terminal insertions into a 4070A amphotropic envelope construct designated CAEP-P that was modified to include a unique Pst 1 cloning site as described in Hall et al, Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor, Hum Gene Ther 11 :983-993 (2000).
  • VEGF 95 identifies nucleotide positions 1-15 of SEQ ID NO:8 (i.e., nucleotide positions 95-109 of SEQ ID NO:3)
  • VEGF 589 identifies nucleotide positions 481-495 of SEQ ID NO:8 (i.e., nucleotide positions 575-589 of SEQ ID NO:3)
  • VEGF 424" identifies nucleotide positions 313-330 of SEQ ID NO:4 (i.e., nucleotide positions 407-424 of SEQ ID NO:3).
  • the level of expression of the retroviral env protein gp70 and the gag protein p30 in 293T cell lysates of wild type CAE env protein, a chimeric env protein bearing a VEGF isoform (V110-BA; V121-BA, V165-BA; V110-CAEP; V165-CAEP; brackets) was evaluated by Western blotting with and without co-expression with WT CAE env (V110-BA+CAE; V121-BA+CAE; V165-BA+CAE; V110-CAEP+CAE; V165-CAEP+CAE; brackets).
  • Fig 3 Binding of retroviral vectors displaying VEGF/env constructs to KSYl Kaposi sarcoma cells.
  • ATCC human KSYl Kaposi sarcoma endothelial cells
  • Test vectors were prepared with ecotropic CEE (rodent-specific) envelope partners, which do not by themselves recognize/infect human cells.
  • Fig 4. Cytotoxicity of retroviral vectors bearing VEGF env escort constructs.
  • A:100X;B:400X Normal morphological appearance of KSYl cells 24 hrs after transduction with a retroviral vector bearing a chimeric VEGF env escort construct (VI 10-BA) co-expressed with WT CAE env.
  • C,E: 100X; D,F:400X Morphological appearance of disintegrating KSYl cells 24 hrs after transduction with retroviral vectors bearing only the VI 10-BA and V121-BA env escort constructs respectively.
  • G:200X Morphological appearance of normal HUVE cells 24 hrs after transduction with a retroviral vector bearing a chimeric VEGF env escort construct (VI 10-BA) co-expressed with WT CAE env;
  • H: 200X Morphological appearance of disintegrating HUVE cells 24 hrs after transduction with retroviral vectors bearing only the VI 10-BA env escort constructs.
  • Fig.5 Cytocidal activity of targeted VEGF virionoids in KSYl cells. 4 x 10 5 KSYl cells were transduced with a vector bearing either WT env or a virionoid bearing a VEGF congener. The cell count obtained 24 h after transduction, plotted on the vertical axis, is expressed as a function of type of VEGF congener, plotted on the horizontal axis.
  • B Inhibitory activity of targeted VEGF virionoids in KSYl cells. 3 x 10 4 KSYl cells were transduced with a vector bearing either WT env or a virionoid bearing a VEGF congener.
  • Fig.6 (A) Cytocidal activity of targeted VEGF virionoids in HUVE cells. 1.5 x 10 4 HUVE cells were transduced with a vector bearing either WT env or a virionoid bearing a VEGF congener. The cell count obtained 24 h after transduction, plotted on the vertical axis, is expressed as a function of type of VEGF congener, plotted on the horizontal axis.
  • Fig. 6B Inhibitory activity of targeted VEGF virionoids in HUVE cells.
  • VEGF coding sequence inserts with cohesive ends were cloned into the CEE+ ⁇ hinge (ecotropic)-envelope (env) construct (Wu, BW, Cannon PM, Gordon EM, Hall FL, Anderson WF, [1998] Characterization of the proline-rich region of murine leukemia virus envelope protein, J Virol 72: 5383-5391), designated CEEC, which was modified from CEE+ by substitution of a coding sequence for an amphotropic proline rich hinge region (PRR; SEQ ID ⁇ O:l; see Table 1A), such that at least 90% of the amino acid residues of the receptor binding region of the surface protein have been removed, and the hypervariable polyproline region of the ecotropic gp70 protein is replaced by the hypervariable polyproline region of the amphotropic gp70 protein (SEQ ID NO:2; see Table 2A), the coding sequence of which contains three unique restriction sites
  • the MLV-based env construct was cut with BstEII and Avrll and the linearized env plasmid was verified by restriction analysis on agarose gels and purified by the Gene Clean method (Bio 101, Vista, CA) prior to ligation with the respective VEGF insert and T4 DNA Ligase (New England Biolabs, Beverly, MA) for either 3 hours at room temperature or overnight at 4°C
  • the VEGF receptor binding domain flanked by glycine linkers replaced the entire receptor binding region of the MLV ecotropic env surface (SU) protein, between the BstEII site at the amino terminus and the Avrll site located proximal to the transmembrane (TM) domain.
  • Plasmid DNA was extracted from selected transformed clones using QIAprep Miniprep Kits (Qiagen, Valencia, CA). Each construct was confirmed by enzyme digestion and analysis of the respective inserts, followed by direct DNA sequence analysis.
  • nucleotide positions 1-592 of SEQ ID NO:3 correspond to nucleotide positions 41-632 of GenBank Accession M32977 (Human heparin-binding vascular endothelial growth factor [VEGF] coding sequence, Leung,D.W. et al, Vascular endothelial growth factor is a secreted angiogenic mitogen, Science 246:1306-1309 [1989]).
  • Nucleotide positions 17-94 of SEQ ID NO:3 i.e., SEQ ID NO: 10
  • SEQ ID NO:l l encode a VEGF signal peptide (SEQ ID NO:l l)(Table 7 below).
  • Retroviral vectors bearing wild-type (WT) env and/or VEGF- bearing "escort" proteins were assembled using a three-plasmid or four-plasmid transient transfection system as described in Soneoka, Y., P.M. Cannon, E.E. Ramsdale, J.C Griffiths, R. Gaetano, S. M. Kingsman, and A.J. Kingsman (1995) A transient three-plasmid expression system for the production of high titer retroviral vectors, Nucl. Acid Res. 23, 628-633), respectively, depending on whether or not a wild type (WT), amphotropic or ecotropic env protein was co-expressed.
  • WT wild-type
  • gag-pol The packaging components gag-pol, the WT env, the chimeric env, and a retroviral vector bearing a nuclear-targeted ⁇ - galactosidase expression construct were placed on separate plasmids, each containing the SV40 origin of replication. Ten ⁇ g of each plasmid were co-transfected by the calcium phosphate method into 293T cells, which express SV40 large T antigen. In particular, the plasmids used were:
  • pcgp (described and designated pHIT660 in Soneoka, Y., et al., A transient three-plasmid expression system for the production of high titer retroviral vectors, Nucl. Acid Res. 23, 628-633 [1995]; pcgp is a plasmid encoding viral gag-pol);
  • pCAE (described in Morgan RA et al, Analysis of the functional and host range- determining regions of the murine ecotropic and amphotropic retrovirus envelope proteins, J Virol 67:4712-4721 [1993]; pCAE is a plasmid containing a polynucleotide sequence encoding the wild type amphotropic gp70 env protein), or (ii) pCEE (described in MacKrell, AJ, et al, Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding, J Virol 70:1768-1774 [1996]; pCEE is a plasmid containing a polynucleotide sequence encoding the wild type ecotropic gp70 env protein), or (iii) pCEEC (described in Liu L et al., Incorporation of tumor vasculature targeting motifs (TVTMs) into MLV env
  • pESCORT (described in Liu, L et al, Incorporation of tumor vasculature targeting motifs (TVTMs) into MLV env escort proteins enhances retroviral binding and transduction of human endothelial cells, J Virol, 74:5320-5328 [2000]; a plasmid encoding the modified ecotropic env protein (CEEC) and a targeting peptide [in this case, human VEGF or VEGF receptor-binding fragments thereof]; and
  • pcnBg (a retroviral vector bearing a nucleus targeted ⁇ -galactosidase gene; designated pHITl 12 in Soneoka [1995]).
  • the producer cells were designated "293T” (deposited with ATCC [Manasas, VA] as SD-3515).
  • viral particles were purified from soluble proteins and cell debris on a 20% sucrose gradient (in PBS), and the virion-associated proteins were subjected to Western analysis using anti-gp70 and anti-p30 antibodies (Zhu et al., (1998) Mutational analysis of the fusion peptide of Moloney murine leukemia virus transmembrane protein pi 5E, J Virol 72:1632-1639). Determination of viral titers.
  • Retroviral vector- mediated gene transfer of antisense cyclin Gl inhibits proliferation of human osteogenic sarcoma cells, Cancer Research 55:5493-5498), as determined by light microscopy. Briefly, 2.5 x 10 4 NIH 3T3 cells were plated in each well of 6-well plates one day prior to transduction.
  • KSYl Kaposi sarcoma cells CRL-114478
  • HUVE human umbilical cord vascular endothelial cells CC-2517
  • ATCC American Type Cell Culture Collection
  • Clonetics San Diego, CA
  • 5 x 10 6 KSYl or HUVE cells were suspended in RPMI 1640 in a microcentrifuge tube, and were spun down for 15 sec, after which time 1 ml of test vector supernatant was added (viral titers were generally normalized to ⁇ 1 x 10 6 cfu/ml). The mixture was incubated with gentle shaking at room temperature for 30 min.
  • the cells were washed twice with D10 (DMEM+10%FBS) medium, and then resuspended in 300 ⁇ l in the presence of a rat monoclonal 83 A25 antibodies directed against the C-terminus of the gp70 MLV env protein (Evans, LH, Morrison FG, Malik J, Portis J and Brittt WJ (1990) A neutralizable epitope common to the envelope glycoprotein of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses, J Virol 64: 6176-6183) and incubated at room temperature for one hr.
  • D10 DMEM+10%FBS
  • A25 antibodies directed against the C-terminus of the gp70 MLV env protein (Evans, LH, Morrison FG, Malik J, Portis J and Brittt WJ (1990) A neutralizable epitope common to the envelope glycoprotein of ecotropic, polytropic, xenotropic, and amphotropic
  • the cells were again washed twice with D10 medium, and then incubated in 500 ⁇ l 1 :2500 HRP-goat anti-rat IgG (Zymed Laboratories Inc.) at RT for 30 min.
  • the cells were washed and then incubated in 500 ⁇ l 1 :1000 rat peroxidase anti- peroxidase antibody (Stemberger Monoclonals, Inc.) at room temperature for 30 min.
  • the cells were resuspended in 100 ⁇ l TMB single solution (Zymed Laboratories Inc.), and transferred to a 96-well ELISA plate, where the intensity of the color reaction (blue) was read at OD650 nm on a Rainbow Spectra ELISA reader (TECAN US, Inc., NC). Transduction of human endothelial cells.
  • KSYl or HUNE cells were cultured on 1% gelatin-coated dishes in RPMI 1640 supplemented with either 2% (KSYl) or 10% (HUVE) fetal calf serum, 1% sodium pyruvate, 1% essential amino acids, 1% non-essential amino acids, 1 mM glutamine, and 1% penicillin-streptomycin.
  • KSYl 2%
  • HUVE fetal calf serum
  • sodium pyruvate fetal calf serum
  • essential amino acids fetal calf serum
  • 1% non-essential amino acids 1 mM glutamine
  • penicillin-streptomycin penicillin-streptomycin.
  • 2 x 10 4 KSYl or 1.5 x 10 4 HUVE cells in 3 ml RPMI-2% (2%FBS in RPMI 1640) were plated into each gelatin-coated well in a 6-well plate, and allowed to attach overnight at 37°C. The following morning, medium was replaced
  • the cultures were transduced with 1 ml of each test vector supernatant normalized for equivalent viral titers in the presence of polybrene (8 ⁇ g/ml) at 37 D C for 30 minutes. Thereafter, 2.5 ml fresh RPMI-2% was added to the cultures which were incubated overnight at 37 C C Medium was then replaced with fresh medium, and the cultures were further incubated at 37 ⁇ C for another 24 hrs. The cells were then stained with X-Gal to visualize the presence of nuclear- targeted ⁇ -galactosidase activity under light microscopy. To quantify the resulting transduction efficiency, the number of ⁇ -galactosidase positive cells (cells with blue-staining nuclei) was divided by the total cell number per well and expressed as % transduction efficiency.
  • Cytotoxicity Assays A standard assay for inhibitory activity in transduced KSYl or HUVE cells was conducted as previously described ( Skotzko et al., (1995) Retroviral vector-mediated gene transfer of antisense cyclin Gl (CYCG1) inhibits proliferation of human osteogenic sarcoma cells, Cancer Research 55:5493-5498). Briefly, either 3 x 10 4 KSYl cells or 1.5 x 10 4 HUVE cells were exposed to 1 ml of the test vector or medium as control in the presence of Polybrene (8 ⁇ g/ml) for 2 h, with periodic rocking.
  • the transduced cells were evaluated for their proliferative potential by counting the number of viable cells in triplicate cultures at 24 h after transduction without G418 selection. Cytocidal activity was verified by a comparative decrease in cell number in the test vector-treated cultures compared to control vector- or control medium-treated cultures. The mean cell number in test vector- treated cultures at 24 h was then compared to that of control medium-treated cultures and expressed as % inhibitory activity, using the following formula:
  • % inhibitory activity # of cells (medium-treated cultures ' ) - # of cells (test vector- treated cultures ' ) X 100
  • VEGF-bearing envelope constructs Five distinct VEGF-bearing envelope constructs were selected for comparative evaluation (see Figure 1). Upon transient transfection, all of the 5 envelope proteins were expressed well in human 293T retroviral vector producer cells, each exhibiting an apparent molecular mass of about 70 to 80 kDa. As seen in Figure 2 (panel A), the expression of the envelope "escort" proteins was not impaired by co-transfection and co-expression of wild-type envelope proteins, which confer vector tropism and infectivity (i.e., ecotropic, CEE or amphotropic, CAE). Each of the VEGF "escort" proteins could be detected in purified viral particles, however, notable differences in incorporation efficiencies were observed (see Figure 2, panel B).
  • VEGF 165 the largest VEGF isoform
  • VEGF 165 the largest VEGF isoform
  • the co- expression of a wild-type CAE envelope was seen to facilitate the incorporation of the modified "escort" protein, presumably due to structural complementation of the tertiary structures (Zhao, Y, Lee S and Anderson WF, (1997) Functional interactions between monomers of the retroviral envelope protein complex, J Virol 71:6967-6972, Anderson, et al., (1998) Human Gene Therapy, Nature (Suppl) 392, 25-30).
  • VEGF insertions into CAEP yielded viral particles of varying infectivity, whereby the particles bearing the smaller congener (VEGFl lO) had markedly reduced infectivity while the larger VEGF isoform (VEGF 165) was moderately to highly infectious (Table 1).
  • KSYl Kaposi sarcoma endothelial cells which exhibit a constitutive (autocrine) expression of both VEGF ligand and VEGF-receptors (Masood, R, Cai J, Zheng T, Smith DL, Naidu Y and Gill PS (1997) Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma, Proc Nat'l Acad Sci USA 94:979-984), and compared the cell binding ability of the "escort" constructs with that of both the amphotropic (CAE) and ecotropic (CEE) envelopes, the latter of which does not infect human cells.
  • CAE amphotropic
  • CEE ecotropic
  • each of the VEGF-bearing escort constructs exhibited a striking gain of cell binding function.
  • the VEGF-bearing insert constructs also exhibited pronounced cell binding affinities, which was greater than that of the CAE (amphotropic) envelope-bearing vectors (data not shown).
  • test vector supernatants were prepared for cellular transduction studies ⁇ with the exception that an amphotropic e «v partner (CAE) was utilized to enable the transfection of human cells — and then normalized for equivalency of titer, based on the transduction of NIH 3T3 cells (see Table 1).
  • CAE amphotropic e «v partner
  • single enveloped vectors bearing VEGF-110 and VEGF-121 but not VEGF-165 "escort proteins" demonstrated cytocidal activity in KSYl cells (p ⁇ 0.001 and 0.005 respectively), with the VEGF-110 vector exhibiting greater inhibitory activity (67.3 + S.D. 0.94%) than the VEGF-121 vector (48.7 ⁇ 12.28%; p ⁇ 0.05; Figure 5B).
  • VEGF-bearing vector particles bind to endothelial cells via NEGF receptor-mediated mechanisms, and that in the absence of a fusogenic envelope, this physical contact without internalization of the viral particles is inherently cytotoxic.
  • the present invention is not dependent on any particular mechanism, the observed contact cytotoxicity could be due to interference with normal NEGF signal transduction events or by disturbance of membrane stability.
  • Growth factor and/or adhesion receptors that are selectively expressed on surfaces of activated endothelial cells provide an advantageous locus for targeting drugs and gene therapy vectors to angiogenic tissues.
  • Previous studies identified a series of fibronectin-derived ⁇ GR-bearing congeners that served to enhance the transduction of KSYl cells in vitro (Liu et al., (2000) Incorporation of tumor vasculature targeting motifs (TVTMs) into ML V env escort proteins enhances retroviral binding and transduction of human endothelial cells, J Nirol, 74:5320-5328).
  • TVTMs tumor vasculature targeting motifs
  • VEGF receptor-binding ligands when expressed in the context of MLV envelope proteins enhance both endothelial cell binding and endothelial cell transduction.
  • the single enveloped vectors incorporating all VEGF congeners as escort proteins exhibited profound cytotoxicity in HUVE cell cultures and varying degrees of cytotoxicity in KSYl cell cultures (see Figures 4-6), while no overt cytotoxicity was noted in murine ⁇ IH3T3 cells.
  • VEGF110-CAEP non- infectious VEGF/env insert construct
  • VEGF165-CAEP infectious VEGF/env insert construct
  • cytocidal virionoids which exhibit cell-specific selectivity, which function in the absence of gene delivery, and which may exemplify a novel viral-based targeting approach to antiangiogenesis. Therefore, whether the clinical objective is to deliver a therapeutic (cytocidal) construct to tumor vasculature by VEGF-directed retroviral particles or to eliminate the tumor vasculature by contact cytotoxicity (see Figure 4), the present study provides the first proofs of principle that this approach is indeed feasible.
  • the cytocidal virionoids of the present invention can be aimed at translating the cell specific targeting properties of these VEGF-bearing retroviral vectors to effective inhibition of tumorigenesis.
  • VEGF-bearing peptide congeners presented in the context of MLV env "escort" proteins, including strategic linkers and cloning sites, and were determined to be suitable for protein expression, retroviral vector production, and cell-binding kinetics.
  • These VEGF-bearing env "escort” proteins were further demonstrated to function as targeting elements which serve to increase the cell binding affinity and transduction efficiency of the chimeric retroviral vectors, illustrating a potential utility for improving gene delivery in therapeutic angiogenesis and or antiangiogenesis/anticancer strategies.
  • MOLECULE TYPE polynucleotide
  • NAME/KEY polynucleotide encoding hypervariable polyproline region of amphotropic gp 70 protein
  • Nucleotide positions 1-592 of SEQ ID NO:3. Includes coding sequence of VEGF signal peptide (nt. positions 17-94), VEGF 165 (nt. positions 95-589), and "tga" stop codon at nt. positions 590-592).
  • VEGF 110 coding sequence SEQ DO NO:4 and peptide (SEQ ID NO:5) gca ccc atg gca gaa gga gga ggg cag aat cat cac gaa gtg gtg aag 48 Ala Pro Met Ala Glu Gly Gly Gly Gin Asn His His His Glu Val Val Lys 1 5 10 15 ttc atg gat gtc tat cag cgc age tac tgc cat cca ate gag ace ctg 96 Phe Met Asp Val Tyr Gin Arg Ser Tyr Cys His Pro lie Glu Thr Leu 20 25 30 gtg gac ate ttc cag gag tac cet gat gag ate gag tac ate ttc aag 144 Val Asp lie Phe Gin Glu Tyr Pro Asp Glu lie Glu Tyr lie Phe Lys 35 40 45 cca tec tgt gtg ccc ctg atg atg
  • VEGF 165 coding sequence SEQ ID NO:8 and peptide (SEQ ID NO:9) gca ccc atg gca gaa gga gga ggg cag aat cat cac gaa gtg gtg aag 48 Ala Pro Met Ala Glu Gly Gly Gly Gin Asn His His His Glu Val Val Lys 1 5 10 15 ttc atg gat gtc tat cag cgc age tac tgc cat cca ate gag ace ctg 96 Phe Met Asp Val Tyr Gin Arg Ser Tyr Cys His Pro lie Glu Thr Leu 20 25 30 gtg gac ' ate ttc cag gag tac cet gat gag ate gag tac ate ttc aag 144 Val Asp lie Phe Gin Glu Tyr Pro Asp Glu lie Glu Tyr lie Phe Lys 35 40 45 cca tec tgt gtg ccc ctg
  • Coding sequence (SEQ ID NO: 10)for VEGF signal peptide (SEQ ID NO: 1 1) atg aac ttt ctg ctg tct tgg gtg cat tgg age etc gcc ttg ctg etc 48

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Abstract

La présente invention a trait à une particule rétrovirale non infectieuse porteur d'un peptide VEGF artificiel. La particule virale est privée d'enveloppe fusogénique et est capable de liaison par l'intermédiaire du récepteur de VEGF aux cellules endothéliales d'une espèce présélectionnée. La présente invention a trait également à un procédé d'inhibition sélective d'angiogenèse tumorale chez un sujet mammalien. Le procédé comprend l'administration au sujet d'une quantité efficace d'un peptide VEGF qui se lie de manière spécifique à un récepteur de VEGF d'une espèce mammalienne présélectionnée. La particule se lie de manière sélective à une cellule endothéliale vasculaire du sujet porteuse d'un récepteur de VEGF sans délivrance de matériau génétique à partir de la particule. La liaison est cytotoxique à la cellule et produit une inhibition sélective d'angiogenèse dans un tumeur chez le sujet.
PCT/US2003/011142 2002-04-11 2003-04-11 Virionoides cytocides inhibiteurs d'angionenese WO2003087329A2 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044938A1 (fr) * 1997-04-10 1998-10-15 University Of Southern California Proteines modifiees se fixant a des composants de matrice extracellulaires
WO1999055893A1 (fr) * 1998-04-29 1999-11-04 University Of Southern California Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee
US6037329A (en) * 1994-03-15 2000-03-14 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6037329A (en) * 1994-03-15 2000-03-14 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
WO1998044938A1 (fr) * 1997-04-10 1998-10-15 University Of Southern California Proteines modifiees se fixant a des composants de matrice extracellulaires
WO1999055893A1 (fr) * 1998-04-29 1999-11-04 University Of Southern California Vecteurs retroviraux incluant des proteines d'escorte a enveloppe modifiee

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