EP0854929A1 - Virus recombines comprenant une proteine pouvant etre clivee par une protease - Google Patents

Virus recombines comprenant une proteine pouvant etre clivee par une protease

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Publication number
EP0854929A1
EP0854929A1 EP96931908A EP96931908A EP0854929A1 EP 0854929 A1 EP0854929 A1 EP 0854929A1 EP 96931908 A EP96931908 A EP 96931908A EP 96931908 A EP96931908 A EP 96931908A EP 0854929 A1 EP0854929 A1 EP 0854929A1
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Prior art keywords
viral
protease
heterologous polypeptide
particle
cells
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EP96931908A
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German (de)
English (en)
Inventor
Stephen James Russell
François-Loic COSSET
Frances Joanne Morling
Bo Harald Kurt Nilson
Kah - Whye Peng
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Medical Research Council
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Medical Research Council
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Priority claimed from GBGB9519691.1A external-priority patent/GB9519691D0/en
Priority claimed from GBGB9523225.2A external-priority patent/GB9523225D0/en
Priority claimed from GBGB9604562.0A external-priority patent/GB9604562D0/en
Application filed by Medical Research Council filed Critical Medical Research Council
Publication of EP0854929A1 publication Critical patent/EP0854929A1/fr
Withdrawn legal-status Critical Current

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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A—HUMAN NECESSITIES
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    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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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

Definitions

  • the invention relates to recombinant viral particles incorporating protease cleavable proteins and to various applications of the recombinant particles.
  • Retroviral envelope glycoproteins mediate specific viral attachment to cell surface receptors and subsequently trigger fusion between the viral envelope and the target cell membrane.
  • All retroviral envelope spike glycoproteins examined to date are homooligomers containing two to four heterodimeric subunits (Doms et al. 1993 Virology 193, 545). Each subunit comprises a large extraviral glycoprotein moiety (SU) noncovalently attached at its C-terminus to a smaller transmembrane polypeptide (TM) that anchors the complex in the viral membrane.
  • SU extraviral glycoprotein moiety
  • TM transmembrane polypeptide
  • SU comprises two domains connected by a proline-rich hinge, the N-terminal domain conferring receptor specificity and exhibiting a high degree of conservation between murine leukemia viruses (MLVs) with different host ranges (Battini et al. 1992 J. Virol 66, 1468-1475).
  • MLVs murine leukemia viruses
  • Moloney MLV envelopes confer an ecotropic host range because they attach selectively to a peptide loop in the murine cationic amino acid transporter (CAT-1), found only on cells of mouse and rat origin (Albritton et al. 1989 Cell 57, 659-666).
  • 4070A MLV envelopes attach to an epitope on the ubiquitous RAM-1 phosphate symporter that is conserved throughout many mammalian species, and confer an amphotropic host range (Miller et al. PNAS 91 , 78-82; VanZeijl et al. 1994 PNAS 91, 1168-1172).
  • retroviral vectors with 4070A envelopes infect human cells promiscuously, whereas vectors with Moloney envelopes fail to infect human cells.
  • the SU and TM polypeptides are derived from a single chain precursor glycoprotein that undergoes proteolytic maturation in the Gogi compartment during its transport to the cell surface.
  • Uncleaved envelope precursor glycoproteins can be incorporated into viruses but are unable to trigger membrane fusion.
  • the requirement for proteolytic maturation/activation is a feature common to the fusogenic membrane glycoproteins of many virus families and is most commonly mediated by the ubiquitous Golgi compartment serine protease, furin.
  • a general method has been disclosed which allows the display of a (glyco)polypeptide on the surface of a retroviral vector as a genetically encoded extension of the SU glycoprotein (WO 94/06920, Medical Research Council).
  • the polypeptide is fused (by genetic engineering) to the N-terminal part of the SU glycoprotein such that the envelope protein to which it has been grafted remains substantially intact and the fused nonviral polypeptide ligand is displayed on the viral surface.
  • a virus displaying such a chimaeric envelope protein might be capable of multivalent attachment both to the natural virus receptor (via the N-terminal domain of SU) and to the cognate receptor for the displayed polypeptide.
  • EGF epidermal growth factor
  • the displayed polypeptide may sterically hinder the interaction between the N-terminal domain of SU and the natural virus receptor.
  • ecotropic and amphotropic vectors displaying EGF could bind to EGF receptors but were thereafter sequestered into a non-infectious entry pathway, giving greatly reduced titres on EGF receptor-positive cells, but normal titres on EGF receptor-negative cells.
  • EGF receptor-negative cells which were fully susceptible to the engineered retroviral vector, showed reduced susceptibility when they were genetically modified to express EGF receptors. The reduction in susceptibility was in proportion to the level of EGF receptor expression.
  • soluble EGF was added to competitively inhibit virus capture by the EGF receptors, gene transfer was restored.
  • the engineered vector is capable of binding to the natural virus receptor or to the receptor for EGF; attachment to the natural virus receptor leads to infection of the target cell, whereas the attachment to the EGF receptor does not lead to infection of the target cell.
  • the two binding reactions (4070A envelope protein to RAM-1, and EGF to EGF receptor) proceed in competition and the infectivity of the virus for the target cells is reduced in proportion to the efficiency with which the EGF-EGF receptor binding reaction competes virus away from RAM-1.
  • the degree to which gene transfer can be inhibited by this mechanism depends on the relative affinities of the two binding reactions (envelope protein to natural receptor and non-viral ligand to its cognate receptor), the relative densities of the two receptors on the target cell surface, and the relative densities of the nonviral ligand and the intact envelope protein on the viral surface. Inhibition of gene transfer is additionally influenced by intrinsic properties of the receptor for the non-viral ligand, such as the distance it projects from the target cell membrane, its mobility within the target cell membrane and its half life on the cell surface after engagement of ligand.
  • chimaeric envelopes displaying the N-terminal domain from 4070A MLV SU as an N-terminal extension of Moloney MLV SU can apparently bind to RAM-1 (the receptor for 4070A SU) but not to ecoR (the receptor for Moloney SU); it may be possible that the displayed domains from 4070A SU may form a trimeric cap over the Moloney SU trimer, completely masking its receptor binding sites.
  • the invention provides a recombinant viral particle capable of infecting a eukaryotic cell, the viral particle comprising: a substantially intact viral glycoprotein fused, via an intervening linker region, to a heterologous polypeptide displayed on the surface of the particle, which heterologous polypeptide modulates the ability of the viral panicle to infect one or more eukaryotic cell types and is cleavable from the viral glycoprotein by a protease acting selectively on a specific protease cleavage site present in the linker region, such that cleavage of the heterologous polypeptide from the viral glycoprotein allows the glycoprotein to interact normally with its cognate receptor on the surface of a target cell.
  • Such a panicle is of considerable benefit in the targeted delivery of nucleic acid sequences, which may be present within the panicle, to specific desired target cells, such as is required for gene therapy.
  • the invention provides a nucleic acid construct, comprising a sequence encoding a fusion protein, the fusion comprising a substantially intact viral glycoprotein fused, via an intervening linker region, to a heterologous polypeptide, wherein the fusion protein is capable of being incorporated into a viral particle capable of infecting an eukaryotic cell, and further wherein the heterologous polypeptide modulates the ability of the viral particle to infect one or more eukaryotic cell types, but cleavage of the heterologous polypeptide from the fusion protein allows the viral glycoprotein to interact normally with its cognate receptor on the surface of the eukaryotic cell.
  • the invention provides a nucleic acid sequence library comprising a plurality of the nucleic acid constructs defined above, wherein at least part of the sequence encoding the intervening linker region is randomised in each construct, such that each construct comprises one of a plurality of different linker regions which are represented in the library.
  • the invention also provides a library of the viral panicles defined above, each panicle comprising a single nucleic acid construct from the nucleic acid library defined above.
  • substantially intact as used herein is intended to refer to a viral glycoprotein which retains all of its domains so as to conserve post-translational processing, oligomerisation (if any), viral incorporation and fusogenic properties.
  • certain alterations e.g. point mutations, deletions, additions
  • the gluycoprotein may lack a few (e.g. about 1 to 10) amino acid residues, especially at the N terminus, but will otherwise be generally the same size as the wild-type protein and possess substantially the same biological properties as the wild-type protein.
  • the intervening linker region will preferably be quite short, typically comprising from 4 to 30 amino acid residues, more typically 5 to 10 residues.
  • a short linker is preferred, because this will tend to maximise the modulation of infection effected by the heterologous polypeptide.
  • a suitable linker region may be present as a natural part of the heterologous displayed polypeptide.
  • the viral particle may be any virus capable of infecting one or more eukaryotic cell types, but conveniently will be a viral particle suitable for use in gene therapy, such as an adenovirus or a retrovirus (especially a C-type retrovirus).
  • the viral glycoprotein will typically comprise a viral envelope glycoprotein, or may be a chimeric polypeptide comprising sequences conesponding to different viral glycoproteins but which, in total, consitute a substantially intact, functional protein.
  • the heterologous polypeptide may be a short amino acid sequence (say, a peptide of about 10-20 residues, especially if the sequence undergoes oligomerisation, e.g. a leucine zipper peptide sequence) but more typically will comprise 30 or more amino acid residues. Generally, but not essentially, the polypeptide will comprise a functional binding domain.
  • the heterologous polypeptide when fused to the viral glycoprotein via the linker region, modulates the ability of the viral particle to infect one or more eukaryotic cell types. Specifically, the presence of the heterologous polypeptide serves to inhibit the process of infection of a eukaryotic target cell mediated by the viral glycoprotein.
  • heterologous is intended to refer to any polypeptide which is not naturally fused or otherwise bound to the viral glycoprotein.
  • the heterologous polypeptide may or may not possess specific binding affinity for a surface component of a target cell.
  • the heterologous polypeptide has affinity for a cell surface component, binding to which will not lead to infection of the cell by the virus.
  • a variety of different examples can be envisaged.
  • a eukaryotic cell expresses a receptor for the viral glycoprotein (binding to which allows the virus to infect the cell) and a non-permissive receptor for the heterologous polypeptide, with inhibition of infection resulting simply from competition between the viral glycoprotein and the heterologous polypeptide for binding to their respective receptors on the target cell.
  • the conformational arrangement of the respective receptors and their ligands is such that binding of the heterologous polypeptide to its receptor causes steric hindrance, such that binding of the viral glycoprotein to its receptor, or fusion of the virus and the cell, is blocked.
  • the heterologous polypeptide does not bind to a non-permissive receptor on the target cell, but the presence of the heterologous polypeptide serves to create steric hindrance sufficient to prevent binding of the viral glycoprotein to its receptor, or may allow binding to occur but inhibits subsequent fusion of the viral particle with the target cell, such that infection of the cell by the viral particle is inhibited at the binding and/or fusion stage.
  • the heterologous polypeptide is capable of forming oligomers when displayed on the surface of the viral particle.
  • the oligomer will be a dimer or, more preferably, a trimer.
  • Such oligomerisation may allow for efficient inhibition of the interaction between the substantially intact viral glycoprotein and its receptor, which inhibition may be removed by proteolytic cleavage of the oligomerised heterologous polypeptide from the viral glycoprotein.
  • the intervening linker may also undergo oligomerisation.
  • the heterologous polypeptide oligomerises with the same stoichiometry as that of the viral glycoprotein.
  • Vascular endothelial growth factor (VEGF) and rumour necrosis factor (TNF) are both proteins which are known to oligomerise and have high affinity for cell surface ligands. Effective (oligomer-forming, and preferably ligand-binding) portions of these proteins may be particularly suitable for use as heterologous polypeptides in accordance with the present invention.
  • the heterologous polypeptide is cleavable from the viral glycoprotein by the selective action of a protease (i.e. a molecule capable of cleaving a peptide bond) which cleaves the linker region at a protease cleavage site.
  • the cleavage site represents a unique peptide sequence not present, or at least not accessible to the protease, in the viral glycoprotein, although a similar site may be present in the heterologous polypeptide (this is generally preferably avoided, as proteolytic attack on the heterologous polypeptide may affect its functioning).
  • the size, and number, of the protease cleavage sites in the linker region may be varied with advantage. Thus, for example, the presence of two or more cleavage sites, recognised by the same or by respective proteases could facilitate cleavage, whilst the use of one long cleavage site will tend to enhance specificity of cleavage.
  • proteases are involved in a number of physiological and/or pathological processes, such as tissue remodelling, wound healing, inflammation and tumour invasion, and such proteases would be of use in the present invention.
  • Specific classes of protease which would be of use include: serine proteases (such as plasminogen/plasmin enzymes); cysteine proteases; and matrix metalloproteinases (MMPs) of various types, (such as Gelatinase A and membrane-type MMP [or MT-MMP]).
  • the protease which serves to cleave the heterologous polypeptide from the viral glycoprotein is preferably selectively secreted by the cell to which it is desired to target the viral particle, or at least the tissue in which the target cell is located. It is preferred that the protease will be secreted only by cells of the target cell type or, less preferably, only by cells (other than the target cells) remote from the tissue containing the target cell. This confers an extra degree of specificity, which is desirable when the particle is used for targeted gene delivery.
  • the present invention allows for two-step targeting, in which a first level of specificity may be imposed by the heterologous polypeptide (e.g.
  • a second level of specificity may be imposed by selective cleavage of the heterologous polypeptide by proteases secreted by, or in the same tissue as, the target cell.
  • the relevant protease may be added exogenously, such that if the viral particle is used for targeted gene delivery in a patient, the protease may be administered (e.g. by injection) to the tissue in which the target cell is located.
  • Accessibility of the protease cleavage site to the relevant protease may also be varied. It has been found by the present inventors that use of a short intervening linker region (e.g. 5 amino acid residues) tends to restrict accessibility of the cleavage site, and use of a larger linker region (e.g. 15 to 20 residues) tends to increase accessibility of the cleavage site. This phenomenon is presumably due to seric hindrance of the cleavage site due to the proximity of the viral glycoprotein and/or the heterologous polypeptide. Accordingly, it should also be possible to modify accessibility of the cleavage site, as desired, by varying the size of the heterologous polypeptide.
  • a short intervening linker region e.g. 5 amino acid residues
  • a larger linker region e.g. 15 to 20 residues
  • the cleavage site is accessible to the relevant protease before the viral particle becomes bound to an eukaryotic cell, whilst in an alternative embodiment the cleavage site is inaccessible to the protease until the viral particle has become bound to a eukaryotic cell.
  • the cleavage site may be made accessible by a conformational change occurring as a result of binding of the heterologous polypeptide to its cognate receptor.
  • the viral glycoprotein binding to its cognate receptor may make the cleavage site accessible, cleavage of the heterologous polypeptide then allowing fusion of the viral particle to the eukaryotic target cell.
  • the invention provides for a method of selectively delivering a nucleic acid to a target eukaryotic cell present among non-target cells, comprising: administering to the target and non-target cells a recombinant viral particle capable of infecting eukaryotic cells, the particle comprising the nucleic acid to be delivered, and a fusion protein comprising a substantially intact viral glycoprotein fused, via an intervening linker region, to a heterologous polypeptide displayed on the surface of the particle, which heterologous polypeptide modulates the ability of the particle to infect one or more eukaryotic cell types and being cleavable from the glycoprotein by a protease acting selectively on a specific protease cleavage site present in the linker region, such that cleavage of the heterologous polypeptide from the glycoprotein occurs preferentially at, or in the vicinity of, the target cell and allows the viral glycoprotein to interact normally with its cognate receptor on the surface of the target cell.
  • the method may be performed in vitro, for example to deliver a lethal nucleic acid to fibroblasts in tissue culture, which cells often outgrow a slower-growing, more differentiated cell type in culture.
  • the method may be performed as a method of gene therapy, in vivo or may be performed ex vivo, on cells which are then re-introduced into a human or animal subject.
  • Preferential cleavage of the protease cleavage site may occur only when the viral particle is bound to the target cell, or when the viral particle is adjacent to the target cell and thus exposed to a protease secreted by the target cell.
  • protease may well be preferred to add the relevant protease exogenously, after administration of the viral particle, so as to ensure sufficient concentration of the protease and as another aid to specificity of delivery (by local administration of the protease). It is already known that some proteases may be safely given in vivo (e.g. those enzymes, such as urokinase, streptokinase and tPA, given to patients with myocardial infarcts).
  • those enzymes such as urokinase, streptokinase and tPA, given to patients with myocardial infarcts.
  • the invention also provides, in a further aspect, a method of screening nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease.
  • a method of screening nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease.
  • many viral envelope glycoproteins are processed through the cellular export pathway of the eukaryotic cell in which they are synthesised, generally leading to cleavage, which cleavge is essential for production of an infectious viral particle.
  • the invention therefore provides a method of screening nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease present in the export pathway of an eukaryotic cell, comprising: causing the expression of a plurality of nucleic acid sequences in eukaryotic cells, each sequence encoding a substantially intact viral glycoprotein fused to a heterologous polypeptide via a randomised intervening linker region, the presence of the heterologous polypeptide serving to inhibit the (binding or fusion) interaction of the viral glycoprotein with its cognate receptor, and wherein each nucleic acid sequence further comprises a packaging signal allowing for viral incorporation, such that those intervening linkers which are recognised by a protease present in the export pathway of the eukaryotic cells will allow for cleavage of the heterologous polypeptide from the viral glycoprotein, resulting in the production of an infectious viral particle; and recovering those nucleic acid sequences directing the expression of such cleavable link
  • Nucleic acid sequence determination may optionally be performed, to deduce those amino acid sequences which are recognised by an export protease.
  • a modification of the above method will allow for the screening of nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease present in the eukaryotic cell import pathway.
  • the presence of a heterologous polypeptide may, in some embodiments, still allow for binding of the viral glycoprotein to its cognate receptor, but will prevent fusion of the viral particle with the eukaryotic cell to which it is bound. Cleavage of the heterologous polypeptide by a protease in the cellular import pathway will then allow infection of the cell.
  • the invention provides for a method of screening nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease, comprising: causing the expression of a plurality of nucleic acid sequences in eukaryotic cells, each sequence encoding a substantially intact viral glycoprotein fused to a heterologous polypeptide via a randomised intervening linker region, the presence of the heterologous polypeptide serving to inhibit the fusion of a viral particle with a eukaryotic cell to which it is bound, and wherein each nucleic acid sequence further comprises a packaging signal allowing for viral incorporation; enriching the viral particles so produced for those which retain the heterologous polypeptide (and so are non-infectious); and contacting the enriched particles with a susceptible eukaryotic cell comprising, or in the presence of, a protease such that those intervening linkers which are recognised by the protease will allow for cleavage of the heterolog
  • the enrichment step is required because of the possibility that the heterologous polypeptide may be cleaved from the viral glycoprotein by an export pathway protease during synthesis of the panicles.
  • a number of possible enrichment techniques will be readily apparennt to those skiled in the an with the benefit of the present teaching.
  • the viral particles prior to infection of the susceptible cells, the viral particles could be subjected to an affinity enrichment technique - the particles could be passed through an antibody affinity column, wherein the antibody has affinity for the heterologous polypeptide.
  • Those particles which retain the heterologous polypeptide will be bound to the column, whilst those in which the heterologous polypeptide was cleaved during export from the producing cell will pass straight through the column.
  • the bound particles may be eluted (e.g. by competition with free heterologous polypeptide, or the part thereof recognised by the antibody, or by alteration of pH or other factors) and then used to infect the susceptible "indicator" cells.
  • Figure 1 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figures 2 and 3 are photographs of Western blots demonstrating viral incorporation of certain chimeric polypeptides and their sensitivity to Factor Xa protease;
  • Figure 4 is a photograph showing the infectivity of various ⁇ -galactosidase transducing viruses on target cells with or without Factor xa treatment, as judged by assay on X-gal containing plates;
  • Figure 5 is a schematic representation of how two-step targeting of gene delivery might be achieved using the present invention.
  • Figure 6A is a photograph of a Western blot demonstrating viral incorporation of certain chimeric polypeptides and their sensitivity to Factor Xa protease;
  • Figure 6B is a bar chart illustrating the infectivity of certain recombinant viruses in the presence or absence of Factor Xa;
  • Figure 7 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figure 8A is a photograph of two Western blots, the upper one comparing electrophoretic mobility of various chimeric polypeptides, the lower one comparing the amount of protein present;
  • Figure 8B is a photograph of a Western blot comparing the sensitivity to Factor Xa protease of various chimeric polypeptides
  • Figure 8C is a photograph of a Western blot comparing processing of certain chimeric polypeptides
  • Figure 9 is a panel of photographs comparing the growth of of a recombinant virus on NIH 3T3 and A431 cells, with or without Factor Xa treatment;
  • Figure 10 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figure 11 shows three Tables, A, B and C, illustrating the titre (in enzyme forming units, "e.f.u. ") of various recombinant viruses on NIH 3T3 or A431 cells in the absence (-) or presence ( +) of Factor Xa protease;
  • Figure 12 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figure 13 A is a photograph of a Western blot demonstrating viral incorporation of various chimeric polypeptides
  • Figure 13B is a photograph of a Western blot comparing the sensitivity of various chimeric polypeptides in the presence (+) or absence (-) of pro-gelatinase A, with (+) or without (-) pre-activation of the protease by p-aminopheny .mercuric acetate (APMA);
  • Figure 14 is a bar chart showing how infectivity of a recombinant virus is dependent upon concentration of pro-gelatinase A;
  • Figure 15 is a bar chart comparing the infectivity of three different recombinant viruses on HT 1080 or A431 cells;
  • Figure 15 A is a photograph comparing the growth of a recombinant virus on HT 1080 or A431 cells;
  • Figure 16 is a panel of four photographs (I, II, III and IV) comparing the infectivity of various viruses on HT 1080 (H) or A431 (A) cells;
  • Figure 17 is a photograph of a gel for detection of gelatinolytic activity.
  • Figures 18 and 19 are schematic representations of retroviral vector constructs coding for chimeric envelopes.
  • Tropism-modifying binding domains were anchored to murine leukaemia virus (MLV) envelopes via factor Xa-cleavable linkers to generate retroviral vectors whose tropism could be regulated by factor Xa protease.
  • the binding domains could not be cleaved from vector particles by factor Xa when the linker was fused to amino acid +7 of Moloney MLV SU but could be efficiently cleaved when fused to amino acid + 1 of Moloney or 4070 A MLV SU glycoproteins.
  • Vectors displaying a cleavable EGF domain were selectively sequestered on EGF receptor-expressing cells, but their infectivity was fully restored when the EGF domain was cleaved from the vector particles with factor Xa. Partial restoration of infectivity was observed when only a fraction of the envelope proteins were cleaved. Conversely, vectors that displayed a cleavable RAM-1 binding domain fused to Moloney MLV SU had an expanded host range that was reversible upon treatment with factor Xa. It is suggested that retroviral vectors with engineered binding specificities whose tropism is regulated by exposure to specific proteases may facilitate novel strategies for targeting retroviral gene delivery.
  • MLV-derived retroviral vectors are versatile gene delivery vehicles whose host range can be varied by incorporation of different envelope spike glycoproteins (Miller, 1992 Curr. Top. Microbiol. Immunol. 158, 1; Vile & Russell, 1995 British Medical Bulletin. 51, 12; Weiss, in Retroviridae, J. Levy, Ed. (Plenum Press, 1993), pp. 1-108). Retroviral envelope spike glycoproteins mediate virus attachment to specific receptors on the target cell surface and subsequently trigger fusion between the lipid membranes of virus and host cell.
  • the envelope spike glycoproteins of murine leukaemia viruses are homotrimers in which each of the three heterodimeric subunits comprises a large extraviral glycoprotein moiety (SU) attached at its C-terminus to a smaller transmembrane polypeptide (TM) that anchors the complex in the viral membrane (March et al. , 1974 Virology 60, 595; Ikeda et al. , 1975 J. Virol. 16, 53; Kamps et al., 1991 Virology 184, 687).
  • SU extraviral glycoprotein moiety
  • TM transmembrane polypeptide
  • SU consists of two domains connected by a proline-rich hinge, the N-terminal domain conferring receptor specificity and exhibiting a high degree of conservation between MLVs with different host ranges (Battini et al. , 1992 J. Virol. 66, 1468; Morgan et al. , 1993 J. Virol. 67, 4712; Battini et al. , 1995 J. Virol. 69, 713).
  • Moloney MLV envelopes confer an ecotropic host range because they attach selectively to a peptide loop in the murine cationic amino acid transporter (CAT-1), found only on cells of mouse and rat origin (Albritton et al.
  • 4070A MLV envelopes attach to an epitope on the ubiquitous RAM-1 phosphate symporter that is conserved throughout many mammalian species and confer an amphotropic host range (Miller et al. , 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 78; VanZeijl et al. , 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 1168; Kavanaugh et al. , 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 7071).
  • retroviral vectors with 4070A envelopes infect human cells promiscuously whereas vectors with Moloney envelopes fail completely to infect human cells.
  • ecotropic vectors displaying a RAM-1 receptor-binding domain from 4070 A SU were able to infect RAM-1-positive human cells whereas amphotropic vectors displaying epidermal growth factor (EGF) could bind to EGF receptors but were thereafter sequestered into a noninfectious entry pathway, giving greatly reduced titres on EGF receptor-positive cells, but normal titres on EGF receptor-negative cells.
  • EGF epidermal growth factor
  • each construct is a schematic representation of the N-terminal region of the expressed envelope glycoprotein monomer; Open circles indicate N-terminal receptor-binding domain of the (ecotropic) Moloney MLV SU glycoprotein, filled squares indicate the N-terminal receptor binding domain of the (amphotropic) 4070 A MLV SU glycoprotein, grey triangles represent EGF, and factor Xa cleavage sites are denoted with arrows.
  • LTR long terminal repeat
  • L envelope signal peptide
  • p polyadenylation sequence. The NotI cloning site is also shown.
  • chimaeric envelopes and a control ecotropic (Moloney) envelope were expressed in TELCeB6 cells which express MLV gag-pol core particles and an nlsLacZ retroviral vector (Cosset et al., 1995 J. Virol. 69, 7430-7436).
  • Virus-containing supernatants from the transfected TELCeB6 cells were harvested, filtered (0.45 ⁇ m), digested with 0 or 4 ⁇ g/ml factor Xa protease for 90 minutes and ultracentrifuged to pellet the viral particles.
  • Retroviral particles incorporating chimaeric envelopes were analyzed by Western immunoblotting ( Figure 2) before (-) or after (+) treatment with factor Xa protease.
  • Lanes A, B and C were loaded with pelleted retroviral vectors incorporating Mo, EXMo1 , and EXMo7 envelopes, respectively.
  • the different envelope expression constructs were transfected (as described in Sambrook et al., Molecular cloning, A laboratory manual, (Cold Spring Habour, N.Y., 1989) pp. 16.33-16.36) into TELCeB6 packaging cells and stable phleomycin (50 ⁇ g/ml) resistant colonies were expanded and pooled. Cells were grown in DMEM supplemented with 10% fetal calf serum and when confluent transferred from 37°C to 32°C and incubated for 72 hrs.
  • Supernatants containing retroviral particles were harvested after overnight (16 hrs) incubation in 10 mis serum-free DMEM at 32°C and filtered (0.45 ⁇ m) before being incubated with 0 or 4 ⁇ g/ml of factor Xa (Promega) for 90 minutes at 37°C in the presence of 2.5 mM CaCk The supernatants were centrifuged at 30 000 rpm in a SW40 rotor (Beckman) for 1 hour at 4°C and the pelleted viral particles were resuspended in 100 ⁇ l phosphate buffered saline.
  • EGF was not cleaved from the EXMo7 envelope by factor Xa (Fig. 2, lane C), suggesting that the cleavage site was not accessible to the protease when inserted in this position.
  • EMo1 and EXMo1 coding for chimaeric envelopes in which EGF is fused to amino acid + 1 (rather than +7) of Moloney SU by a linker comprising 3 alanines, or 3 alanines and the IEGR factor Xa cleavage site (see Figure 1).
  • EMo1 and EXMo1 chimaeric envelopes were incorporated into virions and analysed on immunoblots after treatment with 0 or 4 ⁇ g/ml factor Xa protease for 90 minutes.
  • FIG. 2 shows that EXMo1 envelopes were cleaved by factor Xa to yield an SU cleavage product whose mobility was indistinguishable from unmodified Moloney SU. Control EMo1 envelopes which lack the factor Xa cleavage site were not cleaved.
  • Retroviruses displaying these chimaeric envelopes could bind to EGF receptors but were thereafter sequestered into a noninfectious entry pathway, giving greatly reduced titres on EGF receptor-positive cells, but near-normal titres on EGF receptor-negative cells.
  • the expression plasmids FBMoS ALF and FB4070ASALF (described by Cosset et al. , 1995 J. Virol. 69, cited above) coding for unmodified Moloney and 4070A MLV envelopes are referred to in the text as Mo and A respectively. Construction of EA, EMo7 (previously called EMO) and AMO expression plasmids was also described by Cosset et al., (cited above).
  • PCR primers NotXMo7Back, NotMo1Back and NotXMo1Back were used with primer envseq7 to amplify modified envelope fragments from Mo (FBMoSALF) which were digested with NotI and BamHI and cloned into the NotI/ BamHI-digested backbone of EMo7.
  • PCR primers NotA1Back and NotXA1Back were used with primer 4070Afor to amplify modified envelope fragments from A (FB4070ASALF) which were digested with NotI and BamHI and cloned into the NotI/BamHI-digested backbone of EA.
  • AMo1 and AXMo1 constructs were generated by cloning the Ndel-Notl fragment from AMO into the Ndel-Notl-digested backbones of EMo1 and EXMo1 , respectively. The correctness of all constructs were confirmed by DNA sequencing.
  • Oliogonucleotides used were:
  • Figure 3 shows that the IEGR sequence in the interdomain linker of the expressed EXA1 envelopes was correctly recognized and cleaved by factor Xa whereas there was no cleavage of control EA1 envelopes.
  • Figure 3 an immunoblot of the recombinant amphotropic retroviral particles before (-) or after (+) treatment with factor Xa protease: lanes A, B and C were loaded with pelleted retroviral vectors incorporating A, EA1 and EXA1 envelopes, respectively. The analysis was performed as described above for Figure 2.
  • EGF receptor-expressing cell lines A431 ATCC CRL1555), HT1080 (ATCC CCL121), and EJ (Bubenik, et al., 1973 Int. J. Cancer 11, 765) were grown in DMEM supplemented with 10% fetal calf serum (Gibco-BRL) at 37°C in an atmosphere of 5 % CO 2 .
  • Jurkat T cells ATCC CRL8805 were grown in RPMI supplemented with 10% fetal calf serum at 37°C in an atmosphere of 5% CO 2 .
  • target cells were seeded at 2 ⁇ 10 5 cells/well in six-well plates and incubated at 37°C overnight.
  • Producer cell supernatants containing ⁇ -galactosidase-transducing retroviruses were filtered (0.45 ⁇ m) after overnight incubation at 32°C in serum free medium.
  • Supernatant dilutions in 2.5 ml serum-free medium were incubated with target cells for 2 hours in the presence of 8 ⁇ g/ml polybrene.
  • the retroviral supernatant was then removed and the cells were incubated with regular medium for 48-72 hours.
  • X-Gal staining for detection of ⁇ -galactosidase activity was performed as previously described (Takeuchi et al. , 1994 J. Virol. 68, 8001).
  • Viral titre (enzyme forming units/ml) was calculated by counting blue stained colonies microscopically with the use of a grid place underneath the 6 well plates.
  • Both vectors incorporating EA1 or EXA1 envelopes could infect EGF receptor-negative Jurkat cells but were selectively sequestered on EGF receptor-expressing human cells, although EXA1 was sequestered less completely than EA1 (Table 1).
  • EXA1 was sequestered less completely than EA1 (Table 1).
  • soluble EGF was added as competitor to prevent the vectors from binding to EGF receptors their infectivity on EGF receptor positive cells could be fully restored (Table 1), confirming that sequestration was mediated specifically through binding of the engineered envelopes to EGF receptors.
  • Factor Xa protease is capable of binding directly to procoagulant phospholipid on the surface of an enveloped virus (Pryzdial & Wright, 1994 Blood 84, 3749-3757) and might therefore go on to become stably associated with phospholipid in the engineered vector particles after cleaving their EXA1 envelopes.
  • a control experiment was therefore performed to confirm that the restoration of infectivity of vectors incorporating EXA1 envelopes on A431 cells was due to cleavage of EGF, and not mediated by panicle-associated factor Xa protease.
  • Retroviral vectors with engineered binding specificity whose tropism is regulated by exposure to specific proteases may facilitate novel strategies for targeting retroviral gene delivery.
  • Vectors incorporating EXA1 envelopes were therefore treated with factor Xa and titrated on EGF receptor-expressing A431 cells.
  • Complete cleavage of the fused EGF domain with 4 ⁇ g/ml factor Xa for 90 minutes completely restored the infectivity of vectors with EXA1 envelopes but had no effect on the infectivity of vectors carrying EA1 envelopes (Figure 4).
  • Figure 4 illustrates factor Xa-mediated infection of A431 cells with chimaeric EGF-4070A MLV vector particles.
  • Filtered supernatants containing ⁇ -galactosidase-transducing retroviruses were preincubated with 0 (-) or 4 (+) ⁇ g/ml concentrations of factor Xa (Promega) for 90 minutes at 37°C with 2.5 mM added CaCl 2 .
  • the treated supernatants were then used for target cell transduction, as described above. X-gal-stained plates were photographed without magnification.
  • cleavage of the chimaeric envelope is preceded by its attachment to the target cell via the engineered binding domain. Therefore, to determine whether EGF receptor-bound vector particles that were cleaved at the cell surface could go on to infect their target cells, we loaded the vectors onto EGF receptor-positive A431 cells and EJ cells, washed the cells, and then treated them with factor Xa protease. Table 3 shows that, when sequestered onto EGF receptors and then cleaved by factor Xa protease, the vectors incorporating EXA1 , but not EA1 envelopes, proceeded to infect their target cells.
  • proteases that may be of interest in this respect such as the proteases that co-operate in degrading the extracellular matrix during tumour invasion (Poustis-Delpont et al., 1992 Cancer Research 52, 3622-3628; Vassalli & Pepper, 1994 Nature 370, 14-15; Sato et al. , 1994 Nature 370, 61-65; and Chen et al., 1995 Breast Cancer Res. Treat. 31, 217-226); haematopoietic differentiation antigens that are also membrane proteases (Shipp & Look, 1993 Blood 82, 1058-1070) or the membrane protease that has been implicated in the entry pathway of HIV (Murakami et al., 1991 Biochim. Biophys. Acta 1079, 79-284).
  • MLV-derived retroviral vectors are versatile gene delivery vehicles whose host range properties are determined by membrane glycoproteins which mediate their attachment to specific receptors and subsequently trigger fusion.
  • the envelope glycoproteins of the murine leukaemia virus (MLV) are displayed as a homotrimeric complex on the surface of the virus (Fass et al. , Nature Structural Biology 3:465-469; Kamps et al., Virology 184:687-694).
  • Each subunit of the trimer consists of two parts, SU and TM.
  • the SU (surface) component is entirely extraviral and is attached to the retrovirus via the smaller TM component, which anchors the complex in the viral membrane (Pinter et al., Virology 91:345-351).
  • the N-terminal domain of the SU glycoprotein confers receptor specificity and exhibits a high degree of conservation between MLVs with different host ranges (Battini et al., J. Virol. 69:713-719).
  • Moloney MLV envelopes confer an ecotropic host range because they bind to a murine cationic amino acid transporter (Albritton et al., J. Virol. 67:2091-2096; Albritton et al. , Cell 57:659-666).
  • 4070A MLV envelopes attach to the RAM-1 phosphate transporter which is conserved throughout many mammalian species, to confer an amphotropic host range (Kavanaugh et al. , Proc. Natl. Acad. Sci. USA 91:7071-7075). After binding to target cell receptors has occurred, the trimeric SU-TM complex is thought to undergo a large conformational rearrangement which triggers the process of fusion between the viral and target cell membranes.
  • step one the retroviral vector attaches to the target cell via an engineered binding domain
  • step two the engineered linker that tethers the virus to the binding domain is cleaved by a specific protease
  • the uncleaved vector therefore retains the ability to infect non target cells through the Ram-1 receptor.
  • envelope modifications that would completely inhibit the infectivity of uncleaved vectors but would permit full restoration of infectivity upon exposure to a selected protease.
  • the unmodified envelopes of 4070A MLV and Moloney MLV were encoded by the expression plasmids FB4070ASALF (A) and FBMoSALF (Mo), respectively (Cosset et al., 1995 J. Virol. 69, 7430-7436)).
  • the constructs AMo1 and AXMo1, which code for chimaeric envelopes in which the RAM-1 receptor binding domain from 4070A SU is fused to amino acid + 1 of Moloney SU by a factor Xa protease-cleavable (AAAIEGR) or non-cleavable (AAA) linker have been described previously (Nilson et al., Gene Ther. 3:280-286).
  • EA1 and EXA1 coding for chimaeric envelopes in which EGF is fused to amino acid + 1 of 4070A SU by a linker comprising three alanines, or three alanines and the IEGR factor Xa cleavage site, have also been described (Nilson et al., Gene Ther. 3:280-286).
  • plasmids pEGSlXA1 and pEGS3XA1 were first produced in which there is a 12 amino acid (AAAGGGGSIEGR, Seq ID No. 8) or 22 amino acid (AAAGGGGSGGGGSGGGGSIEGR, Seq ID No. 9) linker, respectively, between the 4070A MLV envelope and the displayed EGF domain.
  • PCR primers NotGSlXA1back and NotGS3XA1back (respectively) were used with primer 4070Afor to amplify modified envelope fragments from EXA1 which were digested with Notl and BamHI and cloned into the Notl/BamHI-digested backbone of EA1.
  • Figure 7 is a diagramatic representation of plasmid constructs coding for chimaeric envelope glycoproteins in which the helical peptides AA, VL and II were fused to residue + 1 of the 4070A MLV SU.
  • the general format is shown diagramatically and the amino acid sequence (single letter code) of the helical peptides and the linkers between these peptides and the SU protein are shown in detail.
  • LTR long terminal repeat;
  • L envelope signal peptide.
  • Amino acid residues at the a and d positions of the heptad repeat are shown in bold.
  • PCR primers Gal4 VLback and Gal4 VLfor were used to produce PCR fragments by priming off each other and then outer primers Gal4back and Ga14 for were used to amplify the fragment further.
  • the PCR products were digested with SfiI and Notl and cloned into the SfiI/Notl-digested backbones of EXA1 , pEGSlXA1 and pEGS3XA1.
  • PCR primers Gal4 AAback and Gal4 AAfor were used to produce PCR fragments by priming off each other and then outer primers Ga14back and Ga14 for were used to amplify the fragments further.
  • the PCR products were digested with SfiI and Notl and cloned into the SfiI/Notl-digested backbones of EXA1 and pEGS3XA1.
  • PCR primers Gal4 Ilback and Gal4 Ilfor were used to produce PCR fragments by priming off each other and then outer primers Gal4back and Gal4for were used to amplify the fragments further.
  • the PCR products were digested with Sfil and Notl and cloned into the SfiI/Notl-digested backbones of EXA1 , pEGS1XA1 and pEGS3XA1. The correct sequence of all constructs was verified by DNA sequencing.
  • GP+Env AM12 cells (Markowitz et al., Virology 167:400-406) were derived from the murine cell line NIH 3T3 and express the MLV-A envelope which blocks the RAM-1 receptor by interference.
  • NIH 3T3, GP+Env AM 12 and the human cell line A431 (Giard et al., J. Natl. Cancer Inst. 51, 1417-1421), were grown in DMEM supplemented with 10% fetal calf serum.
  • the different envelope expression constructs were transfected into TELCeB6 packaging cells (Cosset et al., J. Virol. 69:7430-7436) by calcium phosphate precipitation (Takeuchi et al., J. Virol.
  • Virus producer cells were lysed in a 20mM Tris-HCl buffer (pH 7.5) containing 1 % Triton X-100, 0.05% SDS, 5mg/ml sodium deoxycholate, 150mM NaCl and ImM PMSF. Lysates were incubated for 10 mins at 4°C and were centrifuged for 10 mins at 10,000 ⁇ g to pellet the nuclei. Virus samples were obtained by ultracentrifugation of filtered viral supernatants (10ml) at 30 000 rpm in a SW40 rotor (Beckman, USA) for 1 hr at 4°C. The pelleted viral particles were resuspended in 100 ⁇ l PBS.
  • RLV Rausher leukaemia virus
  • CA RLV p30 capsid protein
  • Blots were developed with horseradish peroxidase-conjugated rabbit anti-goat antibodies (DAKO, Denmark) and an enhanced chemiluminescence kit (Amersham Life Science, UK).
  • Target cells were seeded at 2 ⁇ 10 5 cells/well in six-well plates and incubated at 37°C overnight.
  • Producer cell supernatants containing ⁇ -galactosidase-transducing retroviruses were filtered (0.45 ⁇ m) after overnight incubation at 32°C in serum-free medium.
  • the harvested supernatants were incubated with 0 or 4 ⁇ g/ml of factor Xa (Promega) for 90 minutes at 37°C in the presence of 2.5mM CaCl,.
  • Supernatant dilutions in 2ml serum-free media were incubated with target cells for 6 hrs in the presence of 8 ⁇ g/ml polybrene.
  • the retroviral supernatant was then removed and the cells were incubated with regular medium for 48-72 hrs.
  • X-Gal staining for detection of ⁇ - galactosidase activity was performed as previously described (Tatu et al. , EMBO J. 74: 1340-1348).
  • Viral titre (enzyme forming units/ml) was calculated by counting blue stained colonies microscopically with the use of a grid placed underneath the 6 well plates.
  • AMo1 and AXMo1 are previously described chimaeric envelopes in which the RAM-1 receptor binding domain from 4070A SU is fused to aminoacid + 1 of Moloney SU by a noncleavable (AAA) or factor Xa-cleavable (AAAIEGR) linker (Nilson et al., Gene Ther. 5:280-286).
  • Viruses incorporating the AMo1 and AXMo1 envelopes were pelleted, cleaved with 0 or 4 ⁇ g/ml factor Xa protease and then analysed on immunoblots using an anti-envelope antiserum as a probe.
  • Figure 6 A is an immunoblot of pelleted recombinant retroviral particles incorporating Mo, AMo1 or AXMo1 envelopes before (-) or after (+) treatment with factor Xa protease, probed with antiserum to the SU glycoprotein.
  • Figure 6B shows the results when the target cell line GP+Env AM12 was infected with harvested producer cell supernatants containing ⁇ -galactosidase-transducing retroviruses (AMo1 , AXMo1 , Mo and A) with or without treatment with factor Xa protease. Detection of ⁇ -galactosidase activity was performed by X-gal staining and titres were expressed as e.f.u./ml.
  • Such a block would be expected to be reversible by cleaving the Ram-1 binding domain from the vector and, in keeping with this prediction, the infectivity of the AXMo1 vector was fully restored on Rec-1 positive, Ram-1 deficient cells when the Ram-1 targeting domain was cleaved from its surface with factor Xa protease (Fig. 6B).
  • the helical peptides that were chosen for these studies were variants of the dimeric GCN4 leucine zipper peptide with systematic V, L, I or A (single letter aminoacid code) substitutions in the a and d positions of the heptad repeat that are known to force the formation of trimeric coiled coils (VL and II peptides) or to prevent oligomerisation (AA peptide) (Harbury, et al., Science 262: 1401-1407).
  • VL and II peptides trimeric coiled coils
  • AA peptide oligomerisation
  • the spacing between the 4070A SU glycoprotein and the displayed peptide motifs was therefore varied by insertion of linkers comprising amino acids AAAIEGR, Seq ID No. 20), AAAGGGGSIEGR (Seq ID No. 8) or AAAGGGGSGGGGSGGGGSEEGR (Seq ID No. 9), where the highlighted sequence is known to be recognised and cleaved by Factor Xa (Nilson et al., Gene Ther. 5:280-286).
  • AA, VL and II chimaeric envelopes and a control amphotropic (4070A) envelope were stably transfected into TELCeB6 cells which express MLV gag-pol core particles and an nls LacZ retroviral vector (Cosset et al., J. Virol. 69:7430-7436).
  • Virus-containing supernatants were harvested from these stably transfected TELCeB6 cells and ultracentrifuged to pellet the viral particles. Pellets were than analysed on immunoblots for the presence of viral core proteins and envelope proteins (Fig. 8A).
  • Figure 8 illustrates the viral incorporation and cleavage of chimaeric envelopes expressing factor Xa-cleavable helical peptides as N-terminal extensions of the 4070 A MLV SU.
  • Figure 8A is an immunoblot of pelleted retroviral particles incorporating chimaeric envelopes. The lane contents are as follows: 1 :VLXA1, 2:VLGS1XA1, 3:VLGS3XA1, 4.AAXA1, 5:AAGS3XA1, 6:IIXA1, 7:IIGS1XA1, 8:IIGS3XA1, and 9:A.
  • the top immunoblot was probed with an anti-SU antiserum and the lower one with an anti-p30 antiserum to detect the p30 CA protein.
  • Figure 8B shows the Factor Xa-mediated cleavage of chimaeric envelopes and takes the form of an immunoblot of pelleted recombinant amphotropic retroviral particles incorporating A, VLXA1, AAXA1 , IIXA1 or EXA1 envelopes before (-) or after (+) treatment with factor Xa protease, probed with anti-SU antiserum.
  • Figure 8C is an immunoblot of cell lysates prepared from the virus producing TELCeB6 transfectants A, VLXA1, AAXA1, IIXA1 and the control, untransfected TELCeB6, probed with anti-SU antiserum.
  • viral pellets were digested with 0 or 4 ⁇ g/ml factor Xa protease and then analysed on immunoblots as before.
  • Figure 8B shows that there is a mobility shift when expressed envelopes VLXA1, AAXA1 and the control EXA1 , have been cleaved with factor Xa protease, indicating that the helical peptides are indeed cleaved from the SU. Due to the low levels of incorporation of the IIXA1 chimaeric envelope, cleavage can not be seen for this vector. This immunoblot also indicates that the chimaeric envelope AAXA1 was incorporated 10 times more efficiently than VLXA1.
  • FIG. 8C shows that the unprocessed precursors of all three chimaeric envelopes are detectable in the cell lysates.
  • the VL and II envelope precursors are less abundant than the AA precursor.
  • the processing of the VL and II precursors to mature SU is severely impaired relative to the processing of the AA precursor indicating that these chimaeric envelopes are not efficiently transported from the endoplasmic reticulum to the Golgi compartment.
  • FIG. 9 shows the reversible inhibition of infection by cleavage of the chimaeric envelope, VLXA1, expressing a factor Xa-cleavable, N-terrninal oligomerizing peptide and is a magnified view of virally infected cells after X-gal staining.
  • Chimaeric envelope VLXA1 shows strong inhibition of infectivity on NIH 3T3 and A431 cells, which is reversible on addition of factor Xa.
  • control vectors displaying the AA peptide gave titres comparable to that of the wild type amphotropic vector and the titres did not change after factor Xa cleavage indicating that the AA peptide does not significantly interfere with the functions of the underlying 4070A envelope.
  • the vectors displaying the trimerising VL and II helical peptides gave greatly reduced titres on both cell lines which were enhanced as much as 2000-fold by factor Xa cleavage.
  • the Ram-1 binding domain from the homotrimeric 4070A SU glycoprotein can inhibit Rec-1 mediated infection by the homotrimeric Moloney SU glycoprotein when grafted to its N-terminus.
  • short trimeric leucine zipper peptides but not a monomeric helical peptide, can inhibit Ram-1 mediated infection by the 4070A envelope when fused to its N-terminus.
  • factor Xa protease to cleave the trimeric N-terminal extensions from the virally incorporated envelopes, it was possible to reverse the block to Rec-1 or Ram-1 mediated infection.
  • the masking of envelope functions by these inhibitory N-terminal extensions is a consequence of their assembly into a trimeric complex at the tip of the SU glycoprotein trimer to which they are grafted.
  • the VL, II and AA peptides that we fused to the 4070A envelope are mutants of the GCN4 leucine zipper in which the conserved, buried residues that direct dimer formation have been substituted with valine, leucine, isoleucine or alanine residues (Harbury et al., Science 262: 1401-1407).
  • the VL mutant oligomerises to form extremely stable (T m 95 °C) two- and three-stranded alpha-helical coiled coil structures whereas the II mutant forms exclusively three-stranded coiled coils which are even more stable (T m > 100°C) than the VL structures.
  • T m 95 °C extremely stable
  • the II mutant forms exclusively three-stranded coiled coils which are even more stable (T m > 100°C) than the VL structures.
  • In the AA peptide all of the hydrophobic core residues of the GCN4 leucine zipper were substituted with alanines
  • Retroviral incorporation of chimaeric envelopes displaying the VL and II peptides was significandy impaired relative to chimaeric envelopes displaying the control AA peptide, which showed only a slight reduction in incorporation compared to unmodified 4070A envelopes.
  • the VL chimaeric envelopes were approximately ten-fold less abundant in viral pellets than the AA chimaeric envelopes, and the II chimaeric envelopes were so poorly incorporated that they were not visible on immunoblots of pelleted virions.
  • VL and II chimaeric envelopes It is currently unclear what is responsible for the impaired expression of the VL and II chimaeric envelopes. Neither protein appears to be toxic to the virus producing cells since there was no difference in the number or size of stably transduced TELCeB6 clones that were obtained after transfecting the different (oligomerising or control) chimaeric envelope expression plasmids (data not shown).
  • An alternative possibility might be that the low intracellular abundance of the VL and II envelope precursors is due to their premature oligomerisation in the endoplasmic reticulum.
  • VL or II chimaeric envelopes showed inhibition of infection on NIH3T3 and A431 cells, which was reversible on cleaving the peptides from the vectors with factor Xa protease. Titres were not restored completely to wild type levels due to the reduced levels of incorporation of these envelopes.
  • the VL and II peptides therefore function as oligomerising peptide adaptors which mask the functions of the retroviral envelope glycoprotein to which they are fused.
  • the inhibition of infection may be as a result of the oligomerizing peptide blocking binding of the vector to its target cells by masking the underlying binding domain.
  • the presence of an oligomerizing peptide may prevent dissociation of the envelope trimer, blocking fusion.
  • binding studies were uninformative so we are unable to determine which of these mechanisms is more dominant.
  • Vectors pEGF LVA1 and pEGF LVXA1 display an oligomerising peptide, LV, fused to residue + 1 of 4070A SU with a non cleavable (SAA) or factor Xa protease-cleavable (SAAIEGR, Seq ID No. 21) linker and also display the EGF binding domain.
  • SAA non cleavable
  • SAAIEGR factor Xa protease-cleavable
  • PCR primers Gal4 LV, Gal4 LVbak and Gal4 LVfor were used for assembly of the PCR fragment coding for the oligomerising peptide, LV (Harbury et al., 1993 Science 262, 1401-1407).
  • Gal4 LV 5'-GAC AAG CTA GAG GAA GTA CTT AGC AAG CTC TAC CAT GTC GAG AAC GAA CTT GCT CGA GTT AAG AAG-3' (Seq ID No. 22);
  • Gal4 LVback (containing NotI site), 5'-GGC ATT CAT GCG GCC GCA ATG AAG CAA GTG GAA GAC AAG CTA GAG GAA GTA C-3' (Seq ID No. 23);
  • Gal4 LVfor (containing Eagl site), 5'-GCA AAT CTG CGG CCG ACT CTC CCA GAA GCT TCT TAA CTC GAG CAA GTT C-3' (Seq ID No. 24).
  • the murine cell line NIH 3T3, and the human cell line A431 were grown in DMEM supplemented with 10% fetal calf serum.
  • the envelope expression constructs were transfected into TELCeB6 packaging cells by calcium phosphate precipitation and stable phleomycin (50mg/ml) resistant colonies were expanded and pooled.
  • Cells were grown in DMEM supplemented with 10% fetal calf serum and when confluent transferred from 37°C to 32°C and incubated for 72hrs.
  • Supernatants containing retroviral panicles were harvested after overnight (16hrs) incubation at 32°C in 10mls serum-free DMEM for infections. All supernatants were filtered (0.45 ⁇ m) before use.
  • Target cells were seeded at 2 ⁇ 10' cells/well in six-well plates and incubated at 37°C overnight.
  • the harvested supernatants containing ⁇ -galactosidase-transducing retroviruses were incubated with 0 or 4 ⁇ g/ml of factor Xa (Promega) for 90 minutes at 37°C in the presence of 2.5mM CaCL.
  • Supernatant dilutions in 2ml serum-free media were incubated with target cells for 6 hrs in the presence of 8 ⁇ g/ml polybrene.
  • the retroviral supernatant was then removed and the cells were incubated with regular medium for 48-72 hrs.
  • X-Gal staining for detection of ⁇ -galactosidase activity was performed and viral titre (enzyme forming units/ml) was calculated by counting blue stained colonies microscopically with the use of a grid placed underneath the 6 well plates.
  • MMPs matrix metalloproteinases
  • Matrix metalloproteinases are important for angiogenesis, tissue remodelling, inflammation and wound healing, and they play a crucial role in various pathological processes including cancer invasion and metastasis and the destruction of articular cartilage in rheumatoid arthritis (Liotta et al., 1991 Cell 64, 327; Woessner Jr. , 1991 FASEB J.
  • MMPs include matrilysin, collagenases1-3, stromelysinsl-3, gelatinases A and B and a group of 4 membrane-type MMPs (MT-MMP) which are anchored to cell membranes (Sato et al. , 1994 Nature 370, 61; Takino et al.,
  • MMPs are secreted as zymogen forms and require activation before they can exert their proteolytic activities.
  • the net activities of the enzymes are also regulated by the three tissue inhibitors of MMPs (TTMPs).
  • the MMPs co-operate with one another in a cascade pathway to cause degradation of the extracellular matrix.
  • Gelatinase A (GLA; MMP-2) and the MT- MMPs are of special interest with respect to tumour invasion.
  • Pro-GLA is secreted by stromal fibroblasts and concentrated on tumour cell membranes, especially at the invasive front of the tumour (Afzal et al., 1996 Lab. Invest. 74, 406; Nomura et al. , 1996 Int. J.
  • GLA is often found to be elevated in invasive or metastatic tumours.
  • MMPs as promising targets for novel therapeutic agents and there are several general or specific MMP-inhibitors that are currently being tested for their usefulness in treatment of MMP-linked diseases in a number of clinical trials (Hodgson 1995 Biotech. 13, 554; Eccles et al. , 1996 Can. Res.
  • This example describes the generation of targeted retroviral vectors whose infectivity for human EGF receptor-expressing cancer cells is strongly activated by membrane-associated MMPs.
  • chimaeric envelope expression constructs were generated in which a cDNA coding for the 53 amino acid receptor binding domain of EGF was linked to the N-terminal codon of the 4070A murine leukemia virus (MLV) SU envelope glycoprotein via short non-cleavable or protease-cleavable linkers.
  • MLV murine leukemia virus
  • a and E.X.A have an EGF cDNA, flanked by SfiI and NotI restriction sites, inserted at codon + 1 of the N-terminus of wild type 4070A MLV SU (surface protein gp 70) envelope, with a linker of either 3 alanines (E.A.) or 3 alanines and the IEGR Factor Xa cleavage sequence (E.X.A.) between the domains.
  • Figure 12 is a schematic representation of the chimaeric envelope expression constructs, E.A, E.G 4 S.A, E.X.A and E.MMP.A.
  • the envelope constructs were transfected into TELCeB6 complementing cells, virus-producing clones were pooled and expanded in 10% FCS-DMEM selection medium containing 50 ⁇ g/ml phleomycin. Arrows indicate potential site of cleavage by respective proteases.
  • the E.A and E.G 4 S.A chimaeric envelopes contained non-cleavable linkers AAA and AAAGGGGS (Seq ID No. 25) respectively (single letter amino acid code), the E.X.A envelope contained a Factor Xa-cleavable linker AAAIEGR and the E.MMP.A envelope contained the linker AAAPLGLWA (Seq ID No. 26) in which the highlighted sequence is known to be recognised and cleaved by GLA and by MT1-MMP (Ye et al. , 1995 Biochem. 34, 4702; Will et al. , J. Biol. Chem. 277, in press) ( Figure 12).
  • the chimaeric envelope constructs and a wild type 4070A envelope expression construct were stably transfected into TELCeB6 complementing cells which express Moloney MLV gag-pol proteins and the nlsLacZ retroviral vector, as described in the preceding examples.
  • infectious enveloped vector particles capable of transferring the lacZ marker gene are rescued into the culture supernatant.
  • Viral supernatants were harvested from confluent plates of pooled transfected TELCeB6 cells and the viral particles were pelleted by ultracentrifugation and immunoblotted using an anti-envelope antiserum as probe. Immunoblotting was performed as described in the preceding examples. The results are shown in Figure 13 A, B.
  • Figure 13B is an immunoblot demonstrating cleavage of MMP-cleavable linker in E.MMP.A by purified p-aminophenylmercuric acetate (APMA)-activated gelatinase A (GLA).
  • APMA p-aminophenylmercuric acetate
  • GLA p-aminophenylmercuric acetate
  • APMA p-aminophenylmercuric acetate
  • E.X.A, E.G 4 S.A or E.MMP.A viral pellets were incubated with PBS, APMA (final concentration 2 mM) or APMA-activated GLA (32 ⁇ g/ml) for 30 min at 37°C].
  • E.MMP.A-SU On treatment of E.MMP.A-SU with activated GLA, a band with the same mobility as the wild type 4070A-SU was recovered, indicating that the EGF domain could be efficiently cleaved from this chimaeric envelope without further GLA-mediated degradation (Fig. 13B).
  • the E.G 4 S.A and E.X.A chimaeric envelopes were unaffected by treatment with GLA indicating that cleavage was specific for the MMP-sensitive linker (not shown).
  • E.A, E.G 4 S.A and E.MMP.A vectors on A431 cells were low between 10 2 -10 3 efu/ml and were not greatly increased by treatment with Factor Xa protease (not shown).
  • A431 cells in 10% FCS-DMEM were seeded, at a density of 3 ⁇ 10 4 per individual well, in a 24-well tissue culture plate (Corning, New York) overnight at 37°C. The media were removed the next day and the cells were washed once in serum-free DMEM. Varying amounts (final concentration 2-40 ⁇ g/ml) of pro-GLA were mixed with 200 ⁇ l of filtered E.MMP.A viral supernatant after which the mixture was added to A431 cells and incubated at 37°C for 6 h. At the end of 6 h, the media was removed and cells were washed once in serum-free DMEM.
  • FIG. 14 is a graph showing that increase in titre (efu ⁇ 10 -4 /ml) of the E.MMP.A MMP-sensitive vector on A431 cells is correlated with the amount of pro-gelatinase A (pro-GLA) added onto the cells.
  • HT1080 is a human fibrosarcoma cell line that constitutively produces MT1-MMP and pro-GLA (Okada et al. , 1995 Proc. Natl. Acad. Sci. 92, 2730).
  • Figure 15 is a graph showing the titre of EGF chimaeric vectors on A431 and HT1080 cells.
  • Figure 15A shows the high infectivity of E.MMP.A vector on HT 1080 cells compared to on A431 cells as indicated by the number of blue ⁇ -galactosidase positive colonies.
  • One ml out of 10 ml filtered E.MMP.A viral supernatant was incubated with
  • the infectivity of the vectors on A431 cells was low in the absence of exogenous pro-GLA.
  • the infectivity of the MMP-cleavable vector E.MMP.A was activated by two orders of magnitude compared to the MMP-resistant control vectors E.G 4 S.A and E.X.A (Fig. 15, 15A).
  • the higher titre of the MMP-dependent E.MMP.A vector must be due to its cleavage by MMPs produced endogenously by HT1080 cells.
  • the MMP-activatable E.MMP.A vector could selectively target the MMP-expressing HT1080 cells in preference over A431 cells, we allowed the vector to infect both cell types on the same petri dish simultaneously.
  • the coverslips coated with the cells were placed in a 10 cm petri dish (Falcon) and E.G 4 S.A (1: 1.5 dilution), E.MMP.A (1:1.5) or 4070A (1:20) supernatants were added onto the petri dishes with 8 ⁇ g/ml polybrene for 6 h at 37°C. At the end of the incubation period, the media was removed and the cells were incubated in 10% FCS-DMEM for 72 h before X-gal staining.
  • E.MMP.A vector grown on HT1080 (H) cells and A431 (A) cells is shown in I and II, with the control E.G 4 S.A vector in III and the wild type 4070 A vector in IV.
  • E.MMP.A infected HT1080 cells preferentially over A431 cells.
  • the wild type 4070 A vector and E.G 4 S.A vector with the non-cleavable linker showed no such preference (Fig. 16).
  • the MMP-activatable E.MMP.A vector did not infect A431 cells more efficiently in the presence of HT1080 cells than in their absence. This suggests that soluble GLA released into the medium from the HT1080 cells does not play a significant role in activation of the vector.
  • TIMP-1 and TIMP-2 a synthetic inhibitor
  • CT 1339 a synthetic inhibitor
  • TIMP-1 at a final concentration of 10 ⁇ g/ml
  • TIMP-2 5 ⁇ g/ml
  • CT 1339 1 mM
  • the inhibitors were added to 200 ⁇ l of diluted (1:10) E.MMP.A or undiluted E.G 4 S.A viral supernatants.
  • the mixture was then added onto A431 or HT1080 cells, which had been washed once in serum free DMEM, and the cells were incubated for 6 h at 37°C.
  • the cells were washed once in serum free DMEM, incubated for 72 h in 10% FCS-DMEM after which they were stained with X-gal.
  • the E.MMP.A supernatant was diluted to obtain a titre that would allow accurate counting of the number of transduced colonies.
  • Inhibition studies on A431 cells were performed with 200 ⁇ l undiluted E.MMP.A or E.G 4 S.A in presence of 16 ⁇ g/ml pro-GLA.
  • Table 5 Influence of MMP inhibitors on the titre of vectors on A431 and HT1080 cells.
  • TIMP-1 displays only weak activity against the MT1-MMP expressed on HT1080 cells (Fig. 17. described below). These experiments therefore point to a central role for the MT-MMP in HT1080-mediated activation of the E.MMP.A vector.
  • Figure 17 is a gelatin zymogram showing the effect of TIMP-1 or a synthetic MMP- inhibitor, CT 1339 on cellular activation of endogenous pro-GLA on HT 1080 cells.
  • the E.MMP.A viral supernatant was incubated on HT 1080 cells for 6 h at 37°C in the absence of any inhibitors (lane 1), in the presence of 10 ⁇ g/ml (lane 2) or 30 ⁇ g/ml TIMP-1 (lane 3), and 1 ⁇ M (lane 4) or 10 ⁇ M (lane 5) CT 1339.
  • the targeting strategy that we have pursued may have interesting parallels with the mechanism of HIV entry in which primary virus attachment to CD4 leads to a conformational rearrangement or proteolytic cleavage in gp120, and secondary virus attachment to one of the recently characterised HIV co-receptors (Feng et al. , 1996 Science 272, 872; Deng et al., 1996 Nature 381, 661; Handley et al., 1996 J. Virol. 70, 4451).
  • C-type retroviral vectors with engineered SU glycoproteins could therefore be developed as model systems to probe the entry mechanisms that are employed by naturally occurring viruses, such as HIV.
  • Retroviral display of trimeric binding domains, TNF alpha and CD40 ligand Retroviral display of trimeric binding domains, TNF alpha and CD40 ligand.
  • chimaeric envelopes bearing TNF alpha or CD40 ligand as an N-terminal extension can be incorporated into retroviral vector particles where it appears that the trimeric binding domain forms a cap over the envelope glycoprotein to which it is fused.
  • the amphotropic infectivity of the vectors incorporating these chimaeric envelopes is therefore low but is greatly enhanced by cleaving the trimeric ligand from their surface.
  • the TELCeB6 cell line has been described in the preceding examples.
  • the NIH 3T3, A431 (human squamous carcinoma; ATCC CRL1555) and HT1080 (human fibrosarcoma; ATCC CCL121) cell lines were grown in DMEM (Gibco-BRL, UK) supplemented with 10% fetal calf serum (FCS; PAA Biologicals, UK), benzylpenicillin (60 mg/ml) and streptomycin (100 mg/ml) at 37°C in an atmosphere of 5% CO 2 .
  • the human tumour necrosis factor-alpha (TNF-a)-4070A SU chimaeric envelope expression vectors TNF-a.A. TNF-a.GS.A, TNF-a.X.A, TNF-a.XA, andTNF-a.MMP.A have an TNF-a cDNA (Wang et al. , 1995 Science, 228: 149-154), flanked by SfiI and NotI restriction sites, inserted at codon + 1 of the N-terminus of wild type 4070 A MLV SU envelope by different linkers (Fig. 18).
  • the TNF-a.A vector is linked via a 3 alanine (AAA) linker; TNF-a.GS.A via a non-cleavable AAAG 4 S linker; TNF-a.X.A via Factor Xa protease cleavable linker (AAAIEGR) and TNF-a.MMP.A via an MMP-cleavable linker (AAAPLGLWA) (single letter amino acid code).
  • the Factor Xa protease cleaves IEGR after the arginine residue and the PLGLWA linker is susceptible to gelatinase A (MMP-2) and MT-MMP between the giycine and leucine residues.
  • the CD40L-4070A SU chimaeric envelope expression vectors have part of the CD40L cDNA, flanked by SfiI and NotI restriction sites, inserted at codon + 1 of the N-terminus of 4070A MLV by the 4 different linkers as mentioned above.
  • the vectors are termed CD40L.A, CD40L.GS.A, CD40L.X.A and CD40L.MMP.A (Fig. 19).
  • a PCR derived SfiI-NotI DNA fragment encoding the 155 amino acids of the trimeric human TNF-a was generated using a cDNA template and two primers, sTNFback
  • the SfiI-NotI PCR fragment encoding the 145 amino acids of the soluble extracellular domain of the trimeric CD40L (Gly 116-Leu 261; Karpusas et al., 1995 Structure, 5: 1031-1039) was generated using a cDNA template (ATCC 79813) and two primers: sCD40Lb (5' > CCG GTA CCG GCC CAG CCG GCC GGT GAT CAG AAT CCT CAA ATT GC, Seq ID No. 31) with a SfiI site and
  • nCD40Lf (5' > AAG TCT TAG CGG CCG CGA GTT TGA GTA AGC CAA AGG, Seq ID No. 32) with a NotI site.
  • the respective PCR fragments were digested with SfiI and NotI restriction enzymes and cloned into the SfiI-NotI digested EA.1 backbone or EXA.1 to obtain T ⁇ F-a.A or CD40L.A. and T ⁇ F-a.X.A or CD40L.X.A, respectively (Nilson et al. , 1996 Gene Therapy 5: 280-286).
  • TNF-a.GS.A or CD40L.GS.A and TNF-a.MMP.A or CD40L.MMP.A
  • SfiI-NotI digested TNF-a or CD40L PCR fragments were cloned into SfiI-NotI digested E.GS.A or E.MMP.A backbones, respectively (Peng et al., A gene delivery system activatable by disease-associated matrix metalloproteinases, submitted). The sequences of the constructs were checked and verified by DNA sequencing.
  • TNF-a and CD40L envelope expression plasmids were stably transfected by calcium phosphate precipitation (Sambrook et al. , 1989, Molecular cloning: A laboratory manual) into the TELCeB6 packaging cells.
  • Transfected cells grown in 10% FCS-DMEM at 37°C, were selected with 50 ⁇ g/ml phleomycin (Sigma, Poole, Dorset, UK). Resistant colonies were pooled and expanded, and before harvest, the confluent cells were tranfened to 32°C for 72 h.
  • the viral supernatants were then harvested and filtered (0.45 ⁇ m, Acrodisc, Gelman Sciences MI, USA) after overnight incubation of the confluent cells with serum free DMEM at 32°C. These filtered supernatants were then used either for immunoblotting, binding or infection assays.
  • the viral particles were pelleted by ultracentrifugation of the filtered viral supernatant (Beckman, USA) at 30,000 rpm for 1 h at 4°C in a SW 40 rotor. The pellet was then resuspended in 100 ⁇ l cold PBS and stored at -70°C till further analysis.
  • the viral complementing cells were grown to confluency on petri dishes (10 cm in diameter), washed once in cold PBS and then incubated for 10 min at 4°C with cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 % Triton-X, 0.05% sodium dodecyl sulfate, 5 mg/ml sodium doxycholate and 1 mM PMSF.
  • the lysed cells were scraped from the plates and the suspension centrifuged at 10,000 ⁇ g for 20 min to pellet the nuclei. Thirty ⁇ l of the supernatant was used for electrophoresis and immunoblotting.
  • the TNF-a and CD40L chimaeric vectors were tested for infectivity on NIH 3T3, A431 and HT1080 cells.
  • the cells were seeded overnight at 37°C at a density of approximately 1 ⁇ 10 5 cells per individual well in a 6-well tissue culmre plate (Corning, New York). The medium was removed the next day and cells were washed once in serum-free DMEM. An aliqout (1 ml) of the filtered viral supernatant was used to infect the cells in the presence of 8 ⁇ g/ml polybrene. At the end of the 6 h incubation period, the medium was removed and the cells washed once in serum-free DMEM and 10% FCS-DMEM was added.
  • the titre of the TNF-a-4070A vectors on NIH3T3 and HT1080 cells were low (Table 6). This low level of infectivity could be due to the low level of chimaeric envelope expression. However, it could also be due to the display of the trimeric TNF-a on the 4070A-SU. The trimer was able to block the infectivity of the amphotropic vector, which would be otherwise be highly infective on the murine NIH 3T3 cells, which do not bear the human TNF-a receptor. e
  • the infectivity of the CD40L-4070A chimaeras are significandy lower than that of the wild type on NIH 3T3, A431 and HT1080 cells (Table 8), indicating that the display of CD40L on the envelope is blocking the infectivity of the vector.

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Abstract

On décrit une particule virale recombinée pouvant infecter une cellule eucaryote et comprenant: une glycoprotéine virale, sensiblement intacte, fusionnée, via une région intermédiaire de liaison, à un polypeptide hétérologue présenté sur la surface de la particule, lequel polypeptide module la capacité de la particule virale à infecter un ou plusieurs types de cellules eucaryotes et peut être clivé à partir de la glycoprotéine virale par une protéase agissant de manière sélective sur le site de clivage spécifique des protéases, présent dans la région de liaison, de telle manière que le clivage du polypeptide hétérologue à partir de la glycoprotéine virale permette à cette dernière d'interagir normalement avec son récepteur correspondant sur la surface d'une cellule cible.
EP96931908A 1995-09-27 1996-09-27 Virus recombines comprenant une proteine pouvant etre clivee par une protease Withdrawn EP0854929A1 (fr)

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US6942853B2 (en) * 2001-01-09 2005-09-13 Queen Mary And Westfield College Latent fusion protein
US20060068421A1 (en) * 2004-08-05 2006-03-30 Biosite, Inc. Compositions and methods for phage display of polypeptides
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US9926352B2 (en) * 2014-03-03 2018-03-27 Serendipity Biotech Inc. Chimeric dystrophin-VSV-G protein to treat dystrophinopathies
US10385101B2 (en) 2014-08-08 2019-08-20 Vlp Therapeutics, Llc Virus like particle comprising modified envelope protein E3
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