WO2015097268A1 - Construction chimérique pour le diagnostic et le traitement des cancers induits par le papillomavirus - Google Patents

Construction chimérique pour le diagnostic et le traitement des cancers induits par le papillomavirus Download PDF

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WO2015097268A1
WO2015097268A1 PCT/EP2014/079279 EP2014079279W WO2015097268A1 WO 2015097268 A1 WO2015097268 A1 WO 2015097268A1 EP 2014079279 W EP2014079279 W EP 2014079279W WO 2015097268 A1 WO2015097268 A1 WO 2015097268A1
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chimera
sequence
pdz
fusion polypeptide
polypeptide
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PCT/EP2014/079279
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Gilles TRAVE
Juan Ramon RAMIREZ RAMOS
François DERYCKERE
Juline POIRSON
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Universite De Strasbourg
Centre National De La Recherche Scientifique
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22071—Demonstrated in vivo effect

Definitions

  • the present disclosure relates to a chimeric fusion polypeptide composed of the E6-binding PDZ1 domain and the E6-binding LxxLL consensus sequence.
  • the chimera acts as a strong bi-functional ligand of E6, which binds simultaneously to the two oncogenic interaction surfaces of E6.
  • This chimeric construct is both suited to capture in vitro high-risk mucosal E6 proteins for diagnosis applications, and to block their oncogenic function in vivo.
  • Papillomaviruses infect the cutaneous or mucosal epithelia of vertebrates, with almost 200 PV types so far identified and sequenced (Bernard et al., Virology 2010). Whereas most papillomaviruses produce epithelial hyperplasias, a subset of types known as "high-risk mucosal Human papillomaviruses" (hrm-HPV s) have been recognized as the causative infectious agent for cervical cancer (Zur Hausen, Semin Cancer Biol 1999), and are also involved in a significant proportion of head and neck tumors (Gillison et al., Vaccine 2012).
  • HPV 16 Human Papillomavirus 16 is the most prevalent and best studied PV type associated with cervical cancer. HPV carcinogenesis is primarily linked to two PV oncoproteins, E6 and E7 (Ghittoni et al., Virus Genes 2010; Wise-Draper & Wells, Front Biosci. 2008). Two main oncogenic activities of hrm-HPV E6 have been extensively described both functionally and structurally. On the one hand, hrm-HPV E6 recruits the ubiquitin ligase E6AP and the tumor suppressor p53, leading to ubiquitin-mediated degradation of p53.
  • Hrm-HPV E6 interacts with, and sometimes promotes the destruction of, several cellular proteins containing "PDZ" domains, which regulate cell-cell adhesion, cell polarity and apoptosis.
  • Hrm-HPV E6 captures its target PDZ domains by means of a short "PBM” (PDZ Binding Motif) situated at the extreme C-terminus of E6.
  • PBM PDZ Binding Motif
  • E6 possesses two interaction surfaces responsible for two oncogenic activities of E6: formation of the E6-E6AP complex that degrades p53, and capture of PDZ containing proteins.
  • the PDZ-LxxLL chimera also appears to specifically recognize, with a strong binding affinity, wild-type E6 oncoproteins of hrm-HPV types.
  • the inventors have also demonstrated that a recombinant adenovirus expressing the PDZ-LxxLL chimera, when transduced in HPV -positive cell lines, induced the restoration of higher protein levels of P53 and of its transcriptional target p21 in HPV -positive cells as well as cleavage of Caspase 3, a typical marker of apoptosis induction. Furthermore, the transduced chimera specifically provoked death and detachment of HPV -positive cells. The transduced PDZ domain alone also up-regulated P53 and apoptosis markers and provoked cell death, albeit less efficiently than the chimera.
  • the present invention concerns, in a first aspect, a chimeric fusion polypeptide comprising a first amino acid sequence comprising a PDZ domain and a second amino acid sequence comprising a LxxLL consensus sequence.
  • the present invention concerns, in a second aspect, the chimeric fusion polypeptide for use in the treatment of Papillomavirus-induced cancers.
  • the present invention concerns, in a third aspect, the chimeric fusion polypeptide for a use in the diagnosis of Papillomavirus-induced cancers.
  • the present invention also deals with a polynucleotide encoding the polypeptide of the present invention, a nucleic acid construct comprising said polynucleotide, or a recombinant expression vector comprising the same, and uses thereof.
  • the E6 F47R 4C4S construct is a soluble mutant of HPV16 E6 (Zanier et al., Structure 2012) which contains one [Phe ->Arg] mutation (F47R) in the N-terminal domain and four [Cys ->Ser] mutations in the C-terminal domain.
  • the PDZ1 construct is the highly soluble construct, which was used to solve the NMR of structure of MAGI-1 PDZ1 domain in complex with E6 C-terminus (Charbonnier et al., J. Mol. Biol. 2011).
  • the chimeric fusion construct comprises said PDZ1 sequence fused at its C-terminus to a three-alanine linker followed by a 12- residue peptide corresponding to residues 403-414 of the E6AP, which contains the LxxLL consensus motif and which was previously used to solve the structure of the E6-E6AP complex (Zanier, Charbonnier et al., Science 2013).
  • the e6ap peptide ('e6ap' is written in small case letters to avoid confusion with the full-length protein E6AP) contains the triple alanine linker followed by residues 403-414 of ubiquitin ligase E6AP.
  • the e6ct peptide corresponds to the 11 last residues at the C-terminus of HPV16 E6.
  • Figure 2. Building up a chimeric bivalent binder of hrm-HPV E6.
  • a. Crystal structure of the E6-LxxLL complex (Zanier, Charbonnier et al., Science 2013).
  • the helical LxxLL motif of E6AP grey
  • the C-terminus PBM of E6 (e6-ct) is extended and flexible in its unbound state.
  • b NMR structure of the MAGI1 PDZ2/6/ e6ct peptide complex (Charbonnier et al, J. Mol. Biol. 2011).
  • the E6 PBM inserts into the peptide-binding groove of the PDZ domain.
  • c Model structure of the chimeric PDZ-LxxLL chimera.
  • d Model structure of the chimera bound to E6. The long linker between folded PDZ core and LxxLL motif allows simultaneous binding of PDZ to PBM and of LxxLL to the E6 pocket.
  • the few peaks of the chimera that do not find a match in PDZ1 spectrum must correspond to residues of the fused e6ap peptide plus a few PDZ residues at the junction with the e6ap peptide.
  • the few cross-peaks in the chimera's spectrum that find a match in the e6ap spectrum correspond to the e6ap moiety of the chimera.
  • the few peaks of the chimera that match the e6ap peptide peaks coincide with the ones that do not find a match in the PDZ1 spectrum (compare to panel A).
  • FIG. 5 Detection of chimera / E6 complex by NMR.
  • E6 HSQC spectrum of free chimera and of chimera / E6 complex.
  • most of the amide peaks of the chimera are displaced to new positions in the spectrum, indicating a dramatic change in the conformation and/or chemical environment of the chimera.
  • most cross peaks are widened, indicating an increase in molecular mass. Both observations unambiguously demonstrate that the chimera forms a specific complex with the E6 protein.
  • the peaks of the e6ap peptide essentially match to those peaks of the chimera-e6ct spectrum, which do not overlap with peaks of the chimera-E6 spectrum (instances indicated by red arrows). Therefore, when the chimera is bound to the e6ct peptide, which does not provide aLxxLL-binding pocket, the e6ap moiety of the chimera adopts a free conformation equivalent to that of the e6ap peptide.
  • the "blue-only" peaks of panel A do not match to peaks of the free e6ap peptide (see blue arrows). This confirms that these peaks likely correspond to peaks of the e6ap moiety bound to the LxxLL-pocket of E6.
  • FIG. 9 Provides the affinity enhancement induced by fusing PDZ to LxxLL: isothermal titration calorimetry (ITC) analysis.
  • ITC isothermal titration calorimetry
  • U20S (HPV-), SiHa (HPV16+) and Hela (HPV18+) cells were transduced for 48 hours with adenoviruses expressing GFP, myc-tagged PDZ or myc-tagged PDZ-LxxLL chimera.
  • Expression of p53, p21, PDZ and Chimera was analyzed by Western-blotting using appropriate monoclonal antibodies. P53 and P21 levels are specifically increased in SiHa and HeLa cells transduced by PDZ and chimera.
  • SiHa (HPV 16+) and Hela (HPV 18+) cells were transduced for 48 hours with adenoviruses expressing LacZ, myc-tagged PDZ or myc-tagged PDZ-LxxLL chimera, and were analyzed by immunofluorescence using appropriate antibodies.
  • Cleaved caspase 3 a marker of apoptosis, is specifically observed in cells producing PDZ and chimera. Most cells positive for active caspase 3 have smaller nuclei with brighter DAPI staining, which is also a hallmark of apoptosis.
  • the verb "to comprise” is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. According to restricted embodiments of the invention, the verb “to comprise” can also be interpreted as the expression “to consist essentially of or “to consist of, where the specified features are included only, unless otherwise stated.
  • hrm-HPVs refers to high-risk mucosal Human papillomaviruses, such as HPVs types 16, 18, 33, 35, 39, 52, 53, 58, or 66.
  • PDZ refers to the PDZ domain that is a common structural domain of 80-90 amino-acids found in the signaling proteins of bacteria, yeast, plants, viruses and animals, the term “PDZ” also refers to DHR (Dig homologous region) or GLGF (glycine-leucine-glycine -phenylalanine) domains.
  • PDZ domain-containing proteins There are about 266 human PDZ domains, though since several PDZ domain-containing proteins hold several domains, the number of PDZ domain-containing proteins is close to 140.
  • Some of the well-studied PDZ domain-containing proteins include hDlg, hScrib, MAGI-1, MAGI-2, and MAGI-3 (for "Membrane-Associated Guanylate kinase homology proteins with an inverted domain structure"), PSD95, TIP-1 and MUPP1.
  • MAGI-1 contains six PDZ domains. The second of the 6 PDZ domains, sometimes named PDZ1, or also named MAGI1 PDZ2/6, was the one used to build the chimeric polypeptide of the invention.
  • the papillomavirus is a mammalian papillomavirus, for example a human papillomavirus.
  • the papillomavirus genome is composed of six early (El , E2, E4, E5, E6, and E7) ORFs, two late (LI and L2) Open-Reading Frames (ORFs), and a non-coding long control region (LCR).
  • ORFs Open-Reading Frames
  • LCR non-coding long control region
  • E6 and E7 proteins inactivate two tumor suppressor proteins, p53 (inactivated by E6) and pRb (inactivated by E7).
  • E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in mammalians, including cervical or head and neck cancers.
  • Mammalian E6 proteins are cysteine-rich proteins consisting of two zinc- binding domains named E6N and E6C.
  • a "host cell” as used herein refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and the like.
  • Heterologous as used herein means "of different natural origin" or represents a non-natural state. For example, if a host cell is transformed with a DNA or gene derived from another organism, particularly from another species, that gene is heterologous with respect to that host cell and also with respect to descendants of the host cell which carry that gene. Similarly, heterologous refers to a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. a different copy number, or under the control of different regulatory elements.
  • a vector molecule is a nucleic acid molecule into which heterologous nucleic acid may be inserted which can then be introduced into an appropriate host cell.
  • Vectors preferably have one or more origin of replication, and one or more site into which the recombinant DNA can be inserted.
  • Vectors often have convenient means by which cells with vectors can be selected from those without, e. g., they encode drug resistance genes.
  • Common vectors include plasmids, viral genomes, and (primarily in yeast and bacteria) "artificial chromosomes.” Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
  • isolated means that the material is removed from its original environment (e. g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • transcriptional control sequence refers to DNA sequences, such as initiator sequences, enhancer sequences, and promoter sequences, which induce, repress, or otherwise control the transcription of protein encoding nucleic acid sequences to which they are operably linked.
  • transcriptional control sequences are any of those transcriptional control sequences normally found associated with a mammalian gene.
  • non-human transcriptional control sequence is any transcriptional control sequence not found in the human genome.
  • polypeptide is used interchangeably herein with the terms “polypeptides” and “protein(s)”. It includes the variants or any modification of amino acid sequences as described below.
  • a chimeric fusion polypeptide comprising an amino sequence comprising a PDZ domain and an amino acid sequence comprising a LxxLL consensus sequence.
  • the PDZ domain is a common structural domain of 80-90 amino- acids involved in cell polarity and adhesion.
  • the LxxLL consensus sequence is an amphipathic (hydrophobic- acidic) helix sequence comprising the LxxLL consensus sequence, in which some Leucins may be substituted by other hydrophobic amino acids and at least one x position should be a negatively charged amino acid (such as glutamic acid -E- or aspartic acid -D).
  • amphipathic helices with LxxLL consensus often mediate protein-protein interactions regulating cell motility, cell adhesion, and gene expression.
  • the chimeric fusion polypeptide both captures the C-terminus of mammalian E6 protein via the peptide -binding groove the PDZ domain, and inserts its own C-terminus, said e6ap helical motif, into the LxxLL-binding pocket of mammalian E6 protein.
  • the invention relates to a chimeric fusion polypeptide comprising an amino sequence comprising a LxxLL consensus sequence, an amino sequence comprising a PDZ domain, and optionally at least one additional linker.
  • the linker may be positioned between the amino sequence comprising a LxxLL consensus sequence and the amino sequence comprising a PDZ domain.
  • the linker is preferably a sequence of amino acids. Any amino acid sequence known to those skilled in the art may be used as the linker sequence so long as the linker sequence does not reduce, inhibit or otherwise interfere with functioning of the chimeric fusion polypeptide of the invention.
  • the linker sequence may include, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (such as 25) amino acids, preferably selected from amino acids with hydrophobic side chains, such as for instance Alanine, Leucine, or Isoleucine.
  • a particularly suitable linker sequence may be, for example, AAA, as defined below.
  • the additional linker sequence separates the amino sequence comprising a LxxLL consensus sequence and the amino sequence comprising a PDZ domain.
  • sequence comprising a LxxLL consensus sequence and the amino sequence comprising a PDZ domain are not separated by additional linker sequence but are directly adjacent to one another.
  • chimeric fusion polypeptide means that the PDZ domain and LxxLL consensus stem from different parent polypeptides.
  • a chimeric protein may also be called bi-functional molecule or chimera.
  • the PDZ domain comprises the amino acid sequence of SEQ ID NO:l
  • SEQ ID NO: 1 GAMGKPFFTRNPSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQIKSLVLD GPAALDGKMETGDVIVSVNDTCVLGHTHAQVVKIFQSIPIGASVDLELCRGYPLPFDPDDPNT SLVTSVAILDKEP
  • SEQ ID NO: l sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: l , wherein said sequence preferably binds to mammalian E6 protein.
  • the chimeric fusion polypeptide comprises an amino sequence comprising PDZ domain and an amino sequence comprising LxxLL consensus sequence separated by an additional linker sequence comprising from 1 to 25 amino acids.
  • a linker comprising 1 to 25 amino acids links PDZ Domain to LxxLL consensus sequence.
  • the linker comprises, preferably consists of, the amino acid sequence AAA.
  • the chimeric fusion polypeptide comprises, or consists of, or consists essentially of, an amino sequence comprising a LxxLL consensus sequence having at least 80% of sequence identity with SEQ ID NO: 2, and an amino sequence comprising a PDZ domain sequence having at least 80% of sequence identity with SEQ ID NO: 1, and optionally at least one additional linker, such as defined above.
  • the chimeric fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 3
  • the chimeric fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 4 (SEQ ID NO: 4
  • the chimeric fusion polypeptide comprises an amino sequence comprising LxxLL consensus sequence and an amino sequence comprising PDZ domain separated by an additional linker sequence comprising at 1 to 25 amino acids.
  • the chimeric fusion polypeptide comprises the PDZ sequence fused at its C-terminus or N-terminus, preferably at its C-terminus, to a three-alanine linker followed by a 12- residue peptide corresponding to residues 403-414 of the E6AP, which contains the acidic LxxLL consensus motif.
  • the chimeric fusion polypeptide comprises 12-residue peptide corresponding to residues 403-414 of the E6AP, which contains the acidic LxxLL consensus motif fused at its C- terminus or N-terminus, preferably at its C-terminus, to a three-alanine linker followed by a PDZ sequence.
  • the chimeric fusion polypeptide further comprises at least one additional sequence in addition to the PDZ domain and the LxxLL consensus sequence, wherein said additional sequence is appropriate for interacting with E6.
  • the chimeric fusion polypeptide comprises one additional sequence and is thus tri-functional.
  • said sequences may correspond to at least part of the sequence of known ligands of E6.
  • sequence identity between two amino acid sequences is described by the parameter "percentage of identity".
  • percentage of identity is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods, for instance, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • the percentage of identity may be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B).
  • the sequence identity is determined with respect to the full-length of the amino acid sequences of interest.
  • Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one may use, for example, the tool "Emboss needle” for pairwise sequence alignment of proteins providing by EMBL-EBI and available on
  • amino acid modifications which may be also termed “amino acid changes”, herein include amino acid mutations such as substitution, insertion, and/or deletion in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • amino acid insertion or “insertion” is meant the addition of an amino acid at a particular position in a parent polypeptide sequence.
  • amino acid deletion or “deletion” is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.
  • amino acid substitutions may be conservative.
  • a conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and/or hydrophobicity).
  • R-group residue having a side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:
  • valine-leucine -isoleucine phenylalanine- tyrosine
  • lysine-arginine alanine -valine
  • asparagine-glutamine a group consisting of: valine-leucine -isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine -valine, and asparagine-glutamine.
  • a parent polypeptide as used herein, it is meant an unmodified polypeptide that can be subsequently modified to generate a variant.
  • the parent polypeptide sequence may be a naturally-occurring PDZ domain sequence.
  • a variant polypeptide, polypeptide variant or variant:_as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification.
  • a variant is a variant of a naturally-occurring PDZ domain sequence and contain at least one functional or structural characteristic of a naturally- occurring PDZ domain sequence, such as binding to the E6 oncogenic protein of HPV.
  • a variant comprises from 1 to 30 amino acid modifications, preferably from 1 to 20 amino acid modifications.
  • the variant may have from 1 to 20 amino acid changes, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid changes as compared to its parent.
  • the variants may comprise one or several amino acid substitutions, and/or, one or several amino acid insertions, and/or one or several amino acid deletions. In some embodiments, the variant may comprise one or several conservative substitutions, e.g. as shown hereinabove. It should be understood that the definition of polypeptide of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, variants or derivatives of polypeptides of the invention that are described herein are also provided.
  • the derivatized polypeptides can comprise any moiety, molecule or substance that imparts a desired property to the polypeptide, such as increased half-life in a particular use.
  • the derivatized polypeptide can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the polypeptide for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses).
  • a detectable (or labeling) moiety e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (such as a magnetic or electrodense (e.g., gold) be
  • Examples of molecules that can be used to derivatize a polypeptide as described above include albumin (e.g., human serum albumin), polyethylene glycol (PEG), dextran, poly(n-vinyl pyrrolidone), propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
  • albumin e.g., human serum albumin
  • PEG polyethylene glycol
  • dextran poly(n-vinyl pyrrolidone)
  • propropylene glycol homopolymers polypropylene oxide/ethylene oxide co-polymers
  • polyoxyethylated polyols polyoxyethylated polyols
  • polyvinyl alcohols polyvinyl alcohols
  • the moiety can be a moiety facilitating its cellular uptake or entry, in particular a PTD (protein transduction domain); a homing peptide; a stabilizing agent such as PEG (polyethyleneglycol), oligo-N-methoxy- ethylglycine (NMEG), albumin, an albumin-binding protein or an immunoglobulin Fc domain; an affinity tag such as an immune-tag, biotin, lectin, or chelator; a detectable label such as an optical tag, a chelated lanthamide, a fluorescent dye, or a FRET acceptor/donor; a targeting moiety or a combination thereof.
  • the polypeptide can be part of a protein fusion.
  • PTD generally comprises a certain amino acid sequence of 10 to 20 amino acids (Matsushita and Matsui, (2005), J Mol Med 83, 324-328; Vives et al, Biochimic et Biophysica Acta, 2008, 1786, 126-138).
  • PTD is mainly composed of basic amino acids such as arginine or lysine
  • representative examples of the PTD include arginine rich peptides such as poly R 8 (RRRRRRRR, SEQ ID No 5) or (RRPRRPRRPRRPRRP, SEQ ID No 6), antennapedia or penetratin peptide such as (RQIKIWFQNRRMKWKK, SEQ ID No 7) or HIV-Tat (YGRKKRRQRRR, SEQ ID No 8).
  • the present invention relates to an isolated polynucleotide encoding the polypeptide of the present invention.
  • the nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in single stranded form or in duplex form or a mixture of the two. It can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to one skilled in the art, including chemical synthesis, recombination, and mutagenesis.
  • a polynucleotide encoding the polypeptide of the invention may be selected from the group consisting of nucleic acids that encode a chimeric fusion polypeptide as defined above, including any of the particular embodiments described above, said polypeptide comprising an amino sequence comprising a PDZ domain and an amino acid sequence comprising a LxxLL consensus sequence.
  • the polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO: 9 (SEQ ID NO: 9
  • the present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a chimeric fusion polypeptide according to the present disclosure, operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • control sequence(s) is/are heterologous to the polynucleotide encoding the polypeptide of the invention.
  • a polynucleotide may be manipulated in a variety of ways to provide for expression of the chimeric fusion polypeptide of the invention. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may include a promoter that is recognized by a host cell or an in vitro expression system for expression of a polynucleotide encoding the polypeptide of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • the present invention also relates to recombinant expression vectors comprising a nucleic acid construct as disclosed above, or a polynucleotide encoding the polypeptide of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptides at such sites.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • Virus vectors utilized in the present invention include, but are not limited to (a) retroviral vectors, including but not limited to vectors derived from Moloney murine leukemia virus (MoMLV); (b) adenovirus vectors; (c) adeno-associated vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; and (i) vaccinia virus vectors.
  • retroviral vectors including but not limited to vectors derived from Moloney murine leukemia virus (MoMLV);
  • adenovirus vectors adeno-associated vectors;
  • herpes simplex virus vectors SV40 vectors;
  • polyoma virus vectors polyoma virus vectors;
  • papillomavirus vectors papillomavirus vectors; (h) picornavirus vectors;
  • the recombinant vector is positioned adjacent to and under the control of, or is operably linked to, an expression promoter.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide encoding the polypeptide according to the present disclosure operably linked to one or more control sequences that direct the production of the polypeptide of the present invention.
  • a construct or vector comprising a polynucleotide encoding the polypeptide according to the present disclosure is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self -replicating extra-chromosomal vector as described earlier.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell depends upon the gene encoding the polypeptide and its source.
  • the host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote (Gram-positive or Gram-negative bacterium) or a eukaryote, such as a mammalian, insect, plant, or fungal cell, in particular yeast cell.
  • a prokaryote Gram-positive or Gram-negative bacterium
  • a eukaryote such as a mammalian, insect, plant, or fungal cell, in particular yeast cell.
  • the cell can thus be a mammalian cell, for example COS, CHO (US 4,889,803; US 5,047,335).
  • the cell is non-human and non-embryonic.
  • the polypeptide of the invention could be produced by a non-human transgenic animal, for instance in the milk produced by the animal.
  • the cell can be a plant cell.
  • the polypeptide of the invention could be produced by a transgenic plant. Any method known in the art for introducing DNA into a host cell can be used.
  • the host cells of the invention may be used for producing the polypeptide of the invention.
  • the present invention also relates to methods for producing the polypeptide of the present invention by using a host cell according to the invention.
  • the invention relates to a method for producing a polypeptide of the inventon, said method comprising:
  • the polypeptide may have one or several features as fully-described above.
  • the cell is cultivated in a nutrient medium suitable for production of the polypeptides of the invention using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation may take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. If the polypeptide is secreted into the nutrient medium, the enzyme can be recovered directly from the medium.
  • polypeptide If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the polypeptide may be recovered using methods known in the art.
  • the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, and/or precipitation.
  • the polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography, electrophoretic procedures, or extraction to obtain substantially pure polypeptide.
  • the polypeptide is not recovered, but rather a host cell of the present invention expressing the polypeptide, or a lysate thereof, is used as a source of the polypeptide.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a chimeric fusion polypeptide, or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode a polypeptide of the invention, according to the invention, optionally with a pharmaceutically acceptable carrier or excipient.
  • composition refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
  • the pharmaceutical composition comprises a chimeric fusion polypeptide, a polynucleotide, a nucleic acid construct, a recombinant expression vector, or a recombinant host cell according to the invention and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure is also directed to pharmaceutical compositions comprising the above- described chimeric fusion polypeptide that can bind to mammalian E6 protein, or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode a polypeptide of the invention, in a pharmaceutically acceptable carrier.
  • compositions comprising the above-described chimeric fusion polypeptide or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode the chimeric fusion polypeptide of the invention, for the treatment of mammalian papillomaviruses-induced cancers, in a pharmaceutically acceptable carrier, preferably by prevention of degradation of p53 by high-risk mucosal E6 protein.
  • pharmaceutically acceptable carrier refers to any solvent, diluent, or other liquid vehicle, dispersing or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant and others.
  • Any conventional pharmaceutical vehicle can be used in the context of the present invention, unless it is incompatible with the compounds of the invention, for example if it produces an undesirable biological effect or else if it interacts adversely with another component of the pharmaceutical composition.
  • materials that can serve as pharmaceutically acceptable vehicles comprise, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid or potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulphate, disodium phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, ethylene-polyoxypropylene polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as maize starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth in powder form, malt; gelatin; talc; excipients such
  • compositions according to the invention can be in various forms, notably in a form selected from the group comprising tablets, capsules, coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres and injectable solutions and solid lipid nanoparticles.
  • compositions according to the invention can be administered by various routes.
  • routes of administration we may mention the oral, rectal, cutaneous, pulmonary, nasal, sublingual route, the parenteral route notably intradermal, subcutaneous, intramuscular, intravenous, intraarterial, intraspinal, intraarticular, intrapleural, intraperitoneal, ocular, inhalation, transdermal, epidural, intrabronchial, intrabursal, intracameral, intracardiac, intracerebral, intracavernous, intracerebro ventricular, intracisternal, intragastric, intralesional, intralymphatic, intraosseous, intraspinal, intrathecal, intratracheal, intraduodenal, intratympanic, intraurethral, intrauterine, intravaginal, intravesical, intravitreal, sublabial, rectal, subconjunctival, retrobulbar, intratumoral in particular subconjunctival or retrobulbar, routes.
  • the pharmaceutical compositions of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, or by means of a catheter.
  • the agents are delivered to a tissue comprising cancerous tissue in the subject.
  • Dosages and desired drug concentrations of the disclosed pharmaceutical compositions may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician.
  • the term therapeutically effective amount as used herein refers to the amount needed to perform the particular treatment for a disease such as, for example, a cervical cancer.
  • nucleic acids comprising a sequence encoding the polypeptide of the invention are administered for the treatment of mammalian papillomaviruses-induced cancers, by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
  • the nucleic acid produces its encoded protein that mediates a therapeutic effect preferably by prevention of degradation of p53 by high-risk mucosal E6 protein. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • the therapeutic comprises a nucleic acid that is part of an expression vector that expresses the chimeric polypeptide of the invention in a suitable host.
  • such a nucleic acid has a promoter operably linked to the coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific.
  • nucleic acid molecule in which the polypeptide coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the polynucleotide of the invention.
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the polypeptide of the invention.
  • This can be accomplished by any of numerous methods known in the art, e. g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e. g., by infection using a defective or attenuated retroviral or other viral vector (adenovirus, adeno-associated virus and lentivirus), or by direct injection of naked DNA, or by use of microparticle bombardment (e.
  • a gene gun Biolistic, Dupont
  • coating with lipids or cell-surface receptors or transfecting agents encapsulation in liposomes, microparticle s, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (which can be used to target cell types specifically expressing the receptors), etc.
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor.
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA (as described herein) for expression, by homologous recombination.
  • a viral vector that contains the polynucleotide of the invention is used.
  • a retroviral vector can be used. These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
  • the polynucleotide of the invention be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
  • Adenoviruses are other viral vectors that can be used in gene therapy.
  • Adeno-associated virus AAV has also been proposed for use in gene therapy.
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient directly or after encapsulation.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcellmediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the resulting recombinant cells can be delivered to a patient by various methods known in the art.
  • epithelial cells are injected, e. g., subcutaneously.
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e. g., hematopoietic stem or progenitor cells
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e. g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • a polynucleotide of the invention is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem-and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.
  • stem cells include but are not limited to hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells, and neural stem cells.
  • Epithelial stem cells or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures. In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining of the gut provide for a rapid renewal rate of this tissue.
  • ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture. If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e. g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used. Retinal stem cells.
  • HSC hematopoietic stem cells
  • any technique which provides for the isolation, propagation, and maintenance in vitro of HSC can be used in this embodiment of the invention.
  • Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic.
  • Non-autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host/patient.
  • the invention refers to the use of a chimeric fusion polypeptide, or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode a polypeptide of the invention, or of a pharmaceutical composition according to the invention in the treatment of Papillomavirus-induced cancers.
  • the invention also relates to a chimeric fusion polypeptide, or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode a polypeptide of the invention or a pharmaceutical composition according to the invention, for use in the treatment of at least one Papillomavirus-induced cancer.
  • the invention also relates to a method for the treatment of at least one Papillomavirus-induced cancer, comprising the administration to a subject in need of such treatment of at least one chimeric fusion polypeptide, or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode a polypeptide of the invention or a pharmaceutical composition according to the invention.
  • the invention also relates to the use of a chimeric fusion polypeptide, or a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell able to encode a polypeptide of the invention or a pharmaceutical composition according to the invention, for the preparation of a medicament for the treatment of at least one Papillomavirus-induced cancer.
  • Papillomavirus-induced cancer refers to uncontrolled and abnormal cell multiplication and all the known diseases caused by such an uncontrolled and abnormal cell multiplication due to papillomavirus infection, and all the known diseases, head and neck cancer, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, and HPV -positive oropharyngeal cancer.
  • Papillomavirus-induced cancer refers more specifically to head and neck cancer or cervical cancer.
  • the term treatment refers to both therapeutic treatment and preventative measures, wherein the object is to interfere with the effects of oncogenic E6 protein on cellular function, more particularly to prevent degradation of p53 by mucosal E6 protein, more particularly by high risk mucosal E6 protein, such as E6 of HPVs 16, 18, 33, 35, 39, 52, 53, 58, or 66 types.
  • the treatment includes interfering with growth of cancer caused by HPV in a subject.
  • the treatment of the present invention prevents growth of cancer in a subject for which oncogenic E6 protein is detected.
  • the treatment of the present invention can slow or stop growth of Papillomavirus- induced cancer.
  • Non-limiting examples of therapeutic endpoints include partial remission, complete remission, a reduction in tumor size, stable tumor size, slowing of tumor growth, reducing the frequency of metastasis, prevention of metastasis for a period exceeding at least 3 months, at least 6 months, at least 9 months or at least 12 months, extension of expected life expectancy, prevention of recurrence of a cancer, extension of the expected time necessary for recurrence of cancer, and reducing the frequency or severity of one or more consequences of cancer, such as pain, edema, etc.
  • Continued administration beyond a given therapeutic endpoint can be utilized as a maintenance therapy to prevent cancer relapse.
  • the chimeric fusion polypeptide or polynucleotide may also be employed in combination therapies. That is, the compositions presently disclosed can be administered concurrently with, prior to, or subsequent to, one or more other desired compositions, therapeutics, treatments or medical procedures.
  • the particular combination of therapies administered will be determined by the attending physician and will take into account compatibility of the treatments and the desired therapeutic effect to be achieved. It will be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition, treatment or procedure, or alternatively may be administered separately.
  • compositions may be employed in combination with chemotherapeutic agents.
  • chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL®); betaTapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topote
  • compositions may be employed in combination with at least one antiviral agent.
  • antiviral agent as used herein is intended to mean an agent that is effective to inhibit the formation and/or replication of a virus in a mammal, including but not limited to agents that interfere with either host or viral mechanisms necessary for the formation and/or replication of a virus in a mammal.
  • antiviral agent as used herein is intended to mean an agent use in the control or prevention of human papillomavirus infection.
  • the antiviral agent can be an interferon, imiquimod, cidofovir, formaldehyde, glutaral, cimetidine, S-fluorouracil, trichloroacetic acid, bleomycin, podofilox or podophyllum.
  • the compositions may be employed in combination with human papillomavirus preventative vaccines therapy.
  • HPV preventive vaccines therapy include Gardasil® and Cervarix®.
  • the invention further relates to the use of a chimeric fusion polypeptide, polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell according to the invention for a use in the diagnosis (more specifically in vitro, ex vivo and/or in vivo diagnosis) of at least one papillomavirus-induced cancer, such as in particular head and neck cancer, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, or HPV -positive oropharyngeal cancer.
  • papillomavirus-induced cancer such as in particular head and neck cancer, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, or HPV -positive oropharyngeal cancer.
  • the chimeric fusion polypeptide, construct, recombinant vector or host cell is used for the diagnosis of papillomavirus-induced cancers, such as in particular head and neck cancer, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, or HPV-positive oropharyngeal cancer.
  • papillomavirus-induced cancers such as in particular head and neck cancer, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, or HPV-positive oropharyngeal cancer.
  • the invention deals with a method for determining if a subject is infected or not with an oncogenic strain of human papilloma virus (HPV), the method comprising detecting the presence of E6 protein in a sample from a subject using the chimeric polypeptide of the present invention, wherein the presence of oncogenic E6 protein indicates that the subject is infected with an oncogenic strain of HPV (on the contrary the absence of oncogenic E6 protein indicates that the subject is not infected with an oncogenic strain of HPV).
  • This method stems from the binding of the subject oncogenic E6 protein with the chimeric polypeptide of the present invention.
  • the chimeric fusion polypeptide or polynucleotide of the invention is used for detecting high-risk or possibly high-risk mucosal Human papillomaviruses (hrm-HPVs), and more particularly HPV 16, 18, 33, 35, 39, 52, 53, 58, or 66 types.
  • hrm-HPVs high-risk or possibly high-risk mucosal Human papillomaviruses
  • the detection or diagnosis may be employed to detect any strain of oncogenic HPV, and more particularly HPV 16, 18, 33, 35, 39, 52, 53, 58, or 66, and more specifically to detect the E6 protein from that strain.
  • One embodiment of the present invention includes the detection of oncogenic E6 proteins using the chimeric polypeptide of the invention.
  • Oncogenic E6 proteins can be detected by their ability to bind to chimeric polypeptide of the invention. This could be developed into a single detection stage approach or more favorably as a two-stage or "sandwich" approach for increased sensitivity and specificity.
  • the polypeptide of the invention may also bind endogenous cellular proteins. Thus, binding or frequency of binding must be compared to control tissues or cells that do not contain E6 oncoproteins.
  • the chimeric fusion polypeptide of the present invention may bind specifically to E6 polypeptide with high binding affinity.
  • a second stage detectable label may be used, for example a labeled antibody directed to one of the constituents of E6 protein.
  • the chimeric fusion polypeptide may be labeled, so as to be directly detectable, or will be used in conjunction with secondary detectable label which will specifically bind the compound for example, for detection or diagnosis purposes.
  • Labels of interest may include dyes, enzymes, chemiluminescers, particles, radioisotopes, or other directly or indirectly detectable agent.
  • the determination of the presence or the quantity (absolute or relative) of E6 protein can be carried out by various techniques, well known in the art, for example sandwich assays, lateral flow assay, immuno-enzymatic reactions, such as ELISA, RIA, EIA, etc.
  • detectable label refers to an atom (e.g., radionuclide), molecule (e.g., fluorescein), or complex, that is or can be used to detect (e.g., due to a physical or chemical property), indicate the presence of a molecule or to enable binding of another molecule to which it is covalently bound or otherwise associated.
  • label also refers to covalently bound or otherwise associated molecules (e.g., a biomolecule such as an enzyme) that act on a substrate to produce a detectable atom, molecule or complex.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Labels useful in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, and the like), radiolabels (e.g.,(3)H, (125) 1 , (35)S, (14)C, or (32)P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and others commonly used in ELISAs), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes
  • radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence- activated cell sorting).
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal generating system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal generating system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal generating system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally-occurring anti- ligands.
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Means of detecting labels are well known to those of skill in the art.
  • the label is a radioactive label
  • means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging.
  • the label is a fluorescent label
  • it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
  • the fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected by observing the color associated with the label. It will be appreciated that when pairs of fluorophores are used in an assay, it is often preferred that they have distinct emission patterns (wavelengths) so that they can be easily distinguished.
  • Diagnosing or detecting the presence of pathogens requires collection of samples appropriate to the organism.
  • tissue for testing more specifically said tissue may be collected from the cervix, penis, anus, or throat of a subject using a specific tool or technique, such as scrape, swab or biopsy technique.
  • the subject to be diagnosed or treated may be a mammal, e.g., a dog or cat, a rodent (e.g., mouse, guinea pig, or rat), or primate (e.g., a human, chimpanzee, or monkey).
  • the subject will be a human, particularly a male or female.
  • the subject may show symptoms of papilloma virus infection, may be suspected of being infected by papilloma virus (e.g., may contain cells that are cytologically consistent with such an infection) or may have already tested positive for papilloma virus.
  • the subject may have no indication of papilloma virus infection, and the above methods may be employed as part of a routine screen.
  • the invention relates to a kit or pharmaceutical pack that can be used in the above-described methods. More specifically, said kit or pack comprises a chimeric fusion polypeptide, a polynucleotide, a nucleic acid construct, a recombinant expression vector, a recombinant host cell according to the invention, more specifically which is able to encode a polypeptide of the invention, or a pharmaceutical composition comprising the same, in one or more containers.
  • kits or packs may optionally contain instructions for using the kit or the pack, as to treat at least one Papillomavirus-induced cancer, such as in particular head and neck cancer, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, or HPV-positive oropharyngeal cancer.
  • the compositions of the present invention may be employed in conjunction with other therapeutic compositions.
  • Other therapeutic compositions include cytotoxic drugs, particularly those which are used for cancer therapy.
  • a kit according to the invention comprises the chimeric fusion polypeptideor a recombinant vector, and one or more of the ingredients for detection of mammalian papillomavirus E6 protein, such as specific antibodies capable of recognizing specifically each high-risk mucosal HPV type, and optionally instructions for using the kit.
  • the E6 construct used in this study was the soluble mutant of HPV 16 E6 (151 residues) called E6 F47R 4C4S, and containing the following mutations: F47R, C80S, C97S, CI IS, CMOS (K. Zanier et al., Structure 2012). Cloning, expression and purification were performed as described in our previous works (K. Zanier et al, Structure 2012) (K. Zanier et al., Protein Expr Purif 2007). Briefly, the DNA sequence of the E6 construct was cloned in the Ncol and Kpnl sites of the E.
  • coli overexpression vector pETM-41 (provided by Gunter Stier, EMBL-Heidelberg) containing an N-terminal His6-MBP tag followed by a TEV cleavage site.
  • Overexpression of unlabelled full-length E6 fused to MBP was carried out overnight in BL21 DE3 cells at 15°C in LB medium.
  • MBP-E6 fusion proteins was purified using amylose affinity chromatography and separated from soluble aggregates by extensive ultracentrifugation as described previously (K. Zanier et al., Protein Expr Purif 2007) (ould M'hamed ould Sidi et al, Protein Expr Purif. 2011).
  • Buffer A 50 mM Tris, pH 6.8, 400 mM NaCl and 2 mM DTT was used throughout the purification. Monomeric E6 sample was isolated by further fractionation on a Superdex 75 16/60 gel-filtration column equilibrated in buffer A. E6 protein was exchanged to buffer B (see below) prior to RMN and ITC titration experiments.
  • MAGI-1 PDZ2/6 domain The wild-type PDZ2/6 domain corresponding to residues 456-580 of human MAGI-1, was cloned, expressed and purified as previously described (Charbonnier S. et al., Protein Expression Purif. 2008). Briefly, the coding sequence was inserted in the Ncol and Kpnl sites of the kanamycin-resistant E. coli overexpression vector pETM-41 (provided by Gunter Stier, EMBL-Heidelberg) containing an N- terminal His6-MBP tag followed by a TEV cleavage site. Both unlabelled and 15N-labelled samples were prepared as extensively described in Charbonnier et al. (Charbonnier S. et al., Protein Expression Purif.
  • the same buffer was used for the ITC and NMR experiments.
  • the gene of the chimera was constructed by raising a PCR fragment from the MAGI-1 PDZ2/6 domain (residues 456-580 of human MAGI-1), eliminating the original STOP codon at the 3' end of the PDZ2/6 construct, and was complemented by appropriate hybridized oligomers encoding for the AAAELTLQELLGEER extension, followed by a STOP codon (see also figures 1 and 2 describing the design of the chimera).
  • the full gene encoding for the chimera has the following DNA and protein sequences: (including a small N-terminal tag, left over after TEV proteolysis):
  • SEQ ID NO: 3 Protein:
  • the DNA sequence was cloned into the kanamycin-resistant pETM-30 vector (kindly provided by Gunter Stier, EMBL-Heidelberg) allowing the producion of the chimera fused to the C-terminus of 6his-GST tag via a linker sensitive to the TEV protease.
  • the construct was overexpressed at 15°C, either in LB or 15N-labelled minimal medium as previously described (Charbonnier et al., Prot Expr Purif 2008).
  • the purification protocol was reminiscent of that used for the PDZ2/6 construct with the following modifications: 1) Gluthatione affinity resin was used instead of amylose resin, 2) his-GST tag and his-tagged TEV protease were eliminated after TEV cleavage by flowing three times the TEV digested sample through Ni-NTA resin, 3) after the Ni-NTA step the sample was concentrated and loaded on a Superdex 75 16/60 gel-filtration column equilibrated in buffer B.
  • the concentration of E6 F47R 4C4S stock solution was determined by absorbance at 280 nm using as a molar absorption coeffient the following value: 21025cm "1 M "1 .
  • the concentrations of Chimera, PDZ2/6 and E6ct(l l) and E6ap(15) peptide stock solutions were determined by *H ID NMR PULCON method.
  • each titration began by addition of 0,3 ⁇ (non used for data adjustement) followed by 20 additions of 2 ⁇ during 4 seconds at intervals of 180 seconds.
  • the data of each titration were collected by the means of the 1TC 200 software.
  • the MicroCal Origin 7.0 program was used for data analysis to correct the baseline and to perform corrections using the control experiments.
  • the thermodynamic parameters ⁇ , AS, K f and n, were calculated using the one binding site model. Structural modeling
  • the chimeric E6 was first assembled using the software PyMol, the building utility in the software was used to move each part of the chimera in order to adjust the orientation of each chain.
  • the PDB file was then created using the CHARMM program and missing hydrogens added with the HBUILD facility (Brunger A. T. et al., Proteins 1988).
  • the final structure was then minimized with 50 steps of the steepest descent method in order to eliminate wrong contacts that could have resulted upon building of the conformation.
  • the coding sequences for Chimera-myc, PDZ-myc and e6ap-myc were PCR-amplified from the construct described above using primers.
  • the AttB flanking sequences in the PCR products allowed to recombine with pDONR207 using BP gateway recombination (Invitrogen).
  • the obtained clones were checked by sequencing before LR recombination with pAd/CMV/V5-DEST.
  • the adenovirus vectors (Ad Chimera myc, Ad PDZ myc and Ad e6ap myc) were produced after transfection of 293A cells as previously described (M. Lagrange M. et al., Biochem Biophys Res Commun. 2007).
  • AttB2 myc Chimera antisense SEQ ID NO: 11 ACCACTTTGTACAAGAAAGCTGGGTTCAGCCCAGATCCTCTTCTGAGATGAGTTTTTGTTC GCCGCCGCCACGTTCCTCGCCCAGCAATTC
  • AttB2 myc PDZ antisense SEQ ID NO: 12 ACCACTTTGTACAAGAAAGCTGGGTTCAGCCCAGATCCTCTTCTGAGATGAGTTTTTGTTC GCCGCCGCCTGGTTCTTTGTCCAAAATGGCCAC
  • Cells were seeded on glass coverslips in six-well plates and transduced at a MOI of lOOpfu/cell. After 48h, cells were fixed with 4% paraformaldehyde (in PBS) for 20 minutes at room temperature and permeabilized for 10 minutes with 0.1%Triton X100 in PBS. Primary antibodies against the myc epitope (9E10 monoclonal antibody) or P53 (Rabbit monoclonal antibody, cell signaling) were diluted in DMEM 10% FCS and incubated for 1 hour.
  • HEK-293T cells were grown and maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% FCS and 50 ⁇ g/ml of gentamycin at 37°C with 5% C0 2 and 95% humidity.
  • DMEM Dulbecco's Modified Eagle's Medium
  • ORFs encoding for the E6 protein from 17 HPV genotypes were amplified by PCR and cloned into vector pDONR207 by recombinatorial cloning system (Gateway recombinational cloning system, In itrogen). The resulting Entry clones were then transferred into Gateway compatible GPCA destination vector pSPICA-N2. ORFs encoding for PDZ domain of MAGI-1, e6ap peptide and chimera were also amplified by PCR and cloned into vector pDONR207 by recombinatorial cloning system (Gateway recombinational cloning system, Invitrogen).
  • GPCA vectors pSPICA-Nl and pSPICA-N2 (both derived from the pCiNeo mammalian expression vector) respectively express the Glucl and Gluc2 complementary fragments of the Gaussia princeps luciferase linked to the N-terminal ends of tested proteins by a flexible hinge polypeptide of 20 amino acid residues.
  • a Kozak consensus translation start sequence was included at the N-terminal end of the fusion protein (Cassonnet P. et al., Nat Methods. 2011). Sequence of DNA constructs were verified by GATC DNA sequencing.
  • Hela ws cells were seeded in white 96-well plates at a concentration of 2xl0 4 cells per well. After 24 h, cells were transfected using JetPEI® (Polyplus transfection) with 100 ng of pSPICA-N2 expressing E6 and 100 ng of pSPICA-Nl expressing PDZ domain, E6AP peptide or chimera. At 24 h post- transfection, cells were washed with 50 ⁇ of PBS and harvested with 40 ⁇ per well of Renilla Lysis Buffer (Promega, E2820) for 30 min.
  • JetPEI® Polyplus transfection
  • Gaussia princeps luciferase enzymatic activity was measured using a Berthold Centro LB960 luminometer by injecting 50 ⁇ per well of luciferase substrate reagent (Promega, E2820) and counting luminescence for 10 seconds. GPCA experiments were performed in triplicate. Results were expressed as a fold change normalized over the sum of controls, specified herein as Normalized Luminescence Ratio (NLR) (Cassonnet P. et al., Nat Methods. 2011).
  • NLR Normalized Luminescence Ratio
  • Example 1 Design of a PDZ-LxxLL fusion capable of binding simultaneously to the two binding surfaces of E6.
  • the inventors designed a chimeric PDZ-LxxLL fusion protein (Fig. 1 & Fig. 2) combining, from N- terminus to C-terminus, the MAGI1 PDZ1 construct described in their previous structural work (Charbonnier et al., J.Mol.Biol. 2011), a short three-residue alanine linker, and the LxxLL motif of E6AP (sequence ELTLQELLGEER, SEQ ID NO: 2) described in their recent structural analysis of the E6-E6AP complex (Zanier, Charbonnier et al., Science 2013).
  • Example 2 Cloning, production and purification of the PDZ-LxxLL fusion.
  • the construct was cloned into a bacterial overexpression vector, allowing the production of the PDZ- LxxLL construct fused to the C-terminus of GST (Gluthatione S transferase) via a TEV-sensitive linker.
  • the GST-PDZ-LxxLL construct was overexpressed in E. coli at 15°C, affinity purified on gluthatione affinity column, separated from the GST carrier protein by TEV proteolysis, and finally isolated by gel-filtration (Fig. 3).
  • the construct was highly soluble and was therefore amenable to NMR and calorimetric analysis.
  • the molecular mass of the isolated protein was experimentally determined by mass spectroscopy, and turned to be identical to the expected theoretical mass of the planned chimeric construct.
  • Example 3 Characterization of the PDZ-LxxLL chimera by NMR.
  • the PDZ-LxxLL construct was 15N-labelled and submitted to [1H,15N] HSQC measurements (Fig.4 A and B, blue spectrum).
  • the spectrum is highly pronounced of the HSQC spectrum of the MAGI1 - PDZ1 construct (Fig.4 A, red spectrum), with the exception of a few additional cross-peaks observed in the fusion construct.
  • Fig.4 A red spectrum
  • Example 4 Analysis of the chimera / HPV16 E6 interaction by NMR. Formation of the chimera / E6 complex: The inventors mixed a sample of 15N-labelled PDZ-LxxLL chimera with a non-labelled sample of HP16 E6 protein (the soluble mutant F47R 4C4S, which is amenable to structural and biophysical analysis by contrast to the wild-type E6 protein). The E6 molecule was provided in slight molar excess (1.2 / 1) to ensure full titration of the PDZ- LxxLLconstruct. The superimposition of the spectra of PDZ-LxxLL chimera alone and PDZ-LxxLL chimera in presence of E6 protein (Fig.
  • the inventors constituted a chimera-E6 complex by mixing 15N-labelled chimera with unlabelled E6 in a slight stoichiometric excess, as already described before (see Fig 6B). Next, they titrated the pre-formed complex with a 10-fold stoichiometric excess of e6ap peptide (SEQ ID NO: 17). The inventors then recorded a spectrum of the resulting chimera-E6-e6ap mixture.
  • Comparison of chimera-E6 and chimera-e6ct complexes The inventors next compared the spectra of the chimera-e6ct and chimera-E6 complexes. Remarkably, most cross peaks in the two spectra were superimposable, to the exception of a few cross peaks, which likely correspond, as discussed before, to the SEQ ID NO: 17 extension, either free in the chimera-e6ct complex, or bound to the LxxLL motif- binding pocket of E6 in the chimera-E6 complex. This result further confirms that the PDZ-LxxLL chimera captures the C-terminal motif of E6 in the same manner as the PDZ domain does.
  • the PDZ and LxxLL moieties When combined within the chimera, both retain their capability to interact with their cognate binding sites (E6 C-terminus and LxxLL binding pocket of E6, respectively).
  • E6 When E6 binds to the chimera, it induces on the PDZ moiety of the chimera exactly the same structural change as the one it induces on the isolated PDZ domain.
  • the inventors performed isothermal titration calorimetry (ITC) to determine the binding affinity of the bivalent chimera to E6, and to compare it to the binding affinities of the isolated PDZ and LxxLL moieties. Several control experiments were also performed.
  • ITC isothermal titration calorimetry
  • a condition for detecting binding signal by ITC for a protein A - protein B complex is that the concentration of protein A in the cuvette should be at least 2-3 fold superior the K D of the complex, whereas the concentration of protein B in the titrating solution should be 10-fold superior to the concentration of protein A.
  • the titrating protein (E6) could not be raised at higher concentrations than 122 ⁇ because of its aggregating tendencies.
  • ITC data The ITC data unambiguously demonstrate that: 1) The fusion of two mild-affinity binders of the E6 protein, respectively the PDZ1 domain of
  • MA Gil protein and the LxxLL (or e6ap) motif of E6AP has generated a high-affinity binder of E6.
  • the strong binding affinity of the chimera-E6 complex is specifically conferred by the genetic fusion that was operated between PDZ and LxxLL moieties in the chimera, since no enhancement of binding affinity was observed when the interaction of E6 with a mixture of unfused PDZ domain and e6ap peptide was probed.
  • Example 6 Effect of Adeno virus-mediated intracellular expression of the PDZ-LxxLL chimera on p53 levels in HPV -positive Hela cells:
  • the inventors constructed recombinant noninfectious adenoviruses expressing the PDZ-LxxLL chimera, as well as control adenoviruses expressing either the PDZ domain or the LxxLL (e6ap) peptide. All constructs were equipped with a myc tag to enable their detection. Next, the inventors transduced separate batches of Hela cells with the three recombinant adenoviruses. Hela cells are a HPV 18 -positive cell line originating from a cervical cancer.
  • HPV18 E6 which (like all hrm-HPV E6 proteins) interacts with the ubiquitin ligase E6AP to promote the accelerated destruction of p53.
  • the inventors also hypothesized that, since the chimera contains the e6ap peptide, it should lock the E6AP-binding pocket of E6 and competitively block the interaction of HPV18 E6 with E6AP within the transduced Hela cells.
  • the inventors After having transduced the three cell batches with the three different adenoviruses, the inventors detected by immunofluorescence, via the myc-tag fused to the recombinant constructs, the cells expressing these constructs. While inventors could observe cells expressing either the PDZ domain or the chimera; they did not observe cells expressing the e6ap peptide. This may be due to the small size of that peptide, which may promote its rapid turnover. The inventors also analyzed the expression status of p53 in the cells expressing either the chimera or the PDZ domain. Whereas the cells expressing the PDZ domain displayed a hardly detectable p53 signal, the cells expressing the chimera displayed a highly enhanced p53 signal (Fig. 11 A).
  • Example 7 Additional cellular data To probe the potential of the chimera as an in vivo inhibitor of E6 oncogenic functions, Hela (HPV18 positive) and Caski (HPV16 positive) cell lines originating from cervical or head and neck cancers were transduced with a recombinant non-infectious adenovirus (AdV) expressing the PDZ-LxxLL chimera. hrm-HPV positive cells naturally express E6 oncoproteins, which interact with the ubiquitin ligase E6AP to promote the accelerated destruction of P53 and the subsequent inactivation of pro- apoptotic pathways.
  • AdV non-infectious adenovirus
  • the transduced chimera induced the restoration of higher protein levels of P53 and of its transcriptional target p21 in HPV -positive cells (Fig. 11 A) as well as cleavage of Caspase 3, a typical marker of apoptosis induction (Fig. 11B). Furthermore, the transduced chimera specifically provoked death and detachment of HPV -positive cells (Fig. 11 C and D). The transduced PDZ domain alone also up-regulated P53 and apoptosis markers and provoked cell death, albeit less efficiently than the chimera (Fig. 11 C and D).

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Abstract

L'invention concerne un polypeptide de fusion chimérique comprenant le domaine PDZ de liaison à E6 et la séquence consensus Lxx LL de liaison à E6. La chimère agit en tant que ligand bifonctionnel fort de E6, qui se lie simultanément à deux surfaces d'interaction oncogène de E6. La construction chimérique selon l'invention est adaptée à la capture de protéines E6 des muqueuses à haut risque dans des applications diagnostiques et thérapeutiques.
PCT/EP2014/079279 2013-12-23 2014-12-23 Construction chimérique pour le diagnostic et le traitement des cancers induits par le papillomavirus WO2015097268A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002000A1 (fr) * 1994-07-08 1996-01-25 New England Medical Center Hospitals, Inc. Proteines de liaison des proteines e6
WO2004022006A2 (fr) * 2002-09-09 2004-03-18 Arbor Vita Corporation Procede de diagnostic du cancer du col de l'uterus
WO2004076646A2 (fr) * 2003-02-27 2004-09-10 Arbor Vita Corporation Methodes et compositions destinees a traiter le cancer du col uterin
EP2108657A1 (fr) * 2008-04-08 2009-10-14 DKFZ Deutsches Krebsforschungszentrum Peptides pour inhiber l'oncoprotéine HPV-E6
WO2010127324A2 (fr) * 2009-04-30 2010-11-04 Arbor Vita Corporation Protéine contenant un domaine pdz chimérique pour une détection virale

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002000A1 (fr) * 1994-07-08 1996-01-25 New England Medical Center Hospitals, Inc. Proteines de liaison des proteines e6
WO2004022006A2 (fr) * 2002-09-09 2004-03-18 Arbor Vita Corporation Procede de diagnostic du cancer du col de l'uterus
WO2004076646A2 (fr) * 2003-02-27 2004-09-10 Arbor Vita Corporation Methodes et compositions destinees a traiter le cancer du col uterin
EP2108657A1 (fr) * 2008-04-08 2009-10-14 DKFZ Deutsches Krebsforschungszentrum Peptides pour inhiber l'oncoprotéine HPV-E6
WO2010127324A2 (fr) * 2009-04-30 2010-11-04 Arbor Vita Corporation Protéine contenant un domaine pdz chimérique pour une détection virale

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DAVID BILDER ET AL, NATURE CELL BIOLOGY, vol. 2, no. 7, 1 July 2000 (2000-07-01), pages E114 - E114, XP055176579, ISSN: 1465-7392, DOI: 10.1038/35017119 *
J. M. SPANGLE ET AL: "Activation of Cap-Dependent Translation by Mucosal Human Papillomavirus E6 Proteins Is Dependent on the Integrity of the LXXLL Binding Motif", JOURNAL OF VIROLOGY, vol. 86, no. 14, 2 May 2012 (2012-05-02), pages 7466 - 7472, XP055176084, ISSN: 0022-538X, DOI: 10.1128/JVI.00487-12 *
KAZUNORI NAGASAKA ET AL: "Human homolog of Drosophila tumor suppressor Scribble negatively regulates cell-cycle progression from G1 to S phase by localizing at the basolateral membrane in epithelial cells", CANCER SCIENCE, vol. 97, no. 11, 1 November 2006 (2006-11-01), pages 1217 - 1225, XP055176591, ISSN: 1347-9032, DOI: 10.1111/j.1349-7006.2006.00315.x *
SHIN TAKIZAWA ET AL: "Human scribble, a novel tumor suppressor identified as a target of high-risk HPV E6 for ubiquitin-mediated degradation, interacts with adenomatous polyposis coli", GENES TO CELLS, vol. 11, no. 4, 1 April 2006 (2006-04-01), pages 453 - 464, XP055176651, ISSN: 1356-9597, DOI: 10.1111/j.1365-2443.2006.00954.x *
SUSANNE DYMALLA ET AL: "A novel peptide motif binding to and blocking the intracellular activity of the human papillomavirus E6 oncoprotein", JOURNAL OF MOLECULAR MEDICINE, SPRINGER, BERLIN, DE, vol. 87, no. 3, 21 December 2008 (2008-12-21), pages 321 - 331, XP019681201, ISSN: 1432-1440 *

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