WO2008097854A2 - Procédé et substances pour traiter une infection par le vph - Google Patents

Procédé et substances pour traiter une infection par le vph Download PDF

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WO2008097854A2
WO2008097854A2 PCT/US2008/052819 US2008052819W WO2008097854A2 WO 2008097854 A2 WO2008097854 A2 WO 2008097854A2 US 2008052819 W US2008052819 W US 2008052819W WO 2008097854 A2 WO2008097854 A2 WO 2008097854A2
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fadd
hpv
polypeptide
binding
protein
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PCT/US2008/052819
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WO2008097854A3 (fr
WO2008097854A4 (fr
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Penelope J. Duerksen-Hughes
Maria Filippova
Sandy S. Tungteakkhun
Jonathan W. Neidigh
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Loma Linda University
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Publication of WO2008097854A4 publication Critical patent/WO2008097854A4/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • E6 is best known for its ability to associate with the cellular protein E6AP, which together with E6 forms the E3 ubiquitin-protein ligase complex responsible for directing the degradation of p53. This ability of E6 is essential for ensuring cellular survival and for promoting viral propagation.
  • E6 interacts with a wide array of other cellular proteins.
  • protein partners include those involved in the regulation of transcription and DNA replication such as CBP/p300, IRF-3, hMcm7, E6TP1, and AD A3; proteins involved in apoptosis such as Bak, and c-Myc; proteins involved with epithelial organization and differentiation such as paxillin, E6BP/ERC-55, zyxin, and fibulin-1; proteins involved in cell-cell adhesion, polarity, and proliferation control such as hDLG, hScrib, MAGI-I, MAGI-2, and MAGI-3; and proteins involved in DNA repair such as XRCCl and O-6-methylguanine-DNA methyltransferase.
  • the extrinsic pathway of apoptosis is initiated by binding of a ligand such as TNF- ⁇ or Fas-L to its respective cell surface receptor, giving rise to formation of the death-inducing signaling complex (DISC).
  • DISC death-inducing signaling complex
  • FADD adaptor protein
  • DDs death domains
  • FADD death effector domain
  • DED death effector domain
  • E6 binds to this set of proteins via their PDZ domains.
  • the significance of this domain in E6 binding has been demonstrated through the creation of deletion and site-directed mutants. Manipulation of the residues that make up the PDZ domain render E6 unable to interact with its respective partners. Therefore, to date, two E6-binding motifs have been identified. Interestingly, these motifs are absent from the sequence of FADD, although these two proteins do indeed interact in both an in vitro as well as an in vivo system. This points to the existence of an additional binding motif for E6.
  • the polypeptide includes a FADD protein fragment having an amino acid sequence corresponding to amino acid residues one to 23 of SEQ ID NO:1.
  • a minimal E-6 binding domain having an amino acid sequence corresponding to amino acid sequences 15 to 19 of SEQ ID NO:1 and a synthetic peptide comprising a minimal E- 6 binding domain and a trp-cage to stabilize the molecule (SEQ ID NO:3).
  • a conjugate comprising: (a) a polypeptide as described herein; and (b) a solid particle, carrier protein or label linked to the polypeptide.
  • an expression vector comprising a promoter operably linked to a polynucleotide encoding the polypeptide containing an E6-binding domain.
  • the expression vector may further comprise a polynucleotide encoding a heterologous protein fused in frame with said polypeptide.
  • host cell transformed with the expression vector, which is capable of expressing a polypeptide containing the E6-binding domain or a related fusion protein.
  • composition is suitable for use in treating an HPV infection, comprising an effective amount of the polypeptide and a pharmaceutically acceptable carrier, wherein the effective amount is an amount sufficient to inhibit E6 binding to FADD.
  • composition comprises the expression vector described herein, which can express a polypeptide containing an E6-binding domain, and a carrier.
  • the HPV-infected cell is infected with a high risk strain of HPV, such as HPV type 16 or type 18 and/or the HPV-infected cell is a cancer cell.
  • N-terminal amino acids of FADD amino acid one to 83 of SEQ ID NO:1, Dl (SEQ ID NO:4), D2 (amino acids 80 to 83 of SEQ ID NO:1), SELT (SEQ ID NO:5), SSLS (SEQ ID NO:6), and SLT2 (SEQ ID NO:7).
  • Figure 2 depicts the SELT sequence that is conserved in E6AP (SEQ ID NO: 1
  • FADD amino acid residues 9 to26 of SEQ ID NO:1
  • Figure 2B shows the residues that mediate E6 binding reside in the N-terminus of FADD.
  • Glutathione bead-bound GST- E6 was used to pull-down bacterially expressed and purified FADD variants D2, Dl, SELT, SSLS, and SLT2.
  • Top panel following incubation of the indicated FADD constructs with GST-E6 (lanes 5-9), beads were washed, bound proteins eluted from the beads, and the proteins separated by SDS-PAGE. After the proteins were transferred to a membrane, the membrane was blotted with antibodies against FADD. Lanes 1-4 shows that equivalent amounts of the indicated FADD variant were used for each incubation. Input for protein D2 was demonstrated on a separate membrane. The same membrane was stripped and reblotted with antibodies against GST (bottom panel).
  • Figure 3 shows the five amino acid mutations in SLT2 inhibit
  • Figure 3A shows that introducing five mutations in FADD blocks E6 binding in cells and prevents E6-mediated FADD degradation.
  • U2OS cells stably expressing epitope-tagged hemagglutinin (HA)-E6, U2OSE64A and U2OSE612, were transfected with pcDNA FADD (lanes 1-2) or pcDNA SLT2 (lanes 3-4), coding for either the wild type FADD protein or the mutant SLT2 protein, respectively. After forty-eight hours, cell lysates were prepared, proteins separated by SDS-PAGE and the subsequent membrane was blotted with antibodies against FADD to analyze FADD expression (top panel). The same membrane was stripped and reblotted with antibodies against ⁇ -actin to demonstrate that equivalent amounts of Iy sate were used for analysis (bottom panel).
  • HA epitope-tagged hemagglutinin
  • Figure 4 shows that the five amino acid mutations in SLT2 inhibit procaspase 8 binding and the mediation of apoptosis.
  • Figure 6A shows E6 cannot bind to SLT2, SLT3, or SLT4 and thus, does not mediate the accelerated degradation of the mutant FADD proteins.
  • Top panel U2OSE6tet24 cells cultured with 0 or 100 ng/ml doxycycline were transfected with plasmids encoding wild type FADD, the SLT2 mutant, the SLT3 mutant, or the SLT4 mutant FADD protein. Twenty-four hours post-transfection, cell lysates were prepared for immunoblot and subsequent membranes were blotted with antibodies against FADD to analyze FADD content.
  • Densitometric analysis of protein bands was performed using the Chemilmager 4400TM (Alphalnnotech Corp.). Error bars represent the standard deviation obtained from three separate experiments.
  • Figure 6C shows the amino acids mutated in SLT3 and SLT4 do not eliminate the ability of FADD DED to bind to procaspase 8 DED.
  • Sequences encoding procaspase 8 DED, wild type FADD DED, the SLT2 mutant, the SLT3 mutant, or the SLT4 mutant FADD DED were cloned into the bait or prey plasmids of a mammaliam two-hybrid kit (Stratagene).
  • the indicated combination of plasmids, along with a plasmid encoding the luciferase reporter gene were transfected into cells that express E6 under the control of the tetracycline/doxycycline response element. Cells were treated with the indicated concentrations of doxycycline to regulate E6 expression. Luciferase expression was measured using a luminometer to detect chemiluminescence. Measurements were made in triplicate, and the error bars represent the standard deviation.
  • Figure 6D shows the amino acids mutated in SLT3 and SLT4 do not inhibit the ability of cells to undergo apoptosis.
  • U2OS cells were transfected with either pcDNA (empty vector), pcDNA FADD, pcDNA SLT2, pcDNA SLT3, or pcDNA SLT4. Twenty-four hours post-transfection, cell viability was measured via the MTT assay. Measurements were made in triplicate, and the error bars represent the standard deviation.
  • Figure 7 shows the peptides can be used to block the interaction between E6 and FADD in vitro.
  • Figure 7A shows two peptide inhibitors designed and synthesized to obstruct binding between E6 and FADD based on the localization of the E6 binding domain to the N-terminus of FADD.
  • the relevant amino acid sequences are set forth in the Sequence Listing as follows: FADD (amino acid residues 9 to26 of SEQ ID NO:1), which display a short region of homology with E6AP, Peptide A (SEQ ID NO:3) and Peptide B (SEQ ID NO: 11).
  • Figure 7B shows peptide inhibitors that mimic the E6 binding domain on FADD can inhibit E6/FADD interaction.
  • Glutathione bead-bound GST-E6 was used to pull-down bacterially expressed and purified wild type FADD (Dl) protein in the absence (lane 2) or presence of peptides B (lanes 3-4) or A (lanes 5-6). Two different concentrations of the peptides were used for the pull-down, 10 ⁇ M and 25 ⁇ M.
  • Top panel following SDS-PAGE separation of proteins and transfer to membranes, blots were probed with antibodies against FADD.
  • Bottom panel the same membrane was stripped and reprobed with antibodies against GST.
  • Lane 1 shows the amount of protein Dl used for each pull-down.
  • FIG 8A shows E6 binding was localized to the N-terminal 23 amino acids of FADD DED.
  • the pcDNA3 FADD plasmid was digested with the DNA restriction enzymes Sac I and Xba I to remove the region of FADD that lies outside the E6 binding domain. This gave rise to the pcDNA 23 amino acid (aa) plasmid, which expresses a fusion protein containing the amino acid residues set forth in SEQ ID NO: 12.
  • Figure 8B shows expression of the implicated E6 binding domain on FADD re-sensitizes cells to Fas-induces apoptosis.
  • E6-binding domain refers to a polypeptide domain demonstrated herein to be sufficient to specifically or selectively bind the human papilloma virus E6 oncoprotein.
  • biologically active fragment refer to a peptide or polypeptide in accordance with the above description of polypeptides containing an E6-binding domain, wherein the peptide interacts specifically with E6, obstructs binding between E6 and FADD, regulates E6-mediated FADD degradation and/or regulates the cellular response to apoptotic signals in E6 expressing cells.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • heterologous as it relates to proteins, denotes an amino acid sequence not normally associated with another peptide, polypeptide or protein and is not a naturally occurring component of the other protein found in nature.
  • heterologous as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally associated with a region of a recombinant construct, and/or are not normally associated with a particular cell.
  • a “heterologous" region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
  • a heterologous region of a construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene).
  • a host cell transformed with a construct which is not normally present in the host cell would be considered heterologous for purposes of this invention.
  • induce expression refers to an increase in the transcription and/or translation of a gene or cDNA.
  • a polypeptide containing the E-6 binding domain in accordance with the present invention, should generally be less than 65 amino acid residues in length or at least five or six amino acid residues in length, and may contain up to about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30 about 35, about 40, about 45, about 50 about 55 or about 60 residues or so.
  • An FADD protein fragment containing an E6-binding domain should generally be at least five or six amino acid residues in length, and may contain up to about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or about 23 residues or so.
  • Preferred embodiments of the polypeptide include a fragment of 5 to 23 consecutive amino acid residues contained within the first 23 amino residues of SEQ ID NO:1.
  • a particularly preferred embodiment of the polypeptide includes an FADD fragment having an amino acid sequence corresponding to amino acid residues one to 23 of SEQ ID NO:1.
  • the polypeptide contains one or more conservative amino acid substitutions to amino acids residues one to 14 and/or amino acids 20 to 23 of SEQ ID NO:1.
  • a polypeptide containing an E6-binding domain further comprising additional amino acid residues unrelated to the FADD protein.
  • the polypeptide may further comprise a "tag," such as tat peptide, derived from the HIV tat protein, or the 3E10FV fragment of an anti-DNA antibody, to facilitate entry of the polypeptide into cells.
  • the FADD fragment may be appended at the N- terminus or the C-terminus to include additional residues that provide structural stability, such as a trp-cage or the SEDE sequence. See, e.g., Nommeh et al., Nature Structural Biology 9: 425 (2002), which is incorporated herein by reference in its entirety.
  • This peptide includes a minimal E6 binding domain, i.e., LSSSE, and a trp cage, i.e., WLKGGPSSGRPPS.
  • a polypeptide within the scope of this invention can be an altered form of an FADD fragment providing generally of course that the essential biological activity of the polypeptide remains substantially unchanged.
  • altered form refers to a peptide that has been treated to change its naturally occurring structure.
  • An altered form can include a conjugate, which can be prepared, for example, by covalent modification of an FADD peptide fragment, by crosslinking an FADD peptide fragment to an insoluble support matrix, or by crosslinking an FADD peptide fragment to a carrier protein.
  • a polypeptide within the scope of this invention can be a fusion protein containing a FADD peptide fragment attached to a heterologous protein.
  • the heterologous protein has an amino acid sequence that is not substantially similar to any portion of the FADD protein. More particularly, the heterologous protein does not include the DED (amino acid residues 27 to 59 of SEQ ID NO:1) or DD (amino acid residues 100 to 179 of SEQ ID NO:1) domains of FADD.
  • the heterologous protein can be fused to the N-terminus or C-terminus of the E-6 binding fragment of FADD.
  • Peptidomimetics are commonly understood in the pharmaceutical industry to include non-peptide drugs having properties analogous to those of the mimicked peptide.
  • the principles and practices of peptidomimetic design are known in the art and are described, for example, in Fauchere J., Adv. Drug Res. 15: 29 (1986); and Evans et al., J. Med. Chem. 30: 1229 (1987).
  • Peptidomimetics which bear structural similarity to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • such peptidomimetics have one or more peptide linkages optionally replaced by a linkage, which may convert desirable properties such as resistance to chemical breakdown in vivo.
  • Peptidomimetics may exhibit enhanced pharmacological properties (biological half life, absorption rates, etc.), different specificity, increased stability, production economies, lessened antigenicity and the like which makes their use as therapeutics particularly desirable.
  • E6-binding domain mimetics or binding molecules may be carried out by those of skill, using known methods of rational drug design.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs, which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.
  • Covalent modifications can be introduced into a peptide by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Covalent modification of polypeptides using organic derivatizing agents is well known to those of skill in the art.
  • cysteinyl residues can be reacted with ⁇ haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Histidyl residues can be derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0, or with para- bromophenacyl bromide at pH 6 in 1 M sodium cacodylate.
  • Lysinyl and amino terminal residues can be reacted with succinic or other carboxylic acid anhydrides.
  • Arginyl residues can be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
  • Spectral labels can be introduced into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane; most commonly, N- acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3- nitro derivatives, respectively.
  • Carboxyl side groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimides (R'- N-- C-- N-- R') such as 1- cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3 (4 azonia 4,4- dimethylpentyl) carbodiimide.
  • carbodiimides R'- N--- C--- N-- R'
  • carbodiimides Rosinyl or glutamyl
  • aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues can be deamidated to the corresponding glutamyl and aspartyl residues.
  • the vector can be a cloning vector for maintaining nucleic acid molecules, or an expression vector.
  • a variety of cloning and expression vectors are well known to those of skill in the art. Examples include plasmid vectors, single or double stranded phage vectors, single or double stranded DNA or RNA viral vectors, or artificial chromosomes, such as BAC and YAC.
  • An expression vector contains a nucleotide sequence encoding a polypeptide comprising an E6-binding domain, as described herein, operably linked to a promoter. Promoters, terminators and other regulatory regions suitable for controlling transcription and translation in a variety of prokaryotic and eukaryotic host cells are well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989).
  • Longer peptides or polypeptides can be produced by standard recombinant DNA techniques.
  • a DNA fragment encoding an FADD fragment can be cloned in a commercially available expression vector that already contains a heterologous protein, with the result being the FADD fragment fused in-frame to the heterologous protein.
  • a nucleic acid encoding an FADD fragment containing an E6-binding domain and/or a component described herein may be used, for example, to produce a polypeptide in vitro or in vivo for the various compositions and methods of the present invention.
  • a nucleic acid encoding a polypeptide containing an E6-binding domain or a fusion protein is a component of, for example, a vector in a recombinant cell.
  • the nucleic acid may be expressed to produce a peptide or polypeptide comprising an E6-binding domain.
  • the peptide or polypeptide may be secreted from the cell, or as part of or within the cell.
  • One embodiment of the present invention further provides a host cell containing an expression vector of this invention.
  • the host cell can be a mammalian cell, plant cell, insect cell, yeast and other fungi, or bacteria. Suitable host cells for various expression vectors are well known to those of skill in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • An expression vector can be introduced into a suitable host cell by techniques such as calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques well known to those in the art (Sambrook, et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989).
  • compositions of the present invention are prepared in conventional dosage unit forms by the incorporation of one or more of the polypeptides with an inert, non-toxic pharmaceutical "carrier" moiety according to accepted methodologies, in a non-toxic concentration sufficient to produce the desired physiological activity in a mammal and, in particular, a human subject.
  • the composition contains the active ingredient in a biologically active, but non-toxic, concentration. The concentration utilized will be dependent upon such factors as the overall specific biological activity of the ingredient, specific biological activity desired, as well as the condition and body weight of the subject.
  • the pharmaceutical carrier or vehicle employed may be, for example, a solid or liquid and a variety of pharmaceutical forms may be employed.
  • a solid carrier when a solid carrier is utilized, the preparation may be plain milled, micronized in oil, tabulated, placed in a hard gelatin or enterically-coated capsule in micronized powder or pellet form, or in the form of a troche, lozenge, or suppository.
  • the solid carrier, containing the polypeptide can also be ground up prior to use.
  • the preparation When utilized in a liquid carrier, the preparation may be in the form of a liquid, such as an ampule, or as an aqueous or non-aqueous liquid suspension.
  • the active ingredient may be formulated using bland, moisturizing bases, such as ointments or creams.
  • suitable ointment bases include, but are not limited to, petrolatum plus volatile silicones, lanolin, and water in oil emulsions such as Eucerin.®. (Beiersdorf).
  • suitable cream bases include, but are limited to, Nivea Cream.®. (Beiersdorf), cold cream (USP), Purpose Cream.®. (Johnson & Johnson), hydrophilic ointment (USP), and Lubriderm.®. (Warner-Lambert) .
  • the active ingredient may be applied internally at or near the affected site.
  • a solid or gelled medium which is sufficiently permeable to allow the release of the active ingredient, preferably in a timed-release manner, may be utilized for such internal application.
  • gels include, but are not limited to, hydrogels such as polyacrylamide, agarose, gelatin, alginate, or other polysaccharide gums.
  • the active ingredient may be imbedded in a solid material, such as filter paper, which is capable of absorbing and subsequently releasing the active ingredient, at the appropriate time and location.
  • a polypeptide comprising an E6-binding domain according to the present invention may be provided for use in any suitable form appropriate to the protocol of administration and/or the needs of a patient.
  • the peptides may for example be provided, either singly or in combination, in lyophilized or freeze dried solid forms.
  • E6-binding domain such as amino acid residues 15 to 19 or one to 23 of SEQ ID NO: 1, as well as other small peptide derivatives that constitute a "minimal" E6-binding domain, are demonstrated herein to modulate the biological functions associated with the E6-FADD interaction.
  • the biological activity of a polypeptide or fusion protein can be determined using assays well known to one of skill in the art, with reference to the present disclosure.
  • assays well known to one of skill in the art, with reference to the present disclosure.
  • the ability of a particular peptide, polypeptide or protein to selectively bind and/or interact with E6 oncoprotein can be determined by a protein binding assay.
  • protein binding assays include the in vitro pull down assay, AlphaScreenTM analysis or mammalian two hybrid assay (Stratagene), described in further detail in the examples of the present disclosure.
  • the ability of a particular peptide, polypeptide or protein, in accordance with certain embodiments of the present invention, to inhibit or obstruct binding to E6 oncoprotein can be determined by a competitive protein-binding assay, wherein the peptide, polypeptide or protein is included as an inhibitor of another E6-binding protein.
  • the ability of a particular peptide, polypeptide or protein to regulate, modulate or restore the cellular response to apoptotic signals can be determined by performing cell viability assays, well known in the art with reference to the present disclosure, following the application of apoptosis inducing stimuli.
  • the present invention provides a method of treating HPV-infected cells, which includes the step of contacting an HPV-infected cell, which expresses E6, with a polypeptide containing and E6-binding domain, but not the DED or DD of FADD.
  • the E6-binding domain of the polypeptide selectively binds to the E6 oncoprotein.
  • This selective binding of the E6- binding domain to the E6 oncoprotein inhibits binding of E6 to cellular FADD containing a DED and DD.
  • a normal response includes any increase in response to apoptotic stimuli compared to an untreated E6- expressing cell.
  • the first step of the method is transfecting an HPV-infected cell with an expression vector, which expresses an encoded polypeptide containing the E6-binding domain.
  • the present invention provides therapeutic or prophylactic methods for treating HPV and the like, which are accomplished by the administration of an effective amount of a therapeutic agent capable of restoring the normal response to apoptotic signal.
  • Therapeutic or prophylactic indications can include treatment of (prevention and/or reduction of the severity) of HPV-associated lesions or cancers.
  • administer includes applying the compositions containing the polypeptide or expression vector to HPV-infected cells or tissues sufficiently proximal to the affected site such that the polypeptide is effective at ameliorating the condition.
  • the methods are well suited to the treatment of cancerous or precancerous cells.
  • the methods are applied to keratinocytes, more particularly to cells of anogenital SCC (squamous cell carcinoma) and its precursor, high-grade dysplasia, also known as high-grade intraepithelial neoplasia (IN) or high- grade squamous intraepithelial lesions (SIL). Warts may also be treated using the invention.
  • SCC squamous cell carcinoma
  • SIL high-grade squamous intraepithelial lesions
  • the methods of this invention need not be limited to the treatment of cancerous or pre-cancerous cells.
  • the methods are well suited in the treatment of any condition in which HPV infection is a component of the etiology.
  • the methods of this invention can be used to target HPV 6, and/or HPV 11 the human papillomaviruses associated with genital warts.
  • transfection reagents suitable for transfection cells by topical application are also well known to those of skill in the art and many are commercially available (e.g. cationic lipids, lipofectamineTM, ChariotTM, etc.).
  • CH-Il (Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan)), monoclonal antibodies directed against ⁇ -actin (Sigma, St. Louis, MO US) and HA (Roche Applied Science), peroxidase-coupled monoclonal antibodies against GST, peroxidase-coupled anti-rabbit polyclonal antibodies (Roche Applied Science), and rabbit polyclonal antibodies against FADD (Santa Cruz Biotechnology) were used as previously reported.
  • ATCC Manassas, VA US
  • McCoy's 5A medium Invitrogen, Carlsbad, CA US
  • 10% fetal bovine serum Invitrogen
  • penicillin 100 U/ml
  • streptomycin 100 mg/ml
  • pHA-E6 S and pHA-E6 AS plasmids have been described previously, and respectively contain either the sense or the antisense versions of epitope-tagged E6 (HA-E6) under the control of the CMV promoter.
  • pcDNA3-FADD plasmid (referred to as pcDNA FADD in this disclosure) served as the basis for all of the additional FADD-expressing constructs disclosed in this disclosure.
  • the caspase 8 death effector domain (DED) sequence was obtained from cDNA prepared from U2OS cells by PCR amplification using primers 5'-ACTTCAGCAGAAATCTTTATGATATTGGGGAAC-S' (SEQ ID NO: 13) and 5'-GAGATTGTCATTACCCCACACA-S' (SEQ ID NO: 14), and was cloned in-frame with the activation domain of the pAD plasmid.
  • FADD FADD and its variants in the pBD plasmid (Stratagene) for use in the mammalian two-hybrid assay
  • the appropriate region of FADD was cloned in-frame with the binding domain of the pBD plasmid.
  • One tablet of protease inhibitor mixture (Roche Applied Science) was added per 10 ⁇ l of lysis buffer just prior to use. The protein concentration in cleared lysates was measured using the Bio-Rad Protein Assay (Bio-Rad).
  • U2OS cells capable of expressing variable amounts of HA-E6, regulated by the dose of doxycycline present in the media were created using the Tet-off system (Clontech) following the manufacturer's protocol with some modifications. Cultures of these cells were grown in the indicated concentrations of the drug, which were maintained throughout the duration of the experiment.
  • Mammalian Two-hybrid Assay [0114] The mammalian two-hybrid binding assay was performed as directed by the manufacturer (Stratagene). Transfection of the indicated combination of vectors and subsequent luciferase activity measurements were performed according to techniques known to those with skill in this art.
  • the binding assays were performed using white 384-well Optiplates (PerkinElmer) in a total volume of 25 ⁇ l. Proteins were diluted in a buffer containing PBS, 0.1% BSA, and 0.05% tween-20. The AlphaScreen kits (Histidine coated acceptor beads and Glutathione coated donor beads) were obtained from PerkinElmer. 5 ⁇ l of the dilution buffer was added to the plate followed by the addition of 5 ⁇ l of each of the interacting protein partners.
  • the E6 oncoprotein of HPV 16 mediates the accelerated degradation of many of its protein partners, and one such protein is FADD. Since in vitro pull-down assays reveal that the SLT2 mutant does not bind to E6, the next step was to determine whether these mutations could also block E6-mediated degradation in an in vivo system. This was assessed by transfecting U2OS cells stably transfected with epitope-tagged hemagglutinin (HA)-E6, U2OSE64A and U2OSE612, with pcDNA FADD, coding for the wild type FADD protein, or with pcDNA SLT2, coding for the mutant FADD (SLT2) protein.
  • HA epitope-tagged hemagglutinin
  • SLT2 mutant FADD
  • sequences encoding procaspase 8 DED, wild type FADD DED, or the SLT2 mutant FADD DED were cloned into the bait or prey plasmids of a mammalian two-hybrid system, along with a plasmid encoding the luciferase reporter gene.
  • Each set of plasmids was transfected into cells that express E6 under the control of the tetracycline/doxycycline response element.
  • the amount of E6 expressed by these U2OSE6tet24 cells can be regulated by the concentration of tetracycline or doxycycline added to the culture media (TetOff System, Clontech).
  • FIG. 5A illustrates the sequences of the constructs used to perform the next set of in vitro pull-down assays with GST-E6.
  • the SLT3 mutant also seems to exhibit a similar pattern of expression to SLT2, suggesting that the amino acids mutated here also impair protein turnover.
  • the SLT4 mutant behaves as the wild type protein as seen by diminished protein expression over time, indicating functional protein turnover. Therefore, although the mutations in both SLT3 and SLT4 prevent E6 binding, only the protein encoded by the SLT4 construct allows for normal protein turnover of FADD. This implies that the three amino acids manipulated in SLT4 are unique to the mediation of oncoprotein binding to FADD.
  • FIG. 4B reveals that the mutations in SLT2 prevent the engagement of procaspase 8 DED.
  • Figure 6C shows that whereas procaspase 8 DED cannot associate with SLT2, the amino acid changes in SLT3 and SLT4 do not inhibit procaspase 8 DED interaction with FADD DED.
  • the 23 amino acids in the N-terminus of FADD appear to mediate oncoprotein binding based upon the mutations made in the various constructs. Over-expressing the region of FADD involved in E6 binding may competitively inhibit E6 binding to endogenous FADD in E6-expressing cells. This would leave FADD free to facilitate apoptosis once the apoptotic cascade of events is initiated.
  • cells expressing either the sense (U2OSE612) or anti-sense (U2OSE6 AS) version of E6 were transfected with the pcDNA vector or with a plasmid that codes for expression of the N-terminal 23 aa of FADD, pcDNA 23 aa ( Figure 8A).
  • the Molluscum contagiosum virus produces the MC159 protein that binds to cellular FADD and inhibits Fas-induced apoptosis. Additionally, some viruses have developed mechanisms to evade the host immune system by producing homologues of cytokines and chemokines as well as their receptors. For example, members of the poxvirus family produce soluble decoy receptors that inhibit the initial ligand-receptor interaction at the cell membrane. The cowpox virus, for instance, produces CrmB, CrmC, CrmD, and CrmE, TNFR-2 orthologs that bind TNF. The viruses referred to thus far produce proteins aimed at inhibiting one step in the cell death pathway.
  • Viruses such as HPV
  • HPV have developed means by which to avoid elimination by the host immune response in order to continue to persist and to propagate.
  • the ability of the oncoprotein E6 to protect infected cells from undergoing apoptosis through its interactions with the TNF receptor and the death effector domains of both procaspase 8 and FADD enhance the prospective oncogenicity of the virus. This points to the significance of E6 in promoting virus survival post-infection.
  • HPV-induced cervical cancer a quadrivalent vaccine, Gardasil® manufactured by Merck & Co., intended to prevent against infection with the HPV strains 6, 11, 16, and 18 and a bivalent vaccine, Cervarix® manufactured by GlaxoSmithKline, intended to prevent against infection with strains 16 and 18.
  • These prophylactic vaccines are designed to induce the generation of antibodies against the Ll and L2 capsid proteins of HPV in the hopes that these antibodies will neutralize future virus infection.
  • These vaccines if administered before exposure to HPV, are reported to be highly effective at protecting women from infection. However, they are not therapeutic and papillomavirus infections often remain undetectable for prolonged periods of time.
  • therapeutic vaccines designed to target the early non- structural proteins of the virus, such as E6, may be more effective once infection has occurred.
  • the finding of the present invention of a novel E6 binding domain on FADD, a key player in viral oncogenesis, in conjunction with demonstrating the successful use of peptide inhibitors to block E6 activity provide agents that can be used for therapeutic approaches for cervical cancer. Additionally, since some head and neck cancers have been found to contain high-risk HPV types, these agents also aid in the design of therapeutic approaches for these cancers as well.

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Abstract

La présente invention concerne de nouveaux polypeptides et compositions capables de se lier à l'oncoprotéine E6 du papillomavirus humain (VPH) et des procédés d'emploi des nouveaux polypeptides et compositions de l'invention pour traiter une infection par le VPH. Sous un aspect, l'invention concerne des vecteurs d'expression codant les polypeptides, qui contiennent un domaine de liaison E6 dérivé d'un fragment FADD, et des cellules hôtes contenant les vecteurs.
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US12059413B2 (en) 2016-11-02 2024-08-13 The Research Foundation For The State University Of New York Methods of inhibiting viruses using compositions targeting TSG101-ubiquitin interaction

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US6558950B1 (en) * 1997-03-03 2003-05-06 Mcgill University Methods and reagents for modulating apoptosis
US20070197444A1 (en) * 2006-02-17 2007-08-23 Nastech Pharmaceutical Company Inc. Phage displayed cell binding peptides

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US12059413B2 (en) 2016-11-02 2024-08-13 The Research Foundation For The State University Of New York Methods of inhibiting viruses using compositions targeting TSG101-ubiquitin interaction

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