WO2005115434A1 - Compositions therapeutiques et methodes de traitement du cancer de la peau induit par le papillomavirus humain - Google Patents

Compositions therapeutiques et methodes de traitement du cancer de la peau induit par le papillomavirus humain Download PDF

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WO2005115434A1
WO2005115434A1 PCT/EP2005/005618 EP2005005618W WO2005115434A1 WO 2005115434 A1 WO2005115434 A1 WO 2005115434A1 EP 2005005618 W EP2005005618 W EP 2005005618W WO 2005115434 A1 WO2005115434 A1 WO 2005115434A1
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pdcd4
protein
hpv
binding
pharmaceutical composition
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PCT/EP2005/005618
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Herbert Pfister
Baki AKGÜL
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Cell Center Cologne Gmbh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • compositions and methods for treating HPV induced skin cancer are provided.
  • the present invention relates to the technical field of virology and therapy of viral diseases.
  • the present invention relates to a pharmaceutical composition for treating or preventing a tumor comprising a compound capable of specifically inhibiting or blocking the binding of human papillomaviruses (HPV) protein E7 to programmed cell death 4 (Pdcd4) protein, and optionally a pharmaceutically acceptable carrier.
  • HPV human papillomaviruses
  • Pdcd4 programmed cell death 4
  • the present invention further concerns a method for identifying and obtaining an anti-tumor agent comprising the steps of subjecting candidate compounds to a sample comprising a first binding partner which comprises at least a Pdcd4 protein binding domain and which is able to bind HPV E7; a second binding partner which comprises at least a protein binding domain of HPV E7 under conditions allowing complex formation of binding partners; and determining whether complex formation occurs in the presence or absence of one or more candidate compounds.
  • the present invention relates to a method of screening drug candidates for the treatment of a tumor disorder comprising contacting a stable cell line which is genetically engineered and/or exposed to express Pdcd4 or a derivative thereof and HPV protein E7 or a derivative thereof; and evaluating the effect of said drug candidate(s) on said cell line.
  • Non-melanoma skin cancer is the most common human cancer in the Caucasian population, with about 1 million cases in the USA and more than 40,000 cases occurring annually in the UK (Miller and Weinstock, J. Am. Acad. Dermatol. 30 (1994), 774-778; Stern, Arch. Dermatol. 135 (1999), 843-844).
  • BCC Basal cell carcinoma
  • SCC squamous cell carcinoma
  • HPVs Human papillomaviruses
  • HPVs Human papillomaviruses
  • EV epidermodysplasia verruciformis
  • the major target of the E6 protein of high-risk mucosal HPNs is p53, whose functions are disrupted after its E6-promoted proteolysis (Werness et al., Science 248 (1990), 76-79).
  • the E7 protein of high-risk mucosal HPNs functions in cellular transformation by interaction principally with pRb (Munger et al., J. Nirol. 63 (1989), 4417-4421) and other cellular targets (for a review see Smola-Hess and Pfister, "Interaction of papillomaviral oncoproteins with cellular factors" in: Structure-Function Relationships of Human Pathogenic Viruses, A. Holzenburg and E.
  • HPV E7 can uncouple cellular differentiation and proliferation and hence, retain differentiating keratinocytes in a DNA replication competent stage.
  • E7 from cervical HPVs can immortalize human foreskin keratinocytes in vitro, alone or in cooperation with E6.
  • the E7 genes of cutaneous skin cancer-associated HPV types 8 and 47 failed to induce any detectable transformation of rodent cells (Iftner et al., J. Virol.
  • HPV5 and 8 gave rise to transformed cell lines (Yamashita et al, Oncogene 8 (1993), 2433-2441).
  • the E7 of the plantar wart-specific HPV1 (a low risk papillomavirus) fully transformed the mouse fibroblast cell line C127 (Schmitt et al, J. Virol. 68 (1994), 7051-7059).
  • HPN Human papillomaviruses
  • ⁇ MSC non-melanoma skin cancer
  • This invention relates to cancer therapeutics. More particularly, this invention relates to the interception of human papillomaviruses (HPN) protein E7 binding to programmed cell death 4 (Pdcd4) protein, which binding prevents Pdcd4 from functioning as a tumor suppressor.
  • the present invention is directed to a pharmaceutical composition for treating or preventing a tumor comprising a compound capable of specifically inhibiting or blocking the binding of human papillomaviruses (HPN) protein E7 to programmed cell death 4 (Pdcd4) protein, and optionally a pharmaceutically acceptable carrier. It is proposed that HPNs are involved in non-melanoma skin cancer ( ⁇ MSC).
  • the programmed cell death protein 4 (Pdcd4) is a 60 kDa cellular R ⁇ A-binding protein that was originally identified by screening for upregulated genes during apoptosis. Recently it has been demonstrated that Pdcd4 acts as transformation (tumor)-suppressor in keratinocytes.
  • the present invention relates to a method for identifying and obtaining an anti-tumor agent comprising the steps of subjecting candidate compounds to a sample comprising a first binding partner which comprises at least a Pdcd4 protein binding domain and which is able to bind HPV E7; a second binding partner which comprises at least a protein binding domain of HPV E7 under conditions allowing complex formation of binding partners; and determining whether complex formation occurs in the presence or absence of one or more candidate compounds.
  • the present invention also concerns a method of screening drug candidates for the treatment of a tumor disorder comprising contacting a stable cell line which is genetically engineered and/or exposed to express Pdcd4 or a derivative thereof and HPV protein E7 or a derivative thereof; and evaluating the effect of said drug candidate(s) on said cell line.
  • the therapeutic use of the identified drug in accordance with the present invention may be accompanied by the use of further therapeutic agents such as Pdcd4. It is an object of the present invention to provide a use of HPV E7 protein for the preparation of a composition for inhibiting Pdcd4 apoptotic signal transduction pathway.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising Pdcd4 or a fragment thereof having tumor suppressor activity and an anti-tumor agent identified in accordance with the present invention.
  • the present invention relates to an anti-tumor agent identifiable by any one of the methods of the present invention, which can belong to different classes of antineoplastic drugs such as small molecules, peptides and antibodies.
  • a Pdcd4 protein or fragment thereof having tumor suppressor activity and being incapable of binding to HPV E7 and its therapeutic use is provided.
  • HPV8-E7 protein binds to Pdcd4.
  • HPV8 early proteins El, E2, E6 and E7 were expressed as GST fusion proteins, purified and equal amounts were used to measure binding to in vitro translated, radiolabeled Pdcd4.
  • the bound Pdcd4 was assessed by SDS-PAGE and autoradiography. Input Pdcd4 and binding to control GST protein is also shown.
  • Fig.2 The E7 proteins of HPV5, 8, 15 and 77 are binding to Pdcd4.
  • the various E7 proteins were expressed as GST fusion proteins, purified and equal amounts were used to measure binding to in vitro translated, radiolabeled Pdcd4.
  • the bound Pdcd4 was assessed by SDS-PAGE and autoradiography. Input Pdcd4 and binding to control GST protein is also shown.
  • the present invention has identified the tumor suppressor protein programmed cell death 4 (Pdcd4) as a target for skin tumors associated human papillomavirus (HPV) E7 protein.
  • the present invention relates to the prevention or treatment of non-melanoma skin cancer (NMSC) by reconstituting the biological function of a tumor suppressor in human papillomavirus (HPV) infected cells.
  • NMSC non-melanoma skin cancer
  • HPV human papillomavirus
  • the present invention relates to a pharmaceutical composition for treating or preventing a tumor comprising a compound capable of specifically inhibiting or blocking the binding of human papillomaviruses (HPV) protein E7 to programmed cell death 4 (Pdcd4) protein, and optionally a pharmaceutically acceptable carrier.
  • the present invention is based on the observation that the E7 protein of human papillomaviruses found in non-melanoma-skin cancers interacts with the transformation suppressor Pdcd4.
  • the Pdcd4 gene also known as MA-3, TIS, H731, DUG and APOP-3) was originally identified by screening for genes whose expression is upregulated during apoptosis (Shibahara et al., Gene 166 (1995), 297-301). However, most of the recent interest in the gene arose from the observation, that Pdcd4 acts as transformation suppressor in mouse keratinocytes (Cmarik et al., PNAS 96 (1999), 14037-14042).
  • Pdcd4 expression was found to be high in a keratinocyte cell line that was resistant to transformation by a tumor promoter and low in a related cell line that was efficiently transformed in the presence of the tumor promoter.
  • the Pdcd4 protein has recently been shown to interact with eucaryotic translation initiation factors elF4A and elF4G (Yang et al., Mol. Cell. Biol. 23 (2003), 26-37), suggesting that it might be involved in regulating protein translation.
  • the present invention provides a pharmaceutical composition for treating or preventing a tumor comprising a compound capable of specifically inhibiting or blocking the binding of human papillomaviruses (HPV) protein E7 to programmed cell death 4 (Pdcd4) protein, and optionally a pharmaceutically acceptable carrier.
  • HPV human papillomaviruses
  • Pdcd4 programmed cell death 4
  • Pdcd4 is also known as "programmed cell death for Isoform 1", “programmed cell death 4", "nuclear antigen H731” and “APOP-3". Furthermore, it is believed that Pdcd4 is the human homologue of mouse apoptosis-inducible protein MA-3. As has been described in Yang et al, Mol. Cell. Biol. 23 (2003), 26-37, Pdcd4 is a transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1 -dependent transcription required for transformation.
  • MA-3 or TIS is a mouse protein associated with apoptosis (Shibahara et al., Gene 166 (1995), 297-301; Onishi and Kizaki, Biochim. Biophys. Res. Commun. 228 (1996), 7-13).
  • the nucleotide sequence of the mouse proteins predicts an amino acid sequence of 469 residues.
  • MA-3 is highly expressed in thymus and is present in all apoptosis-inducible cell lines including thymocytes, T cells, B cells, and pheochromocytoma (Shibahara et al, supra).
  • TIS expression is down-regulated in the RVC lymphoma cells incubated with an topoisomerase I inhibitor, an anti-tumor drug, and the low expression level of TIS may be a contributing factor to the cytotoxicity of the topoisomerase inhibitors (Onishi and Kizaki, supra).
  • compound is taken to include both organic compounds such as peptides, as well as inorganic compounds such as ion chelators.
  • Antibodies e.g., polyclonal or monoclonal antibodies directed against E7 protein, the Fab, Fab', F(ab') fragments of such antibodies, as . well as single-chain anti-E7 antibodies can also be considered as compounds of the present methods.
  • inhibitor capable of specifically inhibiting or blocking
  • inhibitor includes substances which reduce the binding of HPV E7 protein to Pdcd4 and these which nullify it altogether.
  • inhibitor includes substances which antagonize the HPV E7 protein inhibitory effect on Pdcd4 suppressor activity.
  • Pdcd4 polypeptides can be generated in accordance with the present invention, which have been modified to loose their binding to HPV E7 protein while retaining their tumor suppressor activity.
  • Inhibitors include competitive as well as non- competitive inhibitors.
  • a competitive inhibitor (or competitive blocker) interacts with or near the site specific for the HPV E7 binding to Pdcd4.
  • a non-competitive inhibitor or blocker inactivates the HPV E7 binding function by interacting with a site other than the Pdcd4 binding site.
  • the inhibitor is a small chemical agent which directly interacts with HPV E7 protein. Therefore, there will preferably be a direct relationship between the molar amount of compound required to inhibit the binding of HPV E7 and Pdcd4 and the molar amount of HPV E7 protein in the cell.
  • tumor refers to benign as well as to malignant neoplasias in their respective stages.
  • the first stage of neoplastic progression is an increased number of relatively normal appearing cells, the hyperplastic stage.
  • hyperplastic stage There are several stages of hyperplasia in which the cells progressively accumulate and begin to develop an abnormal appearance, which is the emergence of the dysplastic phase.
  • epithelial dysplasias cells resemble immature epithelial cells, and during this phase of epithelial neoplastic progression, an increasing percentage of the epithelium is composed of these immature cells.
  • invasive cancers develop in epithelia severely affected by dysplasia.
  • treatment means obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • the compound to be used in the pharmaceutical composition of the present invention is capable of interfering with the binding of HPV E7 to Pdcd4.
  • Pdcd4 binding could be shown for the E7 protein of HPV5, 8, 15 and 77 in in vitro binding assays; see Figs. 1 and 2 and corresponding Examples 1 and 2.
  • HPV5, 8, 15 and 77 belong to the subgroup B (HPV4-related and Epidermodysplasia Verruciformis [EV] HPV); see Pfister in J. Natl. Cancer Inst. Monogr. 31 (2003), 52-56, Chapter 8, Human papillomavirus and skin cancer.
  • HPVs human papillomaviruses
  • NMSC non- melanoma skin cancer
  • EV inherited multifactorial disease
  • NMSC non- melanoma skin cancer
  • EV inherited multifactorial disease
  • NMSC non- melanoma skin cancer
  • EV inherited multifactorial disease
  • NMSC non- melanoma skin cancer
  • EV inherited multifactorial disease
  • Several isolates which appear to constitute new types have been found in skin lesions of renal transplant patients (Berkhout et al., Journal of Clinical Microbiology 33 (1995), 690-695; Shamanin et al., Cancer Research 54 (1994), 4610-4613).
  • SCC squamous cell carcinomas
  • the pharmaceutical composition of the present invention is designed to be applied to a subject before, during or after renal transplantation.
  • Inhibitors may be peptides, proteins, nucleic acids, antibodies, small organic compounds, peptide mimics, aptamers or PNAs (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198; Gold, Ann. Rev. Biochem. 64 (1995), 736-797).
  • said polypeptide, peptide or pepditomimetic is derived from a protein binding domain of Pdcd4.
  • amino acid sequences of Pdcd4 critical for subunit association can be identified and synthetic peptides containing these amino acid sequences selectively prevent binding of E7 to Pdcd4 resulting in the maintenance of its tumor suppressor function.
  • Anti HPV activity can be verified in cell culture as well as in a murine model of HPV induced skin tumor.
  • a similar strategy has been described for a potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo; see Liuzzi et al, Nature 372 (1994), 695-698.
  • peptide libraries of the entire human Pdcd4 protein can be employed in order to screen for peptides that are useful as an inhibitor in accordance with the present invention.
  • a corresponding approach has been described for compounds that are capable of interfering with the binding between p53 and MDM2, wherein peptide libraries of the human p53 protein have been employed in order to identify appropriate agents; see international application WO96/02642, in particular at the Materials and Methods section.
  • said inhibitor is a nucleic acid molecule which is designed for the expression of said inhibitor in target cells. This embodiment is particularly envisaged if the inhibitor is derived from Pdcd4, for example a peptide which comprises or mimics the E7 binding site of native Pdcd4.
  • said inhibitor may be a functional Pdcd4 tumor-suppressor protein which has been modified to be no longer a target for HPV E7 protein binding.
  • the nucleic acid sequence encoding the inhibitor is operably linked to a regulatory sequence directing its expression to epithelial cells.
  • a regulatory sequence when describing the relationship between two polynucleotide sequences, means that they are functionally linked to each other.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
  • promoter elements as well as enhancers may be used.
  • expression regulation sequences are derived from genes that are expressed primarily in the tissue or cell type chosen.
  • the genes from which these expression regulation sequences are obtained are expressed substantially only in the tissue or cell type chosen, although secondary expression in other tissue and/or cell types is acceptable if expression of the recombinant DNA in the transgene in such tissue or cell type is not detrimental to the animal.
  • the recombinant nucleic acid molecules will usually also comprise downstream expression regulation sequences to supplement tissue or cell-type specific expression.
  • the downstream expression regulation sequences include polyadenylation sequences (either from the endogenous gene or from other sources) and sequences that may affect RNA stability as well as enhancer and/or other sequences which enhance expression.
  • said regulatory sequence comprises an epithelial cell specific promoter.
  • Particularly useful for targeting the expression of inhibitor nucleic acid sequences to epithelial cells are the promoters from genes encoding keratin. Keratin are proteins that are expressed in epithelial tissues, and specific keratin proteins, identified by a number e.g., keratin-5 are exclusively expressed not only in certain epithelia, but also in selected cells populating the epithelia.
  • the epidermis is composed of layers of cells (keratinocytes) which produce specific types of keratin proteins.
  • the basal cells produce keratin 5 and 14 (K5 and K14), whereas the more mature, terminally differentiated keratinocytes, e.g., the suprabasal keratinocytes, produce K10 and Kl. Promoters from other keratin genes, such as K8 and K19 are useful in direct expression to epithelia in the bladder or intestines.
  • the basal cell specific keratin-14 (K14) promoter has been used to overexpress the growth factor TGF- ⁇ in the epidermis (Vassar et al., Cell 64 (1991), 365-380). These animals displayed a transient, neonatal hyperproliferation that disappeared in adults. Other workers have used promoters from genes specific to suprabasal cells to express growth factors, cytokines, and oncogenes in these cells; see, Cheng et al., Genes Dev. 6 (1992), 1444-1456; Guo et al., EMBO J. 12 (1993), 973-986; Turksen, Proc. Natl. Acad. Sci. USA. 89 (1992), 5068-5072; and Vassar et al., Proc. Natl. Acad. Sci. USA 86 (1989), 1563-1567).
  • the regulatory sequence directs the expression of said nucleic acid sequence in basal epithelial cells.
  • a basal cell keratin promoter e.g., K5 or K14
  • the keratin-14 (K14) promoter is particularly preferred.
  • Jiang et al. identified a 300 bp controlling segment of the K14 promoter conferring epithelial-specific expression, while the K5 promoter was studied by Byrne and Fuchs (Mol. Cell. Biol. 13 (1993), 3176 - 3190).
  • said nucleic acid molecules are preferably contained in a vector that can be an expression, a gene transfer or gene targeting vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • the present invention provides a method for intercepting the binding between Pdcd4 and E7 in a subject by administering to the subject, an effective amount of a nucleic acid molecule coding for a peptide which prevents the binding between Pdcd4 and E7.
  • such nucleotide sequence is provided in an expression vector.
  • Preferred expression vectors for use in a therapeutic composition include any appropriate gene therapy vectors, such as nonviral (e.g., plasmid vectors), retroviral, adenoviral, herpes simplex viral, adeno-associated viral, polio viruses and vaccinia vectors.
  • nucleic acid molecule can be delivered "naked” by direct injection into the blood stream or to the desired tissue or organ of a subject.
  • the vector can be combined with a lipid compound which facilitates the uptake of the molecule by cells.
  • the lipid compound includes liposome, lipofectins, cytofectins, lipid-based positive ions, and may be introduced into the body fluids, the blood stream, or a selected tissue site.
  • Liposome mediated gene therapy is well known in the art and is described by, e. g., Lesoon-Wood et al., Human Gene Ther. 6 (1995), 395; Tsan et al, Am. J. Physiol 268 (1995), 11052; Vieweg et al, Cancer Res. 5585 (1995), 2366; Trivedi et al., J. Neurochem. 64 (1995), 2230; Hickman et al., Human Gene Ther.
  • DNA carriers which can facilitate the uptake of a desired vector by the target cells include nuclear protein, or ligands for certain cell receptors, which can be combined with a vector in engineered vesicles for delivery.
  • the introduction and gene therapeutic approach should, preferably, lead to the expression of a functional copy of the target gene of the invention.
  • the inhibitor as defined hereinabove or identified and obtained according to the method of the present invention described farther below may also be used for the treatment or prevention of a tumor which does not apparently involve HPV infection.
  • the tumor suppressor Pdcd4 is not only targeted by HPV E7 protein but by other viral or cellular gene products as well.
  • the E7 protein-binding site of Pdcd4 might also interact with other proteins leading to the inactivation of the suppressor function of Pdcd4.
  • an inhibitor which is capable of interfering with the binding of HPV E7 protein and Pdcd4, or a Pdcd4 protein modified to no longer bind HPV E7 protein could also be used to treat or prevent a tumor which is due to inactivated Pdcd4 suppressor function but unrelated to HPV.
  • treating a tumor it is meant that the tumor growth or metastasis is significantly inhibited, as indicated by reduced tumor volumn or reduced occurrences of tumor metastasis.
  • Tumor growth can be determined, e.g., by examining the tumor volume via routine procedures (such as obtaining two-dimensional measurements with a dial caliper).
  • Tumor metastasis can be determined by examining the appearance of tumor cells in secondary sites or examining the metastatic potential of biopsied tumor cells in vitro using various laboratory procedures.
  • the tumors which can be treated by using the methods of the present invention may include, but are not limited to, melanoma, lymphoma, plasmocytoma, sarcoma, glioma, thymoma, leukemia, breast cancer, prostate cancer, colon cancer, esophageal cancer, brain cancer, lung cancer, ovary cancer, cervical cancer, hepatoma, and other neoplasms known in the art.
  • the present invention contemplates particularly Pdcd4- related tumors.
  • the term "Pdcd4- related" refers to tumor cells in which wild-type (wt) Pdcd4 is absent, disabled or otherwise mutated.
  • the present invention is preferably applied in embodiments concerning HPV infection and thus for the treatment of NMSC.
  • the present invention relates to a mefhod for inducing growth arrest or apoptosis in tumor cells containing wild type Pdcd4 and HPV E7 protein, comprising interfering with the interaction of Pdcd4 and HPV E7 in vivo or in vitro.
  • the treatment or prevention of a tumor and method for inducing growth arrest or apoptosis in tumor cells comprises
  • step (b) introducing into said cells, tissue or organ a nucleic acid molecule encoding and capable of expressing the inhibitor in vivo; (c) preparing the cells, tissue or organ obtained in step (b) for reintroduction into the same subject or different subject.
  • recombinant DNA molecules and expression vectors such as those described above may be used.
  • the present invention relates to a method for identifying and obtaining an anti-tumor agent comprising the steps of (a) subjecting candidate compounds to a sample comprising (i) a first binding partner which comprises at least a Pdcd4 protein binding domain and which is able to bind HPV E7; (ii) a second binding partner which comprises at least a protein binding domain of HPN E7 under conditions allowing complex formation of the binding partners; and (b) determining whether one or more candidate compounds inhibit complex formation.
  • the production of the proteins or binding peptides can be done in vitro and in vivo according methods well known in the art; see, e.g., Examples 1 and 2. Furthermore, the sources for Pdcd4 and E7 protein, respectively, as well as methods for their preparation are well known in the art.
  • the nucleotide and amino acid sequence of Pdcd4 is described in the prior art; see, for example, GenBank accession no. ⁇ M_014456 and references Yoshinaga et al., Pathol. Int. 49 (1999), 1067-1077; Soejima et al, Cytogenet. Cell Genet. 87 (1999), 113-114; Azzoni et al., J. Immunol. 161 (1998), 3493-3500; Shibahara et al., Gene 166 (1995), 297-301.
  • the nucleotide and amino acid sequence of mouse Pdcd4 is described in the prior art; see, for example, GenBank accession no.
  • nucleotide and amino acid sequence of the rat homolog of Pdcd4 DUG is also described in the prior art; see, for example, GenBank accession no. NM_022265.
  • a sample containing E7 proteins can be contacted with a test compound for a period of time sufficient to allow binding of the test compound to E7. Then, the sample is contacted with Pdcd4, and the amount of complexes formed between E7 and Pdcd4 can be measured and compared to the amount of complexes formed in the absence of the test compound. A decrease in the value determines the test compound as a compound which prevents the binding of E7 to Pdcd4.
  • An alternative format to carry out the method of the present invention can include as a first step, contacting a sample containing E7 with Pdcd4 for a time sufficient to allow Pdcd4 to bind to E7 and measuring the amount of complexes formed between E7 and Pdcd4 in the sample. Afterwards, the sample is contacted with a test compound for an appropriate period of time. Compounds which displace Pdcd4 from the prior formed Pdcd4-E7 complexes can be identified as a compound which prevents the binding of E7 to Pdcd4.
  • biochemical assays and immunoassays can be employed for measuring the amount of the E7-Pdcd4 complexes formed in a sample.
  • the proteins can be conveniently labeled with, e.g., isotope, biotinyl group, or fluorescein, to facilitate the detection.
  • the assays can be carried out in a variety of formats, such as immunoprecipitation, Far Western blot analysis, RIA, ELISA, forward or reverse sandwich assays, peptide competition binding assays, and the like.
  • the assays can be readily adapted to provide screens at a large scale, for example, from synthetic combinatorial peptide libraries, by carrying out the process in a 96-well format. Automated screening techniques can be applied in these circumstances as would be understood in the art.
  • the method of the present invention can also be a simple binding assay such as those described in Examples 1 and 2, wherein the in vitro transcribed and translated Pdcd4 protein and recombinant expressed GST HPV8 E7 protein are incubated with a candidate compound prior to subjecting the sample on SDS-PAGE.
  • a “variant" of Pdcd4 refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • altered nucleic acid sequences encoding Pdcd4 include those with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent Pdcd4. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding Pdcd4, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding Pdcd4.
  • the encoded protein may also be "altered” and contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent Pdcd4. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of Pdcd4 is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine, glycine and alanine, asparagine and glutamine, serine and threonine, and phenylalanine and tyrosine.
  • the first binding partner comprises human Pdcd4 protein or at least the HPV E7 protein binding domain thereof.
  • the E7 protein of HPV5, 8, 15 and 77 is preferred as the second binding partner in the screening method of the present invention.
  • complex formation can be tested in SDS-PAGE.
  • gel-shift assay procedures such as those described for identifying inhibitors of the interaction between p53 and dm2; see international application WO97/11367.
  • At least one of the components in the method of the present invention is detectably labeled.
  • a variety of techniques are available for labeling biomolecules and are well known to the person skilled in the art and are considered to be within the scope of the present invention; see also supra and the Examples 1 and 2.
  • Commonly used labels comprise, inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, ⁇ - galactosidase, alkaline phosphatase), radioactive isotopes (like 32 P or 125 I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).
  • fluorochromes like fluorescein, rhodamine, Texas Red, etc.
  • enzymes like horse radish peroxidase, ⁇ - galactosidase, alkaline phosphatase
  • radioactive isotopes like 32 P or 125 I
  • biotin digoxygenin
  • colloidal metals chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).
  • Labeling procedures like covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases) are well known in the art.
  • Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
  • the second binding partner is a fusion protein comprising glutathion-S transferase and/or the first binding partner is labeled with S 35 -methionine; see also Examples 1 and 2.
  • agents that are capable of interfering with HPV E7 protein binding to Pdcd4 can be identified by their capability of disrupting protein-protein interactions in a cell-based assay, for example the 2- or 3 -hybrid system.
  • a cell-based assay for example the 2- or 3 -hybrid system.
  • the mammalian reverse 2-hybrid system as described in international application WO02/50259 may be used, which is also amenable to high throughput screening of compounds that disrupt protein-protein interactions.
  • a more complex system for the identification of specific protein- protein interactions, which may also be useful in order to identify Pdcd4 variants that are no longer capable of binding HPV E7 protein is described in international application WO97/47763.
  • the screening methods of the invention are preferably performed with the drug candidates provided as a collection of compounds.
  • the number and/or diversity of compounds within said collection is successively reduced in repeated screening rounds.
  • Such collections are also commercially available, for example from Pharmacopeia, Inc. or Chemical Diversity Labs, Inc.
  • the present invention also envisages the combination of the screening methods. While cell- based in vitro methods are more amenable to high throughput assay formats, the in vivo screening in animals will provide more precise results. Thus collection of drugs with high diversity can be tested in the in vitro assays of the present invention employing high throughput techniques and after reducing the diversity in several round of high throughput screening the obtained low diversity collection is then tested in animals while reducing the diversity further until a single compound is identified.
  • Methods for clinical compound discovery comprises for example ultrahigh-throughput screening (Sundberg, Curr. Opin. Biotechnol. 11 (2000), 47-53) for lead identification, and structure-based drug design (Verlinde and Hoi, Structure 2 (1994), 577-587) and combinatorial chemistry (Salemme et al., Structure 15 (1997), 319-324) for lead optimization.
  • said drug candidate(s) are small molecule(s). Methods for the synthetization, optimization and testing of small molecules are well known in the art.
  • said drug candidate(s) are antibodies, preferably antibody conjugate(s).
  • Antibodies play an increasing role in the treatment of disorders due to their high specificity. Once a target molecule with a critical role in the development of the particular disorder is identified it is possible to generate either polyclonal or monoclonal antibodies binding to it. The antibody can inhibit the detrimental function of the molecule either directly for instance by binding to the functional core or by assigning the molecule to be degraded.
  • Particularly preferred test compounds to be screened by the present methods are peptides which are made to resemble the E7 binding site on Pdcd4 and analogs thereof.
  • the E7 binding site refers to the part on a Pdcd4 protein which interacts with a HPV E7 protein and is responsible for the binding of the Pdcd4 protein to the E7 protein.
  • the binding site is believed to be constituted by at least about 6, preferably at least about 8 or 9, or more preferably, at least about 12, amino acid residues of Pdcd4 which are contiguous in the primary sequence.
  • the amino acids constituting the binding site are noncontiguous in the primary sequence, but are in the functional vicinity of each other in the tertiary structure of Pdcd4.
  • Those skilled in the art can map the E7 binding site on Pdcd4 by using a variety of modern molecular biology techniques, such as systematic mutagenesis in combination with 3-D modeling.
  • a peptide fragment can be as short as 6 amino acids in length, preferably, at least about 8 amino acid in length, more preferably, at least about 12 amino acid in length, to mimic the binding site on Pdcd4.
  • analogs it means variants of a peptide in issue.
  • the variations include substitutions, insertions or deletions of one or more amino acid residues, or modifications of the side chains of the amino acid residues.
  • analogs of a peptide can include homologous peptides from other mammalian species, peptides containing non-natural amino acid residues, peptides having chemical modifications on the side groups of amino acid residues, as well as peptides artificially designed to resemble the three dimensional structure of the binding site on human Pdcd4.
  • Pdcd4 A variety of techniques are available to those skilled in the art to make various fragments or analogs of Pdcd4. Such techniques include standard chemical synthesis, recombinant expression, and structural modeling (also called mimetics). The sequences of Pdcd4 from a number of mammalian species are highly conserved and are available to those skilled in the art, e.g., via Databases such as GenBank; see also supra.
  • Mimetics is a well-known technique involving the design of compounds which contain functional groups arranged in a manner mimicking that of the original, lead compound. Mimetics is desirable where the synthesis of the original compound is difficult, or where the original compound is unsuitable for a particular method of administration (e.g., orally) as such compound may tend to be degraded too quickly by proteases in the alimentary canal. Mimetic design, synthesis and testing can avoid laborious screenings of a large number of molecules for a target property.
  • Drugs and agents identified in accordance with the present invention may further be modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.
  • peptide mimetics and/or computer aided design of appropriate derivatives and analogues can be used, for example, according to the methods described above.
  • Methods for the lead generation in drug discovery also include using proteins and detection methods such as mass spectrometry (Cheng et al. J. Am. Chem. Soc. 117 (1995), 8859-8860) and some nuclear magnetic resonance (NMR) methods (Fejzo et al., Chem. Biol. 6 (1999), 755-769; Lin et al., J. Org. Chem. 62 (1997), 8930-8931). They may also include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, J. Med. Chem.
  • the present invention also relates to the anti-tumor agents identifiable and obtained by any one of the methods of the present invention.
  • the antineoplastic compounds that may be identified by the methods provided by the present invention are often compounds that are capable of inducing apoptosis of particular cells.
  • the methods of the invention thus further comprise the step of identifying a candidate that has the ability to induce death or apoptosis of cancer cells.
  • the present invention relates to the use of HPV E7 protein as described above for the preparation of a composition for inhibiting Pdcd4 apoptotic signal transduction pathway.
  • This embodiment is particularly suited for conditions which are due to aberrant, i.e. enhanced expression or activity of Pdcd4 protein.
  • any one of the above-described HPV E7 proteins or derivatives thereof or their encoding nucleic acid molecules in any form may be used for inhibiting Pdcd4 tumor-suppressor activity, thereby interfering with the apoptotic signal transduction pathway.
  • kits comprising any one of the above-described HPV E7 proteins, Pdcd4 proteins, or derivatives thereof, or assay components useful for performing any one of the above-described methods of the present invention.
  • the kit may comprise specific reagents such as those described hereinbefore and would typically but not necessarily comprise a proximityalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant protein or antibody suitable for detecting for example complexed proteins.
  • the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
  • the present invention also contemplates pharmaceutical compositions which include, as an active ingredient, an E7-Pdcd4 binding intercepting compound as described hereinabove.
  • an active compound i.e., such as binding-intercepting peptides or antibodies, or the nucleic acid molecules encoding such peptides or antibodies
  • the active compound can be used in combination with one another, or with other anti- tumor agents that are available in the art.
  • the active compounds can be suitably administered in combination with pharmaceutically acceptable carriers.
  • the carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the active ingredient contained therein, its use in practicing the methods of the present invention is appropriate.
  • carriers include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • the active ingredients can be combined with the carrier in any convenient and practical manner, e.g., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like, and if necessary, by shaping the combined compositions into pellets or tablets. Such procedures are routine for those skilled in the art.
  • Dosages of a compound in accordance with the present invention depend on the disease state or condition being treated and other clinical factors, such as weight and condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration.
  • the precise dosage of a compound to be therapeutically effective can be determined by those skilled in the art.
  • the therapeutically effective dosage of a compound can be in the range of about 0.5 pg to about 2 grams per unit dosage form.
  • a unit dosage form refers to physically discrete units suited as unitary dosages for mammalian treatment: each unit containing a predetermined quantity of the active material calculated to produce the desired theraputic effect in association with any required pharmaceutical carrier.
  • the methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
  • These compounds can be administered via standard routes, including the oral, ophthalmic nasal, topical, parenteral injections (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular), as well as direct injection to a preselected tissue site or as dermal pad, gel, etc.
  • parenteral injections e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular
  • direct injection e.g., direct injection to a preselected tissue site or as dermal pad, gel, etc.
  • Example 1 E7 of HPV8 interacts with Pdcd4 Experimental data have proved a central transforming role for HPN in cervical carcinoma (zur Hausen, ⁇ atl. Cancer Inst. 92 (2000), 690-698).
  • High-risk mucosal HPN types encode two oncoproteins, E6 and E7, which inactivate the function of the retinoblastoma protein and the p53 tumor suppressor protein and which have been shown to immortalize cells in culture.
  • the pathomechanisms of skin infection with HPN are not entirely clear. Jackson et al., Genes Dev.
  • Pdcd4 for programmed cell death protein 4
  • Pdcd4 is a novel transformation suppressor that is highly expressed in a keratinocyte cell line resistant to transformation and weakly expressed in a related cell line that could efficiently be transformed. Upregulation of Pdcd4 rendered the cells resistant to transformation whereas antisense-mediated downregulation of Pdcd4 resulted in higher transformation rates (Cmarik et al., Proc. Natl. Acad. Sci. USA 96 (1999), 14037-14042).
  • Pdcd4 contains two conserved helical MA-3 domains,' which are present in the eukaryotic translation initiation factors eIF4G I and elFG II and are involved in protein- protein interactions (Kang et al., Biochem. Biophys. Res. Commun. 293 (2002), 617-621; G ⁇ ke et al., Biochem. Biophys. Res. Commun. 297 (2002), 78-82).
  • HPV8 proteins were expressed as GST-fusion proteins in E.coli.
  • the coding sequences of HPV8-E1, -E2, -E6 and -E7 were amplified by PCR using appropriate primers and cloned into the pGEX2T vector (Enzenauer et al., Intervirology 41 (1998), 80-90).
  • HPV16 nt. 559-860
  • HPV77 nt. 524-793
  • Escherichia coll (BL21 LysS) cells harbouring the GST-fusion vectors were induced for 2 h at 37 °C with lmM IPTG.
  • the cells were lysed by sonification in buffer A (50 mM Tris, pH 7.9; 10 % Glycerol; 0.1 % NP-40; 1 mM DTT) containing 500 mM KC1.
  • the insoluble material was removed by centrifugation. 1 ml of the extracts were then incubated for 1 h at 4 °C with 100 ⁇ l of glutathione-Sepharose beads.
  • the beads were collected by centrifugation and washed three times with 1 ml of buffer A containing 1.0 M KC1 and twice with 1 ml of buffer A containing 100 mM KC1.
  • the bound protein was detected by staining with Coomassie blue following SDS-PAGE.
  • Identical amounts of immobilized GST proteins were incubated with 35 S-methionine-labeled Pdcd4.
  • the immobilized GST fusion proteins were washed as described above and incubated with in vitro translated proteins in a 100 ul volume for 2 h at 4 °C with nutation. The beads were then washed three times for 10 min. with buffer A containing 100 mM KCl.
  • HPV1 and 10 are recognized as benign cutaneous types.
  • HPV5, 8 and 15 all belong to the group of EV-specific HPV types and are associated with the malignant conversion of EV-tumors to SCCs (Pfister, Rev. Physiol. Biochem. Pharmacol. 99 (1984), 111-118).
  • HPV77 is found in benign, premalignant and malignant lesions in renal transplant patients (Purdie et al, EMBO J. 18 (1999), 5359-5369).
  • HPV16 is one of the high- risk mucosal HPV types, which are associated with cervical cancer. As shown in Fig.2, the E7 oncoprotein of the EV-associated HPVs 5, 8 and 15 as well as HPV77, are binding to Pdcd4. Unlike these E7 proteins, the E7 protein of the low risk cutaneous HP VI, the low-risk cutaneous HPV10 and the high-risk mucosal HPV16 do not complex with Pdcd4. These results indicate that the property to bind Pdcd4 is only shared by E7 proteins of HPVs found in NMSC.

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Abstract

Selon l'invention, la protéine E7 du papillomavirus humain (PVH) cutané perturbe la fonction de la protéine 4 de la mort cellulaire programmée des suppresseurs de tumeurs (Pdcd4). La présente invention concerne des compositions et des méthodes utiles dans le traitement des tumeurs, en particulier des tumeurs de la peau, consistant à intercepter l'interaction entre la protéine E7 du PVH et Pdcd4. L'invention concerne également des procédés d'identification de composés qui perturbent la liaison entre la protéine E7 du PVH et Pdcd4.
PCT/EP2005/005618 2004-05-24 2005-05-24 Compositions therapeutiques et methodes de traitement du cancer de la peau induit par le papillomavirus humain WO2005115434A1 (fr)

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CN105859846A (zh) * 2015-01-20 2016-08-17 温州医科大学 对hpv16 e7具有结合亲和力的多肽及其用途

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105859846A (zh) * 2015-01-20 2016-08-17 温州医科大学 对hpv16 e7具有结合亲和力的多肽及其用途
CN105859846B (zh) * 2015-01-20 2019-07-12 温州医科大学 对hpv16 e7具有结合亲和力的多肽及其用途

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