WO2003014140A1 - Compositions, methodes et composes faisant intervenir un papillomavirus humain, destines a la detection et au traitement d'un cancer - Google Patents

Compositions, methodes et composes faisant intervenir un papillomavirus humain, destines a la detection et au traitement d'un cancer Download PDF

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WO2003014140A1
WO2003014140A1 PCT/US2001/025095 US0125095W WO03014140A1 WO 2003014140 A1 WO2003014140 A1 WO 2003014140A1 US 0125095 W US0125095 W US 0125095W WO 03014140 A1 WO03014140 A1 WO 03014140A1
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hpv
patient
cancer
sequence
group
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Paul L. Hermonat
Suzanne V. Klimberg
Yong Liu
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Board Of Trustees Of The University Of Arkansas
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • 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
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464401—Neoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • TITLE Compositions, Methods and Products Comprising Human Papillomavirus for Detecting and Treating a Cancer.
  • the present invention relates to products, compositions, methods and apparatus for identification of cancers and pre-cancerous cellular changes.
  • the present invention relates to products, compositions, methods and apparatus for identification of breast cancers or pre-cancerous cellular changes in breast tissues.
  • the present invention relates to products, compositions, methods and apparatus for treatment of cancers and pre-cancerous cellular changes.
  • the present invention relates to products, compositions, methods and apparatus for treatment of breast cancer and pre- cancerous cellular changes in breast tissues.
  • Breast cancer is the most common form of cancer in women in the United States. It is estimated that in the year 2000, 182,800 new cases of female invasive breast cancer will be diagnosed, and 40,800 women will die from the disease. All women are at risk for breast cancer, with this risk increasing as a woman ages. Women are generally considered to be at increased risk for developing breast cancer if they have one or more of the following risk factors: a) a family history of breast cancer, b) a previous diagnosis of a malignant breast tumor or other gynecological cancers, c) hormonal factors, or d) not having had any children or having the first child later in their child bearing years. Even so, the majority of all breast cancers occur in women who apparently do not have identifiable risk factors.
  • Breast cancer cannot currently be prevented. But detecting and treating it at an early stage, when the tumor is small and has not spread beyond the breast, can increase the chances of survival significantly. However, not all breast cancers are currently detected at this early stage. Therefore, screening for breast cancer has become a critical aspect in the overall management of this disease.
  • the techniques currently used to screen for breast cancer and other breast conditions include monthly breast self examination, mammography, and clinical breast examination. Also, genetic testing can be performed for BRCAl and BRCA2 genes in women who have a strong family history of breast cancer, since these genes are associated with approximately 5 to 10 percent of breast cancer cases. In spite of this genetic knowledge, the genetic changes involved in the vast majority of breast cancers remains largely undetermined.
  • HPV 16 DNA is present in 65% of CX CAs, and with the other HPV types, more than 90% of CX CAs contain HPV DNA.
  • the E6 and E7 genes of HPV 16s can cause contact-inhibited cells to lose this phenotype.
  • E6 and E7 interact with the cellular anti-oncogenes RB 105 and p53, respectively, leading to their inactivation.
  • HPV-16 is a central etiologic agent and risk factor in the development of cervical/genital cancer.
  • HPVs have also been found in oral, penile, and vulvar cancer. It appears that whatever tissue site HPVs are known to infect, they cause pathology. Usually the pathology is limited to a tissue hyperplasia or papilloma. However, there is a significant risk that this higher than normal active cell growth may become an outright malignancy.
  • compositions, products and methods for screening a patient for breast cancer and/or pre-cancerous cellular changes in the breast There is another need in the art for improved compositions, products and methods for screening a patient for breast cancer and/or pre-cancerous cellular changes in the breast .
  • compositions, methods and products for screening a patient for cancer and/or pre-cancerous cellular changes It is another object of the present invention to provide for compositions, products and methods for screening a patient for breast cancer and pre-cancerous cellular changes in the breast .
  • a method of screening a patient for a cancer generally comprises performing an amplification technique on a sample from a biopsy taken from a patient.
  • the sample comprises nucleic acid, and the amplification technique is directed to specific amplification of a portion of a human papillomavirus (HPV) sequence contained therein.
  • HPV human papillomavirus
  • the method further includes probing for the presence of an HPV sequence in the amplified sequence using an HPV specific probe.
  • a method of screening a patient for a cancer generally comprises contacting cellular material together with an HPV specific probe.
  • the cellular material is generally extracted from a sample, such as a biopsy, taken from a patient.
  • the cellular material may be any purified or non-purified cellular material such as, for example, deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) , polypeptides, or a combination thereof.
  • the cellular material may be purified, either partially or wholly, using any of the methods well known in the art.
  • the probe used in the screening methods of the invention may be specific to any HPV selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the screening method further comprises contacting the cellular material with a second HPV specific probe, wherein the first and second HPV are different from one another and are selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the screening method further comprises contacting the cellular material with a second HPV specific probe, wherein the first HPV specific probe is specific to HPV 16 and the second HPV specific probe is specific to at least one HPV selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • a method of treating a patient generally comprises administering a composition comprising an effective amount of an antisense HPV DNA sequence to a patient .
  • the antisense HPV DNA sequence is expressed from a viral expression vector, such as an adeno-associated vector.
  • the HPV may be any member of the HPV family, such as, for example, HPV16, HPV18, HPV31, HPV35, HPV 5, HPV58, and any combination thereof.
  • a method of treating a patient generally comprises administering an effective amount of a composition to a patient, wherein the composition comprises an agent that inhibits expression of at least one HPV gene.
  • a method of treating a patient generally comprises administering an effective amount of a composition to a patient, wherein the composition comprises an agent that specifically inhibits an HPV protein.
  • HPV proteins to target for inhibition include the HPV16 E6 protein and the HPV16 E7 protein.
  • Inhibition of a protein can be by any of the methods known in the art, such as, targeting with an antibody, inhibition of post- translation modification, inhibition of protein stability and half-life.
  • a preferred agent for use in the treatment method of the present invention is an antibody specific for interaction with an epitope of an HPV protein, such as HPV16 E6 protein or HPV16 E7 protein.
  • a method of treating a patient comprises transfecting dendritic cells (DCs) into a patient, wherein the dendritic cells have been altered to stably produce an HPV antigen.
  • DCs dendritic cells
  • a recombinant retrovirus such as for example an adeno-associated virus (AAV) that has been genetically manipulated to comprise a portion of an HPV antigen- encoding gene is used to infect monocyte precursors which are then induced to differentiate into DCs. Differentiation of monocytes in DCs may be accomplished by treating the monocytes with at least one cytokine .
  • AAV adeno-associated virus
  • kits useful for screening a patient for a cancer comprising a probe that is specific for the detection of an HPV family member.
  • the HPV-specific probe may be a single-stranded oligonucleotide sequence, a double- stranded oligonucleotide sequence, a polypeptide, or any combination thereof.
  • the HPV may be any HPV family member including, HPV16, HPV18, HPV31, HPV35, HPV45, HPV58, and any combination thereof.
  • the HPV is HPV16 or HPV18.
  • the probe may be used on any sample derived from a patient .
  • Figure 1 is a Polymerase Chain Reaction (PCR) /Dot Blot Hybridization analysis for HPV-16/18/31 "super" probe.
  • Figure 2 provides results from a PCR/Dot blot analysis for HPVs using an LI targeting primer set and probing with HPV- 16 sequences.
  • Figure 3 is a Polymerase Chain Reaction (PCR) /Dot Blot Hybridization analysis for HPVs in breast cancer specimens using an LI targeting primer set and probing with HPV- 18 probe.
  • PCR Polymerase Chain Reaction
  • Figure 4 is a Polymerase Chain Reaction (PCR) /Dot Blot Hybridization analysis for HPVs in breast cancer specimens using an LI targeting primer set and probing with HPV-31 probe.
  • PCR Polymerase Chain Reaction
  • Figure 5 provides results from a PCR/Dot blot analysis for HPVs using an E6-E7 junction targeting primer set and probing with HPV-16 sequences.
  • Figure 6 provides results from a PCR/Dot blot analysis for HPVs using an E6-E7 junction targeting primer set and probing with HPV-18 sequences.
  • Figure 7 is a PCR/Dot Blot Hybridization analysis for contaminating plasmids by using a primer set which targets the pBR322/ColEl ori region.
  • Figure 8A shows a structural map of the AAV/NE6/NE0 (a.k.a. dl6-95/E6 p5 /NEO SV40 ) virus with the names of the components at the top.
  • Figure 8B shows the analysis of various 293/vector producer cell lines.
  • Figure 8C shows a titering analysis of the AAV/E6/Neo virus stock used in this study.
  • Figure 8D shows a graphic description of the experimental protocol .
  • Figure 9 provides E6 mRNA expression in infected DC.
  • Figure 10 provides the efficiency of Mo/DC-pulsing analyzed by intracellular staining.
  • Figure 11 shows the amount of chromosomal integration by AAV/E6/Neo in DC.
  • Figure 12 shows the early appearance of priming rosettes during AAV-mediated priming.
  • Figure 13 shows the cytotoxic response resulting from AAV vector and DOTAP-protein lipofection after 7 days of priming.
  • Figure 14 is a two-color flow cytometric characterization of primed cell populations.
  • Figure 15 is the characterization of DC at day 7 under different conditions.
  • Figure 16 provides the characterization of CD80 in pulsed DC.
  • a method of screening a patient for a cancer generally comprises performing an amplification technique on a sample from a biopsy taken from a patient.
  • the sample comprises cellular material, preferably nucleic acid, and the amplification technique is directed to specific amplification of a portion of a marker for a cancer.
  • the marker is a human papillomavirus (HPV) sequence.
  • the cancer is breast cancer in any stage of development .
  • the method may further include probing for the presence of an HPV sequence in the amplified sequence using an HPV specific probe.
  • the methods of the invention may comprise amplification of at least one HPV sequence selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, or any combination comprising at least HPV sequences.
  • Suitable combinations include: HPV16 and at least any one of the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58; HPV18 and at least any one of the group consisting of HPV16, HPV31, HPV 33, HPV35, HPV45, HPV58; and at least any two of the group consisting of HPV 18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the methods of the invention may comprise probing for at least one HPV sequence selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58, or any combination comprising at least HPV sequences.
  • Suitable combinations include: HPV16 and at least any one of the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58; HPV18 and at least any one of the group consisting of HPV16, HPV31, HPV 33, HPV35, HPV45, HPV58; and at least any two of the group consisting of HPV 18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the samples used in the present invention may be obtained from a biopsy from a cervical intraepithelial neoplasia III (CIN III) positive patient or a CIN III negative patient.
  • CIN III cervical intraepithelial neoplasia III
  • the sample is obtained from a biopsy from a patient who is not afflicted with CIN III (a non-CIN III patient) .
  • the sample may first be tested/assayed for the presence of any CIN Ill-marker known in the art .
  • the sample may be derived from the patient by any method known in the art, such as, for example, any well known method for obtaining a biopsy, including the recently reported technique of breast duct lavage (Dooley W.C. et al., Lancet, 2001, 357 (9265) : 1335-6; Evron, E. et al., Obstetrics & Gynecology, 2001, 97(4) :S2, both of which are incorporated herein by reference) .
  • Numerous methods for obtaining a sample via biopsy are known in the art and include for example bite, brush, cone, cytological, aspiration, endoscopic, excisional, exploratory, incisional, percutaneous, punch, and surface biopsy.
  • Breast duct lavage also referred to as ductal lavage and intraductal lavage, is a relatively non- invasive procedure and enables the retrieval of breast epithelial cells that line the ductal/lobular systems of all milk ducts.
  • the technique of ductal lavage comprises use of a microcatheter which is inserted into the milk ducts through the nipple surface orifices. Saline is then flushed through the ducts to wash out epithelial cells for collection and further evaluation.
  • Amplification methods are well known in the art and include techniques such as, for example, polymerase chain reaction (PCR) amplification and reverse transcription PCR (RT-PCR), as well as others.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription PCR
  • the amplified products may be detected and analyzed using any of the numerous techniques well known in the art.
  • the amplification technique used herein may be specific for amplification of a portion of at least one HPV sequence selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the amplification technique used herein may be specific for amplification of a portion of at least two HPV sequences selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the amplification technique used herein may be specific for amplification of a portion of HPV16 and at least one HPV sequence selected from the group consisting of HPV18, HPV31, HPV 33, HPV35, HPV45, HPV58.
  • the method generally comprises contacting cellular material together with an HPV specific probe.
  • the cellular material is generally extracted from a sample, such as a biopsy, taken from a patient.
  • the cellular material may be any purified or non-purified cellular material such as, for example, deoxyribonucleic acid (DNA) , ribonucleic acid (RNA), polypeptides, or a combination thereof.
  • the cellular material may be purified, either partially or wholly, using any of the purification methods well known in the art .
  • the HPV probes useful in the screening methods of the present invention may be any type of probe useful in detecting the presence of HPV.
  • These probe types include, but are not limited to, a single-stranded or double-stranded oligonucleotide sequence complementary to the plus or minus strand of an HPV DNA sequence, a single-stranded or double-stranded oligonucleotide sequence complementary to a portion of an HPV mRNA sequence, and an antibody specific to an epitope of an HPV protein.
  • Suitable examples of HPV proteins include, but are not limited to, the HPV16 E6 or HPV16 E7.
  • Oligonucleotide sequences specific to HPV sequences are known in the art, such as, for example, those disclosed in Breast Cancer Rch. Trtmt, 53 / 121-135, Anticancer Rch. 19;5057-5062, J. Gen.Virol. 76:1057-1062, J.Pathol 165: 301-309, and J. Clin.Microbiol. 34: 2095-2100, with all of these articles herein incorporated by reference. Oligonucleotides may also be designed according to the Los Alamos National Laboratory Database nomenclature for the different HPV genomes, incorporated herein by reference .
  • the screening methods of the present invention may be performed on a sample from any organism/patient capable of developing cancer.
  • the method of the present invention is performed on a samples taken from a mammal, more preferably a human.
  • the patients on which the methods of the invention are used may be CIN III positive or CIN III negative. In a preferred embodiment, the patients are not afflicted with CIN III (CIN III negative) .
  • the screening methods described herein are useful in detecting numerous types of cancer, such as, for example, breast, dermal, oral, penile, vulvar cancer, and any combination thereof.
  • the screening methods of the present invention are useful in detecting a cancer in any stage of development .
  • the method generally comprises administering a composition comprising an effective amount of an antisense HPV sequence to a patient.
  • the size of the sequence is not limited and can range in size from that of an oligonucleotide to that of a transcript.
  • the antisense HPV sequence may comprise DNA, RNA, ribosomal RNA, or any combination thereof.
  • the HPV may be any member of the HPV family, non-limiting examples of which include, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV58, and any combination thereof.
  • Preferred HPVs include HPV 16 and HPV 18.
  • Non-limiting examples of combinations include: HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31, HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.
  • the HPV sequence may be expressed from a recombinant expression vector.
  • Suitable vectors are known in the art and include, for example, mammalian expression vectors and viral vectors .
  • viral vectors suitable for use in the present invention include: retroviruses; adenoviruses; adenoviral/retroviral chimeras; adeno- associated viruses; herpes simplex virus I or II; parvovirus; and reticuloendotheliosis virus.
  • Other possible viral vectors may be derived from poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as chimeric vectors incorporating favorable aspects of any two or more of the above viruses.
  • the antisense HPV sequence is expressed from a recombinant viral expression vector, such as an adeno-associated vector.
  • Still another embodiment of the present invention provides for a method of treating a patient afflicted with a cancer.
  • the treatment method generally comprises administering an effective amount of a composition to a patient, wherein the composition comprises an agent that inhibits expression of at least one HPV gene.
  • the patients of the present invention may be CIN III positive or CIN III negative. Preferably, the patient is negative for CIN III.
  • Mechanisms for inhibiting the expression of a gene are numerous and well known in the art and include, but are not limited to, inhibiting gene transcription, inhibiting the messenger RNA (mRNA) of a gene, inhibiting translation of an mRNA, inhibiting post-translational modification of a gene product, and inhibiting a gene product . These inhibition methods may be direct or indirect . Any of these mechanisms may be used in the present invention.
  • mRNA messenger RNA
  • Agents that inhibit expression of at least one HPV gene suitable for use in the present invention include, an oligonucleotide or longer stretch of nucleic acid comprising antisense HPV DNA, RNA or ribosomal RNA, and an oligonucleotide or longer stretch of DNA, RNA, or ribosomal RNA comprising a sequence complementary to the plus or the minus strand of HPV DNA.
  • the HPV targeted in the present invention may be any HPV family member, non- limiting examples of which include, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV58, and any combination thereof.
  • Preferred HPVs include HPV 16 and HPV 18.
  • Non-limiting examples of combinations include: HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31, HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.
  • compositions of the present invention may be by any method known in the art.
  • administration of the present invention to a recipient may be by a route selected from oral, parenteral (including, subcutaneous, intradermal, intramuscular, and intravenous) and rectal.
  • the compositions of the present invention may be administered via localized delivery to the targeted tissue, such as, for example, breast tissue in the case of breast cancer.
  • a modified breast duct lavage technique also known as ductal lavage, may be used for localized directly of the anticancer compounds and compositions of the present invention to a breast duct and breast epithelial cells .
  • a microcatheter may be inserted into a nipple surface orifice and subsequently into a milk duct.
  • the microcatheter may be used for localized delivery of a composition of the invention directly to the breast duct and breast epithelial cells.
  • Yet another embodiment of the present invention provides for a method of treating a patient afflicted with a cancer.
  • the cancer may be any cancer in any stage of development.
  • the cancer is breast cancer.
  • the patient may be CIN III positive or CIN III negative.
  • the patient is negative for CIN III.
  • the treatment method generally comprises administering an effective amount of a composition to a patient, wherein the composition comprises an agent that specifically inhibits an HPV protein.
  • HPV proteins to target for inhibition include the HPV16 E6 protein and the HPV16 E7 protein.
  • Inhibition of a protein can be by any of the methods known in the art, such as, inhibition of gene expression, targeting a protein with an antibody, inhibition of post-translation modification, and inhibition of protein stability and half-life.
  • a preferred agent for use in the treatment method of the present invention is an antibody specific for interaction with an epitope of an HPV protein, such as HPV16 E6 protein or HPV16 E7 protein.
  • Additional inhibitory agents suitable for use in the compositions and methods of the invention include agents wherein the agent is a DNA, cDNA, RNA, ribosomal RNA, or polypeptide sequence.
  • Suitable examples of such agents include, an antisense HPV sequence which inhibits transcription or translation of a HPV gene or gene product, transcription factors which decrease expression of an HPV gene, factors which affect translation of an HPV mRNA, factors which decrease the stability/half-life of an HPV mRNA molecule, factors which decrease the stability/half-life of an HPV polypeptide, and factors which interact with an HPV polypeptide, such as a polypeptide encoding an antibody which specifically interacts with an epitope of a HPV.
  • the material and methods for producing these types of inhibitors are known in the art and are included in the present invention.
  • expression vectors expressing a sequence inhibitory to transcription of a HPV gene or expressing a sequence inhibitory to translation of a HPV mRNA are within the scope of the HPV inhibitors defined herein.
  • Expression vectors suitable for the present invention may comprise an antisense HPV sequence, or a sequence encoding a negative regulator of transcription of a HPV gene .
  • Even still another embodiment of the present invention provides for a method of treating a patient afflicted with a cancer.
  • the method generally comprises transfecting dendritic cells (DCs) , primed T cells or a combination thereof, into a patient, wherein the dendritic cells have been altered to stably produce an HPV antigen.
  • DCs dendritic cells
  • the basis for using DC cells for human immunotherapy has recently been established as described in ⁇ Young, J.W. , and Inaba, K. (1996) DCs as adjuvants for
  • DCs are presently believed to be the most effective antigen presenting cells for activating naive T cells.
  • Blood monocytes can be induced to differentiate in to DCs by treatment with at least one cytokine .
  • DCs when pulsed with antigen (Ag) results in a class I restricted cytotoxic response against the Ag.
  • antigen proteins to be targeted are transfected into DCs for immune stimulation. These DC protocols may be used herein.
  • both the antigen to be presented by the Des, and the cytokines used to generate the DCs degrade with time as a reflection of their half-lives.
  • a more effective technique for pulsing/treating the DCs may be the in si tu generation/production of the antigen protein such as at least a portion of an HPV protein, and/or a cytokine within and by the DC itself.
  • the antigen protein such as at least a portion of an HPV protein
  • a cytokine within and by the DC itself.
  • the infected monocytes are then induced to differentiate into DCs by treatment with at least one cytokine.
  • suitable cytokines include, but are not limited to IL-2, IL-4, other interluekins, GM-CSF, TNF, INF, and any combination thereof.
  • the dendritic cells may also stably produce a cytokine from a recombinant vector.
  • a recombinant retrovirus such as, for example, an adeno-associated virus (AAV) that has been genetically manipulated to comprise a portion of an HPV antigen-encoding gene is used to infect monocyte precursors which are then induced to differentiate into DCs.
  • AAV adeno-associated virus
  • an AAV-HPV is used to infect monocytes which are then induced to differentiate into DCs.
  • recombinant retroviral vectors may comprise the general purpose p5 transcriptional promoter to express the antigen genes.
  • Recombinant adeno-associated virus rAAVs
  • rAAVs Recombinant adeno-associated virus
  • these stocks may then be CsCl purified and titered by quantitating the amount of encapsidated genomes (virus) by Southern blot .
  • HPVs useful in the present invention include, HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV58, and any combination thereof.
  • Preferred HPVs include HPV 16 and HPV 18.
  • Non-limiting examples of combinations include: HPV 16 with at least one of HPVI8, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31, HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16, HPV18, HPV31, HPV33, HPV35, HPV 5, and HPV58.
  • the DCs stably produce HPV 16 E6, HPV 16 E7 or both. Any portion of the HPV, full- length or not, may be utilized herein.
  • the treatment methods of the present invention may be performed on any organism having a cancer. Preferably, the methods of the present invention are performed on a human. The patient may be positive for CIN III or negative for CIN III. In a preferred embodiment, the screening methods are carried out on patients who have tested negative for CIN III, and/or are not afflicted with cervical intraepithelial neoplasia (CIN III) .
  • the treatment methods described herein may be useful in treating numerous types of cancer, such as, for example, breast, dermal, oral, penile, vulvar cancer, and any combination thereof. In addition, the treatment methods of the present invention may be against a cancer in any stage of development .
  • compositions and methods of the present invention are suitable for any individual afflicted with a cancer. Suitable individuals include mammals such as, humans, dogs, cats, horses, cows, sheep, goats, pigs, rats and mice. As mentioned, preferably the patient is human.
  • the compositions and methods of the present invention are also suitable for use in any tissue or cell line that serves as a model for the study of cancer. Thus the present invention is useful to medical and health care professionals including, medical doctors, and veterinarians, as well as research scientists. It should be noted that the present invention encompasses any and all methods for screening a patient for a cancer, wherein the method comprises detection of an HPV. Any and all methods for treating a patient having a cancer, wherein the method comprises inhibition of an HPV are also within the scope of the invention.
  • compositions useful in the methods of the present invention further comprise a pharmaceutically acceptable carrier/vehicle.
  • pharmaceutically acceptable carriers/vehicles include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, propylene glycol, polyethylene glycol, vegetable oil, injectable organic esters such as ethyloleate, water, saline solutions, parenteral vehicles such as sodium chloride and Ringer's dextrose, glycerol, lipids, alcohols.
  • compositions of the present invention may be in any form known in the art, such as an orally digestible form, a sterile injectable form, forms suitable for delayed release, and forms that are enterically coated.
  • Compositions of the invention may be in solid forms, including, for example, powders, tablets, pills, granules, capsules, sachets and suppositories, or may be in liquid forms including solutions, suspensions, gels and emulsions.
  • the compositions and methods of the present invention may be administered to a recipient/patient as a single dose unit, or may be administered in several dose units, for a period ranging from one day to several years.
  • the dose schedule is dependent upon at least the severity of the glomerular disorder, as well as the mode of administration.
  • the effective dose of the compositions of the present invention is further dependent upon the body weight (BW) of the recipient/patlent and also upon the chosen inhibitor.
  • BW body weight
  • kits for screening a patient for a cancer comprises a probe that is specific for the detection of an HPV family member.
  • the HPV-specific probe may be a single-stranded oligonucleotide sequence, a double-stranded oligonucleotide sequence, a polypeptide, or any combination thereof.
  • the HPV may be any HPV family member, non-limiting examples of which include, HPV16, HPV18, HPV31, HPV33, HPV35, HPV 5, HPV58, and any combination thereof.
  • Preferred HPVs include HPV 16 and HPV 18.
  • Non-limiting examples of combinations include: HPV 16 with at least one of HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58; HPV 18 with at least one of HPV16, HPV31, HPV33, HPV35, HPV45, and HPV58; both HPV16 and HPV18 with at least one of HPV31, HPV33, HPV35, HPV45, and HPV58, and at least any two of HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58.
  • the kit of the present invention is useful in screening any organism capable of developing a cancer.
  • the sample to be screened is derived from a human patient.
  • the patient may be CIN III positive or CIN III negative, preferably the patient is CIN III negative.
  • the kit of the present invention may be useful in detecting a cancer that is in any stage of development, and may be useful in detecting any cancer, such as, for example breast, dermal, oral, penile, vulvar cancer, and any combination thereof.
  • compositions for treating a patient having a cancer comprising an effective amount of an HPV sequence.
  • the size of the sequence is not limited.
  • the HPV sequence of the composition may comprise single-stranded nucleic acids, double-stranded nucleic acids, polypeptides, and any combination thereof .
  • the HPV sequence may be any one or any combination of HPV family members. Suitable HPV family members include but are not limited to, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 45, HPV58, and any combinations thereof.
  • Suitable combinations include: HPV16 and any one of the group consisting of HPV 18, HPV 31, HPV 33, HPV 35, HPV 45, and HPV58; HPV 18 and any one of the group consisting of HPV 16, HPV 31, HPV 33, HPV 35, HPV 45, and HPV 58; HPV 16 and HPV 18; HPV 16 and HPV 18 and any one of the group consisting of HPV 31, HPV 33, HPV 35, HPV 45, HPV 58, and any combinations thereof.; HPV 16, HPV 18 and HPV 33, and any one of the group consisting of HPV 31, HPV 35, HPV 45, HPV 58, and any combinations thereof, and at least any two of the group consisting of HPV 16, HVP 18, HPV 31, HPV 33, HPV 45 and HPV 58.
  • Example 1 Analysis of breast cancer tissue for presence of HPV via PCR.
  • total DNA was isolated from breast cancer tissues and analyzed for the presence of HPV by use of PCR amplification.
  • the amplification targeted the LI gene and was broad spectrum, thus allowing for amplification of many different HPV types.
  • PCR Polymerase Chain Reaction
  • An HPV Ll-targeting consensus primer set has been described elsewhere and was designed to amplify a 450- base segment of HPV LI gene sequence (Bauer, H.M. , et al . , JAMA 1991; 265: 472-7). These primers enable PCR amplification of most genital HPV types.
  • a second PCR primer set targeting E6-E7 region has also been described by elsewhere (Fujinaga, Y, et al . , J. Gen. Virol. 1991; 72: 1039-1044). These primers allowed for the amplification of most of the cancer associated HPVs.
  • PCR amplification was carried out as described by Hermonat et al . (Hermonat P; et al . , Virus Genes 1997; 14:13-17). Positive controls included various amounts of the indicated cloned HPV genome, while negative controls included all reagents except specimen DNA.
  • PCR amplification was carried out with pBR322 plasmid targeting primers.
  • the primers were designed to amplify a 414 -base segment of pBi?322 sequence (upstream primer 5 ' - ATACCTGTCCGCCTTTCTC-3 ' , and downstream primer 5 ' - AATCTGCTGCTTGCAA AC-3 ' ) , containing the origin of replication ( ori ) .
  • Amplification of DNA samples was carried out in 100 ⁇ l reactions using approximately 5 ⁇ g of the total cellular DNA, 0.2 mM of each dNTP, 1 ⁇ M of each primers, and 2.5 U of Taq iPolymerase according to the suppliers instructions (Fisher Scientific Co., Pittsburgh, PA.) instructions. After 5 minutes at 94° C, each sample was subjected to the following amplification cycle: 55 seconds at 94° C, 1 minutes at 60° C, and 50 seconds at 72° C for 35 cycles, then 10 minutes at 72° C in the final cycle .
  • Primer-a-Gene Labeling System Promega Co.
  • the membranes were analyzed with the radiolabeled full length HPV or pBR322 DNA sequences as indicated.
  • the membranes were soaked in hybridization solution (100 ⁇ g of denatured salmon sperm DNA, 1%SDS, 1M NaCL, 10% dextran sulfate) and incubated at 65° C overnight. After hybridization, the membranes were first washed by 2 X SSC twice at room temperature for 10 minutes, and then washed by 2 X SSC and 1%SDS twice at 55° C (super probe) or 65° C (all others) for 30 minutes.
  • Figure 1 provides the results of an assay in which PCR products were dot blotted and probed with an HPV - 16/18/31 "super" -probe.
  • Figure 1 of the breast cancer specimens, six (B2, B4 , B10, B13 , B15, B17) were positive for HPV.
  • the PCR products were probed with a probe specific for HPV-16 only ( Figure 2) , a probe specific for HPV-18 only ( Figure 3) , and a probe specific for HPV-31 ( Figure 4) only.
  • Figure 2 the results for the HPV-16-specific probe revealed that specimens B2 , B4, and B13 gave a strong signal, suggesting that B2 , B4 , and B13 are most likely HPV-16.
  • HPV DNA In cervical cancer the HPV DNA is often chromosomally integrated. To determine the state of the HPV DNA in breast cancer, 10 ⁇ gs of genomic DNA was digested with Bam HI or Xho I. HPV-16 and HPV contain a single Bam HI site, and no Xho I sites. The restricted DNA, along with undigested DNA, were agarose gel electrophoresed, Southern blotted and probed with 32 P-HPV- 16 DNA. The Southern Blot revealed hybridization of the probe with an 8 kb band, consistent with episomal DNA, . Only in the cervical swab specimen C2 was there significant evidence of chromosomal integration of the HPV DNA.
  • Example 2 Construction of the AAV/E6/Neo genome , generation of virus stocks, and titering of virus stocks .
  • the AAV/E6/Neo genome was constructed as a plasmid, in a similar manner to the construction of the AAV/GM- CSF/Neo viral genome as described by Liu (Liu. Y., et al., J. Inf. Cytok. Res. 2000; 20:21-30), incoporated herein by reference. However, instead of the GM-CSF gene, the HPV-16 E6 open reading frame was cloned by PCR amplification using Pfu polymerase and ligated into the vector. A structural map of the AAV/E6/Neo vector used in this study is shown in Figure 8A.
  • the E6 gene is expressed from the AAV p5 promoter, which is known to be active in DC.
  • An AAV/E7/Neo vector was also made in this study (not shown) .
  • the E7 gene is expressed from the AAV p5 promoter.
  • the virus solution treated by DNase I was incubated with 0.5% deoxycholic acid (Sigma Co.) for 30 minutes at 37°C. After filteration the solution was applied on a heparin-agarose column (Sigma Co.) . The matrix was washed twice with 25ml of 0.254M NaCl-PBS, pH 7.4, and then eluated with 15ml of 0.554M NaCl-PBS, pH 7.4. The eluate was then concentrated to about 1 ml using a Millipore Biomax-IOOK NMWL filter device and cetrifugation. 50 . Purity of the viral preparation (100 ul) was assessed on 4-20% SDS-polyacrylamide gel run.
  • the proteins were detected by Coomassie staining.
  • the titer of purified virus was calculated by dot blot and determined to be 1X10 11 encapsidated genomes per ml.
  • AAV/E6/Neo virus stock was generated by the two step process mentioned above, and a comparison of various G418 resistant producer cell lines, by dot blot hybridization, is shown in Figure 8B.
  • the titering of the non-purified virus stock, in encapsidated genomes (eg) per ml of this virus stock, by dot blot hybridization is shown in Figure 8C (about 10 11 eg/ml) .
  • CA1 patient 1
  • CA2 patient 2
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood monolayer
  • the normal donor had a haplotype of HLA Al, compatible with the target cancer cells.
  • the PBMCs were inoculated into six-well culture plates and incubated with two milliliters of AIM-N medium for two hours at 37 C° and 5% C0 2 , . At that time non- adherent cells were removed by carefully washing the monolayer three times with phosphate buffered saline (PBS, pH 7.0) .
  • PBS phosphate buffered saline
  • the adherent Mo were infected (pulsed) with 0.5 ml of virus stock ( ⁇ 5X10 10 encapsidated genomes) when using the non-purified virus, or 10 7 encapsidated genomes when using the purified virus. After two hours incubation the medium/virus solution was removed, the cells were washed with AIM-V, and finally fed with AIM-V medium.
  • the infection protocol is outlined in Figure 8D.
  • the Mo/DC precursors were infected with 0.5 ml of virus stock or lysate at days 0, 3, and 5. Throughout this time period the Mo/DC culture was treated with human GM-CSF
  • GST-E6 protein was generated in a similar manner to previous generation of GST-E7. 34
  • the Mo/DC were lipofected (pulsed) with 15 ⁇ g of GST-E6 on day 5 as previously described (Santin, A. D., et al . , J. Virol., 1999: 73: 5402-5410).
  • the treatment of the protein pulsed Mo/DC with cytokines was the same as the virus infected DC.
  • E6 mRNA expression was measured by RT-PCR amplification along with a cellular mRNA control.
  • Total RNA was isolated from mock (lysate) infected and AAV/E6/Neo infected Mo/DC using Trizol reagent (GIBCO BRL Life Technologies Inc.), according to the manuf cturer's protocol and treated with 5U/ g of RNase-free DNase I
  • reaction buffer [1 ⁇ g mRNA; 50mM Tris-HCI, pH8.3 ; 75mM KCl; 3mM MgCl 2 ; lOmM DTT; 0.5 ⁇ g oligo(dT) 15 (Promega Co.); 0.5mM each of the
  • PCR amplification of the cDNA was performed in 100 ⁇ l reaction volume which contained 2.5U Tag DNA polymerase (Fisher Scientific Co.); lOmM Tris-HCI, pH8.3 ; 50mM KCl; 2mM MgCl 2 ; 0.2mM each of the four dNTPs; 1 ⁇ M of each upstream and downstream primer specific for the cDNA template and 10 ⁇ l cDNA template.
  • the E6 primer set used was 5 ' -ACCACAGTTATGCACAGAGC-3 ' and 5- AGGACACAGTGGCTTTTGAC-3' , which targeted amplification of the HPV-16 sequences from nt 139 to 420.
  • a control RT- PCR analysis of expression of the housekeeping gene TFIIB was also undertaken with the primer set 5'- GTGAAGATGGCGTCTACCAG-3 ' and 5 ' -GCCTCAATTTATAGCTGTGG-3 ' , which amplified nt 356-1314 of that mRNA..
  • a direct PCR was also undertaken. The products were then analyzed on an agarose gel, stained with ethidium bromide, and visualized by ultraviolet light.
  • the positive contro was the PCR product resulting from the Aav/E6/NEO vector plasmid as a template.
  • Another control was PCR analysis of RNA from cells infected by AAV/E6/Neo virus.
  • a final control included the analysis of cellular TF IX B RNA. Note that only RNA from cells infected with AAV/E6/Neo virus resulted in an appropriate E6 RT-PCR sized product, while mock and PCR amplification of the RNA from cells infected by AAV/E6/Neo virus did not give a product, indicating lack of contaminating DNA.
  • the protocol used was adapted from that described by Pala et al . , (Immunology 2000; 100:209-216).
  • the Mo/DC were infected with virus or lipofected with protein as described above. Cells were then treated with IL-4 and GM-CSF under standard conditions. Seven days after infection/lipofection the cells were harvested, washed and fixed with 2% paraformaldehyde in PBS for 20 min at room temperature. The cells were washed and permeabilized with PBS/1% BSA/0.5% saponin (S-7900, Sigma) for 10 min at room temperature.
  • Cells were stained with anti-HPV-16/18 E6 (Chemicon Inc., Temecula, CA; Cat no MAB874) plus FITC-anti-mouse-Ig (Becton Dickinson Inc., cat no 554001) and analyzed by flow cytometry .
  • Chromosomal integration of the AAV/E6/Neo genome was undertaken by vector-chromosome junction PCR amplification and Southern blot analysis as previously described (Liu. Y. , et al . , J. Inf. Cytok. Res. 2000; 20:21-30). Chromosomal integration of the AAV/E6/Neo vector in DC was observed. Chromosomal integration, while not essential for gene expression from AAV vectors, does signifies a permanent genetic alteration of the DC, and is a desirable "gold standard" for viral transduction.
  • the positive control lane contained 100 ng of Eco RV digested AAV/GM-CSF/Neo plasmid (6.7 & 1.3 Kb) .
  • the negative control lane contained products from a PCR reaction with DNA mock infected cells. Note that multiple Neo-positive bands result from the infected DC population indicating chromosomal integration by the vector, and that multiple vector-positive cell clones are present in the population. As shown in Figure 11, multiple vector-chromosomal junction products were observed in the AAV/E6/Neo infected DC, but not in mock infected DC. Unfortunately, the vector must integrate close to an Alu I element in order to be identified by this technique. In any case, these data indicate that at least some of the viral genomes are able to chromosomally integrate in the DC population.
  • Chromium release assay of CTL activity Chromium release assay of CTL activity.
  • Non-adherent PBMCs T cells and B cells were washed and resuspended in AIM-V at 10-20 x 10 s cells/well in 6- well culture plates (Costar, Cambridge, MA) with rAAV or GST-E6 pulsed DC (ratios from 20:1, responders : dendritic) .
  • the cultures were supplemented with recombinant human GM-CSF (500 U/ml) and recombinant human IL-2 (10 U/ml) .
  • the cells were assayed for tumor cell killing activity in a 6-hour chromium-51 release assay as previously described (Santin, A. D., et al .
  • HLA class I Al compatible primary cervical tumor cells was 51 Cr-labeled and used as targets as previously described (Santin, A. D., et al., J. ' Virol., 1999: 73: 5402-5410).
  • monoclonal anti-Class I Mabs were used to block cytotoxicity.
  • the 51 Cr-labeled tumor targets were pre- incubated with Mabs specific for monomorphic HLA class I W6/32 (50ug/ml) (hybridoma obtained from the ATCC, Rockville, MD) .
  • the effector cells and 51 Cr-labeled targets were then incubated in a final volume of 200 ul for 6 hours at 37°C with 5% C0 2 .
  • FACS flourescent antibody cell sorting
  • T cells For the analysis of T cells, at day 12 of the experiment the primed T cell populations were analyzed for surface markers. A panel of mAbs recognizing the following antigens was used: anti-CD4, anti-CD8, anti- CD56 (Pharmingen, San Diego, CA) . Control irrelevant isotype-matched FITC- or PE- conjugated mAbs were obtained from Becton-Dickinson. These cells were greater than 95% viable as assessed by trypan blue exclusion. Cell suspensions were counted and distributed into 12X75 mm tubes. Mouse monoclonal antibodies were diluted in cold assay buffer and the final pellet was resuspended in 500- ⁇ l volume.
  • Tubes were incubated for 30 minutes followed by two washes with assay buffer and the final cell pellet was resuspended in 500 ⁇ ls of assay buffer for subsequent analysis.
  • Cells were analyzed with a fluorescence activated cell sorter (FACS; Becton- Dickinson) with a 15 mW argon laser with an excitation of 488 nm. Fluorescent signals were gated on the basis of cell dimension (i.e. forward and right angle light scattering typical of PBL activated. Gated signals (5,000-10,000) were detected at 585 BP filter and analyzed using Cell Quest software (Becton-Dickinson) .
  • FACS fluorescence activated cell sorter
  • Mouse monoclonal antibodies were diluted in cold assay buffer (PBS, pH 7.2, supplemented with 0.1% FBS) and added in a 50 ⁇ l volume. For direct fluorescence, tubes were incubated for 30 min followed by two washes with assay buffer and the final cell pellet was resuspended in 500 ⁇ l assay buffer for subsequent analysis .
  • cold assay buffer PBS, pH 7.2, supplemented with 0.1% FBS
  • AAV-mediated pulsing of DC results in rapid and effective T cell priming.
  • Adherent Mo were mock, GST-E6, or AAV/E6/Neo virus pulsed as before ( Figure 8D) , .
  • Each of these cultures were then treated with GM-CSF and IL-4 as prescribed by Sallusto and Lanzavecchia (1994) and Romani et al . (1994) for generating DC.
  • the resulting DCs were then incubated with non-adherent peripheral blood lymphocytes.
  • representative pictures were taken of the cultures at low and high power. Note that the virus treated DC-T cell cultures exhibited much higher levels of rosetted cell clusters, suggesting stronger DC-T cell interaction.
  • DC-T cell incubation and priming require 2 to 3 weeks to allow for significant cytotoxic T lymphocyte activity (CTL) .
  • AAV-pulsing of DCs may require only 7 days of priming.
  • FIG. 13A Shows a representative experiment of cytotoxic response resulting from the indicated pulsing techniques Mo/DC, and T cells from a normal individual against HLA Al matched primary cervical cancer cells (CA1) and a primary multiple myeloma (MM) .
  • CA1 normal individual against HLA Al matched primary cervical cancer cells
  • MM primary multiple myeloma
  • Figure 13B shows a representative experiment of cytotoxic response against a second HLA Al matched primary cervical cancer (CA2) . Again, note that the addition of the class I blocking antibody W632 greatly inhibits killing.
  • DC were pulsed with 10 7 purified encapsidated genomes of AAV/E6/Neo (approximately 10 "3 virus used in the Figure 13 experiments) .
  • a CTL assay was then carried out on the CA1 cervical cancer primary target as described in Figure 13.
  • the resulting killing for AAV/E6/Neo, AAV/E6/Neo plus anti-class I antibodies, GST-E6, and lysate control pulsing of DC was 36.1+/-1.4%, 0.4+/-0.3%, 3.1+/-1.4%, and 2.0+/-0.5%, respectively.
  • Example 3 Higher CD8/CD4 and lower CD56/CD8 cell ratios result with AAV-mediated pulsing/priming.
  • T cell populations which resulted from AAV-transduction or protein lipofection, was observed.
  • An effective CTL response while requiring CD8+ T cells as an effector of lysis, also requires CD4+ helper T cells.
  • Flow cytometric analysis was used to determine the phenotype of the population of the lysate, GST-E6 pulsed, and AAV/E6/Neo pulsed T cell populations.
  • Figure 14A shows the CD8 and CD4 prevalence within the primed population resulting from three different techniques as indicated (on the right) , as well as an FL1-H, FL2-H control (left) .
  • Figure 14B shows the CD56 and CD8 ratios in the same experimental situations as A.
  • CD56 The expression of CD56 was also observed. Some consider this marker as being specific for natural killer T cells. However, others have reported that some CD8+ T cells do express CD56 and do exhibit HLA class I restricted killing. In spite of this confusion, all of T cell populations tested herein exhibited low CD56 expression. However, what is noteworthy in this analysis is the very high level of CD8+ T cells in the case of AAV/E6/Neo pulsing, confirming the CD8/CD4 analysis ( Figure 14A) .

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Abstract

L'invention concerne des méthodes destinées à la détection d'un cancer chez un patient, consistant à détecter un HPV dans une biopsie effectuée sur le patient. L'invention concerne également des compositions et des composés destinés à la détection et au traitement d'un cancer chez un patient, ainsi que des méthodes destinées au traitement d'un patient atteint d'un cancer.
PCT/US2001/025095 2001-08-09 2001-08-09 Compositions, methodes et composes faisant intervenir un papillomavirus humain, destines a la detection et au traitement d'un cancer WO2003014140A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010125420A1 (fr) * 2009-04-30 2010-11-04 Instituto Nacional De Cancerologia Procédé de détection et d'identification de variants du vph16 au moyen d'amorces et de sondes
CN112920267A (zh) * 2021-03-09 2021-06-08 北京康乐卫士生物技术股份有限公司 一种抗人乳头瘤病毒31型的单克隆中和抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4777239A (en) * 1986-07-10 1988-10-11 The Board Of Trustees Of The Leland Stanford Junior University Diagnostic peptides of human papilloma virus
US5238171A (en) * 1991-05-13 1993-08-24 Kanto Yakin Kogyo K.K. Brazing method by continuous furnace
US5501947A (en) * 1990-07-19 1996-03-26 Royal Free Hospital School Of Medicine PCR diagnosis of human papilloma virus type 16
US5629146A (en) * 1988-10-28 1997-05-13 Ferring Ab Method for detection of human papillomavirus (HPV) for diagnostic purposes
US6066624A (en) * 1993-08-26 2000-05-23 Baylor College Of Medicine Gene therapy for solid tumors using adenoviral vectors comprising suicide genes and cytokine genes
WO2000039557A2 (fr) * 1998-12-28 2000-07-06 Pro Duct Health, Inc. Dispositifs, methodes et systemes de prelevement de matiere dans un canal galactophore
US6265154B1 (en) * 1996-10-25 2001-07-24 Abbott Laboratories Nucleic acid primers and probes for detecting oncogenic human papillomaviruses

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4777239A (en) * 1986-07-10 1988-10-11 The Board Of Trustees Of The Leland Stanford Junior University Diagnostic peptides of human papilloma virus
US5629146A (en) * 1988-10-28 1997-05-13 Ferring Ab Method for detection of human papillomavirus (HPV) for diagnostic purposes
US5501947A (en) * 1990-07-19 1996-03-26 Royal Free Hospital School Of Medicine PCR diagnosis of human papilloma virus type 16
US5238171A (en) * 1991-05-13 1993-08-24 Kanto Yakin Kogyo K.K. Brazing method by continuous furnace
US6066624A (en) * 1993-08-26 2000-05-23 Baylor College Of Medicine Gene therapy for solid tumors using adenoviral vectors comprising suicide genes and cytokine genes
US6265154B1 (en) * 1996-10-25 2001-07-24 Abbott Laboratories Nucleic acid primers and probes for detecting oncogenic human papillomaviruses
WO2000039557A2 (fr) * 1998-12-28 2000-07-06 Pro Duct Health, Inc. Dispositifs, methodes et systemes de prelevement de matiere dans un canal galactophore

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010125420A1 (fr) * 2009-04-30 2010-11-04 Instituto Nacional De Cancerologia Procédé de détection et d'identification de variants du vph16 au moyen d'amorces et de sondes
CN112920267A (zh) * 2021-03-09 2021-06-08 北京康乐卫士生物技术股份有限公司 一种抗人乳头瘤病毒31型的单克隆中和抗体及其应用

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