HK1070391B - Methods and compositions relating to hpv-associated pre-cancerous and cancerous growths, including cin - Google Patents

Description

Methods and compositions relating to HPV-associated precancerous and cancerous growths, including CIN

Background

This application claims priority from U.S. provisional patent application 60/306,809 filed on 20/7/2001, which is incorporated herein by reference in its entirety. The U.S. government may have rights in this invention pursuant to grant numbers CA65561 and CA77378 from the National Cancer Institute (NCI) and grant number CA16672 from the national health institute (NIH).

1. Field of the invention

The present invention relates generally to immunology, virology and oncology. And more particularly to methods of diagnosing and treating the occurrence and recurrence of precancerous and cancerous growths or lesions, including Cervical Intraepithelial Neoplasia (CIN), caused by Human Papillomavirus (HPV).

2. Description of the related Art

In women worldwide, cervical cancer is the second malignancy, accounting for 15% of all female cancer patients (Parkin et al, 1993). Cervical cancer is one of the most prevalent neoplasms of the female reproductive tract in the united states. Laboratory and epidemiological studies have focused on the etiological role of some classes of Human Papillomaviruses (HPV) in the pathogenesis of cervical neoplasia (Brinton, 1992; Munoz et al, 1992). Generally, HPV DNA is detected in more than 79% of samples from women diagnosed with cervical disease. The most common HPV type is HPV16, which is found in high-grade squamous intraepithelial lesions and cancers (Lorincz et al, 1992). Epidemiological findings support a correlation between cervical tumors and HPV, a relationship that is more pronounced in HPV type 16 (Morrison et al, 1991; Koutsky et al, 1992; and Munoz et al, 1992). Tumors are formed by abnormal growth of cells, which often results in invasion of normal tissues, such as primary tumors, or in dissemination to distant tissues, such as metastases.

The E6 and E7 genes of HPV16 are often co-expressed and they are abnormally abundant in viral transcripts of live tissue of HPV 16-positive cervical cancer (Wettstein, 1990; Seedorf et al, 1987). There is ample evidence that coexpression of the open reading frames of E6 and E7 is necessary and sufficient for malignant transformation of a variety of mammalian cells (Munger et al, 1989). Furthermore, continuous expression of the E6 and E7 regions of the viral genome appears to be essential for the maintenance of the malignant phenotype (von Knebel Doeberitz et al, 1988).

Some HPV infected patients develop cervical tumors, while others do not. A large proportion of spontaneous regression was also observed, indicating host immunity. The response to the introduction of a Cytotoxic T Lymphocyte (CTL) establishes the primary defense mechanism against viral infection; sometimes, a virus-specific CTL response may be fully protective without concomitant antibody response (Sastry et al, 1992; Bevan, 1989; Lukacher, 1984). Based on the existing literature concerning the relationship between the widespread growth of anti-HPV antibodies, especially those directed against the E7 oncoprotein, and the severity of cervical disease (Cason et al, 1992; Hamsikova et al, 1994; Jha et al, 1993), it was shown that HPV-specific humoral responses may not be protective against HPV-associated cervical disease (Nakagawa et al, 1996). On the other hand, an increased incidence of HPV-associated cervical tumors has been reported in individuals with CMI deficiency, demonstrating the involvement of T-cells in controlling the formation of human HPV-associated tumors (Nakagawa et al, 1996; Tsukui et al; 1996; Feltkamp et al, 1993 and Clerici et al, 1997). In patients with invasive cervical cancer, a reduction in IL-2 production and proliferative response to mitogens such as PHA and concanavalin-A has been observed (Park et al, 1992). A number of in vitro and in vivo strategies have been described to identify peptides that induce T cell activity in mice and humans from the E6, E7 peptides of HPV-16, and the LI protein (Feltkamp et al, 1993; Strang et al, 1990; Tindel et al, 1991; Shepherd et al, 1992; Stauss et al, 1992; Kast et al, 1993). Typically, induction of virus-specific CTLs can be achieved by infection with a virus or recombinant virus that expresses a viral gene product. The viral gene product is processed and presented as a peptide on the surface of infected cells, binds to MHC class I molecules, and is recognized by CTL (Unanuue, 1989).

In addition, research has focused on the identification and characterization of HIV peptides that elicit a virus-specific CTL response. Townsend et al, elucidated the concept of using T-cell epitopes in proteins as vaccine candidates, while their group demonstrated the effect of short synthetic peptides in influenza nucleoprotein as epitopes on CTL responses (Townsend et al, 1986). The present inventors and others have reported the in vivo production of virus-specific CTLs using synthetic peptides (Kast et al, 1991; Aichele et al, 1990; Deres et al, 1989; Sastrain et al, 1992; Sastrain et al, 1994; Casement et al, 1995) against influenza, lymphocytic choriomeningitis, Sendai virus and HIV.

More than 90% of cervical cancers express Human Papillomavirus (HPV) E6 and E7 proteins. These unique antigens are ideal targets for the development of Cytotoxic T Lymphocytes (CTLs) for anticancer immunotherapy. Synthetic peptides corresponding to the E6 and E7 oncoproteins of HPV-16 have been identified as effective for inducing HPV-specific CTL responses in vivo (Sarkar et al, 1995). Recently, Nakagawa et al reported that systemic T cell proliferative responses and CTL responses to HPV-16 peptides and proteins could be detected in many virginals and sexually-living women without cervical lesions, but not in women with disease (Nakagawa et al, 1997). Similarly, Tsukui et al reported that TH lymphocyte responses to HPV antigens, and in particular IL-2 production, were greater in cytologically normal women than in women with a varying degree of progression of cervical neoplasia (Tsukui et al, 1996). Moreover, Clerici et al found that production of TH1 cytokines (IL-2 and IFN-. gamma.) potentially potentiating CMI was defective in women infected with large amounts of HPV, and that progression of CIN was associated with a shift in production of TH1 to TH2 cytokines (Clerici et al, 1997). TH activity was determined using a long-term in vitro stimulation protocol, Kadish et al reported that lymphoproliferative responses to specific HPV peptides were associated with clearance of HPV and degeneration of CIN (Kadish et al, 1997). On the other hand, de Gruijil et al reported that T cell proliferation responses against the HPV 16E 7 peptide are associated with persistent infection of HPV, but that antigen-specific IL-2 production is associated with both viral clearance and the progression of cervical lesions (de Gruijil et al, 1996).

Common clinical treatment regimens for CIN patients include ablation or ablative therapy. However, further studies have demonstrated that a significant number of patients will relapse. Currently, there is no clear understanding of the occurrence of a precancerous or cancerous growth, recurrence or recovery condition in patients who have received ablation or resection therapy for CIN. There is a need for better and improved methods for effectively treating pre-cancerous or cancerous growth and damage diseases associated with HPV.

Summary of The Invention

The present invention is based on the observation that cell-mediated immune (CMI) responses to E6 and/or E7 peptides of Human Papillomavirus (HPV) in patients infected with HPV correlate with their prognosis. For the urogenital tract, particularly for the pre-cancerous or cancerous growth of the cervix, patients exhibiting a cell-mediated immune response at a reduced risk than those not exhibiting cell-mediated immunity; in other words, patients who show a positive cell-mediated immune response, particularly against the E6 and/or E7 peptides, will have a good prognosis in terms of developing HPV-associated precancerous or cancerous growth. Alternatively, a patient who exhibits no or low CMI response to HPV E6 or E7 proteinaceous compounds is at greater risk of poor prognosis with respect to the physiological effects of HPV infection outcome. Thus, the present invention relates to compositions and methods for identifying patients at risk for HPV-associated hyperproliferative conditions, including warts, CIN and malignancies or other pre-cancerous or cancerous growths; the invention is particularly suited for assessing the likelihood of a patient's recurrence of a hyperproliferative condition. Herein, the terms "growth" and "damage" are used interchangeably. Also, the term "precancerous or cancerous growth" refers to growth associated with HPV. In addition to precancerous or cancerous growths or lesions of the cervix, such tumors or lesions may also appear as neoplastic growths or lesions in the urogenital tract, including the perineum, vulva, and penis. Patients suitable for use in the present method may include any mammal capable of being infected with HPV virus; in some embodiments, the patient is specified as a human, male or female.

In some embodiments, the present invention relates to methods for determining the likelihood that a patient infected with human papillomavirus will develop or relapse to a precancerous or cancerous growth. In some cases, the patient has been treated for tumor growth. The method comprises the following steps: obtaining a sample from a patient, incubating the sample with at least one HPV E6 or E7 peptide; and determining cell-mediated immune (CMI) responses of the sample against the peptides. A cell-mediated immune response to either the E6 or E7 peptides, or a combination thereof, indicates a reduced risk of relapse than a human not exhibiting such a response. One pre-cancerous growth frequently observed in the development of cervical cancer is cervical intraepithelial neoplasia or CIN. In some embodiments of the invention, the methods of the invention may be applicable to patients at any stage of CIN (CIN1, CIN2, CIN3 or Squamous Intraepithelial Lesions (SIL), low-grade SIL (L-SIL) and high-grade SIL (HSIL)). Further, in other embodiments, the methods may be applicable to patients with more severe hyperproliferative growths other than CIN, such as malignant or cancerous growths. In the present invention, the term "recurrence" refers to the appearance of a precancerous or cancerous growth, or the recurrence of an initial growth, or the appearance of an initial precancerous or cancerous growth after regression, elimination, or treatment. As used herein, the term "incubating" refers to exposing or contacting a sample to a composition comprising a peptide.

The claimed method is suitable for infection with human papillomavirus. The human papillomavirus may be of a high grade or high risk type, such as HPV16, 18, 31, 45,56 or 58. In some embodiments, the human papillomavirus is HPV 16. In other embodiments, the human papillomavirus is a medium risk type, such as HPV33, 35, 37, 51, 52, 59, 66 or 68. In still further embodiments, the HPV is a low risk or low grade type associated with warts, such as types 6, 11, 26, 40, 42, 43, 44, 53, 55, 62, or 66.

The sample comprises cells capable of eliciting a cell-mediated immune response. In some embodiments, the sample is a blood sample or a serum sample, while in other embodiments, the sample is obtained by lavage, smear or swabbing of an area suspected of infection or having been infected, such as an area of the vagina, cervix or penis. Peripheral Blood Mononuclear Cells (PBMCs) can lead to a cell-mediated immune response, and any sample containing such cells can also be used in the methods of the invention. In some embodiments, it is contemplated that cells from the sample are cultured in culture medium after obtaining and prior to the assay. It is contemplated that the sample may be cultured in the culture medium for 1,2, 3, 4,5, 6,7, 8, 9, 10, 11, 12 or more hours and up to 1,2, 3, 4,5, 6 or 7 days, and up to 1,2, 3, 4,5 or more weeks, and up to 1,2, 3, 4,5, 6,7, 8, 9, 10, 11 or 12 months prior to the assay. It is also contemplated that the cells are cultured in culture medium prior to the assay and/or stored at less than zero degrees centigrade. The cells themselves or cell culture supernatant (broth, incomplete cells) can be used for subsequent analysis of cell-mediated immune responses. In some embodiments, cells are first cultured in culture medium for 2-8 hours (in some cases 6 hours) and then analyzed for intracellular cytokines using flow cytometry. In other embodiments, cells are first cultured in medium for 2 days to 20 days (in some cases 15 days) and then assayed for chromium release to determine Cytotoxic T Lymphocyte (CTL) activity.

The methods of the invention involve determining whether a patient exhibits a cell-mediated immune response to an HPV peptide. In a particular embodiment, the peptides are E6 or E7 peptides, meaning that their amino acid sequence is at least 90% identical over its length to the contiguous amino acid sequence of an E6 or E7 polypeptide. Specifically, consider the use of 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more SEQ ID NOs: 19 (E6 from HPV 16) or SEQ ID NO: 20 (E7 from HPV 16). It is contemplated that in some embodiments, only one sequence of peptide is detected (e.g., the E6 peptide, or another example, the E7 peptide), while in other embodiments, multiple sequences may be detected. In one embodiment, the E6 peptide and the E7 peptide are used in the present invention. In other embodiments, at least two E6 peptides (referring to at least two different E6 sequences), at least two E7 peptides (referring to at least two different E7 sequences), or both are detected simultaneously. It is contemplated that 1,2, 3, 4,5, 6,7, 8, 9, 10, 12, 13, 14, 15 or more of the E6 or E7 peptides may be used, as may any combination of E6 or E7 peptides. In a more particular manner, the E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R, or G10S, or a combination thereof. In particular embodiments, the following E6 peptides will be used alone or as a mixture including one or more of the following peptides, K9L, E10I, C10R, Q15L, or VIOC. In yet other embodiments, the E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C, or D20C, or a mixture thereof. In particular embodiments, the following E7 peptides Q19D, R9F, R9V, L9V, G10C are used alone or as a mixture. In addition, included in the following peptides are mixtures of at least one E6 peptide and one E7 peptide: K9L, E10I, C10R, Q15L, V10C, Q19D, R9F, R9V, L9V or G10C. In some embodiments, it is specifically contemplated to exclude one or more peptides from the above mixture. It is specifically contemplated that compositions related to the diagnostic methods of the invention may also be suitable for use in the prophylactic and therapeutic methods of the invention.

The methods of the invention relate to cell-mediated immune (CMI) responses against human papillomaviruses. There are different approaches to identify and evaluate cell-mediated immune responses (as distinguished from serum or antibody-mediated immune responses). In an embodiment of the invention, proliferation of T cells is measured. T cell proliferation can be assayed by measuring incorporation of tritiated thymidine. A proliferative response having a SI value range equal to or greater than 2.0 for at least one E6 or E7 peptide is considered positive and indicates that the patient is at reduced risk of pre-cancerous or cancerous growth or recurrence of the lesion. A proliferative response to at least one E6 or E7 peptide having an SI value equal to or greater than 3.0 is indicative of a cell-mediated response, thereby identifying improved prognostic progression in pre-cancerous or cancerous growth in the patient. In other words, patients with SI below 2.0, including SI values of zero, are considered to have low or no cell-mediated immune response to the E6 or E7 peptides and are considered to have an increased risk of developing or relapsing their precancerous or cancerous growth.

Cell-mediated responses can also be measured by nonradioactive methods such as MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) dye or reduction assays, a viable cell (T cell proliferation) colorimetric assay (daCosta et al, 1999), or alamar Blue assay, another colorimetric assay for IL-2-responsive cells (Gloeckner et al, 2001; KWack et al, 2000).

In another embodiment of the invention, determining the cell-mediated immune response comprises measuring the amount of a TH1 or TH2 cytokine. Even if the patient does not show a CMI response from a T cell proliferation assay, the increased risk of relapse is still associated with the production of TH2 cytokines, such as IL-10. Patients who show a response to the E6 and/or E7 peptides producing TH1 cytokines, such as IFN-. gamma.and IL-2, are observed to have a reduced risk of relapse. In some examples, the amount of TH1 cytokine is measured, such as IL-2, Interferon (IFN) γ, Tumor Necrosis Factor (TNF) α, or THF- β, IL-3, IL-12, IL-15, IL-16, IL-17, or IL-18. In a specific embodiment, the amount of IL-18 is measured. In further examples, the amount of TH2 cytokine is measured, such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13 or IL-14.

The CMI response can be determined by an immunoassay, such as ELISA or radioimmunoassay or by flow cytometry. In some embodiments, a sample from a patient may be tested more than once, in duplicate samples or different tests. In some embodiments, there may be multiple samples from a patient. The multiple samples may be of the same type, such as multiple blood samples, or they may be of different types, such as one blood sample and one vaginal swab sample.

Patients suitable for use in the methods of the invention include those who have not yet been diagnosed with HPV but who are considered to have HPV, patients who have been infected with HPV but who have not yet shown signs of HPV infection, patients who are known to have HPV infection, patients who have a precancerous or cancerous growth in or around the cervix or other genitourinary area and who are not known to themselves to have HPV infection, patients who have been successfully or unsuccessfully treated for a precancerous or cancerous growth, and at least one patient who has a recurrence of a precancerous or cancerous growth. Precancerous or cancerous growth or damage refers to highly proliferative cells that cannot be controlled in growth, including pre-stage neoplasias, such as CIN and neoplasias (benign and malignant), involving squamous epithelial cells and indeterminately significant Atypical Squamous Cells (ASCUS). Patients with more than one tumor or lesion are considered. The treatment of any tumor involves both surgical (ablation or resection) procedures and conventional cancer treatment against HPV. Such treatments include chemotherapy, radiation therapy, hormonal therapy, immunotherapy, administration of calcium phosphocholine, Thiovir analogs (BioKeys), podofilox, podophyllum, trichloroacetic acid (TCA), or 5 fluorouracil (5-FU), intralesional or intraransal interferon, Imiquimid cream. Ablation techniques include the use of liquid nitrogen, electrosurgery or electrodissection, surgical resection, or laser techniques. Successful treatment refers to treatment that completely removes any signs of a tumor, while partially successful treatment refers to affecting a tumor such that its size or growth rate is reduced, or preventing enlargement of a tumor, or if there are multiple tumors, reducing the number of tumor growths. Patients who have been infected with HPV may then show no signs of HPV infection. However, it is believed that such patients still experience the potential for pre-cancerous or recurrent growth of the cancer, as in patients with continued HPV infection.

In some embodiments of the invention, the patient is evaluated for a determination of HPV infection. In particular, the identification of the HPV serotype is also included or is part of the initial determination of infection. In a further embodiment, the patient is assessed for pre-cancerous or cancerous growth and, in the case of cancer, whether the tumor is benign or malignant.

The methods of the invention include embodiments wherein the sample is obtained from the patient at least one month after treatment for the pre-cancerous or cancerous growth. The patient may be treated for at least one pre-cancerous or cancerous growth, such as by some form of ablation.

The invention also includes methods of treatment for use with the diagnostic methods of the invention. In some embodiments of the invention, patients are identified that have an increased risk of recurrent progression of a precancerous or cancerous growth. Measures may be taken that are not considered until the patient is considered to have an increased risk. In some embodiments, patients who have not been otherwise treated are treated prophylactically for precancerous or cancerous growth or are examined frequently, or both. Prophylactic treatment is treatment in the absence of precancerous or physiological signs of cancer growth; "methods of treatment" include medical treatment of a physiological condition exhibited by a patient. Prophylactic treatment includes the use of HPV infections and HPV-associated precancerous and cancerous growth treatments, as described above.

In some embodiments, the prophylactic methods of protecting against or reducing the risk of pre-cancerous and cancerous growth progression comprise immunotherapy with the E6 and E7 peptides of HPV disclosed herein. If the patient is identified as having a low or no cell-mediated immune response to, inter alia, the E6 or E7 peptides, or to a combination of these peptides, the peptide sequence of E6 or E7 is administered to the patient to elicit a CMI response. These peptides include any of the E6 or E7 peptides, including in particular all or part of the peptides of table 3. Also, peptides from E6 or E7 polypeptides, as discussed in the diagnostic methods of the invention, can also be administered in a prophylactic method. It is contemplated that the patient will be administered a composition containing one or more peptide sequences, and in some embodiments, will also include an adjuvant, a liposome-based composition, or both. In other embodiments, the patient is administered the peptide more than once.

In some embodiments, there is a method of preventing recurrence of a precancerous or cancerous growth (e.g., CIN) in a patient infected with HPV by identifying the patient as at risk for recurrence of an HPV-associated tumor growth using the methods disclosed in the methods of the present invention; and methods of preventing or treating any recurrence. Methods of treatment include surgery (ablation or resection), as well as the conventional anti-HPV cancer therapies described above. In some embodiments, the method is an immunotherapy treatment comprising at least one E6 or E7 peptide from HPV as described above.

In addition, the invention includes a kit for determining the likelihood of recurrence of a precancerous or cancerous growth in a patient who has been infected with HPV and treated for tumor growth, the kit comprising, in suitable reagent containers, at least one E6 or E7 peptide from HPV, and an antibody capable of detecting a cell-mediated immune response against the peptide. In some embodiments, the antibody is attached to a non-reactive structure to which the sample is applied, such as a plate with wells. In other embodiments, the non-reactive structure has a film that can be adhered or otherwise attached to the structure. In some embodiments, the kit can be used in an enzyme-linked immunospot (ELISPOT) assay to detect, in some embodiments, the number of cytokine-secreting cells. In other embodiments, the kit comprises an anti-TH 1 or TH2 cytokine antibody disclosed herein. Other means include detection reagents for detecting the contained antibodies. The detection reagent is any compound that is capable of detecting another compound, including reagents that are capable of visual detection, such as colorimetric detection reagents.

The use of "a" or "an" in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but also mean "one or more", "at least one", "one or more".

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

The figures attached hereto form part of the present specification and are included to further illustrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments of the invention.

FIG. 1A-B proliferation of PBMCs of four different groups of women studied, each with a different response to different synthetic peptides E6 and E7. Group 1 women Normal (CIN)^(-)/HPV^(-)N-6), group 2 women had just diagnosed HPV associated CIN (CIN)⁽⁺⁾/HPV⁽⁺⁾N-31), group 3 post-treatment no disease (Recur)^(-)N-22), and group 4 is relapsed (reccur (+), n-10). PBMCs from four different groups of women were assayed for proliferative response to peptides from oncoprotein E6 or E7 of HPV-16. A. Show each one ofThe Stimulation Index (SI) values of the individual patients for the various peptides tested are based on³[H]The increase in fold over media control for thymidine incorporation peptide treated samples was calculated. B. Summary of positive reactions of groups to E6, E7 peptide or both peptides. The number of groups is shown on the x-axis and the percentage of positive reactions is shown on the y-axis.

FIGS. 2A-D. group 3 (Recur)^(-)) And group 4 (Recur)⁽⁺⁾) Proliferative responses to PBMCs of synthetic peptides derived from HPV-16 oncoproteins E6 and E7. PBMCs from these groups of women were tested for proliferative responses against the 7 and 8 peptides from HPV-16 oncoproteins E6 and E7, respectively, versus control peptides (controls). Representative data showing SI values are shown from two patients in groups 3 (panels a and B) and 4 (panels C and D), respectively.

Figure 3 PBMCs from group 3 women stimulated TH1 cytokine production in response to selected E6 and E7 peptides. Group 3 (Recur)^(-)) And group 4 (Recur)⁽⁺⁾) PBMCs from women were cultured for two days in the presence of E6 peptide Q15L and E7 peptide Q19D, and the presence of various TH1(IL-2, IFN-. gamma., IL-12) and TH2(IL-4, IL-10) cytokines was detected in the respective supernatants by ELISA. The amount of cytokines produced by each patient tested in groups 3 and 4 was shown after adjustment against the culture medium.

Description of illustrative embodiments

Human Papillomavirus (HPV) infection is a major risk factor for cervical cancer, and there is a link between strong HPV-specific cell-mediated immunity and a lesser degree of CIN severity. Common clinical treatment strategies for CIN patients include ablation or ablative treatment, however subsequent studies indicate that a significant number of patients experience relapse. Currently, there is no clear understanding of the recurrence or absence of disease in these patients. Prediction and therapeutic or prophylactic treatment of CIN is critical for infected, non-infected and relapsed patients. Most treatments have been tested and implemented, but they have not been able to rule out disease or prevent disease recurrence. There are a considerable number of patients with CIN relapses, but there is no method to test the likelihood of relapse.

The present invention provides a method for determining the likelihood of recurrence of CIN as a prognostic and biomarker for HPV infection and treatment of CIN patients. The methods involve detecting and analyzing cell-mediated immune responses against HPV oncoprotein peptides, such as E6 and E7. The method also helps identify patients infected with HPV who are at risk of recurrence of CIN. Further, the present invention utilizes a targeted delivery system, kit and immunotherapy to prevent recurrence of CIN and to diagnose patients at high risk for CIN.

I.HPV

Human Papillomaviruses (HPV) were first identified as an important cofactor in cervical neoplasia and cancer progression. However, HPV infection is not sufficient to cause cervical cancer. Not all women infected with HPV develop cervical cancer. Women are usually treated for cervical dysplasia detected in the annual Pap smear. Despite the presence of pap smear tests, epidemiological studies continue to suggest that HPV is the only largest risk factor for the development of cervical tumors and cancer. Cervical Intraepithelial Neoplasia (CIN) is a cervical cancer caused by Human Papillomavirus (HPV). HPV is associated with the development of cervical cancer, particularly HPV types 16, 18, 31, 45,56 and 58. They constitute the high-grade/high-risk types of HPV. Intermediate/medium risk types include HPV33, 35, 37, 51, 52, 59, 66 and 68. Other low grade/low risk types associated with warts are types 6, 11, 26, 40, 42, 43, 44, 53, 54, 55, 62 and 66. These lower forms are not malignant in nature. The HPV genome is currently present in CIN in episomal (incomplete, circular) form, whereas in invasive cervical cancer the genome is usually integrated completely into the host DNA. The high-risk type-expressed E6 and E7 oncoproteins of HPV play a key role in the malignant transformation of cervical squamous epithelium, as they are able to bind and then inactivate two important tumor suppressor genes, p53 and the retinoblastoma gene (Rb). Inactivation of these tumor suppressor proteins is a key factor in the oncogenic potential of HPV.

A. Diagnosis and treatment of cervical cancer

Human papillomaviruses have been identified as being associated with the development of cervical cancer, a condition in which cervical epithelial neoplasia (CIN) exhibits malignancy through several stages. Despite the existence of pap smear tests, epidemiological studies continue to suggest that HPV is the only greatest risk factor for developing CIN, and many studies continue to seek host and/or viral markers that can help identify HPV-infected women at risk for CIN. Equally important to this is the likelihood of recurrence of CIN in the treated patient. Follow-up studies of patients undergoing ablation or ablative therapy have shown a significant number of patient relapses. Therefore, it is very important to be able to evaluate the recurrence probability of CIN. Predicting recurrence may allow the physician to consider the choice of prevention or treatment.

Since the first reports of HPV association with cervical cancer in the early eighties of the twentieth century (zur Hausen, 1994), it has been generally accepted that high-risk HPV types contribute to the initiation and progression of pre-invasive intraepithelial lesions to cancer. In fact, it has been noted that HPV infection culminates in a distinct cytopathy on a Pap smear and is characterized by a perinuclear clearance of the associated nuclear allotype (Kurman et al, 1994). In revised Bethesda terms, these HPV alterations in combination with mild dysplasia are defined as LSIL (Kurman and Solomon, 1994). The effectiveness of HPV testing is complicated by the need to distinguish between low-risk (L-SIL) and high-risk (H-SIL) HPV types (only the latter causing the important risks associated with dysplasia → cancer progression), and the actual risk of progression. In previous cases, new hybrid capture assays for HPV have distinguished high-risk HPV types (Sherman et al, 1995; Poijak et al, 1999; Clavel et al, 2000). In addition to the method of the present invention, DNA image cell counting can also be used to diagnose patients with cervical lesions (Lorenzato et al, 2001).

1. Pap smear

In the past fifty years, Pap smears ("Pap Smear") have been the basis for reducing mortality from cervical cancer. The pap smear is effective because it identifies the earliest stages of cervical cancer. It is currently estimated that 6-7 million pap smears are made in the United states each year. Pap smears thus become the standard for testing for cervical cancer. Despite its wide acceptance in the medical community, studies have shown that pap smear screening fails to detect 50-80% of low grade cancer lesions and even 15-30% of high grade cancer lesions.

The first step in any cytological diagnostic procedure is to obtain suitable pap smear cells for observation. In a conventional pap smear test, a cytologist detects samples of exfoliated cells, which are obtained from scraped cells of the cervical lining, smeared on a slide, and stained with papanicolaou stain. Cytologists detected cells of abnormal morphology on stained smears, indicating the presence of a malignant condition. The term "malignant condition" refers to the presence of dysplasia, including carcinoma of the Adenocarcinoma (AIS) in situ, invasive Carcinoma (CA), tumor, or similar malignant or neoplastic cells.

In the methods of the invention, the exfoliated cell sample is from a patient with or without a malignant condition. These samples may be obtained by rotating a cervical sampling device, such as a swab, spatula, or cytobrush along a portion of the cervical or vaginal mucosa to obtain a cell sample. Suitable samples will contain endocervical cells with squamous and/or glandular cells.

The exfoliated cell sample is typically spread on a glass slide, forming a thin layer of the sample on the surface of the slide. However, manual observation for cellular abnormalities or automated analysis of cytological material can be optimized by preparing a "monolayer" of cells on a sample slide. The definition of "monolayer" is essentially a two-dimensional layer of evenly distributed cellular material, consisting primarily of single cells and small cell clusters.

When performing pap smear tests, the gynecologist collects the exfoliated cells from the cervical surface and places them on a slide, which is handed to the cytologist for further testing. Cytologists then observe the cells placed on the slides and look for abnormal cells. If abnormal cells are found, the pap smear is considered positive. If there are no abnormal cells, the pap smear is considered negative. Pap smear screening is often used as a practical and economical method for early cervical cancer detection. In the present invention, HPV positivity is determined by Virapap/Viratype analysis (technologies Inc., Gaithersburg, Md.).

2. Colposcopy

Pap smear processing is designed for primary screening, while colposcopy and its associated methods are commonly used to determine pap smear abnormalities, cancer grade and potential cancer lesions. Since its introduction in 1925, colposcopy has been widely recognized as a follow-up diagnostic method for patients with a potential for cervical abnormalities as detected by pap smears. It is generally considered to be very effective in evaluating patients with pap smear abnormalities using colposcopy, and has become the medical standard for this condition in western society. An estimated 4 million colposcopy examinations are performed in the united states each year.

3. Fluorescence spectroscopy

Another method for detecting precancerous and cancerous growth or damage is fluorescence spectroscopy, which is capable of rapidly, non-invasively, quantitatively detecting biochemical and morphological changes in tissues as they become tumors. The altered biochemical and morphological state of tumor tissue is reflected as a measurable property of fluorescence. U.S. patents 6,258,576 and 6,135,965 discuss diagnosis of cervical squamous epithelial (CIN) lesions and are specifically incorporated by reference.

3. Treatment of precancerous and cancerous growths

Treatment means elimination, reduction or delay of tumor growth. Cancer growth can be treated by ablation or ablation methods. In addition to the immunotherapy described in detail below, the following methods of treatment or prevention may also be used in the methods of the present invention.

i) Chemotherapy

Cancer therapy also includes a variety of combination therapies based on a combination of both chemical and radiation therapy. Combination chemotherapy, such as Cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine hydrochloride, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea (nitrosurea), actinomycin D, rubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, paclitaxel, gemcitabine, norvegen, farnesyl protein transferase inhibitors, carboplatin (transplatinum), 5 fluorouracil, vincristine, vinblastine and methotrexate, or any analogue or derivative variant of the above drugs.

ii) radiotherapy

Other factors that cause DNA damage and widely used methods include so-called gamma-ray, X-ray and/or radioisotope targeted delivery to tumor cells. Other forms of DNA damaging factors such as microwaves and UV radiation are also included. All of these factors are likely to affect a wide range of DNA damage, precursor DNA, and DNA replication and repair, as well as chromosome assembly and maintenance. The dose values of the X-rays range from 50 to 200 roentgens per day for a period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dosage value of the radioisotope varies widely and it depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by the tumor cells.

When used with respect to a cell, the terms "contacting" and "exposure" herein describe the process by which a therapeutic or diagnostic peptide or polynucleotide, or an agent for chemotherapy or radiotherapy, is delivered to, or placed directly in juxtaposition to, a target cell. To achieve cell lethality or stasis, the two agents are delivered to the cell in a combined total amount effective to kill the cell or prevent its division.

iii) genes

In another embodiment, the secondary treatment employs gene therapy, wherein the therapeutic polypeptide is administered before, after or simultaneously with the chimeric polypeptide of the invention. Delivery of the chimeric polypeptide in combination with a second vector encoding the following gene products will produce a combined anti-hyperproliferative effect on the target tumor. Alternatively, a single vector encoding both genes may be used. The invention encompasses a variety of proteins, including cell proliferation inducers (e.g., growth factor receptors), cell proliferation inhibitors (e.g., tumor suppressors), and cell death modulators.

iv) ablation method

Most people with cancer typically undergo some type of surgery, including prophylactic, diagnostic or staging, therapeutic or palliative surgery. Therapeutic surgery is a treatment for pre-cancerous or cancerous conditions that may be used in conjunction with other therapies, such as chemotherapy, radiation therapy, hormone therapy, gene therapy, immunization and/or additional therapies as described herein.

Curative surgery includes resection in which all or part of the precancerous or cancerous tissue is physically cut, excised, and/or destroyed. Tumor resection refers to at least the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and gene misregistration-controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with the removal of superficial cancers, precancers, or incidental certain normal tissues.

After removal of all or part of the cancer cells, tissue or tumor, a cavity is formed in the body. Treatment is accomplished by perfusion, direct injection or local administration of additional anti-cancer therapy in the area. Such treatment may be repeated, for example, every 1,2, 3, 4,5, 6 or 7 days, or every 1,2, 3, 4 and 5 weeks, or every 1,2, 3, 4,5, 6,7, 8, 9, 10, 11 or 12 months. Such treatment may also vary in dosage.

v) other pharmaceutical agents

Other agents are also contemplated for use in conjunction with the methods of the present invention to improve the therapeutic effect. These additional agents include immunomodulatory agents, agents that affect upregulation of cell surface receptors and GAP junctions, agents that inhibit cell growth and differentiation, inhibitors of cell adhesion, or agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents. Immunomodulators include tumor necrosis factor, interferon alpha, beta, gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 β, MCP-1, RANTES, and other chemokines. Further included is the ability to enhance the apoptosis-inducing capacity of the present invention by establishing an autocrine or paracrine effect on hyperproliferative cells, an increased modulation of cell surface receptors or their ligands, such as Fas/Fas ligand, DR4 or DR 5/TRAIL. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effects of the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiating agents may be used in combination with the present invention to enhance the anti-hyperproliferative effects of the methods. It is also contemplated that the use of cell adhesion inhibitors improves the therapeutic efficacy of the present invention. Contemplated inhibitors of cell adhesion are inhibitors of Focal Adhesion Kinases (FAKs) and lovastatin. In addition, other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in conjunction with the present invention to increase therapeutic efficacy.

Hormone therapy may also be used in combination with the methods of the invention or any of the other cancer therapies described above. Hormone therapy is used to treat certain cancers, such as breast, prostate, ovarian or cervical cancer, to reduce or prevent the action of certain hormones, such as testosterone or estrogen. This therapy is often used in combination with at least one other cancer therapy as a treatment or to reduce the risk of metastasis.

v) antiviral agents

Patients infected with HPV may be treated with antiviral agents alone or in combination with anti-cancer therapies. "antiviral agent" refers to a class of compositions that prevent or inhibit viral infection; preventing or inhibiting progression of viral infection; reducing viral infectivity; preventing, inhibiting or reducing the physiological symptoms of viral infection; preventing or reducing the incidence of viral activation; inhibiting a viral host cell; inducing apoptosis in the host cell; systemic or local clearance of virus; inducing virus inactivation; or any combination of the above.

anti-HPV agents include the administration of drugs such as calcium phosphocholine, Thiovir, Thiovir analogs (BioKeys), Podophyllotoxin, trichloroacetic acid (TCA), 5-fluorouracil (5-FU), intralesional or intraransal interferon, or Imiquimid cream. Other reagents are disclosed in U.S. patents 6,245,568, 6,238,659, and 6,214,874.

Selection, Synthesis and use of proteins and peptides

In the present invention, the peptides are used in diagnostic and therapeutic methods. These peptides are related to HPV16 oncoprotein.

A. Protein composition

In certain embodiments, the present invention relates to novel compositions comprising at least one protein molecule having a peptide sequence as follows: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and the polypeptide SEQ ID NO: 20 and SEQ ID NO: 21. as used herein, "protein molecule," "protein composition," "protein compound," "protein chain," or "proteinaceous substance" generally includes, but is not limited to, a protein of more than 200 amino acids or the full-length endogenous sequence translated from a gene; a polypeptide of more than about 100 amino acids, and/or a peptide from about 3 to about 100 amino acids. All of the above "protein" terms may be used interchangeably herein.

In certain embodiments, at least one protein molecule comprises an amino acid length of, but not limited to, 1,2, 3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 120, 130, 150, 190, 180, 170, 140, 220, 170, 180, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or more amino molecule residues, and any variable range therein. The peptides of the invention include 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or up to and including 100 peptides from SEQ ID NOS: 1-21 adjacent amino acids. SEQ ID NOS: 1-10 peptides from HPV E6 polypeptide, SEQ ID NOS: 11-19 are derived from the E7 polypeptide of HPV. SEQ ID NOS: 20 and 21 are polypeptides from HPV oncoproteins E6 and E7, respectively. GenBank accession number AF327851(SEQ ID NO: 26) for E6 in HPV16 and accession number U76404(SEQ ID NO: 27) for E7 in HPV16 are specifically incorporated by reference. As can be seen from table 3, peptides specifically contemplated as part of the present invention include the following E6 peptides: K9L (amino acids 18-26 of SEQ ID NO: 26), E10I (amino acids 23-34 of SEQ ID NO: 26), C10R (amino acids 37-46 of SEQ ID NO: 26), Q15L (amino acids 43-57 of SEQ ID NO: 26), VIOC (amino acids 49-58 of SEQ ID NO: 26), P9L (amino acids 66-74 of SEQ ID NO: 26), P10I (amino acids 102-111 of SEQ ID NO: 26), Q20P (amino acids 97-116 of SEQ ID NO: 26), R16R (amino acids 131-146 of SEQ ID NO: 26), G10S (amino acids 141-150 of SEQ ID NO: 26), or combinations thereof. From table 3, it is further clear that peptides specifically contemplated as part of the present invention include the following E7 peptides: T10Q (amino acids 7-15 of SEQ ID NO: 27), M9T (amino acids 12-20 of SEQ ID NO: 27), D9L (amino acids 14-22 of SEQ ID NO: 27), Q19D (amino acids 44-62 of SEQ ID NO: 27), R9F (amino acids 49-57 of SEQ ID NO: 27), R9V (amino acids 66-74 of SEQ ID NO: 27), L9V (amino acids 82-90 of SEQ ID NO: 27), G10C (amino acids 85-94 of SEQ ID NO: 27), D20C (amino acids 75-94 of SEQ ID NO: 27), or a combination thereof.

As used herein, an "amino molecule" refers to any amino acid, amino acid derivative or amino acid analog known to those skilled in the art. In certain embodiments, the residues of the protein molecule are contiguous, absent any sequence of non-amino molecules interrupting the amino molecule residues. In other embodiments, the sequence may include one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the protein molecule may be interrupted by one or more non-amino molecule moieties.

Thus, the term "protein composition" includes amino molecule sequences containing at least one amino acid of the 20 common amino acids found in naturally synthesized proteins, or including but not limited to at least one modified or unusual amino acid shown in table 1 below.

TABLE 1 modified and unusual amino acids
				Abbreviations	Amino acids	Abbreviations	Amino acids
Aad	2-aminoadipic acid	EtAsn	N-Ethyl asparagine
				Baad	3-aminoethandioic acid	Hyl	Hydroxylysine
Bala	Beta-alanine, beta-aminopropionic acid	AHyl	Isohydroxylysine
				Abu	2-aminobutyric acid	3Hyp	3-hydroxyproline
4Abu	4-aminobutyric acid, gamma-aminobutyric acid (piperinic acid)	4Hyp	4-hydroxyproline
				Acp	6-aminocaproic acid	Ide	Isodesmoic (lysine) acid
Ahe	6-amino heptanoic acid	AIle	Isoleucine
				Aib	2-Aminoisobutyric acid	MeGly	N-methylglycine, sarcosine
Baib	3-Aminoisobutyric acid	MeIle	N-methylisoleucine
				Apm	2-amino-pimelic acid	MeLys	6-N-methyl lysine
Dbu	2, 4-diaminobutyric acid	MeVal	N-methylvaline
				Des	Catenin (lysine)	Nva	Norvaline
Dpm	2, 2' -diaminopimelic acid	Nle	Norleucine
				Dpr	2, 3-diaminopropionic acid	Orn	Ornithine
EtGly	N-ethylglycine

In certain embodiments, the protein composition comprises at least one protein, polypeptide, or peptide. In further embodiments, the protein composition comprises a biocompatible protein, polypeptide or peptide. The term "biocompatible" as used herein means that it does not produce a significant untoward reaction when administered or dosed in the manner and dosage described herein to a given organism. Organisms include, but are not limited to, such malaise or adverse reactions such as significant toxicity or adverse immune reactions. In a preferred embodiment, the composition comprising a biocompatible protein, polypeptide or peptide is typically a mammalian protein or peptide or a synthetic protein or peptide that is substantially free of toxins, pathogens or harmful immunogens.

Protein compositions may also be prepared by any technique known in the art, including expression of proteins, polypeptides or peptides by standard molecular biology techniques, isolation of proteinaceous compounds from natural sources, or chemical synthesis of proteinaceous materials. Nucleotide and protein, polypeptide and peptide sequences for different genes have been disclosed and can be obtained from computer databases by one of ordinary skill in the art. One of the databases is the national center for Biotechnology information Genbank and GenPept database (http:// www.ncbi.nlm.nih.gov /). The coding regions of these known genes can be amplified and/or expressed using techniques disclosed herein or known in the art.

In certain embodiments, the protein composition may be purified. Generally, "purified" refers to a particular protein, polypeptide, or peptide composition that has been fractionated to remove various other proteins, polypeptides, or peptides, and the composition substantially retains its activity and can be assayed, such as by protein assays for a particular or desired protein, polypeptide, or peptide, as known to those of ordinary skill in the art.

Virtually any protein, polypeptide or peptide-containing ingredient can be used in the compositions and methods of the invention. However, the proteinaceous material is preferably biocompatible. In certain embodiments, it is contemplated that forming a more viscous composition facilitates more precise or easier application of the composition to the tissue and maintains contact with the tissue throughout the procedure. In this case, it is contemplated to use peptide compositions, or more preferably, polypeptide or protein compositions. Viscosity ranges include, but are not limited to, about 40 to 100 poise. In certain aspects, a viscosity of 80 to 100 poise is preferred.

B. Selection, synthesis and use of peptides

Peptide sequences corresponding to the E6 and E7 oncoproteins of HPV16 were selected based on the amphipathic structure and information associated with known T cell epitopes described in the literature.

The peptides of the invention can be synthesized in solution or on a solid support according to conventional methods. Various automated synthesizers are commercially available and used according to known procedures. Typically, small synthetic peptide sequences of less than 100 residues in length are typically prepared by stepwise solid phase synthesis. This solid phase synthesis utilizes an insoluble resin to support the growth of the oligomer. The subunit sequences which are destined to comprise the polymer of interest are co-reacted in sequence on the support. In the initial reaction the terminal amino acid is attached to a solid support either directly or via a linker. The terminal residue is in turn reacted with a series of further residues such as amino acids or blocked amino acid moieties to give a growing oligomer attached to the solid phase via the terminal residue. At each step of the synthesis scheme, unreacted reactants are washed away or removed from contact with the solid phase. This cycle continues with the preselected residue sequence until the desired polymer is fully formed, but remains in contact with the solid phase. The polymer is then cleaved from the solid support and purified for use. The general synthetic scheme described above was invented by r.b. Merrifield and used to prepare certain peptides (Merrifield, 1986). These peptides can be synthesized in a modified Vega 250 automated peptide synthesizer (Vega biochemicals, Tucson, AZ) or using the "pocket method" mentioned by Houghten (Houghten, 1985). See also, e.g., Stewart and Young, (1984); tam et al, (1983); and Barany and Merrifield (1979), which are incorporated herein by reference.

Short peptide sequences, or libraries of overlapping peptides, corresponding to the selected region, typically consisting of 6 to 35 to 50 amino acids, can be readily synthesized and then screened by screening methods designed to identify reactive peptides. The invention includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21, or a peptide comprising at least 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or up to about 100 consecutive amino acid residues.

The compositions used in the present invention may include a peptide modified to enhance its activity or to render it biologically protective. As disclosed in us patent 5,028,592 (incorporated herein by reference), the bioprotective peptides have certain advantages when administered to a patient over non-protected peptides, which typically exhibit enhanced pharmacological activity.

The compositions useful in the present invention may also comprise peptides comprising all L-amino acids, all D-amino acids, or mixtures thereof. The use of D-amino acids may provide additional resistance to proteases naturally occurring in humans and may be less immunogenic and therefore expected to have a longer biological half-life.

Protein purification

Peptides and proteins from HPV can be purified by a variety of methods. Generally, "purified" refers to a particular protein, polypeptide, or peptide composition that has been fractionated to remove various other proteins, polypeptides, or peptides, and which substantially retains its activity, either by protein assay as described below, or by evaluating the protein, polypeptide, or peptide of interest by methods well known to those of ordinary skill in the art.

Protein purification techniques are well known to those skilled in the art. These techniques include, at one level, coarse fractionation of the cell components with polypeptide and non-polypeptide fractions. After separating the polypeptide from other proteins, the polypeptide of interest may be further purified by chromatographic and electrophoretic techniques to achieve partial or complete purification (purification to homogeneity). Analytical methods which are particularly suitable for the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing electrophoresis. Particularly effective methods for purifying peptides are fast protein liquid chromatography or even HPLC.

Certain aspects of the invention relate to purification, and in particular embodiments, substantially to purification of encoded proteins and peptides, particularly peptides from E6 or E7 oncoproteins. The term "purified protein or peptide" as used herein refers to a component that can be separated from other components, wherein the protein or peptide is purified to any degree relative to its native state. Thus a purified protein or peptide is also a protein or peptide that leaves its natural environment.

Generally, "purified" refers to protein or peptide compositions that have been fractionated to remove various other components, and which compositions substantially retain their expressed biological activity. And the use of the term "substantially purified" means that the protein or peptide forms the major component of the composition, such as comprising about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein of the composition.

One skilled in the art will appreciate different methods of quantifying the purity of a protein or peptide based on the present disclosure. These methods include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptide in a fraction by SDS/PAGE analysis. The preferred method of assessing the purity of a fraction is to calculate the specific activity of this fraction and compare it with the specific activity in the initial extract to calculate the purity, here estimated by the "fold purification" (-fold purification number). The actual unit used to represent the activity value will, of course, depend on the particular assay technique chosen after purification and whether the expressed protein or peptide exhibits detectable activity.

Various techniques suitable for protein purification are well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibody, etc., or denaturation by heating, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxyapatite, and hydrophilic chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is well known in the art, the order in which the purification steps are performed may be varied, or certain steps may be omitted, and still provide a suitable method for obtaining a substantially purified protein or peptide.

It is generally not necessary to always provide the protein or peptide in its purest state. In fact, in certain embodiments, substantially impure products are also available. Partial purification can be accomplished using a combination of fewer purification steps, or using different forms of the same conventional purification scheme. For example, cation exchange column chromatography separation using an HPLC apparatus generally results in a higher level of purification than the same technique using a low pressure chromatography system. Methods that exhibit a relatively low degree of purification are advantageous for the overall recovery of the protein product or to maintain the activity of the expressed protein.

It is known that migration of polypeptides is sometimes evident in different SDS/PAGE situations (Capaldi et al, 1977). Thus, under different electrophoresis conditions, it is known that purified or partially purified expression products will exhibit different molecular weights.

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary peak resolution. This is achieved by using very fine particles and high pressure to maintain sufficient flow rate. The separation can be completed in about a few minutes or to an hour. In addition, only a small amount of sample is required, since the particles are very small and densely packed, and only a small fraction of the bed volume is void volume. The concentration of the sample also need not be high because the band is narrow and the sample has only a small spread.

Gel chromatography or molecular sieve chromatography is a particular type of separation chromatography method based on molecular size. The theory behind the gel chromatography separation technique is that the column is made of inert material particles containing small pores, which separate large molecules from small molecules according to their molecular size as the solution passes through or by the pores. The only factor that determines the flow rate is the size of the molecules, as long as the material from which the particles are made does not absorb the molecules. Thus, as long as the properties are relatively unchanged, the molecules elute from the column in order of decreasing size. Gel chromatography is best for separating molecules of different sizes, since the separation is independent of all other factors, such as PH, ionic strength, temperature, etc. And the method has no absorption, less zone diffusion and simple correlation between elution volume and molecular weight.

Affinity chromatography is a chromatographic procedure that relies on a particular affinity between the substance being separated and the molecule to which it specifically binds. This is a receptor-ligand type interaction. The column material is synthesized by covalently binding one of the binding partners therein to an insoluble matrix. The column material is then able to specifically adsorb the species in solution. Elution occurs by changing the conditions to conditions where no binding occurs (e.g., changing the PH, ionic strength and temperature).

A particular type of affinity chromatography used for the purification of sugar-containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to various polysaccharides and glycoproteins. Lectins are usually attached to agarose by cyanogen bromide. "Glucan-coupled concanavalin A" was the first application and has been widely used to isolate polysaccharides and glycoproteins from this group, and other lectins include lentil lectin, used to purify the N-acetylglucosamine residue wheat germ lectin and the mantle snail (Helixpomatia) lectin. The lectin itself is purified by affinity chromatography with sugar ligands. Lactose has been used to purify lectins from castor beans and peanuts; maltose is used for extracting lectin from semen lablab album and semen Canavaliae; N-acetyl-D-galactosamine is used for purifying lectin from soybean; n-acetylglucosamine binds to lectins from wheat germs; d-galactosamine is used to bind lectins from clams and L-fucose from lotus.

The matrix should be a substance that does not itself absorb molecules to any significant degree and has a wide range of chemical, physical and thermal stability. The ligands should be coupled in such a way that their binding is not affected. The ligand should also provide relatively tight binding. The elution of the substance should be carried out as far as possible without damaging the sample or the ligand. One of the most common affinity chromatography is immunoaffinity chromatography. The production of antibodies suitable for use in the present invention is discussed below.

Nucleic acids

A. Screening of DNA

In the present invention, nucleic acid screening is used not only to screen infected samples, but also to discover the likelihood of disease recurrence. Screening means relying on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided that there is a suitable probe. Oligonucleotide probes corresponding to a portion of the sequence encoding the protein of interest can be chemically synthesized. This requires knowledge of the amino acid sequence of the short oligopeptide. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to carry out a mixed addition reaction when the sequence is degenerate. Including heterogeneous mixing of denatured double-stranded DNA. For this screening, hybridization is preferably carried out on single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in detecting cDNA clones derived from sources where there are very small amounts of mRNA sequences associated with the polypeptide of interest. In other words, by using stringent hybridization conditions that avoid non-specific binding, it is possible to allow, for example, self-visualization of a particular cDNA by hybridizing the target DNA to a single probe in the mixture that is fully complementary to it (Wallace et al, 1981). Double-stranded molecules that are both stable and selective can be formed using probes or primers of 13 to 100 nucleic acids, particularly probes or primers between 17 to 100 nucleic acids in length, or in some aspects of the invention, up to 1 to 2 kilobase pairs or longer. Molecules having complementary sequences of greater than 20 bases in contiguous length are generally preferred to enhance stability and/or selectivity of the resulting hybrid molecule. It is generally preferred that the nucleotide molecules designed for hybridization have one or more complementary sequences of 20 to 30 nucleotides or as long as desired. Such fragments are readily prepared, for example, by direct synthesis by chemical means or by recombinant introduction of the selectable sequence into a recombinant vector.

Thus, the nucleic acid sequences of the invention may be used, depending on their ability, to selectively form double-stranded molecules having complementary sequences to DNA and/or RNA, or to provide primers for amplification of DNA or RNA from a sample. Depending on the application, it may be desirable to use a variety of hybridization conditions to achieve varying degrees of selectivity of probes and primers for target sequences.

For applications requiring high selectivity, it is typically desirable to form hybrids using relatively high stringency conditions. For example, relatively low salt and/or high temperature conditions, such as providing a NaCl concentration of 0.02M to about 0.10M and a temperature of about 50 ℃ to about 70 ℃. Such highly stringent conditions allow little, if any, mismatch between the probe or primer and the template or target strand, and are particularly suitable for isolating a particular gene or detecting a particular mRNA transcription. Conditions can be rendered more stringent by the addition of increased amounts of formamide.

For certain applications, such as site-directed mutagenesis, it is understood that conditions of lower stringency are preferred. In this case, hybridization occurs, although the sequences of the hybridized strands are not perfectly complementary, but are mismatched at one or more positions. By increasing the salt concentration and/or lowering the temperature, the conditions may become less stringent. For example, medium stringency conditions are those in which the NaCl concentration is from 0.1 to 0.25M at about 37 ℃ to about 55 ℃, while low stringency conditions can provide a salt concentration of from about 0.15M to about 0.9M, and a temperature in the range of from about 20 ℃ to 55 ℃. Hybridization conditions can be conveniently controlled depending on the desired results.

In other embodiments, hybridization can be performed under conditions such as 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl₂1.0mM dithiothreitol at a temperature between about 20 ℃ and about 37 ℃. Other hybridization conditions used include about 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl₂And a temperature range of between about 40 ℃ to about 72 ℃.

In certain embodiments, it is advantageous to use the nucleic acid sequences determined according to the invention in combination with a suitable method (e.g., a label) to determine hybridization. A variety of suitable indicators are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which can be used for detection. In a preferred embodiment, it is advantageous to use a fluorescent label or an enzymatic label, such as urease, alkaline phosphatase or peroxidase, without the use of radioactive or other environmentally unfriendly reagents. In the case of enzymatic reagents, it is known that colorimetric indicators can provide a visual or spectrophotometrically detectable detection reagent to detect specific hybridization with a sample containing complementary nucleic acids.

In general, it is contemplated that the probes or primers described herein may be used as reagents in hybridization solutions, such as in PCR^TMFor detecting the expression of the corresponding gene, and also for embodiments using a solid phase. In embodiments involving a solid phase, the DNA (or RNA) under test is absorbed or affixed to a selected matrix or surface. The immobilized single-stranded nucleic acid is then hybridized with the selected probe under the desired conditions. The choice of such conditions will depend on the particular circumstances (e.g., depending on the G + C content, the type of target nucleic acid, the source of the nucleic acid, the size of the hybridization probe, etc.). According to the specific purposeThe use of (A) to optimize hybridization conditions is well known to those skilled in the art. After elution of the hybridized molecules to remove non-specifically bound probe molecules, hybridization is detected and/or quantified by determining the amount of bound label. Typical solid phase hybridization methods have been disclosed in U.S. Pat. Nos. 5,843,663, 5,900,481, and 5,919,626. Other hybridization methods that can be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. These and other related references identified in this section of the specification are hereby incorporated by reference.

B. Synthesis of DNA

When the complete sequence of the amino acid residues of the polypeptide product of interest is known, the synthesis of a DNA sequence is a frequently chosen method. When the complete sequence of amino acid residues of the polypeptide product of interest is not known, it is not possible to directly synthesize a DNA sequence, and the method of choice is to synthesize a cDNA sequence. The standard procedure for isolating a cDNA sequence of interest is to form a cDNA library carried by a plasmid or phage obtained by reverse transcription of mRNA enriched in donor cells having high levels of gene expression. When synthesized using polymerase chain reaction techniques, even rare expression products can be cloned. Where the major portion of the amino acid sequence of the polypeptide is known, DNA/DNA hybridization of a labeled single or double stranded DNA or RNA probe sequence identical to that which is supposed to be present in the target cDNA can be used, this hybridization being carried out on a cloned copy of the cDNA which has been denatured to single stranded form.

1. Biological chip

Methods for isolating arrays of biomolecules on various supports (called biochips) have been developed and used in DNA synthesis, sequencing, mutation studies, gene expression analysis, and gene discovery. The biochip can be used in the present invention because it enables us to identify markers of pathological states, here HPV infections, which may have subsequent diagnostic value.

The use of biochips involves hybridizing labeled molecules or collections of molecules to targets immobilized on the biochip. The tagged molecule is typically a cDNA copy of the mRNA of the cell or tissue. In this case, the copy number of each different type of cDNA reflects the copy number of the corresponding mRNA species in the original isolate. In general, the intensity of hybridization to the target immobilized on the biochip is proportional to the cDNA concentration, and thus measurement of the hybridization density allows the relative amount of mRNA in the initial isolate to be extrapolated. The relative amount of the same mRNA in different mRNA isolates can be determined by comparing the hybridization density to the same target between two samples. These measurements can be used to identify specific cell types or pathological conditions that can have subsequent diagnostic value. Alternatively, a sudden increase in a particular mRNA in a given disease may indicate a possible target for drug attack, thus providing a new therapeutic target.

C. Nucleic acid amplification reaction

Nucleic acid molecules can be detected by using a variety of techniques, including amplification reactions. The invention includes the use of such amplification reactions to detect cell-mediated immune responses or to identify patients infected with HPV and/or having pre-cancerous or cancerous growth. For example, cell-mediated immune responses can be detected by RT-PCR of TH1 or TH2 cytokines disclosed herein.

1. Polymerase Chain Reaction (PCR)^TM)

The nucleic acid used as a template for amplification is isolated from the cells contained in the biological sample according to standard procedures (Sambrook, 1989). The nucleic acid may be genomic DNA or cellular RNA in part or in whole. When RNA is used, it is necessary to convert the RNA to cDNA.

Selective hybridization corresponds to K under conditions permitting selective hybridization_ATPA primer pair of a nucleic acid of a channel protein or a mutant thereof is contacted with the isolated nucleic acid. The term "primer" as defined herein includes any nucleic acid capable of priming synthesis of a nascent nucleic acid in a template-dependent process. Typically, the primer is an oligonucleotide of 10 to 20 base pairs in length, although longer sequences may also be used. Guiding deviceThe substance may be supplied in single-stranded or double-stranded form, but single-stranded forms are preferred.

Once hybridized, the nucleic acid: the primer complex is contacted with one or more enzymes that promote template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles", are performed until a sufficient number of amplification products are produced.

Next, the amplification product is detected. In some applications, detection may be performed visually. Alternatively, detection may involve indirect identification of the product by chemiluminescence, radioscintillation or fluorescent labeling with the introduction of a radioactive label or even by means of electrical or thermal pulse signals (Affymax process).

A number of template-dependent methods can be used to amplify the marker sequences present in a given template sample. The best known method of amplification is the polymerase chain reaction (known as PCR)^TM) This process is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, all of which are incorporated herein by reference in their entirety.

Briefly, in PCR^TMTwo primer sequences complementary to opposite complementary strand regions of the marker sequence are prepared. Excess deoxynucleoside triphosphates are added to the reaction mixture with a DNA polymerase (e.g., Tap polymerase). If a marker sequence is present in the sample, the primer will bind to the marker and the polymerase will extend the primer along the marker sequence by adding nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primer will separate from the label to form a reactant, the excess primer will bind to the label as well as the reactant, and the process is repeated.

For quantifying the amount of amplified mRNA or preparing cDNA from the mRNA of interest, reverse transcriptase PCR may be used^TM(RT-PCR^TM) And (3) an amplification process. Methods for reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989. Alternative reverse transcription methods utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO90/07641, filed 1990 on 21/12, which is hereby incorporated by reference. Polymerase chain reaction is a well-known method in the art。

2. Other nucleic acid amplification reactions

Another amplification method is the ligase chain reaction ("LCR"), disclosed in EPA No.320308, which is incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, each pair binding opposite complementary strands of the target sequence in the presence of the target sequence such that they are adjacent. In the presence of a ligase, the two probe pairs will be joined into a single sequence. By temperature cycling (as in PCR), the bound linker sequence is separated from the target and then used as a "target sequence" to link excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR to bind probe pairs to target sequences.

Qbeta replicase may also be used as an alternative amplification method of the present invention, as described in PCT application PCT/US87/00880, which is incorporated herein by reference in its entirety. In this method, a replication sequence of RNA having a region complementary to a target is added to a sample in the presence of RNA polymerase. The polymerase will copy the replicated sequence, which is then detected.

The invention also provides for the amplification of nucleic acids using isothermal amplification methods that employ restriction endonucleases and ligases to effect amplification of a target molecule that comprises nucleotides 5' - [ α -thio ] -triphosphate on one strand of the cleavage site.

Strand Displacement Amplification (SDA) is another method of performing isothermal amplification of nucleic acids involving multiple cycles of strand displacement and synthesis, i.e., nick translation. A similar approach, known as the Repair Chain Reaction (RCR), involves annealing several probes in the amplified target region, and then allowing the repair reaction to occur in the presence of only two of the four bases. Two additional bases may be added as biotinylated derivatives for detection. A similar method is also applied in SDA. Target-specific sequences can also be detected using the Cycling Probe Reaction (CPR). In CPR, probes having the 3 'and 5' sequences of non-specific DNA and the intermediate sequence of specific RNA hybridize to DNA present in the sample. After hybridization, the reaction was treated with RNase H and the products of the probes were identified as the different products released after digestion. The original template is annealed to another cycling probe and the reaction repeated.

Still further amplification methods may be used in the present invention and are disclosed in UK application 2202328 and PCT application PCT/US89/01025, both of which are incorporated herein by reference in their entirety. At the beginning of use, "modified" primers were used for PCR^TMClass, which relies on template and enzyme synthesis. The primer may be modified by labeling the capture moiety (e.g., biotin) and/or the detection moiety (e.g., enzyme). In subsequent uses, an excess of labeled probe is added to the sample. In the presence of the target sequence, the probe binds and is catalytically cleaved. After cleavage, the target sequence is released intact to bind excess probe. Cleavage of the labeled probe indicates the presence of the target sequence.

Other nucleic acid amplification methods include transcription-based amplification systems (TAS), including nucleic acid sequence-based amplification (NASBA) and 3SR Gingeras et al, PCT application WO 88/10315, incorporated herein by reference. In NASBA, nucleic acids prepared for amplification can be extracted by standard phenol/chloroform extraction, thermal denaturation of clinical samples, treatment with lysis buffer and small spin columns to separate DNA and RNA or guanidinium chloride to extract RNA. These amplification techniques involve annealing primers with target specific sequences. After polymerization, the DNA/RNA hybrid is digested by RNase H, and the double stranded DNA molecules are annealed again by heating. Under either condition, the single-stranded DNA is completely double-stranded by the addition of the second target specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by RNA polymerases such as T7 or SP 6. In the isothermal cycling reaction, RNA is reverse transcribed into single-stranded DNA, which is then converted into double-stranded DNA, and then transcribed again with an RNA polymerase such as T7 or T6. Whether the synthesis product is truncated or intact, the target specific sequence is indicated.

Davey et al (EPA No.329822, incorporated herein by reference in its entirety) disclose a nucleic acid amplification method that includes the cyclic synthesis of single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which can be used in the present invention. ssRNA is the template for the first primer oligonucleotide, which is extended by reverse transcriptase (RNA dependent DNA polymerase). RNA is then separated from the resulting DNA by the action of ribonuclease H (RNase H, a ribonuclease specific for RNA in a DNA or RNA pair): removal in the RNA duplex. The resulting ssDNA is the template for the second primer and also includes the sequence of the RNA polymerase promoter (exemplified by T7RNA polymerase) 5' to its sequence homologous to the template. The primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of e.coli DNA polymerase I) to yield a double-stranded DNA ("dsDNA") molecule having the same sequence as the RNA between the original primers and further containing a promoter sequence at one end. The primer sequence can be used to prepare many RNA copies of the DNA by an appropriate RNA polymerase. These copies are then re-entered into the cycle resulting in very rapid amplification. With the appropriate choice of enzyme, this amplification can be performed under isothermal conditions without the need to add the enzyme in each cycle. Because of the cyclic nature of this process, the initiation sequence may be selected to be either in DNA or RNA form.

Miller et al (PCT application WO 89/06700, incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on hybridizing promoter/primer sequences to a target single-stranded DNA ("ssDNA") and then transcribing many RNA copies of the sequence. This protocol does not cycle, i.e., does not generate new templates from the synthesized RNA transcripts. Other amplification methods include "RACE" and "single-sided PCR" (Frohman, 1990, incorporated by reference).

Methods based on the ligation of two (or more) oligonucleotides that amplify a di-oligonucleotide in the presence of a nucleic acid containing the resulting "di-oligonucleotide" sequence may also be used in the amplification step of the present invention.

D nucleic acid detection

After any amplification, it is desirable to separate the amplification product from the template and/or excess primer. Detection of nucleotides can be used to identify cell-mediated immune responses, and patients infected with HPV and/or having pre-cancerous or cancerous growth.

In one embodiment, the amplification products are separated by agarose, agarose acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989). The amplification product can be excised from the gel and eluted for isolation. Using a low-melting agarose gel, the separation band can be removed by heating the gel, and the nucleic acid can then be extracted.

Nucleic acid isolation may also utilize chromatographic separation techniques well known in the art. A wide variety of chromatographic techniques may be used in the practice of the invention, including adsorption chromatography, partition chromatography, ion exchange chromatography, hydroxyapatite chromatography, molecular sieve chromatography, reverse phase chromatography, column chromatography, paper chromatography, thin layer chromatography, gas chromatography and HPLC.

In certain embodiments, amplification products may be visualized. Typical visualization methods include staining the gel with ethidium bromide and visualizing the bands under ultraviolet light. Alternatively, if the amplification products are globally labeled with radiolabeled or fluorescently labeled nucleotides, the separated amplification products can be visualized by exposure to x-ray film or under appropriate excitation spectra.

In one embodiment, the labeled nucleic acid primer is contacted with the amplified marker sequence after isolation of the amplification product. The primer is preferably conjugated to a chromophore, but may also be radiolabeled. In another embodiment, the primer is conjugated to a binding partner, such as an antibody or biotin, or other binding partner with a detectable moiety.

In a particular embodiment, detection is performed by hybridization of a labeled primer by southern blotting. Techniques involving southern blot hybridization are well known to those skilled in the art (see Sambrook et al, 1989). The above examples are described in U.S. Pat. No. 5,279,721, which discloses an apparatus and method for automated electrophoresis and nucleic acid transfer, incorporated herein by reference. Instruments that allow electrophoresis and blotting without the need for additional handling of the gel are ideal for practicing the methods of the invention.

HPV infection can also be detected by catalytic amplification of the colorimetric signal of DNA hybridization in situ (CSAC-ISH) (GenPoint system, DAKO) (Birner et al, 2001).

Other methods for practicing nucleic acid detection of the present invention are disclosed in U.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992,5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and 5,935,791, all of which are incorporated herein by reference.

Cell-mediated immunity (CMI)

Some of the methods claimed herein exploit T cell responses by using them as a diagnostic indicator of relapse or as a prophylactic treatment to prevent the development of CIN. More specifically, these methods determine CMI responses against E6 and E7 oncoprotein synthetic peptides of HPV 16. The E6 and E7 genes of HPV16 are often co-expressed and they are most abundant in the viral transcripts of HPV 16-positive cervical cancer biopsies (Wettstein, 1990; Seedorf et al, 1987). There is ample evidence that coexpression of the open reading frames of E6 and E7 is necessary and sufficient for malignant transformation of various mammalian cells (Munger et al, 1989). Furthermore, continuous expression of the E6 and E7 regions of the viral genome appears to be essential for maintenance of the malignant phenotype (von Knebel Doeberitz et al, 1988).

Viral infection of most immunocompetent mammals results in a cell-mediated immune response against cells infected with the virus, and the ultimate effect is cytolysis. During viral infection, viral proteins are synthesized in the cell in order to encapsulate new viral particles. Some of these endogenous viral proteins are also degraded and transported to the class I antigen presentation pathway where foreign antigens bound to MHC class I molecules are bound. The peptide-MHC complex is then transported to the surface of cells presenting the foreign antigen, via the self-MHC, to reach cytotoxic T Cells (CTLs).

CTLs are specific antigen effector cells. The study of lymphocyte surface markers can be used to analyze for the presence of such T cell surface markers by a variety of methods well known to those of ordinary skill in the art, including the use of immunofluorescence and flow cytometry. Once the foreign antigen is recognized, the CTL lyses the target cell, destroying its integrity, either by molecular interactions that induce apoptosis or by secreting microporous-forming enzymes that create holes in the plasma membrane. Thus, CTL-mediated immune responses play a critical role in the clearance of cells infected with the virus.

The ability of CTL effector cells to lyse virus-infected target cells is regulated by genetic and antigenic limitations. The target cells must carry the same or equivalent viral antigen as the virus that originally induced the CTL. The target cells and the induced CTLs must also bear the same MHC class I molecules.

A. Peripheral Blood Mononuclear Cells (PBMC)

The proliferative responses were obtained from PBMCs. There are a few methods available for PBMC isolation.

Monocytes were isolated from non-rosette cells by attachment to glass or polyethylene tissue culture vessels with or without collagen coating in RPMI1640 containing 20% Fetal Calf Serum (FCS) and were endotoxin free as determined by limulus ameboclysis assay; alternatively, autologous serum, or normal donor serum at 10% AB + is used as the serum source. Non-adherent cells were removed by gentle washing. Using this method, adherent cells typically have > 90% monomeric cells. If tissue analysis suggests significant contamination with T cells, B cells, or NK cells, these contaminating cells are removed with a mixture of antibodies, including anti-leu 5B, anti-leu 12 and anti-leul 1B and young rabbit complement (Rossen et al, 1985).

5% CO in a Teflon coated vessel humidified at 37 ℃₂After 1 hour or more of attachment in air, monocytes are released for suspension culture (crown et al, 1987). For the case of cells plated on the surface of collagen coating, 1mg/ml collagenase type 1 was added to the medium. Cells were released by incubation in Dulbecco phosphate buffered saline containing 5% FCS and EDTA, without calcium and magnesium, for 15 minutes or more. Incubation with EDTA was performed on ice. Use of a disposable cell scraper to aid in cell removal. Scraped cells were washed twice in calcium and magnesium free Dulbecco's PBS and cultured in Teflon culture tanks in RPMI1640 and 10% AB + human serum as described by Crowe et al (1987).

A second strategy for isolating peripheral blood mononuclear cells is the Percoll density gradient method according to Hester and Walker (1981) to increase the concentration of mononuclear cells in non-rosette cell populations. Monocyte-enriched populations, if required treated with the antibody cocktail and complement described above, to remove contaminating T cells, B cells and NK cell remnants as much as possible. Monocytes recovered by this method were cultured directly in Teflon-coated vessels without the "activation" that had to occur when monocytes started to attach to the surface. However, the Percoll density gradient step and/or exposure to antibodies and complement may also "activate" these cells, possibly in a different manner.

A third strategy for isolating peripheral blood mononuclear cells is to use the refractive properties of the mononuclear cells to sort them based on forward angle light scattering using the high speed cell sorting function of flow cytometry. It has the potential to produce cells of high purity without being affected by contact with antibodies or complement.

B.T cell response

T cell responses can be detected by various methods known to those of ordinary skill in the art. Some of the analytical methods will be described in detail below.

1.³[H]Thymidine incorporation assay

The proliferation response of PBMCs from different samples can be as standard in the open literature³[H]Thymidine incorporation assays were determined (Nehete, 1996; Nehete, 1995). Significance of proliferative responses of T cells to individual E6 and E7 peptides (in terms of stimulation index SI]) The cells that can be exposed to the peptide exceeded those of the control group without peptide addition³[H]Fold increase in thymidine incorporation was calculated. When the SI value is at least 2.0, including at least 2.1, 2.2, 2.3, 2.4,a positive reaction is considered to be caused when the reaction is carried out at 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 or more. Generally, the SI value is calculated by measuring the amount of radioactivity (cpm) in cells incubated with the peptide in the medium, divided by the amount of radioactivity in cells incubated without the peptide (medium only).

2. By using⁵¹[Cr]Dissolution

Cell-mediated lympholysis (CML) can be used to indicate T cell responses. The target cells may be exposed to radioactive chromium-51 (C) (b)⁵¹[Cr]) And (4) marking. Released into the culture medium⁵¹[Cr]The amount of (a) is proportional to the level of cell-mediated lysis.

3. Production of interferon-gamma

Gamma interferon (gamma interferon), also known as type II immune interferon, is produced by T cells and NK cells. It is critical for helper T cell development. Being the initial macrophage activating factor, it is a powerful cytokine in cell-mediated immunity. Interferon gamma increases the expression levels of MHC class I and II molecules, which enhance antigen presentation and other recognition responses. It also amplifies the effects of TNF- α and increases the expression of vascular endothelial cell surface adhesion molecules which allow T cells to adhere and extravasate.

4. Tetramer analysis

Tetramer analysis methods are well known to those skilled in the art. See Altman, 1996.

5. Cytokine production

Cytokines are proteins that play an important role in the immune response and in the differentiation pathways of different cell types. They play a key role in T cell regulation and development and include interferon-gamma, interleukin 1(IL-1), IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, lymphotoxin, MIF, TGF-beta, TNF-alpha and other chemotactic cytokines. TH1 cytokines include IL-2, Interferon (IFN) γ, Tumor Necrosis Factor (TNF) α, or TNF- β, IL-3, IL-12, IL-15, IL-16, IL-17, or IL-18. TH2 cytokines include IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-14 or IL-18. Assays for cytokines are well known in the art, some of which are disclosed herein.

6. Cytokine analysis

TH1 and TH2 cytokines can be detected by ELISA, Radioimmunoassay (RIA) or flow cytometry (FACS). Various useful immunoassay procedures have been described in the scientific literature, such as Doolittle and Ben-Zeev, 1999; gulbis and Galand, 1993; and De Jager et al, 1993, all incorporated herein by reference.

7. Immunoassay

In more specific embodiments, the invention relates to binding, purification, removal, quantification and/or other immunoassay methods that are generally used to detect proteins, polypeptides or peptides of biological components. In some embodiments, the immunoassay is used to detect a cell-mediated immune response against an HPV peptide. Some of the mentioned immunodetection methods include enzyme linked immunosorbent assays (ELISA), Radioimmunoassays (RIA), immunoradiometric assays, fluoroimmunoassay, chemiluminescent assays, bioluminescent assays, and western blots. Various useful immunoassay procedures have been disclosed in the scientific literature such as Doolittle MH and Ben-Zeev O, 1999; gulbis B and GalandP, 1993; and De Jager R et al, 1993; all incorporated herein by reference.

Generally, the immunological binding methods comprise obtaining a sample suspected of containing proteins, polypeptides and/or peptides and contacting the sample according to the invention with an antibody under conditions effective to form an immune complex, as the case may be. For example, in the present invention, the E6 or E7 peptides can be used to stimulate T cell responses in cells. Antibodies may be directed against cytokines produced as a result of a cell-mediated immune response, or against cytokine receptors on T cells. Alternatively, antibodies against CD69 or CD45, or both, may be used.

These methods include the purification of proteins, polypeptides and/or peptides from a sample of an organelle, cell, tissue or organism. In these instances, the antibody removes proteins, polypeptides and/or peptides from the sample. The antibody is preferably attached to a solid support, such as a column matrix, and a sample suspected of containing protein, polypeptide and/or peptide antigen components is applied to the immobilized antibody. The unwanted components will be washed off the column, leaving the antigen immunocomplexed to the immobilized antibody, which will be eluted.

For the detection of cytokine responses, the biological sample to be tested may be any sample suspected of containing cytokines, for example, a tissue section or specimen, a homogeneous tissue extract, cells, organelles, any of the above isolated and/or purified forms of the antigen-containing components, or even any biological fluid in contact with cells or tissues, including blood and/or plasma, although tissue samples or extracts are preferred.

Contacting the selected biological sample with the antibody under effective conditions for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is typically a simple addition of the antibody component to the sample and incubation of the mixture for a sufficient period of time to allow the antibody to form (i.e., bind to) immune complexes with any antigen present. Thereafter, the sample-antibody complex, such as a tissue section, ELISA plate, dot blot or western blot, is typically washed to remove any non-specifically bound antibody species, while allowing only the test antibody to remain specifically bound to the primary immune complex.

In general, detecting immune complex formation is well known in the art and can be accomplished by a number of methods. These methods are generally based on the detection of labels or markers, such as any radioactive, fluorescent, biological and enzymatic labels. U.S. patents, including 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, which are incorporated herein by reference, relate to the use of these tags. Of course, additional benefits may be obtained by utilizing a second binding ligand, such as a second antibody and/or biotin/avidin ligand binding scheme, as is known in the art.

The antigen antibody used for detection may itself be linked to a detectable label, and we then need only detect this label, thereby determining the amount of primary immunocomplexes in the mixture. Alternatively, the first antibody bound to the primary immune complex may be detected by a second binding ligand having binding affinity for the antibody. In this case, the second binding ligand may be bound to a detectable label. The second binding partner itself is typically an antibody, which may be referred to as a "second" antibody. Under effective conditions, the primary immune complex is contacted with the labeled second binding partner or antibody for a sufficient time to form a second immune complex. The second immune complex is then typically washed away any non-specifically bound labeled second antibody or ligand, and the retained label is then detected in the second immune complex.

Further methods include detecting the primary immune complex in a two-step process. A second immune complex is formed with a second binding partner, such as an antibody, having binding affinity for the antibody, as described above. After rinsing, the second immune complex is contacted with a third binding ligand or antibody having binding affinity for the second antibody, again under conditions effective for a time sufficient for a third immune complex to occur. The third ligand or antibody is linked to a detectable label, enabling detection of the tertiary immune complex thus formed. Such a system may provide signal amplification if desired.

One immunoassay method designed by Charles Cantor uses two different antibodies. The first step is a biotinylated monoclonal or polyclonal antibody for detection of the target antigen, and the second step is an antibody which is then used to detect biotin associated with the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the antibodies of the first step. Some antibodies bind to the antigen to form biotinylated antibody/antigen complexes if the target antigen is present. The antibody/antigen complex is then amplified by incubating it in a continuous solution of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, each step adding biotin sites to the antibody/antigen complex. The amplification step is repeated until an appropriate level of amplification is obtained, where the sample is incubated in a solution containing a second step antibody against biotin. The second antibody is labeled, e.g., with an enzyme, which can detect the presence of the antibody/antigen complex by histoenzymology using a chromogenic substrate. Upon appropriate magnification, macroscopic conjugation occurred.

Another known immunoassay method utilizes an immuno-PCR (polymerase chain reaction) method. The PCR method is similar to the Cantor method up to the step of incubation with biotinylated DNA, however, the method does not use multiple cycles of streptavidin and biotinylated DNA incubation, but rather washes the DNA/biotin/streptavidin/antibody complex with low PH or high salt buffer that releases the antibody. The resulting eluate was subjected to a PCR reaction using appropriate primers and appropriate controls. The method provides a useful means of identifying a group of women infected with HPV as having cervical cancer or CIN.

Another method for determining whether a patient infected with HPV has a precancerous or cancerous growth is by hybrid capture, as disclosed in Birner et al, 2001 and Clavel et al, 2000, both of which are incorporated by reference.

The immunoassay method of the present invention has significant utility for the diagnosis and prognosis of diseases such as various diseases specifically expressing, for example, cancer-specific gene products and the like. Biological and/or clinical samples suspected of being infected with HPV causing CIN are used herein. However, these embodiments also apply to non-clinical samples, such as in the determination of titers in antigen or antibody samples, such as in the selection of hybridomas.

a.ELISA

As noted above, immunoassay, in its simplest and/or straightforward sense, refers to a binding assay. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and/or Radioimmunoassays (RIA) well known in the art. Immunohistochemical detection using tissue sections is also very useful. However, it will be readily appreciated that detection is not limited to these techniques, and/or that techniques such as western blotting, dot blotting, FACS detection, and the like may also be used.

In a typical ELISA, antibodies (including antigens) of the invention directed against the products of a cell-mediated immune response are immobilized on a selected surface exhibiting protein affinity properties, such as wells of a polystyrene microtiter plate. The component to be tested, e.g., a clinical sample, suspected of containing the antigen is then added to the well. Bound antigen can be detected after binding and/or washing to remove non-specifically bound immune complexes. Detection is achieved by adding another antibody linked to a detectable label. This ELISA is a simple "sandwich ELISA" and detection can also be achieved by adding a secondary antibody followed by a third antibody having binding affinity for the secondary antibody, the third antibody being linked to a detectable label.

In another typical ELISA, a sample suspected of containing an antigen is immobilized on the surface of a well and/or is then contacted with an antibody produced by the anti-cell mediated immune response product (including the antigen). Bound antibodies are detected after binding and/or washing to remove non-specifically bound immune complexes. Where the original antibody is bound to a detectable label, the immune complex can be detected directly. Similarly, the immune complex may be detected using a second antibody having binding affinity for the first antibody, the second antibody having a detectable label attached thereto.

Another ELISA in which the antigen is immobilized involves the use of antibody competition in the assay. In such an ELISA, labeled antibodies against an antigen are added to the wells, allowed to bind, and/or detected by their labeling. During incubation with coated wells, the amount of antigen in an unknown sample can be determined by mixing the sample with a labeled antibody against the antigen. The presence of the antigen in the sample has the effect of reducing the amount of antibody that can bind to the antigen on the well, thereby reducing the ultimate signal. This method is also suitable for detecting antibodies against an antigen in an unknown sample, wherein unlabeled antibody binds to the antigen-coated wells and the amount of antigen that can bind to the labeled antibody is also reduced.

Regardless of the format used, the ELISA has certain common features such as coating, incubation and binding, washing to remove non-specifically adsorbed species, and detection of bound immune complexes. These will be described below.

When a plate is coated with antigen or antibody, the wells in the plate are typically incubated with a solution of the antigen or antibody overnight or over several hours. The wells of the plate were then washed to remove incompletely adsorbed material. Any remaining available surface of the wells is then "coated" with non-specific proteins. Such non-specific proteins are antigenically neutral to the antisera tested and include Bovine Serum Albumin (BSA), casein or milk powder solutions. This coating can block non-specific adsorption sites on the immobilized surface, thereby reducing the background caused by non-specific adsorption of antisera on the surface.

In ELISA, it may be more common to use secondary or tertiary detection methods rather than direct methods. Thus, after binding of proteins or antibodies to the wells, the plate is coated with a non-reactive material to reduce background, washed to remove unbound material, and the immobilized surface is contacted with a biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. A labeled second binding ligand or antibody is required to detect the immune complex, and the second binding ligand or antibody is linked to a labeled third antibody or third binding ligand.

By "under effective conditions that allow immune complexes (antigen/antibody) to form" is meant that the conditions preferably include antigen and/or antibody diluted with a solution such as BSA, bovine propanglobulin (BGG) or Phosphate Buffered Saline (PBS)/Tween. These added reagents also tend to help reduce non-specific background.

"suitable" conditions also means that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Typical incubation steps are from about 1 to 2 to 4 hours or so, preferably at a temperature of about 25 ℃ to 27 ℃, or such as overnight at about 4 ℃.

After all incubation steps of the ELISA, the contact surfaces were washed to remove non-complexed material. Preferred washing methods include washing with solutions such as PBS/Tween, or borate buffer. After specific immune complexes are formed between the sample to be tested and the original binding substance and subsequently washed, the presence of even minute amounts of immune complexes can be detected.

To provide a method of detection, the second or third antibody will be conjugated to a label for detection. Preferably, the label is an enzyme which develops a colour when incubated with a suitable luminescent substance. Thus, it may be desirable to contact or incubate the first or second immune complexes with urease, glucose oxidase, alkaline phosphatase or peroxidase-conjugated antibodies for a period of time and under conditions suitable for development of further formed immune complexes (e.g., incubation for 2 hours at room temperature in a solution containing PBS, such as PBS-Tween).

After incubation with labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified, as in the case of enzymatic labeling, by reaction with a luminescent substrate such as urea, or bromocresol purple, or 2, 2' -azino-bis- (3-ethyl-benzthiazoline-6-sulfonic Acid (ABTS), or H₂O₂And (5) culturing. And then quantified by measuring the degree of color produced, e.g., with a spectrophotometer for visual spectroscopy.

b. Immunohistochemical method

The antibodies may also be used in conjunction with fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for Immunohistochemical (IHC) studies. Methods for preparing tissue blocks from these particular specimens have been successfully used in previous IHC studies of various predictive factors, and/or are well known to those skilled in the art (Brown et al, 1990; Abbondanzo et al, 1990; Allred et al, 1990).

Briefly, frozen sections were prepared by rehydrating 50ng of frozen "minced" tissue in Phosphate Buffered Saline (PBS) in small plastic capsules at room temperature; centrifuging and precipitating the particles; resuspending it in a viscous embedding medium (OCT); turning over the capsule and/or centrifuging again for precipitation; rapidly freezing in isopentane at 70 deg.C; cutting the plastic capsule and/or removing the frozen columnar tissue; securing the columnar tissue to a cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent sections can be prepared by a similar method, including rehydrating a 50mg sample in a plastic microcurent ultracentrifuge tube (microfuge tube); precipitating; resuspend in 10% formalin for 4 hours to fix; cleaning/precipitating; resuspend in 2.5% warm agar; precipitating; cooling in ice water to solidify the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the blocks in paraffin; and/or cut into 50 consecutive permanent sections.

c. Fluorescence sorting of cell profiles

Proteins can also be detected by flow cytometry, as described in Fujishima et al, 1996. In an example of this method, cells are fixed and then incubated with a monoclonal antibody against the expressed protein to be detected. The bound antibody is then contacted with labeled antibody IgG (for example) for detection. A typical label is FITC. Then detecting the fluorescence intensity with a flow cytometer, such as Ortho cyclon, Ortho diagnostics, or FACScan; becton dickinson.

FACS can initially separate a subpopulation of cells based on their light scattering properties as they pass through a laser beam. Forward light scattering (FALS) is related to cell size, and right angle light scattering is related to cell concentration, cell contour and nuclear to cytoplasmic ratio. Since the cells are labeled with fluorescently labeled antibodies, they can be further detected by fluorescence intensity and setting up positive and negative windows on the FACS to collect bright and dark fluorescence. Cells were sorted at a flow rate of about 3000 cells per second and positive and negative cells were collected.

d. Western blot

The compositions of the invention are useful for immunoblot and western blot analysis. The peptides can be used to stimulate cytokine production by cells. The antibodies of the invention can be used as high affinity primary reagents for the detection of proteins immobilized on a solid support, such as nitrocellulose, nylon, or combinations thereof. In combination with an immunoprecipitation reaction, followed by gel electrophoresis, it can be used as a single-step reagent for detecting an antigen against which a second reagent for detecting the antigen causes an adverse background. This method is particularly useful when the antigen being investigated is an immunoglobulin (excluding the use of immunoglobulins which bind to cell wall components of the bacteria), the antigen being investigated cross-reacts with the detection reagent, or they migrate with the same relative molecular weight as the cross-reaction signal.

Immunological-based detection methods for synergistic western blotting include secondary antibodies that are enzymatically-, radiolabelled-, or fluorescently-labeled to the anti-protein moiety, and are considered herein to be of particular interest.

VIII immunotherapy

As a method of infection or cancer treatment, immunotherapy is based on the premise that antigen-bearing tumor cells stimulate the production of specific antibodies and/or Cytotoxic T Lymphocytes (CTLs).

A. Type of immunotherapy

As described below, immunotherapy of cancer can be broadly classified into adoptive type, passive type and active type.

1. Passive immunization

There are many different approaches to passive immunization. They can be broadly divided into the following categories: injection of antibody only; injecting an antibody conjugated with a toxin or a chemotherapeutic agent; injecting a radioisotope-conjugated antibody; injecting an anti-idiotype antibody; and finally, the bone marrow is cleared of tumor cells.

Preferably, human monoclonal antibodies are used in passive immunization because they produce little or no side effects in the patient. However, its use is somewhat limited by the lack and so far only the human monoclonal antibodies have been administered intralesionally. Human monoclonal antibodies against ganglioside antigens have been administered into wounds of patients with cutaneous recurrent melanoma (Irie & Morton, 1986). Regression was observed in 6 of 10 patients, injected daily or weekly to the lesion site. In another study, two human monoclonal antibodies were injected into the lesion site with some success (Irie et al, 1989).

It is preferred to administer more than one monoclonal antibody directed against two different antigens or even antibodies with multiple antigen specificities. Treatment regimens also include administration of lymphokines or other immune enhancers, as described in Bajorin et al (1988). The remainder of the specification further details the development of human monoclonal antibodies.

2. Active immunization

In active immunization, antigenic peptides, polypeptides or proteins, or autologous or heterologous tumor cell compositions or "vaccines", are usually administered together with a unique bacterial adjuvant (Ravintranath & Morton, 1991; Morton & Ravintranath, 1996; Morton et al, 1992; Mitchell et al, 1990; Mitchell et al, 1993). In immunotherapy of melanoma, patients who elicit high IgM response often survive better than patients who do not elicit or elicit low IgM antibodies (Morton et al, 1992). IgM antibodies are often transient antibodies, with the conventional exception of anti-ganglioside or anti-sugar antibodies.

3. Adoptive immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor-infiltrating lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2, or genetically transformed to necrose the tumor, and re-administered (Rosenberg et al, 1988; 1989). To achieve this, an immunologically effective amount of active lymphocytes is administered to an animal, or human patient, in combination with an adjuvant-integrated antigenic peptide composition as described herein. Most preferably, the active lymphocytes are patient's own cells that have been earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro. This form of immunotherapy has produced some cases of regression of melanoma and renal cancers, but the percentage of responses is less than the percentage of those that do not.

B. Vaccine injection

The invention includes immunotherapies that induce or improve cell-mediated immune responses using E6 and E7 peptides from HPV. This approach is particularly significant in patients who do not produce or produce low CMI responses to E6 and/or E7 peptides of HPV. Comprises all or part of SEQ ID NO: peptides or polypeptides of 1 to 19 amino acids are clinically important as effective vaccines for the treatment and prevention of HPV infection, including the prevention of pre-cancerous and cancerous growths associated with HPV, and the induction of cell-mediated immune responses in patients.

Once produced, synthesized and/or purified, the peptides and polypeptides of the invention can be formulated as vaccines for administration to patients. The peptides, polypeptides and vaccines of the invention may also be combined with other vaccines or vaccine components, such as other additional antigens, to stimulate the immune response of the antigen. In this embodiment, preferred additional antigens are those that are suggested to be specifically or preferentially expressed under cancerous and hyperproliferative conditions. Additional antigens and vaccines contemplated for use in conjunction with the peptides, polypeptides and vaccines of the present invention include those described in U.S. patent nos. 5,840,317 and 5,882,654, which are incorporated herein by reference.

One of ordinary skill in the art will envision some potential therapeutic agents and delivery means for testing. For example, potential anti-HPV and anti-tumor agents may be natural products or artificially designed synthetic molecules. Furthermore, this model provides a means to select effective agents from a group of known and novel compounds. The dosage and mode of delivery of any particular potential therapeutic agent can be determined based on established guidelines for the preparation of pharmaceutically active ingredients. For example, the test compound may be administered intravenously, intradermally, intramuscularly, topically, orally, or by other pharmaceutically effective means. Using animals produced by the methods of the invention, researchers are for the first time able to evaluate prophylactic and therapeutic agents against high risk human papillomavirus-induced diseases, possibly including viral replication and transmission. Including chemotherapeutics, gene therapy, antisense suppression strategies, or prophylactic or therapeutic vaccines. Many methods are known for evaluating the results of laboratory tests for proposed therapies.

Depending on the host species, various adjuvants may also be used to increase the immune response, including, but not limited to, Freund (complete and incomplete), inorganic gelling agents such as aluminum hydroxide, surface active substances such as lysolecithin, polyether polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and adjuvants very useful to humans such as BCG (bacterium Calmetter-Guerin) and Corynebacterium parvum. Adjuvants, in addition to being used in the immunotherapy of the invention, may also be used in assays that enhance cell-mediated immune responses within the scope of the invention.

C. Directional conveying system

In some embodiments of the claimed invention, for the detection of virus-specific T cell responses, HPV polypeptides or peptides may be delivered to target cells, expressing viral protein fragments on their surface, for the purpose of generating T cell responses. Different methods of delivery include perfusion, transfection of expression constructs, viral vectors, and other methods disclosed below.

1. Transfer by perfusion

One embodiment of the claimed invention transfers peptides or compositions of peptides into cells by perfusion. Continuous perfusion of the expression construct and the viral construct is also included. The amount of construct or peptide delivered in continuous perfusion may be determined by the amount of uptake desired. An example of perfusion is disclosed where the initial concentration is 10⁶Cell cultures of individual cells/ml can be labeled first, washed, and then cultured in 100ug of synthetic peptide for two hours.

2. Expression vector

Delivery of the therapeutic peptide can be accomplished using an expression vector. In embodiments of the invention, HPV polypeptides and peptides are delivered to target cells using expression constructs. In the course of this application, the term "expression construct" is intended to encompass any genetic construct comprising a nucleic acid encoding an HPV polypeptide. "viral vector" refers to an expression construct derived primarily from viral sequences. For efficient expression of the construct, the polynucleotide encoding the HPV polypeptide will be under transcriptional control of a promoter. "promoter" refers to a DNA sequence recognized by the synthetic machinery of a host cell or introduced synthetic machinery necessary to initiate transcription of a particular gene. The phrase "under transcriptional control" refers to a promoter at the correct position relative to a polynucleotide to control initiation of an RNA polymerase and expression of the polynucleotide.

The term promoter is used herein to refer to a set of transcriptional control motifs clustered at the RNA polymerase II start site. Many considerations as to how the promoter is organized have been derived from analysis of some viruses, including HSV thymidine kinase (tk) and SV40 early transcription units. By extension of recent work, these studies have shown that promoters consist of discrete functional motifs, each consisting of approximately 7-20bp of DNA, and contain one or more transcriptional activator or repressor recognition sites.

At least one motif in each promoter functions to locate the initiation site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking the TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late gene, the discrete elements covering the start site themselves help to fix the start position.

Additional promoter elements regulate the frequency of transcription initiation. Typically, they are located in a region 30-110bp upstream of the start site, although many promoters have recently been shown to contain functional elements also downstream of the start site. The spacing between promoter elements is flexible such that promoter function is preserved when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter, the individual elements may act synergistically or independently to activate transcription.

The particular promoter used to control expression of the HPV polynucleotide is not critical, so long as it is capable of expressing the polypeptide in the target cell. Thus, when targeting human cells, it is preferred to position the region encoding the polynucleotide adjacent to and under the control of a promoter capable of expression in human cells. Generally, such promoters include human promoters or viral promoters. It is also contemplated that expression of the polynucleotide may be obtained using other viral or mammalian cell or bacteriophage promoters (well known in the art), provided that the level of expression is sufficient to induce a T cell response.

By using promoters with well-known properties, the expression level and pattern of the transfected polynucleotide can be optimized. For example, promoters selected to be active in specific cells, such as tyrosinase (melanoma), alpha-fetoprotein and albumin (liver tumor), CC10 (lung tumor) and prostate specific antigen (prostate tumor), allow tissue-specific expression of HPV polynucleotides. Further, selection of promoters that are modulated in response to specific physiological signals allows for the expression of inducible HPV polypeptide constructs.

Enhancers were originally discovered as genetic elements that enhance transcription from promoters located at distant sites of the same DNA molecule. In classical studies on the regulation of transcription in prokaryotes, this ability to function remotely is almost unprecedented. Later work showed that the DNA region with enhancer activity organized much like a promoter. That is, they are composed of many individual elements, each of which binds to one or more transcribed proteins.

The fundamental difference between enhancers and promoters is operational. All enhancer regions as a whole must be able to stimulate distant transcription, which is not necessary for the promoter region or its constituent elements. On the other hand, a promoter must have one or more elements that directly initiate RNA synthesis, particularly at a particular site or at a particular orientation, while an enhancer lacks these features. Promoters and enhancers often overlap and are in close proximity, often having a similar motif organization on the surface.

In addition, any promoter/enhancer combination (according to the Eukaryotic promoter data Base EPDB) may be used to drive expression of the HPV polynucleotide construct. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from a bacteriophage promoter if an appropriate bacteriophage polymerase is provided, either as part of the delivery complex, or as an additional gene expression vector.

In certain embodiments of the invention, delivery of the expression vector within the cell can be identified in vitro or in vivo by a marker contained within the expression vector. For transfected cells that allow for the identification of expression, the marker will produce a recognizable change. Typically, drug selectable markers are included to aid in cloning and selection of transformants. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic) or Chloramphenicol Acetyl Transferase (CAT) (prokaryotic) may be used. Immunological markers may also be used. The use of a selectable marker is not critical, so long as it is capable of being expressed with the polynucleotide encoding the HPV polypeptide. Further examples of selectable markers are known to those skilled in the art.

One typically accomplishes proper polyadenylation of the transcript by including a polyadenylation signal. The nature of the polyadenylation signal is not important to the successful practice of the present invention, and any such sequence may be used. The SV40 polyadenylation signal has been used by the inventors because it is convenient and known to function well in the use of target cells. Also as an element of the expression construct is a terminator. These elements can be used to enhance signal levels and reduce read-through from the construct to other sequences.

3. Viral vectors

In some embodiments of the invention, the expression construct comprises a virus or a modified construct derived from a viral gene. The ability of certain viruses to undergo body-mediated endocytosis into cells, and in some cases, to integrate with cell chromosomes, has made them attractive candidates for gene transfer to mammalian cells. However, since it has been demonstrated that direct uptake of naked DNA vaccines, and receptor-mediated DNA complexes (discussed below), the uptake of the expression vector need not be viral, but can be any plasmid, cosmid, or phage construct capable of supporting expression of the encoded gene in mammalian cells, such as pUC or Bluescript^TMSeries of plasmids.

a. Retroviruses

Retroviruses are subdivided into three main groups: tumor viruses, such as murine leukemia virus; lentiviruses, and foamy viruses. Retroviruses are single-stranded RNA viruses characterized by their ability to convert their RNA to double-stranded DNA by reverse transcription in infected cells (Coffin, 1990). The resulting DNA is then integrated into the chromosome of the cell as a provirus and directs the synthesis of viral proteins. This integration results in the retention of the viral gene sequence in the recipient cell and its progeny.

The retroviral genome contains three genes gag, pol, and env encoding the capsid protein, polymerase, and envelope components, respectively. The sequence found upstream of the gag gene, called ψ, functions as a signal that will wrap the genome into the virus. Two Long Terminal Repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. They contain strong promoter and enhancer sequences and must also be integrated into the host cell genome (Coffin, 1990).

To construct retroviral vectors, nucleotides encoding HPV polypeptides are inserted into certain viral sequences of the viral genome to produce replication-defective viruses. Alternatively, a mutated HPV virus that does not result in HPV infection may be used. To generate virus particles, packaging cell lines were constructed containing the gag, pol and env genes, but no LTR and ψ components (Mann, 1983). When a recombinant plasmid containing human cDNA is introduced into the cell line (e.g., by calcium phosphate precipitation) together with retroviral LTR and the ψ sequence, the ψ sequence allows the RNA transcript of the recombinant plasmid to be packaged into virus particles and then secreted into a medium (Nicolas and Rubenstein, 1988; Mann, 1983). The medium containing the recombinant retrovirus is then collected, optionally concentrated, for gene transfer. Retroviruses infect many types of cells. However, integration and stable expression require host cell division (passkind, 1975).

A new approach designed to allow specific targeting of retroviral vectors has recently been developed, which is based on chemical modification of retroviruses by chemical addition of lactose residues to the viral envelope. This modification may allow specific infection of hepatocytes by the sialoglycoprotein receptor.

A different approach to targeting recombinant retroviruses was devised in which anti-retroviral envelope proteins and biotinylated antibodies against specific cellular receptors were applied. The antibodies were coupled via the biotin component by using streptavidin (Roux, 1989). By using antibodies against major histocompatibility complex class I and class II antigens, they showed that human cells bearing those surface antigens were infected in vitro with exogenous viruses. (Roux, 1989).

b. Adenoviral vectors

Human adenovirus is a double-stranded DNA tumor virus with a genome length of approximately 36kb (Tooze, 1981). Adenoviruses are widely studied and well characterized as model systems for eukaryotic gene expression, making the development of adenoviruses an attractive gene transfer system. Such viruses are relatively easy to grow and replicate and exhibit a wide host range in vivo and in vitro. In lysing infected cells, adenovirus can shut down protein synthesis in the host, direct the cellular machinery to synthesize large quantities of viral proteins, and produce large quantities of virus.

The E1 region of the genome includes E1A and E1B and some cellular genes, E1A and E1B encode proteins responsible for regulating transcription of viral genes. Expression of E2 (including E2A and E2B) synthesizes viral replication functions such as DNA binding proteins, DNA polymerases, and the terminal proteins that initially initiate replication. The E3 gene product prevents cell lysis by CTL and tumor necrosis factor and shows importance for virus proliferation. Functions associated with E4 include DNA replication, late gene expression, and host cell shut-down. Late gene products mainly include viral capsid proteins, which are expressed only after the occurrence of most of one major transcription process from a major late promoter. The Major Late Promoter (MLP) showed high efficiency in the late phase of infection (Stratford-Perricaudet and Perricaudet, 1991).

Since only a small portion of the viral genome shows cis requirements (Tooze, 1981), adenovirus-derived vectors show excellent potential for replacing large DNA fragments when cell lines related to, for example, 293 cells are used. Ad 5-transformed human embryonic kidney cell line (Graham, 1977) has been developed to provide the necessary viral proteins in trans. The nature of adenoviruses makes them good candidates for in vivo targeting of cells (Grunhaus and Horwitz, 1992).

Particular advantages of using the adenovirus system to deliver foreign proteins to cells include: (i) the ability to replace relatively large fragments of viral DNA with foreign DNA; (ii) stability of adenovirus recombinant structure; (iii) safety of adenovirus administration to humans; (iv) there is no known association between adenoviral infection and cancer or malignancy; (v) the ability to obtain high titer viral recombinants; (vi) high infectivity of adenovirus.

Typically, adenoviral gene delivery systems are based on recombinants, which are engineered adenoviruses that are incapable of replication due to a partial genome deletion, such as deletion E1, but retain their infectious capacity. When deletions are added to the adenovirus genome, sequences encoding relatively large heterologous proteins may be expressed. For example, adenoviruses deleted for the E1 and E2 regions can carry 10kb of foreign DNA and can be produced in 293 cells at high titers (Stratford-Perricaudet and Perricaudet, 1991). Accidental permanent expression of transgenes following adenovirus infection has also been reported.

AAV vectors

Adeno-associated virus (AAV) is an attractive vector system for cell transduction in accordance with the present invention, since it integrates at high frequency and infects non-dividing cells, which makes it useful for gene delivery to mammalian cells, e.g., in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host infection spectrum (Lebkowski, 1988; McLaughlin, 1988; Laughlin, 1986; Tratschin, 1984). Details regarding the production and use of rAAV are described in U.S. patent No. 5,139,941 and U.S. patent No. 4,797,368, both incorporated herein by reference.

Studies demonstrating the use of AAV for gene delivery include LaFace et al (1988); zhou et al (1993); flotte et al (1993); and Walsh et al (1994). Recombinant AAV vectors have been successfully used for in vitro and in vivo transduction of marker genes (Kaplitt, 1994; Shelling and Smith, 1994; Yoder, 1994; Zhou, 1994; Samulski, 1989; Lebkowski, 1988; McLaughlin, 1988; Tratschin, 1985; Hermonat and Muzyczka, 1984) and genes involved in human diseases (Luo, 1994; Walsh, 1994; Wei, 1994; Flotte, 1992; Ohi, 1990). Recently, AAV vectors have allowed phase I human trials for cystic fibrosis treatment.

AAV is a dependent parvovirus that requires a concerted infection with other viruses (one of the members of adenovirus or herpes virus) in cultured cells for effective infection (Muzyczka, 1992). In the absence of helper virus coinfection, the wild-type AAV genome integrates through its ends into human chromosome 19, where it resides in a latent state as a provirus (Samulski, 1991; Kotin, 1990). However, rAAV is not restricted to chromosome 19 by integration unless AAV Rep proteins are also expressed (Shelling and Smith, 1994). When cells that transport AAV proviruses are doubly infected with helper cells, the AAV genome is "rescued" from the chromosome or recombinant plasmid, thus establishing a generally effective infection (Muzyczka, 1992; Kotin, 1990; Samulski, 1989; McLaughlin, 1988).

Typically, recombinant AAV viruses (rAAV) are made by co-transfecting a plasmid containing two AAV terminal repeats flanking the gene of interest (McLaughlin, 1988; Samulski, 1989; each incorporated herein by reference), and an expression plasmid containing the wild-type AAV coding sequence without terminal repeats, such as pIM45(McCarty, 1991; incorporated herein by reference). The cells may also be infected or transfected with an adenovirus or plasmid carrying the adenoviral genes for the desired helper functions of the AAV. rAAV viral stocks prepared in this manner are contaminated with adenovirus, which must be physically separated from the rAAV particles (e.g., by cesium chloride density centrifugation). Alternatively, adenoviral vectors containing AAV coding regions or cell lines containing AAV coding regions and some or all of the adenoviral helper genes can be used (Clark, 1995; Yang, 1994). Cell lines carrying rAAV DNA as an integrating provirus can also be used (Flotte, 1995).

d. Other viral vectors as expression constructs

Other viral vectors may be used as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Coupar, 1988; Ridgeway, 1988; Baichwal and Sugden, 1986) and herpes viruses may be used. These viruses provide several attractive properties for a variety of mammalian cells (Horwich, 1990; Friedmann, 1989; Coupar, 1988; Ridgeway, 1988; Baichwal and Sugden, 1986).

With the recent knowledge of hepatitis b virus, new insights have been generated regarding the structural functions of different viral sequences. In vitro experiments have shown that despite a deletion of 80% of the genome, the virus retains its ability to rely on helper virus packaging and reverse transcription (Horwich, 1990). This means that a large part of the genome can be replaced by foreign genetic material. Hepatectomy and survival (integration) are particularly attractive properties for liver-targeted gene transfer. Chang (1991) recently introduced the chloramphenicol transacetylase (CAT) gene into the duck hepatitis B virus genome at the polymerase site, surface and pre-surface coding regions. It was transfected synergistically with wild-type virus in avian hepatoma cell lines. The medium containing the high titer virus recombinant was used to infect primary duckling hepatocytes. At least 24 days after transfection, stable CAT gene expression was detected (Chang, 1991).

e. Non-viral transfer method

The present invention also contemplates non-viral methods for transferring expression vectors to cultured mammalian cells. They include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe, 1990) DEAE-dextrose (Gopal, 1985), electroporation (Tur-Kaspa, 1986; Potter, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley, 1979) and lipofectamine-DNA complexes, sonication of cells (Fechheimer, 1987), bombardment of genes with high velocity microparticles (Yang, 1990), polycations (Boussif, 1995), and receptor-mediated transfection (Wu and Wu, 1988; Wu and Wu, 1987). Some of these techniques may be successfully adapted for in vivo or ex vivo applications.

D. Colloidal dispersion system

The colloidal dispersion constitutes a directional transport vehicle. These dispersions include macromolecular complexes, nanocapsule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that are used as delivery vehicles in vitro and in vivo. It has been shown that Large Unilamellar Vesicles (LUVs) in the range of 0.2-4.0um can encapsulate a sufficient amount of macromolecular aqueous buffer. RNA, DNA and intact virions can be encapsulated into an aqueous interior and delivered to cells in a biologically active form (Fraley, et al). In addition to mammalian cells, liposomes have been used to deliver polynucleotides in plant, yeast and bacterial cells. In order for liposomes to be effective gene delivery vehicles, the following characteristics are required: (1) the target genes are wrapped with high precision without destroying their biological activity; (2) preferentially and sufficiently bind to target cells as compared to non-target cells; (3) efficient transport of the aqueous contents of vesicles to the target cytoplasm; and (4) correct and efficient expression of genetic information (Manning, et al, Biotechniques, 6: 682, 1988). Embodiments of the invention provide that the synthetic peptide may be formulated as a liposome. The composition of liposomes is typically a combination of phospholipids, especially high phase transition temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other phospholipids may also be used. The physical properties of liposomes depend on PH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, wherein the lipid moiety contains 14-18 carbon atoms, especially 16-18 carbon atoms, and is saturated. Exemplary phospholipids include egg lecithin, dipalmitoyl lecithin, distearoyl lecithin.

Targeting of liposomes can be based on structural classification and mechanistic factors. The structural classification is based on a level of selectivity, such as organ-specific, cell-specific and organelle-specific. The distinction of the mechanistic target is based on whether it is active or passive. In organs containing sinusoidal capillaries, passive targeting exploits the natural tendency of liposomes to distribute to reticuloendothelial system (RES) cells. In contrast, active targeting involves modification of liposomes by combining them with specific ligands such as monoclonal antibodies, sugars, glycolipids or proteins, or by modifying the composition or size of the liposomes to target organs and cell types, rather than localization to naturally occurring sites.

The surface of the oriented transport system can be modified in various ways. In a targeted delivery system of liposomes, lipid groups may be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable contact with the liposome bilayer. Various linking groups may be used to bind the proton chain to the targeting ligand.

E. Pharmaceutical composition

The invention includes the use of synthetic peptides in forming pharmaceutical compositions. Generally, a pharmaceutical composition includes an effective amount of one or more protein sequences, nucleic acids or antibodies or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and components that, when properly administered to an animal, such as a human, do not produce adverse, allergic, or other untoward reactions. In light of the present disclosure, those skilled in the art will be aware of the preparation of Pharmaceutical compositions comprising at least one protein sequence, nucleic acid or antibody or additional active ingredient as exemplified by Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990), incorporated herein by reference. In addition, for administration to animals (e.g., humans), it is understood that the preparation should meet the biological standards for sterility, pyrogenicity, general safety, and purity required by the FDA.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (such as antibacterial agents, antifungal agents), absorption delaying agents, salts, preservatives, anesthetics, drug stabilizers, binders, excipients, dispersants, lubricants, sweeteners, flavoring agents, dyes, and the like, or combinations thereof, as known to those of ordinary skill in the art (see, for example, Remington's pharmaceutical Sciences, 18th edition, Mack printing company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

The protein sequence, nucleic acid or antibody may comprise different types of carriers depending on whether it is administered in the form of a solid, liquid or aerosol, and also depending on whether sterility is required when administered in some manner, such as by injection. The invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intrathecally, rectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularly, intraparenaceously, intrapericardially, orally, topically, with aerosol, injection, infusion, continuous infusion, direct local perfusion bathing of target cells, via a catheter, via lavage, in emulsion, in a lipid composition (e.g., liposomes), or by other means or combinations of the foregoing methods well known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, ed. mack printing company, 1990, incorporated herein by reference).

The amount of the composition of the present invention administered to an animal patient depends on physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, the patient's primary disease and the route of administration. In any event, the skilled physician in charge of administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual patient.

In certain embodiments, the pharmaceutical composition can comprise, for example, about 0.1% of the active compound. In other embodiments, for example, an active compound may comprise from about 2% to about 75% of the unit weight, or from about 25% to about 60%, or any range derivable therein. In other non-limiting examples, the dose can comprise from about 1 microgram/kg/body weight, 5 microgram/kg/body weight, 10 microgram/kg/body weight, 50 microgram/kg/body weight, 100 microgram/kg/body weight, 200 microgram/kg/body weight, 350 microgram/kg/body weight, 500 microgram/kg/body weight, 1 milligram/kg/body weight, 5 milligram/kg/body weight, 10 milligram/kg/body weight, 50 milligram/kg/body weight, 100 milligram/kg/body weight, 200 milligram/kg/body weight, 350 milligram/kg/body weight, 500 milligram/kg/body weight, to about 1000 milligram/kg/body weight or more, but may be any range derivable therein. Based on the amounts described above, a range of from about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 micrograms/kg/body weight, and the like, can be administered in non-limiting examples derivable from the numbers listed herein.

In any event, the composition can include various antioxidants to prevent oxidation of one or more components. In addition, the action of microorganisms can be prevented by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, sodium mercurous thiosalicylate, or combinations thereof.

The protein sequence, nucleic acid or antibody may be formulated into the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, e.g., formed with the free amino groups of the protein, or with inorganic acids such as hydrochloric or phosphoric acids, or certain organic acids such as acetic, oxalic, tartaric or mandelic acid. The salt forms with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or iron hydroxides; or an organic base such as isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in the form of a liquid, the carrier can be a solvent or dispersion medium, including but not limited to water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), lipids (such as triglycerides, vegetable oils, liposomes), and combinations thereof. Maintaining proper fluidity, for example, by the use of a coating such as phosphatidylcholine; by maintaining the desired particle size by dispersion in a carrier such as a liquid polyol or lipid; by using a surfactant such as hydroxypropyl cellulose; or a combination of these. In many cases, it is preferred to include isotonic agents, such as sugars, sodium chloride or combinations thereof.

In other embodiments, eye drops, nasal solutions or sprays, or aerosols or inhalants may also be administered in the present invention. Certain compositions are generally designed to be compatible with a target tissue type. In a non-limiting example, the nasal solution is an aqueous solution, typically designed to be applied to the nasal passages in drops or sprays. Nasal solutions are prepared to resemble nasal secretions in many ways and thereby maintain normal ciliary function. Thus, in preferred embodiments, the aqueous nasal solutions are generally isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, if desired, the formulation may include an antimicrobial preservative similar to those used in ophthalmic formulations, pharmaceuticals, or suitable pharmaceutical stabilizers. For example, various commercial nasal formulations are known and include pharmaceuticals, such as antibiotics or antihistamines and the like.

In certain embodiments, administration of the protein sequence, nucleic acid or antibody may be by some route, such as orally. In these embodiments, the solid composition may include, for example, a solution, a suspension, an emulsion, a tablet, a pill, a capsule (e.g., a hard or soft shell capsule), a sustained release formulation, an oral composition, a lozenge, an elixir, a suspension, a syrup, a wafer, or a combination thereof. The oral composition can be directly combined with food. Preferred carriers for oral administration include inert diluents, ingestible food carriers, or combinations thereof. In other aspects of the invention, the oral compositions may be prepared as syrups or elixirs. A syrup or elixir may also contain, for example, at least one active ingredient, a sweetener, an antiseptic, a flavoring, a dye, an antiseptic, or a combination thereof. .

In certain preferred embodiments, the oral composition may include one or more binders, excipients, dispersants, lubricants, flavoring agents, and combinations thereof. In certain embodiments, the composition may include one or more of the following agents: binders, such as, gum tragacanth, gum acacia, corn starch, gelatin or combinations thereof; excipients, such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; dispersing agents, such as, corn starch, potato starch, alginic acid or combinations thereof; lubricants, such as magnesium stearate; a sweetening agent, such as sucrose, lactose, saccharin, or combinations thereof; flavoring agents, such as peppermint, wintergreen oil, cherry flavoring, orange flavoring, and the like; or combinations of the foregoing. When the unit dosage form is a capsule, it may include, in addition to materials of the above type, a carrier, such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the unit dosage form.

Another dosage form suitable for other modes of administration includes suppositories. Suppositories are solid dosage forms of various weights and shapes, usually administered by insertion into the rectum, vagina or urethra. After insertion, the suppository softens, melts or dissolves into the body fluid in the cavity. In general, for suppositories, conventional carriers may be used, such as polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed of mixtures including, for example, active ingredients from about 0.5% to about 10%, and preferably from about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, after filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. When sterile powders are used to prepare sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional ingredient desired from a previously sterile-filtered liquid medium. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient salt or sugar prior to injection. It is also envisaged to prepare high concentration compositions for direct injection, where the use of DMSO as solvent is considered to result in very rapid penetration, delivering high concentrations of active ingredient to a small area. The compositions must be stable under the conditions of manufacture and storage and be preserved under conditions of microbial contamination, such as bacterial and fungal contamination. It will be appreciated that endotoxin contamination should be controlled to a minimum level, such as less than 0.5ng/mg protein.

In certain embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, such as aluminum monostearate, gelatin or combinations thereof.

Lipid-based compositions are also relevant to the present invention, as the peptide can be administered to a patient as an immunotherapeutic. These will be discussed in detail below.

In certain embodiments, the present invention relates to a novel composition comprising one or more lipids in combination with at least one peptide. Lipids are substances which are characteristically insoluble in water and extractable with organic solvents. Lipids include, for example, fat droplets that naturally occur in the cytoplasm, and such classes of compounds known to those skilled in the art, which contain long chain aliphatic hydrocarbons and other derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Of course, other compounds known to those skilled in the art as lipids, other than those specifically described herein, are also included in the compounds and methods of the present invention.

Lipids may be naturally occurring or synthetic (e.g., artificially designed or produced). However, lipids are typically biological substances. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids (lysolipids), glycosphingolipids, glycolipids, sulfides (sulfolipids), lipids with ether and lipid linked fatty acids and polymerizable lipids, and combinations thereof.

Lipid types

Neutral fats include glycerol and fatty acids. Typical glycerol is a three carbon alcohol. Fatty acids typically consist of long carbon chains with an acidic moiety (e.g., carboxylic acid) at the end. Typical fatty acid carbon chains may be of any length, however, preferred carbon chain lengths are from about 2, 3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 to about 30 or more carbon atoms, and any range derived therefrom. However, the chain moiety of the fatty acid preferably ranges from about 14 to 24 carbon atoms, with about 16 to about 18 carbon atoms being particularly preferred in certain embodiments. In certain embodiments, the fatty acid carbon chain may include an odd number of carbon atoms, and in certain embodiments preferably includes an even number of carbon atoms. Fatty acids containing only single bonds in the carbon chain are referred to as saturated, while fatty acids containing at least one double bond in the carbon chain are referred to as unsaturated.

Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, arachidonic acid, ricinoleic acid, tuberculostearic acid, lactobionic acid. The acidic groups of one or more fatty acids are covalently bonded to one or more hydroxyl groups of glycerol. Thus, a monoglyceride consists of one glycerol and one fatty acid, a diglyceride consists of one glycerol and two fatty acids, and a triglyceride consists of one glycerol and three fatty acids.

Phospholipids typically include a glycerol or sphingosine moiety, a phosphate ion group that produces amphoteric compounds, and one or more fatty acids. Types of phospholipids include, for example, glycerophospholipids, in which the phosphate group is attached to the first carbon atom of glycerol in a diglyceride; and sphingomyelins (e.g., sphingomyelin), in which the phosphate group is esterified with the amino alcohol of sphingosine. Another example of a sphingomyelin is sulfatide, which includes a sulfate ion group that renders the molecule amphiphilic. Of course, phospholipids also include more chemical groups, for example, alcohols linked to phosphate groups. These alcohol groups include serine, ethanolamine, choline, glycerol and inositol. Thus, particular phosphoglycerides include phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol or phosphatidylinositol. Other phospholipids include phosphatidic acid or diacetyl phosphoric acid. In one aspect, the phosphatidylcholine includes dioleoylphosphatidylcholine (e.g., cardiolipin), egg phosphatidylcholine, dipalmitoylphosphatidylcholine, monomealmitoylphosphatidylcholine, monostearoylphosphatidylcholine, monooleoylphosphatidylcholine, dibutyrylphosphatidylcholine, dipentanoylphosphatidylcholine, didecanoylphosphatidylcholine, diheptanoylphosphatidylcholine, dioctanoylphosphatidylcholine, or distearoylphosphatidylcholine.

Glycolipids are related to sphingomyelins, but contain a sugar group attached to the primary hydroxyl group of the sphingolipid instead of a phosphate group. One type of glycolipid is called a cerebroside, which contains a sugar group (e.g., glucose or galactose) attached to a primary hydroxyl group. Another example of a glycolipid is a ganglioside (e.g., monosialoganglioside, GM1), which contains about 2, 3, 4,5, 6,7 or so sugar groups, which may be branched, attached to a primary hydroxyl group. In another embodiment, the glycolipid is a ceramide (e.g., lactosylceramide).

Steroids are four-membered ring system derivatives of phenanthrene. Steroids typically have regulatory functions in cells, tissues and organisms, and include, for example, the progestagen hormones and related compounds (e.g., progesterone), glucocorticoids (e.g., hydrocortisone), mineralocorticoids (e.g., aldosterone), androgens (e.g., testosterone) and estrogens (e.g., estrone) series. Cholesterol is another example of a steroid and generally provides a structural rather than a regulatory function. Vitamin D is another example of a sterol which involves the absorption of calcium from the small intestine.

Terpenes are lipids that contain one or more five carbon isoprene groups. Terpenes have various biological functions including, for example, vitamin a, coenzyme Q and carotenoids (e.g., lycopene and beta-carotene).

B. Charged and neutral lipid compositions

In certain embodiments, the lipid component of the composition is or is otherwise uncharged. In one embodiment, the lipid component of the composition comprises one or more neutral lipids. In another aspect, the lipid component of the composition may be substantially free of anionic and cationic lipids, such as certain phospholipids (e.g., phosphatidyl choline) and cholesterol. In certain aspects, the uncharged or otherwise uncharged lipid composition comprises about 95%, 96%, 97%. 98%, 99%, 100% of lipid components without any charge, substantially uncharged lipids, and/or lipid mixtures with equal amounts of positive and negative charges.

In other aspects, the lipid composition can be charged. For example, according to the present invention, charged phospholipids may be used to prepare lipid compositions and may have a positive net charge or a negative net charge. In a non-limiting example, diacetyl phosphate can be used to negatively charge the lipid composition, and stearamide can be used to positively charge the lipid composition.

C. Production of lipids

As is well known to those of ordinary skill in the art, lipids can be obtained from natural sources, commercial products, or chemical synthesis. For example, phospholipids may be obtained from natural sources such as egg or soy phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, cardiolipin and plant or bacterial phosphatidylethanolamine. In another example, lipids suitable for use according to the present invention may be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma Chemical co, dicetyl phosphate ("DCP") is available from K & K laboratories (Plainview, NY), cholesterol is available from Calbiochem-Behring, dimyristyl phosphatidylglycerol ("DMPG") and other Lipids available from Avanti Polar Lipids, Inc. In certain embodiments, the lipid stock in chloroform or chloroform/methanol may be stored at about-20 ℃. Preferably, chloroform is used as the only solvent, since it is more readily evaporated than methanol.

D. Lipid composition structure

In a preferred embodiment of the invention, the peptide is linked to a lipid. The lipid-linked peptide may be dispersed in a solution containing lipids, solubilized with lipids, emulsified, mixed, associated, covalently bound to lipids, contained in suspension in lipids, contained or mixed with micelles or liposomes, or linked to lipids or lipid structures. The lipid or lipid/chimeric polypeptide combination composition of the present invention is not limited to any particular structure. For example, they may also be merely dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. In another example, they may be present in a bilayer structure, such as a micelle, or with a "collapsed" structure. In another non-limiting example, lipo-transamine (Gibco BRL) -chimeric polypeptides or superfect (qiagen) -chimeric polypeptide complexes are also contemplated.

In certain embodiments, the lipid composition may comprise about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% or any range derived therefrom of a particular lipid, lipid or non-lipid component, such as a drug, protein, sugar, nucleotide or other substance disclosed herein or known to those skilled in the art. In a non-limiting example, the lipid composition can include from about 10% to about 20% neutral lipid, from about 33% to about 34% cerebrosides, and about 1% cholesterol. In another non-limiting embodiment, the liposomes can comprise from about 4% to about 12% terpenes, wherein about 1% of the micelles are the particular lycopene, and the remaining about 3% to about 11% of the liposomes are composed of other terpenes; and further comprising from about 10% to about 35% phosphatidylcholine and about 1% drug. Thus, the lipid compositions of the present invention may include any lipid, lipid type or other ingredient in any combination or percentage range.

1. Emulsion formulation

Lipids may be included in the emulsion. Lipid emulsions are essentially permanent heterogeneous liquid mixtures obtained from two or more liquids that are normally insoluble in each other by mechanical agitation or by the addition of small amounts of known emulsifier-adding substances. Methods for preparing lipid emulsions and additional ingredients to be added are well known in the art (e.g., Modern pharmaceuticals, 1990, herein incorporated by reference).

For example, one or more lipids are added to ethanol or chloroform or any other suitable organic solvent and stirred by manual or mechanical techniques. The solvent was then evaporated from the mixture, leaving a dry lipid glaze. The lipids are resuspended in an aqueous medium, such as phosphate buffered saline, to give an emulsion. To obtain emulsified lipids with a more homogeneous size distribution, the mixture can be sonicated using conventional sonication techniques, further emulsified using microfluidization (e.g., using a mierofluid, Newton, Mass.), and/or extruded at high pressure (e.g., 600psi) using an Extruder Device (Lipex Biomembranes, Vancouver, Canada).

2. Micelle

Lipids may be contained in micelles. Micelles are clusters or aggregates of lipid compositions, usually in the form of lipid monolayers, and are prepared by using any micelle production procedure known to those skilled in the art (e.g., Canfield et al, 1990; El-Gorab et al, 1973; Shinoda et al, 1963; and Fendler et al, 1975, each of which is incorporated herein by reference). For example, one or more lipids are typically suspended in an organic solvent, the solvent is evaporated, the lipids are resuspended in an aqueous solution, sonicated, and then centrifuged.

3. Liposomes

In a particular embodiment, the lipid consists of liposomes. "liposomes" is a generic term that encompasses a variety of single or multi-layered lipid vehicles formed by the production of closed lipid bilayers or aggregates. Liposomes can be characterized as a membrane-bubble structure with bilayer membranes, typically comprising a phospholipid, and an internal medium, typically comprising an aqueous composition.

Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Multilamellar liposomes form naturally when phospholipids-containing lipids are suspended in excess aqueous solution. Prior to the formation of closed structures, the lipid components undergo self-rearrangement and trap water and dissolved solutes between lipid bilayers (Ghosh and Bachhawat, 1991). Molecules that are lipophilic or carry lipophilic regions may also be solubilized or incorporated into the lipid bilayer.

In certain less preferred embodiments, naturally derived phospholipids, such as egg or soy phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, cardiolipin and plant or bacterial phosphatidylethanolamine are preferably not used as the major phospholipids, i.e., do not constitute 50% or more of the total phospholipid composition or liposomes, because the resulting liposomes are unstable and are leaky.

In particular embodiments, the lipid and/or chimeric polypeptide can, for example, be encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linker molecule associated with the liposome and chimeric polypeptide, captured by a liposome, complexed with a liposome, and the like.

a. Preparation of liposomes

As is well known to those of ordinary skill in the art, liposomes for use in accordance with the present invention can be prepared by various methods. When a phospholipid is dispersed in water, it can form various structures other than liposomes depending on the ratio of lipid to water. At low ratios, liposomes are the preferred structure.

For example, phospholipids (Avanti Polar Lipids, Alabaster, AL), such as neutral phospholipids, Dioleoylphosphatidylcholine (DOPC) are dissolved in t-butanol. The lipid is then mixed with the chimeric polypeptide, and/or other ingredients. Tween20 was added to the lipid mixture such that Tween20 was 5% by weight of the composition. An excess of t-butanol is added to the mixture such that the volume of t-butanol is at least 95%. The mixture was vortex mixed, frozen in a dry ice/acetone bath and lyophilized overnight. The lyophilized formulation was stored at-20 ℃ and could be used for three months. When needed, the lyophilized liposomes were reconstituted in 0.9% saline. Microparticles for blocking the chimeric polypeptides were obtained with Tween20, and their average particle size was about 0.7 to 1.0 um.

Alternatively, liposomes can be prepared in containers by mixing lipids in a solvent, e.g., glass, pear-shaped flasks. The volume of the container should be more than ten times the volume of the desired liposome suspension. The solvent was removed in vacuo at approximately 40 ℃ by using a rotary evaporator. The solvent is typically removed within about 5 minutes to 2 hours, depending on the desired volume of the liposomes. The composition may be further dried in a dryer. Dried lipids are typically discarded after about a week due to a tendency to deteriorate over time.

The dried lipids were reconstituted with 25-50mM phospholipids in sterile, pyrogen-free water by shaking until all lipid sheets were resuspended. The aqueous liposomes were then aliquoted, filled into vials, lyophilized and sealed in vacuo.

In alternative methods, liposomes can be prepared according to other known experimental procedures (see, e.g., Bangham et al, 1965; Gregoriadis, 1979; Deamer and Uster 1983; Szoka and Papahadjollous, 1978, each of which is incorporated by reference in relevant part). These methods differ in their respective abilities to capture aqueous materials and their respective gap to lipid ratios.

The dried lipids or lyophilized liposomes prepared as above can be dehydrated and reconstituted in a solution of the inhibitory peptide and diluted with a suitable solvent to a suitable concentration, such as DPBS. The mixture was then vigorously shaken in a vortex mixer. Unencapsulated added substances, such as pharmaceutical agents (including but not limited to hormones, drugs, nucleotide compositions, etc.) were removed by centrifugation at 29,000Xg, and the liposome pellet was washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, such as about 50-200 mM. The amount of encapsulated additional substance or active agent can be determined according to standard methods. After the amount of additional substance or active agent encapsulated in the liposome preparation is determined, the liposomes can be dissolved in an appropriate concentration and stored at 4 ℃ until use. Pharmaceutical compositions comprising liposomes generally include a sterile pharmaceutically acceptable carrier or diluent, such as water or saline.

Liposomes can vary in size depending on the method of synthesis. Liposomes of the invention can be of various sizes. In certain embodiments, the liposomes are small, such as having an outer diameter of less than about 100nm, about 90nm, about 80nm, about 70nm, about 60nm, or less than about 50 nm. In preparing such liposomes, any of the methods described herein or known to those of ordinary skill in the art can be used. Other non-limiting examples of preparing liposomes are described in U.S. Pat. Nos. 4,728,578, 4,728,575,4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706; international applications PCT/US85/01161 and PCT/US 89/05040; british patent application GB 2193095A; mayer et al, 1986; hope et al, 1985; mayhewet al.1987; mayhew et al, 1984; cheng et al, 1987; and Gregoriadis, 1984, each incorporated herein by reference).

Liposomes suspended in aqueous solutions are generally spherical vesicles having one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel arrangement of molecules represented by the formula XY, where X is a hydrophilic moiety and Y is a hydrophobic moiety. In aqueous suspensions, the concentric layers are arranged such that the hydrophilic portions tend to remain in contact with the aqueous phase and the hydrophobic regions tend to self associate. For example, when the aqueous phase is present both inside and outside the liposome, the lipid molecules may form a bilayer, known as a lamella, arranged as XY-YX. Lipid aggregates can be formed when the hydrophilic and hydrophobic portions of more than one lipid molecule begin to associate with each other. The size and shape of these aggregates depends on many different variables, such as the nature of the solvent and the presence of other compounds in solution.

After (I) reverse phase evaporation (II) dehydration-rehydration (III) detergent dialysis and (IV) membrane hydration, the lipid formulation product is usually obtained by sonication or continuous drainage of the liposome mixture. In one aspect, the method of preparing liposomes in certain embodiments comprises thermal sonication, and continuous discharge through a filter or membrane of reduced pore size, thereby resulting in a small, stable liposome structure. This preparation only yields liposomes/chimeric polypeptides or liposomes of appropriate and uniform size, which are structurally stable and give rise to great activity. This technique is well known to those skilled in the art (see, e.g., Martin, 1990).

Once produced, the lipid structures can be used to encapsulate compounds that are toxic (e.g., chemotherapy) or unstable in circulation (e.g., nucleic acids). The physical properties of liposomes depend on PH, ionic strength and/or the presence of divalent cations. Liposomes can exhibit low permeability to ionic and/or polar substances, but an increase in the temperature of the phase transition process significantly alters their permeability. Phase transitions range from dense, ordered structures (known as the colloidal state) to loosely packed, unordered structures (known as the liquid state). This phenomenon occurs at a characteristic phase transition temperature and/or results in an increase in permeability to ions, sugars and/or drugs. Liposomal encapsulation has resulted in reduced toxicity and increased serum half-life of these compounds (Gabizon et al, 1990).

Liposomes affect cellular delivery of agents by four different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system, such as macrophages and/or neutrophils; adsorption to the cell surface by non-specific weak hydrophobic and/or electrostatic forces, and/or by specific interactions with cell surface components; fusing with the plasma membrane by inserting the lipid bilayer of the liposome into the plasma membrane, while releasing the liposome contents into the cytoplasm; and/or by transferring liposomal lipids to cell membranes and/or subcellular membranes, and/or vice versa, without the need to associate any liposomal contents. While more than one mechanism may be operating simultaneously, the liposomal formulation may alter which mechanism is effective.

Treatment of many diseases uses lipid-based gene transfer strategies to augment conventional therapies or to establish new therapies, especially for hyperproliferative diseases. Advances in liposome formulation have improved the efficiency of gene transfer in vivo (Templeton et al, 1997) and it is contemplated that liposomes can be prepared by these methods. Another method of liposome-based preparation for delivery of nucleic acids has been described (WO 99/18933).

In another liposomal formulation, an ampholytic vehicle, called a solvent-diluted microcarrier (SDMC), is capable of incorporating a specific molecule into the bilayer of a lipid carrier (U.S. patent 5,879,703). SDMC can be used to transport lipopolysaccharide, polypeptides, nucleic acids, and the like. Of course, any other method of preparing liposomes can be used by the skilled person to obtain the desired liposome formulation in the present invention.

b. Targeting ligands

The targeting ligand may be either immobilized on the hydrophobic portion of the complex or linked to a reactive end group on the hydrophilic portion of the complex. The targeting ligand may be attached to the liposome by a linkage to a reactive group, such as at the distal end of the hydrophilic polymer. Preferred reactive groups include amino, carboxyl, acyl and thiol groups. The binding of the targeting ligand to the hydrophilic polymer can be performed by organic chemical methods well known to those skilled in the art. In certain embodiments, the total concentration of targeting ligands can be from about 0.01 to about 10% mol.

A targeting ligand is any ligand that is specific for a particular component of the target region. Preferred targeting ligands include proteins such as polyclonal or monoclonal antibodies, antibody fragments or chimeric antibodies, enzymes or hormones, or carbohydrates such as mono-, oligo-and polysaccharides (see Heath et al, 1986). For example, the disialoganglioside GD2 is a tumor antigen that has been identified as a tumor of neuroectodermal origin, such as neuroblastoma, melanoma, small cell lung carcinoma, glioma and certain sarcomas (Mujoo et al, 1986, Schulz et al, 1984). Liposomes containing monoclonal antibodies against the asialoglycoganglioside GD2 have been used to assist in targeting the liposomes to cells expressing tumor antigens (Montaldo et al, 1999; Pagan et al, 1999). In another non-limiting example, antibodies specific for breast and gynecological cancer antigens are described in U.S. patent 5,939,277, which is incorporated herein by reference. In a further non-limiting example, prostate cancer specific antibodies are disclosed in U.S. patent 6,107,090, which is incorporated herein by reference. Accordingly, it is contemplated that antibodies described herein or known to those of ordinary skill in the art may be used in conjunction with the compositions and methods of the present invention to target specific tissues and cell types. In certain embodiments of the invention, contemplated targeting ligands interact with integrins, proteoglycans, glycoproteins, receptors and transporters. Suitable ligands include any substance specific to cells of the target organ or specific to the structure of the target organ exposed to the circulation due to a local pathology such as a tumor.

In certain embodiments of the invention, a targeting moiety (ligand) of an antibody or cyclic peptide is attached to the lipid complex in order to enhance transduction of the cell, increase transduction of the target cell, or to limit transduction of unwanted cells. Such methods are well known in the art. For example, liposomes that specifically target mammalian central nervous system cells have been further described (U.S. patent 5,786,214, incorporated herein by reference). The liposomes consist essentially of N-glutaryl phosphatidylethanolamine, cholesterol, and oleic acid, wherein a monoclonal antibody specific for glia is conjugated to the liposomes. A monoclonal antibody or antibody fragment can be used for targeted delivery to specific cells, tissues or organs in an animal such as brain, heart, lung, liver, etc.

Further, the chimeric polypeptide may be delivered to a target cell by receptor-mediated delivery and/or targeting vectors containing lipids or liposomes. They take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that occurs on target cells. This method of delivery additionally increases the degree of specificity of the invention in view of the cell type-specific distribution of the various receptors.

Thus, in certain aspects of the invention, the ligand is selected to correspond to a receptor specifically expressed in the target cell population. The cell-specific chimeric polypeptide delivery and/or targeting vector may comprise a specific binding partner associated with the liposome. The chimeric polypeptide to be delivered is encapsulated in a liposome and the specific binding partner is functionally bound to the liposome membrane. Such that the liposome specifically binds to the receptor of the target cell and delivers the contents into the cell. This system has shown its function using, among other things, Epithelial Growth Factor (EGF) for the receptor-mediated transport of nucleic acids into cells showing upregulation of EGF receptors.

In certain embodiments, the receptor-mediated transport and/or targeting vector comprises a cellular receptor-specific ligand and a peptide. Others include cell receptor-specific ligands to which the peptide to be delivered is operatively linked. For example, several receptors have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al, 1990; Perales et al, 1994; Myers, EPO0273085), which demonstrates the operability of this technique. In another example, specific transport in another mammalian cell type has been disclosed (Wu and Wu, 1993; incorporated herein by reference).

In still further embodiments, the specific binding partner may comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactyl ceramide, a galactose-terminal asialoganglioside, has been incorporated into liposomes and increased insulin gene uptake by hepatocytes has been observed (Nicolau et al, 1987). Asialoglycoproteins containing terminal galactose residues, asialofetuin, have also been shown to target liposomes to the liver (Spanjer and Scherphof, 1983; Hara et al, 1996). When bound to the backbone of a polypeptide, the saccharides mannoyl, trehalayl or N-acetylglucosamine bind to the high affinity mannose (manose) receptor (U.S. Pat. No. 5,432,260, specifically incorporated herein by reference in its entirety). The cell-or tissue-specific transformation constructs of the invention can be specifically delivered to the target cells in a similar manner.

In another example, lactoyl ceramide and peptides targeting LDL receptor-associated proteins, such as apolipoprotein E3 ("Apo E"), are used to target liposomes to the liver (Spanjer and Scherphof, 1983; WO 98/0748).

Folate and folate receptors have also been disclosed for cell targeting (us patent 5,871,727). In this example, vitamin folic acid is conjugated to the complex. Folate receptors have high affinity for their ligands and are overexpressed in several mammalian cell lines, including lung, breast and brain tumors. Anti-folate, such as methotrexate, can also be used as a targeting ligand. Transferrin-mediated delivery systems target a wide range of replicating cells that express transferrin receptors (gililand et al, 1980).

c. Liposome/nucleic acid composition

In certain embodiments, the liposome/chimeric polypeptide can include nucleic acid, such as an oligonucleotide, polynucleotide, or nucleic acid construct (e.g., an expression vector). When a bacterial promoter is used in the DNA construct to be transfected into eukaryotic cells, it is advantageous to include a suitable bacterial polymerase in the liposomes.

This technique has applicability in the present invention when the liposome/chimeric polypeptide composition is contemplated to include a cell or tissue specific nucleic acid. In certain embodiments, the lipid-based non-viral formulation provides another approach to viral gene therapy. Although many cell culture studies have demonstrated lipid-based non-viral gene transfer, there are limitations to systemic gene delivery through lipid-based formulations. The major limitation of non-viral lipid-based gene delivery is the toxicity of cationic lipids, which comprise the non-viral delivery vehicle. The in vivo toxicity of liposomes accounts for the difference in gene transfer results between in vitro and in vivo. Another factor contributing to this pair of data is the difference in liposome stability with and without serum proteins. The interaction between liposomes and serum proteins has a surprising effect on the stability of liposomes (Yang and Huang, 1997). Anionic liposomes attract and bind negatively charged serum proteins. Liposomes coated with serum proteins are solubilized or absorbed by macrophages, allowing their clearance from the circulation. Current in vivo liposome delivery methods use aerosolization, subcutaneous, intradermal, intratumoral or intracranial injection to avoid toxicity and stability problems associated with cationic lipids in the circulation. The interaction between liposomes and plasma proteins is a significant cause of differences in gene transfer effects between in vitro (Felgner et al, 1987) and in vivo (Zhu et al, 1993; Philip et al, 1993; Solodin et al, 1995; Liu et al, 1995; Thierry et al, 1995; Aksentijevich et al, 1996).

d. Administration of lipids

The actual dosage of the lipid composition (e.g., liposome-chimeric polypeptide) administered to a patient will depend on physical or physiological factors, such as body weight, severity of the condition, the patient's primary disease and the route of administration. In view of these factors, the dosage of the lipid composition appropriate for a particular patient and/or course of treatment can be readily determined.

The invention may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intrathecally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, intravaginally, mucosally, intrapericardially, orally, topically and/or by aerosol, injection, continuous infusion, direct local infusion of bathing target cells or by catheter and/or lavage.

IX. kit

Certain embodiments of the present invention relate to diagnostic and therapeutic kits. The peptides may be utilized in the form of a kit to determine the likelihood of a patient with HPV developing and relapsing into a precancerous or cancerous growth. The kit may determine the stimulation of T cell lymphocyte proliferation and/or the increase in T helper 1 cytokine production. The components of the various kits may be stored in suitable containers. The container typically comprises at least one vial, test tube, flask, bottle, syringe or other container into which the peptide reagent is placed, preferably dispensed appropriately. The kit also includes a second container for holding a sterile, pharmaceutically acceptable buffer or other solution. Kits of the invention also typically include a vial-tight container for commercial sale, such as an injection or blow-molded plastic container, wherein the desired vial is retained.

The kit may contain a reagent (detection reagent) for detecting the interaction between the sample and the antibody. The reagents may be provided with radioactive, fluorescent or enzymatic labels. The kit may comprise a known radiolabeled reagent capable of binding to or interacting with an antibody which allows the detection and/or measurement of a cell-mediated immune response. Generally, these methods comprise first obtaining a sample suspected of containing such proteins, peptides or antibodies, contacting the sample with the antibodies or peptides according to the methods of the invention, and, as the case may be, detecting the formation of immune complexes under conditions effective to allow immune complex formation.

In some embodiments, one or more of the E6 and/or E7 peptides are included in a suitable container. The sample may be contacted or incubated with the peptide and the sample then tested for a cell-mediated immune response against the peptide. Thus, in some embodiments, the kit contains a non-reactive structure, which in some embodiments may be plastic or some other synthetic material. The non-reactive structure may be a container for holding the sample, such as a container with a small aperture. Containers with multiple apertures are also included as part of the present invention. In some cases, the structure is scored or attached with a film. A well cut by membrane streaking can be used to incubate the E6 or E7 peptides and then the same container is used to detect cell-mediated immune responses. This process can be performed by using antibodies that detect cell-mediated immune responses. Such antibodies include antibodies against TH1 or TH2 cytokines, cytokine receptors, or any other receptor on T cells indicative of a cell-mediated immune response. Detection reagents are also included in the kit.

The reagents of the kit may be supplied as liquid solutions, attached to a solid support or as dry powders. When the reagent is supplied in the form of a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent is supplied attached to a solid support, the solid support can be a chromatographic medium, a multi-well test plate, or a slide. When the reagent to be supplied is a dry powder, the powder is supplied by reconstitution with the addition of a suitable solvent.

In general, detection of immune complex formation is well known in the art and can be obtained by using a number of methods. For example, the present invention contemplates the use of ELISA, RIA, immunoblots (e.g., dot blot), ELISPOT, indirect immunofluorescence techniques, and the like. Typically, immune complex formation is detected by the use of a label, such as a radioactive label or an enzymatic label (e.g., alkaline phosphatase, horseradish peroxidase, or the like). Of course, we have found that there are additional advantages to using a second binding ligand, such as a second antibody or a biotin/avidin ligand binding configuration, as is well known in the art.

For detection purposes, virtually any sample suspected of containing a cell-mediated immune response can be used, as the case may be. The sample may comprise cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other non-cellular compositions suspected of containing cells capable of producing a cell-mediated immune response. In general, kits consistent with the present invention include at least one E6 or E7 peptide and antibodies against proteinaceous compositions associated with cell-mediated immunity, as well as immunodetection reagents and containers holding the antibodies, peptides, and reagents. The immunodetection reagent typically comprises a label linked to an antibody or antigen, or to a second binding ligand. Exemplary ligands may include a second antibody against a first antibody or antigen or a biotin or avidin (or streptavidin) ligand linked to a label. Of course, as mentioned above, many typical markers are known in the art and all such markers can be used in connection with the present invention.

The container typically includes a vial in which the antibody, antigen or detection reagent can be placed, preferably the container is appropriately aliquoted. Kits of the invention also typically include means for tightly holding containers for antibodies, antigens, and reagents for commercial sale. The container may comprise an injection or blow-molded plastic container with the desired vial retained therein.

In one embodiment, the diagnostic kit comprises probes and primers for use in a method of detecting nucleic acids. All materials and reagents necessary for the detection of peptide markers in biological samples are assembled together into a kit. This will typically comprise primers that are pre-selected for a particular marker. Also included are enzymes suitable for amplifying nucleic acids, including various polymerases (RT, Taq, etc.), as well as deoxynucleotides and buffers to provide the reaction mixture necessary for amplification.

Such kits typically contain in appropriate manner in separate containers a variety of individual reagents and enzymes, as well as primer pairs for various markers. Preferred primers for amplifying nucleotides are selected to amplify the nucleotide sequence of SEQ ID NO: 1-19 or the complement thereof.

In another embodiment, such a kit comprises a nucleic acid sequence specific for a polypeptide having a sequence as set forth in SEQ ID NO: 1-19 or a sequence complementary thereto. In a suitable manner, such kits usually contain various individual reagents and enzymes, as well as hybridization probes, in different containers.

In other embodiments, the invention relates to an immunoassay kit for use in the above immunoassay method. Since the peptide is typically a protein, polypeptide or peptide, the peptide is preferably included in the kit. The immunoassay kit thus comprises the peptide, and optionally an immunoassay reagent, in a suitable container.

The immunodetection reagents of the kit may take one or more forms, including those detectable labels associated with or linked to a given antibody. Detectable labels associated or bound with the second binding partner are also contemplated. Typically the second ligand is a second antibody having binding affinity for the first antibody.

The kit may further comprise a suitable aliquot of the wild-type or mutant protein, polypeptide or peptide composition, labeled or unlabeled, for use in generating a standard curve for the detection assay. The kit also comprises the antibody-labeled conjugate, either in a fully conjugated form, in an intermediate form, or as a separate moiety conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in lyophilized form.

The therapeutic kit of the invention is a kit comprising the peptide sequence of seq id no: 1-19 peptide kits. Such kits typically contain, in a suitable container, a pharmaceutically acceptable preparation of a polypeptide, peptide, biofunctional equivalent, immunological fragment, functional region, inhibitory factor, antibody, gene, polynucleotide, nucleic acid or complement, or a vector which expresses any of the above in a pharmaceutically acceptable form. The kit may have only one container, or each compound may have a different container.

When the components of the kit are supplied in one or more liquid solutions, the liquid solution is an aqueous solution, with sterile aqueous solutions being particularly preferred. The peptide component may also be prepared in the form of an injectable composition. In this case, the container itself may be a syringe, pipette, or other similar instrument from which the formulation may be applied to the infected site of the body, injected into the animal, or even applied to and mixed with other components of the kit.

However, the components of the kit may also be supplied in the form of a dry powder. When the agent or ingredient is provided as a dry powder, the powder is reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may be supplied in another container.

The container of the kit generally comprises at least one vial, test tube, flask, bottle, syringe or other container in which the antibody can be placed, and preferably is suitably aliquoted. When a polypeptide or peptide, or a second or third binding ligand or additional component is supplied, the kit will also typically include a second, third or other additional container in which such ligand or component may be placed. The kits of the invention also typically include a means for tightly holding the containers of antibodies, antigens and any other reagents for commercial sale. The container may comprise an injection or blow-molded plastic container in which the desired vial is retained.

Regardless of the number or type of containers, the kits of the invention may also contain or be packaged with instruments to assist in injection/administration or placement of the final peptide in the body. Such an instrument may be a syringe, pipette, forceps, or any medically approved delivery tool.

Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which have been disclosed by the inventors herein function well in the practice of the invention, and as such, are considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Materials and methods

Patient's health

Embodiments of the invention comprise a group of study populations selected from patients diagnosed at the colposcopic examination of the Anderson cancer center, university of Texas, M.D. Patient consent was obtained and all procedures were performed according to protocols approved by the ethics committee in the study institute. These women were 17 years of age or older and had no pregnancy and no history of immunization. Four groups of women were identified in this study. Group 1 consisted of six women with no CIN cytological or histological diagnosis and with HPV negative tests (CIN)^(-)/HPV^(-)). Group 2 included 31 histological diagnoses with CIN and HPV positive tests (CIN)⁽⁺⁾/HPV⁽⁺⁾) The woman of (1). Groups 3 and 4 were selected from women who were treated for at least 6 months with CIN ablation or resection at the colposcopic clinic prior to the study. Women in groups 3 and 4 were (CIN) prior to CIN treatment⁽⁺⁾/HPV⁽⁺⁾). However, these women were only evaluated for disease at the time of enrollment, i.e., after a minimum of 6 months following CIN treatment. Group 3 consisted of 22 women with no signs of relapse CIN (Recur)^(-)) Group 4 included 10 women with histologically diagnosed recurrent CIN (Recur)⁽⁺⁾). HPV positivity was determined using the Virapap/Viratype assay (Technologies Inc., Gaithersburg, Md.). In this procedure, dot blot hybridization of HPV RNA was performed using exfoliated cervical epithelial cells from a cervical swab. The detection method comprises the use of³²A P-labeled DNA probe set that identifies HPV types: 6/11, 16/18, and 31/33/35. Cells were isolated and processed according to the manufacturer's instructions. At the time of the study, this test was used as part of the standard care procedure at the time of colposcopy. HPV positivity was further determined by PCR using a primer from paraffin-coated panel as described previouslyDNA extracted from biopsies (Ting et al, 1990; Schiffman et al, 1991). Consensus primers for PCR analysis were derived from the LI open reading frame of papillomaviruses (MY11, GCMCAGGGWCATAAYAATGG (SEQ ID NO: 23) and MY09, CGTCCMARRGGAWACTGATC (SEQ ID NO: 24): where M ═ a + C, R ═ a + G, W ═ a + T, and Y ═ C + T). HPV-16 positivity was confirmed using a specific oligonucleotide probe CATACACCTCCAGCACCTAA (SEQ ID NO: 25). Clinical traits, including HPV status, of the subjects studied are listed in table 2.

Investigating the characteristics of the subject

Traits	Total of	Group 1	Group 2	Group 3	Group 4
						Number of patients average age range ethnic white race Spanish Africa-American Asian HPV status negative-Positive HPV-16 initial diagnosis of other HPV types negative CIN1CIN2&3	693117-5452(75.4％)8(12.6％)8(12.6％)1(1.4％)6(8.7％)63(91.3％)57(90.5％)6(9.5％)6(8.7％)4(5.8％)59(85.5％)	63117-433(50％)3(50％)0(0％)30(0％)6(100％)0(0％)0(0％)0(0％)6(100％)0(0％)0(0％)	313121-5026(83.9％)1(3.2％)3(9.7％)1(3.2％)0(0％)31(100％)31(100％)0(0％)0(0％)0(0％)31(100％)	223218-5415(68.2％)4(18.2％)3(13.6％)0(0％)0(0％)22(100％)17(77.3％)5(22.7％)0(0％)4(18.2％)18(81.8％)	102720-398(80％)0(0％)0(0％)0(0％)0(0％)10(100％)9(90％)1(10％)0(0％)0(0％)0(100％)

Injecting: in group 3, the diagnosis of CIN was negative at the time of enrollment, while in group 4, the diagnosis of CIN2/3 was positive.

Peptides

Peptide sequences corresponding to the E6 and E7 oncoproteins of HPV-16 were selected based on the known amphipathic structure and information of T cell epitopes described in the relevant literature. Table 3 lists peptides used in the present study. All peptides were prepared as reported earlier (Sarkar et al, 1995) using either the Merrifield solid phase method (Merrifield, 1963) or on a modified Vega 250 automated peptide synthesizer (Vega Biochemicals, Tucson, AZ) or using the "bag" method described by Houghten, 1985. In most experiments, peptides with a purity of approximately 70-80% were used, and in some experiments peptides with a purity > 95% were used with the same results. In addition to the E6 and E7 peptides, we used the peptide from the c-mos proto-oncogene [ amino acid 158-170, STRTPEDSNSLGT (SEQ ID NO 22) ] as a negative control. Stock solutions of the peptides were prepared in PBS (pH7.0) and filter sterilized.

T cell proliferation assay

Heparinized blood was collected from study participants by venipuncture and PBMCs were separated by centrifugation on a Ficoll-Hypaque density gradient (Histopaque-1073; Sigma Chemical Co., St. Louis, Mo.). After stimulation with PHA, c-mou peptide, or the respective E6 and E7 peptides, the PBMC proliferation response in different individuals was determined using the previously described [ 2 ], [³H]Thymidine incorporation assays (Nehete et al, 1996) (Table 4). Briefly, each sample was on a 96-well microtiter plateThree portions were inoculated and humidified at 37 ℃ with 5% CO₂Cultured in the air for 7 days. At the last 16-18 hours, 1. mu. Ci of³[H]Thymine (6.7 Ci/mmol; ICN Biomedicals, Inc., Costa Mesa, Calif.). Cells were collected on filter strips for evaluation³[H]Incorporation of thymine. The specific radioactivity of the cells treated with the various additives was calculated in each case by subtracting the counts per minute (cpm) values of cells cultured only in the medium. Data from pre-experiments showed that at 5ug/ml, each peptide produced consistent levels of proliferation. Significance of T cell proliferative responses to individual E6 and E7 peptides (in terms of stimulation index SI]) It can be calculated that the cells exposed to the peptide exceeded those of the control group to which no peptide was added³[H]Fold increase in thymidine incorporation. When the SI value > 3.0, it is considered a positive response, it can be used in all statistical analyses to determine the significance of the proliferative response in relation to the disease remission or disease relapse status. In all experiments, the data for the triplicate samples were comparable with a standard error of < 10%. In the four study groups tested, none of the women showed a proliferative response specific to the control c-mos peptide (SI < 2.0).

TABLE 3

Amino acid sequences of E6 and E7 peptides from HPV-16

Peptides	Residue of	Sequence of
			E6 peptide
K9L(SEQ ID NO：1)	(amino acid 18-2)6)	KLPQLCTEL
			E101(SEQID NO：2)	(amino acids 25 to 34)	ELQTTIHDII
C10R(SEQ ID NO：3)	(amino acids 37-46)	CVYCKQQLLR
			Q15L(SEQ ID NO：4)	(amino acids 43-57)	QLLRREVYDFAFRDL
V10C(SEQ ID NO：5)	(amino acids 49-58)	VYDFAFRDLC
			P9L(SEQ ID NO：6)	(amino acids 66-74)	PYAVCDKCL
P10I(SEQ ID NO：7)	(amino acid 102-111)	PLCDLLIRCI
			Q20P(SEQ ID NO：8)	(amino acids 97-116)	QQYNKPLCDLLIRCINCQKP
R16R(SEQ ID NO：9)	(amino acid 131-	RWTGRCMSCCRSSRTR
			G10S(SEQ ID NO：10)	(amino acids 141-	GRCMSCCRSS
			E7 peptide
T10Q(SEQ ID NO：11)	(amino acids 7 to 15)	TLHEYMLELQ
			M9T(SEQ ID NO：12)	(amino acids 12 to 20)	MLDLQPETT
D9L(SEQ ID NO：13)	(amino acids 14 to 22)	DLQPETTDL
			Q19D(SEQ ID NO：14)	(amino acids 44-62)	QAEPDRAHYNIVTFCCKCD
R9F(SEQ ID NO：15)	(amino acids 49-57)	RAHYNIVTF
			R9V(SEQ ID NO：16)	(amino acids 66-74)	RLCVQSTHV
L9V(SEQ ID NO：17)	(amino acids 82 to 90)	LLMGTLGIV
			G10C(SEQ ID NO：18)	(amino acids 85-94)	GTLGIVCPIC
D20C(SEQ ID NO：19)	(amino acids 75-94)	DIRTLEDLLMGTLGIVCPIC

aa, amino acids

Cytokine analysis

Cryopreserved PBMCs were used in this assay. PBMC (1X 10)⁵) The peptide was incubated with various HPV peptides in RPMI-1640 medium (containing 10% fetal bovine serum) at 37 ℃ for 48 hours in three identical wells of a 96-well round bottom plate. After centrifugation, the supernatant (100ul) was removed from each well and stored frozen at-70 ℃ in another 96-well plate. The plates were then thawed and supernatants were assayed for various cytokines (IFN-. gamma., IL-2, IL-4, IL-10, and IL-12) using a cell screening immunoassay kit (Biosource International, Camarillo, Calif.) according to the manufacturer's instructions.

Statistical analysis

Using Pearson X²And Fisher exact test (exact test) to assess the differences in SI values between patient groups. For statistical analysis, a significant proliferative response was defined as SI ≧ 3.0. Statistics ofSignificance was set at p < 0.05.

Example 2

A total of 69 women aged 17 to 54 years (31 years on average) participated in the study. Of these 69 women, 52 caucasians, 8 each of african americans and hispanic, 1 asian (table 2). PBMCs from these women were analyzed for proliferative responses against synthetic peptides corresponding to the antigenic sequences of HPV-16 oncoproteins E6 and E7 (Table 3) (FIG. 1A).

Analysis of the proliferative responses specific to the various E6 and E7 peptides in different four groups of patients showed that: majority of patients in group 3 (Recur)^(-)) All seven tested E6 peptides and 7/8E 7 peptide showed positive reactions (SI ≧ 3.0) (FIG. 1A). On the other hand, in group 2 (CIN)⁽⁺⁾/HPV⁽⁺⁾) Only 5/31 showed a response to any of the peptides tested, group 1 (CIN)^(-)/HPV^(-)) And group 4 (Recur)⁽⁺⁾) Did not respond to any of the peptides tested. Fig. 1B summarizes the above.

The relationship between proliferative responses to E6 and/or E7 peptides and post-treatment disease conditions in women in groups 3 and 4 is shown in Table 4. Although group 4 (Recur)^(-)) Does not show a response to any of the tested E6 or E7 peptides, but in group 3 64% of patients showed a significant proliferative response to the E6 peptide (p 0.001), 82% to the E7 peptide (p < 0.001), and 86% to at least one of the E6 or E7 peptides (p < 0.001). There was no difference in the proliferative responses of common mitogens such as PHA (p 0.912, data not shown) between groups 3 and 4, indicating that the innate immune status of these patients was not compromised. These conclusions strongly suggest that the proliferative response to synthetic peptides from the E6 and E7 oncoproteins of HPV-16 is associated with disease-free conditions following CIN treatment.

The inventors determined high level proliferation responses against two peptides from E6((Q15L and V10C) and E7(Q19D and R9F), respectively, typical proliferation responses are shown in FIG. 2 according to SI values, from which they were derived, respectivelyProliferative responses of 2 patients from groups 3 and 4 to 7 synthetic peptides of the E6 oncoprotein and 8 synthetic peptides of the E7 oncoprotein of HPV-16. Comparison of proliferative responses against these four peptides showed statistically significant differences between women in groups 3 and 4 (table 5). Although no proliferative response was observed to these peptides in group 4, a total of 11 women showed a response to peptide Q15L (p ═ 0.006), 10 to peptide VIOC (p ═ 0.006), 13 to peptide Q19D (p ═ 0.002), and 10 to peptide R9F (p ═ 0.013) in group 3. As shown in table 3,9 of the 10 amino acids in the E6 peptide V10C overlapped with the Q15L peptide. Similarly, the 9 amino acids of the E7 peptide R9F overlapped with the amino acids of the Q19D peptide. Proliferative responses specific for these four peptides were included together in group 3 (Recur)^(-)) All responses observed (women of 19/22). These results indicate that the peptides Q15L and Q19D in HPV-16 oncoproteins E6 and E7, respectively, are likely to be immunodominant regions for HPV-specific cellular immune responses.

In a sub-sample of the population studied, the inventors also tested whether these peptides also induced the production of various TH1 and TH2 cytokines. From the third group (Recur)^(-)) 8 women and fourth group (Recur)⁽⁺⁾) Cryopreserved PBMCs from 6 women were stimulated in vitro with peptides Q15L and Q19D. The amounts of the various TH1 cytokines (IL-2, IL-12, and IFN- □), and TH2 cytokines (IL-4 and IL-10) in the culture supernatants, after correction in the relatively unstimulated cultures, are shown in FIG. 3. Third group (Recur)^(-)) PBMCs from women in 7 out of 8 (87.5%), and 5 out of 8 (62.5%) showed production of IFN- □ and IL-2 in response to both Q15L and Q19D, respectively (Table 6). In addition, IL-12 production in response to Q15L was observed in position 3 of these 8 women, whereas Q19D mediated IL-12 production was evident in position 6 of 8 women. On the other hand, PBMCs from this group of women did not secrete IL-4 in response to stimulation with peptides Q15L or Q19D, whereas only 3 women showed IL-10 production in response to either peptide. And the third group (Recur)^(-)) In contrast to women, group IV (Recur)⁽⁺⁾) The women in (2) highlighted IL-10 production (5/6 due to Q15L, 6/6 due to Q19D). This panel was observed when PBMCs were stimulated with either of the two test peptidesOf 6 women showed IL-4 production at position 1 (Table 6). Overall, these results show that the third group (Recur)^(-)) Patients in (A) mainly showed TH1 cytokine production (IL-2, IFN-. gamma.and IL-12), and fourth group (Recur)⁽⁺⁾) The women in (A) although not showing a specific proliferative response directly against HPV peptide, showed production of IL-10, a TH2 cytokine.

TABLE 4

Proliferative responses to all synthetic peptides of HPV-16 oncoproteins E6 and/or E7 and CIN therapy

Correlation between later disease states

Proliferative responses	Group 3 (No disease)	Group 4 (recurrence)	Significance of
				The E6 peptide isaWhether or notbThe E7 peptide isaWhether or notbAny of the E6 or E7 peptides isaWhether or notb	14(64％)8(36％)18(82％)4(18％)19(86％)3(14％)	010(100％)010(100％)010(100％)	p＝0.001p＜0.001p＜0.001

^aSI≥3.0 ^bSI＜3.0

TABLE 5

Proliferative response to specific synthetic peptides of HPV-16 oncoproteins E6 and/or E7 and CIN therapy

Correlation between later disease states

Proliferative responses	Group 3 (no disease) (n ═ 22)	Group 4 (relapses) (n ═ 10)	Significance of
				E6 peptide Q15L isaWhether or notbV10C isaWhether or notbThe E7 peptide Q19D isaWhether or notbR9F isaWhether or notb	11(50％)11(50％)11(50％)11(50％)13(59％)9(41％)10(46％)12(54％)	010(100％)010(100％)010(100％)010(100％)	P＝0.006P＝0.006P＝0.002P＝0.013

^aSI≥3.0 ^bSI＜3.0

TABLE 6

Response to HPV-16^aE6 and E7 oncoprotein synthetic peptide stimulated

PBMC cytokine production in groups 3 and 4 patients

Cytokineb	Group 3 (n ═ 8)		Group 4 (n ═ 6)
					Q15Lc	Q19Dd	Q15L	Q19D
	IFN-γIL-2IL-12IL-4IL-10	7/85/83/80/83/8	7/85/86/80/83/8	0/60/60/61/65/6					0/60/60/61/66/6

^aNumber of positive patients/number of test persons

^bThe positivity of cytokine production is based on the value of the sensitivity of the detection kit for each cytokine, in terms of concentration pg/ml: IL-2 ═ 8.7, IFN-y ═ 4.0, IL-12 ═ 1.0, IL-4 ═ 2.0, and IL-10 ═ 5.0.

^cQ15L peptide from E6 oncoprotein in HPV-16

^dQ19D peptide from E7 oncoprotein in HPV-16

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred modes, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps and in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, certain agents which are both chemically and physically related may obviously be substituted for the agents described herein while the same or similar results would be achieved. It will be apparent to those skilled in the art that all such similar substitutes and modifications are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Sequence listing

<110>SASTRY，K.JAGANNADHA

TORTOLERO-LUNA，GUILLERMO

FOLLEN，MICHELE

<120> methods and compositions relating to the growth of HPV-associated precancerous carcinomas (including CIN)

<130>UTFC：560CN

<140>PCT/US02/23198

<141>2002-07-13

<150>60/306,809

<151>2001-07-20

<160>27

<170>PatentIn Ver.2.1

<210>1

<211>9

<212>PRT

<213> human papilloma virus

<400>1

Lys Leu Pro Gln Leu Cys Thr Glu Leu

1 5

<210>2

<211>10

<212>PRT

<213> human papilloma virus

<400>2

Glu Leu Gln Thr Thr Ile His Asp Ile Ile

1 5 10

<210>3

<211>10

<212>PRT

<213> human papilloma virus

<400>3

Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg

1 5 10

<210>4

<211>15

<212>PRT

<213> human papilloma virus

<400>4

Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu

1 5 10 15

<210>5

<211>10

<212>PRT

<213> human papilloma virus

<400>5

Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys

1 5 10

<210>6

<211>9

<212>PRT

<213> human papilloma virus

<400>6

Pro Tyr Ala Val Cys Asp Lys Cys Leu

1 5

<210>7

<211>10

<212>PRT

<213> human papilloma virus

<400>7

Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile

1 5 10

<210>8

<211>20

<212>PRT

<213> human papilloma virus

<400>8

Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn

1 5 10 15

Cys Gln Lys Pro

20

<210>9

<211>16

<212>PRT

<213> human papilloma virus

<400>9

Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg

1 5 10 15

<210>10

<211>10

<212>PRT

<213> human papilloma virus

<400>10

Gly Arg Cys Met Ser Cys Cys Arg Ser Ser

1 5 10

<210>11

<211>10

<212>PRT

<213> human papilloma virus

<400>11

Thr Leu His Glu Tyr Met Leu Glu Leu Gln

1 5 10

<210>12

<211>9

<212>PRT

<213> human papilloma virus

<400>12

Met Leu Asp Leu Gln Pro Glu Thr Thr

1 5

<210>13

<211>9

<212>PRT

<213> human papilloma virus

<400>13

Asp Leu Gln Pro Glu Thr Thr Asp Leu

1 5

<210>14

<211>19

<212>PRT

<213> human papilloma virus

<400>14

Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys

1 5 10 15

Lys Cys Asp

<210>15

<211>9

<212>PRT

<213> human papilloma virus

<400>15

Arg Ala His Tyr Asn Ile Val Thr Phe

1 5

<210>16

<211>9

<212>PRT

<213> human papilloma virus

<400>16

Arg Leu Cys Val Gln Ser Thr His Val

1 5

<210>17

<211>9

<212>PRT

<213> human papilloma virus

<400>17

Leu Leu Met Gly Thr Leu Gly Ile Val

1 5

<210>18

<211>10

<212>PRT

<213> human papilloma virus

<400>18

Gly Thr Leu Gly Ile Val Cys Pro Ile Cys

1 5 10

<210>19

<211>20

<212>PRT

<213> human papilloma virus

<400>19

Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val

1 5 10 15

Cys Pro Ile Cys

20

<210>20

<211>151

<212>PRT

<213> human papilloma virus

<400>20

Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys

1 5 10 15

Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr

20 25 30

Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg

35 40 45

Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp

50 55 60

Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys

65 70 75 80

Tyr Ser Val Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu

85 90 95

Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro

100 105 110

Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile

115 120 125

Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg

130 135 140

Thr Arg Arg Glu Thr Gln Leu

145 150

<210>21

<211>590

<212>PRT

<213> human papilloma virus

<400>21

Met Ser Leu Pro Gly Gly Arg Gly Thr Val Lys Ile Glu Thr Arg Glu

1 5 10 15

Arg Ile Trp Val Arg Arg Val Asn Gly Glu Thr Gly Val Tyr Asp Thr

20 25 30

Arg Ala Gly Ser Phe Glu Thr Val Ser Cys Gln Glu Phe Glu Ala Ala

35 40 45

Ala Asp Thr Val Pro Ser Val Pro Val Phe Cys Asp Arg Cys Phe Gly

50 55 60

Thr Ser Leu Tyr Glu Val Pro Leu Thr Gly Phe Gly Thr Phe Val Val

65 70 75 80

Gly Thr Cys Cys Ile Phe Ser Pro Gly Asp Pro Val Asp Asp Pro Ser

85 90 95

Ile Pro Ala His Met Arg Lys Tyr Gln Gln Pro Ile Glu Ala His Gln

100 105 110

Thr Met Val Gln Val Ala Pro Gly Thr Leu Lys Tyr Ser His Gln Ile

115 120 125

Pro Met Gly Lys Val Leu Gly Tyr Trp His Val His Met Glu Asp Arg

130 135 140

Val Tyr Leu Asn Met Ile Gly Gly Ile Asp Glu Ser Glu Asp Thr Gly

145 150 155 160

Lys Arg Cys Val Glu Thr Phe Thr Glu Ala Asp Ile Pro Cys Ala Leu

165 170 175

Ser Leu Gly Thr Leu Asp Val Gly Leu Asn Glu Val Ile Leu Glu Cys

180 185 190

Ser Val Val Val Ile Pro Ala Arg Arg Gly Cys His Ala Lys Leu Phe

195 200 205

Thr Arg Asp Thr Val Ser Asp Gly Leu Glu Lys Phe Cys Phe Gln Ser

210 215 220

His Ala Thr Leu Pro Pro Thr Leu Leu Ala Ser Phe Gly Ser Thr Ser

225 230 235 240

Glu Ser Pro Glu Arg Lys Thr Phe Tyr Glu Ala His Val Asp Ala Leu

245 250 255

Asn Asn Tyr Ile Lys Leu Leu Arg Thr Ile Tyr Ser His Lys Gly Glu

260 265 270

Thr Glu Ile Glu Gln Tyr Leu Ile Glu Gly Ser Lys Leu Tyr Ser Glu

275 280 285

Leu Ile Gly Glu Pro Ser Arg Val Leu Asp Ala Thr Met Lys Ala Ala

290 295 300

Gln Ile Ala Glu Pro Gln Thr His Thr Gly Gly Ala Asp Arg Gln Arg

305 310 315 320

Pro Gln Arg Pro Asp Gly Ile Pro Tyr Ser Val Pro Asp Arg Phe Pro

325 330 335

Met Thr Gly Tyr Pro Phe Ala Pro Gln Phe Cys Gly Asp Pro Gly Leu

340 345 350

Val Ser His Tyr Asn Pro Phe Val Pro Pro Gln Ser Phe Gly Gln Gly

355 360 365

Tyr Gly Pro Glu Arg Val Gly Gly Tyr Tyr Pro Gln Pro Pro Asn Pro

370 375 380

Tyr Val Leu Pro Ile Ser Tyr Gly Gln Gln Pro Tyr Pro Gly His Pro

385 390 395 400

Gln Pro His Gly His His Gln Gln Arg Ser Gly Gly Gly Asp Leu Lys

405 410 415

Ala Glu Leu Ile Glu Thr Leu Gly Leu Ala Pro Lys Thr Asn Ala Val

420 425 430

Gln Glu Ser Leu Lys Ser Phe Ile Ser Glu Ile Leu Glu Ser Glu Leu

435 440 445

Lys Asn Cys Gly Ile Lys Arg Ala Ala Gly Asn Ile Glu Arg Asn Cys

450 455 460

Asp Val Asp Glu Glu Pro Pro Arg Thr Lys Arg Ala Arg Pro Glu Pro

465 470 475 480

Lys Thr Ala Val Glu Ala Ile Val Arg Ala Pro Tyr Gly Asp Phe Asp

485 490 495

Ser Thr Ala Leu Thr Thr Lys Ile Gly Gln Val Ser Asp Thr Val Glu

500 505 510

Lys Leu Asn Lys Val Ile Glu Thr Leu Leu Thr Gln Ser Ser Ala Gln

515 520 525

Pro Ala Pro Leu Ser Thr Pro Ala Gln Ala Ala Pro Val Gln Pro Ser

530 535 540

Leu Pro Gln Pro Val Pro Glu Pro Leu Ala Pro Gln Glu Pro Pro Pro

545 550 555 560

Pro Gly Thr Ser Ala Pro Thr Leu Glu Ala Ser Leu Pro Gln Gln Lys

565 570 575

Pro Val Val Ser Lys Gly Ala Phe Glu Thr Leu Met Asn Leu

580 585 590

<210>22

<211>13

<212>PRT

<213> mouse

<400>22

Ser Thr Arg Thr Pro Glu Asp Ser Asn Ser Leu Gly Thr

1 5 10

<210>23

<211>20

<212>DNA

<213> human papilloma virus

<220>

<221> modified base

<222>(3)

<223> M ═ A, C, or R

<220>

<221> modified base

<222>(9)

<223> W ═ A and T

<220>

<221> modified base

<222>(15)

<223> Y ═ C and T

<400>23

gcmcagggwc ataayaat gg 20

<210>24

<211>20

<212>DNA

<213> human papilloma virus

<220>

<221> modified base

<222>(6)

<223> M ═ A, C, or R

<220>

<221> modified base

<222>(8)..(9)

<223> R ═ A and G

<220>

<221> modified base

<222>(13)

<223> W ═ A and T

<400>24

cgtccmarrg gawactgatc 20

<210>25

<211>20

<212>DNA

<213> human papilloma virus

<400>25

catacacctc cagcacctaa 20

<210>26

<211>98

<212>PRT

<213> human papillomavirus type 16

<400>26

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Ser Asp Ser Ser

20 25 30

Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp

35 40 45

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr

50 55 60

Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu

65 70 75 80

Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln

85 90 95

Lys Pro

<210>27

<211>151

<212>PRT

<213> human papillomavirus type 16

<400>27

Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Leu Cys

1 5 10 15

Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr

20 25 30

Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg

35 40 45

Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp

50 55 60

Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys

65 70 75 80

Tyr Ser Val Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu

85 90 95

Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro

100 105 110

Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile

115 120 125

Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg

130 135 140

Thr Arg Arg Glu Thr Gln Leu

145 150

Claims (37)

1. Use of at least one HPV E6 or E7 peptide in the manufacture of a reagent for determining the likelihood of recurrence of a precancerous or cancerous growth in a patient infected with Human Papillomavirus (HPV) or suspected of being infected with HPV, wherein the patient also has or had a precancerous or cancerous growth on or around the cervix, comprising:

a) incubating a sample from the patient with at least one HPV E6 or E7 peptide; and

b) determining a cell-mediated immune response of the sample against the peptide;

wherein the sample contains peripheral blood mononuclear cells capable of producing a cell-mediated immune response, and wherein the E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R or G10S, the E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C or D20C.

2. The use of claim 1, wherein the sample is incubated with at least two E6 peptides or at least two E7 peptides.

3. The use of claim 1, wherein the sample is incubated with the E6 peptide of HPV.

4. The use of claim 1, wherein the E6 peptide is K9L, E10I, C10R, Q15L, V10C, or a combination thereof.

5. The use of claim 1, wherein the sample is incubated with the E7 peptide of HPV.

6. The use of claim 1, wherein the E7 peptide is Q19D, R9F, R9V, L9V, G10C, or a combination thereof.

7. The use of claim 1, wherein the sample is incubated with at least one E6 peptide and at least one E7 peptide.

8. The use of claim 1, wherein the patient is known to be infected with HPV.

9. The use of claim 1, further comprising determining whether said patient is infected with HPV.

10. The use of claim 1, wherein the patient has or has had a pre-cancerous growth.

11. The use of claim 10, wherein the precancerous growth is Cervical Intraepithelial Neoplasia (CIN).

12. The use of claim 1, wherein the patient has no more pre-cancerous or cancerous growths.

13. The use of claim 12, wherein the patient has no more pre-cancerous growth.

14. The use of claim 13, wherein the precancerous growth is CIN.

15. The use of claim 1, wherein the sample is blood.

16. The use of claim 1, wherein the sample is obtained by a vaginal swab.

17. The use of claim 1, further comprising incubating the sample in a medium after obtaining the sample.

18. The use of claim 1, wherein the assay comprises contacting the sample with the peptide and measuring T cell proliferation of the sample.

19. The use of claim 18, wherein proliferation of said T cells is determined by measuring incorporation of tritiated thymidine.

20. The use of claim 19, wherein the sample has an SI value greater than or equal to 2.0, indicative of a cell-mediated immune response.

21. The use of claim 20, wherein the sample has an SI value greater than or equal to 3.0, indicative of a cell-mediated immune response.

22. The use of claim 1, wherein the assay comprises measuring the amount of a TH1 or TH2 cytokine.

23. The use of claim 22, wherein the amount of TH1 cytokine is measured.

24. The use of claim 23, wherein the TH1 cytokine is IL-2, IFN- γ, TNF- α or TNF- β.

25. The use according to claim 22, wherein the amount of TH2 cytokine is determined.

26. The use of claim 25, wherein the TH2 cytokine is IL-4, IL-5, IL-10, or IL-13.

27. The use of claim 22, wherein the TH1 or TH2 cytokine is measured by an immunoassay.

28. The use of claim 27, wherein the immunoassay is an ELISA or radioimmunoassay.

29. The use of claim 22, wherein the TH1 or TH2 cytokine is measured with a flow cytometer.

30. The use of claim 1, wherein the sample is determined more than once.

31. The use of claim 30, wherein the samples are determined using different assays.

32. The use of claim 1, further comprising obtaining a second sample from the patient and detecting a cell-mediated immune response of the second sample against at least one E6 or E7 peptide of HPV.

33. The use of claim 1, wherein the sample is obtained from the patient at least one month after treating a precancerous or cancerous growth thereof.

34. The use of claim 1, wherein the patient has undergone ablative treatment of a precancerous or cancerous growth in the urogenital tract of the patient.

Use of an E6 or E7 peptide in the manufacture of an agent for identifying an HPV infected patient at risk of recurrent precancerous or cancerous growth, comprising:

a) incubating a blood sample from a patient with an E6 or E7 peptide;

b) evaluating the sample for a cell-mediated immune response to the peptide;

wherein the E6 peptide is K9L, E10I, C10R, Q15L, V10C, P9L, P10I, Q20P, R16R or G10S, the E7 peptide is T10Q, M9T, D9L, Q19D, R9F, R9V, L9V, G10C or D20C.

36. The use of claim 1, wherein the human papillomavirus is of a higher order type.

37. The use of claim 36, wherein the human papillomavirus is HPV 16.