Trial Information

HPV and Cervical Cancers Human papillomaviruses (HPV) are small, nonenveloped DNA viruses
which induce epithelial tumors of skin or mucosa. The majority of tumors are benign, show
limited growth and usually regress spontaneously. However, a number of human
papillomaviruses induce tumors that may eventually progress to carcinomas. The genital HPV
types 16 and 18, and less frequently, types 31, 33, 35, 45, 51 and 56, have been implicated
in the etiology of cervical and other anogenital cancers. Approximately 500,000 women
worldwide develop cervical cancer yearly and it is the second leading cause of death from
cancer in women [1]. In developed countries, cancer of the cervix ranks behind cancers of
the breast, lung, uterus, and ovaries and accounts for 7% of all female cancers. In the
United States, there are about 4,800 deaths annually from cervical cancer [2]. The evidence
linking HPVs to anogenital cancer comes from epidemiologic and laboratory studies. More than
90% of cervical cancers and their precursors, so-called cervical intraepithelial neoplasia
(CIN), contain human papillomavirus (HPV) DNA sequences [3]. The HPV types found in cancer
cells have transforming activity in in vitro studies [4] and the viral transforming
proteins, E6 and E7, are consistently expressed in cervical cancer cell lines [5] and in
HPV-associated cancers of patients [6]. In HPV-associated malignant transformation, viral
DNA may be integrated into the cellular DNA and integration often results in deletion of
large sectors of the viral genome. Late genes (L1 and L2) and some early genes (E1 and E2)
are usually lost, leaving E6 and E7 as the only open reading frames frequently found in
carcinomas. Expression of E6 and E7 is likely to overcome the regulation of cell
proliferation normally mediated by proteins like p53 and Rb, allowing uncontrolled growth
and providing the potential for malignant transformation [7].

HPV Oncogenic Proteins, E6 and E7, as Ideal Targets for the Development of Antigen-Specific
Immunotherapies or Vaccines for HPV-Associated Cervical Malignancies E6 and E7 represent
ideal targets for the development of antigen-specific immunotherapies or vaccines for
HPV-associated malignancies. First, more than 90% of cervical cancers have been associated
with HPVs, particularly type 16, and E6 and E7 are consistently expressed in most cervical
cancers. Second, while most tumor specific antigens are derived from normal proteins or
mutated protein, E6 and E7 are completely foreign viral proteins, and potentially may harbor
more antigenic peptides/epitopes than a mutant protein (i.e. p53) or a reactivated embryonic
protein (i.e. MAGE-1). Third, since E6 and E7 are required for the induction and maintenance
of malignant phenotype of cancer cells [8], cells of cervical cancer cannot evade an immune
response through antigen loss. Without functional E6 and E7, these cells would cease to be
tumorigenic. Therefore, E6 and E7 proteins represent ideal targets for developing
antigen-specific immunotherapies or vaccines for cervical cancer.

Various forms of vaccines, such as vector-based vaccines, tumor-based vaccines, DNA based
vaccines and protein/peptide-based vaccines have been described in experimental systems
targeting HPV-16 E6 and/or E7 proteins [9, 10]. For example, Meneguzzi et al. reported that
inoculation of rats with vaccinia recombinants expressing HPV-16 E6 or E7 retarded or
prevented tumor development in 25-47% of rats challenged with a tumorigenic rat cell line
co-transfected with HPV-16 and activated ras [9]. In addition, Chen et al. demonstrated
that immunization of mice with syngeneic non-tumorigenic cells transfected with the HPV-16
E7 gene confers protection against transplanted HPV-16 E7 positive syngeneic tumor cells
[11]. Feltkamp et al. identified a CTL epitope in HPV-16 E7 using H-2Kb and H-2Db MHC class
I-peptide-binding studies. Immunization with this peptide rendered mice resistant to a
challenge with HPV-16 transformed tumor cells [12]. Furthermore, chimeric
papillomavirus-like particles (CVLPs) consist of HPV-16 L1-E7 (Nieland et al., personal
communication) or HPV-16 L1/L2-E7 (Greenstone et al., personal communication) chimeric
proteins has been used as therapeutic vaccines against HPV-16 E7 expressing tumors in murine
models. More recently, a phase I/II clinical trial were performed in eight patients with
late stage cervical cancer using a live recombinant vaccinia virus expressing the E6 and E7
proteins of HPV 16 and 18 (TA-HPV) [13]. In that study, no significant clinical side-effects
or environmental contamination by live TA-HPV were observed [13].

Importance of Cell Mediated Immune Responses in Controlling both HPV Infections and
HPV-Associated Neoplasms Several lines of evidence suggest that cell mediated immune
responses are important in controlling both HPV infections and HPV-associated neoplasms (for
review, see [14]). First, the prevalence of HPV-related diseases (infections and neoplasms)
is increased in transplant recipients [15] and human immunodeficiency virus (HIV) infected
patients [16], both of whom are known to have impaired cell mediated immunity. Second,
animal studies have demonstrated that immunized animals are protected from papillomavirus
infection and from the development of neoplasia. Immunization also facilitates the
regression of existing lesions [17-19]. Third, infiltrating CD4+ (T helper cells) and CD8+
(cytotoxic /suppressor T cells) T cells have been observed in spontaneously regressing warts
[20] and fourth, warts in patients who are on immunosuppressive therapy often disappear when
this treatment is discontinued (for review, see [21].

Cellular Immune Responses to HPV The understanding of T-cell mediated immunity to HPV
infections was facilitated by identification of MHC class I and class II epitopes of HPV
proteins. Several groups have attempted to map murine [22-24] and human [25, 26] T helper
(Th) cell epitopes on HPV proteins. Several groups have also tried to map murine [12, 27-30]
as well as human [31-34] cytotoxic T-lymphocyte (CTL) epitopes on HPV proteins. Kast et al.
have identified several high affinity binding peptides of HPV-16 E6 and E7 proteins for
human HLA-A alleles [32]. Furthermore, HPV-specific CTLs recognizing HPV E6 and E7 proteins
have been demonstrated in peripheral blood of cervical cancer patients [13, 35], in healthy
donors [33, 36] and in patients with CIN lesions [34, 37, 38]. Furthermore, infiltration of
cervical cancer tissue with HPV-specific CTLs has been recently described[39].

Cell-mediated immune responses in HPV-infected lesions can be demonstrated by in vivo skin
tests [40, 41], in vitro CTL assays [35, 37, 39] and in vitro lymphoproliferative response
[25, 26, 42-48]. For instance, Hopfl et al. have used bacterially-expressed HPV-16 proteins
for skin tests in patients with CIN lesions and have found specific skin responses to the
virion protein L1 and not the E4 protein [40]. In patients with CIN lesions, HPV-specific
CTLs have been identified in PBMC [35, 37] and in cervical tissues [39]. The in vitro
lymphoproliferative responses in patients with CIN lesions has been actively investigated.
For example, de Gruijl et al. reported that T cell proliferative responses against HPV-16 E7
oncogenic protein were most prominent in CIN patients with a persistent HPV infection [45].
However, Kadish et al. reported that lymphoproliferative responses to specific HPV-16 E6 and
E7 peptides appeared to be associated with the clearance of HPV infection and the regression
of CIN lesions [46].

Importance of Helper T Cell Functions in Generating Effective Antitumor Responses Increasing
evidence has suggested that inadequate antitumor responses can result from a failure of the
helper arm of the immune response. The events leading to the activation of CTL are tightly
regulated in order to protect against the development of inappropriate immune responses to
self antigens or exaggerated responses to foreign antigens. This regulation is mediated by
lymphokines produced by CD4+ T helper cells. CD4+ T helper cells are critical to the
generation of potent antitumor immune responses. CD4+ T cells have been shown to be
instrumental in generating immune responses against several solid malignancies in murine
[49, 50] and in human [51, 52]. Several mouse tumors that are transfected with syngeneic MHC
class II genes become very effective vaccines against subsequent challenge with wild type
class II negative tumors [53, 54]. In addition, as crucial memory cells in the T cell arm of
the immune system, CD4+ cells may be able to provide long term immunity against specific
antigens [55, 56].

Role of Cytokines in Cell-Mediated Immunity Cell mediated immunity is regulated by cytokines
which are secreted by T helper cells. In general, T helper cells can be classified as Th1
and/or Th2 cells based on the different types of cytokines they secrete. Th1 cells secrete
interleukin (IL) 2 and interferon gamma (IFN-). Th2 cells produce IL-4, IL-5, IL-10 and
IL-13. The Th1 lymphocytes are the most important effector cells in inflammatory reactions
associated with vigorous delay-type hypersensitivity but low antibody production, as occurs
in contact dermatitis and in viral or intracellular bacterial infections (for review, see
[57, 58]). The functional phenotype of most Th2 cells may account for both the persistent
production of certain antibody isotypes, particularly IgG1 and IgE, and the eosinophilia
observed in human helminthic infections and allergic disorders. Lymphocyte mediated
protection from viral infections as well as control of tumors is thought to be mediated by
Th1 cytokine responses and impaired by Th2 cytokine responses. The IL-2 and IFN- producing
Th1 response is likely to be the major component that contributes to the development of cell
mediated immunity against HPV infections and HPV-associated neoplasms.

Chimeric E7-specific vaccines can control the HPV16 E7-expressing tumor model With
cooperating with Prof. TC Wu in Johns Hopkins Medical Institutes, we have successfully
developed several chimeric DNA, RNA, and virus-vector vaccines to prevent and treat HPV16
E7-expressing tumor in the animal model [59-61]. We found that these E7-chimeric DNA
vaccines are capable of preventing and treating the growth of murine model tumors expressing
E7. These positive results from the preclinical murine models have encouraged us to focus on
the development of cancer vaccine and immunotherapy and apply these vaccines to human
subjects.

However, it is very important to set up various E7-specific immunologic assays of human
being to evaluate the effect of cancer vaccine or immunotherapy in the future clinical
trials. So we would like to provide this proposal to address on the development of HPV 16
E7-specific immunologic assays in human being. There are several aims in this project: 1) to
develop and utilize assays to measure CTLs to HPV 16 E7 proteins, 2) to develop and utilize
assays to measure T helper (Th) responses to HPV 16 E7 antigens.