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. 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. 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. The HPV types found in cancer cells have transforming
activity in in vitro studies and the viral transforming proteins, E6 and E7, are
consistently expressed in cervical cancer cell lines and in HPV-associated cancers of
patients. 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.

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 proteins, 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 phenotypes of cancer cells, 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. 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. 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.
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. 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 have been used as
therapeutic vaccines against HPV-16 E7 expressing tumors in murine models. More recently, a
phase I/II clinical trial was 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). In that study, no significant clinical side-effects or environmental contamination
by live TA-HPV were observed.

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. First, the prevalence of
HPV-related diseases (infections and neoplasms) is increased in transplant recipients and
human immunodeficiency virus (HIV) infected patients, 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. Third, infiltrating CD4+
(T helper cells) and CD8+ (cytotoxic/suppressor T cells) T cells have been observed in
spontaneously regressing warts and fourth, warts in patients who are on immunosuppressive
therapy often disappear when this treatment is discontinued.

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 and human T helper (Th) cell epitopes on HPV proteins. Several
groups have also tried to map murine as well as human 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. Furthermore, HPV-specific CTLs
recognizing HPV E6 and E7 proteins have been demonstrated in peripheral blood of cervical
cancer patients, in healthy donors and in patients with CIN lesions [Nakagawa, 1997 #562].
Furthermore, infiltration of cervical cancer tissue with HPV-specific CTLs has been recently
described.

Cell-mediated immune responses in HPV-infected lesions can be demonstrated by in vivo skin
tests, in vitro CTL assays and in vitro lymphoproliferative responses. 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. In patients with CIN lesions, HPV-specific CTLs have been identified in PBMC and in
cervical tissues. The in vitro lymphoproliferative responses in patients with CIN lesions
have 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. 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.

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 [Fearon, 1990 #833; Golumbek, 1991 #834] and in human populations [Topalian, 1994
#396; Topalian, 1994 #397]. 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 [Ostrand-Rosenberg, 1990 #177; James, 1991 #272; Chen, 1993 #213]
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 [Tew, 1990 #624; Sprent,
1994 #625].

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-gamma). 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 [Mosmann, 1989 #168; Romagnani, 1992
#130]). 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-gamma
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. By
cooperating with Prof. TC Wu at the Johns Hopkins Medical Institutes, we have successfully
developed several chimeric DNA, RNA, and virus-vector vaccines to prevent and treat HPV16
E7-expressing tumors in animal models. 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 a 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
beings to evaluate the effect of a cancer vaccine or immunotherapy in future clinical
trials. So we would like to provide this proposal to address the development of HPV 16
E7-specific immunologic assays in human beings. There are several aims in this project:

1. to develop and utilize assays to measure CTLs to HPV 16 E7 proteins, and

2. to develop and utilize assays to measure T helper (Th) responses to HPV 16 E7 antigens.