Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-09-03
2001-05-22
Hauda, Karen M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C424S204100, C424S192100, C424S186100, C536S023720, C514S04400A
Reexamination Certificate
active
06235523
ABSTRACT:
FIELD OF THE INVENTION
The invention is concerned with immunotherapy of cancer, specifically cervical cancer.
BACKGROUND OF THE INVENTION
Cervical cancer is the second most common cause of cancer-related deaths in women worldwide. There is both epidemiological and experimental data which links the etiology of cervical cancer to infection with human papilloma virus (HPV) types 16 and 18. The HPV virus is prevalent in 35 to 40% of young women. Although treatment of early stage disease is relatively successful, recurrent disease is found in 15% of the patients. The outcomes of patients with recurrent disease are relatively poor. Hence, there is a need for a novel therapeutic approach (refs. 1, 2, 3—various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure).
The strong association of HPV infection and cervical cancer suggests that a viral antigen-specific immunotherapeutic approach may be a feasible strategy in the treatment of cervical cancer. The goal of specific immunotherapy is to stimulate the immune response of a tumour-bearing patient to attack and eradicate tumour lesions. This strategy has been made feasible with the identification of tumour associated antigens (TAA). The strong association between HPV-16 infection and cervical cancer has made this disease a good candidate for immunotherapeutic intervention (ref. 4).
In HPV DNA-positive cervical cancers, the E6 and E7 oncogenic proteins are expressed. Experimental evidence suggests that these two proteins are responsible for the carcinogenic progression of cervical cancers as their expression leads to a transformed and immortalized state in human epithelial cell cultures (ref. 5). Therefore, these two proteins are potential candidates for antigen-specific immunotherapy in HPV-induced cervical cancers and are evaluated herein.
Although many questions remain regarding the nature of immunity to natural HPV infection, and, in turn, to cervical cancer, it is clear that there is an immune component as immunosuppressed individuals are at higher risk for developing a cervical malignancy (ref. 6). Furthermore, this immunity is most likely mediated by the cellular arm of the immune response. Extensive cellular infiltrates are observed upon examination of spontaneous regressions of cervical tumours (ref. 7). Thus, an antigen-specific cellular response appears to be required to treat cervical cancer patients.
Although the nature of the outcome of an immunotherapeutic strategy has been identified, the ability to induce this type of response using current vaccine technology is limited. Prophylactic vaccine development for HPV has focussed on recombinant subunit preparations consisting of L1 and L2 virion structural proteins. In eukaryotic cells, L1 (major capsid protein) organizes itself into papillomavirus-like particles (VLPS) (ref. 17). Although L1 alone is sufficient for assembly of VLPs, the coexpression of L2 (minor capsid protein) provides for greater capsid production (ref. 18). By contrast, therapeutic vaccine development has typically been directed to the expression of wild type E6 and/or E7 protein. Expression vectors employed include vaccinia virus (for example, as described in U.S. Pat. No. 5,744,133, (ref. 19, 20), alphavirus (for example U.S. Pat. No. 5,843,723), or other poxviruses (for example, U.S. Pat. No. 5,676,950). Therefore, a DNA vector encoding HPV antigens implicated in carcinogenic progression of disease was determined to be the optimal method by which a successful immunotherapeutic strategy could be achieved.
However, as previously noted the E6 and E7 HPV antigens are putatively oncogenic and thus immunization with a DNA construct encoding either or both of these proteins could result in the induction of further malignancy (refs. 5, 10, 11). Therefore, in order to minimize toxicity risks, a genetically detoxified E7 molecule was encoded herein in a DNA construct. This detoxified molecule is modified through the deletion of the retinoblastoma (Rb) binding region (refs. 8, 9). Another method of achieving antigen-specific immunity without the concomitant risks of oncogenic transformation is the use of an epitope strategy where only key parts of the molecule are administered to induce a specific immune response (refs. 12 to 16). This approach was used herein in the design of the DNA-polyepitope construct where a number of T-cell epitopes derived from both E6 and E7 are linked together. A comparison of these two approaches was made herein in a murine model of HPV-associated cervical cancer.
SUMMARY OF THE INVENTION
According to the invention, there are provided novel DNA constructs for the administration of HPV antigens to a host to provide an immune response in the host. The invention extends to methods of immunotherapy of HPV-caused tumor, particularly cervical cancer.
Antigens chosen for immunotherapy of HPV DNA-positive cervical cancers may be those expressed by such cancers. One such HPV antigen is the E7 antigen, which has previously been shown to have a protective ability in a vaccinia vector system.
Potential toxicity concerns exist with the use of a native form of E7 in a vaccine due to its ability to bind to the Rb protein and thus promote an oncogenic state. A novel detoxified version of E7 was constructed herein by deletion of the Rb binding site and incorporated into pCMV-3 (
FIG. 2
) to provide vector pCMV-dE7 (
FIG. 1
) for immunization.
The detoxified E7 coding sequence was prepared from the unmodified coding sequence by replacing approximately 210 bp with DNA formed from annealed oligos. The substitute sequence omitted a stretch of 18 nucleotides encoding the region of E7 involved in complex formation with cellular retinoglastoma (Rb) family proteins, namely amino acids 21 to 26 from the native E7 protein.
Immunization with the pCMV-dE7 construct resulted in significant protection from tumor outgrowth following engraftment of live C3 tumor cells expressing the wild-type E7 molecule, showing that the E7 DNA construct can be successfully used to stimulate protective immunity without any associated toxicity risks.
Another such HPV antigen is the E6 antigen. The antigens may be provided as full-length proteins or in the form of specific T-cell epitopes in the DNA constructs.
To evaluate the T-cell approach, a synthetic mini-gene was prepared containing nucleotide sequences encoding T-cell epitopes from both E6 and E7 proteins of HPV-16 (FIG.
5
A). A DNA construct (pCMV3-HPVT#1) containing the mini-gene (
FIG. 6
) was used to immunize mice. Mice immunized with the DNA construct of
FIG. 6
were 100% protected from tumor outgrowth.
The results from these studies indicate that DNA immunization can be used successfully to protect against live C3 tumor challenge and thus may be effective in the clinical treatment of cervical cancer.
Accordingly, in one aspect of the present invention, there is provided a vector comprising a nucleic acid molecule encoding at least one non-toxic T-cell epitope of the E6 and/or E7 antigen of a strain of human papilloma virus (HPV) associated with cervical cancer, such as HPV-16, and a promoter operatively coupled to the nucleic acid molecule for expression of the nucleic acid molecule in a host to which the vector is administered.
The promoter preferably is a cytomegalovirus promoter. The nucleic acid molecule may be contained within plasmid pCMV-3 which contains the immediate early cytomegalovirus promoter including enhancer and intron sequences, along with the bovine growth hormone polyA tail and a kanamycin resistance gene. The elements of pCMV-3 are shown in FIG.
2
.
The nucleic acid molecule, in one embodiment, is an E7 antigen coding sequence detoxified to prevent oncogene replication in the host. The detoxification may be effected in any convenient manner, including remov
Cao Shi-Xian
Gajewczyk Diane M.
Klein Michel H.
Moingeon Phillipe
Persson Roy
Connaught Laboratories Limited
Hauda Karen M.
Nguyen Quang
Sim & McBurney
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