Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2001-05-30
2003-06-24
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S199100, C424S204100, C435S235100, C435S320100, C435S477000
Reexamination Certificate
active
06582693
ABSTRACT:
The present invention relates to recombinant vaccinia virus derived from the vaccinia virus Ankara (MVA) encoding and capable of expressing the E2 gene of Bovine papillomavirus, and the use of such recombinant in the treatment of lesions caused by papillomaviruses.
BACKGROUND OF THE INVENTION
Cervical Carcinoma
In man neoplasic transformation has been linked to the presence of papillomaviruses. They can induce diseases, from warts up to condilomas, and lesions, which can progress to malignant neoplasias. Approximately one million people get infected with papilloma virus every year, and in developing countries 50,000 women die annually due to cervical cancer.
In humans, it is well known that cervical cancer correlates with the presence of human papillomavirus (HPVS) particularly types 16, 18, 31, 33 and 35
3,4,5,6,7,8,9,10,11
. In contrast, HPV types 6, 11, 42, 43 are found only in the anogenital tract and are not associated with cervical cancer
3, 7, 12, 13
.
Cervical carcinoma is the second most common cancer among women worldwide. More than 95% of all cervical carcinomas contain DNA of some papillomavirus (HPV)
3
with types 16 or 18 accounting for about 50% and 30% of all cases, respectively.
Most benign lesions (papillomas) generally regress on their own, but in some cases, some of them undergo malignant transformation. Papillomavirus are also found infecting a wide variety of vertebrates, and it has been described that they produce tumors in these animals as well
4,5,6,7,14
.
Normally human papillomavirus infects and replicates in the form of an unintegrated circular episome in keratinocytes of genital mucosa and perigenital skin. The papillomavirus E2 and E1 gene products regulate viral DNA replication. The E2 gene product can also activate or repress transcription of different HPVs promoters
15,16
. In particular, the papillomavirus protein E2 is known to down regulate the P105 promoter, from HPV18, which controls transcription of the E6 and E7 oncogenes, which are expressed in a variety of cervical human tumors. The best studied HPVs, types 16 and 18 persist extracromosomally in precancerous lesions, but frequently get integrated into the cellular genome of cancer cells
16
. This event makes the papillomavirus E2 gene disrupted or inactivated leading to a derepression of the E6 and E7 oncogenes. Expression of these oncogenes appears to be a critical step in the maintenance of the transformed stage and progression to invasive carcinoma. This mechanism, however, is not necessarily the only one operating to induce the malignant stage, as suggested by recent studies where HPV integration was not detected in at least 30% of the analyzed cancers
13,17
.
Since cervical cancer is a serious health problem and because there is a strong dependence with the infection by HPV, it is thought that the induction of a protective stage against these viruses would help in preventing the appearance of cervical tumors. Based on this idea, different strategies to develop a safe vaccine or immunotherapeutic agent against cervical cancer have been tried. Other methods, such as radiotherapy and chemotherapy have, of course been used to reduce papillomas, and cancer tumors. However, these methods work only efficiently during the first stages of tumor development. At later times it becomes very difficult to treat cervical tumors because of the tumor size and the side effects that anti-cancer drugs may have.
Because of the strong relationship between HPV infection and cervical cancer, many approaches have been tried in an effort to develop a successful therapy for cervical cancer. It has been shown that antisense oligonucleotides from partial or complete full-end clones of E6 and E7 genes are able to inhibit tumor cell growth in vitro and in vivo
18,19,20
by down regulating the E6 and E7 oncogenes. Cloned antigens, recognized by therapeutic antitumor lymphocytes (TAA), such as the B7-1 or B7-2 molecules, have been used in the construction of recombinant anticancer vaccines
21
.
Using a recombinant vaccinia virus expressing the B7-1 molecule, it was found that the number of pulmonary metastases decreased in mice bearing tumor
21
. These molecules interact wit the T lymphocyte ligands CD28 and CTLA-4, and initiate a cascade of effects mediated, at least in part by up-regulation of interleukin 2 production
22,23,24
.
The findings that the E2 protein of papillomavirus is capable of promoting cell growth arrest and stopping cell proliferation by inducing apoptosis of human cancer cells
15,25,26
, and that immunized animals with recombinant E2 proteins are capable of inducing tumor regression and decreasing the number of new papilloma foci formed
27
, have lead to a novel approach for cervical cancer therapy. Namely, the introduction of the E2 gene into vaccinia virus was to efficiently direct the E2 protein against HPV tumors.
Inserting an antigen in a vaccinia virus (poxyirus) increases the expression of this molecule in the infected cell, therefore stimulating more efficiently the immune system
28,29
. The purpose of using different antigens expressed in vaccinia virus is to try to enhance the immune response against these specific antigens. For these reasons it is thought that recombinant poxvirus are excellent candidates for a new type of vaccine and also for new therapeutic strategies.
Knowing these difficulties to treat cervical cancer it was decided to use an attenuated vaccinia virus known as Modified Vaccinia Ankara (MVA). This virus was developed and tested as a safe smallpox vaccine
30
. It was also found to be a virulent for normal or immunosuppressed animals, without side-effects in 120,000 humans inoculated for priming vaccination
30,31,32
. One reason that makes the MVA very safe is that viral expression and recombinant mechanisms are impaired for this virus. It has also been demonstrated that MVA is an excellent vector for expressing foreign genes, such as the
Escherichia coli
Lac Z or the page T7 polymerase
33,34
in infected cells. Moreover, MVA is capable of infecting most, if not all, the human cell lines tested up to now. Because of these characteristics, at the present time, the most successful strategy for vaccine development involves the use of vaccinia virus vectors.
This approach has already been used to protect animals against polyoma virus (PY) infection
35
. In these reports, it was demonstrated that treatment of rats with live recombinant vaccinia viruses expressing tumor-specific antigens from PY could prevent cognate tumor development and in some cases could also induce regression of preexisting tumors. Also an MVA recombinant virus expressing the haemaglutinin and nucleoprotein genes (HA and NP, respectively) of influenza virus (A/PR/8/34HA) was found to fully protect mice against a lethal influenza virus challenge
36
.
According to the invention, a DNA sequence which codes for the E2 gene of Bovine papillomavirus is introduced using DNA recombination techniques within a naturally occurring DNA sequence of the MVA genome which is non-essential for virus replication. This recombinant virus will be helpful for the therapy of lesions induced by papillomaviruses.
OBJECT OF THE INVENTION
The present invention is to provide a recombinant MVA virus that expresses a papillomavirus antigen and is used as a therapeutic method to treat lesions generated by papillomaviruses.
SUMMARY OF THE INVENTION
The scope of this invention covers the necessary methods and the technology to express a specific gene in a viral expression vector (vaccinia virus strain MVA) specifically in a recombinant viral vector that carries the E2 gene of the bovine papilloma virus. This recombinant vector is capable of efficiently expressing the E2 gene inside any mammalian cell as well as in chicken fibroblast cells.
The recombinant MVA E2 virus as designed basically to be used in the therapeutic treatment of early/late lesions that are produced by infections with papilloma virus capable of generating tumors. This virus was mainly designed to inoculate directly into lesions produced by human papilloma vir
Lemery, S.A. de C.V.
Mosher Mary E.
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