Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2001-09-06
2003-07-29
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S205100, C424S184100, C424S186100, C435S069100, C435S069300, C536S023720
Reexamination Certificate
active
06599508
ABSTRACT:
FIELD OF THE INVENTION
1. The invention relates to recombinantly produced papilloma virus-like particles, proteins, fusion proteins as well as processes for the formation and purification of these particles, proteins and fusion proteins.
BACKGROUND OF THE INVENTION
2. Infections with certain (high-risk) types of genital papilloma viruses in humans (HPV), e.g. HPV 16, 18 or 45 are held to be the main risk factor for the formation of malignant tumours of the anogenital tract. Of these, cervical carcinoma is by far the most frequently occurring. According to an estimate aby the WHO, about half a million new cases of this disease occur annually. Because of this frequent occurrence, the connection between HPV infection and cervical carcinoma has been the best investigated:
a) Precursor lesions of cervical carcinoma (cervical intraaepithelial neoplasia: CIN) are caused by papilloma virus infections.
b) The genomes of certain HPV types (e.g. 16, 18, 33, 35, 45) have been proven to occur in more than 95% of tumour biopsies as well as in cell lines derived from them.
Depending on the geographic origin of the tumours, 5-70% of these contain HPV 16.
c) In all subsequently examined cases the ORFs E6 and E7 are transcribed (Wettstein et al., in Pfister H. (ed): Papilloma viruses and human cancer, pp. 155 to 179, Boca Raton, 1990).
d) The proteins E6 and E7 can be proven in all cervicalcarcinoma cell-lines as well as in in vitro transformed human keratinocytes, and the majority of patients with cervical carcinoma have E6- or E7- specific antibodies.
e) The constitutive expression of the E6/E7 proteins is necessary to maintain the transformed condition of HPV-positive tumours.
f) The E6- and E7 genes of HPV 16 and HPV 18 are biologically active in the following experimental systems:
Induction of cellular DNA synthesis in human cells;
Transformation of human keratinocytes and other cells in culture;
Tumour formation in transgenic mice.
3. Other HPV types (principally HPV 6 and 11) cause benign genital warts (condylomata acuminata) and are only extremely rarely associated with malignant tumours (low-risk types).
4. As a rule, genital papilloma viruses in humans are transmitted during intercourse and in most cases lead to persistent infection in the anogenital mucous membrane. This led to the conclusion that primary infections induce only an insufficient immune response, or that the virus has developed possibilities of avoiding the immune surveillance in the infected cells. On the other hand there are good indications suggesting that the immune system is involved during primary manifestation or during the malignant progression of papilloma virus infections. (For an overview see Altmann et al. (1994) in Minson A., Neil J., McCrae M. (eds): Viruses and cancer, Cambridge University Press, pp. 71 to 80).
a) In the case of animal papilloma viruses (rabbit papilloma virus and bovine papilloma virus), the clinical manifestation of primary infections can be avoided by vaccination with viral structural proteins or with wart extracts (autologous vaccines).
b) Rodents are protected by vaccination with HPV 16 E6- or E7-positive vaccinia recombinants or by synthetic peptides prior to tumour formation after inoculation of HPV 16- transformed autologous cells.
c) Regression of warts is often systemic and in the case of animal papilloma viruses can be induced by the transfer of lymphocytes of regressor animals.
d) In the case of immuno-suppressed patients (e.g. kidney transplantees or HIV-infectd persons), the incidence of genital warts, CIN and anogenital cancer is increased.
5. This led to the conclusion that papilloma virus-specific vaccinations aimed at preventing the primary infection and the occurrence of genital cancer should be possible.
6. 1. Avoidance of HPV infections is suitable by vaccination with the structural proteins L1 and L2 of the papilloma virus (prophylactic vaccination).
7. Because papilloma viruses cannot be propagated to adequate titres in cell cultures or other experimental systems, the viral proteins can only be produced by means of recombinant vectors. Recently, virus-like particles (VLPs) which, after expression of the viral structure proteins L1 and L2 (or L1 on its own), are formed in recombinant vaccinia or baculo virus, have been described. Purification of the VLPs can be achieved very simply by means of centrifugaton in CsCl- or sucrose gradients.
8. WO 93/02184 describes a method which provides papilloma virus-like particles (VLPs), which are used for diagnostic applications or as a vaccine against infections caused by the papilloma virus.
9. WO 94/00152 describes a recombinantly produced L1 main capsid protein which mimics the conformational neutralising epitope on human and animal papilloma virions. These recombinant proteins can be used as vaccines which protect against papilloma virus-infections.
10. 2. Treatment of cervical carcinoma or precursor lesions by immunotherapy assisted by early papilloma virus-proteins (principally E6 or E7) which are expressed in the persistently infected cells (therapeutic vaccination).
11. It is assumed that by this vaccination, cytotoxic T-cells are activated against persistently infected genital lesions. The target population are patients with HPV associated pre-malignant or malignant genital lesions.
12. Early HPV proteins are produced by expression in
E. coli
or eukaryotic vectors (e.g. baculo virus or yeast).
Purification is however rendered difficult by the low solubility and as a rule requires a combination of ion exchange chromatography, gel filtration and affinity chromatography.
13. PCT patent application WO 93/20844 discloses that the E7 protein of the papilloma virus from HPV or BPV is therapeutically effective in the regression (not however in the prevention) of papilloma virus-tumours in mammals. In addition, preferred antigenic proteins fragment sequences are described.
14. So far, however, no VLPs were described which are suitable both for prophylactic and therapeutic vaccination. The last-mentioned processes have the disadvantage that, for example, early HPV proteins, because of their low solubility, can only be cleaned with difficulty.
15. A high particle production would be particularly desirable, in particular in view of a vaccine for prophylactic and therapeutic vaccination.
16. A disadvantage for the process described so far was that it was not possible to produce VLPs after expression of L1 in
E. coli.
DESCRIPTION OF THE INVENTION
17. It is therefore the object of the present invention, to make available recombinantly produced proteins as well as VLPs which are suitable as a vaccine for prophylactic and therapeutic vaccination, as well as processes for the production of these proteins and VLPs. Equally, simple purification of the recombinant proteins obtained should be possible. Also production of VLPs after expression of L1 in
E. coli
should be possible.
REFERENCES:
patent: WO 93/02184 (1993-02-01), None
patent: WO 93/20844 (1993-10-01), None
patent: WO 94/00152 (1994-01-01), None
Jochmus et al, Archives of Medical Research, 1999, vol. 30, pp. 269-274.*
Michel et al, Virology, 2002, vol. 294, pp. 47-59.*
Altmann, et al., “Towards HPV Vaccination,” in Minson A., Neil J., McCrae M. (eds): Viruses and Cancer, Cambridge University Press, pp. 71-80 (1994).
Wettstein, et al., “State of Viral DNA and Gene Expression in Benign VS. Malignant Tumors,” in Pfister H. (ed): Papilloma viruses and Human Cancer, Boca Raton, 8:155-179 (1990).
Gissmann Lutz
Muller Martin
Painstil Jeanette
Zhou Jian
Loyola University of Chicago
Marshall Gerstein & Borun
Salimi Ali R.
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