Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
2000-02-18
2002-01-29
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S185100, C424S186100, C424S204100, C536S023400, C536S023720, C530S350000, C435S069300, C435S069700, C435S325000, C435S252300, C435S320100
Reexamination Certificate
active
06342224
ABSTRACT:
The present invention relates to fusions proteins, comprising a protein or part of a protein that provides T helper epitopes and an antigen from a human-papilloma virus that find utility in the treatment or prophylaxis of human papilloma induced tumours. In particular the invention relates to fusion proteins comprising an E6 or E7 protein from HPV strain 16 or 18 linked to protein D from Heamophilius influenza B.
Papillomaviruses are small naked DNA tumour viruses (7.9 kilobases, double strand), which are highly species-specific. Over 70 individual human papillomavirus (HPV) genotypes have been described. Papillomaviruses are classified on the basis of species of origin (human, bovine etc.) and of the degree of genetic relatedness with other papillomaviruses from the same species. HPVs are generally specific for the skin or mucosal surfaces and have been broadly classified into “low” and “high” risk on the basis of rare and common, respectively, detection in abnormal or tumour tissue. Low risk HPVs usually cause benign lesions (warts or papillomas) that persist for several months or years. High risk HPVs are associated with cancer. The strongest positive association between an HPV virus and human cancer is that which exist between HPV 16 and 18 and cervical carcinoma. More than ten other HPV types have also been found in cervical carcinomas including HPV 31 and HPV 33 although at less frequency.
Genital HPV infection in young sexually active women is common and most individuals either clear the infection, or if lesions develop, these regress. Only a subset of infected individuals has lesions which progress to high grade intraephithelial neoplasia and only a fraction of these progress further to invasive carcinoma.
The molecular events leading to HPV infection have not been clearly established. The lack of an adequate in vitro system to propagate human papillomaviruses has hampered the progress to a best information about the viral cycle.
Today, the different types of HPVs have been isolated and characterised with the help of cloning systems in bacteria and more recently by PCR amplification. The molecular organisation of the HPV genomes has been defined on a comparative basis with that of the well characterised bovine papillomavirus type 1 (BPV1).
Although minor variations do occur, all HPVs genomes described have at least seven early genes, E1 to E7 and two late genes L1 and L2. In addition, an upstream regulatory region harbors the regulatory sequences which appears to control most transcriptional events of the HPV genome.
E1 and E2 genes are involved in viral replication and transcriptional control, respectively and tend to be disrupted by viral integration. E6 and E7 are involved in viral transformation. E5 has also been implicated in this process.
In the HPVs involved in cervical carcinoma such as HPV 16 and 18, the oncogenic process starts after integration of viral DNA. The integration results in the inactivation of genes coding for the capsid proteins L1 and L2 and loss of E2 repressor function leads to deregulation of the E6/E7 open reading frame installing continuously overexpression of the two early proteins E6 and E7 that will lead to gradually loss of the normal cellular differentiation and the development of the carcinoma. E6 and E7 overcome normal cell cycle by inactivating major tumor suppressor proteins, p53 and pRB, the retinoblastoma gene product, respectively.
Carcinoma of the cervix is common in women and develops through a pre-cancerous intermediate stage to the invasive carcinoma which frequently leads to death. The intermediate stages of the disease is known as cervical intraepithelial neoplasia and is graded I to III in terms of increasing severity (CIN I-III).
Clinically, HPV infection of the female anogenital tract manifests as cervical flat condylomas, the hallmark of which is the koilocytosis affecting predominantly the superficial and intermediate cells of the cervical squamous epithelium.
Koilocytes which are the consequence of a cytopathic effect of the virus, appear as multinucleated cells with a perinuclear clear haloe. The epithelium is thickened with abnormal keratinisation responsible for the warty appearance of the lesion.
Such flat condylomas when positive for the HPV 16 or 18 serotypes, are high-risk factors for the evolution toward cervical intraepithelial neoplasia (CIN) and carcinoma in situ (CIS) which are themselves regarded as precursor lesions of invasive cervix carcinoma.
The natural history of oncogenic HPV infection presents 3 consecutive phases, namely:
(1) a latent infection phase,
(2) a phase of intranuclear viral replication with product of complete virions, which corresponds to the occurrence of koilocytes. At this stage, the HPV is producing its full range of proteins including E2, E5, E6, E7, L1 and L2.
(3) a phase of viral integration into the cellular genome, which triggers the onset of malignant transformation, and corresponds to CIN II and CIN III/CIS with progressive disappearance of koilocytes. At this stage, the expression of E2 is down-regulated, the expression of E6 and E7 is enhanced. Between CIN II/III and CIN III/Cervix carcinoma the viral DNA changes from being episomal in the basal cells to integration of E6 and E7 genes only (tumoral cells). 85% of all cervix carcinomas are squamos cell carcinomas most predominantly related to the HPV16 serotype. 10% and 5% are adenocarcinomas and adenosquamos cell carcinomas respectively, and both types are predominantly related to HPV 18 serotype. Nevertheless other oncogenic HPV's exist.
International Patent Application No. WO 96/19496 discloses variants of human papilloma virus E6 and E7 proteins, particularly fusion proteins of E6/E7 with a deletion in both the E6 and E7 proteins. These deletion fusion proteins are said to be immunogenic.
The present invention provides compositions comprising either an E6 or E7 or an E6/E7 fusion protein linked to an immunological fusion partner having T cell epitopes.
In a preferred form of the invention, the immunological fusion partner is derived from protein D of Heamophilus influenza B. Preferably the protein D derivative comprises approximately the first 1/3 of the protein, in particular approximately the first N-terminal 100-110 amino acids. The protein D may be lipidated (Lipo Protein D). Other immunological fusion partners include the non-structural protein from influenzae virus, NSI (hemagglutinin). Typically the N terminal 81 amino acids are utilised, although different fragments may be used provided they include T-helper epitopes.
In another embodiment the immunological fusion partner is the protein known as LYTA. Preferably the C terminal portion of the molecule is used. Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gen {Gene, 43 (1986) page 265-272} an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of
E.coli
C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798}. As used herein a preferred embodiment utilises the repeat portion of the Lyta molecule found in the C terminal end starting at residue 178. A particularly preferred form incorporates residues 188-305.
Accordingly, the present invention in preferred embodiment provides fusion proteins comprising Protein D-E6 from HPV 16, Protein D-E7 from HPV 16 Protein D-E7 from HPV 18, Protein D-E6 from HPV 18, and Protein D E6 E7 from both HPV 16 and 18. The protein D part preferably comprises the first 1/3 of protein D. It will be appreciated that other E6 and E7 proteins may be utilised from other HPV subtypes.
The proteins of the present invention preferably are expressed in
E. coli.
In a pref
Bruck Claudine
Cabezon Silva Teresa
Fernande Delisse Anne-Marie Eva
Gerard Catherine Marie Ghislaine
Lombardo-Bencheikh Angela
Foley Shanon A.
King William T.
Kinzig Charles M.
Majarian William R.
Mosher Mary E.
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