WO1993020844A1 - Papillomavirus e7 protein - Google Patents

Papillomavirus e7 protein Download PDF

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Publication number
WO1993020844A1
WO1993020844A1 PCT/GB1993/000679 GB9300679W WO9320844A1 WO 1993020844 A1 WO1993020844 A1 WO 1993020844A1 GB 9300679 W GB9300679 W GB 9300679W WO 9320844 A1 WO9320844 A1 WO 9320844A1
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Prior art keywords
protein
papillomavirus
formulation according
fragment
bpv
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PCT/GB1993/000679
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French (fr)
Inventor
Maria Saveria Campo
William Fleming Hoggan Jarrett
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Cancer Research Campaign Technology Ltd.
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Publication of WO1993020844A1 publication Critical patent/WO1993020844A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the use of papillomavirus E7 protein in medicine, particularly for the regression of papillomavirus disease tumours particularly in mammals including humans; and to pharmaceutical formulations comprising the E7 protein.
  • Papillomaviruses induce a variety of lesions both in humans and in animals. Some papillomas, albeit benign, are themselves a clinical problem, such as laryngeal papillomas of children (Steinberg and Abramson, 1985) or penile papillomas of bulls (Jarrett, 1985a) , and others are known to be a risk factor in the pathogenesis of cancer, as in the case of flat lesions of the cervix or penile condylomata in humans (zur Hausen, 1978) and papillomas of the alimentary canal in cattle (Jarrett et al, 1978) .
  • human papillomavirus types HPV-16 and HPV-18 and bovine papillomavirus type 4 are potentially carcinogenic. Therefore both in human and veterinary medicine an antiviral vaccine, particularly a therapeutic one inducing lesion rejection, would be of major importance.
  • Vaccination studies in humans present several problems: first of all experimentation is ethically unacceptable and, secondly, very limited amounts of virus are available as some lesions, in particular those of the cervix, do not produce viral progeny, and no in vitro system is yet available which allows vegetative replication of virus.
  • the bovine system is an excellent model for the human one, given the several similarities between the two: multiple virus types with high lesion specificity (Ca po et al, 1981; Jarrett et al, 1984) , homology of genetic structure (Danos et al, 1984) and progression of some lesions to malignancy (Jarrett et al, 1978) .
  • the bovine system also presents several advantages: cofactors in oncogenesis are known (Jarrett et al, 1978; Campo and Jarrett, 1986) and, above all, direct experimentation is possible (Jarrett, 1985a) .
  • the present invention resides in the discovery that the papillomavirus E7 protein is therapeutically effective in the treatment of papillomavirus disease and particularly for the regression of tumours.
  • the present invention provides the use of papillomavirus E7 protein in medicine, particularly for the therapy of papillomavirus disease.
  • the invention also provides a pharmaceutical formulation for the therapy of papillomavirus disease, which comprises; papillomavirus E7 protein in admixture with a pharmaceutically acceptable carrier.
  • the invention further provides papillomavirus E7 protein for use in the production of a medicament for use in medicine, particularly for use in the therapy of papillomavirus disease.
  • the invention still further provides a method of treating a mammal for the therapy of papillomavirus disease, which comprises the administration of papillomavirus E7 protein to the mammal.
  • the E7 protein has been found to be antigenic and to lead to the production of antibodies. However, no protective effect against viral infection and tumour production is observed. Instead, the protein is found to have a therapeutic effect in reducing the size of tumours and speeding their regression.
  • the therapeutic effect of the E7 protein is limited to the respective papillomavirus type.
  • the therapy will be applicable to papillomavirus infections of mammals, including humans and animals.
  • the present work has been done on E7 protein of bovine papillomavirus BPV-4 (which generally affects the alimentary canal; Campo et al, 1980) .
  • BPV-4 bovine papillomavirus
  • HPV-16 human papillomavirus HPV-16
  • the therapy is useful for the regression of tumours, for example the removal of warts from the udders (BPV-2) or mouth (BPV-4) of milk cows, and for the treatment of horses and donkeys.
  • the E7 protein is generally produced by recombinant DNA techniques.
  • a plasmid containing the gene coding for the E7 protein may be transfected into a bacterium such as E. coli and cultured.
  • the entire E7 protein as it exists in nature may be employed, or an antigenic fragment or fragments thereof may be used providing that the antigenicity and therapeutic effectiveness is retained.
  • the antigenic fragment may be an immunogenic epitope.
  • Immunodominant B and T cell epitopes are shown in Figure 4.
  • Bl, B2 and B3 map to amino acids 1-30, 79-98 and 51-69 respectively.
  • Tl and T2 map to amino acids 20-50 and 70-88 respectively.
  • the whole of the E7 protein or epitope may be employed, or with additions deletions or substitutions which do not substantially effect its therapeutic effectiveness.
  • the E7 protein is preferably administered as a fusion protein, for example fused to beta-galactosidase or glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • the co-protein may also be antigenic and assist in providing general stimulation of the immune system.
  • the E7 protein will usually be administered in the form of a pharmaceutical formulation.
  • the formulation contains a pharmaceutically acceptable carrier.
  • the carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the pharmaceutical formulation is preferably formulated for parenteral administration, including subcutaneous, intramuscular and intravenous injection; or as a suppository or pessary.
  • parenteral administration the formulation may be presented as a sterile solution or suspension in a suitable liquid vehicle, which may also contain preservatives and materials for rendering the formulation isotonic.
  • the formulations may be presented in unit-dose or multi-dose containers.
  • the carrier will generally be apyrogenic. Each dose will generally contain 100 to 10,000 micrograms of the E7 protein.
  • the protein may be administered together with an adjuvant, such as Freund's incomplete adjuvant, as an oil-in-water emulsion or using other adjuvant systems known in the art such as LI01 and DDA as used in Pilacinksi et al (1986) .
  • adjuvant such as Freund's incomplete adjuvant
  • Aluminium gel made up for example from equal volumes of 3% aluminium hydroxide and 2% aluminium phosphate
  • Figure 1 shows the geno ic organisation of BPV-4 and for comparison HPV-16.
  • Figure 3 shows the results of vaccination with BPV-4 E7 protein in terms of the development of papillomas of size greater than 2 mm in control animals and in vaccinated animals. The mean tumour number of papillomas per animal is plotted against weeks after challenge; and
  • Figure 4 shows the B and T cell epitopes of BPV-4 and E7 protein as mapped.
  • the open reading frame (ORF) encoding the E7 peptide was isolated by digesting the BPV-4 genome cloned in pAT 153 (Campo and Coggins, 1982) with Bam HI.
  • the open reading frame encoding the E7 peptide (nt 647-1009 of the BPV-4 genome; Jackson et al, 1991) was isolated as a fragment encompassing nt 652-1250. The fragment contains the E7 ORF ATG and termination codons.
  • the fragment was cloned in the pUR and pGEX vector series (Ruther and Muller-Hill, 1983) ; Smith and Johnson, 1988) giving rise to plasmids pURE7 and pGEXE7, which produce beta-galactosidase (b-gal)-E7 fusion and glutathione S-transferase (GST)-E7 fusion proteins respectively.
  • the plasmids produced were transfected into E. Coli JM 109.
  • Peptide for vaccination was prepared from mid-log phase cultures induced for 1 hours in L-broth supplemented with 100 ug/ml ampicillin and containing ImM IPTG.
  • Bacterial pellets resuspended in lysozyme buffer (50mM TRIS-HC1 pH 8.0, lOmM McCl 2 , 50mM glucose, 1 mg/ml lysozyme) were left at 20°C for 10 min, when EDTA was added to 50mM.
  • the calves receiving the E7 vaccine were given a 1ml PBS suspension containing 1 mg of either of the two E7 fusion proteins plus 1 ml of Freund's incomplete adjuvant into the right quadriceps muscle. This was repeated four weeks later as a boost.
  • BPV-4 was purified from alimentary papillomas (Campo et al, 1980) and the concentration of viral particles was estimated by the electron microscope assay (Jarrett et al, 1990a) . Each calf was challenged at ten sites in the palate in two parallel rows of five sites each with 10 11 virus particles four weeks after vaccination.
  • the b-gal -E7 and the GST-E7 fusion proteins were characterized i munologically. They were injected into rabbits or mice and the antisera were tested against the fusion proteins in Western blot assays. The antisera were reactive with both types of fusion proteins. Therefore the fusion proteins present their immunodominant epitopes effectively to the host immune system. Vaccination with E7 inhibits papilloma growth and promotes tumour rejection.
  • Vaccination of cattle with E7 is followed by vigorous humoral and cellular immune response to the vaccine. Both responses appear much earlier and have a greater amplitude in the vaccinated animals than in the control calves. Indeed, some of the control animals never developed detectable antibodies to E7 throughout the course of the experiment.
  • the T cell response is particularly strong especially when compared to control animals and may explain the efficacy of the vaccine, although the possible contribution of anti-E7 antibodies cannot be discounted.
  • Vaccine E7 is therefore successfully presented to both effector arms of the immune system while viral E7 is poorly presented.
  • the immunodominant B- and T-cell epitopes encoded by E7 have been mapped by the use of synthetic overlapping peptides in ELISA and cell proliferation assays respectively.
  • Bl maps to amino acids 1-30 at the N-terminus, B2 to amino acids 79-98 at the C-terminus and B3 to amino acids 51-69 in the middle portion of the protein. Bl and B2 are more often recognised than B3 (10/14, 11/14 and 6/14 animals respectively) .
  • Tl maps to amino acids 20-50 and T2 to amino acids 70-88; Tl appears to be recognised more often than T2 (2/3 and 1/3 animals respectively) .
  • BPV-4 E7 The regions of BPV-4 E7 where the epitopes have been mapped have amino acid and functional homology to HPV-16 E7 (Rb-binding domain I and II and Zn-binding domain) and the same areas in HPV-16 E7 have been shown to contain B- and T-cell epitopes (see Comerford et al., Krchnak et al., Tindle et al. (1990) and (1991)).
  • the immunological homology between conserved areas of BPV-4 and HPV-16 E7 proteins gives weight to the suggestion that the former is a predictive indicator of the latter.
  • Ciba Campo MS and Jarrett WFH (1986) Papillomavirus infection in cattle: viral and chemical cofactors in naturally occurring and experimentally induced tumours.
  • Papillomavirus Genomes Sequences and Consequences.
  • Jarrett WFH (1985) The natural history of bovine papillomavirus infection. Advances in viral oncology. Ed: G.Klein, vol 5, pp83-102.Jarrett WFH, McNeil PE, Gri shaw WTR, Sel an IE and Mclntyre WIM (1978) High incidence area of cattle cancer with a possible interaction between an environmental carcinogen and a papillomavirus. Nature, 274, 215-217.
  • Tindle RW Tindle RW
  • Fernando GJ Tindle RW
  • Sterling JC Frazer IH.
  • a "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes. Proc. Natl. Acad. Sci. 88,5887-5891.

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Abstract

Papillomavirus E7 protein from HPV or BPV is effective therapeutically in the regression (but not prevention) of papillomavirus tumours in mammals. Preferably the protein or fragment thereof is in the form of a coprotein with beta-galactosidase or GST. Preferred antigenic protein fragment sequences are also described.

Description

PAPILLOMAVIRUS E7 PROTEIN
TECHNICAL FIELD
The present invention relates to the use of papillomavirus E7 protein in medicine, particularly for the regression of papillomavirus disease tumours particularly in mammals including humans; and to pharmaceutical formulations comprising the E7 protein.
BACKGROUND OF THE INVENTION
Papillomaviruses induce a variety of lesions both in humans and in animals. Some papillomas, albeit benign, are themselves a clinical problem, such as laryngeal papillomas of children (Steinberg and Abramson, 1985) or penile papillomas of bulls (Jarrett, 1985a) , and others are known to be a risk factor in the pathogenesis of cancer, as in the case of flat lesions of the cervix or penile condylomata in humans (zur Hausen, 1978) and papillomas of the alimentary canal in cattle (Jarrett et al, 1978) . In particular, human papillomavirus types HPV-16 and HPV-18 and bovine papillomavirus type 4 are potentially carcinogenic. Therefore both in human and veterinary medicine an antiviral vaccine, particularly a therapeutic one inducing lesion rejection, would be of major importance. Vaccination studies in humans present several problems: first of all experimentation is ethically unacceptable and, secondly, very limited amounts of virus are available as some lesions, in particular those of the cervix, do not produce viral progeny, and no in vitro system is yet available which allows vegetative replication of virus. The production of viral proteins in bacteria and the use of synthetic peptides have circumvented this last problem and have allowed the ongoing analysis of the immune response to papillomavirus infection (see for instance Jenison et al, 1988: Jochmus-Kudielka et al, 1989; Tindle et al, 1990, Dillner, 1990 and Strang et al, 1990) . Whilst investigation into the feasibility of a human papillomavirus vaccine is still at an early stage, effective prophylactic vaccines, both natural (Jarrett et al, 1990a) and genetically engineered (Pilachinski et al, 1986; Jarrett et al, 1991) have already been produced against bovine papillomaviruses, and regression of Shope papillomas has been achieved by vaccinating rabbits with tumour tissue extracts (Evans et al, 1962) . The bovine system is an excellent model for the human one, given the several similarities between the two: multiple virus types with high lesion specificity (Ca po et al, 1981; Jarrett et al, 1984) , homology of genetic structure (Danos et al, 1984) and progression of some lesions to malignancy (Jarrett et al, 1978) . The bovine system also presents several advantages: cofactors in oncogenesis are known (Jarrett et al, 1978; Campo and Jarrett, 1986) and, above all, direct experimentation is possible (Jarrett, 1985a) .
It has recently been shown that vaccination with bovine papillomavirus type 2 (BPV-2) successfully prevented infection by the same virus (Jarrett et al, 1990a), but not by other virus types (Jarrett et al,1990b) Prevention was accompanied by production of neutralising antibodies in the serum of vaccinated animals, indicating that neutralising epitopes are present in the virus.
Our earlier British patent application GB9113809.9 discloses therapeutic activity of L2 protein of bovine papilloma virus BPV-2 (Jarrett et al, 1991; Campo, 1991) .
International patent publication W092/05248 discloses the use of the E7 protein of human papilloma virus HPV.
SUMMARY OF THE INVENTION
Generally speaking, the present invention resides in the discovery that the papillomavirus E7 protein is therapeutically effective in the treatment of papillomavirus disease and particularly for the regression of tumours.
Thus, the present invention provides the use of papillomavirus E7 protein in medicine, particularly for the therapy of papillomavirus disease.
The invention also provides a pharmaceutical formulation for the therapy of papillomavirus disease, which comprises; papillomavirus E7 protein in admixture with a pharmaceutically acceptable carrier.
The invention further provides papillomavirus E7 protein for use in the production of a medicament for use in medicine, particularly for use in the therapy of papillomavirus disease.
The invention still further provides a method of treating a mammal for the therapy of papillomavirus disease, which comprises the administration of papillomavirus E7 protein to the mammal.
The E7 protein has been found to be antigenic and to lead to the production of antibodies. However, no protective effect against viral infection and tumour production is observed. Instead, the protein is found to have a therapeutic effect in reducing the size of tumours and speeding their regression.
Generally speaking, the therapeutic effect of the E7 protein is limited to the respective papillomavirus type. Thus, for general therapeutic applications, especially where the particular papillomavirus type is unknown, it may be desirable to employ a mixture of E7 proteins from a variety of papillomavirus types.
Generally, the therapy will be applicable to papillomavirus infections of mammals, including humans and animals. The present work has been done on E7 protein of bovine papillomavirus BPV-4 (which generally affects the alimentary canal; Campo et al, 1980) . However, because of substantial sequence homology between BPV-4 and human papillomavirus HPV-16 (see Figures 1 and 2; Jaggar et al, 1990) it can be predicted that E7 protein will also demonstrate therapeutic effectiveness in tumour regression against HPV-16 and possibly HPV-18 human carcinogenic viral types. In this way pre-cancerous lesions may be treated. In animals, the therapy is useful for the regression of tumours, for example the removal of warts from the udders (BPV-2) or mouth (BPV-4) of milk cows, and for the treatment of horses and donkeys.
The E7 protein is generally produced by recombinant DNA techniques. In particular, a plasmid containing the gene coding for the E7 protein may be transfected into a bacterium such as E. coli and cultured. The entire E7 protein as it exists in nature may be employed, or an antigenic fragment or fragments thereof may be used providing that the antigenicity and therapeutic effectiveness is retained.
In particular the antigenic fragment may be an immunogenic epitope. Immunodominant B and T cell epitopes are shown in Figure 4. Bl, B2 and B3 map to amino acids 1-30, 79-98 and 51-69 respectively. Tl and T2 map to amino acids 20-50 and 70-88 respectively. The whole of the E7 protein or epitope may be employed, or with additions deletions or substitutions which do not substantially effect its therapeutic effectiveness.
The E7 protein is preferably administered as a fusion protein, for example fused to beta-galactosidase or glutathione S-transferase (GST) . This stabilises the E7 protein and may assist purification thereof. The co-protein may also be antigenic and assist in providing general stimulation of the immune system.
The E7 protein will usually be administered in the form of a pharmaceutical formulation. The formulation contains a pharmaceutically acceptable carrier. The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Since the protein is broken down in the stomach, oral administration is not preferred. The pharmaceutical formulation is preferably formulated for parenteral administration, including subcutaneous, intramuscular and intravenous injection; or as a suppository or pessary. For parenteral administration the formulation may be presented as a sterile solution or suspension in a suitable liquid vehicle, which may also contain preservatives and materials for rendering the formulation isotonic. The formulations may be presented in unit-dose or multi-dose containers. The carrier will generally be apyrogenic. Each dose will generally contain 100 to 10,000 micrograms of the E7 protein.
In order to enhance the therapeutic effect of the protein, it may be administered together with an adjuvant, such as Freund's incomplete adjuvant, as an oil-in-water emulsion or using other adjuvant systems known in the art such as LI01 and DDA as used in Pilacinksi et al (1986) . Aluminium gel (made up for example from equal volumes of 3% aluminium hydroxide and 2% aluminium phosphate) may also be an advantageous adjuvant system. DESCRIPTION OF PREFERRED EMBODIMENTS.
Embodiments of the present invention will now be described by way of example only with reference to the following experimental protocol.
The attached drawings show the following. Figure 1 shows the geno ic organisation of BPV-4 and for comparison HPV-16.
The percentages amino acid homology are: El = 55.3 E2 = 43.6 E4 - 32.6 E7 = 53.6 LI = 66.0 L2 = 47.8 The BPV-4 genome is 7265 base pairs long; the HPV-16 genome is 7904 base pairs long. The open reading frames are represented by boxes. El-8=early genes; Ll-2=late genes.
Figure 2 shows the homology between the E7 proteins of BPV-4 and HPV-16. Identical amino acids are linked by a continuous line and similar amino acids by a broken line. Region 1 and 2=retinoblastoma protein binding domain; Zn++= zinc fingers domains; hydrophobic = stretch of hydrophobic amino acids;
Figure 3 shows the results of vaccination with BPV-4 E7 protein in terms of the development of papillomas of size greater than 2 mm in control animals and in vaccinated animals. The mean tumour number of papillomas per animal is plotted against weeks after challenge; and
Figure 4 shows the B and T cell epitopes of BPV-4 and E7 protein as mapped.
EXAMPLE 1 (Production of BPV-4 E7 and vaccination)
MATERIALS AND METHODS Calves
Nineteen 20-week old calves of several different breeds were obtained from a papilloma-free source. They were randomly assigned to two groups, group A of eight animals and group B of eleven animals. They were housed in separate, clean, well ventilated pens in the isolation unit of the Department of Veterinary Pathology, Glasgow. All the calves were bled on arrival for haematological analysis and to obtain pre-inoculation serum samples. The experiment was started when the calves were approximately 26 weeks old.
Production of E7 peptide in Escherichia coli The open reading frame (ORF) encoding the E7 peptide was isolated by digesting the BPV-4 genome cloned in pAT 153 (Campo and Coggins, 1982) with Bam HI. The open reading frame encoding the E7 peptide (nt 647-1009 of the BPV-4 genome; Jackson et al, 1991) was isolated as a fragment encompassing nt 652-1250. The fragment contains the E7 ORF ATG and termination codons. The fragment was cloned in the pUR and pGEX vector series (Ruther and Muller-Hill, 1983) ; Smith and Johnson, 1988) giving rise to plasmids pURE7 and pGEXE7, which produce beta-galactosidase (b-gal)-E7 fusion and glutathione S-transferase (GST)-E7 fusion proteins respectively. The plasmids produced were transfected into E. Coli JM 109. Peptide for vaccination was prepared from mid-log phase cultures induced for 1 hours in L-broth supplemented with 100 ug/ml ampicillin and containing ImM IPTG. Bacterial pellets resuspended in lysozyme buffer (50mM TRIS-HC1 pH 8.0, lOmM McCl2, 50mM glucose, 1 mg/ml lysozyme) were left at 20°C for 10 min, when EDTA was added to 50mM. Following cell lysis by the addition of Triton XlOO to 1% (v/v) , the respective fusion peptide was pelleted at 39000 g for 30 min and resuspended by boiling and sonication in 5% SDS, 50mM B-mercaptoethanol, 50mM TRIS-HC1, pH 8.0 Purity of 90-95% was achieved by preparative SDS PAGE, the final yields being up to 2 mg of product per gm wet weight of cells. The protein was stored at -20°C before use, but prolonged storage caused degradation. In this way, (beta-gal) -E7 and (GST) -E7 fusion proteins were produced.
Experimental design
The eleven animals in group A were vaccinated with E7, while the eight animals in group B were kept as controls. Vaccination (group A)
The calves receiving the E7 vaccine were given a 1ml PBS suspension containing 1 mg of either of the two E7 fusion proteins plus 1 ml of Freund's incomplete adjuvant into the right quadriceps muscle. This was repeated four weeks later as a boost.
Virus challenge
BPV-4 was purified from alimentary papillomas (Campo et al, 1980) and the concentration of viral particles was estimated by the electron microscope assay (Jarrett et al, 1990a) . Each calf was challenged at ten sites in the palate in two parallel rows of five sites each with 1011 virus particles four weeks after vaccination.
RESULTS
Characterization of fusion protein
The b-gal -E7 and the GST-E7 fusion proteins were characterized i munologically. They were injected into rabbits or mice and the antisera were tested against the fusion proteins in Western blot assays. The antisera were reactive with both types of fusion proteins. Therefore the fusion proteins present their immunodominant epitopes effectively to the host immune system. Vaccination with E7 inhibits papilloma growth and promotes tumour rejection.
All the eleven vaccinated animals were vaccinated before challenge with virus. The vaccinated animals had high titre anti-E7 antibodies in their sera before virus injection, whereas the control animals developed anti-E7 antibodies only thirteen weeks post challenge and the magnitude of their response was much smaller than in the vaccinated animals. Likewise, E7-activated T-lymphocytes were detected in the vaccinated animals much earlier than in the controls, and in addition the response of control T-lymphocytes to E7 stimulation was very sluggish. These results confirm the immunological activity of E7 fusion proteins also in cattle.
Following challenge, all animals developed tumours at the ten sites of injection, showing that the virus was equally efficatious in both sets of animals and that all animals were equally susceptible to virus infection. In other words, no protection effect was seen with the E7 vaccine. However, fifteen weeks after challenge, most papillomas developed to full size and actively produced virus in the control animals. In contrast, in the vaccinated animals on average only one papilloma per animal achieved full size and the papillomas regressed five weeks earlier than the controls. E7 vaccination achieved two goals: inhibition of papilloma development and early regression of the lesions. Figure 3 shows the vaccination results. The numbers of papillomas of size greater than 2mm is higher for the control group than for the vaccinated group.
EXAMPLE 2 (Epitope Mapping; of BPV-4 E7)
Vaccination of cattle with E7 is followed by vigorous humoral and cellular immune response to the vaccine. Both responses appear much earlier and have a greater amplitude in the vaccinated animals than in the control calves. Indeed, some of the control animals never developed detectable antibodies to E7 throughout the course of the experiment. The T cell response is particularly strong especially when compared to control animals and may explain the efficacy of the vaccine, although the possible contribution of anti-E7 antibodies cannot be discounted. Vaccine E7 is therefore successfully presented to both effector arms of the immune system while viral E7 is poorly presented. The immunodominant B- and T-cell epitopes encoded by E7 have been mapped by the use of synthetic overlapping peptides in ELISA and cell proliferation assays respectively. As shown in Figure 4 Bl maps to amino acids 1-30 at the N-terminus, B2 to amino acids 79-98 at the C-terminus and B3 to amino acids 51-69 in the middle portion of the protein. Bl and B2 are more often recognised than B3 (10/14, 11/14 and 6/14 animals respectively) . Tl maps to amino acids 20-50 and T2 to amino acids 70-88; Tl appears to be recognised more often than T2 (2/3 and 1/3 animals respectively) . An interesting observation derives from these results. The regions of BPV-4 E7 where the epitopes have been mapped have amino acid and functional homology to HPV-16 E7 (Rb-binding domain I and II and Zn-binding domain) and the same areas in HPV-16 E7 have been shown to contain B- and T-cell epitopes (see Comerford et al., Krchnak et al., Tindle et al. (1990) and (1991)). The immunological homology between conserved areas of BPV-4 and HPV-16 E7 proteins gives weight to the suggestion that the former is a predictive indicator of the latter.
REFERENCES
Campo MS (1991) Vaccination against papillomavirus.
Cancer Cells 3, 421-426.
Campo MS, Moar MH, Jarrett WFH and Laird HM (1980) A new papillomavirus associated with alimentary cancer in cattle. Nature 286, 180-182.
Campo MS and Coggins LW (1982) Molecular cloning of bovine papillomavirus genomes and comparison of their sequence ho ologies by heteroduplex mapping. Journal General
Virology, 63, 255-264.
Campo MS and Jarrett WFH (1986) Papillomavirus infection in cattle: viral and chemical cofactors in naturally occurring and experimentally induced tumours. Ciba
Foundation Symposium 120; Papillomaviruses. ppll7-131.
Campo MS, Moar MH, Laird HM and Jarrett WFH (1981)
Molecular heterogeneity and lesion site specificity of cutaneous bovine papillomviruses. Virology, 113, 323-335.
Comerford SA, McCance DJ, Dougan G and Tite JP (1991)
Identification of T- and B-cell ^.epitopes in the E7 protein of human papillomavirus type 16. Journal of
Virology, 65, 4681-4690.
Danos 0, Giri I, Thierry F and Yaniv M (1984)
Papillomavirus Genomes: Sequences and Consequences. The
Journal of Investigative Dermatology, 83, 7s-lls.
Dillner J (1990) Mapping of linear epitopes of human papillomavirus type 16: the El, E2, E4, E5, E6 and E7 open reading frames. International Journal of Cancer, 46,
703-711.
Evans CA, Gorman LR, Ito Y and Weiser RS (1962) Antitumour immunity in the Shope papilloma-carcinoma complex of rabbits. I. Papilloma regression induced by homologous and autologous tissue vaccines. Journal of the National Cancer Institute, 29, 277-285.
Jackson ME, Pennie WD, McCaffery RE, Smith KT, Grindlay GJ and Campo MS (1991) The B sub-group bovine papillomaviruses lack an identifiable E6 ,open reading frame. Molecular Carcinogenesis, 4, 382-387. Jaggar RT, Pennie WD, Smith KT, Jackson ME and Campo MS (1990) Cooperation between bovine papillomavirus type 4 and ras in the morphological transformation of primary bovine fibroblasts. Journal of General Virology, 71, 3041-3046..
Jarrett WFH (1985) The natural history of bovine papillomavirus infection. Advances in viral oncology. Ed: G.Klein, vol 5, pp83-102.Jarrett WFH, McNeil PE, Gri shaw WTR, Sel an IE and Mclntyre WIM (1978) High incidence area of cattle cancer with a possible interaction between an environmental carcinogen and a papillomavirus. Nature, 274, 215-217.
Jarrett WFH, Campo MS, O'Neill BW, Laird HM and Coggins LW (1984) . A novel bovine papillomavirus (BPV-6) causing true epithelial papillomas of the mammary gland skin: a member of a proposed new subgroup. Virology, 136, 255-264. Jarrett WFH, O'Neil BW, Gaukroger JM, Laird HM, Smith KT and Campo MS (1990a) Studies on vaccination against papillomaviruses; a comparison of purified virus, tumour extract and transformed cells in prophylactic vaccination. The Veterinary Record, 126, 449-452.
Jarrett WFH. O'Neil BW, Gaukroger JM, Smith KT, Laird HM and Campo MS (1990b) Studies on vaccination against papillomaviruses; the immunity after infection and vaccination with bovine papillomaviruses of different types. The Veterinary Record, 126, 473-475.
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Chandrochud LM, Grindlay GJ, McGarvie GM and Campo MS
(1991) Studies on vaccination against papillomaviruses; prophylactic and therapeutic vaccination with recombinant structural proteins. Virology, 184, 33-42.
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(1988) Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins. Journal of Virology, 62, 2115-2123.
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Krchnak V, Vagner J, Suchankova A, Krcmar M, Ritterova L and Vonka V, (1990) Synthetic peptides derived from E7 region of human papillomavirus type 16 used as antigens in ELISA. Journal of General Virology, 71,2719-2724. Pilacinski WP, Glass an DL, Glassman KF, Reed DE, Lum MA,
Marshall RF, Muscoplat CC and Faras AJ (1986)
Immunization against bovine papillomavirus infection.
Ciba Foundation Symposium 120; Papillomaviruses. ppl36-148.
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Smith DB and Johnston KS. (1988) Single-step purification of polypeptides expressed in E coli as fusions with glutathione S-transferase. Gene 67, 31-40 Steinberg BM and Abra son AL (1985) Laryngeal papillomas. Clinics in Dermatology, 3, 130-138.
Strang G, Hickling JK, Mclndoe GAJ, Howland K, Wilkinson D, Ikeda H and Rothbard JB. (1990) Human T-cell responses to human papillomavirus type 16 LI and E6 synthetic peptides: identification of T-cell determinants, HLA-DR restriction and virus type specificity. Journal General Virology, 71, 423-431. Tindie RW, Smith JA, Geysen HM, Selvey LA and Frazer IH
(1990) Identification of B epitopes in human papillomavirus type 16 E7 open reading frame protein.
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(1991) A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes. Proc. Natl. Acad. Sci. 88,5887-5891.
Zur Hausβn H (1978) Condyloma acuminata and human genital cancer. Cancer Research, 36, 794. SEQUENCE LISTING (FIGURE 2)
SEQ ID NO
SEQUENCE TYPE : protein sequence
SEQUENCE LENGTH 98 amino acids
STRANDEDNESS single
TOPOLOGY linear
MOLECULAR TYPE E7 protein
ORIGINAL SOURCE
ORGANISM Bovine papilloma virus BPV-4
USE therapy
DEPOSIT
FEATURES aa 4-15 and 23-36 = presumed retinoblastoma protein binding domain aa 47-50 and 80-83 = presumed zinc fingers domains aa 54-73 = presiuned hydrophobic stretch. SEQUENCE LISTING (FIGURE 4)
SEQ ID NO
SEQUENCE TYPE protein sequence
SEQUENCE LENGTH 98 amino acids
STRANDEDNESS single
TOPOLOGY linear
MOLECULAR TYPE E7 protein
ORIGINAL SOURCE
ORGANISM Bovine papilloma virus BPV-4
USE therapy
DEPOSIT
Figure imgf000021_0001

Claims

1. A pharmaceutical formulation for the therapy of papillomavirus disease^ which comprises papillomavirus E7 protein or therapeutically effective fragment thereof in admixture with a pharmaceutically acceptable carrier.
2. A formulation according to claim 1 wherein the E7 protein is a bovine papillomavirus protein.
3. A formulation according to either preceding claim wherein the E7 protein is BPV-2 or BPV-4 protein.
4. A formulation according to any preceding claim comprising a therapeutically effective E7 fragment which has the sequence amino acids 1-30, 79-98, 51-69, 20-50 or 70-88 as set out in Figure 4; or with additions, deletions o substitutions thereto which do not substantially effect the therapeutic effectiveness thereof.
5. A formulation according to any preceding claim wherein the E7 protein is present in the form of a fusion protein with a different co-protein.
6. A formulation according to claim 5 wherein the co-protein in the E7 fusion protein is beta-galactosidase.
7. A formulation according to claim 5 wherein the co-protein in the E7 fusion protein is glutathione S-transferase (GST) .
8. A formulation according to any preceding claim wherein the E7 protein or fragment thereof is produced by recombinant DNA techniques.
9. A formulation according to any preceding claim in the form of an injectable formulation, wherein the carrier is a pharmaceutically acceptable injection vehicle.
10. A formulation according to any preceding claim which further comprises an adjuvant selected from Freund's incomplete adjuvant, and aluminium gel.
11. A transformed bacterial cell producing recombinant E7 protein or therapeutically effective fragment thereof.
12. Use of papillomavirus E7 protein or effective fragment thereof in medicine for the therapy of papillomavirus disease.
13. Use of papillomavirus E7 protein or effective fragment thereof in the production of a medicament for use in the therapy of papillomavirus disease.
14. A method of treating a mammal for the therapy of papillomavirus disease, which comprises the administration " of papillomavirus E7 protein or effective fragment thereof to the mammal in a therapeutically effective dosage.
PCT/GB1993/000679 1992-04-08 1993-04-01 Papillomavirus e7 protein WO1993020844A1 (en)

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WO1999055876A2 (en) * 1998-04-30 1999-11-04 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Polypeptide with immunogenic properties and with a protein with modified biological functions
US6228368B1 (en) 1997-10-06 2001-05-08 Loyola University Of Chicago Papilloma virus capsomere formulations and method of use
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