AU755242B2 - Medicament for preventing or treating papilloma virus-specific tumors - Google Patents
Medicament for preventing or treating papilloma virus-specific tumors Download PDFInfo
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Description
1 Medicament for the avoidance or treatment of papillomavirusspecific tumour The present invention relates to a medicament for the avoidance or treatment of human papillomaviruses (HPV)-specific tumour comprising at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, the fusion protein containing no papillomavirus-unspecific epitopes.
Papillomaviruses, also called wart viruses, are double-stranded DNA viruses having a genome size of approximately 8000 base pairs and an icosahedral-like capsid having a diameter of about 55 nm. To date, more than 100 different human papillomavirus types are known, of which some, e.g. HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-52 or HPV-58, can cause malignant tumours and others, e.g. HPV-6, HPV-11 or HPV-42, can cause benign tumours.
Electron-microscopic analysis of BPV-1 and HPV-1 shows that the viruses are constructed from 72 pentameric capsomers, which in turn consist of five LI molecules (Baker, T. et al. (1991) Biophys. 60, 1445).
The genome of the papillomaviruses can be subdivided into three areas: the first area concerns a non-coding region which contains regulation elements for the transcription and replication of the virus. The second region, the so-called E (early) 20 region, contains various protein-coding sections E1-E7, of which, for example, the E6 protein and the E7 protein is responsible for the transformation of epithelial cells and the 0 El protein controls the DNA copy number. The E6 region and E7 region are so-called oncogenes, which are also expressed in malignantly degenerate cells. The third region, also called the L (late) region, contains oo .0 *0 0 0.
*OoO [R:\LIBVV]0261 0.doc:ais WO 99/48518 2 PCT/EP99/01996 two protein-coding sections LI and L2, which code for structural components of the virus capsid. The LI protein is present to over 90% in the viral capsid, the ratio of Ll:L2 in general being 30:1.
HPV-6 and HPV-11 have been held responsible, inter alia, for genital warts; some papillomavirus types such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-52 and HPV-58 are associated with malignant tumours of the anogenital tract. In over 50% of the cases, HPV-16 is connected with cervical cancer (carcinoma of the cervix). HPV-16 is therefore the main risk factor for the formation of cervical neoplasias. In addition, the immune system plays an important role in the progress of the disease. Thus cellular immune responses and in particular antigen-specific T lymphocytes are supposedly important for the defence mechanism. It has furthermore been found that in high-grade cervical intraepithelial neoplasias (CIN II/III) and cervical tumour the E7 gene is expressed constitutively in all layers of the infected epithelium. The E7 protein is therefore considered as a potential tumour antigen and as a target molecule for activated T cells (see, for example, WO 93/20844). The E7-induced cellular immune response in the patient, however, is apparently not strong enough to influence the course of the disease.
The immune response can possibly be amplified by suitable vaccines.
It has now been possible to show that the expression of the LI gene or the coexpression of the Ll and L2 gene forms virus-like particles (VLPs). It was possible to use the VLPs for the formation of neutralizing antibodies in various animal systems. The formation of virus-neutralizing antibodies, however, is of relatively low clinical importance if the virus infection has already taken place, since for the i; elimination of virus-infected cells a virus-specific
F-
WO 99/48518 3 PCT/EP99/01996 cytotoxic T-cell (CTL) response appears to be necessary. So-called chimeric papillomavirus-like particles (CVLPs) were therefore developed which consist of a chimeric Ll-E7 protein (Miller, M. et al.
(1997) Virology, 234, 93): some CVLPs induce an E7-specific CTL response in mice, although experiments failed to induce antibodies by immunization of mice with CVLPs against E7 (Muller, M. et al. (1997), supra). In addition, neutralizing antibodies of HPVassociated disorders in patients appear to limit the immune response to administered Ll protein (Muller, M.
et al. (1997), supra). CVLPs, however, are still of interest for the development of a vaccine, as the E7 proteins of tumour cells presented via MHC molecules of class I would represent target molecules of CTLs.
Peng et al. (1998) Virology, 240, 147 now describe CVLPs consisting of C-terminally truncated Ll of the bovine papillomavirus (BPV) and HPV-16E7 49 -57, which induce E7-specific cytotoxic T cells after inoculation of C57B1/6 mice and protect against the growth of E7-expressing tumours. Greenstone et al. (1998) Proc.
Natl. Acad. Sci. USA, 95, 1800 describe CVLPs consisting of HPV-16L1 plus HPV-16L2 fused to the fulllength HPV-16E7 protein, which protect against the growth of epithelial E7-expressing tumour cells after immunization of C57B1/6 mice, cytotoxic T cells, however, not being detected and thus the induction of the immune response appearing to be less efficient.
VLPs and CVLPs are in general prepared by means of genetic engineering by expression of the corresponding genes coding for one or more L proteins or L and E proteins in suitable expression systems. The corresponding genes are described, for example, in Kirnbaum, R. et al. (1994) J. Virol., 67, 6929-6936 or tR obtainable via the EMBL databank. The accession numbers WO 99/48518 4 PCT/EP99/01996 are, for example, for HPV18: PAPHPV18; for HPV31: PAPPPH31; for HPV33: PAPPPH33 or for HPV58: PAPPPH58.
Suitable expression systems are, for example, yeasts modified by genetic engineering, e.g. Saccharomyces (cerevisiae), Pichia (pastoris), Kluyvermyces (lactis), Schizosaccharomyces (pombe) or Hansenula (polymorpha) (Carter, J. J. et al. (1991), Virology, 182, 513), insect cells, such as, for example, Trichoplusia ni High Five (see, for example, Muller et al. (1997), supra) or prokaryotic cells (see, for example, WO 96/11272). In the case of the production of the particles in prokaryotic cells, these are in general deposited in the cell and form so-called inclusion bodies, which then have to be renatured and brought into solution. For use of the particles or capsids produced by genetic engineering or their precursors, the so-called capsomers, further purification steps are necessary after expression.
A crucial disadvantage of the active compounds against HPV described in the literature is, however, that on the one hand they exhibit only a slight action and on the other hand that it has not been possible to date to show any effective immunotherapy of papillomavirusspecific tumour.
It was therefore the object of the present invention to make available a medicament with which human papillomavirus-specific tumour can be avoided or treated effectively, which can be produced simply and which appears to be suitable for licensing as a medicament.
It has now surprisingly been found that the medicament according to the invention is effective against HPV- >y specific tumour.
According to a first embodiment of the present invention there is provided a medicament when used for the prevention or treatment of human papillomavirus (HPV)specific tumours, the medicament comprising at least one fusion protein from at least one L1 protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, the fusion protein containing no papillomavirus-unspecific epitopes.
According to a second embodiment of the present invention there is provided a process for the production of a medicament comprising at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, the fusion protein containing no papillomavirus-unspecific epitopes, wherein a cell comprising an expression vector containing at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses is cultured under suitable conditions, the expression product is isolated and, if
S**
appropriate, suitable additives and excipients are added.
According to a third embodiment of the present invention there is provided use of a fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses for the manufacture of a medicament, for the prevention of HPV-specific tumours or the regression of already existing HPVspecific tumours wherein the fusion protein contains no papillomavirus-unspecific epitopes.
According to a fourth embodiment of the present invention there is provided a method of preventing or treating human papillomavirus (HPV)-specific tumours in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a medicament comprising at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, wherein the fusion protein contains no papillomavirus-unspecific epitopes.
Papillomavirus-unspecific epitopes within the meaning of the present invention are understood in general as meaning epitopes in the fusion protein which are caused by a foreign protein fraction, by post-translational modifications or by misfolding of j qpillomavirus-specific proteins.
[R:\LIBVV]02610.doc:ais The papillomavirus-unspecific epitopes are, for example, a reason for the fact that although neutralizing antibodies or CTL immune responses are induced, the papillomavirus-specific tumour cannot be effectively avoided or controlled, as the immunological action is weakened by unspecific antibodies or CTLs or immunological side effects interfere with the action of the actual active compound.
The medicament according to the invention is preferably effective for the avoidance or treatment of benign or malignant tumour, in particular of carcinoma of the larynx, cervix, penis, vulva or anus, including their preliminary stages, such as, for example, high-grade CIN (cervical intraepithelia neoplasia).
In a further preferred embodiment, the medicament according to the invention contains no adjuvant, i.e. no substance which amplifies the immunity of the papillomavirus-specific protein, as the presence of an L protein in particular, especially of L1, already *eo [R:\LIBVV]02610.doc:ais WO 99/48518 6 PCT/EP99/01996 adequately amplifies the immunity. This property is particularly advantageous in the licensing as a medicament or diagnostic, as the only immunostimulating materials licensed by the licensing authorities at present are aluminium salts. Moreover, undesired side effects are avoided by the omission of adjuvants and/or other excipients and additives.
As already mentioned above, a further significant problem in the use of capsids and capsomers as medicaments is their poor solubility. Thus capsids or capsomers of HPV-16, for example, tend to aggregate, whereby the solubility is significantly reduced. The solubility of the capsids or capsomers, which in some cases is low, leads not only to a loss of yield, but also to complicated use as a medicament.
In a further preferred embodiment, the medicament according to the invention therefore contains as a suitable additive or excipient about 0.3 to about 4 M, preferably about 0.4 to about 3 M, in particular about to 2 M, especially about 1 to about 2 M, of a salt having a pH of about 7.3 to about 7.45, preferably about 7.4.
The advantage of this salt solution is that the fusion protein remains in solution or is present finely divided as a suspension, in general more than about 90%, especially more than about 95%, of the fusion protein remains in solution and also does not deposit for a period of time of at least about 12 hours. The fusion protein is also not significantly sedimentable by centrifugation at a maximum of 5000 g.
The salt is in general an alkali metal or alkaline earth metal salt, preferably a halide or phosphate, in particular an alkali metal halide, especially NaCl WO 99/48518 7 PCT/EP99/01996 and/or KC1. Use of NaCI is particularly preferred for the production of a pharmaceutical formulation.
The pH of the medicament is in general adjusted using a suitable organic or inorganic buffer, such as, for example, preferably using a phosphate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3-morpholino-l-propanesulphonic acid). The choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer is suitable, for example, for injection and infusion solutions.
Suitable further additives and/or excipients which serve, for example, for the further stabilization of the papillomavirus-specific protein in the medicament according to the invention are, for example, detergents, such as, for example, Triton X-100 or sodium deoxycholate, but also polyols, such as, for example, polyethylene glycol or glycerol, sugars, such as, for example, sucrose or glucose, zwitterionic compounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or zwitterionic compounds are preferred. Other additives and/or excipients are protease inhibitors, such as, for example, aprotinin, E-aminocaproic acid or pepstatin A.
Those additives are preferred which induce no immunological side effects.
The terms Ll/L2 protein and E protein are understood within the meaning of the present invention as meaning both the full-length proteins and their mutants, such as, for example, deletion mutants.
WO 99/48518 8 PCT/EP99/01996 In a further preferred embodiment, the fusion protein according to the invention contains a deleted L protein, preferably a deleted L1 and, if appropriate, L2 protein. The deletion has the advantage that particularly active different proteins, for example papillomavirus-specific E protein sequences, can be inserted into the deleted area, whereby the application area of the composition according to the invention can be widened. An L protein having a C terminal deletion and in particular a C-terminally deleted L1 protein is particularly preferred. The C-terminal deletion has the advantage that the efficiency of the formation of virus-like particles can be increased, since the nuclear location signal located at the C terminus is deleted. The C-terminal deletion is therefore preferably up to about 35 amino acids, in particular about 25 to about 35 amino acids, especially about 32 to 34 amino acids. For example, a C-terminal deletion of the HPV-16L1 protein 32 amino acids long is adequate to be able to increase the formation of virus-like particles by at least about In a further preferred embodiment, the E protein is also deleted, especially the E6 and/or E7 protein. It is particularly preferred if the C-terminal part of the E protein is deleted, preferably the C-terminal part of the E7 protein, as these constructs can preferably form capsomers and/or capsids in combination with deleted L protein. Deletions of up to 55 amino acids are particularly preferred, preferably about 5 to about amino acids, in particular about 32 to about 43 amino acids.
A particularly preferred construct is, for example, E7 having the N-terminal amino acids 1 to about 60, as this construct contains a mouse epitope for the 9 activation of cytotoxic T lymphocytes, which is located Sin the area of the amino acids 49-57. Another preferred 9 construct is E7 having the N-terminal amino acids 1 to about 55, which preferably forms capsomers and capsids in combination with deleted L protein, as this construct does not contain E7-specific sequences in the area of the amino acids 56-70, which can interfere with the formation of capsids. An LI protein of HPV-16 C-terminally deleted by 32 s amino acids and which is linked to an E7 protein of HPV-16 having the amino acids 1-55 or 1-60 is particularly preferred. These constructs not only induce neutralizing antibodies or a specific CTL response, but on the one hand prevents the formation of tumours and on the other hand cause regression of already existing tumours in animal experiments. E7 having the amino acids 1-60 especially exhibits a marked prophylactic and therapeutic action in tumours. A particularly preferred embodiment of the present invention is therefore an L1AE7 1 -x fusion protein, preferably in the form of a CVLPs, in particular of HPV16, x being an integer from 55 up to and including 60, and in particular an L1AE7 1 5 or L1AE71- 60 fusion protein.
The present invention therefore also relates to the use of the constructs according to the invention for the production of a medicament, on the one hand for the connection of HPV-specific tumours and on the other hand for the regression of already existing HPVspecific tumours.
For the production of a medicament which is active both prophylactically and therapeutically, it is preferred if the described papillomavirus-specific fusion protein is 20 present in the form of a capsid and/or capsomer, since the immune reaction can additionally be markedly increased by the capsids and/or capsomers and in particular by the fraction of L protein. Preferred fusion proteins which are suitable for capsid and/or o* oo [R:\LIBVV]026 WO 99/48518 10 PCT/EP99/01996 capsomer formation are therefore, for example, fusion proteins from deleted Ll and E7, E6 and/or El.
Capsids within the meaning of the present invention are viral or virus-like structures in a generally icosahedral form, which in general are constructed of 72 capsomers.
Capsomers within the meaning of the present invention are assembled proteins comprising at least one papillomavirus structural protein, preferably Ll or deletions of Ll. For example, 5 fusion proteins according to the invention can be assembled to give a capsomer which in turn can be assembled to give a capsid.
For the production of a human medicament, proteins or peptides of the human papillomavirus (HPV) and preferably of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-52 and/or HPV-58, in particular HPV-16, HPV-18, HPV-31 and/or HPV-45 are suitable for the constructs described. Especially for the production of a combination vaccine, it is advantageous to combine proteins or peptides from various HPV types, for example a combination of HPV-16 and HPV-18 or HPV-18, HPV-31, HPV-45 and HPV-58 in the case of, for example, carcinoma of the cervix or HPV-6 and HPV-11 in the case of, for example, condylomas.
A further subject of the present invention is a process for the production of a medicament according to the invention, in which a suitable cell comprising a suitable expression vector which codes for the fusion protein mentioned is cultured under suitable conditions, the expression product is isolated and, if Sappropriate, suitable additives and/or excipients are added.
WO 99/48518 11 PCT/EP99/01996 The expression vectors can be, for example, prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors are, for expression in E. coli, e.g. the vectors pGEM or pUC derivatives (see, for example, WO 96/11272). Examples of eukaryotic expression vectors are, for expression in Saccharomyces cerevisiae, e.g. the vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768, Carter, J. J. et al. (1991) supra) and, for expression in insect cells, e.g. Baculovirus vectors, in particular the Autographa Californica virus, such as disclosed in EP-Bl-0 127 839 or EP-Bl-0 549 721 (see, for example, also WO 94/20137), and, for expression in mammalian cells, e.g. the vectors Rc/CMV and Rc/RSV or SV40 vectors which are all generally obtainable.
However, commercially obtainable Baculovirus expression systems are also suitable, such as, for example, the Baculo Gold TM transfection kit from Pharmingen or the Bac-to-Bac TM Baculovirus expression system from Gibco BRL. Further suitable expression systems are recombinant vaccinia viruses (see, for example WO 93/02184).
In general, the expression vectors also contain promoters suitable for the respective host cell, such as, for example, the trp promoter for expression in E. coli (see, for example, EP-Bl-0 154 133), the ADH2 promoter for expression in yeasts (Russel et al.
(1983), J. Biol. Chem. 258, 2674-2682), the Baculovirus polyhedrin promoter for expression in insect cells (see, for example EP-Bl-0 127 839 or U.S. 5,004,687) or the early SV40 promoter or LTR promoters, e.g. of MMTV (mouse mammary tumour virus; Lee et al. (1981) Nature 214, 228-232).
Suitable host cells are, for example, the E. coli strains DH5, HB101 or BL21, the yeast strains Saccharomyces, Pichia, Kluyvermyces, Schizosaccharomyces WO 99/48518 12 PCT/EP99/01996 or Hansenula (Carter, J. J. et al. (1991), Virology, 182, 513), the insect cell line Lepidopteran, e.g. from Spodoptera frugiperda, Trichoplusia ni, Rachiplusia ou or Galleria Mellonela or the animal cells COS, C127, Vero, 293 and HeLa, which are all generally obtainable (see, for example, WO 94/00152).
The coding nucleic acids for the individual papillomavirus-specific proteins can be isolated and cloned, for example, from a gene bank by means of a PCR (polymerase chain reaction) amplification. For example, the genome of BPV-1 is generally obtainable under the GenBank Accession No. X02346 or HPV-16 under the GenBank Accession No. K02718. An HPV-16 Ll sequence is also disclosed, for example, in WO 94/05792. The sequence of the 98 amino acid-long HPV16 E7 protein is described, for example, in Seedorf et al. (1985) Virology, 145, 181-185. Another method of obtaining the desired nucleic acids is to isolate the papillomavirusspecific genes directly from warts or tumours by means of PCR. Suitable primers for the E6 and E7 genes from HPV-16 and HPV-18 are disclosed, for example, in WO 93/21958. Further references for the desired nucleic acids are, for example, Kirnbaum, R. et al. (1994), supra or the clones deposited in the EMBL databank already mentioned above.
In a further preferred embodiment, the expression vector is constructed such that the expressed fusion protein is extended by no further amino acids caused by the vector. This is achieved, for example, by removing undesired nucleotides which code for additional amino acids by mutagenesis in a PCR reaction by means of suitable primer oligonucleotides (Ho et a. [sic] (1989) Gene, 77, 51-59). In this way, a fusion protein is obtained which is free of additional amino acids and 7~ thus free of possible additional foreign epitopes which 7can cause immunological side reactions.
WO 99/48518 13 PCT/EP99/01996 After the expression of the described fusion protein, it is preferred to purify this further or to renature it. Examples of chromatographic purification processes are found in Hjorth, R. Moreno-Lopez, L. (1982) J. Virol. Meth. 5, 151; Nakai, Y. et al. (1987) J. Gen.
Virol., 68, 1891 [sic]; Hofmann, K. J. et al. (1995) Virology, 209, 506; Rose, R. C. et al. (1993) J. Virol., 67, 1936, Sasagawa, T. et al. (1995) Virology, 206, 126 or WO 95/31532.
In general, the medicament can be administered orally, parenterally, such as, for example, subcutaneously, intramuscularly or via the mucous membrane, in liquid or suspended form, in the form of an elixir or as capsules, preferably as an injection or infusion solution. In the case of the formulations according to the invention, an adjuvant can be dispensed with, which is particularly advantageous.
A further subject of the present invention therefore relates to the use of the formulation according to the invention as an injection or infusion solution.
Injection solutions are in general used if only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be administered to the body. Infusion solutions are in general used if a larger amount of a solution or suspension, for example one or more litres, are to be administered. Since, in contrast to the infusion solution, only a few millilitres are administered in the case of injection solutions, small differences from the pH and from the osmotic pressure of the blood or the tissue fluid in the injection do not make themselves noticeable or only make themselves ST noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according 14 to the invention before use is therefore in general not necessary. In the case of the administration of relatively large amounts, however, the formulation according to the invention should be diluted briefly before administration to such an extent that an at least approximately isotonic solution is obtained. An example of an isotonic solution is a 0.9% s strength sodium chloride solution. In the case of infusion, the dilution can be carried out, for example, using sterile water while the administration can be carried out, for example, via a so-called bypass.
The figures and the following examples are intended to illustrate the invention in greater detail without restricting it.
Description of the figures Fig. 1 shows an E7-specific CTL response which were produced after immunization with 1 jg of LIE7 1 60 CVLPs. The isolated spleen cells of mice immunized with LiE71- 60 CVLPs (filled symbols) and HBS buffer (open symbols) were stimulated once in vitro with irradiated RMA-E7 cells and tested after five days in a standard four-hour 51 Cr release cytotoxicity test with the following target cells: RMA-E7 cells (squares), E7 49 57 peptide-loaded RMA cells (triangular) and RMA cells (circles). The results are expressed as specific lysis in Fig. 2 shows the result of a titration test, carried out using an E7 specific CTL line I which was obtained after vaccination of a C57BL/6 mouse with 20 pIg of LlE71- 60 CVLPs 20 and in vitro stimulation three times with RMA-E7 transfectants. The peptide concentration was between 100 pg and 1 tg/ml. The target cells used were sCr-labelled E7 49-57 peptide-loaded RMA cells. The ratio of cells to target cells was 30:1.
*0.
*000 [R:\LIBVVIO2610.doc:ais Fig. 3 shows the protection of C57BL/6 mice against the growth of TC-1 tumour cells. The mice (5 per group) were immunized s.c. either with 10 p.g of LlE71- 60 CVLPs (triangular), with 10 ptg of L1AC VLPs (circles) or with HBS buffer (squares). Two weeks later, 6 x 10 4 TC-1 tumour cells per mouse were inoculated into the left flank of the mice The mice were checked twice per week.
Fig. 4 shows the prevention of the growth of TC-1 tumour by LIE71- 60 CVLPs. 6 x 4 TC-1 tumour cells per mouse were inoculated into the left flank of C57BL/6 mice per group). Two weeks later, the mice were immunized with an s.c. injection of 10 Pjg of L1E71- 60 CVLPs (triangles), 10 pg of L1ACVLPs (circles) or HBS buffer (squares).
Examples 1. Preparation of chimeric genes coding for HPV16L1E7 fusion proteins The HPV-16L1 open reading frame (ORF) was excized from the plasmid HPV-16- 114/k-Ll/L2-pSynxtVI (Kimbauer, R. et al. (1994) J. Virol. 67, 6929) using the restriction endonuclease BglII and cloned into the BamHI site in the vector pUC19 (New England Biolabs).
For the preparation of HPV-16L1AC, two primers were constructed which are complementary to HPV-16L1 ORF. The first primer has the sequence
AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC
20 and the second primer
AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACG
Both primers encode an EcoRV restriction enzyme cleavage site In the primers lying downstream, a [R:\LIBVV]02610.doc:ais TAA translation stop codon follows the EcoRV site in order to delete the last 34 amino acids of the HPV16L1 ORF. The PCR reaction was carried out in order to amplify the entire LI ORF and the entire vector. The linear product was cleaved with EcoRV, cirucularized with T4 DNA ligase and transformed E. coli DH5a cells. The clones were analysed for the presence of an EcoRV site. The construct pUCHPV16L1AC obtained was used in order to clone the ORF of HPV16E7 1-50 into the EcoRV site.
For the cloning of the fragment, primers having a 5'EcoRV restriction enzyme cleavage site were used. The following primer pair was used:
AAAAGATATCATGCATGGAGATACACCTACATTGC
and
TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC
The PCR products were cleaved with EcoRV and inserted into the EcoRV site of the modified LI gene.
For the elimination of the EcoRV sites, two PCR reactions were carried out in order to amplify two overlapping fragments of the clone pUC-HPV16LIACE7 1-50. The resulting DNA fragments overlapped in the position of the L1/E7 boundary (Four primer PCR, Ho, S. N. et al (1989) Gene 77, 51). However, the primers did not contain the two EcoRV restriction enzyme cleavage sites. Fragment 1 was prepared using the primers P1 and P2 and fragment 2 using the primers P3 and P4.
20 P1: GTTATGACATACATACATTCTATG (LI) P2: CCATGCATTCCTGCTTGTAGTAAAAATTTGCGTCC (E7) P3: CTACAAGCAGGAATGCATGGAGATACACC (E7) P4: CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCTG (E7) [R:\LIBVV]0261 0.doc:ais WO 99/48518 17 PCT/EP99/01996 A tenth of the purified products was mixed and used as a matrix in the PCR reaction with the primers P1 and P4 exclusively. The resulting product was cleaved using EcoNI (LI) and HindIII (downstream of the stop codon on the primer P4) and used in order to replace an EcoNI/HindIII fragment of the cloned HPV16L1 ORF. The resulting clone therefore differs from the clone HPV16L1ACE7 1-50 by the loss of the two internal EcoRV restriction enzyme cleavage sites and the corresponding non-HPV amino acids Asp and Ile between the LI ORF and E7 and downstream of E7. The first EcoRV site was replaced by the original LI amino acids in this position (AlaGly). The second EcoRV site was replaced by a translation stop signal. This clone (HPV16L1AC*E7 1-52) additionally contains the first 52 amino acids of HPV16E7. Clone HPV16LIAC*E7 1-52 was used for the preparation of the clones HPV16LIAC*E7 1-55, 1-60 and 1-65 with the aid of the primer P1 in combination with P6 and P7.
PS: CATCTGAAGCTTATCAATATTGTAATGGGCTCTGTCCG (E7 1-55) P6 CATCTGAAGCTTACTTGCAAC AAAAGGTTAC.AATATTGTAATGGGCTCT GTCCG (E7 1-60) P7:
CATCTGAAGCTTAAAGCGTAGAGTCACACTTGCAACAAAAGGTTACAA
TATTGTAATGGGCTCTGTCCG (E7 1-65).
HPV16LIAC*E7 1-70 was prepared using the clone HPV16LIAC*E7 1-65 and the primers P1 and P8.
P8:
CATCTGAAGCTTATTGTACGCACAACCGAAGCGTAGAGTCACACTTG
In all cases, EcoNI and HindIII were used in order to replace the corresponding fragments. The clones were 'analysed by DNA sequencing.
30-1 analysed by DNA sequencing.
18 2. Preparation of recombinant Baculoviruses Spodoptera frugiperda (Sf9) cells were used as a monolayer or in suspension culture in TNM-FH insect medium (Sigma, Deisenhofen) with 10% foetal calf serum and 2mM glutamine. Recombinant baculoviruses HPV16L1ACE7 1-x were transfected by cotransfection of 10 jig of the recombinant plasmids and 2 ig of linearized Baculo-Gold DNA (Pharmingen, San Diego, CA) into SF9 cells. Recombinant viruses were purified according to the instructions of the manufacturer. In order to test the expression, 106 Sf9 cells were infected with recombinant Baculovirus and an m.o.i. (multiplicity of infection) of 5 to 10. After the incubation, the medium was removed and the cells washed with PBS (140 mM NaCI, 2.7 mM KC1, 8.1 mM Na 2
PO
4 1.5 mM KH 2
PO
4 pH The cells were then lysed in SDS sample buffer and tested by SDS gel chromatography and immunoblot assay.
3. Purification of virus-like particles For the preparation of CVLPs, Trichoplusia ni (TN) High Five cells were cultured at 27 0 C up to a density of 1-1.5 x 106 cells were per ml in EX-Cell 405 serum-free medium (JRH, Biosciences, Lennexa, KS). A 400 ml culture was harvested and infected with an m.o.i. of 2 to 5 recombinant baculoviruses for one hour with periodic inversions.
Up to 240 ml of medium were added and the cells grew for 3 to 4 days. The cells were then pelleted and resuspended in 10 ml of extraction buffer (10 mM MgCl 2 1-50 mM 20 CaC1 2 150 mM NaCl, 20 mM Hepes, 0.01% Triton (optional), pH 7.4) and sonicated for 45 seconds at 60 watts. After centrifugation at 10,000 rpm in a Sorvall SS34 rotor, the pellet was dissolved in 6 ml of extraction buffer, sonciated for 30 seconds at 60 watts centrifuged again. The *0* 19 supematants were combined and applied to a two-stage gradient of 40% succhrose and 57.5% CsCl. After centrifugation in an SW-28 rotor at 27,000 rpm for two hours, the interphase and the CsCl layer were collected, adjusted to a CsCl density of 1.38 g/ml and centrifuged at 45,000 rpm for 16 hours. The gradients were fractionated and each fraction was tested by Western Blot using anti-HPV16L1mAb Camvirl (Phamingen, San Diego, CA). The reactive fractions were combined and dialysed by means of an ultrafiltration using a Centricon 30 microconcentrator (Amicon Corp. Beverly, MA) against Hepes buffer (lmM Hepes, 149 mM NaC1, 0.5 mM KC1, pH 7.2) and the presence of CVLPs was confirmed by means of transmission electron microscopy. The concentration of L1E7 protein was determined approximately, in an SDS gel which was stained with Coomassie blue, by comparison with BSA standards.
4. Culturing of mouse cells The C57BL/6-derived mouse cells TC-1 are primary pulmonary epithelial cells of C57BL/6 mice, which have been transformed by transfection with the oncogenes HPV-16 E6 and E7 and c-Ha-ras (Lin, (1996), supra). RMA (Ljungrenn Karre (1985) J.
Exp. Med., 162, 1745-1757) and the processing-defective cell line RMA-S are C57BL/6 thymoma cells. The HPV-16 E7-transfected RMA-E7 is described in Speidel, K. et al.
(1997) Eur. J. Immunol., 27 2391-2399. All cells were cultured in RPMI-1640, supplemented with 10% FCS, 2-ME, L-glutamine and antibiotics. 0.8 mg/ml of G418 20 (Gibco BRL, Gaithersburg, MD) was added for the culturing of RMA-E7 transfectants.
TC-1 cells additionally grew in the presence of 0.4 mg/ml of G418, 0.2 mg/ml of hygromycin and 1 mM sodium pyruvate.
S T [R:\LIBVV]02610.doc:ais WO 99/48518 20 PCT/EP99/01996 Production of cytotoxic T-cell (CTL) lines 10-14 days after immunization, the spleen cells were prepared; 2-4 x 107 spleen cells were cocultured with 105 irradiated (200 Gy) syngenic RMA-E7 transfectants per ml. After 5 days, the first 51 Cr-release cytotoxicity test was carried out. For the production of CTL lines, the spleen cells were restimulated weekly in 24-hole plates with 2 x 10 5 irradiated RMA-E7 transfectants per hole as stimulator cells and 5 x 106 irradiated (33 Gy) C57BL/6 spleen cells per hole as feeder cells.
6. 51 Cr-release cytotoxicity test For the 51 Cr-release cytotoxicity test, effector T cells were added to 1 x 104 5Cr-labelled target cells per hole on a 96-hole plate having a different effector cell to target cell ratio The target cells were labelled with Na 2 51 CrO 4 (100 pCi per 2 x 10 6 cells) for one hour at 37 0 C. The peptide having the sequence RAHYNIVTF (amino acid 49-57 of HPV-16 E7; Feltkamp, M.C.W. et al. (1993), Eur. J. Immunol., 23, 2242-2249) was added during this incubation in a concentration of 50 pM. In the peptide titration test with a constant E:T ratio, the 51 Cr-labelled target cells were cultured with decreasing peptide concentrations (1 ig 100 pg per ml) for one hour at 37 0 C before the effector cells were added. After incubation at 37°C for four hours, 50 l of the supernatant per hole were transferred to a Luma plate (Packard) and dried. The radioactivity was measured in a O-counter (Trilux Microbeta, Wallac). The mean specific lysis was calculated according to the following formula: specific lysis (experimental release spontaneous release)/(complete release ,--spontaneous release) x 100.
WO 99/48518 21 PCT/EP99/01996 7. Induction of E7-specific cytotoxic T lymphocytes by CVLPs 11 six to sixteen week-old female C57BL/6 mice were immunized with 1-20 jig of CVLPs without adjuvant by means of a simple s.c. injection. Two weeks later, the spleen cells were prepared and stimulated in vitro with HPV-16 E7-expressing transfectants of the C57BL/6derived tumour cell line RMA (RMA-E7) as stimulator cells. After culturing for five days, the first cytotoxicity test was carried out.
The stimulation of the spleen cells was repeated at weekly intervals. As shown in Fig. 1, immunization with 1 gg of L1ACE7 1 -6 0 CVLPs, i.e. the carboxy-terminal 34 amino acids of HPV-16 Ll were replaced by the amino acids 1-60 of HPV-16 E7, led to a strong E7-specific CTL response.
Altogether, after five days a specific lysis of RMA-E7 transfectants of between 7.7% and 54.7% (average value 33.6%; E:T ratio 8:1-33:1) was observed. The average lysis after the second stimulation was between 35.8% and 57.8% (average value 45.3%; E:T ratio 8:1-44:1). If the mice were only immunized with 1 gg of LlACE71- 60 CVLPs, on average, after a culturing time of 5 days, a lower reactivity of E7-specific CTLs was observed average value 28.5%) than after immunization with 5 gg (17.2-44.7%; average value 33.9%) or 20 Ag (26.0-54.7%; average value 38.5). The spleen cells of the control mice (vaccinated with HBS buffer) showed no E7-specific reactivity after five days (0.3 average value E:T ratio 4:1-9:1).
L1ACE7 1 -6 0 CVLP contains a known H2-Db T-cell epitope of the E7 protein (E7 49 -5 7 Feltkamp, M.C.W. et al. (1993), supra). In order to confirm the obtained E7 specificity Of the CTLs, they were therefore tested against RMA cells which are loaded with the peptide E7 49 57 CTLs which recognize RMA-E7 transfectants showed a specific cytotoxic activity against E7 49 57 RMA or RMA-S target cells. The measured specific lysis varied between 21.2-25% after five days (E:T ratio 8:1-15:1, see Fig. In the peptide titration test carried out, after the third stimulation RMA-S cells were lysed to 70.5% or 71.4% (two CR lines were tested) after loading with E7 49 57 at a concentration of 1 jtg/ml (E:T ratio 40:1, see Fig. Even if RMA-S cells were provided with as little as 100 pg per ml of E7 49 57 peptide, a specific lysis of 36.2% and 47.2% was obtained. CTLs which are loaded with a control peptide, i.e. the influenza virus nucleoprotein-derived peptide having the amino acids 366-374, which represent a Db-CTL epitope, were not -recognized. Spleen cells which were isolated from C57BL/6 mice after treatment with L1ACE71- 55 CVLPs (E7 sequence truncated by five amino acids), did not recognize RMA-E7 cells, even if the mice were vaccinated with an increasing dose of up to 250ug.
From these data, it is concluded that the CTLs were induced by LIACE71- 60 CVLPs, are E7-specific and recognize the peptide E7 4 9 57 with high affinity. In an FACS analysis of representative CTL lines, the CTLs were identified as CD8-positive.
8. Prevention of the growth of a syngenic E7-expression tumour in C57BL/6 mice In order to determine whether the vaccination with L1ACE71- 6 0 CVLPs induce an 20 effective immunity against syngeneic tumour cells (TC-1, Lin, K-Y. (1996), Cancer Res.
56, 21-26), tumour protection experiments were carried out. C57BL/6 mice (3-5 per group) received 1 s.c. injection of 10 itg of L1ACE71- 60 CVLPs, 10 ig of *o [R:\LIBVV]0261 I0.doc:ais WO 99/48518 23 PCT/EP99/01996 L1ACE71- 55 CVLPs, 10 jAg of LIAC VLPs or an equivalent volume of HBS buffer without adjuvant. Two weeks after the immunization, 6 x 104 TC-1 cells, suspended in 200 pl of PBS, were inoculated into the left flank of the mice. The tumour size was measured twice per week for 2 months until the mice were killed.
The results of all experiments are summarized in Table 1. The total tumour growth rate in the mice which were vaccinated with HBS buffer, L1AC VLPs or LlACE71- 5 CVLPs was on average 80.5%. Vaccination with LIAC CVLPs did not protect against the tumour, although delayed tumour growth was observed (see Fig. However, vaccination of mice with LlACE7 1 60 CVLPs protected against tumour growth: only one mouse (1/13) developed a small, slowly growing tumour after 38 days, which regressed within a two-month period.
Spleen cells of HBS or LlAC VLP-vaccinated mice which developed TC-1 tumours showed no or only small levels of an E7-specific CTL response, independently of whether the mice had developed tumours or not. After the 2nd stimulation, an E7-specific lysis of RMA-E7 target cells of between 0-21% in tumour-bearing mice (average value 14%, E:T ratio 5:1-24:1), and of between 0 and 22.2% (average value 10.4%, E:T ratio 7:1-40:1), was observed in tumour-free mice. In contrast to this, in mice which were protected against tumour growth after the immunization with CVLP LlACE7 1 60 an E7specific CTL response of between 29.1 and 49.8% (average value 36.9%, E:T ratio 11:1-24:1) was found after the 2nd stimulation.
It was furthermore analysed whether the vaccination leads to a regression of already existing tumours.
Two weeks after the inoculation of 6 x 104 TC-1 cells I the mice (5 per group) received a single injection of 10 gg of LlACE71- 60 CVLPs, 10 Ag of WO 99/48518 24 PCT/EP99/01996 VLP L1AC or HBS buffer without adjuvant. In this experiment (see Fig. the tumour development of control mice which were either treated with HBS or immunized with VLP L1AC was only 60% (3 of each group, 6/10). However, all mice which were vaccinated with L1ACE71- 60 CVLPs generated an immune response against the established tumours and remained tumourfree two months after the tumour cell injection.
Claims (89)
1. A medicament comprising at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, wherein the fusion protein contains no papillomavirus-unspecific epitopes, when used for the prevention or treatment of human papillomavirus (HPV)-specific tumours.
2. The medicament when used according to claim 1, wherein the tumour is a carcinoma of the larynx, cervix, penis, vulva or anus.
3. The medicament when used according to claim 1 or 2, wherein the medicament contains no adjuvant.
4. The medicament when used according to one of claims 1 to 3, wherein the additive or excipient is about 0.3 to about 4 M, of a salt having a pH of about 7.3 to about
7.45. 5. The medicament when used according to claim 4 wherein the additive or i5 excipient is about 0.4 to about 3 M of a salt having a pH of about 7.3 to about 7.45. 6. The medicament when used according to claim 4 wherein the additive or excipient is about 0.5 to about 2 M of a salt having a pH of about 7.3 to about 7.45. 7. The medicament when used according to claim 4 wherein the additive or excipient is about 1 to about 2 M of a salt having a pH of about 7.3 to about 7.45.
8. The medicament when used according to any one of claims 4 to 7 wherein the pH is about 7.4.
9. The medicament when used according to any one of claims 4 to 8, wherein the salt is an alkali metal or alkaline earth metal salt. The medicament when used according to claim 9 wherein the salt is a halide or phosphate.
11. The medicament when used according to claim 10 wherein the salt is an alkali metal halide.
12. The medicament when used according to claim 11 wherein the salt is NaCI and/or KC1.
13. The medicament when used according to any one of claims 4 to 12, wherein the pH is adjusted using a buffer.
14. The medicament when used according to claim 13 wherein the buffer is a phosphate buffer, tris buffer, HEPES buffer or MOPS buffer. The medicament when used according to any one of the preceding claims, wherein the LI protein is a deleted LI protein. [I:\DAYLIB\LIBVV]02609.doc:ais
16. The medicament when used according to claim 15, wherein the LI protein is a C-terminally deleted LI protein.
17. The medicament when used according to claim 15 or 16, wherein up to about amino acids are deleted from the LI protein. s 18. The medicament when used according to claim 17 wherein about 25 to about amino acids are deleted from the LI protein.
19. The medicament when used according to claim 17 wherein about 32 to 34 amino acids are deleted from the LI protein. The medicament when used according to any one of the preceding claims, wherein the E protein is a deleted E protein.
21. The medicament when used according to claim 20 wherein the deleted E protein is a deleted E6 and/or E7 protein.
22. The medicament when used according to claim 20, wherein the deleted E protein is a C-terminally deleted E protein.
23. The medicament when used according to claim 22 wherein the C-terminally Sdeleted E protein is a C-terminally deleted E7 protein.
24. The medicament when used according to any one of claims 20 to 23, wherein up to about 55 amino acids are deleted.
25. The medicament when used according to claim 24 wherein about 5 to about 55 amino acids are deleted.
26. The medicament when used according to claim 24 wherein about 38 to about 43 amino acids are deleted.
27. The medicament when used according to any one of the preceding claims, wherein the fusion protein is present in the form of a capsid and/or capsomer.
28. The medicament when used according to any one of the preceding claims, wherein the HPV is selected from HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-42, HPV-45, HPV-52 and/or HPV-58.
29. The medicament when used according to claim 28, said medicament being in the form of a combination vaccine, wherein the papillomaviruses are selected from HPV- 16 and HPV-18 or HPV-18, HPV-31, HPV-45 and HPV-58 or HPV-6 and HPV-1 1. The medicament when used according to any one of the preceding claims, wherein the fusion protein is a L1ACE7 1 .x fusion protein, x being an integer from 55 up to and including
31. The medicament when used according to claim 30 wherein said fusion protein 35,1 is in the form of a CVLP. [I:\DAYLIB\LIBVV]02609.doc:ais 27
32. The medicament when used according to claim 30 or 31 wherein said fusion protein is a fusion protein of HPV-16.
33. The medicament when used according to any one of claims 30 to 32, wherein the fusion protein is an L1ACE71- 55 or LlACE71- 60 fusion protein.
34. A process for the production of a medicament comprising at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, the fusion protein containing no papillomavirus-unspecific epitopes, wherein a cell comprising an expression vector containing at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses is cultured under suitable conditions, the expression product is isolated and, if appropriate, suitable additives and excipients are added.
35. The process according to claim 34, wherein the LI protein is a deleted LI protein. 15 36. The process according to claim 35, wherein the LI protein is a C-terminally deleted LI protein.
37. The process according to claim 35 or 36, wherein up to about 35 amino acids are deleted from the LI protein.
38. The process according to claim 37 wherein about 25 to about 35 amino acids are deleted from the LI protein.
39. The process according to claim 37 wherein about 32 to 34 amino acids are deleted from the LI protein.
40. The process according to any one of claims 34 to 39, wherein the E protein is a deleted E protein.
41. The process according to claim 40 wherein the deleted E protein is a deleted E6 and/or E7 protein.
42. The process according to claim 40, wherein the deleted E protein is a C- terminally deleted E protein.
43. The process according to claim 42 wherein the C-terminally deleted E protein is a C-terminally deleted E7 protein.
44. The process according to any one of claims 40 to 43, wherein up to about amino acids are deleted. The process according to claim 44 wherein about 5 to about 55 amino acids A,>ar e deleted. I :\DAYLIB\LIBVV0209docai 28
46. The process according to claim 44 wherein about 38 to about 43 amino acids are deleted.
47. The process according to any one of claims 34 to 46, wherein the HPV is selected from HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-42, HPV-45, HPV-52 and/or HPV-58.
48. The process according to any one of claims 34 to 47, wherein the fusion protein is a LIACE7 1 -x fusion protein, x being an integer from 55 up to and including
49. The process according to claim 48 wherein said fusion protein is in the form of a CVLP.
50. The process according to claim 48 or 49 wherein said fusion protein is a fusion protein of HPV-16.
51. The process according to any one of claims 48 to 50, wherein the fusion protein is an L1ACE71. 55 or L1ACE71- 60 fusion protein.
52. The process according to any one of claims 34 to 51 wherein the expression vector contains a nucleic acid sequence that codes for said fusion protein which is free of additional amino acids.
53. Use of a fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses for the manufacture of a medicament, for the prevention of HPV-specific tumours or the regression of already existing HPV-specific tumours wherein said fusion protein contains no papillomavirus-unspecific epitopes.
54. The use according to claim 53 wherein the L protein is a deleted L protein. 54. The use according to claim 53 wherein the L protein is a deleted LI protein.
55. The use according to claim 54 wherein the LI protein is a C-terminally deleted LI protein.
56. The use according to claim 53 or 54 wherein up to about 35 amino acids are deleted from the LI protein.
57. The use according to claim 56 wherein about 25 to about 35 amino acids are deleted from the LI protein.
58. The use according to claim 56 wherein about 32 to 34 amino acids are deleted from the LI protein.
59. The use according to any one of claims 53 to 58 wherein the E protein is a deleted E protein. The use according to claim 59 wherein the deleted E protein is a deleted E6 E7 protein. 29
61. The use according to claim 59 wherein the deleted E protein is a C-terminally deleted E protein.
62. The use according to claim 61 wherein the C-terminally deleted E protein is a C-terminally deleted E7 protein.
63. The use according to any one of claims 59 to 62 wherein up to about amino acids are deleted.
64. The use according to claim 63 wherein about 5 to about 55 amino acids are deleted. The use according to claim 63 wherein about 38 to about 43 amino acids are deleted.
66. The use according to any one of claims 53 to 65 wherein the fusion protein is present in the form of a capsid and/or capsomer.
67. The use according to any one of claims 53 to 66 wherein the HPV is selected from HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-42, HPV-45, HPV-52 and/or HPV-58.
68. The use according to claim 67 said medicament being in the form of a combination vaccine, wherein the papillomaviruses are selected from HPV-16 and HPV- 18 or HPV-18, HPV-31, HPV-45 and HPV-58 or HPV-6 and HPV-11'.
69. The use according to any one of claims 53 to 68 wherein the fusion protein is a L1ACE7 1 -x fusion protein, x being an integer from 55 up to and including ~70. The use according to claim 69 wherein said fusion protein is in the form of a CVLP.
71. The use according to claim 69 or 70 wherein said fusion protein is a fusion protein of HPV-16.
72. The use according to any one of claims 69 to 71 wherein the fusion protein is an L1ACE71- 55 or L1ACE71- 60 fusion protein.
73. A method of preventing or treating human papillomavirus (HPV)-specific tumours in a patient, said method comprising administering to said patient a therapeutically effective amount of a medicament comprising at least one fusion protein from at least one LI protein of one or more papillomaviruses and at least one E protein of one or more papillomaviruses and, if appropriate, suitable additives and/or excipients, wherein the fusion protein contains no papillomavirus-unspecific epitopes.
74. The method according to claim 73, wherein the tumour is a carcinoma of the S larynx, cervix, penis, vulva or anus. [I:\DAYLIB\LIBVV]02609.doc:ais The method according to claim 73 or 74, wherein the medicament contains no adjuvant.
76. The method according to any one of claims 73 to 75, wherein the additive or excipient is about 0.3 to about 4 M, of a salt having a pH of about 7.3 to about 7.45.
77. The method according to claim 76 wherein the additive or excipient is about 0.4 to about 3 M of a salt having a pH of about 7.3 to about 7.45.
78. The method according to claim 76 wherein the additive or excipient is about to about 2 M of a salt having a pH of about 7.3 to about 7.45.
79. The method according to claim 76 wherein the additive or excipient is about 1 to about 2 M of a salt having a pH of about 7.3 to about 7.45. The method according to any one of claims 76 to 79 wherein the pH is about 7.4 oo••
81. The method according to any one of claims 76 to 80, wherein the salt is an alkali metal or alkaline earth metal salt. 15 82. The method according to claim 81 wherein the salt is a halide or phosphate.
83. The method according to claim 82 wherein the salt is an alkali metal halide.
84. The method according to claim 83 wherein the salt is NaCl and/or KC1. The method according to any one of claims 76 to 84, wherein the pH is adjusted using a buffer.
86. The method according to claim 85 wherein the buffer is a phosphate buffer, tris buffer, HEPES buffer or MOPS buffer.
87. The method according to any one of claims 73 to 86, wherein the L1 protein a deleted LI protein.
88. The method according to claim 87, wherein the L1 protein is a C-terminally deleted L1 protein.
89. The method according to claim 87 or 88, wherein up to about 35 amino acids are deleted from the L1 protein. The method according to claim 89 wherein about 25 to about 35 amino acids are deleted from the L1 protein.
91. The method according to claim 89 wherein about 32 to 34 amino acids are deleted from the L1 protein.
92. The method according to any one of claims 73 to 91 wherein the E protein is a deleted E protein. i 93. The method according to claim 92 wherein the deleted E protein is a deleted 6 and/or E7 protein. [I:\DAYLIB\LIBVV]02609.doc:ais 31
94. The method according to claim 92, wherein the deleted E protein is a C- terminally deleted E protein. The method according to claim 94 wherein the C-terminally deleted E protein is a C-terminally deleted E7 protein.
96. The method according to any one of claims 92 to 95, wherein up to about amino acids are deleted.
97. The method according to claim 96 wherein about 5 to about 55 amino acids are deleted.
98. The method according to claim 96 wherein about 38 to about 43 amino acids are deleted.
99. The method according to any one of claims 73 to 98 wherein the fusion protein is present in the form of a capsid and/or capsomer.
100. The method according to any one of claims 73 to 99 wherein the HPV is selected from HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-42, HPV-45, HPV-52 and/or HPV-58.
101. The method according to claim 100, said medicament being in the form of a combination vaccine, wherein the papillomaviruses are selected from HPV-16 and HPV- 18 or HPV-18, HPV-31, HPV-45 and HPV-58 or HPV-6 and HPV-11. 9S
102. The method according to any one of claims 73 to 101 wherein the fusion protein is a L1ACE7 1 Ix fusion protein, x being an integer from 55 up to and including
103. The method according to claim 102 wherein said fusion protein is in the form of a CVLP.
104. The method according to claim 102 or 103 wherein said fusion protein is a fusion protein of HPV-16.
105. The method according to any one of claims 102 to 104, wherein the fusion protein is an L1ACE71- 55 or L1ACE71- 60 fusion protein.
106. A medicament substantially as hereinbefore described with reference to any one of the examples when used for the prevention or treatment of human papillomavirus (HPV)-specific tumours.
107. A process for the production of a medicament substantially as hereinbefore described with reference to any one of the examples. [I:\DAYLIB\LIBVV]02609.doc:ais
108. A medicament produced by the process as claimed in any one of claims 34 to 52 or 107. Dated 2 October, 2002 MediGene Aktiengesellschaft Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 0:60 S009 [1ADAYLIB\LIBVV]02609.docas
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19812941A DE19812941A1 (en) | 1998-03-24 | 1998-03-24 | Medicines to prevent or treat papilloma virus-specific tumor |
DE19812941 | 1998-03-24 | ||
PCT/EP1999/001996 WO1999048518A2 (en) | 1998-03-24 | 1999-03-24 | Medicament for preventing or treating papilloma virus-specific tumors |
Publications (2)
Publication Number | Publication Date |
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AU3521499A AU3521499A (en) | 1999-10-18 |
AU755242B2 true AU755242B2 (en) | 2002-12-05 |
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Application Number | Title | Priority Date | Filing Date |
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AU35214/99A Ceased AU755242B2 (en) | 1998-03-24 | 1999-03-24 | Medicament for preventing or treating papilloma virus-specific tumors |
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EP (1) | EP1064014B1 (en) |
JP (1) | JP2002509863A (en) |
AR (1) | AR014768A1 (en) |
AT (1) | ATE288279T1 (en) |
AU (1) | AU755242B2 (en) |
CA (1) | CA2323573A1 (en) |
DE (2) | DE19812941A1 (en) |
DK (1) | DK1064014T3 (en) |
ES (1) | ES2237913T3 (en) |
MX (1) | MXPA00009282A (en) |
PT (1) | PT1064014E (en) |
WO (1) | WO1999048518A2 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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SE514982C2 (en) * | 1999-09-30 | 2001-05-28 | Active Biotech Ab | Carrier containing a major capsid protein L1 from human papillomavirus and its use |
DE10059630A1 (en) * | 2000-12-01 | 2002-06-06 | Medigene Ag | Medicines to prevent or treat tumor caused by human papillomavirus type 18 |
JP4614765B2 (en) † | 2002-05-17 | 2011-01-19 | ユニバーシティ・オブ・ケープ・タウン | Chimeric human papillomavirus 16L1 protein containing L2 peptide, virus-like particle prepared therefrom and method for preparing the particle |
JP5186678B2 (en) * | 2005-10-13 | 2013-04-17 | 財団法人ヒューマンサイエンス振興財団 | Antigen kit for transdermal antigen administration |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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DE4435907A1 (en) * | 1994-10-07 | 1996-04-11 | Lutz Prof Dr Gissmann | Recombinant papilloma virus-like particles contg. L1 or L2 proteins |
-
1998
- 1998-03-24 DE DE19812941A patent/DE19812941A1/en not_active Ceased
-
1999
- 1999-03-24 AU AU35214/99A patent/AU755242B2/en not_active Ceased
- 1999-03-24 ES ES99916884T patent/ES2237913T3/en not_active Expired - Lifetime
- 1999-03-24 DK DK99916884T patent/DK1064014T3/en active
- 1999-03-24 AT AT99916884T patent/ATE288279T1/en not_active IP Right Cessation
- 1999-03-24 DE DE59911556T patent/DE59911556D1/en not_active Expired - Fee Related
- 1999-03-24 JP JP2000537565A patent/JP2002509863A/en active Pending
- 1999-03-24 WO PCT/EP1999/001996 patent/WO1999048518A2/en active IP Right Grant
- 1999-03-24 PT PT99916884T patent/PT1064014E/en unknown
- 1999-03-24 CA CA002323573A patent/CA2323573A1/en not_active Abandoned
- 1999-03-24 MX MXPA00009282A patent/MXPA00009282A/en active IP Right Grant
- 1999-03-24 EP EP99916884A patent/EP1064014B1/en not_active Expired - Lifetime
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE4435907A1 (en) * | 1994-10-07 | 1996-04-11 | Lutz Prof Dr Gissmann | Recombinant papilloma virus-like particles contg. L1 or L2 proteins |
Non-Patent Citations (2)
Title |
---|
GREENSTONE, H ET AL.1998. PROC.NAT ACAD SCI USA 95(4) 1800-5 * |
MUELLER, M ET AL. 1997 VIROLOGY, V234, P 94-111 * |
Also Published As
Publication number | Publication date |
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WO1999048518A2 (en) | 1999-09-30 |
PT1064014E (en) | 2005-06-30 |
ES2237913T3 (en) | 2005-08-01 |
AU3521499A (en) | 1999-10-18 |
AR014768A1 (en) | 2001-03-28 |
DE59911556D1 (en) | 2005-03-10 |
EP1064014B1 (en) | 2005-02-02 |
DK1064014T3 (en) | 2005-05-30 |
MXPA00009282A (en) | 2002-12-13 |
EP1064014A2 (en) | 2001-01-03 |
WO1999048518A3 (en) | 1999-12-02 |
CA2323573A1 (en) | 1999-09-30 |
JP2002509863A (en) | 2002-04-02 |
DE19812941A1 (en) | 1999-10-07 |
ATE288279T1 (en) | 2005-02-15 |
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