CA2323526A1 - Formulation having a papilloma virus-specific protein, and the production and use thereof - Google Patents

Abstract

The invention relates to a formulation containing at least one papilloma virus-specific protein with approximately 0.3 to approximately 4 M of a salt with a pH value of approximately 7.3 to approximately 7.45.

Description

Formulation with papillomavirus-specific protein, its preparation and use The present invention relates to a formulation comprising at least one papilloma virus-specific protein with about 0.3 to about 4M of a salt at a pH of about 7.3 to about 7.45.
Papillomaviruses, also called wart viruses, are double-stranded DNA viruses ha-ving a genome size of approximately 8000 base pairs and an icosahedron-like to 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-45, HPV-52 or HPV-58, can cause malignant tumours and others, e.g. HPV-6, HPV-11 or HPV-42, can cause benign tumours.
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
(ear-ly) region, contains various protein-coding sections E 1-E7, of which, for example, the E6 and the E7 protein are responsible for the transformation of epithelial cells 2o and the E 1 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 two protein-coding secti ons L1 and L2, which code for structural components of the virus capsid. The protein is present to over 90% in the viral capsid, the ratio of L1: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-45, HPV-52 and HPV-58 are associated with malignant tumours of the ano-3o genital tract. In over 50% of the cases, HPV-16 is connected with cervical cancer (carcinoma of the cervix). HPV-16 is 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 lay-ers of the infected epithelium. The E7 protein is therefore considered as a potenti-al tumour antigen and as a target molecule for activated T cells. The E7-induced cellular immune response in the patient, however, is apparently not strong enough "' to to influence the course of the disease. The immune response can possibly be am-plified by suitable vaccines.
It has now been possible to show that the expression of the L1 gene or the coex-pression of the L1 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 elimination of virus-infected cells a virus-specific cytotoxic T-cell (CTL) respon-se appears to be necessary. So-called chimeric papillomavirus-like particles (CVLPs) were therefore developed which consist of a chimeric L1-E7 protein (Miiller, M. et al. (1997) Virology, 234, 93): some CVLPs induce an E7-specific CTL response in mice, although experiments failed to induce antibodies against E7 by immunization of mice with CVLPs (Miiller, M. et al. ( 1997), supra). In addition, neutralizing antibodies of HPV-associated disorders in patients appear to limit the immune response to administered L1 protein (Miiller, 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.
3o Peng et al. (1998) Virology, 240, 147 now describe CVLPs consisting of C-terminally truncated L1 of the bovine papillomavirus (BPV) and HPV-16E7a9_5~, which induce E7-specific cytotoxic T cells after inoculation of C57B116 mice and protect against the growth of E7-expressing tumours. Green-stone et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 1800 describe CVLPs consi-sting of HPV-16L1 plus HPV-16L2 fused to the full-length 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 Kirnbauer, R. et al. (1994) J. Virol., 67, 6929-6936 or obtainable via the EMBL databank. The accession numbers are, for example, for HPV 18:
PAPHPV 18; for HPV31: PAPPPH31; for HPV33: PAPPPH33 or for HPV~8:
PAPPPH58. Suitable expression systems are, for example, yeasts modified by genetic engineering, e.g. Saccharomyces (cerevisiae), Pichia (pastoris), Kluyver-myces (lactis), Schizosaccharomyces (pombe) or Hansenula (polymorpha) (Car-ter, J. J. et al. (1991), Virology, 182, 513), insect cells, such as, for example, Trichoplusia ni High Five (see, for example, Miiller, M. 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 significant problem in the use of capsids and capsomers as medicaments is their poor solubility. Thus, for example, capsids or capsomers of HPV-16 tend to ag-gregate, whereby the solubility is significantly reduced. The low solubility of the capsids or capsomers in some cases leads not only to a loss of yield, but also to 3o complicated use as a medicament or diagnostic. Moreover, sometimes degradation of the C terminus of the L 1 protein can be observed, which leads to inhomogene-ous material which is not suitable for licensing as a medicament or diagnostic.
WO 98/44944 describes an HPV antigen formulation comprising a vaccine com-ponent, a salt to physiologically acceptable concentrations and a non-ionic deter-gent to physiologically acceptable concentrations.
It was therefore the object of the present invention to make available a simple and advantageous formulation in which papillomavirus-specific proteins are soluble 1o and stable.
It has now surprisingly been found that the solubility of papillomavirus-specific proteins is dependent on the salt concentration and on the pH of the formulation.
In particular, it was surprising that a composition without addition of an excipient is not stable at neutral pH in an isotonic salt solution of about 100-150 mM
salt, although VLPs or CVLPs are formed in the cytoplasm of the host cells. In additi-on, it was found according to the present invention that at a pH of less than about 5.5 the CVLPs precipitate and at a pH of greater than 9.5 the CVLPs aggregate.
2o One subject of the present invention is therefore a formulation comprising at least one late protein (L protein) of one or more papillomaviruses and/or at least one early protein (E protein) of one or more papillomaviruses and about 0.3 to about 4 M, preferably about 0.4 to about 2.5-3 M, in particular about 0.4-0.5 to about 1-2 M, especially about 1 to about 2 M, of a salt at a pH of about 7.3 to about 7.45, preferably about 7.4, and, if appropriate, suitable additives and/or excipients, preferably without any additive and/or excipient.
The term "formulation" is understood according to the present invention as mea-ning a composition in the form of a solution or a suspension of the papillomavi-3o rus-specific proteins mentioned, where immunoreactive papillomavirus-specific proteins in general and in particular do not significantly sediment at up to at most about 5000 g.
The salt is in general an alkali metal or alkaline earth metal salt, preferably a hali-de or phosphate, in particular an alkali metal halide, especially NaCI and/or KCI.
Use of NaCI is particularly preferred for the production of a pharmaceutical for-mulation.
The pH of the composition is in general adjusted using a suitable organic or inor-1o ganic 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-1-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.
For the use of the composition according to the invention as a medicament or dia-gnostic it is particularly preferred if the papillomavirus-specific proteins contain no papillomavirus-unspecific epitopes, since hereby a papillomavirus-unspecific 2o immune response can be reduced or prevented.
The terms L protein and E protein are understood within the meaning of the pre-sent invention as meaning both the full-length proteins and their mutants, such as, for example, deletion mutants.
In a further preferred embodiment, the composition according to the invention contains a deleted L protein, preferably a deleted L1 and/or L2 protein. The dele-tion has the advantage that different proteins, for example papillomavirus-specific E protein sequences, can be inserted into the deleted area, whereby the application 3o 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 efficien-cy of the formation of virus-like particles can be increased, since the nuclear loca-tion signal located at the C terminus is deleted. The C-terminal deletion is therefo-re 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-16 L1 protein 32 amino acids long and a C-terminal deletion of the BPV-1 L1 protein (bovine papillomavirus type 1) about 26 amino acids long is adequate to be able to increase the formation of virus-like particles by at least about 10-fold.
to 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 about 55 amino acids are particularly preferred, preferably about 5 to about 55 amino acids, in particular about 38 to about 43 amino acids.
A particularly preferred construct is, for example, E7 having the N-terminal ami-no acids 1 to about 60, as this construct contains a mouse epitope for the activati-on of cytotoxic T lymphocytes, which is located in the area of the amino acids 49-57. Another preferred construct is E7 having the N-terminal amino acids I
to about 55, which preferably forms capsomers and capsids in combination with de-leted L protein, as this construct presumably does nut contain E7-specific sequen-ces in the area of the amino acids 56-70, which can interfere with the formation of capsids. An Ll protein of HPV-16 C-terminally deleted by 32 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 prevent the formation of tumours and on the other hand cause regression of already existing tumours in animal ex-periments. E7 having the amino acids 1-60 especially exhibits a marked prophy-lactic and therapeutic action in tumours. A particularly preferred embodiment of -7_ the present invention is therefore an L1~E71_r fusion protein, preferably in the form of a CVLPS, in particular of HPV 16, x being an integer from 55 up to and including 60, and in particular an LlaCE7~_55 or L1~CE7i-6o fusion protein.
For the production of a medicament which is active both prophylactically and therapeutically, it is preferred if the protein is bonded to the E protein, for ex-ample in the form of a fusion protein. It is furthermore preferred if the described papillomavirus-specific proteins are present in the form of a capsid and/or capso-mer, since the immune reaction can additionally be markedly increased by the to capsids and/or capsomers and in particular by the fraction of L protein.
Preferred fusion proteins which are suitable for capsid and/or capsomer formation are there-fore, for example, fusion proteins from deleted L1 and E7, E6 and/or El.
Capsids within the meaning of the present invention are viral or virus-like structu-res in a generally icosahedral form, which in general are constructed of about 72 capsomers.
Capsomers within the meaning of the present invention are assembled proteins comprising at least one papillomavirus structural protein, preferably L 1 or deleti-ons of L1. For example, 5 fusion proteins can be assembled to give a capsomer which in turn can be assembled to give a capsid.
For the production of a human medicament or diagnostic, 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 con-structs described. Especially for the production of a combination vaccine, it is advantageous to combine proteins or peptides from various HPV types, for ex-ample a combination of HPV-16 and HPV-18 or HPV-18, HPV-31, HPV-45 and 3o 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.

_g_ 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). Ex-amples of eukaryotic expression vectors are, for expression in Saccharomyces cerevisiae, e.g. the vectors p426Met25 or p426GALl (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768, Carter, J. J. et al. (1991) supra) and, for expres-sion in insect cells, e.g. Baculovirus vectors, in particular the Autographa Califor-nica virus, such as disclosed in EP-Bl-0 127 839 or EP-B1-0 549 721 (see, for example, also WO 94/20137), and, for expression in mammalian cells, e.g. the l0 vectors Rc/CMV and Rc/RSV or SV40 vectors which are all generally obtainable.
However, commercially obtainable Baculovirus expression systems are also sui-table, such as, for example, the Baculo GoldTT'' transfection kit from Pharmingen or the Bac-to-BacTM Baculovirus expression system from Gibco BRL. Further suitable expression systems are recombinant vaccinia viruses (see, for example t5 WO 93/02184).
In general, the expression vectors also contain promoters suitable for the respecti-ve host cell, such as, for example, the trp promoter for expression in E. coli (see, for example, EP-B1-0 154 133), the ADH2 promoter for expression in yeasts 20 (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).
25 Suitable host cells are, for example, the E. coli strains DHS, HB101 or BL21, the yeast strains Saccharomyces cerevisia, Pichia, Kluyvermyces, Schizosaccharomyces 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 3o 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 (poly-merase chain reaction) amplification. For example, the genome of BPV-1 is gene-rally obtainable under the GenBank Accession No. X02346 or HPV-16 under the GenBank Accession No. K02718. An HPV-16 L1 sequence is also disclosed, for example, in WO 94/05792. The sequence of the 98 amino acid-long HPV 16 E7 protein is described, for example, in Seedorf et al. (1985) Virology, 145, 185. Another method of obtaining the desired nucleic acids is to isolate the papil-lomavirus-specific genes directly from warts or tumours by means of PCR. Sui-1o table 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, Kirnbauer, 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 al. (1989) Gene, 77, 51-59). In this way, a fusion protein is obtained which is free of additional amino acids and thus free of 2o possible additional foreign epitopes which can cause immunological side reacti-ons.
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; Hofmann, K. J. et al. (1995) Virology, 209, 506; Rose, R. C. et al. (1993) J. Virol., 67, 1936, Sasagawa, T. et al. (1995) Viro-logy, 206, 126 or WO 95/31532..
3o Suitable additives and/or excipients which serve, for example, for the further sta-bilization of the papillomavirus-specific protein in the composition 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 com-pounds, such as, for example, amino acids such as glycine or in particular taurine or betaine and/or protein, such as, for example, bovine or human serum albumin.
Detergents, polyols and/or zwitterionic compounds are preferred.
Other suitable additives and/or excipients are protease inhibitors, such as, for ex-ample, aprotinin, E-aminocaproic acid or pepstatin A.
Another subject of the present invention is a process for the production of the formulation according to the invention, in which the papillomavirus-specific pro-tein described above is introduced, for example, into solution comprising about 0.3 to about 4 M, preferably about 0.4 to about 2.5-3 M, in particular about t5 0.4-0.5 to about 1-2 M, especially about 1 to about 2 M, of a salt at a suitable pH
of about 7.3 to about 7.45, preferably about 7.4, and, if appropriate, suitable addi-tives and excipients, andlor is dialysed against the described composition.
The formulation can preferably be stably stored at about 4°C or especially about -80°C over a relatively long period of time, for example 1-2 months or longer.
The formulation according to the invention is suitable as a medicament or diagno-stic. The present invention therefore also relates to the use of the formulation ac-cording to the invention as a medicament or diagnostic. For the immediate use as a medicament or diagnostic, the formulation according to the invention is pre-ferably adjusted to a concentration of about 0.45 M. In particular, it is preferred if the medicament contains no adjuvant, i.e. no substance which amplifies the im-munogenicity of the papillomavirus-specific protein, since the immunogenicity is already adequately amplified, in particular in the presence of an L protein espe-cially of Ll. This property is particularly advantageous in the licensing as a medi-cament or diagnostic, as the only immunostimulating materials at present licensed by the licensing authorities are aluminium salts.

The medicament is particularly suitable for the avoidance and/or treatment of pa-pillomavirus-specific benign or malignant tumour, in particular of malignant tu-mour, such as, for example, carcinoma of the larynx, cervix, penis, vulva or anus, and the diagnostic for the diagnosis of one or more papillomavirus infections.
An example of a diagnostic is the immunodiagnostic known to the person skilled in the art, for example an ELISA for the measurement of papillomavirus-specific antibodies (see, for example, Voller, A. et al. (1976) Bull. World Health Organ., 53, 55-63) or a skin test according to, for example, Hopfl et al. (1991) Lancet. l, 373-374).
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 injec-tion or infusion solution. In the case of the formulations according to the inventi-on, an adjuvant can be dispensed with, which is particularly advantageous.
A further subject of the present invention therefore relates to the use of the for-mulation according to the invention as an injection ar infusion solution.
2o Injection solutions are in general used if only relatively small amounts of a soluti-on 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 con-trast 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 noticeable to an insignificant extent with respect to pain sensation. Dilution of the formulation according 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 isotonic solution can be obtained. An example of an isotonic solution is a 0.9% strength sodium chlori-de 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 significant advantage of the present invention is that the formulation accor-ding to the invention essentially does not lead to precipitation of immuno-reactive papillomavirus-specific protein. In particular, more than about 90%, especially more than about 95%, of the protein remains in solution and does not precipitate 1o for a period of time of at least about 12 hours. The immunoreactive papillomavi-rus-specific protein is also not substantially sedimentable by centrifugation at a maximum of 5000 g. In addition, the formulation remains homogeneous and sta-ble over a relatively long period of time of about 1-2 months and longer.
The figure and the following examples are intended to illustrate the invention in greater detail without restricting it.
Fig. 1 shows graphically the dependence of the solubility of virus-like particles on the salt concentration.
Examples 1. Preparation of chimeric Qenes coding for HPV 16L 1 E7 fusion proteins HPV 16L1~C* E7 1-55 was prepared according to Miiller, M. et al. (1997), supra.
The HPV-16L1 open reading frame (ORF) was in this case excized from the plasmid HPV-16-114/k-L1/L2-pSynxtVI- (Kirnbauer, R. et al. (1994) J. Virol.
67, 6929) using the restriction endonuclease BgIII and cloned into the BamHI site in the vector pUCl9 (New England Biolabs).
For the preparation of HPV-l6Ll~C, two primers were constructed which are complementary to HPV-16L1 ORF. The first primer has the sequence AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC
and the second primer AAAGATATCTAATCTACCTCTACAACTGCTAAACGCA~AAA.AACG.
Both primers encode an EcoRV restriction enzyme cleavage site 5'. In the primers lying downstream, a 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 L1 ORF and the entire vector. The linear product was cleaved with EcoRV, circularized with T4 DNA ligase and transfor-1o med E. coli DHSa cells. The clones were analysed for the presence of an EcoRV
site. The construct pUCHPV16L10C 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:
AAA.AGATATCATGCATGGAGATACACCTACATTGC
and TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC.
2o The PCR products were cleaved with EcoRV and inserted into the EcoRV site of the modified L1 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-HPV16L10CE7 1-50. The resulting DNA fragments overlapped in the position of the L1IE7 boun-dart' (Four Primer PCR, Ho, S. N. et al ( 1989) Gene 77, 51 ). However, the pri-mers did not contain the two EcoRV restriction enzyme cleavage sites. Frag-ment 1 was prepared using the primers P1 and P2 and fragment 2 using the pri-mers P3 and P4.

P 1: GTTATGACATACATACATTCTATG (L 1 ) P2: CCATGCATTCCTGCTTGTAGTAAAAATTTGCGTCC (E7) P3: CTACAAGCAGGAATGCATGGAGATACACC (E7) P4:CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCTG (E7) A tenth of the purified products was mixed and used as a matrix in the PCR
reac-tion with the primers P 1 and P4 exclusively. The resulting product was cleaved using EcoNI (L1) and HindIII (downstream of the stop codon on the primer P4) and used in order to replace an EcoNI/HindIII fragment of the cloned 1o HPV16L10RF. The resulting clone therefore differs from the clone HPVI6Ll~CE7 1-SO by the loss of the two internal EcoRV restriction enzyme cleavage sites and the corresponding non-HPV amino acids Asp and Ile between the L 1 ORF and E7 and downstream of E7. The first EcoRV site was replaced by the original L1 amino acids in this position (AlaGly). The second EcoRV site was t5 replaced by a translation stop signal. This clone (HPVI6Ll~C*E7 1-52) additio-nally contains the first 52 amino acids of HPV 16E7. Clone HPV l6LlOC*E7 1-52 was used for the preparation of the clones HPV16L.10C*E7 1-55 with the aid of the primer P 1 in combination with PS.
2o P5: CATCTGAAGCTTATCAATATTGTAATGGGCTCTGTCCG (E7 1-55) In all cases, EcoNI and HindIII were used in order to replace the corresponding fragments. The clones were analysed by DNA sequencing.
25 2. Preparation of recombinant baculoviruses Spodoptera frugiperda (Sf~) cells were used as a monolayer or in suspension cul-ture in TNM-FH insect medium (Sigma, Deisenhofen) with 10% foetal calf serum and 2 mM glutamine. Recombinant baculoviruses HPV16L1~CE7 1-SS were 3o transfected by cotransfection of 10 pg of the recombinant plasmids and 2 pg of linearized Baculo-Gold DNA (Phanningen, San Diego, CA) into S~ cells. Re-combinant viruses were purified according to the instructions of the manufacturer.
In order to test the expression, 106 Sf~ cells were infected with recombinant Baculovirus and an m.o.i. (multiplicity of infection) of 5 to 10. After the incubati-on, the medium was removed and the cells washed with PBS (140 mM NaCI, 2.7 mM KC1, 8.1 mM Na2P04, 1.5 mM KHzP04, pH 7.2). The cells were then lysed in SDS sample buffer and tested by SDS gel chromatography and immuno-blot assay.
3. Purification of virus-like particles For the preparation of CVLPs, Trichoplusia ni (TN) High Five cells were cultured at 27°C up to a density of 1-1.5 x 106 cells per ml in Ex-Cell 405 serum-free me-dium (JRH, Biosciences, Lennexa, KS). A 400 ml culture was harvested and in-fected with an m.o.i. of 2 to S with recombinant baculoviruses for one hour with periodic inversions. Up to 240 ml of medium were added and the cells grew for to 4 days. The cells were then pelleted and resuspended in 10 ml of extraction buffer (25 mM tris/HCI, pH 7.5; 500 mM NaCI, 1 mM EDTA) 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, sonicated for 30 seconds at 60 watts and centrifuged again. The supernatants were combined and applied to a two-stage gradient of 40% (w/v) sucrose and 57.5% (w/v) CsCI. After centrifuga-tion in an SW-28 rotor at 27,000 rpm for two hours, the interphase and the CsCI
layer were collected, adjusted to a CsCI 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 (Pharmingen, 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 ( 1 mM Hepes, 149 mM NaCI, 0.5 mM KCI, pH 7.2) and the presence of CVLPs was confirmed by means of transmission electron 3o 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. Microdialysis experiments The sample used was a fraction containing virus-like particles which had been isolated from High Five cells by sucrose cushion and caesium chloride equilibri-um ultracentrifugation. The total protein concentration was 0.29 mg/ml and the CVLP concentration 0.17 mg/ml.
40 ml of the corresponding solution were introduced into a 50 ml plastic vessel with a screw closure. On this solution was carefully placed a dialysis filter having a pore diameter of 0.025 Vim, which floats on the liquid during the carrying-out of the dialysis. 30 pl of the pure CVLP solution were pipetted onto this filter and the ~5 vessel was sealed. The vessel was allowed to stand at 4-6°C for at least 12 hours so that the solution of the drop was exchanged for the dialysis solution (50 mM
tris/HCI, pH 7.5 with increasing NaCI concentration). The drop was removed using the piston pipette and it was equalized with 3() p,l of reservoir solution. After centrifugation at 10,000 g (10 min, 4°C), the supernatant was investigated in the 2o ELISA (Kemeny, D. M. (1994) indirect ELISA from: ELISA, use of the enzyme-linked immunosorbent assay in the biologicaUmedicinal laboratory, Gustav Fi-scher Verlag, Stuttgart, p. 111, Test 6.2) using a conformation-specific monoclo-nal antibody against HPV16L1 and in a protein assay. The protein concentration was determined using a bicinchoninic acid assay (Smith, P. K. et al. (1985) Anal.
25 Biochem., 150, 76-85) against bovine serum albumin as a standard. The result is shown in Fig. 1.

Sequence Listing < 110> MediGene Aktiengesellschaft <120> Formulation having a papilloma virus-specific protein, and the pro-duction and use thereof to <150> 198 12 940.8 <151> 1998-03-24 <160> 9 i 5 < 170> FastSEQ for Windows Version 3.0 <210> 1 <211 > 40 <212> DNA
20 <213> artificial sequence <220>
<223> Oligonucleotide PCR-primer that introduces a restriction site.
25 <400> 1 aaagatatct tgtagtaaaa atttgcgtcc taaaggaaac 40 <210> 2 30 <211 > 44 <212> DNA
<213> artificial sequence <220>
35 <223> Oligonucleotide PCR-primer that introduces a restriction site.
<400> 2 aaagatatct aatctacctc tacaactgct aaacgcaaaa aacg 44 <210> 3 <211> 35 <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide PCR-primer that introduces a restriction site.
<400> 3 to aaaagatatc atgcatggag atacacctac attgc 35 <210> 4 <211> 34 <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide PCR-primer that introduces a restriction site.
<400> 4 ttttgatatc ggctctgtcc ggttctgctt gtcc 34 <210> 5 <211> 24 <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide primer for "Four primer PCR"
<400> 5 gttatgacat acatacattc tatg 24 <210> 6 <211> 35 <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide primer for "Four primer PCR"
a5 <400> 6 ccatgcattc ctgcttgtag taaaaatttg cgtcc 35 <210> 7 so <211> 29 i <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide primer for "Four primer PCR"
<400> 7 ctacaagcag gaatgcatgg agatacacc 29 <210> 8 <211> 36 <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide primer for "Four primer PCR"
<400> 8 catctgaagc ttagtaatgg gctctgtccg gttctg 36 <210> 9 <211> 38 <212> DNA
<213> artificial sequence <220>
<223> Oligonucleotide PCR-primer that introduces three additional codons for a C-terminal extension of the coded fusion protein.
<400> 9 catctgaagc ttatcaatat tgtaatgggc tctgtccg 38

Claims (29)

Claims

1. Formulation comprising at least one late protein (L protein) of one or more papillomaviruses and/or at least one early protein (E protein) of one or more papillomaviruses and 0.3 to 4 M of a salt at a pH of 7.3 to 7.45, the stabilization of the protein taking place essentially via the salt concentration and the pH.

2. Formulation according to Claim 1, characterized in that the salt concentration is 0.4 to 3 M at a pH of 7.3 to 7.45.

3. Formulation according to Claim 1 or 2, characterized in that the salt concentration is 0.5 to 2 M at a pH of 7.3 to 7.45.

4. Formulation according to one of Claims 1 to 3, characterized in that the salt concentration is 1 to 2 M at a pH of 7.4.

5. Formulation according to one of Claims 1 to 4, characterized in that the salt is an alkali metal or alkaline earth metal salt.

6. Formulation according to one of Claims 1 to 5, characterized in that the pH is adjusted using a buffer.

7. Formulation according to one of Claims 1-6, characterized in that the protein or proteins mentioned contains no papillomavirus-unspecific epitopes.

8. Formulation according to one of Claims 1-7, characterized in that the L protein is a deleted L protein.

9. Formulation according to Claim 8, characterized in that the L protein is a C-terminally deleted L protein.

10. Formulation according to Claim 8 or 9, characterized in that up to 35 amino acids are deleted from the L protein.

11. Formulation according to one of Claims 1-10, characterized in that the E protein is a deleted E protein.

12. Formulation according to Claim 11, characterized in that the deleted E
protein is a C-terminally deleted E protein.

13. Formulation according to Claim 11 or 12, characterized in that up to 55 amino acids are deleted.

14. Formulation according to one of Claims 1-13, characterized in that the L protein is bonded to the E protein.

15. Formulation according to one of Claims 1-14, characterized in that the protein mentioned is present in the form of a capsid and/or capsomer.

16. Formulation according to one of Claims 1-15, characterized in that the papillomavirus is a human papillomavirus (HPV).

17. Formulation according to Claim 16, characterized in that 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.

18. Formulation according to one of Claims 1-17, characterized in that the formulation has additives and/or excipients which do not significantly contribute to the stabilization of the protein.

19. Formulation according to Claim 18, characterized in that the additives and/or excipients are one or more detergents, polyols and/or zwitterionic compounds.

20. Process for the production of a formulation according to one of Claims 1-19, characterized in that the protein mentioned is incorporated into and/or dialysed against a solution comprising 0.3 to 4 M of a salt at a pH
of 7.3 to 7.45.

21. Process according to Claim 20, characterized in that the protein mentioned is incorporated into and/or dialysed against a solution comprising 0.4 to 3 M of a salt at a pH of 7.3 to 7.45.

22. Process according to Claim 20 or 21, characterized in that the protein mentioned is incorporated into and/or dialysed against a solution comprising 0.5 to 2 M of a salt at a pH of 7.3 to 7.45.

23. Process according to one of Claims 20 to 22, characterized in that the protein mentioned is incorporated into and/or dialysed against a solution comprising 1 to 2 M of a salt at a pH of 7.4.

24. Use of a formulation according to one of Claims 1-18 as a medicament or diagnostic.

25. Use according to Claim 24, characterized in that the formulation contains no adjuvant.

26. Use according to Claim 24 or 25, characterized in that the medicament serves for the avoidance or treatment of papillomavirus-specific tumour.

27. Use according to Claim 26, characterized in that the tumour is a carcinoma of the larynx, cervix, penis, vulva or anus.

28. Use according to one of Claims 24-27, characterized in that the formulation mentioned is used as an injection or infusion solution.

29. Use according to Claim 24, characterized in that the diagnostic serves for the diagnosis of one or more papilomavirus infections.