EP1066321A2

EP1066321A2 - Formulation having a papilloma virus-specific protein, and the production and use thereof

Info

Publication number: EP1066321A2
Application number: EP99917850A
Authority: EP
Inventors: Alexander Burger; Josef Gabelsberger
Original assignee: Medigene AG
Current assignee: Medigene AG
Priority date: 1998-03-24
Filing date: 1999-03-24
Publication date: 2001-01-10
Also published as: AR014969A1; CA2323526A1; JP2002507625A; WO1999048917A3; DE19812940A1; AU3598999A; WO1999048917A2; MXPA00009283A

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 production and use

The present invention relates to a formulation comprising at least one papillomavirus-specific protein with about 0.3 to about 4 M of a salt at a pH of about 7.3 to about 7.45.

The papilloma viruses, also called wart viruses, are double-stranded DNA viruses with a genome size of about 8000 base pairs and an icosahedral capsid with a diameter of approx. 55 nm. To date, more than 100 different types of human papilloma virus are known, some of which, for example, HPV 16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 or HPV-58, malignant tumors and others, e.g. HPV-6, HPV-1 1 or HPV-42, can cause benign tumors

The genome of the papillomaviruses can be divided into three areas. The first area concerns a non-coding region that contains regulatory elements for the transcription and replication of the virus. The second region, so-called E- (early) region, contains different protein-coding sections E1- E7, of which, for example, the E6 and E7 proteins are responsible for the transformation of epithelial cells and the E1 protein controls the DNA copy number. The E6 and E7 regions are so-called oncogenes, which are also malignant in malignant cells Cells are expressed. The third region, also known as the L (late) region, contains two protein-coding sections L1 and L2 which code for structural components of the virus capsid. The Ll protein is present to over 90% in the viral capsid, whereby the Ratio of Ll L2 is generally 30 1

HPV-6 and HPV-11 are blamed for genital warts, some types of papilloma virus such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 and HPV-58 are included malignant tumors of the anogenital tract HPV-16 is associated with cervical cancer in over 50% of cases (Cervical carcinoma) HPV-16 is the main risk factor for the formation of cervical neoplasms. The immune system also plays an important role in the progress of the disease. Cellular immune responses and, in particular, antigen-specific T-lymphocytes are probably important for the defense mechanism that in high-grade cervical intraepithelial neoplasia (CI II / III) and cervical tumor the E7 gene is constitutively expressed in all layers of the infected epithelium. Therefore, the E7 protein is considered as a potential tumor antigen and as a target molecule for activated T cells. induced cellular immune response in the patient is apparently not strong enough to influence the course of the disease. The immune response may be enhanced by suitable vaccines

It has now been shown that the expression of the Ll gene or the co-expression of the Ll and L2 gene forms virus-like particles (VLPs). The VLPs could be used to generate neutralizing antibodies in various animal systems However, virus-neutralizing antibodies are of less clinical importance if the virus infection has already taken place, since a virus-specific cytotoxic T-cell (CTL) response seems to be necessary for the inhibition of virus-infected cells. So-called chimeras were therefore used Papillomavirus-like particles (CVLPs) developed that consist of a chimeric Ll-E7 protein (Muller, 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 immunizing mice with CVLPs (Muller, M et al (1997), supra). Furthermore, neutralizing antibodies from HPV-associated To limit the immune diseases in patients to the administered L1 protein (Muller, M et al (1997), supra) CVLPs are, however, still interesting for the development of a vaccine, since the E7 presented via class I MHC molecules -Proteins from tumor cells were target molecules from CTLs

Peng et al (1998) Virology, 240, 147 now describe CVLPs consisting of C- terminalverkurztem Ll of bovine papillomavirus (BPV) and HPV 16E7 _{_49-57} which specific E7 after immunization of C57BL / 6 mice cytotoxic T cells induce and protect against the growth of E7-expressing tumors Greenstone et al (1998) Proc Natl Acad Sei USA, 95, 1800 describe CVLPs consisting of HPV-16L1 plus HPV-16L2 fused with the full length HPV-16E7 protein, which after immunization of Protect C57Bl / 6 mice against the growth of epithelial E7-expressing tumor cells, although cytotoxic T cells have not been detected and the induction of the immune response thus appears to be less efficient

VLPs or CVLPs are generally produced genetically 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, by Kirnbauer, R et al (1994) J Virol 67 , 6929-6936 or obtainable from the EMBL database. The access numbers are, for example, for HPV18 PAPHPV18, for HPV31 PAPPPH31, for HPV33 PAPPPH33 or for HPV58 PAPPPH58. Suitable expression systems are, for example, genetically modified yeasts, for example Saccharomyces (cerevisiaiae), P (pastoris), Kluyvermyces (lactis), Schizosaccharomyces (pombe) or Hansenula (polymorpha) (Carter, JJ et al (1991), Virology, 182, 513), insect cells, such as, for example, Trichoplusia ni High Five (see, for example, Muller, M et al (1997), supra) or prokaryotic cells (see, for example, WO 96/1 1272). When the particles are produced in prokaryotic cells, they generally precipitate in the cell and form s The aforementioned inclusion bodies (inclusion bodies), which must subsequently be renatured and brought into solution. For the use of the genetically engineered particles or capsids or their precursors, the so-called capsomers, further purification steps are necessary after expression

A major problem with the use of capsids and capsomers as medicaments is their poor solubility. For example, capsids or capsomers of HPV-16 tend to aggregate, as a result of which the solubility is substantially reduced. The partly low solubility of the capsids or capsomers not only leads to a loss of yield, but also to difficult use as a drug or diagnostic. In addition, a degradation of the C-terminus of the Ll protein can sometimes be observed, which leads to inhomogeneous leads material that is not suitable for approval as a drug or diagnostic

WO 98/44944 describes an HPV antigen formulation containing a vaccine component, a salt at physiologically acceptable concentrations and a non-ionic detergent at physiologically acceptable concentrations

The object of the present invention was therefore to provide a simple and advantageous formulation in which papillomavirus-specific proteins are soluble and stable

It has now surprisingly been found that the solubility of papilloma virus-specific proteins is dependent both on the salt concentration and on the pH of the formulation. In particular, it was surprising that at neutral pH in an isotonic salt solution of about 100-150 mM salt Composition without the addition of an auxiliary is not stable, although VLPs or CVLPs are formed in the cytoplasm of the host cells. Furthermore, according to the present invention it was found that the CVLPs fail at a pH value of less than about 5.5 and at a pH value of greater than 9.5 aggregate the CVLPs

The present invention therefore relates to 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 auxiliaries, preferably without any additives and / or auxiliaries

According to the present invention, the term “formulation” is understood to mean a composition in the form of a solution or a suspension of the papillomavirus-specific proteins mentioned, with immunoreactive papilla do not sediment mavirus-specific proteins in general and in particular up to a maximum of about 5000 g

The salt is generally an alkali or alkaline earth metal salt, preferably a halide or phosphate, in particular an alkali halide, especially NaCl and / or KC1. The use of NaCl is particularly preferred for the preparation of a pharmaceutical formulation

The pH of the composition is generally adjusted with a suitable organic or inorganic buffer, such as preferably with a phosphate buffer, Tris buffer (Tris (hydroxymethyl) aminomethane), HEPES buffer ([4- (2 -Hydroxyethyl) -piperazino] -ethanesulfonic acid) or MOPS buffer (3-morpholino-l-propanesulfonic acid). The selection of the respective buffer is generally based 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 diagnostic agent, it is particularly preferred if the papillomavirus-specific proteins do not contain papillomavirus-unspecific epitopes, since this can reduce or prevent a papillomavirus-unspecific immune response

For the purposes of the present invention, the terms L-protein and E-protein mean both the Vollangen 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 deletion has the advantage that other proteins, for example PapiUomavirus-specific E protein sequences, can be inserted into the deleted area , whereby the field of application of the composition according to the invention can be expanded. Particularly preferred is an L protein with a C-terminal deletion and in particular a C-terminal deleted L1 protein. The C-terminal deletion has the advantage that the efficiency of the The formation of virus-like particles can be increased since the nuclear localization signal located at the C-terminus is deleted. The C-terminal deletion is therefore preferably up to approx. 35 amino acids, in particular approx. 25 to approx. 35 amino acids, especially approx. 32 to approx. 34 amino acids For example, a 32 amino acid C-terminal deletion of the HPV-16 L1 protein and an approximately 26 amino acid long C-terminal deletion of the BPV-1 Ll protein (Bovines Papillomavirus type 1) are sufficient to form virus-like particles to be able to increase this at least about ten times

In a further preferred embodiment, the E protein is also deleted, in particular the E6 and / or E7 protein. In particular, it is preferred if the C-terminal part of the E protein is deleted, preferably the C-terminal part of the E7 Proteins, since these constructs can preferably form capsomers and / or capsids in conjunction with deleted L-protein. Deletions of up to approximately 55 amino acids, preferably approximately 5 to approximately 55 amino acids, in particular approximately 38 to approximately 43 amino acids, are particularly preferred

A particularly preferred construct is, for example, E7 with the N-terminal amino acids 1 to about 60, since this construct contains a mouse epitope for the activation of cytotoxic T lymphocytes, which is located in the region of amino acids 49-57. Another preferred construct is E7 with the N-terminal amino acids 1 to about 55, which forms capsomers and capsids in conjunction with deleted L-protein, since this construct presumably does not contain E7-specific sequences in the region of amino acids 56-70, which form the formation of capsids A Ll protein of HPV-16 deleted by 32 amino acids C-terminal, which is linked to an E7 protein of HPV-16 with 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 tumors and on the other hand cause a decline in existing ones Tumors in animal experiments Especially E7 with amino acids 1-60 shows a clear prophylactic as well as therapeutic effect in tumors. A particularly preferred embodiment of the present invention is therefore a LlΔE7ι _-x fusion protein, preferably in the form of a CVLP, in particular of HPV16, where x is an integer from 55 up to and including 60, and in particular an L1ΔCE7] .55 or LlΔCE7ι.6o fusion protein

To produce a medicament which is both prophylactic and therapeutically active, it is preferred to bind the L protein to the E protein, for example in the form of a fusion protein. Furthermore, it is preferred if the described papillomavirus-specific proteins are in the form of a capsid and / or Capsomers are present, since the immune response can be significantly increased by the capsids and / or capsomers and in particular by the proportion of L protein. Fusion proteins which are suitable for capsid and / or capsomer formation are therefore, for example, fusion proteins from deleted L1 and E7, E6 and / or El preferred

For the purposes of the present invention, capsids are viral or virus-like structures in a generally icosahedral form, which are generally composed of approximately 72 capsomers

For the purposes of the present invention, capsomers are assembled proteins containing at least one papillomavirus structural protein, preferably L1 or deletions of L1. For example, 5 fusion proteins can assemble to form a capsomer, which in turn can assemble to form a capsid

Proteins or peptides of human papillomavirus (HPV) and preferably of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV are for the described constructs for the production of a human medicament or diagnostic agent -35, HPV-39, HPV-45, HPV-52 and / or HPV-58, in particular HPV-16, HPV-18, HPV-31 and / or HPV-45 suitable. It is particularly advantageous for the production of a combination vaccine To combine proteins or peptides from different 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, cervical carcinoma or HPV-6 and HPV-11 in the case of eg condylomas

The expression vectors can be, for example, prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors for expression in E. coli, for example, the vectors pGEM or pUC-Derviate (see, for example, WO96 / 11272). Examples of eukaryotic expression vectors for expression in Saccharomyces cerevisiae, for example, the vectors p426Met25 or p426GALl (Mumberg et al (1994) Nucl Acids Res, 22, 5767-5768, Carter, JJ et al (1991) supra) and for expression in insect cells, for example baculovirus vectors, in particular the Autographa Californica virus, as in EP-B1-0 127 839 or EP- B1-0 549 721 (see, for example, also WO94 / 20137), and for expression in sucker cells, for example the vectors Rc / CMV and Rc / RSV or SV40 vectors, all of which are generally available. However, commercially available ones are also suitable Baculovirus expression systems such as the Baculo Gold ™ Transfection Kit from Pharmingen or the Bac-to-Bac ™ Baculovirus Expression Systems from Gibco BRL. Other 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 -B 1-0 154 133), the ADH2 promoter for expression in yeasts ( Rüssel et al (1983), J Biol Chem 258, 2674-2682), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1-0 127 839 or US 5,004,687) or the early SV40 promoter or LTR - Promoters e.g. from MMTV (mouse mammary tumor 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 cerevisia, Pichia, Kluyvermyces, Schizosaccharomyces or Hansenula (Carter, JJ 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, all of which are generally available (see e.g. WO94 / 00152)

The coding nucleic acids for the individual papillomavirus-specific proteins can, for example, be isolated and cloned from a gene bank via PCR (“polymerase chain reaction”) amplification. For example, the genome of BPV-1 is under the GenBank Accession No X02346 or HPV-16 generally available under GenBank Accession No K02718. An HPV- 16 Ll-

Sequence is also disclosed, for example, in WO94 / 05792. The sequence of the 98th

Amino acid long HPV 16 E7 protein is described, for example, by Seedorf et al

(1985) Virology, 145, 181-185. Another method of obtaining the desired nucleic acids is to isolate the papillomavirus-specific genes directly from warts or tumors by means of PCR. Suitable primers for the E6 and E7 genes HPV-16 and HPV-18 are disclosed, for example, in WO93 / 21958. Further references for the desired nucleic acids are, for example, Kirnbauer, R et al (1994), supra and the clones mentioned above in the EMBL database

In a further preferred embodiment, the expression vector is constructed in such a way that the expressed fusion protein is not extended by any further amino acids caused by the vector. This is achieved, for example, by generating unwanted nucleotides by mutagenesis in a PCR reaction using suitable primer oligonucleotides code for additional amino acids, are removed (Ho et a (1989) Gene, 77, 51-59). In this way, a fusion protein is obtained which is free of additional amino acids and thus free of any additional foreign epitopes which can cause immunological side reactions

After the expression of the described fusion protein, it is preferred to further purify or renaturate it. Examples of chromatographic purification processes can be found in Hjorth, R & Moreno-Lopez, L (1982) J Virol Meth, 5, 151, Nakai, Y et al. (1987) J Gen Virol, 68, 1891, Hofmann, KJ et al (1995) Virology, 209, 506, Rose, RC et al (1993) J Virol, 67, 1936, Sasagawa, T et al (1995) Virology, 206, 126 or WO 95/31532

Suitable additives and / or auxiliaries, which serve, for example, to further stabilize 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 Glycerin, sugar, such as B sucrose or glucose, zwitterionic compounds, such as. B amino acids such as glycine or in particular taurine or betaine and / or protein, such as bovine or human serum albumin. Detergents, polyols and / or zwitterionic compounds are preferred

Other suitable additives and / or auxiliaries are protease inhibitors, such as aprotinin, ε-aminocaproic acid or pepstatin A

Another object of the present invention is a process for the preparation of the formulation according to the invention, the PapiUomavirus-specific protein described above, for example in solution containing about 0.3 to about 4 M, preferably about 0.4 to about 2.5 to 3 M, in particular approx. 0.4 - 0.5 to approx. 1 - 2 M, especially approx. 1 to approx. 2 M of a salt with a corresponding pH value of approx. 7.3 to approx. 7.45, preferably approx. 7.4 as well as suitable ones Additives and auxiliary substances are introduced and / or dialyzed against the composition described. The formulation can preferably be stored in a stable manner at about 4 ° C. or above all at about -80 ° C. over a longer period, for example 1-2 months or longer

The formulation according to the invention is suitable as a medicament or diagnostic agent. The present invention therefore also relates to the use of the formulation according to the invention as a medicament or diagnostic agent. For direct use as a medicament or diagnostic agent, the formulation according to the invention is preferably adjusted to a concentration of approximately 0.45 M. In particular it is preferred if the medicament does not contain an adjuvant, ie no substance which enhances the immunogenicity of the papillomavirus-specific protein, since in particular in the presence of an L protein, especially of Ll, the immunogenicity is already sufficiently enhanced. This property is particularly important in the approval as a drug or diagnostic, because the only immunostimulating materials approved by the regulatory authorities are currently aluminum salts

The medicinal product is particularly suitable for avoiding and / or treating papillomavirus-specific benign or malignant tumors, in particular malignant tumors such as, for example, laryngeal, cervical, penile, vulvar or anal carcinoma, and the diagnostic agent for diagnosing one or several pairs pillomavirus infection (s) An example of a diagnostic agent is immunodiagnostics known to the person skilled in the art, for example an ELISA for measuring papillomavirus-specific antibodies (see, for example, Voller, A et al (1976) Bull World Health Organ, 53, 55-63) or a skin test according to e.g. Hopfl et al (1991) Lancet 1, 373-374)

In general, the medicament can be administered orally, parenterally, 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 an adjuvant can be dispensed with, which is particularly advantageous

Another object 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 generally used when only relatively small amounts of a solution or suspension, for example about 1 to about 20 ml, are to be supplied to the organism. Infusion solutions are generally used when a larger amount of a solution or suspension, for example a or several liters, should be administered Since, in contrast to the infusion solution, only a few milliliters are administered with injection solutions, slight deviations from the pH value and from the osmotic pressure of the blood or the tissue fluid during the injection do not, or only insignificantly with regard to the Sensation of pain noticeable. Dilution of the formulation according to the invention before use is therefore generally not necessary. When larger amounts are applied, however, the formulation according to the invention should be diluted shortly before application so that an isotonic solution can be obtained The ionic solution is a 0.9% sodium chloride solution. For infusion, the dilution can be done, for example, with sterile water during the application, for example via a so-called bypass The main advantage of the present invention is that the formulation according to the invention essentially does not lead to the precipitation of immunoreactive papillomavirus-specific protein. In particular, more than about 90%, especially more than about 95% of the protein remain in solution and do not fall for one Period of at least approx. 12 hours from. The immunoreactive PapiUomavirus-specific protein cannot be sedimented significantly by centrifugation at a maximum of 5000 g. In addition, the formulation remains homogeneous and stable over a longer period of approx. 1-2 months and longer

The figure and the following examples are intended to explain the invention in more detail without restricting it

1 graphically shows the dependence of the solubility of virus-like particles on the salt concentration

Examples

1 Production of chimeric genes coding for HPV16LlE7 fusion proteins

HPV 16L1ΔC ^* E7 1-55 was prepared according to Muller, M et al (1997), supra. Here, the HPV-16L1 open reading frame (ORF) was derived from the plasmid HPV-16-114 / k-Ll / L2-pSynxtVr ( Kirnbauer, R et al (1994) J Virol 67, 6929) with the restriction endonuclease BglII and cloned into the vector pUC19 (New England Biolabs) in the BamHI site. Two primers were constructed for the production of HPV-16L1ΔC. 16L1 ORF are complementary. The first primer has the sequence AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC and the second primer AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACG

Both primers code 5 'an EcoRV restriction enzyme interface. In the downstream primers, the EcoRV site is followed by a TAA translation stop codon in order to delete the last 34 amino acids of the HPV16L1 ORF. The PCR reaction was carried out to remove the entire L1 ORF and the whole Vector to amplify. The linear product was digested with EcoRV, circularized with T4 DNA ligase and E coli DH5α cells transformed. The clones were analyzed for the presence of an EcoRV site. The resulting construct pUCHPV16LlΔC was used to isolate the ORF of HPV16E7 1-50 in to clone the EcoRV site

Primers with a 5 PrimcoRV restriction enzyme interface were used to clone the fragment. 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 Ll gene

In order to terminate the EcoRV sites, two PCR reactions were carried out in order to amplify two overlapping fragments of the clone pUC-HPV16LlΔCE7 1-50. The resulting DNA fragments overlapped at the position of the L1 / E7 border ("Four Primer PCR", Ho, SN et al (1989) Gene 77, 51) However, the primers did not contain the two EcoRV restriction enzyme sites. Fragment 1 was produced with the pli P1 and P2 and fragment 2 with the primers P3 and P4

P 1 GTTATGAC ATAC ATAC ATTCTATG (L 1) P2 CCATGCATTCCTGCTTGTAGTAAAAATTTGCGTCC (E7) P3 CTACAAGCAGGAATGCATGGAGATACACC (E7) P4 CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCG

One tenth of the purified products were mixed and used as a template in the PCR reaction with only the primers P1 and P4. The resulting product was cleaved with EcoNI (L1) and HindIII (downstream of the stop codon on the primer P4) and used to make an EcoNI HindIII fragment to replace the cloned HPV16L1 ORF. The resulting clone therefore differs from the clone HPV16L1ΔCE7 1-50 in that it loses the two internal ones EcoRV restriction enzyme interfaces and the corresponding non-HPV amino acids Asp and He between the Ll ORF and E7 and downstream of E7. The first EcoRV site was replaced by the original Ll amino acids at this position (AlaGly). The second EcoRV site was replaced by a translation stop signal this clone (HPV16L1ΔC * E7 1-52) the first 52 amino acids of HPV16E7 clone HPV16L1ΔC * E7 1-52 was used to prepare the clone HPV16L1ΔC * E7 1-55 using the primer P1 in combination with P5

P5 CATCTGAAGCTTATCAATATTGTAATGGGCTCTGTCCG (E7 1-55)

In all cases, EcoNI and HindIII were used to replace the corresponding fragments. The clones were analyzed by DNA sequencing

2 Production of recombinant baculoviruses

Spodoptera frugiperda (Sf9) cells were grown as monolayers or in suspension culture in TNM-FH insect medium (Sigma, Deisenhofen) with 10% fetal calf serum and 2 mM glutamine. Recombinant baculoviruses HPV16L1ΔCE7 1-55 were obtained by cotransfection of 10 μg and the recombinant 2 μg of linearized Baculo-Gold DNA (Pharmingen, San Diego, CA) transfected in Sf9 cells. Recombinant viruses were purified according to the manufacturer's instructions. To test the expression, 10 ⁶ Sf9 cells were mixed with recombinant baculovirus and a moi (“multiplicity of infection ") from 5 to 10 infected. After the incubation, the medium was removed and the cells were treated with PBS (140 mM NaCl, 2.7 mM KC1, 8.1 mM Na ₂ PO ₄ , 1.5 mM KH ₂ PO ₄ , pH 7.2) Washed The cells were then lysed in SDS sample buffer and tested by SDS gel chromatography and immunoblot assay

3 Cleaning virus-like particles For the production of CVLPs, Trichoplusia ni (TN) high five cells were grown at 27 ° C. to a density of 1-1.5 × 10 ⁶ cells per ml in Ex-Cell 405 serum-free medium (JRH, Biosciences, Lennexa, KS) A 400 ml culture was harvested and infected with recombinant baculoviruses for one hour with periodic inversions with a moi of 2 to 5. Up to 240 ml of medium were added and the cells grew for 3 to 4 days. The cells were then pelleted and in 10 ml extraction buffer resuspended (25 mM Tris / HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA) and sonicated for 45 seconds at 60 watts. After centrifugation at 10,000 rpm in the Sorvall SS34 rotor, the pellet was placed in the 6 ml extraction buffer dissolved, sonicated at 60 watts for 30 seconds and centrifuged again. The supernatants were combined and applied to a two-stage gradient composed of 40% (w / v) sucrose and 57.5% (w / v) CsCl after centrifugation in a SW-28 rotor at 27,000 rpm for two For one hour, 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 subjected to Western blot with anti-HPVlόLlmAb Camvirl (Pharmingen, San Diego, CA) The reactive fractions were pooled and ultrafiltered with Centricon 30 microconcentrator (Amicon Corp Beverly, MA) against Hepes buffer (1mM Hepes, 149mM NaCl, 0.5mM KCl, pH 7.2) dialyzed and the presence of CVLPs confirmed by means of transmission electron microscopy. The concentration of LlE7 protein was approximately determined in an SDS gel which was stained with Coomassie blue by comparison with BSA standards

4 microdialysis experiments

A fraction containing virus-like particles isolated from high five cells by sucrose cushion and casium chloride equilibrium ultracentrifuge was used as a sample. The total protein concentration was 0.29 mg / ml and the CVLP concentration was 0.17 mg / ml

40 ml of the corresponding solution were placed in a 50 ml plastic vessel with a screw cap. A dialysis filter was carefully placed on this solution with a pore diameter of 0.025 μm, which floats on the liquid while dialysis is being carried out. 30 μl of the pure CVLP solution were pipetted onto this filter and the vessel was closed. The vessel was left 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 / HCl, pH 7.5 with increasing NaCl concentration). The drop was removed with the piston-stroke pipette and it was balanced with 30 μl reservoir solution. After centrifugation at 10,000 g (10 min, 4 ° C.) the supernatant was determined in the ELISA (Kemeny, DM (1994) Indirect ELISA from ELISA, application of the enzyme linked immunosorbent assay in the biological / medical laboratory, Gustav Fischer Verlag, Stuttgart, p. 111, test 6 2) examined with a conformation-specific monoclonal antibody against HPV16L1 and in a protein assay. The protein concentration was determined with a bicinchonic acid assay (Smith, PK et al (198 5) Anal Biochem, 150, 76-85) against bovine serum albumin as standard. The result is shown in FIG. 1

Claims

claims

Formulation containing 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 approx. 0.4-0.5 to approx. 1-2 M, especially approx. 1 to approx. 2 M of a salt at a pH value of approx. 7.3 to approx. 7.45, preferably approx 7.4, and, if appropriate, suitable additives and / or auxiliaries

Formulation according to claim 1, characterized in that the salt is an alkali or alkaline earth salt, preferably a halide or phosphate, in particular an alkali halide, especially NaCl and / or KCl

Formulation according to claim 1 or 2, characterized in that the pH is adjusted with a buffer, preferably with a phosphate buffer, Tris buffer, HEPES buffer or MOPS buffer

Formulation according to one of claims 1-3, characterized in that the said protein or proteins does not contain any papillomavirus-unspecific epitopes

Formulation according to one of Claims 1-4, characterized in that the L protein is a deleted L protein, preferably a deleted Ll and / or L2 protein

Formulation according to claim 5, characterized in that the L-protein is a C-terminally deleted L-protein, in particular a C-terminally deleted Ll-protein

Formulation according to Claim 5 or 6, characterized in that up to approximately 35 amino acids have been deleted from the L protein, preferably approximately 25 to approximately 35, in particular approximately 32 to approximately 34 amino acids Formulation according to any one of claims 1-7, characterized in that the E protein is a deleted E protein, especially a deleted E6 and / or E7 protein

Formulation according to claim 8, characterized in that the deleted E protein is a C-terminally deleted E-protein, preferably a C-terminally deleted E7 protein

Formulation according to Claim 8 or 9, characterized in that up to approximately 55 amino acids have been deleted, preferably approximately 5 to approximately 55 amino acids, in particular approximately 38 to approximately 43 amino acids

Formulation according to one of Claims 1-10, characterized in that the L protein is bound, preferably fused, to the E protein

Formulation according to one of claims 1-11, characterized in that said protein is in the form of a capsid and / or capsomer

Formulation according to one of Claims 1-12, characterized in that the papillomavirus is a human papillomavirus (HPV)

Formulation according to claim 13, characterized in that the HPV is selected from HPV-6, HPV-1 1, HPV- 16, HPV-18, HPV-3 1, HPV-33, HPV- 35, HPV-39, HPV- 42, HPV 45, HPV-52 and or HPV-58

Formulation according to one of claims 1-14, characterized in that the additives and / or auxiliary substances are one or more detergents, polyols and / or zwitterionic compounds

A process for the preparation of a formulation according to any one of claims 1-15, characterized in that said protein in a solution containing 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 from about 7.3 to about 7.45, preferably from about 7.4, and / or dialyzed

Use of a formulation according to any one of claims 1-15 as a medicament or diagnostic

Use according to claim 17, characterized in that the formulation contains no adjuvant

Use according to claim 17 or 18, characterized in that the medicament serves to avoid or treat papillomavirus-specific tumors

Use according to claim 19, characterized in that the tumor is a larynx, cervix, penis, vulva or anal carcinoma

Use according to one of claims 17-20, characterized in that the said formulation is used as an injection or infusion solution

Use according to claim 17, characterized in that the diagnostic agent is used to diagnose one or more papillomavιrus infections