EP1888620A1

EP1888620A1 - Method for producing virus-type particles containing an active substance

Info

Publication number: EP1888620A1
Application number: EP06707650A
Authority: EP
Inventors: Christoph Reichel; Claus RÜHLAND; Jürgen Hess; Christian Reiser
Original assignee: responsif GmbH
Current assignee: responsif GmbH
Priority date: 2005-05-24
Filing date: 2006-03-28
Publication date: 2008-02-20
Also published as: CA2615415A1; AU2006251454A1; WO2006125490A1; US20090215146A1

Abstract

The invention relates to a method for producing virus-type particles containing an active substance. Proteins, which comprise a first amino acid sequence which is derived from a first virus protein, and fusion proteins are assembled in order to form the virus-type particles. The proteins and the fusion proteins are coexpressed in yeast cells.

Description

description

Process for the preparation of a virus-containing particles containing an active substance

The invention relates to a process for the preparation of virus-containing particles containing an active substance and to the virus-like particles produced by the process.

WO 00/61616 A1 discloses a synthetic biologically active molecule for anchoring an active substance to pentamers of virus protein 1 (VP1) of the polyoma virus. In this case, an amino acid sequence which binds to VPl-pentamers and is derived from the C-terminal end of virus protein 2 (VP2) or 3 (VP3) of the polyoma virus is connected at one end to the active substance.

It is known from WO 99/25837 to produce virus-like particles (VLPs) in yeast by expression of virus protein 1 of the polyoma virus (VP1).

From Palkova, Z. et al. , FEBS Letters 478 (2000), pages 281 to 289 it is known that the expression of virus protein 1 of the polyomavirus in yeast strongly inhibits the growth of yeast cells.

From Buonamassa, D.T. et al. , Virology 293 (2002), pages 335 to 344, the coexpression of the human papillomavirus capsid proteins L1 and L2 and the assembly of these proteins to VLPs in yeasts is known.

From Kiessling, R., Proceedings of the Sixth Annual Walker's Cay Colloquium on Cancer Vaccines and Immunotherapy (2004), Keynote Address, page 4, it is known to express portions of the tumor antigen Her2 / neu at the carboxyterminal end of the VP2 structural capsid protein and to use polyoma VLPs to administer the Her2 / neu VP2 construct to transgenic mice as tumor vaccines.

From Abbing, A. et al. , Journal of Biological Chemistry (2004), Vol. 279, No. 26, pages 27410 to 27421, it is known VPl and a fusion protein having the amino acid sequences of GFP and virus protein 2 of the polyomavirus (VP2), respectively in Expressing E. coli. An assembly of VPl and the GFP-VP2 fusion protein takes place after their isolation in vitro. For this purpose, the proteins to be assembled are introduced in solution in an increased concentration and incubated until VLPs are formed.

A fundamental problem in the expression of VP2 is that VP2 and in particular its C-terminal domain (see FIG. 1) binding to VPl-pentamers is relatively hydrophobic and, when expressed in

E. coli already precipitates in low concentration. Such a hydrophobic domain of VP2 produced in E. coli is often unable to specifically bind to VP1 pentamers. However, expression of recombinant VP2-binding VP2 in E. coli can be promoted by having the amino acid sequence of VP2 together with a hydrophilic amino acid sequence as described in Abbing et al. the amino acid sequence of GFP expressed as a fusion protein.

In the expression of a recombinant fusion protein which, besides the VPl-pentamer interacting domain of VP2, reverses a predominantly hydrophobic amino acid sequence. the problem of proper protein folding exists. Since the VPl-pentamerically interacting domain of VP2 is also hydrophobic, it can interact with the predominantly hydrophobic amino acid sequence and thereby adversely affect protein folding so that the resulting fusion protein can not bind specifically to VPl-pentamers. The expression of such fusion proteins in E. coli often results in the formation of inclusion bodies, which are also referred to as "inclusion bodies". Proteins contained in the inclusion bodies are denatured and non-functional.

From Chen, S.X. et al. , The EMBO Journal (1998), Vol. 17, No. 12, pages 3233 to 3240, a coexpression of VP1 and VP2 or VP3 in E. coli is known. In addition, it is known how the polyoma virus protein VP2 or VP3 interacts with the polyoma virus protein VPl and which regions of the proteins are responsible for it.

Many known tumor antigens contain highly hydrophobic protein regions. These tumor antigens or peptides having sequences of the tumor antigen including these protein regions are often insoluble in water. For immunization of tumor patients with these tumor antigens, they are usually dissolved in dimethyl sulfoxide (DMSO) for administration. This is for example from Gnjatic et al. , Proc. Natl. Acad. Be. USA (2002), Vol. 99, No. 18, pages 11813 to 11818. However, DMSO is considered harmful because of possible side effects such as skin reactions, central nervous system disorders and organ damage to the liver and kidneys.

In addition, a solution of the tumor antigens or peptides in DMSO often also does not lead to a structure as it is present in the native tumor antigens. One against the native Tumor antigens effective immunization can not be ensured.

One feature of chronic disease is that tolerance to specific disease-associated antigens is immune system immune. A prerequisite for a successful immunotherapy of such diseases is therefore to overcome this tolerance. This requires the induction of a B cell response that causes a specific antibody response against the antigens associated with the chronic disease.

For the effective immunological treatment of a chronic disease, such as a tumor, a chronic viral disease, such as an infection with HIV or HCV, or another infectious disease such. As malaria, tuberculosis or schistosomiasis, it is necessary that directed against tumor cells or infected cells cytotoxic T cells are formed. This requires proper and strong activation of T cells. For this purpose, it is not sufficient to apply a tumor antigen or an antigen specific for the infected cells together with an adjuvant. It has been shown that this depends on a frequent repetition of the antigenic motif in a small spatial distance and a good association of the antigen with an immune response stimulating agent, such as VLPs. The company Cytos Biotechnology AG, Switzerland, therefore, links antigens covalently to the outside of recombinantly produced VLPs. The disadvantage here is that the process is complicated and additional purification is required for in vivo administration in order to remove the reagents required for the covalent coupling. The object of the present invention is to eliminate the disadvantages of the prior art. In particular, a method is to be provided which allows the preparation of a drug-containing virus-like particles, wherein the drug is to be presented so that it can induce formation of specific cytotoxic T cells in a mammal or human. Furthermore, the virus-like particles and a use of the particles are to be specified.

According to the invention the object is achieved by the features of claims 1, 17 and 28. Advantageous embodiments of the invention will become apparent from the features of claims 2 to 16 and 18 to 27.

According to the invention, a method is provided for producing virus-like particles containing an active ingredient, wherein proteins which each have a first amino acid sequence derived from a first viral protein and fusion proteins assemble into the virus-like particles. In this case, the first amino acid sequence is an amino acid sequence which is sufficient for the formation of capsid-forming and a second viral protein-specific binding capsomeres. The fusion proteins each have a second amino acid sequence which specifically binds to one of the capsomers and is derived from the second virus protein, and a third amino acid sequence which forms the active substance. The proteins and the fusion proteins are coexpressed in yeast cells.

An amino acid sequence is derived from a protein when it is unchanged from or is different from the complete or incomplete amino acid sequence of the protein by amino acid substitutions, insertions or deletions.

The coexpression of the fusion proteins in the yeast cells ensures that the second amino acid sequence is folded so that it can specifically bind to the capsomeres, even if the third amino acid sequence forming the active substance is predominantly hydrophobic. As a result, virus-like particles which contain the active ingredient can form from the fusion proteins and the capsules formed from the proteins. The active ingredient is arranged so that it often repeats itself at a small distance. The specific binding of the region of the fusion protein formed by the second amino acid sequence to the capsomers ensures good association of the active ingredient with the virus-like particles.

The peculiarity of the method is that the VLPs produced by the method according to the invention in mammals trigger a strong immune response directed against the active ingredient. In particular, cytotoxic T cells are also formed, which attack cells that carry the active ingredient on their surface. Since the VLPs themselves have the effect of an adjuvant, i. H. trigger an immune response directed against the drug, the administration of an additional adjuvant is not required. Compared to the method known from the company Cytos Biotechnology AG, no complex chemical binding of the active ingredient to the outside of the VLPs and no subsequent purification of the VLPs is required. In addition, the active ingredient in the inventive

Process located on the inside of the VLPs and is thus protected from degradation, for example by proteases. In the coexpression of the proteins and the fusion proteins in the yeast cells, capsules are first formed in the yeast cells from the proteins, each of which has the first amino acid sequence. From the capsomeres and the fusion proteins, VLPs form in the yeast cells, which can then be isolated from the hepatic cells.

In contrast to the Chen et al. In the case of co-expression in yeasts, there is no risk in the isolation of the VLPs from the cells used for co-expression from being completely separated from the VLPs by endotoxins. This significantly reduces the expense of cleaning the VLPs containing the active ingredient and of costly toxicological tests. When the purified yeast-produced VLPs are administered to a mammal, there is no risk of triggering fever or anaphylactic shock by endotoxin from the cells used for coexpression. In contrast to an expression in mammalian cells, coexpression in yeast cells furthermore does not entail the risk of contamination by pathogens of human pathogens, such As viruses, bacteria or prions.

A further advantage of the method according to the invention is that all proteins contained in the VLPs are produced and assembled in an organism. As a result, it is not necessary in the production of a drug to provide a separate proof of production for all components according to GMP practice. All it takes is one

Detection of the VLPs produced in yeasts. Since the VLPs are hydrophilic as a whole, they can easily be precipitated in aqueous solution. ger solution be administered. The use of DMSO for administration is not required.

It is particularly advantageous if the first virus protein and / or the second virus protein originate from a virus or can be obtained, wherein the virus is selected from the group of non-enveloped viruses comprising Papovaviridae, in particular polyoma and papillomaviruses, Iridoviridae, Adenoviridae, Parvoviridae, Picornaviridae, in particular polioviruses, Caliciviridae, Reoviridae and Birnaviridae. Preferably, the first virus protein is virus protein 1 of the polyoma virus (VP1) and the second virus protein is virus protein 2 (VP2) or virus protein 3 (VP3) of the polyoma virus. The capsomers are preferably in the form of pentamers, hexamers or heptamers.

The process according to the invention is particularly advantageous if the third amino acid sequence is, at least predominantly, hydrophobic. The hydrophobicity of the amino acid sequence can be determined, for example, by the method known from Kyte, J. and Russell, F.D., Journal of Molecular Biology (1982), Vol. 157, Issue 1, pages 105 to 132. The hydrophilic and hydrophobic properties of each of the 20 amino acid side chains are taken into account. An amino acid sequence is predominantly hydrophobic if the majority of the amino acids forming the sequence are hydrophobic or if a peptide / protein having the amino acid sequence would be sparingly soluble or insoluble in water.

The peculiarity of the coexpression of proteins and fusion proteins in yeast cells is that poorly soluble or insoluble fusion proteins are involved in their formation in yeast situ interact immediately with capsomeres that have been formed from the proteins. This results in soluble complexes which then assemble into VLPs.

Compared to the Abbing et al. known assembly of isolated from E. coli VPl and VP2 fusion protein allows the inventive method for the first time to produce virus-like particles of capsomeres and fusion proteins, wherein the fusion proteins each comprise a predominantly hydrophobic amino acid sequence as an active ingredient. When assembling in the yeast cells, there is no problem of precipitating the fusion proteins formed. The reason for this is probably that the concentration of free, d. H. not bound to the capsomeres, fusion proteins always remains low.

It is advantageous if the third amino acid sequence forms the N-terminus of the fusion protein. This is particularly likely to allow proper folding of the drug, presumably because the folding of the drug is not affected by binding already made to the capsomer due to the N-terminus to C-terminus synthesis. This results in a particularly effective assembly in the yeast cells.

Preferably, in the coexpression of the proteins and the

Fusion proteins, the respective extent of the expression of the proteins and the fusion proteins coordinated so that a maximum in the process of the amount of the active substance-containing virus-like particles is formed. This can be achieved by introducing expression plasmids which code for the proteins and the fusion proteins into the yeast cells in a matched copy number. There may also be nucleic acid sequences which coding for the proteins and the fusion proteins are stably integrated into the genome of the yeast cells in a coordinated copy number. In a preferred embodiment of the method according to the invention, the expression of the proteins takes place under the control of a first promoter, which is contained in a coding for the first plasmid, and the expression of the fusion proteins under the control of a second promoter, which in a coding for the fusion proteins second plasmid is included. In this case, the respective extent of the expression of the proteins and the fusion proteins is coordinated by a suitable choice of the first and the second promoter. By judicious choice of the stoichiometric ratio between the expression of the fusion protein and the first amino acid sequence or the protein, it can be prevented that the fusion protein, the first amino acid sequence or the protein is expressed in the yeast cells in an unnecessary amount. If too much of the fusion proteins are expressed, some of the fusion proteins formed will not be integrated into VLPs. If too much of the first amino acid sequence or protein is expressed, VLPs will be formed which contain little or no drug.

Preferably, the first and / or the second promoter are selected from the group consisting of the promoters of the genes of alcohol dehydrogenase 1 (ADHI), alcohol dehydrogenase 2 (ADH2), orthophosphoric monoester phosphohydrolase (Apase) , Format Dehydrogenase (FOD), Galactokinase (GALl), UDP-Glucose-4-Epimerase (GALlO), Glyceraldehyde-3-Phosphate (GAP), Glyceraldehyde-Phosphate-Dehydrogenase (GAPDH), Alcohol-Oxides (AOX) , Methanol oxidase (MOX), no message in thiamine 1 (NMTl), 3-phosphoglycerate kinase (PGK) and pyruvate kinase (PYKl) and the hybrid promoters GAL10 / PYK1 and ADH2 / GAPDH. The third amino acid sequence preferably comprises a sequence of at least one antigen, of at least one epitope of this antigen or of different epitopes of this antigen. If the third amino acid sequence comprises different epitopes of the antigen, the fusion protein is a so-called multi-epitope construct. The antigen may be a tumor-associated antigen. An antigen is any protein or peptide which can induce an immune reaction in a mammal or human, in particular the formation of cytotoxic T cells. To trigger the immune response, it may be necessary to appropriately present the antigen to the immune system. By a tumor-associated antigen is meant an antigen which is expressed by tumor cells in a different way than by the corresponding non-degenerated cells of the same type or an antigen which has a specific influence on the growth or multiplication of tumor cells. Preferably, the tumor-associated antigen is selected from the group comprising NY-ESO-I, telomerase reverse transcriptase (TERT), p53, MDM2, CYPlB1, HER-2 / neu, CEACAM (Carcinoembryonic antigen-related cell adhesion molecule 5 ) and the apoptosis-inhibiting protein survivin.

In a preferred embodiment of the invention, the antigen is an antigen of an agent of a viral disease or infectious disease. An antigen of a pathogen is an antigen which the pathogen itself has or for which the pathogen has a coding nucleotide sequence. The viral disease or infectious disease can be a chronic viral disease or infectious disease. The antigen may be selected from a group comprising: HIV-associated antigen, HCV-associated antigen antigen, tuberculosis-associated antigen, in particular Ag85A, Ag85B, Rv3407, Esat-6 and Hsp65, malaria-associated antigen, in particular CSP-I, LSA-I, LSA-3 and EXP-1, with a merozoite stage of the malaria pathogen, in particular MSP-I, and bilharzia-associated antigen. The antigen is associated with one of the pathogens if the pathogen expresses the antigen itself or triggers its expression in the affected organism.

For the triggering of an immune response, it is particularly advantageous if the third amino acid sequence forms an MHC class I-specific antigen.

In one embodiment of the invention, the first amino acid sequence derived from the virus protein 1 of the polyomavirus (VP1) does not contain the DNA-binding domain contained in the VP1 and / or the nuclear localization sequence (NLS) contained in the VP1. The DNA-binding domain is contained in the NLS or overlaps with the NLS. The function of NLS is usually to translocate the VPI into the nucleus. The DNA-binding domain can bind any DNA. By omitting the DNA-binding domain or the NLS can be achieved that little or no unwanted DNA from the host organism is packaged in the VLPs. As a result, a higher quality of the VLPs can be achieved. Undesirable side effects of DNA from the host organism are avoided. Tolerability of VLPs when administered to a mammal is improved.

The yeast cells used for coexpression are preferably yeast cells of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Hansenula polyraorpha, Kluyveromyces lactis or Kluyveromyces marxianus.

The invention further relates to virus-like particles, comprising proteins which each have a first amino acid sequence derived from a first virus protein, and fusion proteins. The first amino acid sequence is an amino acid sequence which is sufficient for the formation of capsid-forming and a second viral protein specific binding capsomeres. The fusion proteins each have a second amino acid sequence which specifically binds to one of the capsomers and is derived from the second virus protein, and a predominantly hydrophobic third amino acid sequence which forms an active substance. Such particles with a predominantly hydrophobic third amino acid sequence could not be produced so far. However, their production succeeds when the proteins and the fusion proteins are coexpressed in yeast cells and the particles are formed in the yeast cells. Another way to make them is unknown. Advantageous embodiments of the particles according to the invention result from the preceding embodiments relating to the method according to the invention.

The invention furthermore relates to the particles according to the invention for use as a medicament.

The invention will be explained in more detail with reference to the following embodiments. Show it:

1 is a schematic representation of the virus proteins VP2 and VP3 of the polyomavirus and the VPl-binding domain contained therein, 2 shows a Coomassie-stained SDS-polyacrylamide gel in which samples from fractions 1 to 38 of a cesium chloride gradient have been separated by gel electrophoresis,

Fig. 3 shows a Western blot analysis with VPl-specific antibodies (top) and survivin-specific antibodies (bottom) of fractions 1 to 38 of the cesium chloride gradient and

Fig. 4 is an electron micrograph of the combined VPl-peak fractions 24 to 27 of the cesium chloride gradient in 100,000-fold magnification.

Example 1 :

Production of virus-like particles by coexpression of the virus protein VP1 of the polyoma virus and of a fusion protein from the virus protein VP3 of the polyoma virus and the tumor antigen Her2 / neu

Three yeast expression plasmids are prepared.

The yeast expression plasmid pGCH-VP1 contains the yeast-specific GAL1 promoter, a yeast-specific CYCl transcription termination sequence, a CEN / ARS DNA fragment (origin of replication), a HIS3 selection marker and the coding DNA for the polyomavirus protein VP1. The extracellular domain and the transmembrane domain of Her2 / neu (amino acids 1-683) are translationally fused with VP3. The yeast expression plasmid pGCL-Her2 / neu (1-683) -VP3 contains - the yeast-specific GAL1 promoter, a yeast-specific CYCl transcription termination sequence,

- a CEN / ARS DNA fragment (origin of replication), a LEU2 selection marker and - the coding DNA for the fusion protein from Her2 / neu and VP3, whereby Her2 / neu (amino acids 1-683) at the N-terminus of VP3 ( Amino acids 116-297, see Fig. 1) was fused.

The yeast expression plasmid pGCL-VP3-Her2 / neu (1-683) contains the yeast-specific GAL1 promoter, a yeast-specific CYCl transcription termination sequence,

a CEN / ARS DNA fragment (origin of replication), a LEU2 selection marker and the coding DNA for the fusion protein from VP3 and Her2 / neu, wherein Her2 / neu (amino acids 1-683) to the C-terminus of VP3 ( Amino acids 116-297, see Fig. 1) was fused.

For co-expression of virus-like particles, yeast cells of the strain Saccharomyces cerevisiae JD53 (Ieu2, his3, trpl, Iys2, ura3) are each incubated with the yeast expression plasmid pGCH-VP1 and the yeast expression plasmid pGCL-Her2 / neu (1-683) -VP3 or the yeast expression plasmid pGCL-VP3 -Her2 / neu (1-683) according to the method of Schiestl, RH and Gietz, RD (1989) Current Genetics, Volume 16, pages 339-346. The yeast cells transformed with both plasmids are incubated on agar plates with synthetic SD medium (6.7 g / l YNB (Becton Dikinson GmbH, Heidelberg, Germany), 200 mg / l lysine, 200 mg / l tryptophan, 200 mg / l uracil, 2% glucose) at 30 ⁰ C cultivated. For the production and purification of virus-like particles, the culture of the transformed yeasts is carried out in 1000 ml of SD medium. The cultures are cultured to an optical density (OD ₆₀₀ ) of 4-8. To induce the GALl promoters, the yeast cells are then centrifuged off at 1,000 × g for 2 minutes and the cell pellets are incubated with SG medium (6.7 g / l YNB (Becton Dickinson GmbH, Heidelberg, Germany).

Germany), 200 mg / l lysine, 200 mg / 1 tryptophan, 200 mg / 1 uracil, 2% galactose) to an OD ₆ oo of 2 and cultured for 24 hours at 3O ⁰ C.

The yeasts are then centrifuged for 10 minutes at 1,000 × g and the yeast pellet in 2.5 ml cell disruption buffer (20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.1 × Triton X-ray. 100, 1 mM PMSF) per gram fresh weight of the yeasts. Cell disruption is performed using a bead beamer (Biospec Products, Inc., Bartlesville, OK 74005, USA) using 0.5 mm diameter glass beads. To do this, the cell suspension is treated 15 times at intervals of 15 seconds with the BeadBeater under constant cooling. Subsequently, insoluble residues are separated by centrifugation at 10,000 × g for 20 minutes. For further purification of the virus-like particles to be 6 ml each of the supernatant carefully to 32 ml of a sucrose cushion (45% sucrose, in cell disruption buffer) layered and centrifuged for 4 hours at 4 ⁰ C and 100,000. The pellets formed thereby contain the virus-like particles. The

Pellets are taken up in cell disruption buffer (without PMSF) and insoluble material is removed by centrifugation at 5,000 xg for 10 minutes. The supernatant is on one Cesium chloride step gradient applied. The cesium chloride step gradient is prepared from 4 ml fractions of increasing density (1.23 g / cm ³ , 1.26 g / cm ³ , 1.29 g / cm ³ , 1.32 g / cm ³ , 1.35 g / cm ³ , 1.38 g / cm ³ in 20 mM Tris-HCl, pH 7.6). Subsequent centrifugation is carried out at 100,000 xg and 4 ° C for 36 hours. 1 ml fractions of the gradient are then analyzed by SDS-polyacrylamide gel electrophoresis and Western blot. Purified virus-like particles VPl / VP3-Her2 / neu (1-683) and VPl / Her2 / neu (1-683) -VP3 are detected by negative contrasting with 1% uranyl acetate in the electron microscope.

Example 2:

Production of virus-like particles by coexpression of the virus protein VP1 of the polyomavirus and of a fusion protein from the virus protein VP2 of the polyomavirus and the murine tumor antigen mSurvivin

The virus protein VP2 of the polyomavirus is described in Abbing, A. et al. , 2004, Journal of Biological Chemistry, Vol. 279, pages 27410 to 27421.

Two yeast expression plasmids are prepared. The yeast expression plasmid pGCH-VP1 has already been described in Example 1.

mSurvivin was fused to the N-terminus of VP2. The yeast expression plasmid pmSurv-VP2 contains - a yeast-specific MET25 promoter that encodes

DNA for the fusion protein from mSurvivin (amino acids 1-140) and VP2 (amino acids 252-297, see FIG. 1). followed by a yeast-specific PGK transcription termination sequence, a yeast-specific ADHI promoter, the coding DNA for the fusion protein from mSurvivin (amino acids 1-140) and VP2 (amino acids 252-297, see Figure 1), followed by a yeast -specific ADHI transcription termination sequence, a 2μ (2 micron) DNA fragment of the yeast 2μ plasmid (origin of replication) and - a TRPl selection marker.

For coexpression of virus-like particles, yeast cells of the strain Saccharomyces cerevisiae JD53 (Ieu2, his3, trpl, Iys2, ura3) are incubated with the two yeast expression plasmids according to the method of Schiestl, RH and Gietz, RD (1989) Current Genetics, Volume 16, Pages 339 to 346, transformed.

Cultivation of the hEP cells transformed with both plasmids is carried out as described in Example 1. Subsequently, the yeasts are centrifuged for 10 minutes at 1,000 xg and the yeast pellet in 0.5 ml cell disruption buffer (20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.01% Triton X-100 , 1 mM PMSF) per gram fresh weight of the yeasts. The cell disruption is carried out by means of a hydraulic press according to the principle of the "French Press" (One Shot, Constant Cell Disruption Systems Ltd., Northants, NNI 4SD, Great Britain) in three cycles at 2000 bar.The further processing of the disrupted cells to Purification of the VLPs expressed therein is carried out as described in Example 1.

Fig. 2 shows a Coomassie staining of an SDS-polyacrylamide GeIs, on each of which a sample of fractions 1 to 38 of Cesium chloride gradient has been separated gelelektrophoretisch. Molecular weight markers have been separated on each of the tracks marked M. Purified VPl has been applied as a positive control on the lane designated P. In fractions 24 to 27, purified VPI can be recognized.

Fig. 3 shows a Western blot analysis of fractions 1 to 38 of the cesium chloride gradient. The upper panel shows a Western blot analysis performed with VPI-specific antibodies and the lower panel a Western blot analysis performed with survivin-specific antibodies. Fractions 18 to 27 show both VP1 and survivin-VP2 fusion protein.

Purified virus-like particles VPl / mSurvivin-VP2 are detected by negative contrast with 1% uranyl acetate in the electron microscope. FIG. 4 (enlarged 100,000 times) shows an electron micrograph of the VLPS contained in the fractions 24 to 27 of the cesium chloride gradient.

Example 3:

As an alternative to the purification of the VLPs by means of ultracentrifugation described in Examples 1 and 2, the VLPs are purified after culturing the yeast using classical biochemical methods. For this purpose, the cell pellets in buffer QSA (20 mM ethanolamine, 2 mM EDTA, 6 mM DTT, 50 mM NaCl, 5% glycerol, pH 9.0, 10 ml buffer per gram cell pellet + protease inhibitor + benzonase, final concentration 1 U / ml) resuspended. The cells are disrupted with a French press (3 cycles at 2000 bar). Subsequently, by centrifugation at 75,000 xg for 45 min and 4 ° C cell debris removed. The pH of the supernatant is adjusted to 9.0 by adding 0.1 M NaOH. The first purification is via cation exchange chromatography (POROS (R) 50 HS, Applied Biosystems, Foster City, CA 94404, USA) with a gradient to 100% buffer QSB (20 μM ethanolamine, 2 μM EDTA, 6 μTiM DTT, 1 M NaCl, 5% glycerol, pH 9.0) over 10 column volumes. High VPI concentration fractions are pooled and the conductivity adjusted to 9 ms / cm by addition of buffer QSA. This is followed by purification via anion exchange chromatography (Q Sepharose (R), GE Healthcare, 80807 Munich, Germany) with a gradient to 60% buffer QSB over 8 column volumes. Again, fractions of high VPI concentration are combined. Finally, recapitulation and further purification by gel filtration (Superdex (R) 200, GE Healthcare) in PBS buffer. Fractions containing purified virus-like particles are detected by negative contrast with 1% uranyl acetate in an electron microscope.

Claims

claims

A method of producing virus-like particles containing an active agent, wherein proteins each having a first amino acid sequence derived from a first viral protein and fusion proteins to the virus-like particles, wherein the first amino acid sequence is an amino acid sequence which is responsible for the formation of capsid is sufficient and a second virus protein specific binding capsomeres, the fusion proteins each having a specific binding to each one of the capsomeres derived from the second virus second amino acid sequence and a third amino acid sequence forming the active substance, wherein the proteins and the fusion proteins are coexpressed in yeast cells ,

2. The method according to claim 1, wherein the first viral protein and / or the second viral protein originate / can be derived from a virus, wherein the virus is selected from the group of non-enveloped ) Viruses, comprising Papovaviridae, in particular Polyoma and Papillomaviruses, Iridoviridae, Adenoviridae, Parvoviridae, Picornaviridae, in particular Polioviruses, Caliciviridae, Reoviridae and Birnaviridae.

3. The method according to any one of the preceding claims, wherein the first virus protein is the virus protein 1 of the polyomavirus

(VPl) and the second virus protein is virus protein 2 (VP2) or virus protein 3 (VP3) of the polyomavirus.

4. The method according to any one of the preceding claims, wherein the capsomeres are in the form of pentamers, hexamers or heptamers ren.

5. The method according to any one of the preceding claims, wherein the third amino acid sequence, at least predominantly, is hydrophobic.

6. The method according to any one of the preceding claims, wherein the third amino acid sequence forms the N-terminus of the fusion protein.

7. The method according to any one of the preceding claims, wherein in the coexpression of the proteins and the fusion proteins, the respective extent of the expression of the proteins and the fusion proteins is coordinated so that the largest possible amount of the active substance-containing virus-like particles is formed in the process.

The method of claim 7, wherein the expression of the proteins is under the control of a first promoter contained in a first plasmid coding for the proteins, and the expression of the fusion proteins under the control of a second promoter which is in a coding for the fusion proteins second plasmid is contained, and wherein the respective extent of the expression of the proteins and the fusion proteins by a suitable choice of the first and the second promoter is coordinated.

9. The method according to claim 8, wherein the first and / or the second promoter is / are selected from a group consisting of the promoters of the genes of alcohol dehydrogenase 1 (ADHI), alcohol dehydrogenase 2 (ADH2), orthophosphoric

Monoester phosphohydrolase (apase), formate dehydrogenase (FOD), galactokinase (GALl), UDP-glucose-4-epimerase (GAL10), glyceraldehyde-3-phosphate (GAP), glyceraldehyde-phosphate Dehydrogenase (GAPDH), alcohol oxidase (AOX), methanol oxidase (MOX), no message in thiamine 1 (NMTl), 3-phosphoglycerate kinase (PGK) and pyruvate kinase (PYKl) and the hybrid promoters GAL10 / PYK1 and ADH2 / GAPDH.

10. The method according to any one of the preceding claims, wherein the third amino acid sequence comprises a sequence of at least one antigen, in particular a tumor-associated antigen, from at least one epitope of this antigen or from different epitopes of this antigen.

11. The method of claim 10, wherein the tumor-associated antigen is selected from the group comprising NY-ESO-I, telomerase reverse transcriptase (TERT), p53, MDM2, CYPlB1, HER-2 / neu, CEACAM (Carcinoembryonic antigen -related cell adhesion molecule 5) and the apoptosis-inhibiting protein survivin.

12. The method according to any one of claims 1 to 10, wherein the antigen is an antigen of a pathogen of a, in particular chronic, viral disease or infectious disease.

13. The method of claim 12, wherein the antigen is selected from a group comprising HIV-associated antigen, HCV-associated antigen, tuberculosis-associated antigen, in particular Ag85A, Ag85B, Rv3407, Esat-6 and Hsp65, malaria-associated antigen, in particular CSP- I, LSA-I, LSA-3 and EXP-I, an antigen associated with a merozoite stage of the malaria pathogen, in particular MSP-I, and bilharzia-associated antigen.

14. The method according to any one of the preceding claims, wherein the third amino acid sequence forms an MHC class I-specific antigen.

15. The method according to any one of claims 3 to 14, wherein the viral protein 1 of the polyomavirus (VPl) derived first amino acid sequence does not contain the DNA-binding domain contained in the VPl and / or not contained in VPl Nuclear Lokalis sationssequenz (NLS).

16. The method according to any one of the preceding claims, wherein the yeast cells are yeast cells of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis or Kluyveromyces marxianus.

17. Virus-like particles containing proteins, each having a first amino acid sequence derived from a first virus protein, and fusion proteins, wherein the first amino acid sequence is an amino acid sequence which is for the

Formation of capsid-forming and a second virus protein specific binding capsomeres is sufficient, wherein the fusion proteins each have a specific binding to each one of the capsomers binding derived from the second virus protein second amino acid sequence and an active ingredient forming predominantly hydrophobic third amino acid sequence.

The particle of claim 17, wherein the first virus protein and / or the second virus protein is / are or can be derived from a virus selected from the group of non-enveloped viruses Papovaviridae, in particular polyoma and papillomaviruses, Iridoviridae, Adenoviridae, Par- voviridae, picornaviridae, in particular polioviruses, caliciviridae, reoviridae and birnaviridae.

19. Particles according to claim 17, wherein the first virus protein is the virus protein 1 of the polyoma virus (VP1) and the second virus protein is the virus protein 2 (VP2) or the virus protein 3 (VP3) of the polyoma virus.

20. Particles according to any one of claims 17 to 19, wherein the capsomers are in the form of pentamers, hexamers or heptamers.

21. Particles according to any one of claims 17 to 20, wherein the third amino acid sequence forms the N-terminus of the fusion protein.

22. Particles according to any one of claims 17 to 21, wherein the third amino acid sequence comprises a sequence of at least one antigen, in particular a tumor-associated antigen, from at least one epitope of this antigen or from different epitopes of this antigen.

23. Particles according to claim 22, wherein the tumor-associated antigen is selected from the group comprising NY-ESO-I, telomerase reverse transcriptase (TERT), p53, MDM2, CYPlB1,

HER-2 / neu, CEACAM (Carcinoembryonic antigen-related cell adhesion molecule 5) and the apoptosis-inhibiting protein survivin.

24. Particles according to one of claims 17 to 22, wherein the

Antigen is an antigen of an agent of, in particular chronic, viral disease or infectious disease.

25. The particle of claim 24, wherein the antigen is selected from a group comprising HIV-associated antigen, HCV-associated antigen, tuberculosis-associated antigen, in particular Ag85A, Ag85B, Rv3407, Esat-6 and Hsp65, malaria-associated antigen, in particular CSP-I, LSA-I, LSA-3 and EXP-1, an antigen associated with a merozoite stage of the malaria pathogen, in particular MSP-I, and bilharzia-associated antigen.

26. Particles according to any one of claims 17 to 25, wherein the third amino acid sequence forms an MHC class I-specific antigen.

27. Particles according to any one of claims 19 to 26, wherein the derived from virus protein 1 of the polyomavirus of the first

Amino acid sequence does not contain the DNA-binding domain contained in the virus protein 1 and / or not included in the viral protein 1 nuclear localization sequence (NLS).

28. Particles according to any one of claims 17 to 27 for use as a medicament.