DE102004036913A1 - Use of pentamers for the manufacture of a medicament - Google Patents

Abstract

The The invention relates to a use of pentamers for the production a drug for the treatment of a disease on a tumor or an autoimmune disease in a mammal or human, wherein the pentamers each consist of five Subunits are formed, wherein the subunits each a first amino acid sequence and at least one at the N- or C-terminal end of the first Amino acid sequence subsequent second amino acid sequence wherein the first amino acid sequence consists of an amino acid sequence, which is a structure of the envelope protein of a papovavirus or parvovirus, or one of formation the pentamer is a sufficient part of this amino acid sequence, the second amino acid sequence one amino acid sequence which is at least one epitope of the tumor or at least one the autoimmune disease causing epitope forms, the Tumor is not caused by the papovavirus or parvovirus.

Description

The The invention relates to the use of pentamers for the preparation a drug for the treatment of a tumor disease or autoimmune disease in a mammal or people.

From Öhlschläger, P. et al., Journal of Virology (2003), Vol. 77, No. 8, p. 4635 Until 4645 it is known human papillomavirus (HPV) type 16 L1 capsomers to use L1-specific cytotoxic T lymphocytes and a regression of a tumor in mice too induce. The tumor is one through the human Papillomavirus type 16 caused tumor, which is an epitope of the Kapsomers expressed. It discusses the possibility of HPV-specific Produce vaccines.

Out Brinkman, M. et al., Cancer Cell International 2004, 4 (Suppl I): P18 it is known that heterologous polyomavirus-like particles (PLPs), which are derived from the main envelope protein of polyomavirus VP1 and a foreign CD8 T-cell epitope at its C-terminus are highly immunogenic and mice are resistant to certain melanomas protect can. Furthermore, it is known to prevent mice by a preventive Immunization using pentamer of the envelope protein VP1 of the polyomavirus against a later one Protecting the formation of a tumor. The pentamers have an epitope of the tumor. From these Pentamers formed PLPs were even capable of regression effect existing tumors.

A Immune response, the regression of a Tumor causes significantly different from one through one preventive immunization caused immune response. The preventive Immunization only provides protection against neoplasm a tumor. The immunity builds up in the absence of the tumor. In one against an existing In contrast, tumor-directed immunization will strengthen the immune response influenced by the tumor. The existing tumor has opposite the immune system a privileged position, otherwise he would not be able to survive. Often works the tumor is immunosuppressive. A solid tumor often has mechanisms which prevent it from infiltrating cells of the immune system can be. The immunological differences between an immune response for the treatment of a tumor and an immune response in a preventive Immunization for prevention of a tumor are e.g. van Elsas, A. et al., J. Exp. Med. (2001), Volume 194, Number 4, pages 481-489 known.

to release an immune response causing regression A tumor causes the privileged position of the tumor across from overcome the immune system become. In preventive immunization using pentamers, as described in Brinkman, M. et al., Cancer Cell International 2004, 4 (Suppl I): P18 is described, this is not necessary.

Out Boisgérault, F. et al., Expert Rev. Vaccines (2002), 1 (1), pages 101 to 109 it is known that also referred to as Kapsoide virus-like particles (VLPs), e.g. PLPs, their effect on the immune system probably because of their size and theirs Unfold structure. The uptake by antigen-presenting Cells is by their size and theirs Structure particularly effective.

at Autoimmune diseases is a disorder usually associated with the body's own Antigens existing immune tolerance. For example, the body may use antibodies against exogenous Antigens that then form with the body's own Cross react antigens. It is known that an immune tolerance against such antigens can be restored by the antigen or an epitope of the antigen repeats the body in increasing Amounts are administered. This approach is called hyposensitization designated. However, hyposensitization is not possible in many patients or not enough.

task The present invention is the use of a simple to be provided for the manufacture of a medicament for Treatment of a tumor disease or autoimmune disease.

The The object is solved by the features of claim 1. Expedient refinements result arising from the features of the claims 2 to 12.

The invention provides the use of pentamers for the manufacture of a medicament for the treatment of a disease on a tumor or an autoimmune disease in a mammal or human. The pentamers are each formed from five subunits. The subunits each comprise a first amino acid sequence and at least one subsequent at the N- or C-terminal end of the first amino acid sequence second amino acid sequence. The first amino acid sequence consists of an amino acid sequence which forms a structure of the envelope protein of a papovavirus or parvovirus or of a part of this amino acid sequence sufficient for the formation of the pentamers. The papovavirus may be a papillomavirus or a polyomavirus, especially a simian virus 40. Under the structure the envelope protein is understood to be merely a spatial structure which allows the formation of the pentamers. The amino acid sequence which forms the structure of the envelope protein of the papovavirus or parvovirus is therefore not necessarily identical to the amino acid sequence forming this structure in the papovavirus or parvovirus. It may differ in one or more amino acids. The second amino acid sequence is an amino acid sequence which forms at least one epitope of the tumor or at least one epitope causing the autoimmune disease. The epitope-forming amino acid sequence may be identical to the amino acid sequence by which the epitope is formed in the tumor or autoimmune disease. The second amino acid sequence may be longer than required for epitope formation. It can be of any length as long as it does not prevent pentamer formation. The second amino acid sequence may be the amino acid sequence of a complete protein or a protein fragment. The tumor is not a tumor caused by papovavirus or parvovirus. The tumor may, for example, be a melanoma.

The Inventors have discovered that by the pentamers amazingly triggered an immune response which is the privileged position of the tumor over the Immune system overcomes. This is especially amazing because so far it has been assumed that for Such an immune response requires particles of a certain minimum size are how they z. B. from pentamers of the polyomavirus Kapsoide exhibit. Such capsoids have a diameter of approximately 45 nm, while Pentamere a size of only 8 to 9 nm. About that In addition, the VLPs or capsoids formed from pentamers have one high density of repeating epitopes on. This high epitope density has also been considered important for effective immunization been considered.

Farther The inventors have found that the pentamers are capable are, in the body a payout of inflammatory factors to effect. These factors facilitate infiltration of more solid Tumors through cells of the immune system and thereby can cause regression accelerate these tumors.

in the Regarding the treatment of autoimmune diseases, the Inventors found that by repeated administration of increasing amounts Pentamers a particularly effective and reliable hyposensitization can be achieved.

Of the Advantage of using pentamers over the use of VLPs or PLPs is that much easier to produce are because no assembly of pentamers to capsoids required is. Another advantage is that in the design The Pentamere does not have to be taken care of that too can still assemble to capsoids. The amino acid sequence therefore, the subunit forming the pentamer does not have to be an area included, for the structure of Kapsoide is required. For the construction of Kapsoide is in particular the C-terminus and often also the N-terminus of the amino acid sequence of the envelope protein papovavirus or parvovirus. In the construction the invention used Pentamers can be a bigger part the amino acid sequence, which the coat protein forms, by a foreign sequence, e.g. a tumor epitope, replaced than when using VLPs or PLPs for immunization. Furthermore can attach to the C or N terminus of the coat protein forming amino acid sequence subsequent amino acid sequence an epitope, protein fragment or protein assembly block to capsoid. However, pentamer formation is not impaired.

Preferably it is the coat protein to the VP2 protein a parvovirus, the papillomavirus L1 protein or the VP1 protein of a Polyoma virus, in particular the VP1 protein of a simian virus 40. These envelope proteins have proven to be particularly effective in terms of triggering a Immune reaction by the pentamers proved.

The the structure of the envelope protein the papovavirus or parvovirus-forming amino acid sequence is preferred with which this structure forming in the papovavirus or parvovirus amino acid sequence identical. This promotes error-free formation of pentamers.

Furthermore, it has proven to be advantageous if only the second amino acid sequence follows at the C-terminal end of the first amino acid sequence. The formation of pentamers is favored by the presence of the free N-terminal end of the first amino acid sequence. But it is also possible that at least the second amino acid sequence at the N- and C-terminal end of the first amino acid sequence or at least the second and at the other end of the first amino acid sequence at least one at the N- or C-terminal end of the first amino acid sequence third amino acid sequence connects. The third amino acid sequence is an amino acid sequence which forms at least one further epitope of the tumor or at least one further epitope which causes the autoimmune disease. The further epitope forming amino acid sequence may be identical to the amino acid sequence through which the further epitope in the tumor or in the Au toimmune disease is formed. It may be the amino acid sequence of a complete protein or a protein fragment. The third amino acid sequence can be of any length as long as it does not prevent the formation of pentamer. It has proved to be advantageous if an immunization takes place against at least two different epitopes of the tumor.

The second amino acid sequence may be at least one repetition of the epitope-forming amino acid sequence include. Alternatively or in addition may also be the third amino acid sequence at least a repeat of the further epitope-forming amino acid sequence include. Repetitions of amino acid sequences have proven to be proven beneficial to a stronger Achieve immunization.

The epitope and / or the additional epitope may be an MHC class I specific, in particular an HLA A2 restricted or H-2K ^b restricted, or an MHC class II specific epitope. The MHC class I specific epitope can be used to activate CD8 T cells. The CD8 T cells can then destroy tumor cells carrying the epitope or epitope.

The the epitope or the further epitope-forming amino acid sequence is preferably 7 to 100, in particular 10 to 40, preferably 13 to 19, amino acids long. This length enough for an immunization, without the formation of pentamers essential to impair.

Preferably If the epitope of the tumor is the apoptosis inhibiting Protein survivin or part of it. One against Survivin Immune response causes in an increased number of cells of the tumor a trigger of apoptosis and allows thereby its regression. It may also be alternative to the epitope of the tumor as ErbB2 designated tumor antigen Her-2 / neu or act as part of this tumor antigen. Her-2 / neu is used in many epithelial tumors and in particular at Uterine cancer, breast cancer, Pancreatic adenocarcinoma and colorectal carcinoma expressed. Preferably the part of the tumor antigen consists at least of the extracellular / transmembrane domain of Her-2 / neu, which in particular by the amino acids no. 1 to 683 of the amino acid sequence is formed by Her-2 / neu. The amino acid sequence and the usual numbering of the amino acids of the Her-2 / neu and ErbB2 genes are e.g. from Katoh, M. and Katoh, M., International Journal of Oncology (2003), Vol. 23, pages 831-835. Particularly efficient in the treatment of tumors are pentamers, which are formed of subunits where Her-2 / neu or a part thereof fused to the C-terminus of the VP1 protein of the polyomavirus is.

Especially efficient and simple, the subunits can be genetically engineered, in particular by expression in bacteria, preferably E. coli produce. The subunits are preferably expressed as fusion proteins, each containing the first and the second amino acid sequence. The pentamers can already expressed in the bacteria from the expressed during expression Form subunits. The pentamers are then relatively easy and isolate them from the bacteria in good yield.

The Invention will be explained in more detail with reference to the following starting example. It demonstrate:

1 the result of a photon correlation spectroscopy of chimeric VP1-pentamer particles,

2a , b is a graphic representation of the rate of tumor-free mice and the survival rate of melanoma-bearing mice in early immunotherapy and

3a , b is a graph of tumor size and survival in Molanoma-bearing mice for later immunotherapy.

In C57BL / 6 mice (Charles River WIGA (Germany) GmbH, Sandhofer Weg 7, D-97633 Sulzfeld) are ovalbumin (OVA) expressing tumors by inoculation of Cells of the OVA expressing B16 clone MO5 have been generated. The Cells of the B16 clone MO5 have been obtained from Prof. Dr. med. Kenneth L. Rock (M.D.), University of Massachusetts Worcester Campus, Department of Pathology, 55 Lake Avenue North Worcester, MA 01605, United States. The Preparation of this clone is in Celluzzi, C.M. and Falo, L.D., J. Invest. Dermatol. (1997), Vol. 108 (5), pp. 716-720. The experiments presented can also be done with another clone, which is like the Clone B16 clones MO5 has been produced. OVA is a protein which is indeed expressed by the tumor, but not by the mice themselves. A protein that is also outside of the tumor and of the mice themselves of the tumor is the tyrosinase-related protein 2 (TRP2). Across from TRP2 reject the mice therefore an immune tolerance.

For the treatment of tumors, pentamers based on the polyoma envelope protein VP1 were constructed. The amino acid sequence of the coat protein showed at the C-terminal end a 19 amino acid OVA sequence (VP1-OVA _252-270 ) with the H-2K ^b- restricted epitope OVA _257-264 or a 13 Amino acid long TRP2 sequence (VP1-TRP2 _180-192 ) with the H-2K ^b- restricted epitope TRP2 _180-188 . The preparation of these pentamers was as follows:
Both the TRP2 _180-192 DNA fragment and the OVA _252-270 DNA fragment were generated by hybridization of two corresponding oligonucleotides each with a 5 'SapI restriction site. The two DNA fragments were each cloned into the SapI restriction site of the vector pET9a / VP1. (Abbing, A. et al., J. Biol. Chem. (2004), Vol. 279, pp. 27410-27 421 and Brinkman, M. et al., Lett. Drug Des. Disc. (2004), Vol. 1, pp 137 to 147). The resulting constructs each encode a fusion protein having the TRP2 _180-192 sequence or the OVA _252-270 sequence at the C-terminus of the amino acid sequence of the envelope protein VP1.

The expression, purification and assembly of the pentamers was carried out according to standard methods, as described, for example, in Brinkman, M. et al., Lett. Drug Des. Disc. (2004), Vol. 1, pp. 137-147 or Abbing, A. et al., J. Biol. Chem. (2004), Vol. 279, pp. 27410-27421. The procedure was as follows: After expression of the fusion proteins in E. coli BL21 DE3 for 5 h at 37 ° C, the cells were harvested, resuspended in QS-A buffer (20 mmol / l ethanolamine, 2 mmol / l EDTA, 6 mmol / l DTT, 5% glycerol, 50 mmol / l NaCl, pH 9.0) and lysed. Insoluble cell components were separated by centrifugation and the supernatant was applied to a column filled with an anion exchanger ("Poros 50" from Applied Biosystems, Frankfurter Str. 129B, 64293 Darmstadt, Germany). After elution with a linear sodium chloride gradient of 50 mmol / l to 1 mol / l NaCl, fractions containing fusion protein were further purified via a Q-Sepharose column (Amersham Biosciences Europe GmbH, Munzinger Str. 9, 79111 Freiburg, Germany). Final contaminants were equilibrated by preparative gel filtration using a Superdex 200 column (Amersham Bioscience, Germany), equilibrated in 50 mmol / l Na phosphate, 2 mmol / l EDTA, 6 mmol / l DTT, 5% glycerol, pH 6.8, away. 1 shows that VP1-OVA _252-270 pentamers have formed about 9 nm in diameter.

To generate the tumors, C57BL / 6 mice were each inoculated subcutaneously with 10 ⁵ MO5 cells. Four days later, a first therapeutic immunization was carried out by subcutaneous injection of 50 μg each of VP1-OVA _252-270 , VP1-TRP2 _180-192 or VP1 per animal. Other animals were injected with only phosphate buffered saline (PBS) for control. One week later, the injections were repeated in the same animals.

Out 2a and 2 B It can be seen that treatment of the animals with PBS or VP1-pentamers neither the rate of tumor-free mice ( 2a ) could still increase the survival rate. In contrast, about 40% of the VP1-TRP2 _180-192- treated mice remained tumor-free for more than 60 days ( 2a ) and survived this period ( 2 B ). Of the VP1-OVA _252-270- treated mice, 100 were alive after 60 days ( 2 B ). About 80% of these animals were tumor free at this time ( 2a ). The results obtained with VP1-TRP2 _180-192 show that it is possible by the pentamers to overcome an existing T cell tolerance to the epitope TRP2 _180-188 . The lower survival rate of tumor-free mice achieved with the peptide reflects the lower immunodominant capacity of the TRP2 _180-188 epitope over the OVA _257-264 epitope.

In another experiment, C57BL / 6 mice were injected with 50 μg each of VP1-OVA _252-270 or VP1-TRP2 _180-192 or PBS on day 10 and 17 after inoculation of each 10 ⁵ MO5 cells for control. Tumor size was determined daily. 3a shows that tumor formation is markedly slower in animals treated with VP1-TRP2 _180-192 pentamer or VP1-OVA _252-270 pentamer as compared to the PPS-treated animals. 3b shows a survival rate of 60% after 60 days for the VP1-TRP2 _180-192- treated animals and a survival rate of about 40% for the VP1-OVA _252-270- treated animals. Of the animals treated with PPS, none was alive after 40 days. The result demonstrates that the pentamers used are also immunotherapeutically effective in advanced tumor growth.

Claims (12)

Use of pentamers for the preparation of a Medicament for treating a disease of a tumor or an autoimmune disease in a mammal or human, wherein the pentamers are each formed from 5 subunits, wherein the subunits each have a first amino acid sequence and at least one at the N- or C-terminal end of the first amino acid sequence subsequent comprise second amino acid sequence, wherein the first amino acid sequence from an amino acid sequence, which is a structure of the envelope protein of a papovavirus or parvovirus, or one of formation the pentamer is a sufficient part of this amino acid sequence, the second amino acid sequence an amino acid sequence which is at least one epitope of the tumor or at least one the autoimmune disease causing epitope forms, the Tumor is not caused by the papovavirus or parvovirus. Use according to claim 1, wherein the envelope protein the VP2 protein of a parvovirus, the L1 protein of a papillomavirus or the VP1 protein of a polyomavirus, in particular the VP1 protein of a Simian virus 40, is. Use according to one of the preceding claims, wherein the structure of the envelope protein of the Papovavirus or parvovirus-forming amino acid sequence with this structure is identical in papovavirus or parvovirus-forming amino acid sequence. Use according to one of the preceding claims, wherein only at the C-terminal end of the first amino acid sequence, the second amino acid sequence followed. Use according to any one of claims 1 to 3, wherein the N- and at the C-terminal end of the first amino acid sequence in each case at least the second amino acid sequence or at the N- or C-terminal end of the first amino acid sequence at least the second and at the other end at least one third amino acid sequence connects, where the third amino acid sequence an amino acid sequence which is at least one more epitope of the tumor or at least another epitope causing autoimmune disease. Use according to one of the preceding claims, wherein the epitope-forming amino acid sequence an amino acid sequence is by which the epitope in the tumor or in the autoimmune disease is formed and / or wherein the further epitope forming amino acid sequence an amino acid sequence is formed by which the further epitope in the tumor or in the autoimmune disease becomes. Use according to one of the preceding claims, wherein the second amino acid sequence at least a repeat of the epitope-forming amino acid sequence and / or the third amino acid sequence at least one repetition of the further epitope-forming amino acid sequence comprises. Use according to one of the preceding claims, wherein the epitope and / or the further epitope is an MHC class I specific, in particular an HLA A2 restricted or H-2K ^b restricted, or an MHC class II specific epitope. Use according to one of the preceding claims, wherein the amino acid sequence forming the epitope or the epitope forming the further epitope 7 to 100, especially 10 to 40, preferably 13 to 19, amino acids long is. Use according to one of the preceding claims, wherein the epitope of the tumor by the apoptosis inhibiting protein survivin or a part thereof or the tumor antigen Her-2 / neu or a part thereof, especially the extracellular / transmembrane domain from Her-2 / neu, is formed. Use according to one of the preceding claims, wherein the subunits genetically, in particular by expression in Bacteria, preferably E. coli. Use according to one of the preceding claims, wherein the tumor is a melanoma.