The present invention provides a method for producing a vaccine against cancerous disease, and the vaccine itself. This method involves first using one or more antibodies that are specifically effective against one or more antigens specially expressed by the tumor cells to select one or more mimotopes of said antigens from a phage peptide library. To obtain the vaccine, said mimotopes are conjugated to a macromolecular carrier singly or multiply in the form of their mono-, di-, tri- or oligomers. When administered, the inventively produced vaccine leads to a humoral immune response and thus to the formation of an active immunity as a consequence of vaccination.

This application claims benefit of priority to German Patent Application No. DE 100 41 342.0, filed Apr. 13, 2000 and German Patent Application No. DE 100 18 403.3, filed Aug. 23. 2000.

FIELD OF INVENTION

The present invention relates to a method for producing a vaccine against cancerous diseases and to said vaccine itself.

BACKGROUND OF INVENTION

In recent years there has been a steady increase in cancerous diseases in Western industrial nations. For example, roughly 23,000 men and 29,000 women a year develop colonic and rectal cancer in Germany, the risk of disease rising gradually with age. Malignant lymphomas constitute about 5 percent of all cancer cases, about 9,000 persons a year developing non-Hodgkin's lymphoma in Germany--with an upward trend. Breast cancer even affects about 10 percent of all women in Western industrial nations.

Methods hitherto known for treating cancerous diseases aim above all at early recognition of the illness and at surgical methods or selective destruction of tumor cells. These methods have the disadvantages that they do not permit effective prophylaxis against the genesis of the cancerous disease and that treatment for example by chemotherapy involves very considerable side effects for the patient.

Accordingly, it is the problem of the present invention to provide a vaccine against cancerous diseases that makes it possible to effectively prevent cancerous diseases and thus clearly reduce the risk of such diseases.

SUMMARY OF INVENTION

The invention is based on the finding that such a vaccine can be obtained by a method utilizing antibodies that are effective against an antigen formed by the tumor cells in order to obtain mimotopes of said antigen that can be used to stimulate an endogenous immune response.

The subject matter of the present invention is a method for producing a vaccine against cancerous diseases that is characterized by first using one or more endogenous or synthetic antibodies that are specifically effective against one or more antigens specially expressed by the tumor cells to select one or more mimotopes of said antigens from a phage peptide library and then conjugating said mimotopes to a macromolecular carrier.

The subject matter of the present invention is further a vaccine against cancerous diseases to be produced by said method.

The inventive method obtains vaccines against cancerous diseases even if the nature or structure of the corresponding antigen is unknown or not known in detail.

In addition, the obtained vaccines are phage-free and therefore also very well suited for vaccination in the human system. Thus, the vaccine in particular also permits a prophylaxis against cancerous diseases, i.e., the inventive vaccine is capable of protecting against a potential cancerous disease by active immunization so that it does not arise in the first place. However, the vaccine can also be used to treat an already existing cancerous disease.

In a preferred embodiment, the inventive method uses antibodies that have as such already proved effective against cancerous diseases in clinical tests. Thus, administration of the vaccine induces the formation of endogenous antibodies against those antigens of the cancer cells against which the clinically tested antibodies are effective. This increases the effectiveness of the vaccine.

Conjugation of the mimotopes obtained in the inventive method to the macromolecular carrier can be effected in any desired way, for example, by genetic engineering or chemically, whereby the mimotopes are bound to the carrier by a chemical reaction.

The mimotopes are preferably provided before conjugation to the carrier with a linker, for example, a short-chain oligopeptide. Conjugation to the carrier is then effected via the linker.

In a preferred embodiment, the mimotopes are conjugated to the carrier by genetic engineering, i.e., the vaccine is produced by inserting a DNA or RNA sequence coding for the vaccine into an expression system so that the total vaccine, i.e., the carrier with the mimotopes bound thereto, is expressed.

The mimotopes found with the aid of the antibodies can be conjugated to a macromolecular carrier as mono-, di-, tri- or oligomers. Such conjugations are described, for example, in the publication by Th. H. Turpen, S. J. Reinl, Y. Charoenvit, S. L. Hoffman, V. Fallarme in Bio/Technology 1995, vol. 13, pp. 53 to 57, by the example of conjugating epitopes to macromolecular carriers. The described procedures can be transferred analogously to the conjugation of mimotopes to the macromolecular carrier used in the inventive method. The disclosure of this publication is incorporated herein by reference.

In the stated publication the epitopes are conjugated by genetic engineering. The RNA portions coding for the epitopes are thereby integrated into the RNA sequence of the carrier either singly or one or more times lined up one after the other. This obtains the expression of mono-, di- or oligomeric epitope conjugates. According to the present invention, the RNA or DNA portions coding for the mimotopes are integrated into the RNA or DNA sequence of the carrier as mono-, di-, tri- or oligomeric mimotope sequences.

To further increase the immunogenicity, the mono-, di-, tri- or oligomeric mimotopes can be bound to the macromolecular carrier both singly and in multiple form.

In a preferred embodiment, the present invention provides a method for producing a vaccine against adenocarcinomas of the gastrointestinal tract, carcinoma of the prostate, breast cancer (mastocarcinoma), multiple myeloma, B-lymphoproliferative post-transplant syndrome, B-cell malignoma and chronic lymphatic leukemia. The vaccine produced by the method can counteract the genesis of said kinds of cancer.

The antibodies used for the inventive production of the vaccine against cancerous diseases are specifically effective against antigens specially expressed by tumor cells. Said antibodies may firstly be endogenous ones, as are present, for example, in the blood-serum of affected patients as a result of the humoral immune response to the antigen or antigens. The production or isolation of said antibodies is effected by known, conventional methods.

Secondly, one can also use synthetic antibodies or antibody preparations that have optionally been humanized.

Further, the inventive method can use monoclonal antibodies, as well as polyclonal antibodies.

In one embodiment, the endogenous or synthetic antibodies used in the inventive method include those antibodies that trigger an antibody dependent cellular cytotoxicity (ADCC reaction) or recognize a receptor for a growth factor of the tumor cells acting as an antigen. The use of such antibodies ensures an especially pronounced effect of the vaccine obtained by the inventive method.

The endogenous or synthetic antibodies used in this embodiment preferably include those antibodies that are specifically effective against the HER-2/neu protein expressed by the tumor cells or the epithelial glycoprotein antigen, C017-1A, or the phosphoprotein surface antigen on lymphocytes, CD20, or the epidermal growth factor (EGF) receptor. The use of such antibodies above all guarantees very good effectiveness of the vaccine produced by the inventive method against breast cancer (mastocarcinoma), adenocarcinomas of the gastrointestinal tract, carcinoma of the prostate, multiple myeloma, B-lymphoproliferative post-transplant syndrome, B-cell malignoma and head-neck tumors.

The HER-2/neu protein is a receptor for a growth factor under whose control the tumor cells grow. Antibodies against the HER-2/neu protein are described, for example, in U.S. Pat. No. 5,772,997. The disclosure of this patent print is incorporated herein by reference.

Of the antibodies effective against the HER-2/neu protein, the inventive method preferably uses the clinically tested antibody preparation, herceptin, from Genentech Inc. This antibody preparation, when added to conventional chemotherapy in breast cancer patients, has already brought about a clear improvement of the response rate to the patients' treatment. Herceptin is a humanized monoclonal antibody obtained from mice. Herceptin is effective against the antigen HER-2/neu, which is frequently overexpressed as a growth factor receptor on tumor cells. Specially in breast cancer tumors, the HER-2/neu antigen is expressed specifically on the cell surface in roughly 20 to 30 percent of cases. The administration of herceptin leads to commencement of natural death of the tumor cells expressing HER-2/neu. The inventively produced vaccine is effective particularly against the kinds of cancer in which there is an expression of HER-2/neu on the tumor cells, e.g., against breast cancer.

In a further embodiment, the endogenous or synthetic antibodies used in the inventive method include the clinically tested antibody preparation, Panorex, from GlaxoWellcome. This antibody preparation is also called Edrecolamab. Panorex is a monoclonal antibody obtained from mice. As reported by Riethmuller et al., Lancet 343 (1994) 1177 83, Panorex is directed against the epithelial glycoprotein antigen C017-1A. The disclosure of this publication is incorporated herein by reference. Panorex is effective in particular against adenocarcinomas of the gastrointestinal tract (Punt, Cancer 83 (1998) 679 89; Martin et al., J. Clin. Pathol. 52 (1999) 701 4; Samonigg et al., J. Immunother. 22 (1999) 481), carcinoma of the prostate (Poczatek et al., The Journal of Urology 162 (1999) 1462 6) and against breast cancer (Braun et al., Clin. Cancer Res. 5 (1999) 3999 4004). The disclosure of these publications is incorporated herein by reference. Therefore, the inventively produced vaccine is effective particularly against these kinds of cancer.

In a further embodiment, the endogenous or synthetic antibodies used in the inventive method include the clinically tested antibody preparation, MabThera, from Genentech Inc. This antibody preparation is also called Rituxan (IDEC Pharmaceuticals) or Rituximab (Hoffmann-LaRoche). MabThera is a humanized, monoclonal antibody obtained from mice and directed against the phosphoprotein surface antigen on lymphocytes, CD20. MabThera is effective in particular against multiple myeloma (Treon et al., Ann. Oncol. 11 (2000) 107 11), against B-lymphoproliferative post-transplant syndrome (Milpied et al, Ann. Oncol. 11 (2000) 113 116), against B-cell malignoma (Behr et al., Clin. Cancer Res. 5 (1999) 3304 3314) and against lymphatic leukemia. The disclosure of these publications is incorporated herein by reference. Therefore, the inventively produced vaccine is effective particularly against these kinds of cancer.

In a further embodiment, the endogenous or synthetic antibodies used in the inventive method include the antibody preparation, IMC-C225 (Cetuximab), from ImClone, which is undergoing clinical testing. IMC-C225 (Cetuximab) is a chimerized monoclonal antibody directed against the epidermal growth factor (EGF) receptor. IMC-C225 (Cetuximab) is effective in particular against head-neck tumors (Hueng et al., Cancer Res. 59 (1999), 1935 40; Baselga et al., J. Clin. Oncol. 18 (2000) 904 14). The disclosure of these publications is incorporated herein by reference. The vaccine produced by the inventive method is effective particularly against these kinds of cancer.

The mimotopes selected from the phage peptide libraries or the mimotope conjugates are preferably prepared using a vegetal expression system, such as the tobacco mosaic virus system. In this system, the expression of the mimotopes or mimotope conjugates can be effected by transient infection of the host plants with tobacco mosaic viruses. The thus expressed mimotopes and mimotope conjugates are endotoxin- and phage-free and, thus, particularly suitable for use in the inventive method for producing a vaccine or as the vaccine itself. This expression system is also particularly suitable for producing the mimotopes or mimotope conjugates in a large quantity.

In the following, the inventive method will be described in detail.

The endogenous or synthetic antibodies are used in the inventive method to select from phage peptide libraries suitable peptide mimotopes of the antigens against which antibodies are specifically effective. A survey of phage peptide libraries and associated literature is given by M. B. Zwick, J. Shen and J. K. Scott in Current Opinion in Biotechnology 1998: 427 436. The disclosure of this publication is incorporated herein by reference.

Phage peptide libraries consist of filamentous phages that express different peptides on their surface in a very great variation range. Conventional selection methods are used to find the matching peptide mimotopes from these libraries using the antibodies effective against the special antigen. It is to be noted that the found mimotopes do not have to match the corresponding epitope of the antigen in their chemical nature.

The thus selected mimotopes are characterized by DNA sequencing. According to the pattern of the found sequences, mimotopes are produced as fision protein with macromolecular carrier or synthesized and conjugated chemically to the macromolecular carrier. Said conjugation can be effected for example by connecting an albumen-binding protein (ABP), as is expressed for example by streptococci, with the mimotope protein. The connection of ABP and proteins is described by S. Baumann, P. Grob, F. Stuart, D. Pertlik, M. Ackermann and M. Suter in Journal of Immunological Methods 221 (1998) 95 106. The disclosure of this publication is incorporated herein by reference.

The step of conjugating the mimotopes to macromolecular carrier guarantees that administration of the vaccine induces an immune response of the body, i.e., this step is taken in order to make the mimotopes immunogenic.

The expression of the found mimotope proteins or mimotope conjugate proteins can be effected by systemic transient infection of vegetal expression systems (host plants) such as Nicotiana tabacum or Nicotiana benthamiana by the genomic and infective RNA from recombinant tobacco mosaic viruses (TMV) or by complete recombinant TMV particles.

For this purpose, the DNA sequence coding for the foreign protein is first spliced into a cDNA copy of the TMV located in a plasmid so that this sequence comes under the control of the subgenomic promoter for the original coat protein of the TMV. In the case of the mimotope conjugate, fusion to the ABP is first effected. For this purpose, the cDNA coding for the mimotope is joined to the 3' end of the cDNA coding for the ABP in the same reading frame. The resulting cDNA of the fusion protein ABP mimotope is inserted into a cDNA copy of the TMV genome. Thus this sequence comes under the control of the subgenomic promoter for the original coat protein of the TMV. An RNA transcript of the recombinant TMV genome is then synthesized in vitro. This RNA is infective and is applied to wounded leaves of the abovementioned host plants. Synthesis of the viral proteins and the desired foreign protein occurs in the cytoplasm of the host cells.

This expression method permits the mimotope proteins to be obtained easily in endotoxin-free form even on a large scale.

However, the expression or production of the found mimotype proteins can also be effected by conventional methods, for example expression in E. coli bacteria.

In this case, for conjugating ABP and mimotope protein a single-strand DNA sequence of the selected mimotopes is first obtained and double-strand DNA produced therefrom. This DNA is spliced into the expression vector pSB511. The resulting construct from information for mimotope and ABP is used for transforming competent E. coli XL-1 cells. After amplification and harvest of the cells, the recombinant protein can be purified by NiNTA agarose.
 

Claim 1 of 24 Claims

1. A peptide mimotope comprising a peptide between about 9 and 14 amino acids comprising an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
 

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