EP1272214A1 - Vaccine against cancerous diseases which is based on mimotopes of antigens expressed on tumor cells - Google Patents
Vaccine against cancerous diseases which is based on mimotopes of antigens expressed on tumor cellsInfo
- Publication number
- EP1272214A1 EP1272214A1 EP01925550A EP01925550A EP1272214A1 EP 1272214 A1 EP1272214 A1 EP 1272214A1 EP 01925550 A EP01925550 A EP 01925550A EP 01925550 A EP01925550 A EP 01925550A EP 1272214 A1 EP1272214 A1 EP 1272214A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- mimotopes
- antibodies
- tumor cells
- antibody
- vaccine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates to a method for producing a vaccine against cancer, and this vaccine itself.
- the invention is based on the finding that such a vaccine can be obtained by a process which uses antibodies which are active against an antigen formed by the tumor cells in order to obtain mimotopes of this antigen, by means of which a body's immune response can be stimulated.
- the present invention relates to a method for producing a vaccine against cancer, which is characterized in that first of all using one or more of the body's own or synthetic antibodies, which are specifically active against one or more antigens specifically expressed by the tumor cells, from a phage. Peptide library one or more mimotopes of these antigens are selected and these mimotopes are then conjugated to a macromolecular carrier.
- the present invention furthermore relates to a vaccine against cancer which can be produced by this method.
- Vaccines against cancer diseases are obtained by the method according to the invention, even if the nature or structure of the corresponding antigen is not known or is not known in detail.
- the vaccines obtained are phage-free and therefore ideally suited for vaccination in the human system.
- the vaccine in particular also enables prophylaxis against cancer, i.e. the vaccine according to the invention is able to protect against a potential cancer by active immunization, that is to say not to let it arise in the first place.
- the vaccine can also be used to treat an existing cancer.
- antibodies are used in the method according to the invention which have already proven effective as such in clinical tests against cancer.
- the vaccine when the vaccine is administered, the formation of the body's own antibodies against those antigens of the cancer cells against which the clinically tested antibodies are also effective is induced. This increases the effectiveness of the vaccine.
- the mimotopes obtained in the process according to the invention can be conjugated to the macromolecular carrier in any manner, for example by genetic engineering or chemical means which the mimotopes are bound to the carrier by means of a chemical reaction.
- the mimotopes are preferably provided with a linker such as, for example, a short-chain oligopeptide before the conjugation with the carrier.
- the conjugation to the carrier then takes place via the linker.
- the mimotopes are conjugated to the carrier by genetic engineering, i.e. the vaccine is prepared so that a DNA or RNA sequence coding for the vaccine is incorporated into an expression system so that the entire vaccine, i.e. the carrier with the mimotopes bound to it is expressed.
- the mimotopes found with the help of the antibodies can be conjugated as mono-, di-, tri- or oligomers with a macromolecular carrier.
- 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, Volume 13, pages 53 to 57 using the example of conjugation of epitopes with macromolecular carriers.
- the described procedures can be transferred analogously to the conjugation of the mimotopes used in the method according to the invention with the macromolecular carrier. Reference is hereby made to the disclosure content of this publication.
- RNA sections coding for the epitopes are integrated either individually or one or more times in succession into the RNA sequence of the carrier.
- the expression of mono-, di- or oligomeric epitope conjugates is thereby achieved.
- the RNA or DNA sections coding for the mimotopes are integrated as mono-, di-, tri- or oligomeric mimotope sequences into the RNA or DNA sequence of the carrier.
- the mono-, di-, tri, or oligomeric mimotopes can be bound to the macromolecular carrier either simply or in multiple form.
- the present invention provides a method for producing a vaccine against adenocarcinomas of the gastrointestinal tract, prostate carcinoma, breast cancer (breast carcinoma), multiple myeloma, B-lymphoproliferative post-graft syndrome, B-cell malignancy and chronic lymphatic leukemia available.
- the vaccine produced by the process can counteract the development of these cancers.
- the antibodies used for the production of the vaccine against cancer diseases according to the invention are specifically effective against antigens which are specifically expressed by tumor cells.
- these antibodies can be the body's own, as are present in the blood serum of sick patients as a result of the humoral immune response to the antigen or antigens.
- These antibodies are produced or isolated using known, conventional methods.
- monoclonal antibodies but also polyclonal antibodies can be used in the method according to the invention.
- the body's own or synthetic antibodies used in the method according to the invention include those antibodies which trigger an antibody-dependent cellular cytotoxicity (ADCC reaction) or which recognize a receptor acting as an antigen for a growth factor of the tumor cells.
- ADCC reaction antibody-dependent cellular cytotoxicity
- the use of such antibodies ensures a particularly pronounced effect of the vaccine obtainable by the process according to the invention.
- the body's own or synthetic antibodies used in this embodiment preferably comprise those antibodies which are directed 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 are specifically effective.
- EGF Epidermal Growth Factor
- 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 US 5,772,997. Reference is hereby made to the disclosure content of this patent.
- Herceptin is a humanized monoclonal antibody derived from mice. Herceptin is active against the HER-2 / neu antigen, which is often overexpressed as a growth factor receptor on tumor cells. Especially in breast cancer tumors, the HER-2 / neu antigen is specifically expressed on the cell surface in about 20 - 30% of cases.
- the administration of Herceptin leads to the initiation of the natural cell death of the tumor cells which re-express the HER-2 /.
- the vaccine produced according to the invention is particularly effective against the types of cancer in which HER-2 / neu is expressed on the tumor cells, i.e. e.g. against breast cancer.
- the body's own or synthetic antibodies used in the method according to the invention comprise the clinically proven antibody preparation Panorex from GlaxoWellcome. This antibody preparation is also known as edrecolamab.
- Panorex is a monoclonal antibody derived from mice. As by Riethmüller et al., Lancet 343 (1994) 1177-83 Panorex is directed against the epithelial glycoprotein antigen C017-1A. Reference is hereby made to the disclosure content of this publication. Panorex is particularly effective against adenocarcinomas of the gestrointestinal tract (Punt, Cancer 83 (1998) 679-89; Martin et al., J. Clin. Pathol.
- the body's own or synthetic antibodies used in the method according to the invention comprise the clinically proven antibody preparation MabThera from Genentech Inc.
- This antibody preparation is also referred to as Rituxan (IDEC Pharmaceuticals) or Rituximab (Hoffmann-LaRoche).
- MabThera is a humanized, monoclonal antibody that is obtained from mice and is directed against the phosphoprotein surface antigen on lymphocytes, CD20. MabThera is particularly effective against multiple myeloma (Treon et al., Ann. Oncol. 1 1 (2000) 107-1 1), against B-lymphoproliferative post-graft syndrome (Milpied et al., Ann. Oncol.
- the vaccine produced according to the invention is particularly effective against these types of cancer.
- the body's own or synthetic antibodies used in the method according to the invention include the antibody preparation IMC-C225 (cetuximab) from ImClone, which is in clinical studies.
- IMC-C225 (cetuximab) is a chimerized monoclonal antibody that targets the Epidermal Growth Factor (EGF) receptor.
- EGF Epidermal Growth Factor
- IMC-C225 (cetuximab) is particularly effective against head and neck tumors (Hueng et al., Cancer Res. 59 (1999), 1935-40; Baselga et al., J. Clin. Oncol. 18 (2000) 904-14). Reference is hereby made to the disclosure content of these publications.
- the vaccine produced in the method according to the invention is particularly effective against these types of cancer.
- the representation of the mimotopes or the mimotope conjugates selected from the phage peptide libraries preferably takes place using a plant expression system, such as the tobacco mosaic virus system.
- a plant expression system such as the tobacco mosaic virus system.
- the expression of the mimotopes or the mimotope conjugates can take place by transient infection of the host plants with tobacco mosaic viruses.
- the mimotopes and mimotope conjugates expressed in this way are free of endotoxins and phages and are therefore particularly suitable for use in the process according to the invention for producing a vaccine or as a vaccine itself.
- This expression system is also particularly suitable for the production of the mimotopes or mimotope conjugates in large quantities.
- the body's own or synthetic antibodies are used in the method according to the invention to select suitable peptide mimotopes of the antigens against which antibodies are specifically active from phage peptide libraries.
- An overview of phage peptide libraries and related literature is given by M. B. Zwick, J. Shen and J. K. Scott in Current Opinion in Biotechnologie 1998, 9: 427-436. Reference is hereby made to the disclosure content of this publication.
- Phage-peptide libraries consist of parliamentary phages that express different peptides on their surface in a very wide range of variations.
- the appropriate peptide mimotopes are found by conventional selection methods using the antibodies against the specific antigen from these libraries. It should be noted that the chemical nature of the mimotopes found does not have to match the corresponding epitope of the antigen.
- the mimotopes selected in this way are characterized by DNA sequencing. Following the pattern of the sequences found, mimotopes are produced or synthesized as a fusion protein with a macromolecular carrier and chemically conjugated to the macromolecular carrier.
- This conjugation can take place, for example, in such a way that an albumin-binding protein (ABP), as is expressed, for example, by streptococci, is linked to the mimotope protein.
- ABP albumin-binding protein
- the connection of ABP and proteins is described by S. Baumann, P. Grob, F. Stuart, D. Pertlik, M. Ackermann and M. Suter in the Journal of Immunological Methods 221 (1998) 95-106. Reference is hereby made to the disclosure content of this publication.
- the step of conjugating the mimotopes with the macromolecular carrier ensures that when the vaccine is administered, an immune response of the body is induced, i.e. this step is done to make the mimotopes immunogenic.
- the expression of the mimotope proteins or mimotope conjugate proteins found can be determined by systemic transient infection of plant expression systems (host plants), such as Nicotiana Tabacum or Nicotina benthamiana, using the genomic and infectious RNA from recombinant tobacco mosaic viruses (TMV) or by completely recombinant TMV -Particles are made.
- host plants such as Nicotiana Tabacum or Nicotina benthamiana
- TMV tobacco mosaic viruses
- the DNA sequence coding for the foreign protein is first ligated 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.
- the fusion to the ABP takes place first.
- the cDNA coding for the mimotope is added to the 3 'end of the cDNA coding for the ABP in the same reading frame.
- the resulting cDNA of the ABP-mimotope fusion protein is inserted into a cDNA copy of the TMV genome. 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 is then in vitro Genome synthesized. This RNA is infectious and is applied to wounded leaves of the above host plants.
- the viral proteins and the desired foreign protein are synthesized in the cytoplasm of the host cells.
- the mimotope proteins can also be obtained on a larger scale without problems in endotoxin-free form.
- the expression or production of the mimotope proteins found can also be carried out by conventional methods, such as expression in E. coli bacteria.
- ABP ABP conjugation protein
- mimotope protein a single-stranded DNA sequence of the selected mimotopes is first obtained and double-stranded DNA is generated therefrom. This DNA is ligated into the expression vector pSB 51 1. The resulting construct of information for mimotope and ABP is used to transform competent E. coli XL-1 cells. After the cells have been amplified and harvested, the recombined protein can be purified using NiNTA agarose.
- Herceptin antibody was bound to the monoclonal mouse antibody using its Fc part. Herceptin was incubated in a concentration of 10 ⁇ g / ml PBS for two hours.
- This library contained filamentous Ml 3 phages, in the pVIII gene, which codes for a phage coat protein, inserted nucleotide sequences which code for peptides with a length of 9 amino acid residues.
- This library contained 10 9 individual phage clones, which differed in the sequence of their inserts.
- Phages which bound to the Herceptin using these peptides were detached from the Herceptin again by means of pH reduction. Suitable E. coli host cells were infected with the phage eluted in this way and amplified by the growth of the bacterial cells. This process from the binding to the antibody to the amplification of specifically bound phages is called the panning round. Three rounds of panning were performed and DNA was isolated and sequenced from the resulting phage clones. The most frequently identified sequence coding for an inserted peptide was used as the basis for the synthesis of the vaccine conjugates.
- the peptide fused to the pVIII surface on the phage is not entirely terminal, but 5 amino acid residues of the pVIII protein are still present at its N-terminus. These were in the solid phase synthesis of the Peptides included. Furthermore, the peptide was provided with a linker of the type GlyGlyGlyGlyGly at the C-terminus in order to enable the formation of the native 3D structure also on the conjugation partner. A C-terminal Cys was also added to allow the organic chemical coupling reaction to the carrier proteins.
- the mimotope was provided with the linker GGGGGC at its C-terminus. Accordingly, the amino acid sequence coupled to the coupling partner was AEGEFATLMQIISQGGGGGC (sequence 2).
- Two routes of immunization were used. 15 ⁇ g or 150 ⁇ g of the respective conjugate was used subcutaneously, 150 ⁇ g each intraperitoneally.
- the Gerbu adjuvant was used as adjuvant.
- In the subcutaneously immunized groups there were 5 mice each, in the i. p. immunized groups of 3 mice each.
- the control groups (3 mice each) were given only the conjugation partner with the Gerbu adjuvant.
- Sera from the mouse groups which had been immunized with tetanus toxoid plus sequence 2 were tested for KLH plus sequence 2 alone.
- Sera from the mouse groups that had been immunized with KLH plus sequence 2 were tested for TT plus sequence 2 and TT alone.
- Appropriate rat anti-mouse IgGl and anti-mouse IgG2a were used to determine the isotype of the mimotope-specific antibodies.
- HRP Horseraddish Peroxidase conjugated anti-rat IgG antibody was used to detect the binding. HRP undergoes a color reaction with a substrate. The result of this color reaction can be quantified with the photometer. The absorption coefficient is measured (OD value).
- the titre control was carried out after the third immunization.
- the sera were pooled.
- the dilution was 1: 2500.
- Figures 1 and 2 show the results given in Tables 1 and 2 of immunizations with sequence 2 coupled to KLH.
- the bar groups 1 to 4 correspond as follows:
- Figures 3 and 4 show the results of immunizations with sequence 2 coupled to TT. The meaning of the bars is the same as for Figures 1 and 2.
- Mimotope peptides CWAEMLLPLAC (sequence number 3); RSRLWAVME (sequence number 4); CLADPFIPHGC (sequence number 5) and ATLMQI-ISQ (sequence number 1) were synthesized, chemically coupled to KLH using succinimide-thiopyridine linkers and purified by gel filtration. 200 microg conjugate or 10 10 phage particles (with the mimotope ATLMQIISQ) per dose were used to immunize rabbits. The immunizations were carried out subcutaneously once with complete Freund's adjuvant, followed by five further doses of the antigen in incomplete Freund's adjuvant at 14-day intervals. Blood samples were taken before (preimmune serum) and one week after the last dose.
- mice IgG directed specifically against mimotopes could also be achieved in rabbits.
- the following example shows this:
- Her-2 / neu is recognized in the cell membranes of the SKBR3 cell line in the ELISA by the humanized monoclonal antibody Herceptin.
- this binding is sought to be prevented by preincubation with mimotopes on phages or KLH as a carrier.
- the wells of a 96-well ELISA plate (Nunc) were coated with membrane fractions of the Her-2 / newly overexpressing cell line SKBR3 and as a control for unspecific antibody binding with membrane fractions of the Her-2 / newly negative cell line HTB 132 in PBS overnight.
- Herceptin anti-Her-2 / neu antibody was also treated overnight by preincubation with various antigens: mimotopes, control mimotopes or buffers.
- ELISA was carried out to test the rabbit sera for specific anti-Her 2 / neu antibodies. As already described under point 3.B.), ELISA plates were coated with SKBR3 membranes, blocked and then tested in this case with sera from the immunized rabbits (preimmune serum versus immune serum) in a 1:10 dilution. Bound rabbit IgG was detected with a peroxidase-labeled anti-rabbit IgG antibody and a color reaction was developed and read again by adding substrate.
- This example shows that immunizations with Mimotop-KLH are suitable for inducing antibodies against the natural Her2 / neu on SKBR3 cells.
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Abstract
The invention relates to a method for producing a vaccine against cancerous diseases and to the vaccine itself. The invention provides that, firstly while using one or more antibodies, which are specifically active against one or more antigens specially expressed by the tumor cells, one or more mimotopes of these antigens is/are selected from a phage-peptide library. In order to obtain the vaccine, these mimotopes, in the form of monomers, dimers, trimers or oligomers, are conjugated once or repeatedly with a macromolecular support. The inventively produced vaccines, when administered, lead to a humoral immune response and thus lead to the emergence of an active immunity following the vaccination.
Description
VAKZINE GEGEN KREBSERKRANKUNGEN DIE SICH STÜTZT AUF MIMOTOPE VON AUF TUMORZELLEN EXPRIMIERTE ANTIGENEN VACCINE AGAINST CANCER DISEASES, BASED ON MIMOTOPE OF ANTIGENS EXPRESSED ON TUMOR CELLS
Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung einer Vakzine, gegen Krebserkrankungen, sowie diese Vakzine selbst.The present invention relates to a method for producing a vaccine against cancer, and this vaccine itself.
In den letzten Jahren ist in den westlichen Industrienationen ein stetiges Anwachsen der Krebserkrankungen festzustellen. Beispielsweise erkranken in der Bundesrepublik Deutschland jährlich schätzungsweise 23.000 Männer und 29.000 Frauen an Krebs des Dickdarms und Mastdarms, wobei das Erkrankungsrisiko mit dem Lebensalter allmählich ansteigt. Maligne Lym- phome machen ca. 5% aller Krebsfälle aus, wobei in Deutschland jährlich etwa 9.000 Menschen an Non-Hodgkin-Lymphomen erkranken - bei steigender Tendenz. Von Brustkrebs sind sogar etwa 10% aller Frauen in den westlichen Industrienationen betroffen.There has been a steady increase in cancer rates in western industrialized countries in recent years. For example, in the Federal Republic of Germany, an estimated 23,000 men and 29,000 women develop cancer of the large intestine and rectum annually, with the risk of illness gradually increasing with age. Malignant lymphomas make up about 5% of all cancer cases, with around 9,000 people in Germany contracting non-Hodgkin's lymphomas annually - and the trend is rising. Breast cancer affects approximately 10% of all women in western industrialized countries.
Bisher bekannte Verfahren zur Therapie von Krebserkrankungen zielen vor allen Dingen auf eine frühe Erkennung der Erkrankung und auf operative Methoden bzw. eine möglichst selektive Abtötung der Tumorzellen ab. Diese Verfahren weisen die Nachteile auf, daß eine wirkungsvolle Prophylaxe gegen die Entstehung der Krebserkrankung nicht möglich ist und daß die Behandlung zum Beispiel durch Chemotherapie mit ganz erheblichen Nebenwirkungen für den Patienten verbunden ist.Previously known methods for the treatment of cancer are aimed above all at an early detection of the disease and at operative methods or a selective selective killing of the tumor cells. These methods have the disadvantages that effective prophylaxis against the development of the cancer is not possible and that the treatment, for example by chemotherapy, is associated with very considerable side effects for the patient.
Demgemäß ist es Aufgabe der vorliegenden Erfindung, eine Vakzine gegen Krebserkrankungen zur Verfügung zu stellen, mit Hilfe derer es möglich ist, Krebserkrankungen wirksam vorzubeugen und somit das Risiko einer solchen Erkrankung deutlich zu verringern.Accordingly, it is an object of the present invention to provide a vaccine against cancer, with the help of which it is possible to effectively prevent cancer and thus significantly reduce the risk of such a disease.
Der Erfindung liegt die Erkenntnis zugrunde, daß eine solche Vakzine durch ein Verfahren erhalten werden kann, das Antikörper ausnutzt, die gegen ein von den Tumorzellen gebildetes Antigen wirksam sind, um Mimotope dieses Antigens zu erhalten, mit Hilfe derer eine körpereigene Immunantwort stimuliert werden kann.
Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung einer Vakzine gegen Krebserkrankungen, das dadurch gekennzeichnet ist, daß zunächst unter Verwendung eines oder mehrerer körpereigener oder synthetischer Antikörper, die gegen ein oder mehrere speziell von den Tumorzellen exprimierte Antigene spezifisch wirksam sind, aus einer Phagen- Peptid-Bibliothek ein oder mehrere Mimotope dieser Antigene ausgewählt werden und diese Mimotope dann mit einem makromolekularen Träger konjugiert werden.The invention is based on the finding that such a vaccine can be obtained by a process which uses antibodies which are active against an antigen formed by the tumor cells in order to obtain mimotopes of this antigen, by means of which a body's immune response can be stimulated. The present invention relates to a method for producing a vaccine against cancer, which is characterized in that first of all using one or more of the body's own or synthetic antibodies, which are specifically active against one or more antigens specifically expressed by the tumor cells, from a phage. Peptide library one or more mimotopes of these antigens are selected and these mimotopes are then conjugated to a macromolecular carrier.
Weiter ist Gegenstand der vorliegenden Erfindung eine Vakzine gegen Krebserkrankungen, die nach diesem Verfahren herstellbar ist.The present invention furthermore relates to a vaccine against cancer which can be produced by this method.
Durch das erfindungsgemäße Verfahren werden Vakzine gegen Krebserkrankungen erhalten, selbst wenn die Natur bzw. Struktur des entsprechenden Antigens nicht oder nicht im Detail bekannt ist.Vaccines against cancer diseases are obtained by the method according to the invention, even if the nature or structure of the corresponding antigen is not known or is not known in detail.
Darüber hinaus sind die erhaltenenen Vakzine phagenfrei und somit auch von daher bestens für eine Impfung im Humansystem geeignet. Damit ist durch die Vakzine insbesondere auch eine Prophylaxe gegen Krebserkrankungen möglich, d.h. die erfindungsgemäße Vakzine ist in der Lage, durch aktive Immunisierung vor einer potentiellen Krebserkrankung zu schützen, sie also erst gar nicht aufkommen zu lassen. Die Vakzine kann allerdings auch zur Therapie einer bereits bestehenden Krebserkrankung eingesetzt werden.In addition, the vaccines obtained are phage-free and therefore ideally suited for vaccination in the human system. This means that the vaccine in particular also enables prophylaxis against cancer, i.e. the vaccine according to the invention is able to protect against a potential cancer by active immunization, that is to say not to let it arise in the first place. However, the vaccine can also be used to treat an existing cancer.
In einer bevorzugten Ausführungsform werden im erfindungsgemäßen Verfahren Antikörper eingesetzt, die sich bereits als solche in klinischen Tests gegen Krebserkrankungen wirksam erwiesen haben. Dadurch wird bei Verabreichung der Vakzine die Bildung körpereigener Antikörper gegen solche Antigene der Krebszellen induziert, gegen die auch die klinisch erprobten Antikörper wirksam sind. Damit wird die Wirksamkeit der Vakzine erhöht.In a preferred embodiment, antibodies are used in the method according to the invention which have already proven effective as such in clinical tests against cancer. As a result, when the vaccine is administered, the formation of the body's own antibodies against those antigens of the cancer cells against which the clinically tested antibodies are also effective is induced. This increases the effectiveness of the vaccine.
Die Konjugation der im erfindungsgemäßen Verfahren gewonnenen Mimotope an den makromolekularen Träger kann auf beliebige Weise erfolgen, beispielsweise auf gentechnologischem oder chemischem Weg, bei
dem die Mimotope mit Hilfe einer chemischen Reaktion an den Träger gebunden werden.The mimotopes obtained in the process according to the invention can be conjugated to the macromolecular carrier in any manner, for example by genetic engineering or chemical means which the mimotopes are bound to the carrier by means of a chemical reaction.
Bevorzugterweise werden die Mimotope vor der Konjugation mit dem Träger mit einem Linker wie beispielsweise einem kurzkettigen Oligopeptid versehen. Die Konjugation an den Träger erfolgt dann über den Linker.The mimotopes are preferably provided with a linker such as, for example, a short-chain oligopeptide before the conjugation with the carrier. The conjugation to the carrier then takes place via the linker.
In einer bevorzugten Ausführungsform werden die Mimotope auf gentechnologischem Weg mit dem Träger konjugiert, d.h. die Vakzine wird so hergestellt, daß eine für die Vakzine codierende DNA- oder RNA-Sequenz in ein Expressionssystem eingebaut wird, so daß die gesamte Vakzine, d.h. der Träger mit den daran gebundenen Mimotopen, exprimiert wird.In a preferred embodiment, the mimotopes are conjugated to the carrier by genetic engineering, i.e. the vaccine is prepared so that a DNA or RNA sequence coding for the vaccine is incorporated into an expression system so that the entire vaccine, i.e. the carrier with the mimotopes bound to it is expressed.
Die mit Hilfe der Antikörper aufgefundenen Mimotope können als Mono-, Di-, Tri- oder Oligomere mit einem makromolekularen Träger konjugiert werden. Solche Konjugationen sind zum Beispiel in der Druckschrift von Th.H. Turpen, S.J. Reinl, Y. Charoenvit, S.L. Hoffman, V. Fallarme in Bio/Technology 1995, Band 13, Seiten 53 bis 57 am Beispiel der Konjugation von Epitopen mit makromolekularen Trägern beschrieben. Die beschriebenen Vorgehensweisen lassen sich analog auf die im erfindungsgemäßen Verfahren eingesetzte Konjugation der Mimotope mit dem makromolekularen Träger übertragen. Auf den Offenbarungsgehalt dieser Druckschrift wird hiermit Bezug genommen.The mimotopes found with the help of the antibodies can be conjugated as mono-, di-, tri- or oligomers with a macromolecular carrier. 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, Volume 13, pages 53 to 57 using the example of conjugation of epitopes with macromolecular carriers. The described procedures can be transferred analogously to the conjugation of the mimotopes used in the method according to the invention with the macromolecular carrier. Reference is hereby made to the disclosure content of this publication.
In der genannten Druckschrift wird zur Konjugation der Epitope ein gentechnologischer Weg eingeschlagen. In diesem werden die für die Epitope kodierenden RNA-Abschnitte entweder einzeln oder ein- oder mehrmals hintereinander gereiht in die RNA-Sequenz des Trägers integriert. Dadurch wird die Expression mono-, di- oder oligomerer Epitopkonjugate erreicht. Gemäß der vorliegenden Erfindung werden die für die Mimotope kodierenden RNA- oder DNA-Abschnitte als mono-, di-, tri- oder oligomere Mi- motopsequenzen in die RNA- oder DNA-Sequenz des Trägers integriert.In the publication mentioned, a genetic engineering path is taken for conjugating the epitopes. In this, the RNA sections coding for the epitopes are integrated either individually or one or more times in succession into the RNA sequence of the carrier. The expression of mono-, di- or oligomeric epitope conjugates is thereby achieved. According to the present invention, the RNA or DNA sections coding for the mimotopes are integrated as mono-, di-, tri- or oligomeric mimotope sequences into the RNA or DNA sequence of the carrier.
Um die Immunogenität weiterhin zu erhöhen, können die mono-, di-, trioder oligomeren Mimotope sowohl einfach als auch in multipler Form an den makromolekularen Träger gebunden werden.
In einer bevorzugten Ausführungsform stellt die vorliegende Erfindung ein Verfahren zur Herstellung einer Vakzine gegen Adenocarcinome des Ga- strointestinaltraktes, Prostata Carcinom, Brustkrebs (Mamma Carcinom), Multiples Myelom, B-lymphoproliferatives Post-Transplantat Syndrom, B- Zell Malignom und Chronisch lymphatische Leukämie zur Verfügung. Durch die durch das Verfahren hergestellte Vakzine kann der Entstehung dieser Krebsarten entgegen gewirkt werden.In order to further increase the immunogenicity, the mono-, di-, tri, or oligomeric mimotopes can be bound to the macromolecular carrier either simply or in multiple form. In a preferred embodiment, the present invention provides a method for producing a vaccine against adenocarcinomas of the gastrointestinal tract, prostate carcinoma, breast cancer (breast carcinoma), multiple myeloma, B-lymphoproliferative post-graft syndrome, B-cell malignancy and chronic lymphatic leukemia available. The vaccine produced by the process can counteract the development of these cancers.
Die zur erfϊndungsgemäßen Herstellung der Vakzine gegen Krebserkrankungen verwendeten Antikörper sind gegen speziell von Tumorzellen ex- primierte Antigene spezifisch wirksam. Bei diesen Antikörpern kann es sich zum einen um körpereigene handeln, wie sie in Folge der humoralen Immunantwort auf das oder die Antigene zum Beispiel im Blutserum von erkrankten Patienten vorhanden sind. Die Herstellung bzw. Isolierung dieser Antikörper erfolgt nach bekannten, herkömmlichen Methoden.The antibodies used for the production of the vaccine against cancer diseases according to the invention are specifically effective against antigens which are specifically expressed by tumor cells. On the one hand, these antibodies can be the body's own, as are present in the blood serum of sick patients as a result of the humoral immune response to the antigen or antigens. These antibodies are produced or isolated using known, conventional methods.
Zum anderen können auch synthetische Antikörper oder Antikörperpräparationen verwendet werden, die gegebenenfalls humanisiert wurden.On the other hand, synthetic antibodies or antibody preparations, which have been humanized if necessary, can also be used.
Weiter können im erfindungsgemäßen Verfahren monoklonale Antikörper, aber auch polyklonale Antikörper verwendet werden.Furthermore, monoclonal antibodies, but also polyclonal antibodies can be used in the method according to the invention.
In einer Ausführungsform umfassen die im erfindungsgemäßen Verfahren verwendeten körpereigenen oder synthetischen Antikörper solche Antikörper, die eine Antikörper Abhängige Celluläre Cytotoxizität (ADCC- Reaktion) auslösen oder einen als Antigen wirkenden Rezeptor für einen Wachstumsfaktor der Tumorzellen erkennen. Durch den Einsatz von solchen Antikörpern wird eine besonders ausgeprägte Wirkung der durch das erfindungsgemäße Verfahren erhältlichen Vakzine sichergestellt.In one embodiment, the body's own or synthetic antibodies used in the method according to the invention include those antibodies which trigger an antibody-dependent cellular cytotoxicity (ADCC reaction) or which recognize a receptor acting as an antigen for a growth factor of the tumor cells. The use of such antibodies ensures a particularly pronounced effect of the vaccine obtainable by the process according to the invention.
Bevorzugt umfassen die in dieser Ausführungsform eingesetzten körpereigenen oder synthetischen Antikörper solche Antikörper, die gegen das von den Tumorzellen exprimierte HER-2/neu Protein oder das epitheliale Gly- coprotein Antigen, C017-1A, oder das Phosphoprotein Oberflächenantigen an Lymphozyten, CD20, oder den Epidermal Growth Factor (EGF)- Rezeptor spezifisch wirksam sind. Durch den Einsatz solcher Antikörper ist
vor allen Dingen eine sehr gute Wirksamkeit der durch das erfindungsgemäße Verfahren hergestellten Vakzine gegen Brustkrebs (Mamma Carcinom), Adenocarcinome des Gastrointestinaltraktes, Prostata Carcinom, Multiples Myelom, B-lymphoproliferatives Post-Transplantat Syndrom, B-Zell Malignom und Kopf-Hals-Tumoren gewährleistet.The body's own or synthetic antibodies used in this embodiment preferably comprise those antibodies which are directed 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 are specifically effective. By using such antibodies Above all, a very good effectiveness of the vaccines against breast cancer (breast carcinoma), adenocarcinomas of the gastrointestinal tract, prostate carcinoma, multiple myeloma, B-lymphoproliferative post-graft syndrome, B-cell malignancy and head and neck tumors is guaranteed ,
Das HER-2/neu Protein stellt einen Rezeptor für einen Wachstumsfaktor dar, unter dessen Kontrolle die Tumorzellen wachsen. Antikörper gegen das HER-2/neu Protein sind zum Beispiel in US 5,772,997 beschrieben. Auf den Offenbarungsgehalt dieser Patentschrift wird hiermit Bezug genommen.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 US 5,772,997. Reference is hereby made to the disclosure content of this patent.
Von den gegen das HER-2/neu Protein wirksamen Antikörpern wird im erfindungsgemäßen Verfahren bevorzugterweise die klinisch erprobte Antikörperpräparation Herceptin von Genentech Inc. verwendet. Diese Antikörperpräparation hat bereits bei der Zugabe zur konventionellen Chemotherapie bei Brustkrebspatientinnen eine deutliche Verbesserung der Ansprechrate auf die Behandlung der Patientinnen herbeigeführt. Bei Herceptin handelt es sich um einen humanisierten monoklonalen Antikörper, der aus Mäusen gewonnen wird. Herceptin ist gegen das Antigen HER-2/neu wirksam, das als Wachstumsfaktor-Rezeptor auf Tumorzellen häufig überexprimiert ist. Speziell bei Brustkrebstumoren wird in etwa bei 20 - 30% der Fälle das HER-2/neu-Antigen spezifisch an der Zelloberfläche ex- primiert. Die Verabreichung von Herceptin führt zur Einleitung des natürlichen Zelltods der das HER-2/neu exprimierenden Tumorzellen. Die erfindungsgemäß hergestellte Vakzine ist besonders gegen die Krebsarten wirksam, bei denen es zu einer Expression von HER-2/neu auf den Tumorzellen kommt, d.h. z.B. gegen Brustkrebs.Of the antibodies effective against the HER-2 / neu protein, the clinically proven Herceptin antibody preparation from Genentech Inc. is preferably used in the method according to the invention. This antibody preparation has already brought about a significant improvement in the response rate to the treatment of the patients when added to conventional chemotherapy in breast cancer patients. Herceptin is a humanized monoclonal antibody derived from mice. Herceptin is active against the HER-2 / neu antigen, which is often overexpressed as a growth factor receptor on tumor cells. Especially in breast cancer tumors, the HER-2 / neu antigen is specifically expressed on the cell surface in about 20 - 30% of cases. The administration of Herceptin leads to the initiation of the natural cell death of the tumor cells which re-express the HER-2 /. The vaccine produced according to the invention is particularly effective against the types of cancer in which HER-2 / neu is expressed on the tumor cells, i.e. e.g. against breast cancer.
In einer weiteren Ausführungsform umfassen die im erfindungsgemäßen Verfahren verwendeten körpereigenen oder synthetischen Antikörper die klinisch erprobte Antikörperpräparation Panorex von GlaxoWellcome. Diese Antikörperpräparation wird auch als Edrecolamab bezeichnet. Bei Panorex handelt es sich um einen monoklonalen Antikörper, der aus Mäusen gewonnen wird. Wie von Riethmüller et al., Lancet 343 (1994) 1177-83
berichtet, ist Panorex gegen das epitheliale Glycoprotein Antigen C017-1A gerichtet. Auf den Offenbarungsgehalt dieser Druckschrift wird hiermit Bezug genommen. Panorex wirkt insbesondere gegen Adenocarcinome des Gestrointestinaltraktes (Punt, Cancer 83 (1998) 679-89; Martin et al., J. Clin. Pathol. 52 (1999) 701-4; Samonigg et al., J. Immunother. 22 (1999) 481), Prostata Carcinom (Poczatek et al., The Journal of Urology 162 (1999) 1462-6) und gegen Brustkrebs (Braun et al., Clin Cancer Res. 5 (1999) 3999-4004). Auf den Offenbarungsgehalt dieser Druckschriften wird hiermit Bezug genommen. Daher ist die erfindungsgemäß hergestellte Vakzine besonders gegen diese Krebsarten wirksam.In a further embodiment, the body's own or synthetic antibodies used in the method according to the invention comprise the clinically proven antibody preparation Panorex from GlaxoWellcome. This antibody preparation is also known as edrecolamab. Panorex is a monoclonal antibody derived from mice. As by Riethmüller et al., Lancet 343 (1994) 1177-83 Panorex is directed against the epithelial glycoprotein antigen C017-1A. Reference is hereby made to the disclosure content of this publication. Panorex is particularly effective against adenocarcinomas of the gestrointestinal 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), prostate carcinoma (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). Reference is hereby made to the disclosure content of these publications. The vaccine produced according to the invention is therefore particularly effective against these types of cancer.
In einer weiteren Ausführungsform umfassen die im erfindungsgemäßen Verfahren verwendeten körpereigenen oder synthetischen Antikörper die klinisch erprobte Antikörperpräparation MabThera von Genentech Inc.. Diese Antikörperpräparation wird auch als Rituxan (IDEC Pharmaceuti- cals) oder Rituximab (Hoffmann-LaRoche) bezeichnet. Bei MabThera handelt es sich um einen humanisierten, monoklonalen Antikörper, der aus Mäusen gewonnen wird und gegen das Phosphoprotein Oberflächenantigen an Lymphozyten, CD20, gerichtet ist. MabThera wirkt insbesondere gegen Multiples Myelom (Treon et al., Ann. Oncol. 1 1 (2000) 107-1 1), gegen B- lymphoproliferatives Post-Transplantat Syndrom (Milpied et al., Ann. Oncol. 11 (2000) 1 13-1 16), gegen B-Zell Malignom (Behr et al., Clin. Cancer Res. 5 (1999) 3304-3314) und gegen Lymphatische Leukämie. Auf den Offenbarungsgehalt dieser Druckschriften wird hiermit Bezug genommen. Daher ist die erfindungsgemäß hergestellte Vakzine besonders gegen diese Krebs arten wirksam.In a further embodiment, the body's own or synthetic antibodies used in the method according to the invention comprise the clinically proven antibody preparation MabThera from Genentech Inc. This antibody preparation is also referred to as Rituxan (IDEC Pharmaceuticals) or Rituximab (Hoffmann-LaRoche). MabThera is a humanized, monoclonal antibody that is obtained from mice and is directed against the phosphoprotein surface antigen on lymphocytes, CD20. MabThera is particularly effective against multiple myeloma (Treon et al., Ann. Oncol. 1 1 (2000) 107-1 1), against B-lymphoproliferative post-graft syndrome (Milpied et al., Ann. Oncol. 11 (2000) 1 13-1 16), against B-cell malignancy (Behr et al., Clin. Cancer Res. 5 (1999) 3304-3314) and against lymphatic leukemia. Reference is hereby made to the disclosure content of these publications. Therefore, the vaccine produced according to the invention is particularly effective against these types of cancer.
In einer weiteren Ausführungsform umfassen die im erfindungsgemäßen Verfahren verwendeten körpereigenen oder synthetischen Antikörper die in klinischen Studien befindliche Antikörperpräparation IMC-C225 (Cetuxi- mab) von ImClone. Bei IMC-C225 (Cetuximab) handelt es sich um einen chimärisierten monoklonalen Antikörper, der gegen den Epidermal Growth Factor (EGF)-Rezeptor gerichtet ist. IMC-C225 (Cetuximab) wirkt insbesondere gegen Kopf-Hals-Tumoren (Hueng et al., Cancer Res. 59 (1999),
1935-40; Baselga et al., J. Clin. Oncol. 18 (2000) 904-14). Auf den Offenbarungsgehalt dieser Druckschriften wird hiermit Bezug genommen. Die im erfindungsgemäßen Verfahren hergestellte Vakzine ist besonders gegen diese Krebsarten wirksam.In a further embodiment, the body's own or synthetic antibodies used in the method according to the invention include the antibody preparation IMC-C225 (cetuximab) from ImClone, which is in clinical studies. IMC-C225 (cetuximab) is a chimerized monoclonal antibody that targets the Epidermal Growth Factor (EGF) receptor. IMC-C225 (cetuximab) is particularly effective against head and neck tumors (Hueng et al., Cancer Res. 59 (1999), 1935-40; Baselga et al., J. Clin. Oncol. 18 (2000) 904-14). Reference is hereby made to the disclosure content of these publications. The vaccine produced in the method according to the invention is particularly effective against these types of cancer.
Die Darstellung der aus den Phagen-Peptid-Bibliotheken ausgewählten Mimotope bzw. der Mimotopkonjugate findet bevorzugt unter Verwendung eines pflanzlichen Expressionssystems statt, wie es zum Beispiel das Tabakmosaikvirussystem darstellt. In diesem System kann die Expression der Mimotope bzw. der Mimotopkonjugate durch transiente Infektion der Wirtspflanzen mit Tabakmosaikviren erfolgen. Die solchermaßen expri- mierten Mimotope und Mimotopkonjugate sind endotoxin- und phagenfrei und somit besonders für den Einsatz im erfindungsgemäßen Verfahren zur Herstellung einer Vakzine bzw. als Vakzine selbst geeignet. Dieses Expressionssystem eignet sich auch besonders für die Herstellung der Mimotope bzw. Mimotopkonjugate in größerer Menge.The representation of the mimotopes or the mimotope conjugates selected from the phage peptide libraries preferably takes place using a plant expression system, such as the tobacco mosaic virus system. In this system, the expression of the mimotopes or the mimotope conjugates can take place by transient infection of the host plants with tobacco mosaic viruses. The mimotopes and mimotope conjugates expressed in this way are free of endotoxins and phages and are therefore particularly suitable for use in the process according to the invention for producing a vaccine or as a vaccine itself. This expression system is also particularly suitable for the production of the mimotopes or mimotope conjugates in large quantities.
Im folgenden wird das erfindungsgemäße Verfahren im Detail beschrieben.The method according to the invention is described in detail below.
Die körpereigenen oder synthetischen Antikörper werden im erfindungsgemäßen Verfahren dazu verwendet, um aus Phagen-Peptid-Bibliotheken geeignete Peptid-Mimotope der Antigene, gegen die Antikörper spezifisch wirksam sind, auszuwählen. Einen Überblick über Phagen-Peptid- Bibliotheken und zugehörige Literatur geben M. B. Zwick, J. Shen und J. K. Scott in Current Opinion in Biotechnologie 1998, 9: 427-436. Auf den Offenbarungsgehalt dieser Druckschrift wird hiermit Bezug genommen.The body's own or synthetic antibodies are used in the method according to the invention to select suitable peptide mimotopes of the antigens against which antibodies are specifically active from phage peptide libraries. An overview of phage peptide libraries and related literature is given by M. B. Zwick, J. Shen and J. K. Scott in Current Opinion in Biotechnologie 1998, 9: 427-436. Reference is hereby made to the disclosure content of this publication.
Phagen-Peptid-Bibliotheken bestehen aus fϊlamentösen Phagen, die an ihrer Oberfläche unterschiedliche Peptide in einer sehr großen Variationsbreite exprimieren. Durch herkömmliche Selektionsverfahren werden unter Verwendung der gegen das spezielle Antigen wirksamen Antikörper aus diesen Bibliotheken die passenden Peptid-Mimotope aufgefunden. Dabei ist anzumerken, daß die aufgefundenen Mimotope in ihrer chemischen Natur nicht mit dem entsprechenden Epitop des Antigens übereinstimmen müssen.
Die auf diese Weise ausgewählten Mimotope werden durch DNA Sequenzierung charakterisiert. Nach dem Muster der gefundenen Sequenzen werden Mimotope als Fusionsprotein mit makromolekularem Träger produziert oder synthetisiert und chemisch an den makromolekularen Träger konjugiert. Diese Konjugation kann beispielsweise so erfolgen, daß ein Albuminbindendes Protein (ABP), wie es zum Beispiel von Streptokokken expri- miert wird, mit dem Mimotop-Protein verbunden wird. Die Verbindung von ABP und Proteinen wird von S. Baumann, P. Grob, F. Stuart, D. Pertlik, M. Ackermann und M. Suter im Journal of Immunological Methods 221 (1998) 95- 106 beschrieben. Auf den Offenbarungsgehalt dieser Druckschrift wird hiermit Bezug genommen.Phage-peptide libraries consist of parliamentary phages that express different peptides on their surface in a very wide range of variations. The appropriate peptide mimotopes are found by conventional selection methods using the antibodies against the specific antigen from these libraries. It should be noted that the chemical nature of the mimotopes found does not have to match the corresponding epitope of the antigen. The mimotopes selected in this way are characterized by DNA sequencing. Following the pattern of the sequences found, mimotopes are produced or synthesized as a fusion protein with a macromolecular carrier and chemically conjugated to the macromolecular carrier. This conjugation can take place, for example, in such a way that an albumin-binding protein (ABP), as is expressed, for example, by streptococci, is linked to 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 the Journal of Immunological Methods 221 (1998) 95-106. Reference is hereby made to the disclosure content of this publication.
Durch den Schritt der Konjugation der Mimotope mit makromolekularem Träger wird gewährleistet, daß bei Verabreichung der Vakzine eine Immunantwort des Körpers induziert wird, d.h. dieser Schritt erfolgt, um die Mimotope immunogen zu machen.The step of conjugating the mimotopes with the macromolecular carrier ensures that when the vaccine is administered, an immune response of the body is induced, i.e. this step is done to make the mimotopes immunogenic.
Die Expression der aufgefundenen Mimotop-Proteine oder Mimotopkonju- gat-Proteine kann durch systemische transiente Infektion von pflanzlichen Expressionssystemen (Wirtspflanzen), wie Nicotiana Tabacum oder Nicotina benthamiana mittels der genomischen und infektiven RNA von rekom- binanten Tabakmosaikviren (TMV) oder durch vollständige rekombinante TMV-Partikel erfolgen.The expression of the mimotope proteins or mimotope conjugate proteins found can be determined by systemic transient infection of plant expression systems (host plants), such as Nicotiana Tabacum or Nicotina benthamiana, using the genomic and infectious RNA from recombinant tobacco mosaic viruses (TMV) or by completely recombinant TMV -Particles are made.
Dazu wird zuerst die für das Fremdprotein kodierende DNA Sequenz in eine in einem Plasmid befindliche cDNA Kopie des TMV ligiert, so daß diese Sequenz unter die Kontrolle des subgenomischen Promoters für das ursprüngliche Hüllprotein des TMV gelangt. Im Falle des Mimotop- Konjugats erfolgt zunächst die Fusion an das ABP. Dazu wird die für das Mimotop kodierende cDNA an das 3'- Ende der für das ABP kodierenden cDNA im gleichen Leserahmen angefügt. Die daraus entstehende cDNA des Fusionsproteins ABP-Mimotop wird in eine cDNA Kopie des TMV Genoms eingefügt. Damit gelangt diese Sequenz unter die Kontrolle des subgenomischen Promoters für das ursprüngliche Hüllprotein des TMV. Anschließend wird in vitro ein RNA Transkript des rekombinanten TMV
Genoms synthetisiert. Diese RNA ist infektiv und wird auf verwundete Blätter der o.g. Wirtspflanzen aufgetragen. Im Zytoplasma der Wirtszellen kommt es zur Synthese der viralen Proteine und des gewünschten Fremdproteins.For this purpose, the DNA sequence coding for the foreign protein is first ligated 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, the fusion to the ABP takes place first. For this purpose, the cDNA coding for the mimotope is added to the 3 'end of the cDNA coding for the ABP in the same reading frame. The resulting cDNA of the ABP-mimotope fusion protein is inserted into a cDNA copy of the TMV genome. 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 is then in vitro Genome synthesized. This RNA is infectious and is applied to wounded leaves of the above host plants. The viral proteins and the desired foreign protein are synthesized in the cytoplasm of the host cells.
Durch diese Expressionsverfahren können die Mimotop-Proteine problemlos auch in größerem Maßstab in endotoxinfreier Form erhalten werden.With these expression methods, the mimotope proteins can also be obtained on a larger scale without problems in endotoxin-free form.
Die Expression bzw. Herstellung der aufgefundenen Mimotop-Proteine kann aber auch nach herkömmlichen Methoden, wie zum Beispiel der Expression in E. Coli-Bakterien erfolgen.However, the expression or production of the mimotope proteins found can also be carried out by conventional methods, such as expression in E. coli bacteria.
Dabei wird zur Konjugation von ABP und Mimotop-Protein zunächst eine einzelsträngige DNA-Sequenz der selektionierten Mimotope gewonnen und daraus doppelsträngige DNA erstellt. Diese DNA wird in den Expressionsvektor pSB 51 1 ligiert. Das resultierende Konstrukt aus Information für Mimotop und ABP wird zur Transformation kompetenter E. coli XL-1 Zellen verwendet. Nach Amplifikation und Ernte der Zellen kann das re- kombinante Protein mittels NiNTA Agarose gereinigt werden.For the conjugation of ABP and mimotope protein, a single-stranded DNA sequence of the selected mimotopes is first obtained and double-stranded DNA is generated therefrom. This DNA is ligated into the expression vector pSB 51 1. The resulting construct of information for mimotope and ABP is used to transform competent E. coli XL-1 cells. After the cells have been amplified and harvested, the recombined protein can be purified using NiNTA agarose.
Im folgenden wird die vorliegende Erfindung anhand eines Ausführungsbeispiels und der beigefügten Figuren weiter illustriert.In the following, the present invention is further illustrated using an exemplary embodiment and the attached figures.
Es zeigen:Show it:
Fig. 1 : Ergebnisse der Immunisierung mit dem KonjugatFig. 1: Results of the immunization with the conjugate
AEGEFATLMQIISQGGGGGC-KLH anhand IgGl .AEGEFATLMQIISQGGGGGC-KLH based on IgGl.
Fig. 2: Ergebnisse der Immunisierung mit dem KonjugatFig. 2: Results of the immunization with the conjugate
AEGEFATLMQIISQGGGGGC-KLH anhand IgG2a.AEGEFATLMQIISQGGGGGC-KLH based on IgG2a.
Fig. 3: Ergebnisse der Immunisierung mit dem KonjugatFig. 3: Results of the immunization with the conjugate
AEGEFATLMQIISQGGGGGC-TT anhand IgGl.AEGEFATLMQIISQGGGGGC-TT based on IgGl.
Fig. 4: Ergebnisse der Immunisierung mit dem KonjugatFig. 4: Results of the immunization with the conjugate
AEGEFATLMQIISQGGGGGC-TT anhand IgG2a.
BeispielAEGEFATLMQIISQGGGGGC-TT based on IgG2a. example
1. Identifikation von Herceptin-Mimotopen1. Identification of Herceptin mimotopes
Die Vertiefungen einer Maxisorb-Platte der Firma Nunc wurden mit einem monoklonalen Mausantikörper, der gegen den konstanten Fc-Teil von humanem IgG gerichtet ist, in einer Konzentration von 10 μg/ml PBS über die Dauer von 16 Stunden beschichtet.The wells of a Maxisorb plate from Nunc were coated with a monoclonal mouse antibody which is directed against the constant Fc part of human IgG in a concentration of 10 μg / ml PBS over a period of 16 hours.
Nach Waschen der Vertiefungen und Absättigen freier Bindungsstellen mittels BSA wurde der Herceptin- Antikörper mittels seines Fc-Teils an den monoklonalen Mausantikörper gebunden. Herceptin wurde in einer Konzentration von 10 μg/ml PBS für zwei Stunden inkubiert.After washing the wells and saturating free binding sites using BSA, the Herceptin antibody was bound to the monoclonal mouse antibody using its Fc part. Herceptin was incubated in a concentration of 10 μg / ml PBS for two hours.
Nach weiteren routinemäßigen Waschschritten wurden die Vertiefungen der Maxisorb-Platte mit der Phagenbibliothek überschichtet. Diese Bibliothek enthielt filamentöse Ml 3 Phagen, in deren pVIII Gen, das für ein Phagen- hüllprotein kodiert, Nukelotidsequenzen insertiert waren, die für Peptide einer Länge von 9 Aminosäureresten kodieren. Diese Bibliothek enthielt 109 individuelle Phagenklone, die sich durch die Sequenz ihrer Inserts unterschieden.After further routine washing steps, the wells of the Maxisorb plate were overlaid with the phage library. This library contained filamentous Ml 3 phages, in the pVIII gene, which codes for a phage coat protein, inserted nucleotide sequences which code for peptides with a length of 9 amino acid residues. This library contained 10 9 individual phage clones, which differed in the sequence of their inserts.
Phagen, die mittels dieser Peptide an das Herceptin banden, wurden mittels pH Erniedrigung wieder von dem Herceptin gelöst. Mit den so eluierten Phagen wurden geeignete E. coli Wirtszellen infiziert und durch das Wachstum der Bakterienzellen amplifiziert. Dieser Vorgang von der Bindung an den Antikörper bis hin zur Amplifizierung spezifisch gebundener Phagen wird als Panningrunde bezeichnet. Drei Panningrunden wurden durchgeführt und aus den resultierenden Phagenklonen wurde DNA isoliert und sequenziert. Die am häufigsten identifizierte Sequenz kodierend für ein insertiertes Peptid wurde zur Grundlage der Synthese der Impfstoffkonju- gate verwendet.Phages which bound to the Herceptin using these peptides were detached from the Herceptin again by means of pH reduction. Suitable E. coli host cells were infected with the phage eluted in this way and amplified by the growth of the bacterial cells. This process from the binding to the antibody to the amplification of specifically bound phages is called the panning round. Three rounds of panning were performed and DNA was isolated and sequenced from the resulting phage clones. The most frequently identified sequence coding for an inserted peptide was used as the basis for the synthesis of the vaccine conjugates.
Das an der Phagenoberfläche an das pVIII fusionierte Peptid ist nicht ganz endständig, sondern an seinem N-Terminus sind noch 5 Aminosäurereste des pVIII Proteins vorhanden. Diese wurden in der Festphasensynthese des
Peptides inkludiert. Weiters wurde das Peptid am C-Terminus mit einem Linker der Art GlyGlyGlyGlyGly versehen, um die Ausbildung der nativen 3D Struktur auch am Konjugationspartner zu ermöglichen. Ein C- terminales Cys wurde ebenfalls angefügt, um die Kopplungsreaktion auf organisch-chemischem Wege an die Trägerproteine zu ermöglichen.The peptide fused to the pVIII surface on the phage is not entirely terminal, but 5 amino acid residues of the pVIII protein are still present at its N-terminus. These were in the solid phase synthesis of the Peptides included. Furthermore, the peptide was provided with a linker of the type GlyGlyGlyGlyGly at the C-terminus in order to enable the formation of the native 3D structure also on the conjugation partner. A C-terminal Cys was also added to allow the organic chemical coupling reaction to the carrier proteins.
2. Immunisierungsschema2. Immunization scheme
A) Immunisierung von MäusenA) Immunization of mice
Eines der auf die unter 1. beschriebene Weise aufgefundenen Mimotope mit der Aminosäuresequenz ATLMQIISQ (Sequenz 1) wurde zur Immunisierung benutzt. An dem N-Terminus dieser Sequenz befand sich, wie unter 1. angegeben, noch 5 Aminosäurereste des pVIII Proteins, d.h. die Reste AE- GEF.One of the mimotopes with the amino acid sequence ATLMQIISQ (sequence 1) found in the manner described under 1. was used for the immunization. At the N-terminus of this sequence, as stated under 1., there were still 5 amino acid residues of the pVIII protein, i.e. the remnants AE-GEF.
Vor der Koppelung wurde das Mimotop an seinem C-Terminus mit dem Linker GGGGGC versehen. Dementsprechend war die an den Koppelungspartner angekoppelte Aminosäuresequenz AEGEFATLMQIISQGGGGGC (Sequenz 2).Before coupling, the mimotope was provided with the linker GGGGGC at its C-terminus. Accordingly, the amino acid sequence coupled to the coupling partner was AEGEFATLMQIISQGGGGGC (sequence 2).
Es wurden zwei Koppelungspartner (= Trägerproteine) mit der Sequenz 2 getestet, nämlich das KLH und das Tetanustoxoid. Es wurden zwei Immunisierungsrouten benutzt. Subkutan wurden 15 μg oder 150 μg des jeweiligen Konjugates eingesetzt, intraperitoneal jeweils 150 μg. Als Adjuvans wurde das Gerbu-Adjuvans eingesetzt. In den subkutan immunisierten Gruppen waren jeweils 5 Mäuse, in den i. p. immunisierten Gruppen jeweils 3 Mäuse. Den Kontrollgruppen (jeweils 3 Mäuse) wurden jeweils nur der Konjugationspartner mit dem Gerbu-Adjuvans verabreicht.Two coupling partners (= carrier proteins) with sequence 2 were tested, namely the KLH and the tetanus toxoid. Two routes of immunization were used. 15 μg or 150 μg of the respective conjugate was used subcutaneously, 150 μg each intraperitoneally. The Gerbu adjuvant was used as adjuvant. In the subcutaneously immunized groups there were 5 mice each, in the i. p. immunized groups of 3 mice each. The control groups (3 mice each) were given only the conjugation partner with the Gerbu adjuvant.
Nach der ersten Immunisierung (= Priming) folgten 2 Boosterungen im Abstand von 2 Wochen. Zwei Wochen nach der 2. Boosterung erfolgte eine Titerkontrolle der gegen das Mimotop gerichteten Antikörper. Der Nachweis spezifischer Antikörper erfolgte durch ELISAs;
96 Well Platten wurden mit den entsprechenden Konjugaten (= TT plus Sequenz 2 oder KLH plus Sequenz 2) beschichtet. Als Kontrolle für unspezifische Antikörperbindungen an den Konjugationspartner alleine wurden die Wells mit TT oder KLH beschichtet.After the first immunization (= priming), 2 boosters followed every 2 weeks. Two weeks after the second booster, the titers of the antibodies directed against the mimotope were checked. Specific antibodies were detected by ELISAs; 96 well plates were coated with the corresponding conjugates (= TT plus sequence 2 or KLH plus sequence 2). As a control for unspecific antibody binding to the conjugation partner alone, the wells were coated with TT or KLH.
Seren der Mausgruppen, die mit Tetanustoxoid plus Sequenz 2 immunisiert worden waren, wurden auf KLH plus Sequenz 2 alleine getestet. Seren der Mausgruppen, die mit KLH plus Sequenz 2 immunisiert worden waren, wurden auf TT plus Sequenz 2 und TT alleine getestet. Zur Isotypbestimmung der mimotopspezifischen Antikörper wurden entsprechende Ratten- anti-Maus-IgGl und anti-Maus-IgG2a eingesetzt.Sera from the mouse groups which had been immunized with tetanus toxoid plus sequence 2 were tested for KLH plus sequence 2 alone. Sera from the mouse groups that had been immunized with KLH plus sequence 2 were tested for TT plus sequence 2 and TT alone. Appropriate rat anti-mouse IgGl and anti-mouse IgG2a were used to determine the isotype of the mimotope-specific antibodies.
Zur Detektion der Bindung folgte ein Horseraddish-Peroxidase (HRP) konjugierter anti-Ratte-IgG Antikörper. HRP geht mit einem Substrat eine Farbreaktion ein. Das Ergebnis dieser Farbreaktion kann mit dem Photometer quantifiziert werden. Dabei wird der Absorptionkoeffizient gemessen (OD-Wert).A Horseraddish Peroxidase (HRP) conjugated anti-rat IgG antibody was used to detect the binding. HRP undergoes a color reaction with a substrate. The result of this color reaction can be quantified with the photometer. The absorption coefficient is measured (OD value).
Die Ergebnisse der Immunisierungen sind in den Tabellen 1 bis 4 und Fig. 1 bis 4 gezeigt.The results of the immunizations are shown in Tables 1 to 4 and Figures 1 to 4.
Die Titerkontrolle erfolgte wie bereits oben erwähnt nach der 3. Immunisierung. Die Sera wurden gepoolt. Die Verdünnung betrug 1 :2500.As already mentioned above, the titre control was carried out after the third immunization. The sera were pooled. The dilution was 1: 2500.
Die Figuren 1 und 2 zeigen die in Tabellen 1 bzw. 2 angegebenen Ergebnisse von Immunisierungen mit Sequenz 2 gekoppelt an KLH. Die Balkengruppen 1 bis 4 entsprechen wie folgt:Figures 1 and 2 show the results given in Tables 1 and 2 of immunizations with sequence 2 coupled to KLH. The bar groups 1 to 4 correspond as follows:
(1) präimmune Seren auf Mimotop plus Trägerprotein,(1) preimmune sera on mimotope plus carrier protein,
(2) potimmune (nach der 2. Boosterung) Seren auf Mimotop plus Trägerprotein,(2) potimmune (after the 2nd booster) sera on mimotope plus carrier protein,
(3) präimmune Seren auf Trägerprotein ohne Mimotop,(3) preimmune sera on carrier protein without a mimotope,
(4) postimmune Seren auf Trägerprotein ohne Mimotop.
Die Figuren 3 und 4 zeigen die Ergebnisse von Immunisierungen mit Sequenz 2 gekoppelt an TT. Die Bedeutung der Balken ist wie für Figuren 1 und 2.(4) postimmune sera on carrier protein without a mimotope. Figures 3 and 4 show the results of immunizations with sequence 2 coupled to TT. The meaning of the bars is the same as for Figures 1 and 2.
Die Immunisierungs-Daten zeigen eindeutig, daß durch die subkutane oder i. p. Gäbe von Mimotop konjugiert an KLH oder TT eine Mimotop- speziflsche Antikörperantwort induziert wurde. Diese Antikörperantwort konnte sowohl bei IgGl als auch bei IgG2a beobachtet werden (2. Gruppe von Balken in Figuren 1 bis 4).The immunization data clearly show that the subcutaneous or i. p. If mimotope conjugated to KLH or TT a mimotope-specific antibody response was induced. This antibody response could be observed for both IgGl and IgG2a (2nd group of bars in Figures 1 to 4).
Tabelle 1 : Immunisierung mit Sequenz 2-KLH, Coating mit Sequenz 2-TT, Kontrolle Coating mit TT:Table 1: Immunization with sequence 2-KLH, coating with sequence 2-TT, control coating with TT:
Tabelle 2: Immunisierung mit Sequenz 2-KLH, Coating mit Sequenz 2-TT, Kontrolle Coating mit TT:Table 2: Immunization with sequence 2-KLH, coating with sequence 2-TT, control coating with TT:
Tabelle 3: Immunisierung mit Sequenz 2-TT, Coating mit Sequenz 2-KLH, Kontrolle Coating mit KLH:Table 3: Immunization with sequence 2-TT, coating with sequence 2-KLH, control coating with KLH:
Tabelle 4: Immunisierung mit Sequenz 2-TT, Coating mit Sequenz 2 -KLH, Kontrolle Coating mit KLH: Table 4: Immunization with sequence 2-TT, coating with sequence 2 -KLH, control coating with KLH:
B) Immunisierungen von KaninchenB) Rabbit immunizations
Mimotoppeptide CWAEMLLPLAC (Sequenznummer 3); RSRLWAVME (Sequenznummer 4); CLADPFIPHGC (Sequenznummer 5) und ATLMQI- ISQ (Sequenznummer 1) wurden synthetisiert, mittels Succinimid- Thiopyridin Linker chemisch an KLH gekoppelt und über Gelfiltration gereinigt. Zur Immunisierung von Kaninchen wurden 200 microg Konjugat, respektive 1010 Phagenpartikel (mit dem Mimotop ATLMQIISQ) pro Dosis verwendet. Die Immunisierungen erfolgten subkutan einmal mit komplettem Freund'schem Adjuvans, gefolgt von fünf weiteren Gaben des Antigens in inkomplettem Freund'schem Adjuvans in 14-tägigen Abständen. Blutproben wurden vor (Präimmunserum), als auch eine Woche nach der letzten Gabe genommen.Mimotope peptides CWAEMLLPLAC (sequence number 3); RSRLWAVME (sequence number 4); CLADPFIPHGC (sequence number 5) and ATLMQI-ISQ (sequence number 1) were synthesized, chemically coupled to KLH using succinimide-thiopyridine linkers and purified by gel filtration. 200 microg conjugate or 10 10 phage particles (with the mimotope ATLMQIISQ) per dose were used to immunize rabbits. The immunizations were carried out subcutaneously once with complete Freund's adjuvant, followed by five further doses of the antigen in incomplete Freund's adjuvant at 14-day intervals. Blood samples were taken before (preimmune serum) and one week after the last dose.
Die Kaninchensera wurden auf IgG Induktion gegen das Immunogen (Mimotoppeptide) als auch den Träger KLH alleine im ELISA Assy getestet. Wie schon mit den Mäusen gezeigt, konnten auch in Kaninchen IgG spezifisch gegen Mimotope gerichtet erzielt werden. Dies sei an folgendem Beispiel gezeigt:The rabbit sera were tested for IgG induction against the immunogen (mimotop peptides) as well as the carrier KLH alone in the ELISA assy. As already shown with the mice, IgG directed specifically against mimotopes could also be achieved in rabbits. The following example shows this:
BERICHTGES BLATT (REGEL 91) ISA/EP
Kaninchen immunisiert mit KLH Mimotop:REPORTING SHEET (RULE 91) ISA / EP Rabbits immunized with KLH mimotope:
IgG gegen 1 CWAEMLL RSRLWAV CLADPFIP ATLMQIATLMQIIgG against 1 CWAEMLL RSRLWAV CLADPFIP ATLMQIATLMQI
KLH-Mimotop** PLAC ME HGC ISQ ISQ (Phage)KLH mimotope * * PLAC ME HGC ISQ ISQ (phage)
CWAEMLLPLA 0,43 0,39 0,37 0,32 0,35CWAEMLLPLA 0.43 0.39 0.37 0.32 0.35
RSRLWAVME 0,39 0,34 0,22 0,35 0,85RSRLWAVME 0.39 0.34 0.22 0.35 0.85
CLADPFIPHGC 0,38 0,41 0,35 0,34 0,28CLADPFIPHGC 0.38 0.41 0.35 0.34 0.28
ATLMQIISQ 0,55 0,42 0,42 0,33 0,45ATLMQIISQ 0.55 0.42 0.42 0.33 0.45
KLH 0,29 0,31 0,16 0,29 0,03KLH 0.29 0.31 0.16 0.29 0.03
*) Bindung an KLH-Mimotope (relative OD in ELISA)*) Binding to KLH mimotopes (relative OD in ELISA)
3. Spezifitätsnachweis3. Proof of specificity
A) Inhibitionen von Herceptin mittels Phagenmimotopen im ELISAA) Inhibitions of Herceptin using phage mimotopes in an ELISA
Zum Nachweis der Inhibierung der Bindung von Herceptin und Her-2/neu durch Mimotope wird folgendermaßen vorgegangen.To demonstrate the inhibition of Herceptin and Her-2 / neu binding by mimotopes, the procedure is as follows.
Her-2/neu wird in den Zellmembranen der Zelllinie SKBR3 im ELISA durch den humanisierten monoklonalen Antikörper Herceptin erkannt. Für den ELISA-Inhibitionsassay wird diese Bindung durch Vorinkubation mit Mimotopen an Phagen oder KLH als Träger zu verhindern gesucht.Her-2 / neu is recognized in the cell membranes of the SKBR3 cell line in the ELISA by the humanized monoclonal antibody Herceptin. For the ELISA inhibition assay, this binding is sought to be prevented by preincubation with mimotopes on phages or KLH as a carrier.
Die Vertiefungen einer 96- Well ELISA-Platte (Nunc) wurden mit Membranfraktionen der Her-2/neu überexprimierenden Zelllinie SKBR3 sowie als Kontrolle für unspezifϊsche Antikörperbindungen mit Membranfraktionen der Her-2/neu negativen Zelllinie HTB 132 in PBS über Nacht beschichtet.The wells of a 96-well ELISA plate (Nunc) were coated with membrane fractions of the Her-2 / newly overexpressing cell line SKBR3 and as a control for unspecific antibody binding with membrane fractions of the Her-2 / newly negative cell line HTB 132 in PBS overnight.
Gleichfalls über Nacht wurde Herceptin (anti-Her-2/neu-Antikörper) durch Vorinkubation mit unterschiedlichen Antigenen behandelt: Mimotope, Kontrollmimotope oder Puffer.Herceptin (anti-Her-2 / neu antibody) was also treated overnight by preincubation with various antigens: mimotopes, control mimotopes or buffers.
BERICHTIGES BLATT ( REGEL 91 ) ISA/EP
Am nächsten Tag wurden unspezifische Bindungsstellen durch 3% BSA in PBS blockiert. Auf die beschichtete, blockierte ELISA Platte wurden Herceptin als auch Herceptin nach Vorinkubation mit relevanten und nichtrelevanten Phagenmimotopen bzw. durch Mimotope an KLH als auch an KLH alleine, in Doppelbestimmung aufgetragen. Gebundenes Herceptin konnte mit einem Horseraddish-Peroxidase (HRP)-gekoppelten monoklonalen anti- human-IgG detektiert werden. HRP geht mit dem Substrat eine Farbreaktion ein. Das Ergebnis dieser Farbreaktion kann mit dem Photometer quantifiziert werden. Dabei wird der Absorptionskoeffizient von 450/630 nm bestimmt (OD Wert).CORRECTED SHEET (RULE 91) ISA / EP The next day, non-specific binding sites were blocked by 3% BSA in PBS. Herceptin and Herceptin were applied in duplicate to the coated, blocked ELISA plate after preincubation with relevant and irrelevant phage mimotopes or by mimotopes on KLH as well as on KLH alone. Bound Herceptin could be detected with a horseraddish peroxidase (HRP) coupled monoclonal anti-human-IgG. HRP undergoes a color reaction with the substrate. The result of this color reaction can be quantified with the photometer. The absorption coefficient of 450/630 nm is determined (OD value).
Als Ergebnis stellte sich eine Inhibition der Reaktivität mit dem Her-2/neu der SKBR3 Membranen als eine Verringerung des Signales dar, wenn Herceptin mit relevanten Phagenmimotopen oder Mischungen daraus vorinku- biert worden war (Abbildung ). Im Gegensatz dazu ergab sich nach Vorinkubation durch nichtrelevante Kontrollphagen keine Inhibition der Hercep- tin-Bindung. Gleichfalls keine Reaktion konnte erzielt werden, wenn Membranen der Her-2/neu negativen Zelllinie HTB 132 mit Herceptin inkubiert worden waren. Ein Kontrollantikörper (Rituximab), der gegen das Antigen CD20 gerichtet ist, welches in SKBR3 Membranen nicht exprimiert wird, konnte auf diesen Membranen auch kein Signal erzeugen.As a result, an inhibition of the reactivity with the Her-2 / neu of the SKBR3 membranes turned out to be a reduction in the signal when Herceptin had been pre-incubated with relevant phage mimotopes or mixtures thereof (Figure). In contrast, there was no inhibition of hercepetin binding after preincubation by irrelevant control phages. Likewise, no reaction could be achieved when membranes of the Her-2 / neu negative cell line HTB 132 had been incubated with Herceptin. A control antibody (rituximab), which is directed against the antigen CD20, which is not expressed in SKBR3 membranes, was also unable to generate a signal on these membranes.
Aus diesem Versuch kann geschlossen werden, daß die getesteten Phagen- mimotope, im besonderen Mischungen aus CHPTLLWPDFC (Sequenznummer 6) und CYPSLLLHLPC (Sequenznummer 7), sowie aus RSRLWAVME, CLADPFIPHGC und ATLMQIISQ das natürliche Epitop des Antikörpers Herceptin am Her-2/neu Antigen korrekt wiedergeben.
Inhibitionen mit Phagenmimotopen:From this experiment it can be concluded that the phage mimotopes tested, in particular mixtures of CHPTLLWPDFC (sequence number 6) and CYPSLLLHLPC (sequence number 7), as well as from RSRLWAVME, CLADPFIPHGC and ATLMQIISQ the natural epitope of the antibody Herceptin on the Her-2 / new antigen reproduce correctly. Inhibitions with phage mimotopes:
im ELISA
in the ELISA
Inhibitionen mit Mimotopen an KLH:Inhibitions with mimotopes on KLH:
im ELISA in the ELISA
B) Spezifitätstestung der Kaninchenseren im ELISAB) Specificity testing of the rabbit sera in the ELISA
Zur Testung der Kaninchenseren auf spezifische anti-Her 2/neu Antikörper wurde ein ELISA durchgeführt. Wie schon unter Punkt 3.B.) beschrieben, wurden ELISA Platten mit SKBR3 Membranen beschichtet, blockiert und dann in diesem Fall mit Seren der immunisierten Kaninchen (Präimmunserum versus Immunserum) in einer 1 :10 Verdünnung getestet. Gebundenes Kaninchen IgG wurde mit einem Peroxidase-markiertem anti-Kaninchen IgG Antikörper detektiert und wieder durch Substratzugabe eine Farbreaktion entwickelt und abgelesen.An ELISA was carried out to test the rabbit sera for specific anti-Her 2 / neu antibodies. As already described under point 3.B.), ELISA plates were coated with SKBR3 membranes, blocked and then tested in this case with sera from the immunized rabbits (preimmune serum versus immune serum) in a 1:10 dilution. Bound rabbit IgG was detected with a peroxidase-labeled anti-rabbit IgG antibody and a color reaction was developed and read again by adding substrate.
BERICHTIGES BLATT (REGEL 91) ISA/EP
Kaninchen immunisiert mit KLH-Mimotop zeigte einen spezifischen IgG Titeranstieg gegen SKBR3 Membranen:CORRECTED SHEET (RULE 91) ISA / EP Rabbits immunized with KLH mimotope showed a specific IgG titer increase against SKBR3 membranes:
Präimmun PostimmunPreimmune Postimmune
RSRLWAVME 0,31 0,57RSRLWAVME 0.31 0.57
An diesem Beispiel wird gezeigt, daß Immunisierungen mit Mimotop-KLH geeignet ist, Antikörper gegen das natürliche Her2/neu an SKBR3 Zellen zu induzieren.
This example shows that immunizations with Mimotop-KLH are suitable for inducing antibodies against the natural Her2 / neu on SKBR3 cells.
Claims
AnsprücheExpectations
Verfahren zur Herstellung einer Vakzine gegen Krebserkrankungen, dadurch gekennzeichnet, daß aus einer Phagen-Peptid-Bibliothek unter Verwendung eines oder mehrerer körpereigener oder synthetischer Antikörper, die gegen ein oder mehrere speziell von den Tumorzellen exprimierte Antigene spezifisch wirksam sind, ein oder mehrere Mimotope dieser Antigene ausgewählt werden und diese Mimotope mit einem makromolekularen Träger konjugiert werden.Method for producing a vaccine against cancer, characterized in that one or more mimotopes of these antigens from a phage peptide library using one or more endogenous or synthetic antibodies which are specifically active against one or more antigens specifically expressed by the tumor cells are selected and these mimotopes are conjugated to a macromolecular carrier.
Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß als Antikörper solche ausgewählt werden, die klinische Wirksamkeit gegen Krebserkrankungen besitzen.A method according to claim 1, characterized in that those are selected as antibodies which have clinical efficacy against cancer.
Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die Mimotope über einen Linker an den makromolekularen Träger gekoppelt werden.A method according to claim 1 or 2, characterized in that the mimotopes are coupled to the macromolecular carrier via a linker.
Verfahren nach einem der vorstehenden Ansprüche, dadurch gekennzeichnet, daß die Mimotope gentechnologisch oder chemisch an den makromolekularen Träger konjugiert werden.Method according to one of the preceding claims, characterized in that the mimotopes are conjugated to the macromolecular carrier by genetic engineering or chemical means.
Verfahren nach einem der vorstehenden Ansprüche, dadurch gekennzeichnet, daß die Mimotope als Monomere, Dimere, Trimere oder Oligomere mit dem makromolekularen Träger konjugiert werden.Method according to one of the preceding claims, characterized in that the mimotopes as monomers, dimers, trimers or oligomers are conjugated to the macromolecular carrier.
Verfahren nach einem der vorstehenden Ansprüche, dadurch gekennzeichnet, daß die monomeren, dimeren, trimeren oder oligomeren Mimotope einfach oder mehrfach an den makromolekularen Träger gebunden werden.Method according to one of the preceding claims, characterized in that the monomeric, dimeric, trimeric or oligomeric mimotopes are bound to the macromolecular support one or more times.
Verfahren nach einem der vorstehenden Ansprüche, dadurch gekennzeichnet, daß als Antikörper solche ausgewählt werden, die eine Antikörper Abhängige Celluläre Cytotoxizität auslösen oder einen als
Antigen wirkenden Rezeptor als einen Wachstumsfaktor der Tumorzellen erkennen.Method according to one of the preceding claims, characterized in that those are selected as antibodies which trigger an antibody-dependent cellular cytotoxicity or an as Recognize the antigen-acting receptor as a growth factor of the tumor cells.
Verfahren nach Anspruch 7, dadurch gekennzeichnet, daß als Antikörper solche ausgewählt werden, die das von Tumorzellen exprimierte Antigen HER-2/neu-Protein oder das epitheliale Glycoprotein Antigen, C017-1A, oder das Phosphoprotein-Oberflächenantigen an Lymphozyten, CD20, oder den Epidermal Growth Factor (EGF)- Rezeptor erkennen.Method according to claim 7, characterized in that those selected as antibodies are those which express the antigen HER-2 / neu protein expressed by tumor cells or the epithelial glycoprotein antigen, C017-1A, or the phosphoprotein surface antigen on lymphocytes, CD20, or the Recognize Epidermal Growth Factor (EGF) receptor.
Verfahren nach Anspruch 8, dadurch gekennzeichnet, daß die Antikörper aus den Antikörperpräparationen Herceptin oder Panorex oder MabThera oder IMC-C225 ausgewählt werden.Process according to Claim 8, characterized in that the antibodies are selected from the Herceptin or Panorex or MabThera or IMC-C225 antibody preparations.
Verfahren nach Anspruch 9, dadurch gekennzeichnet, daß als Antikörper die Antikörperpräparation Herceptin verwendet wird.A method according to claim 9, characterized in that the antibody preparation Herceptin is used as the antibody.
Verfahren nach einem der vorstehenden Ansprüche, dadurch gekennzeichnet, daß die Expression der Mimotope oder der Mimotopkonjugate unter Verwendung eines pflanzlichen Expressionssystem erfolgt.Method according to one of the preceding claims, characterized in that the expression of the mimotopes or the mimotope conjugates is carried out using a plant expression system.
Verfahren nach einem der vorstehenden Ansprüche, dadurch gekennzeichnet, daß die Expression der Mimotope oder der Mimotopkonjugate mittels transienter Infektion des pflanzlichen Expressionssystems durch Tabakmosaikviren erfolgt.Method according to one of the preceding claims, characterized in that the expression of the mimotopes or the mimotope conjugates takes place by means of transient infection of the plant expression system by tobacco mosaic viruses.
Vakzine gegen Krebserkrankungen, dadurch gekennzeichnet, daß sie nach einem Verfahren gemäß einem der vorstehenden Ansprüche herstellbar ist.
Vaccine against cancer, characterized in that it can be produced by a method according to one of the preceding claims.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10018403A DE10018403A1 (en) | 2000-04-13 | 2000-04-13 | Preparing vaccine against cancer, useful for treatment or prevention, comprises coupling antibody mimetopes,selected from a phage-display library, to macromolecular carrier |
| DE10018403 | 2000-04-13 | ||
| DE10041342 | 2000-08-23 | ||
| DE10041342A DE10041342A1 (en) | 2000-08-23 | 2000-08-23 | Preparing vaccine against cancer, useful for treatment or prevention, comprises coupling antibody mimetopes,selected from a phage-display library, to macromolecular carrier |
| PCT/EP2001/004251 WO2001078766A1 (en) | 2000-04-13 | 2001-04-12 | Vaccine against cancerous diseases which is based on mimotopes of antigens expressed on tumor cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1272214A1 true EP1272214A1 (en) | 2003-01-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP01925550A Ceased EP1272214A1 (en) | 2000-04-13 | 2001-04-12 | Vaccine against cancerous diseases which is based on mimotopes of antigens expressed on tumor cells |
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| Country | Link |
|---|---|
| US (1) | US7166694B2 (en) |
| EP (1) | EP1272214A1 (en) |
| AU (2) | AU2001252262B2 (en) |
| CA (1) | CA2405290C (en) |
| IL (1) | IL152054A0 (en) |
| WO (1) | WO2001078766A1 (en) |
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| CN102884194B (en) * | 2010-03-11 | 2015-07-22 | 健康研究股份有限公司 | Methods and compositions containing Fc fusiong proteins for enhancing immune responses |
| US20210346484A1 (en) | 2017-12-11 | 2021-11-11 | Medizinische Universitaet Wien | Method of producing a vaccine composition and uses thereof |
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| JP3040121B2 (en) * | 1988-01-12 | 2000-05-08 | ジェネンテク，インコーポレイテッド | Methods of treating tumor cells by inhibiting growth factor receptor function |
| JPH05117165A (en) | 1991-10-24 | 1993-05-14 | Masakazu Ueda | Anticancer medicine |
| US5869445A (en) * | 1993-03-17 | 1999-02-09 | University Of Washington | Methods for eliciting or enhancing reactivity to HER-2/neu protein |
| US5801005A (en) * | 1993-03-17 | 1998-09-01 | University Of Washington | Immune reactivity to HER-2/neu protein for diagnosis of malignancies in which the HER-2/neu oncogene is associated |
| JPH10510988A (en) | 1994-12-14 | 1998-10-27 | ザ スクリップス リサーチ インスティテュート | In vivo activation of tumor-specific cytotoxic T cells |
| WO1997031948A1 (en) * | 1996-03-01 | 1997-09-04 | Novartis Ag | Peptide immunogens for vaccination against and treatment of allergy |
| EP1051483A1 (en) | 1997-12-31 | 2000-11-15 | Pincus, Seth H. | A method of isolating a peptide which immunologically mimics microbial carbohydrates including group b streptococcal carbohydrates and the use thereof |
| CA2330212A1 (en) | 1998-05-08 | 1999-11-18 | Sloan-Kettering Institute For Cancer Research | Compositions and methods for active vaccination |
| JP4658423B2 (en) | 1999-08-03 | 2011-03-23 | ザ オハイオ ステイト ユニバーシティ | Polypeptides and polynucleotides for enhancing immunoreactivity against HER-2 protein |
-
2001
- 2001-04-12 CA CA2405290A patent/CA2405290C/en not_active Expired - Fee Related
- 2001-04-12 AU AU2001252262A patent/AU2001252262B2/en not_active Ceased
- 2001-04-12 US US10/257,603 patent/US7166694B2/en not_active Expired - Lifetime
- 2001-04-12 EP EP01925550A patent/EP1272214A1/en not_active Ceased
- 2001-04-12 AU AU5226201A patent/AU5226201A/en active Pending
- 2001-04-12 IL IL15205401A patent/IL152054A0/en active IP Right Grant
- 2001-04-12 WO PCT/EP2001/004251 patent/WO2001078766A1/en not_active Application Discontinuation
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| Title |
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| See references of WO0178766A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001078766A1 (en) | 2001-10-25 |
| IL152054A0 (en) | 2003-05-29 |
| US7166694B2 (en) | 2007-01-23 |
| US20050100551A1 (en) | 2005-05-12 |
| CA2405290C (en) | 2011-06-21 |
| AU2001252262B2 (en) | 2006-02-02 |
| CA2405290A1 (en) | 2001-10-25 |
| AU5226201A (en) | 2001-10-30 |
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