EP1629275A2 - Method for the selection of epitopes for immunotherapy - Google Patents

Method for the selection of epitopes for immunotherapy

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Publication number
EP1629275A2
EP1629275A2 EP04735707A EP04735707A EP1629275A2 EP 1629275 A2 EP1629275 A2 EP 1629275A2 EP 04735707 A EP04735707 A EP 04735707A EP 04735707 A EP04735707 A EP 04735707A EP 1629275 A2 EP1629275 A2 EP 1629275A2
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EP
European Patent Office
Prior art keywords
ala
val
lys
ile
leu
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EP04735707A
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German (de)
French (fr)
Inventor
Hans Loibner
Gottfried Himmler
Alois Jungbauer
Erich Wasserbauer
Manfred Schuster
Rainer Hahn
Astrid DÜRAUER
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ALTROPUS GMBH
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Igeneon Krebs-Immuntherapie Forschungs- und Entwicklungs-GmbH
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Publication of EP1629275A2 publication Critical patent/EP1629275A2/en
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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention relates to a method for the selection of epitopes for immunotherapy, peptides obtainable by this method and the use of the peptides as vaccines and as diagnostic agents and an immune serum obtainable by this method.
  • Immunotherapy must not only be safe, that is, the vaccines must not have any toxicity, but protective immunity must also be brought about and the longest-lasting immunological memory possible. Especially with cancer, one of the three most common causes of death in industrialized countries, immunotherapy is an important prophylactic and therapeutic approach.
  • Tumor-associated antigens are often the basis for the development of immunotherapeutic agents for the prophylaxis and / or treatment of cancer.
  • TAA are structures that are preferentially expressed on the cell membrane of tumor cells, thereby enabling differentiation from non-malignant tissue and are therefore seen as target points for diagnostic and therapeutic applications of specific antibodies.
  • Examples of tumor-associated carbohydrate structures are the Lewis antigens, which are increasingly expressed in many epithelial cancers. These include Lewis x, Lewis b and Lewis y structures, as well as sialylated Lewis x structures.
  • carbohydrate antigens are GloboH structures, KH1, Tn antigen,, TF antigens, the alpha-1,3-galactosyl epitope (Elektrophoresis (1999), 20: 362; Curr. Pharmaceutical Design (2000 ), 6: 485, Neoplasma (1996), 43: 285).
  • TAA are proteins that are particularly strongly expressed on cancer cells, e.g. CEA, TAG-72, MUC1, Folate Binding Protein A-33, CA125, EpCAM and PSA.
  • Direct therapeutic applications of antibodies against TAA are based on passive immunotherapies, which means that a specific antibody is administered systemically in a suitable amount to cancer patients and has a therapeutic effect only as long as the concentration in the organism is sufficiently high for this.
  • the biological half-life of such agents depends on their structure and ranges from a few hours to several days. It is therefore necessary to carry out repeated applications.
  • xenogenic antibodies eg murine monoclonal antibodies, MAK
  • Another approach to cancer immunotherapy is to selectively activate the cancer patient's immune system to fight malignant cells. This is being attempted using various forms of cancer vaccines. This includes vaccinations with autologous or allogeneic tumor cells, chemically or molecular biologically modified autologous or allogeneic tumor cells, isolated or produced with the help of chemical or molecular biological methods, TAA derived therefrom, recently also vaccinations with DNA coding for TAA or structures derived therefrom, etc.
  • An alternative method is based on the use of anti-idiotypic antibodies for vaccination against cancer. Suitable anti-idiotypic antibodies can immunologically mimic a TAA. As foreign proteins (e.g.
  • anti-idiotypic antibodies can be used as an immunogenic replacement of a tumor antigen for vaccination.
  • a suitable vaccine In contrast to passive immunotherapy with anti-tumor antibodies, very small amounts of a suitable vaccine are in principle sufficient for active specific cancer immunotherapy to induce immunity for months to years, which can be refreshed again after weakening by booster vaccinations.
  • both humoral and cellular immunity can be induced with active immunization, the interaction of which can result in an effective protective effect.
  • TAAs have important functions for cancer cells. They enable the degenerated cells to have characteristic properties for the malignant phenotype, e.g. exercise increased adhesiveness, which are of great importance for the establishment of metastases.
  • antigens can also be expressed on normal cells, where they are responsible for normal functions of these cells. Without claiming to be complete, here are some examples of such antigens:
  • N-CAM Neuronal Cell Adhesion Molecule
  • the Lewis Y carbohydrate antigen which appears on the majority of tumors of epithelial origin, but also plays an important role during the fetal development of epithelial tissues. It has been shown that the expression of this antigen in lung cancer is strongly associated with an unfavorable prognosis, since Lewis Y positive cancer cells obviously have a higher metastatic potential (N. Engl. J. Med. 327 (1992), 14).
  • CEA Carcino Embryonic Antigen
  • Ep-CAM Epidermal Cell Adhesion Molecule
  • epitopes that are particularly well suited for antibody detection, for example because they are located on the surface of a protein, or epitopes that lead to particularly good antibody formation in immune sera. isolate.
  • a simple and quick selection process for such epitopes or isolated peptide sequences of these epitopes would be advantageous for the success of immunotherapies.
  • Mintz et al. Describe a combinatorial approach to this, whereby peptides were isolated from “phage display random peptide libraries" using immunoglobulins isolated from patients with prostate cancer. This "fingerprint” method was used to identify specific prognostic serological markers and isolate their corresponding native antigens. Prior immunotherapy in these patients is not described. The humoral immune system was used to isolate a consensus sequence that can serve as a serological marker for the chances of survival (Nature Biotechn., 2003, 21, 57-63; Nature Biotechnol., 2003, 21, 37ff).
  • Mosolits et al. describe the binding of sera from patients with colorectal cancer to immunogenic regions of the GA733-2 tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • WO 97/15597 describes peptides whose amino acid sequence is derived from EpCAM and which bind to the MHC I molecule.
  • EP 0 326 423 describes vectors and methods for expressing the recombinant human adenocarcinoma antigen.
  • the patients did not receive any immunotherapy, the immune serum did not come from patients who had been treated with an effective vaccine.
  • the present invention is therefore based on the object of providing a method for the selection of epitopes.
  • a method for the selection of epitopes for immunotherapy in which an immune serum is brought into contact with peptide sequences of an antigen of at least 6 amino acids in length and the binding of the immune serum to the peptide sequences is compared with the binding of a presenum.
  • the immune serum can be isolated from subjects who have been immunized with a vaccine.
  • the immunization develops antibodies against the antigen or the antigenic structures with which the immunization was carried out.
  • Preferred antigens are selected from the group of tumor-associated antigens (TAA).
  • TAA tumor-associated antigens
  • TAA are structures that are preferentially expressed on the cell membrane of tumor cells, thereby enabling differentiation from non-malignant tissue and are therefore seen as target points for the diagnostic and therapeutic applications of specific antibodies.
  • the TAA are selected from the group of self-adhesion molecules or cell adhesion molecules, which can have the effect that, for example, tumor cells have strong cell-cell adhesion.
  • An example of a self-adhesion molecule is EpCAM.
  • Cancer cells practically always have, besides other physiological characteristics that distinguish them from normal cells, a different type of glycosylation (Glycoconj. J. (1997), 14: 569; Adv.Cancer Res. (1989), 52: 257; Cancer Res (1996), 56: 5309). Although the changes from tissue to tissue vary, it can be seen that aberrant glycosylation is typical of cancer cells.
  • carbohydrate structures are e.g. all Lewis antigens that are increasingly expressed in many epithelial cancers.
  • Lewis y structures these also include Lewis x and Lewis b structures, as well as sialylated Lewis x structures.
  • Other carbohydrate antigens are Globo-H structures, KH1, Tn antigen and sialyl Tn antigen, TF antigen and the alpha-1,3 galactosyl epitope (Elektrophoresis (1999), 20: 362; Curr. Pharmaceutical Design ( 2000), 6: 485, Neoplasma (1996), 43: 285)
  • TAA are proteins that are particularly strongly expressed by cancer cells, e.g. CEA, TAG-72, MUC1, Folate Binding Protein A-33, CA125, EpCAM, HER-2 / neu, PSA, MART, etc. (Sem. Cancer Biol. (1995), 6: 321).
  • Relevant TAA are often surface antigens of epithelial cells, which increasingly occur in growing cells, such as fetal tissue, and also tumor tissue.
  • a special group of TAA are involved in the adhesion processes of the epithelial cells.
  • Cellular adhesion proteins that are overexpressed on tumor cells include EpCAM, NCAM and CEA.
  • the peptide sequences can be bound to a support using known methods and brought into contact with the immune serum or the preserum.
  • the method according to the invention is preferably carried out via epitope mapping by means of solid phase peptide synthesis and spot synthesis.
  • the peptides are bound to the carrier material directly or by means of spacer sequences.
  • the immune serum and preserum can be purified by methods which are known from the prior art.
  • the purification can be carried out using Protein G Sepharose and the immunoglobulins obtained in this way can be biotinylated or coupled with radioactive substances in order to simplify the detection method.
  • preserum is serum from test subjects who do not receive immunotherapy with an antigen have peptides with which the serum is later brought into contact.
  • immune serum is serum from test subjects who have received immunotherapy with an antigen with the peptide sequences of which the serum is subsequently brought into contact.
  • This can be, for example, an immunization with epitopes against TAA, as mentioned above.
  • Immunotherapy with immunogenic antibodies was particularly preferred, as described for example in EP 1 140 168, EP 1 230 932, EP 0 644 947 and EP 0 528 767.
  • a preferred antibody which is used for active immunotherapy is an anti-EpCAM antibody, as described in WO 00/41722 or A599 / 2003.
  • an effective vaccine is to be understood as a vaccine which is effective in prophylaxis and / or in therapy.
  • the effectiveness of active immunotherapy is preferably demonstrated by the patient developing an immune response against the immunogenic substance.
  • the immune response can be measured by detecting seroconversion in the patient's serum.
  • the seroconversion is determined, for example, by detecting a differential measurement of the binding of the immunoglobulins from the patient's serum (preserum and immune serum) to the antigen used for the immunization.
  • the treatment of patients with the effective vaccine leads to an increase in the survival time or increase in the survival rate in cancer diseases, for example in colorectal or rectal cancer, by at least 10%, preferably at least 20%, preferably at least 30%, particularly preferably at least 50% compared to patients without treatment with the effective vaccine.
  • the different binding patterns of pre- and immune sera to the peptides on the carrier material can be compared.
  • the method is carried out such that the binding of the immune serum to the peptide sequences is stronger than the bond of the presum.
  • the different binding affinity can be shown, for example, by an optically stronger signal which shows the immune serum in comparison to the preserum in the binding to the peptide sequence.
  • the stronger binding pattern can also be due to the fact that the amount of the antibody which binds to this peptide sequence is increased and leads to a stronger binding signal.
  • peptides to which the immune serum has a different binding than the preserum can also be isolated using the claimed method. They are preferably peptides from tumor-associated self-adhesion molecules, for example peptides of the EpCAM protein, preferably peptides from the extracellular domain of the EpCAM protein.
  • peptides with a length of at least 6 amino acids which lie within one of the following amino acid sequences or are selected from one of the following amino acid sequences: Asn Cys Phe ' Val Asn Asn (NCFVNN)
  • they are also peptides with a length of at least 6 amino acids, selected from one of the following amino acid sequences of the EpCAM molecule, which can also be responsible for self-adhesion
  • the length of the peptides depends on their use.
  • the length thereof is preferably at least 6 amino acids and at most 30 amino acids, preferably at most 20 'amino acids, preferably at most 15 amino acids, preferably at most 10 amino acids.
  • the peptides can be bound to carrier molecules.
  • Preferred carrier molecules are antibodies or antibody derivatives or fragments, IgG2a antibodies or fragments, KLH (keyhole limpet hemocyanin), serum albumin etc. Particularly preferred carrier molecules are used which have an immunogenic / effect.
  • immunogenic defines any structure that leads to an immune response in a specific host system.
  • a murine antibody or its fragments can be highly immunogenic in the human organism, especially if it is administered with adjuvants.
  • a vaccine for active immunotherapy can be produced which contains the peptide together with a suitable adjuvant.
  • Vaccine adjuvants such as aluminum hydroxide are suitable for this (Aluminum gel) or phosphate, growth factors, lymphokines, cytokines, for example IL-2, IL-12, GM-CSF, gamma interferon, or complement factors, such as C3d, further liposome preparations or lipopolysaccharide from E. coli (LPS) but also formulations with additional antigens against which the immune system has already given a strong immune response, such as tetanus toxoid, bacterial toxins such as Pseudomonas exotoxins and derivatives of lipid A.
  • additional antigens against which the immune system has already given a strong immune response such as tetanus toxoid, bacterial toxins such as Pseudomonas exotoxins and derivatives of lipid A.
  • Known methods for conjugating or denaturing vaccine components can also be used for vaccine formulation in order to further increase the immunogenicity of the active ingredient.
  • the vaccine containing a peptide according to the invention with a carrier molecule for the active immunization is preferably administered in an amount between 0.01 ⁇ g and 10 mg.
  • the immunogenicity of the vaccine can be increased by xenogenic substances or derivatization of the antibody, which can serve as a carrier molecule for the peptide.
  • the immunogenic dose of the vaccine is preferably between 0.01 ⁇ g and 750 ⁇ g, preferably between 100 ⁇ g and 1 mg, most preferably 100 ⁇ g and 500 ⁇ g.
  • a vaccine that is administered as a depot medication naturally contains significantly higher amounts of immunogenic substance, for example at least 1 mg to 10 mg. The vaccine is released in the body over a longer period of time.
  • the peptide can also be used as a target antigen for passive immunotherapy.
  • an antibody can be produced which contains this peptide as an epitope and binds to EpCAM.
  • this antibody is administered several times at 1 to 2 week intervals.
  • the preferred amount of antibody administered is between 1 mg and 1 g, preferably between 100 mg and 500 mg, and is preferably administered intravenously.
  • an immune serum containing antibodies against epitopes which were obtained by the method according to the invention for the selection of epitopes, is also included.
  • a vaccine against tumor cells expressing EpCAM which are used to form the immune serum leads.
  • This vaccine can contain, for example, peptides, antibodies, antibody derivatives or mimotopes, anti-idiotypic antibodies or plasmids which express the EpCAM protein, together with suitable carrier substances.
  • the peptides obtained by the process according to the invention can also be used as an “antisense , peptide.
  • a peptide that binds to a region necessary for cell adhesion can prevent it.
  • proteins such as EpCAM or CEA, this can mean that the cells can no longer merge to form cells and the formation of metastases is delayed or prevented.
  • a diagnostic for the detection of specific immunoglobulins containing a peptide obtained by the method according to the invention and a detection means for determining the binding of an immune serum can also be provided.
  • test strips to which peptides are coupled can be incubated with immune sera, and the immunoglobulin specificity can be detected on the basis of the staining pattern. After incubation with biotinylated patient serum, the interaction with specific immunoglobulins is demonstrated by a color reaction.
  • the aim is to show a built-up reactivity against specific protein regions of an immunization antigen and to describe the specificity of a built-up immune response. For example, a therapeutic effect can be correlated from the proven reactivity.
  • the peptides can be coupled with biotin groups or radioactive markers and a measurable signal can be read by the radiation striking an X-ray film or radiation-sensitive film and leading to a signal.
  • FIG. 1 shows an example of a result of the EpCAM epitope mapping by means of solid phase synthesis.
  • Figure 2 shows the amino acid sequence of the EpCAM molecule.
  • the transmembrane region and the cytoplasmic region are short S1V.
  • Example 1 Epitope apping using a solid phase peptide synthesis:
  • Protein G Sepharose Fast Flow (Amersham Biosciences). was packed into an HR 5 column (inner diameter 5 mm; Amersham Biosciences). The column was equilibrated at a flow rate of 0.33 ml / min with 5 column volumes of PBS buffer. Then serum was loaded at the same flow rate and washed with a further 5 column volumes. Elution was carried out with 0.2 M acetic acid + 20% ethylene glycol, pH 2.7. The eluate was collected in 1 M sodium bicarbonate buffer and brought to pH 8.6.
  • the spot synthesis was carried out according to a modified method according to Frank (Tetrahedron 48 (1992) 9217-9232). Whatman 540 cellulose was dried in a desiccator overnight. The membrane was then functionalized with 0.2 M Fmoc- ⁇ -alanine, 0.24 M diisopropyl carbodiimide and 0.4 M 1-methyl-imidazole for 3 hours. After washing with 3 ⁇ dimethylfor amide (DMF), the Fmoc group was split off by treatment with 20% piperidine in DMF. The peptides were synthesized using an AutoSpot Robot ASP222 (INTAVIS, Germany). Another ß-alanine spacer was always introduced as the first amino acid.
  • the membrane was treated with 90% trifluoroacetic acid, 3% triisobutylsilane, 1% phenol, 2% water and 4% dichloromethane for 30 minutes.
  • the membrane was then washed 5 times with DCM, 3 times with DMF and methanol and dried.
  • DA ' ⁇ after there was a further cleavage by treatment with 50% trifluoroacetic acid, 3% Triisobutylsilane, 1% phenol, 2% water and 44% dichloromethane for two hours.
  • the mixture was then washed again with DCM, DMF and methanol and the membrane was stored at ⁇ 20 ° C.
  • IgG Since the predominant immune response is in the form of IgG, IgG was purified from the sera via Protein G Sepharose. After acidic elution with 0.2 M acetic acid in the presence of 20% ethylene glycol, the individual fractions were collected in 1M sodium carbonate in order to increase the pH as quickly as possible. These conditions also allow the purified IgG to be biotinylated immediately afterwards without previous buffering.
  • biotinylation reagent Biotinamidocaproate N-hydroxysuccinimideester, NHS-LC-Biotin, ECL protein biotinylation module, RPN 2202, from Amersham Biosciences
  • concentration of the biotinylated IgG was determined by photometric absorption measurement at 280 nm and the material was stored at 4 ° C. after adding 0.2% sodium azide.
  • the membrane development is very similar to that of a Western blot and is built up from the same steps: the cellulose membranes were conditioned in 20% methanol and blocked with 3% BSA / PBS-T. The incubation with the biotinylated sera then took place. After a washing step, incubation was carried out with streptavidin-HRP conjugate (ECL protein biotinylation module, RPN 2202, from Amersham Biosciences), in order then to induce the detectable light development via oxidation of the peroxidase in the presence of an amplifier solution. The membranes were measured immediately after development and with a constant measurement duration. The blank membranes carried were only incubated with dilution buffer instead of biotinylated IgG and then with streptavidin-HRP.
  • a displacement test was carried out in addition to the analysis of the biotinylated preimmune and immune sera.
  • part of the purified preimmune sera was not biotinylated, but only buffered in PBS.
  • the immune serum came from a patient who underwent active immunotherapy with mAbl7-1A, an EpCAM antibody.
  • the immune serum was withdrawn 71 days after the immunization.
  • the preserum was removed on day 1, ie shortly before the start of treatment. It was shown that immune serum binds more strongly to the lomer peptides. de 1-2, 34-36, 42, 45 53, 61, 68-71 while the preserum does not show this bond or shows it only to a small extent.

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Abstract

The invention relates to a method for the selection of epitopes for immunotherapy, peptides obtained by said method, the use of said peptides as vaccines and diagnostics and an immune serum obtainable by said method.

Description

Verfahren zur Selektion von Epitopen zur Immuntherapie Procedure for the selection of epitopes for immunotherapy
Die Erfindung betrifft ein Verfahren zur Selektion von Epitopen zur Immuntherapie, Peptide erhältlich durch dieses Verfahren und die Verwendung der Peptide als Vakzine und als Diagnostikum und ein Immunserum erhältlich durch dieses Verfahren.The invention relates to a method for the selection of epitopes for immunotherapy, peptides obtainable by this method and the use of the peptides as vaccines and as diagnostic agents and an immune serum obtainable by this method.
Immuntherapie muss nicht nur sicher sein, das heißt die Impfstoffe dürfen keine Toxizität aufweisen, sondern es mύss auch eine schützende Immunität herbeigeführt werden und ein möglichst langlebiges immunologisches Gedächtnis erzeugt werden. Besonders bei Krebs, eine der drei häufigsten Todesursachen in den industrialisierten Ländern, ist die Immuntherapie ein wichtiger Prophylaxe- und Therapieansatz.Immunotherapy must not only be safe, that is, the vaccines must not have any toxicity, but protective immunity must also be brought about and the longest-lasting immunological memory possible. Especially with cancer, one of the three most common causes of death in industrialized countries, immunotherapy is an important prophylactic and therapeutic approach.
Tumor-assoziierte Antigene (TAA) sind oftmals Grundlage für die Entwicklung von immuntherapeutischen Mitteln zur Prophylaxe und/oder Behandlung von Krebs. TAA sind Strukturen, die bevorzugt auf der Zellmembran von Tumorzellen exprimiert sind, dadurch eine Unterscheidung zu nicht-malignem Gewebe ermöglichen und daher als Zielpunkte für diagnostische und therapeutische Anwendungen von spezifischen Antikörpern gesehen werden. Beispiele für Tumor-assoziierte Kohlenhydratstrukturen sind die Le- wis-Antigene, die verstärkt in vielen epithelialen Krebsarten exprimiert werden. Dazu gehören Lewis x-, Lewis b- und Lewis y- Strukturen, sowie sialylierte Lewis x-Strukturen. Andere Kohlen- hydrat-Antigene sind GloboH-Strukturen, KH1, Tn-Antigen, ,TF-An- tigen, das alpha-1, 3-Galactosyl-Epitop (Elektrophoresis (1999), 20:362; Curr. Pharmaceutical Design (2000), 6:485, Neoplasma (1996), 43:285).Tumor-associated antigens (TAA) are often the basis for the development of immunotherapeutic agents for the prophylaxis and / or treatment of cancer. TAA are structures that are preferentially expressed on the cell membrane of tumor cells, thereby enabling differentiation from non-malignant tissue and are therefore seen as target points for diagnostic and therapeutic applications of specific antibodies. Examples of tumor-associated carbohydrate structures are the Lewis antigens, which are increasingly expressed in many epithelial cancers. These include Lewis x, Lewis b and Lewis y structures, as well as sialylated Lewis x structures. Other carbohydrate antigens are GloboH structures, KH1, Tn antigen,, TF antigens, the alpha-1,3-galactosyl epitope (Elektrophoresis (1999), 20: 362; Curr. Pharmaceutical Design (2000 ), 6: 485, Neoplasma (1996), 43: 285).
Weitere TAA sind Proteine, die auf Krebszellen besonders stark exprimiert werden, wie z.B. CEA, TAG-72, MUC1, Folate Binding Protein A-33, CA125, EpCAM und PSA.Other TAA are proteins that are particularly strongly expressed on cancer cells, e.g. CEA, TAG-72, MUC1, Folate Binding Protein A-33, CA125, EpCAM and PSA.
Direkte therapeutische Anwendungen von Antikörpern gegen TAA beruhen auf passiven Immuntherapien, das heißt, ein spezifischer Antikörper wird systemisch in geeigneter Menge an Krebspatienten verabreicht und übt eine therapeutische Wirkung nur aus, solange die Konzentration im Organismus dafür genügend groß ist. Die biologische Halbwertszeit solcher Agentien hängt von ihrer Struktur ab und beträgt wenige Stunden bis mehrere Tage. Daher ist es notwendig, wiederholte Applikationen vorzunehmen. Das führt bei Verwendung von xenogenen Antikörpern (z.B. murine monoklonale Antikörper, MAK) aber zu unerwünschten Immunreaktionen, die eine mögliche therapeutische Wirkung neutralisieren und gefährliche Nebenwirkungen (anaphylaktische Reaktionen) bedingen können. Daher können solche Immuntherapeu- tika nur für eine begrenzte Zeit verabreicht werden.Direct therapeutic applications of antibodies against TAA are based on passive immunotherapies, which means that a specific antibody is administered systemically in a suitable amount to cancer patients and has a therapeutic effect only as long as the concentration in the organism is sufficiently high for this. The biological half-life of such agents depends on their structure and ranges from a few hours to several days. It is therefore necessary to carry out repeated applications. When using xenogenic antibodies (eg murine monoclonal antibodies, MAK), however, this leads to undesirable immune reactions, which neutralize a possible therapeutic effect and can cause dangerous side effects (anaphylactic reactions). Therefore, such immunotherapeutics can only be administered for a limited time.
Einem anderen Ansatz für Immuntherapie von Krebs liegt die selektive Aktivierung des Immunsystems von Krebspatienten zugrunde, maligne Zellen zu bekämpfen. Das wird mittels verschiedenster Formen von Krebsvakzinen versucht. Dazu gehören Impfungen mit autologen oder allogenen Tumorzellen, chemisch oder molekularbiologisch modifizierten autologen oder allogenen Tumorzellen, isolierten oder mit Hilfe von chemischen oder molekularbiologischen Methoden hergestellten TAA, daraus abgeleiteten Peptiden, neuerdings auch Impfungen mit DNA, die für TAA oder daraus abgeleiteten Strukturen codieren, etc. Eine alternative Methode beruht auf der Verwendung von anti-idiotypischen Antikörpern zur Vakzinierung gegen Krebs. Geeignete anti-idiotypische Antikörper können ein TAA immunologisch nachahmen. Sie induzieren als Fremdproteine (z.B. murine Antikörper, Ziegen-Antikörper etc.) nach Vakzinierung - im Gegensatz zu den eigentlichen menschlichen Tumorantigenen, die als Selbst-Strukturen oft nur wenig immunogen sind - im Menschen eine starke Immunantwort. Daher können anti-idiotypische Antikörper als immunogener Ersatz eines Tumorantigens zur Impfung verwendet werden.Another approach to cancer immunotherapy is to selectively activate the cancer patient's immune system to fight malignant cells. This is being attempted using various forms of cancer vaccines. This includes vaccinations with autologous or allogeneic tumor cells, chemically or molecular biologically modified autologous or allogeneic tumor cells, isolated or produced with the help of chemical or molecular biological methods, TAA derived therefrom, recently also vaccinations with DNA coding for TAA or structures derived therefrom, etc. An alternative method is based on the use of anti-idiotypic antibodies for vaccination against cancer. Suitable anti-idiotypic antibodies can immunologically mimic a TAA. As foreign proteins (e.g. murine antibodies, goat antibodies etc.) they induce a strong immune response in humans after vaccination - in contrast to the actual human tumor antigens, which are often only slightly immunogenic as self-structures. Therefore, anti-idiotypic antibodies can be used as an immunogenic replacement of a tumor antigen for vaccination.
Im Unterschied zur passiven Immuntherapie mit Anti-Tumorantikörpern genügen für die aktive spezifische Krebs-Immuntherapie im Prinzip sehr kleine Mengen eines geeigneten Impfstoffes, um für Monate bis Jahre eine Immunität zu induzieren, die bei Abschwä- chung durch Booster-Impfungen wieder aufgefrischt werden kann. Darüber hinaus kann bei aktiver Immunisierung sowohl eine humorale als auch eine zelluläre Immunität induziert werden, deren Zusammenspiel eine effektive Schutzwirkung ergeben kann.In contrast to passive immunotherapy with anti-tumor antibodies, very small amounts of a suitable vaccine are in principle sufficient for active specific cancer immunotherapy to induce immunity for months to years, which can be refreshed again after weakening by booster vaccinations. In addition, both humoral and cellular immunity can be induced with active immunization, the interaction of which can result in an effective protective effect.
Zusammenfassend beruht die bisherige Verwendung von Antikörpern oder von deren Derivaten in der Krebs-Immuntherapie im wesentlichen auf zwei Prinzipien:In summary, the previous use of antibodies is based or of their derivatives in cancer immunotherapy essentially on two principles:
• Passive Therapie mit , Antikörpern oder deren Derivaten, die gegen TAA gerichtet sind. Dabei werden Tumorzellen relativ spezifisch zerstört (Immunology Today (2000), 21:403-410; Curr. Opin. Immunol. (1997), 9:717).• Passive therapy with, antibodies or their derivatives that are directed against TAA. Tumor cells are destroyed relatively specifically (Immunology Today (2000), 21: 403-410; Curr. Opin. Immunol. (1997), 9: 717).
• Aktive Immunisierung (Vakzinierung) mit TAA bzw. Antikörpern oder deren Derivaten, die gegen den Idiotyp von Antikörpern mit Spezifität gegen TAA gerichtet sind. Die aktive Impfung löst eine Immunantwort gegen TAA aus. Diese Immunantwort ist somit auch gegen die entsprechenden Tumorzellen gerichtet (Ann. Med. (1999), 31:66; Immunobiol . (1999), 201:1).• Active immunization (vaccination) with TAA or antibodies or their derivatives, which are directed against the idiotype of antibodies with specificity against TAA. Active vaccination triggers an immune response against TAA. This immune response is therefore also directed against the corresponding tumor cells (Ann. Med. (1999), 31:66; Immunobiol. (1999), 201: 1).
Im Verlauf der Entdeckung und nachfolgender Charakterisierung von verschiedensten TAA hat sich herausgestellt, dass diese wichtige Funktionen für Krebszellen haben. Sie ermöglichen den entarteten Zellen, charakteristische Eigenschaften für den malignen Phänotyp wie z.B. vermehrte Adhäsionsfähigkeit auszuüben, die für die Etablierung von Metastasen von großer Bedeutung sind. Allerdings können solche Antigene durchaus in bestimmten Stadien auch auf normalen Zellen exprimiert sein, wo sie für normale Funktionen dieser Zellen verantwortlich sind. Ohne Anspruch auf Vollständigkeit seien hier einige Beispiele für solche Antigene angeführt:In the course of the discovery and subsequent characterization of a wide variety of TAAs, it has emerged that these have important functions for cancer cells. They enable the degenerated cells to have characteristic properties for the malignant phenotype, e.g. exercise increased adhesiveness, which are of great importance for the establishment of metastases. However, in certain stages such antigens can also be expressed on normal cells, where they are responsible for normal functions of these cells. Without claiming to be complete, here are some examples of such antigens:
• N-CAM (Neuronal Cell Adhesion Molecule) , das oft auf Tumoren neuronalen Ursprungs exprimiert ist und homophile Adhäsion bewirkt (J.Cell Biol. 118 (1992), 937).• N-CAM (Neuronal Cell Adhesion Molecule), which is often expressed on tumors of neuronal origin and causes homophilic adhesion (J. Cell Biol. 118 (1992), 937).
• Das Lewis Y Kohlenhydratantigen, das auf der Mehrzahl der Tumoren epithelialen Ursprungs aufscheint, aber auch während der fötalen Entwicklung epithelialer Gewebe eine wichtige Rolle spielt. Es wurde gezeigt, dass die Expression dieses An- tigens in Lungenkrebs stark mit einer ungünstigen Prognose assoziiert ist, da Lewis Y positive Krebszellen offensichtlich ein höheres metastatisches Potential haben (N. Engl. J. Med. 327 (1992) , 14) .• The Lewis Y carbohydrate antigen, which appears on the majority of tumors of epithelial origin, but also plays an important role during the fetal development of epithelial tissues. It has been shown that the expression of this antigen in lung cancer is strongly associated with an unfavorable prognosis, since Lewis Y positive cancer cells obviously have a higher metastatic potential (N. Engl. J. Med. 327 (1992), 14).
• CEA (Carcino Embryonic Antigen) , das häufig auf epithelialen Tumoren des Gastro-Intestinaltraktes vorkommt und als Selbstadhäsionsmolekül identifiziert wurde (Cell 57 (1989), 327). • Ep-CAM (Epithelial Cell Adhesion Molecule) , das auf fast allen Tumoren epithelialen Ursprungs exprimiert ist, aber auch auf vielen normalen Epithelien vorkommt, das als Selbstadhäsionsmolekül charakterisiert wurde und daher als pan-epitheliales Adhäsionsantigen klassifiziert werden kann (J. Cell Biol.125 (1994) , 437) .• CEA (Carcino Embryonic Antigen), which occurs frequently on epithelial tumors of the gastrointestinal tract and has been identified as a self-adhesion molecule (Cell 57 (1989), 327). • Ep-CAM (Epithelial Cell Adhesion Molecule), which is expressed on almost all tumors of epithelial origin, but also occurs on many normal epithelia, which has been characterized as a self-adhesion molecule and can therefore be classified as pan-epithelial adhesion antigen (J. Cell Biol. 125: 437 (1994).
Um eine Immuntherapie möglichst effizient und erfolgreich zu gestalten, ist es aber von Vorteil, jene Epitope, die für die Antikörpererkennung besonders gut geeignet sind da sie beispielsweise an der Oberfläche eines Proteins lokalisiert sind oder Epitope, die in Immunseren zu einer besonders guten Antikörperbildung führen, zu isolieren. Ein einfaches und schnelles Selektionsverfahren solcher Epitope bzw. isolierter Peptidse- quenzen dieser Epitope, wären für den Erfolg von Immuntherapien von Vorteil.In order to make immunotherapy as efficient and successful as possible, however, it is advantageous to use those epitopes that are particularly well suited for antibody detection, for example because they are located on the surface of a protein, or epitopes that lead to particularly good antibody formation in immune sera. isolate. A simple and quick selection process for such epitopes or isolated peptide sequences of these epitopes would be advantageous for the success of immunotherapies.
Einen kombinatorischen Ansatz dazu beschreiben Mintz et al., wobei hier mit Hilfe von Immunglobulinen, isoliert aus Patienten mit Prostata Krebs, aus „Phage display random peptide libraries" Peptide isoliert wurden. Dieses „Fingerprint" Verfahren wurde verwendet, um spezifische prognostische serologische Marker und deren korrespondierende native Antigene zu isolieren. Eine vorhergehende Immuntherapie dieser Patienten wird nicht beschrieben. Das humorale Immunsystem wurde herangezogen, um eine Konsensussequenz zu isolieren, die als serologischer Marker für die Überlebenschancen dienen kann (Nature Biotechn., 2003, 21, 57-63; Nature Biotechnol., 2003, 21, 37ff) .Mintz et al. Describe a combinatorial approach to this, whereby peptides were isolated from "phage display random peptide libraries" using immunoglobulins isolated from patients with prostate cancer. This "fingerprint" method was used to identify specific prognostic serological markers and isolate their corresponding native antigens. Prior immunotherapy in these patients is not described. The humoral immune system was used to isolate a consensus sequence that can serve as a serological marker for the chances of survival (Nature Biotechn., 2003, 21, 57-63; Nature Biotechnol., 2003, 21, 37ff).
Mosolits et al. beschreiben die Bindung von Seren aus Patienten mit Colorektalem Karzinom an immunogene Regionen des GA733-2 Tumor assoziierten Antigens (TAA) . Eine Peptidsequenz von 18 Aminosäuren wurde als immundominantes B Zeilepitop isoliert, da 50% der Patienten Antikörper gegen dieses Peptid aufweisen.Mosolits et al. describe the binding of sera from patients with colorectal cancer to immunogenic regions of the GA733-2 tumor-associated antigen (TAA). A peptide sequence of 18 amino acids was isolated as an immunodominant B cell epitope because 50% of the patients have antibodies against this peptide.
WO 97/15597 beschreibt Peptide deren Aminosäuresequenz aus EpCAM abgeleitet ist und die an das MHC I Molekül binden.WO 97/15597 describes peptides whose amino acid sequence is derived from EpCAM and which bind to the MHC I molecule.
EP 0 326 423 beschreibt Vektoren und Verfahren zur Expression des rekombinanten humanen Adenocarcinom-Antigens . In diesen Publikationen haben die Patienten keinerlei Immuntherapie erhalten, das Immunserum stammte nicht aus Patienten die mit einem wirksamen Impfstoff behandelt worden waren.EP 0 326 423 describes vectors and methods for expressing the recombinant human adenocarcinoma antigen. In these publications, the patients did not receive any immunotherapy, the immune serum did not come from patients who had been treated with an effective vaccine.
Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren zur Selektion von Epitopen zur Verfügung zu stellen.The present invention is therefore based on the object of providing a method for the selection of epitopes.
Die Aufgabe wird durch die Bereitstellung der in den Ansprüchen bezeichneten Ausführungsformen gelöst.The object is achieved by the provision of the embodiments described in the claims.
Erfindungsgemäß wird ein Verfahren zur Selektion von Epitopen zur Immuntherapie bereitgestellt, bei dem ein Immunserum mit Peptidsequenzen eines Antigens von mindestens 6 Aminosäuren Länge in Kontakt gebracht wird und die Bindung des Immunserums an die Peptidsequenzen mit der Bindung eines Präserums verglichen wird.According to the invention, a method for the selection of epitopes for immunotherapy is provided, in which an immune serum is brought into contact with peptide sequences of an antigen of at least 6 amino acids in length and the binding of the immune serum to the peptide sequences is compared with the binding of a presenum.
Das Immunserum kann aus Probanden isoliert werden, die mit einem Impfstoff immunisiert wurden. Durch die Immunisierung werden Antikörper gegen das Antigen bzw. die antigenen Strukturen, mit denen immunisiert wurde, entwickelt. Bevorzugte Antigene sind ausgewählt aus der Gruppe der Tumor assoziierten Antigene (TAA) . TAA sind Strukturen, die bevorzugt auf der Zellmembran von Tumorzellen exprimiert sind, dadurch eine Unterscheidung zu nicht-malignem Gewebe ermöglichen und daher als Zielpunkte für die diagnostische und therapeutischen Anwendungen von spezifischen Antikörpern gesehen werden.The immune serum can be isolated from subjects who have been immunized with a vaccine. The immunization develops antibodies against the antigen or the antigenic structures with which the immunization was carried out. Preferred antigens are selected from the group of tumor-associated antigens (TAA). TAA are structures that are preferentially expressed on the cell membrane of tumor cells, thereby enabling differentiation from non-malignant tissue and are therefore seen as target points for the diagnostic and therapeutic applications of specific antibodies.
In einer alternativen Ausführungsform sind die TAA ausgewählt aus der Gruppe der Selbstadhäsionsmoleküle bzw. Zeiladhäsionsmoleküle, die bewirken können, dass beispielsweise Tumorzellen eine starke Zell-Zell-Adhäsion aufweisen. Ein Beispiel für ein Selbstadhäsions-Molekül ist EpCAM.In an alternative embodiment, the TAA are selected from the group of self-adhesion molecules or cell adhesion molecules, which can have the effect that, for example, tumor cells have strong cell-cell adhesion. An example of a self-adhesion molecule is EpCAM.
Krebszellen haben praktisch immer, neben anderen physiologischen Eigenheiten, die sie von normalen Zellen unterscheiden, eine veränderte Art der Glykosylierung (Glycoconj . J. (1997) , 14:569; Adv.Cancer Res. (1989), 52:257; Cancer Res. (1996), 56:5309). Obwohl die Veränderungen von Gewebe zu Gewebe unterschiedlich sind, kann man feststellen, dass eine aberrante Glykosylierung typisch für Krebszellen ist.Cancer cells practically always have, besides other physiological characteristics that distinguish them from normal cells, a different type of glycosylation (Glycoconj. J. (1997), 14: 569; Adv.Cancer Res. (1989), 52: 257; Cancer Res (1996), 56: 5309). Although the changes from tissue to tissue vary, it can be seen that aberrant glycosylation is typical of cancer cells.
Unter den bekannten Tumor-assoziierten Kohlenhydratstrukturen finden sich z.B. alle Lewis-Antigene, die verstärkt in vielen epithelialen Krebsarten exprimiert werden. Dazu gehören neben Lewis y-Strukturen auch Lewis-x und Lewis b-Strukturen, sowie sialylierte Lewis x-Strukturen. Andere Kohlehydrat-Antigene sind Globo-H Strukturen, KH1, Tn-Antigen und Sialyl Tn-Antigen, TF- Antigen und das alpha-1, 3-Galactosyl-Epitop (Elektrophoresis (1999), 20:362; Curr. Pharmaceutical Design (2000), 6:485, Neo- plasma (1996), 43:285)Among the known tumor-associated carbohydrate structures are e.g. all Lewis antigens that are increasingly expressed in many epithelial cancers. In addition to Lewis y structures, these also include Lewis x and Lewis b structures, as well as sialylated Lewis x structures. Other carbohydrate antigens are Globo-H structures, KH1, Tn antigen and sialyl Tn antigen, TF antigen and the alpha-1,3 galactosyl epitope (Elektrophoresis (1999), 20: 362; Curr. Pharmaceutical Design ( 2000), 6: 485, Neoplasma (1996), 43: 285)
Andere TAA sind Proteine, die von Krebszellen besonders stark exprimiert werden, wie z.B. CEA, TAG-72, MUC1, Folate Binding Protein A-33, CA125, EpCAM, HER-2/neu, PSA, MART, etc. (Sem. Cancer Biol. (1995), 6:321). Relevante TAA sind oftmals Oberflä- chenantigene von epithelialen Zellen, die vermehrt in wachsenden Zellen, wie fötalem Gewebe, und auch Tumorgewebe auftreten. Eine spezielle Gruppe von TAA sind an den Adhäsionsprozessen der epithelialen Zellen beteiligt. Zu den zellulären Adhäsionsproteinen, die auf Tumorzellen überexprimiert werden, zählen EpCAM, NCAM und CEA.Other TAA are proteins that are particularly strongly expressed by cancer cells, e.g. CEA, TAG-72, MUC1, Folate Binding Protein A-33, CA125, EpCAM, HER-2 / neu, PSA, MART, etc. (Sem. Cancer Biol. (1995), 6: 321). Relevant TAA are often surface antigens of epithelial cells, which increasingly occur in growing cells, such as fetal tissue, and also tumor tissue. A special group of TAA are involved in the adhesion processes of the epithelial cells. Cellular adhesion proteins that are overexpressed on tumor cells include EpCAM, NCAM and CEA.
Die Peptidsequenzen können gemäß der vorliegenden Erfindung mit bekannten Verfahren an einen Träger gebunden werden und mit dem Immunserum bzw. dem Präserum in Kontakt gebracht werden. Bevorzugt wird das erfindungsgemäße Verfahren über Epitopmapping mittels Festphasenpeptidsynthese und Spotsynthese durchgeführt. Dazu werden die Peptide direkt oder mittels Spacer-Sequenzen an das Trägermaterial gebunden.According to the present invention, the peptide sequences can be bound to a support using known methods and brought into contact with the immune serum or the preserum. The method according to the invention is preferably carried out via epitope mapping by means of solid phase peptide synthesis and spot synthesis. For this purpose, the peptides are bound to the carrier material directly or by means of spacer sequences.
Das Immunserum und Präserum kann über Verfahren gereinigt werden, die aus dem Stand der Technik bekannt sind. Beispielsweise kann die Reinigung über Protein G Sepharose erfolgen und die so erhaltenen Immunglobuline biotinyliert oder mit radioaktiven Substanzen gekoppelt werden, um das Nachweisverfahren zu vereinfachen.The immune serum and preserum can be purified by methods which are known from the prior art. For example, the purification can be carried out using Protein G Sepharose and the immunoglobulins obtained in this way can be biotinylated or coupled with radioactive substances in order to simplify the detection method.
Bei Präserum handelt es sich erfindungsgemäß um Serum aus Probanden, die keine Immuntherapie mit einem Antigen erhalten haben, mit dessen Peptiden das Serum später in Kontakt gebracht wird.According to the invention, preserum is serum from test subjects who do not receive immunotherapy with an antigen have peptides with which the serum is later brought into contact.
Immunserum ist erfindungsgemäß Serum aus Probanden, die eine Immuntherapie mit einem Antigen erhalten haben, mit dessen Peptidsequenzen das Serum anschließend in Kontakt gebracht wird. Dabei kann es sich beispielsweise um eine Immunisierung mit Epitopen gegen TAA handeln, wie sie oben genannt wurden. Besonders bevorzugt erfolgte eine Immuntherapie mit immunogenen Antikörpern, wie sie beispielsweise in EP 1 140 168, EP 1 230 932, EP 0 644 947 und EP 0 528 767 beschrieben wurden. Ein bevorzugter Antikörper der für die aktive Immuntherapie verwendet wird, ist ein anti-EpCAM Antikörper, wie er in der WO 00/41722 oder A599/2003 beschrieben wurde.According to the invention, immune serum is serum from test subjects who have received immunotherapy with an antigen with the peptide sequences of which the serum is subsequently brought into contact. This can be, for example, an immunization with epitopes against TAA, as mentioned above. Immunotherapy with immunogenic antibodies was particularly preferred, as described for example in EP 1 140 168, EP 1 230 932, EP 0 644 947 and EP 0 528 767. A preferred antibody which is used for active immunotherapy is an anti-EpCAM antibody, as described in WO 00/41722 or A599 / 2003.
Erfindungsgemäß ist unter einem wirksamen Impfstoff ein Impfstoff zu verstehen, der in der Prophylaxe und/oder in der Therapie wirksam ist. Die Wirksamkeit wird bei einer aktiven Immuntherapie vorzugsweise dadurch nachgewiesen, dass der Patient eine Immunantwort gegen die immunogene Substanz entwickelt. Die Messung der Immunantwort kann durch den Nachweis der Serokonversion im Patientenserum erfolgen. Die Serokonversion wird beispielsweise dadurch ermittelt, dass eine differentielle Messung der Bindung der Immunglobuline aus dem Patientenserum (Präserum und Immunserum) an das für die Immunisierung verwendete Antigen nachgewiesen wird.According to the invention, an effective vaccine is to be understood as a vaccine which is effective in prophylaxis and / or in therapy. The effectiveness of active immunotherapy is preferably demonstrated by the patient developing an immune response against the immunogenic substance. The immune response can be measured by detecting seroconversion in the patient's serum. The seroconversion is determined, for example, by detecting a differential measurement of the binding of the immunoglobulins from the patient's serum (preserum and immune serum) to the antigen used for the immunization.
In einer bevorzugten Ausführungsform führt die Behandlung von Patienten mit dem wirksamen Impfstoff zu einer Verlängerung der Überlebenszeit bzw. Erhöhung der Überlebensrate bei Krebserkrankungen, beispielsweise bei colorektalem oder rektalem Krebs, um mindestens 10%, bevorzugt mindestens 20%, bevorzugt mindestens 30% besonders bevorzugt mindestens 50% im Vergleich zu Patienten ohne Behandlung mit dem wirksamen Impfstoff.In a preferred embodiment, the treatment of patients with the effective vaccine leads to an increase in the survival time or increase in the survival rate in cancer diseases, for example in colorectal or rectal cancer, by at least 10%, preferably at least 20%, preferably at least 30%, particularly preferably at least 50% compared to patients without treatment with the effective vaccine.
Die unterschiedlichen Bindungsmuster von Prä- und Immunseren an die Peptide auf dem Trägermaterial können verglichen werden.The different binding patterns of pre- and immune sera to the peptides on the carrier material can be compared.
In einer bevorzugten Ausführungsform ist das Verfahren so ausgeführt, dass die Bindung des Immunserums an die Peptidsequenzen stärker ist als die Bindung des Präserums. Die unterschiedliche Bindungsaffinität kann beispielsweise durch ein optisch stärkeres Signal gezeigt werden, das das Immunserum im Vergleich zu Präserum bei der Bindung an die Peptidsequenz zeigt.In a preferred embodiment, the method is carried out such that the binding of the immune serum to the peptide sequences is stronger than the bond of the presum. The different binding affinity can be shown, for example, by an optically stronger signal which shows the immune serum in comparison to the preserum in the binding to the peptide sequence.
Alternativ dazu kann das stärkere Bindungsmuster auch darin begründet liegen, dass die Menge des Antikörpers, der an diese Peptidsequenz bindet, erhöht ist und zu einem stärkeren Bindungssignal führt.Alternatively, the stronger binding pattern can also be due to the fact that the amount of the antibody which binds to this peptide sequence is increased and leads to a stronger binding signal.
Erfindungsgemäß können mit Hilfe des beanspruchten Verfahrens auch Peptide isoliert werden, an die das Immunserum im Vergleich zu Präserum eine unterschiedliche Bindung aufweist. Bevorzugt handelt es sich um Peptide von tumorassoziierten Selbstadhäsionsmolekülen, beispielsweise um Peptide des EpCAM Proteins, bevorzugt um Peptide aus der extrazellulären Domäne des EpCAM Proteins .According to the invention, peptides to which the immune serum has a different binding than the preserum can also be isolated using the claimed method. They are preferably peptides from tumor-associated self-adhesion molecules, for example peptides of the EpCAM protein, preferably peptides from the extracellular domain of the EpCAM protein.
Besonders bevorzugt handelt es sich um Peptide mit mindestens 6 Aminosäuren Länge, die innerhalb einer der folgenden Aminosäuresequenzen liegen bzw. aus einer der folgenden Aminosäuresequenzen ausgewählt sind: Asn Cys Phe' Val Asn Asn (NCFVNN)It is particularly preferred to have peptides with a length of at least 6 amino acids which lie within one of the following amino acid sequences or are selected from one of the following amino acid sequences: Asn Cys Phe ' Val Asn Asn (NCFVNN)
Ala Gin Asn Thr Val Ile Cys Ser Lys Ala Ala Lys Cys (AQNTVICS- KLAAKC)Ala Gin Asn Thr Val Ile Cys Ser Lys Ala Ala Lys Cys (AQNTVICS- KLAAKC)
Lys Leu Gly Arg Arg Ala (KLGRRA)Lys Leu Gly Arg Arg Ala (KLGRRA)
Glu Ser Gly Leu Phe Lys Ala Lys Gin Cys Asn Gly Thr Ser Thr Cys Trp Cys Val Asn Thr Ala (ESGLFKAKQCNGTSTCWCVNTA) Cys Ser Glu Arg Val Arg (CSERVR) Leu Phe His Ser Lys Lys (LFHSKK)Glu Ser Gly Leu Phe Lys Ala Lys Gin Cys Asn Gly Thr Ser Thr Cys Trp Cys Val Asn Thr Ala (ESGLFKAKQCNGTSTCWCVNTA) Cys Ser Glu Arg Val Arg (CSERVR) Leu Phe His Ser Lys Lys (LFHSKK)
Met Ala Pro Pro Gin Val Leu Ala Phe Gly (MAPPQVLAFG) Gin Val Leu Ala Phe Gly Leu Leu Leu Ala (QVLAFGLLLA) Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile (ITCSER- VRTYWIII)Met Ala Pro Pro Gin Val Leu Ala Phe Gly (MAPPQVLAFG) Gin Val Leu Ala Phe Gly Leu Leu Leu Ala (QVLAFGLLLA) Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile (ITCSER- VRTYWIII)
Thr Tyr Trp Ile Ile Ile Glu Leu Lys His (TYWIIIELKH) Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys (IIELKHKAREK) Ser Leu Arg Thr Ala Leu Gin Lys Glu Ile (SLRTALQKEI) Ala Leu Gin Lys Glu Ile Thr Thr Arg Tyr (ALQKEITTRY) Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr (DPKFITSILY) Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys (IADVAYYFEK) Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly (AYYFEKDVKG) Asp Leu Asp Pro Gly Gin Thr Leu Ile Tyr (DLDPGQTLIY)Thr Tyr Trp Ile Ile Ile Glu Leu Lys His (TYWIIIELKH) Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys (IIELKHKAREK) Ser Leu Arg Thr Ala Leu Gin Lys Glu Ile (SLRTALQKEI) Ala Leu Gin Lys Glu Ile Thr Thr (ALQkeittry) Asp Pro Lys Phe Ile Thr Ser Ile Leu Tyr (DPKFITSILY) Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys (IADVAYYFEK) Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly (AYYFEKDVKG) Asp Leu Asp Pro Gly Gin Thr Leu Ile Tyr (DLDPGQTLIY)
Lys Ala Gly Val Ile Ala Val Ile Val Val (KAGVIAVIVV)Lys Ala Gly Val Ile Ala Val Ile Val Val (KAGVIAVIVV)
Val Ile Ala Val Ile Val Val Val Val Met Ala (IAVIVVVVMA)Val Ile Ala Val Ile Val Val Val Val Met Ala (IAVIVVVVMA)
Met Ala Val Val Ala Gly Ile Val Val Leu (MAVVAGIVVL)Met Ala Val Val Ala Gly Ile Val Val Leu (MAVVAGIVVL)
Ala Gly Ile Val Val Leu Val Ile Ser Arg (AGIVVLVISR)Ala Gly Ile Val Val Leu Val Ile Ser Arg (AGIVVLVISR)
Weiters handelt es sich auch um Peptide mit mindestens 6 Aminosäuren Länge, ausgewählt aus einer der folgenden Aminosäuresequenzen des EpCAM Moleküls, die auch für die Selbstadhäsion verantwortlich sein können sindFurthermore, they are also peptides with a length of at least 6 amino acids, selected from one of the following amino acid sequences of the EpCAM molecule, which can also be responsible for self-adhesion
Met Ala Pro Pro Gin Val Leu Ala Phe Gly Leu Leu Leu Ala (MAPPQV- LAFGLLLA)Met Ala Pro Pro Gin Val Leu Ala Phe Gly Leu Leu Leu Ala (MAPPQV- LAFGLLLA)
Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly (MNGS- KLGRRAKPEG)Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly (MNGS- KLGRRAKPEG)
Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp (WCVN- TAGVRRTDKD)Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp (WCVN- TAGVRRTDKD)
Die Länge der Peptide hängt ab von ihrer Verwendung. Bevorzugt liegt deren Länge bei mindestens 6 Aminosäuren und maximal 30 Aminosäuren, bevorzugt maximal 20' Aminosäuren, bevorzugt maximal 15 Aminosäuren, bevorzugt maximal 10 Aminosäuren.The length of the peptides depends on their use. The length thereof is preferably at least 6 amino acids and at most 30 amino acids, preferably at most 20 'amino acids, preferably at most 15 amino acids, preferably at most 10 amino acids.
Erfindungsgemäß können die Peptide an Trägermoleküle gebunden werden. Bevorzugte Trägermoleküle sind Antikörper oder Antikörperderivate oder -Fragmente, IgG2a Antikörper oder -Fragmente, KLH (Keyhole Limpet Haemocyanin) , Serum Albumin etc. Besonders bevorzugt werden Trägermoleküle verwendet, die immunogen /wirken.According to the invention, the peptides can be bound to carrier molecules. Preferred carrier molecules are antibodies or antibody derivatives or fragments, IgG2a antibodies or fragments, KLH (keyhole limpet hemocyanin), serum albumin etc. Particularly preferred carrier molecules are used which have an immunogenic / effect.
Der Begriff immunogen definiert jede Struktur, die zu einer Immunantwort in einem spezifischen Wirtssystem führt. Beispielsweise kann ein muriner Antikörper oder dessen Fragmente stark immunogen im menschlichen Organismus wirken, besonders wenn dieser mit Adjuvantien verabreicht wird.The term immunogenic defines any structure that leads to an immune response in a specific host system. For example, a murine antibody or its fragments can be highly immunogenic in the human organism, especially if it is administered with adjuvants.
In einer besonderen Ausführungsform kann eine Vakzine für die aktive Immuntherapie hergestellt werden, die das Peptid zusammen mit einem geeigneten Adjuvans enthält.In a particular embodiment, a vaccine for active immunotherapy can be produced which contains the peptide together with a suitable adjuvant.
Denn es hat sich bewährt, die Immunogenität einer Vakzine durch die Verwendung von Adjuvantien zu erhöhen. Dafür sind Vak- zine-Adjuvantien geeignet, wie beispielsweise Aluminiumhydroxid (Alu-Gel) oder -phosphat, Wachstumsfaktoren, Lymphokine, Zyto- kine, etwa IL-2, IL-12, GM-CSF, Gamma Interferon, oder Komplementfaktoren, wie C3d, weiter Liposomenbereitungen oder Lipopolysaccharid aus E. coli (LPS) aber auch Formulierungen mit zusätzlichen Antigenen, gegen die das Immunsystem bereits eine starke Immunantwort gemacht hat, wie Tetanus-Toxoid, Bakterielle Toxine, wie Pseudomonas-Exotoxine und Derivate von Lipid A.It has proven effective to increase the immunogenicity of a vaccine by using adjuvants. Vaccine adjuvants such as aluminum hydroxide are suitable for this (Aluminum gel) or phosphate, growth factors, lymphokines, cytokines, for example IL-2, IL-12, GM-CSF, gamma interferon, or complement factors, such as C3d, further liposome preparations or lipopolysaccharide from E. coli (LPS) but also formulations with additional antigens against which the immune system has already given a strong immune response, such as tetanus toxoid, bacterial toxins such as Pseudomonas exotoxins and derivatives of lipid A.
Zur Impfstoff-Formulierung können auch weiter bekannte Verfahren zur Konjugierung oder Denaturierung von Impfstoff-Bestandteilen eingesetzt werden, um die Immunogenität des Wirkstoffes noch zu erhöhen.Known methods for conjugating or denaturing vaccine components can also be used for vaccine formulation in order to further increase the immunogenicity of the active ingredient.
Die Vakzine enthaltend ein erfindungsgemäßes Peptid mit einem Trägermolekül für die aktive Immunisierung wird bevorzugt in einer Menge zwischen 0.01 μg und 10 mg verabreicht. Die Immunogenität der Vakzine kann noch durch xenogene Substanzen oder De- rivatisierung des Antikörpers, der als Trägermolekül für das Peptid dienen kann, verstärkt werden. Die immunogene Dosis der Vakzine liegt bevorzugt zwischen 0.01 μg und 750 μg, bevorzugt zwischen 100 μg und 1 mg, am meisten bevorzugt 100 μg und 500 μg. Eine Vakzine die als Depotmedikament verabreicht wird, enthält natürlich wesentlich höhere Mengen an immunogener Substanz, beispielsweise mindestens 1 mg bis 10 mg. Dabei wird die Vakzine über einen längeren Zeitraum im Körper freigesetzt.The vaccine containing a peptide according to the invention with a carrier molecule for the active immunization is preferably administered in an amount between 0.01 μg and 10 mg. The immunogenicity of the vaccine can be increased by xenogenic substances or derivatization of the antibody, which can serve as a carrier molecule for the peptide. The immunogenic dose of the vaccine is preferably between 0.01 μg and 750 μg, preferably between 100 μg and 1 mg, most preferably 100 μg and 500 μg. A vaccine that is administered as a depot medication naturally contains significantly higher amounts of immunogenic substance, for example at least 1 mg to 10 mg. The vaccine is released in the body over a longer period of time.
Erfindungsgemäß kann das Peptid aber auch als Zielantigen für eine passive Immuntherapie verwendet werden. Dazu kann beispielsweise ein Antikörper hergestellt werden, der dieses Peptid als Epitop enthält und an EpCAM bindet. Bei der passiven Immuntherapie wird dieser Antikörper mehrmals in Intervallen von 1 bis 2 Wochen verabreicht. Die bevorzugte Menge an verabreichtem Antikörper liegt zwischen 1 mg und lg, bevorzugt zwischen 100 mg und 500 mg, bevorzugt erfolgt die Verabreichung intravenös.According to the invention, however, the peptide can also be used as a target antigen for passive immunotherapy. For this purpose, for example, an antibody can be produced which contains this peptide as an epitope and binds to EpCAM. In passive immunotherapy, this antibody is administered several times at 1 to 2 week intervals. The preferred amount of antibody administered is between 1 mg and 1 g, preferably between 100 mg and 500 mg, and is preferably administered intravenously.
Erfindungsgemäß ist auch ein Immunserum enthaltend Antikörper gegen Epitope umfasst, die nach dem erfindungsgemäßen Verfahren zur Selektion von Epitopen erhalten wurden. Weiters erfindungsgemäß ist auch eine Vakzine gegen EpCAM exprimierende Tumorzellen, die zur Bildung des Immunserums führt. Diese Vakzine kann beispielsweise Peptide, Antikörper, Antikörperderivate oder Mimotope, anti-idiotypische Antikörper oder Plasmide die das EpCAM Protein exprimieren, zusammen mit geeigneten Trägersubstanzen enthalten.According to the invention, an immune serum containing antibodies against epitopes, which were obtained by the method according to the invention for the selection of epitopes, is also included. Also according to the invention is a vaccine against tumor cells expressing EpCAM, which are used to form the immune serum leads. This vaccine can contain, for example, peptides, antibodies, antibody derivatives or mimotopes, anti-idiotypic antibodies or plasmids which express the EpCAM protein, together with suitable carrier substances.
Die Peptide, die nach dem erfindungsgemäßen Verfahren erhalten wurden, können auch als „antisense,-Peptid verwendet werden. Im Falle von Zelladhäsions-Sequenzen kann beispielsweise ein Peptid, das an eine Region bindet, die für die Zelladhäsion notwendig ist, diese verhindern. Im Falle von Proteinen wie EpCAM oder CEA kann dies dazu führen, dass sich die Zellen nicht mehr zu Zellformationen zusammenschließen können und die Bildung von Metastasen verzögert bzw. verhindert wird.The peptides obtained by the process according to the invention can also be used as an “antisense , peptide. For example, in the case of cell adhesion sequences, a peptide that binds to a region necessary for cell adhesion can prevent it. In the case of proteins such as EpCAM or CEA, this can mean that the cells can no longer merge to form cells and the formation of metastases is delayed or prevented.
Auch ein Diagnostikum zum Nachweis spezifischer Immunglobuline enthaltend ein Peptid, das nach dem erfindungsgemäßen Verfahren erhalten wurde, und ein Detektionsmittel zur Bestimmung der Bindung eines Immunserums kann zur Verfügung gestellt werden.A diagnostic for the detection of specific immunoglobulins containing a peptide obtained by the method according to the invention and a detection means for determining the binding of an immune serum can also be provided.
Beispielsweise können Teststreifen, an die Peptide gekoppelt sind, mit Immunseren inkubiert werden und anhand des Färbemusters kann die Immunglobulinspezifität nachgewiesen werden. Nach Inkubation mit biotinyliertem Patientenserum wird die Interaktion mit spezifischen Immunglobulinen durch eine Farbreaktion nachgewiesen. Ziel ist es hierbei, einerseits eine aufgebaute Reaktivität gegen spezifische Proteinregionen eines Immunisierungsantigens aufzuzeigen und andererseits die Spezifität einer aufgebauten Immunantwort zu beschreiben. So kann beispielsweise aus der nachgewiesenen Reaktivität ein therapeutischer Effekt korreliert werden. Als Detektionsmittel können die Peptide mit Biotin-Gruppen oder radioaktiven Markern gekoppelt werden und ein messbares Signal dadurch abgelesen werden, dass die Strahlung auf einen Röntgenfilm oder strahlungsempfindlichen Film trifft und zu einem Signal führt.For example, test strips to which peptides are coupled can be incubated with immune sera, and the immunoglobulin specificity can be detected on the basis of the staining pattern. After incubation with biotinylated patient serum, the interaction with specific immunoglobulins is demonstrated by a color reaction. The aim is to show a built-up reactivity against specific protein regions of an immunization antigen and to describe the specificity of a built-up immune response. For example, a therapeutic effect can be correlated from the proven reactivity. As a detection means, the peptides can be coupled with biotin groups or radioactive markers and a measurable signal can be read by the radiation striking an X-ray film or radiation-sensitive film and leading to a signal.
Figur 1 zeigt beispielhaft ein Ergebnis des EpCAM-Epitopmappings mittels der Festphasensynthese.FIG. 1 shows an example of a result of the EpCAM epitope mapping by means of solid phase synthesis.
Figur 2 zeigt die Aminosäuresequenz des EpCAM Moleküls. Die transmembrane Region und die cytoplasmatische Region sind kur- S1V.Figure 2 shows the amino acid sequence of the EpCAM molecule. The transmembrane region and the cytoplasmic region are short S1V.
B e i s p i e l e :Example:
Beispiel 1 : Epitop apping mittels einer Festphasenpeptidsyn- these :Example 1: Epitope apping using a solid phase peptide synthesis:
Aufreinigung der SerenPurification of the serums
Protein G Sepharose Fast Flow (Amersham Biosciences). wurde in eine HR 5 Säule (innerer Durchmesser 5 mm; Amersham Biosciences) gepackt. Die Säule wurde mit einer Flussrate von 0.33 ml/min mit 5 Säulenvolumen PBS Puffer equilibriert . Danach wurde Serum mit derselben Flussrate geladen und mit weiteren 5 Säulenvolumen nachgewaschen. Die Elution wurde mit 0,2 M Essigsäure + 20% Ethylenglycol, pH 2,7 durchgeführt. Das Eluat wurde in 1 M Na- triumbicarbonatpuffer, aufgefangen und damit auf pH 8,6 gebracht.Protein G Sepharose Fast Flow (Amersham Biosciences). was packed into an HR 5 column (inner diameter 5 mm; Amersham Biosciences). The column was equilibrated at a flow rate of 0.33 ml / min with 5 column volumes of PBS buffer. Then serum was loaded at the same flow rate and washed with a further 5 column volumes. Elution was carried out with 0.2 M acetic acid + 20% ethylene glycol, pH 2.7. The eluate was collected in 1 M sodium bicarbonate buffer and brought to pH 8.6.
Biotinylieren der Seren bzw. der AntikörperBiotinylation of the sera or the antibodies
Ein Aliquot des gereinigten Serums wurde mit NHS-LC-Biotin eine Stunde bei Raumtemperatur inkubiert. Danach wurde die Probe über eine Sephadex G25 Gelfiltrationssäule (PD-10, Amersham Biosciences) umgepuffert, um einerseits das biotinylierte Protein vom freien Biotin zu trennen und weiter in ein für das Testsystem geeignetes Medium zu bringenAn aliquot of the purified serum was incubated with NHS-LC biotin for one hour at room temperature. The sample was then buffered over a Sephadex G25 gel filtration column (PD-10, Amersham Biosciences) in order to separate the biotinylated protein from the free biotin and to bring it further into a medium suitable for the test system
Spotsy heseSpotsy hese
Die Spotsynthese wurde nach einer modifizierten Methode nach Frank (Tetrahedron 48 (1992) 9217-9232) durch geführt. Whatman 540 Cellulose wurde im Exsikkator über Nacht getrocknet. Die Membran wurde dann mit 0.2 M Fmoc-ß-Alanin, 0.24 M Diisopropyl- carbodiimid und 0.4 M 1-Methyl-imidazol für 3 Stunden funktio- nalisiert. Nach Waschen mit 3x Dimethylfor amid (DMF) wurde die Fmoc Gruppe durch Behandlung mit 20% Piperidin in DMF abgespalten. Die Peptide wurden mithilfe eines AutoSpot Robot ASP222 (INTAVIS, Germany) synthetisiert. Als erste Aminosäure wurde immer ein weiterer ß-Alanin Spacer eingeführt. Nach der ersten ß-Alanin Kopplung wurden die restlichen freien Aminogruppen mit 2% Acetanhydride acetyliert. Als Aminosäuren wurden Fmoc geschützte Opfp Ester der Aminosäuren ( O-pentafluorphenyl, No- vabiochem) in einer Konzentration von 0.3 M gelöst in N- Methylpyrolidon verwendet. Pro Zyklus wurde jede Aminosäure zweimal gespotet. Nach jedem Zyklus wurde die Fmoc Gruppe wie oben beschrieben mit Piperidin abgespalten und danach die Membran 6x mit DMF gewaschen. Nach der Synthese der letzten Aminosäure wurde erneut acetyliert und danach die Seitenschutzgruppen abgespalten. Zuerst wurde die Membran mit 90 % Trifluoressigsäu- re, 3 % Triisobutylsilane, 1% Phenol, 2 % Wasser und 4 % Dich- lormethan für 30 Minuten behandelt. Danach wurde die Membran 5x mit DCM, 3 x mit DMF und Methanol gewaschen und getrocknet. Da- '■ nach erfolgte eine weitere Abspaltung durch Behandlung mit 50 % Trifluoressigsäure, 3 % Triisobutylsilane, 1% Phenol, 2 % Wasser und 44 % Dichlormethan für zwei Stunden. Anschließend wurde wieder mit DCM, DMF und Methanol gewaschen und die Membran bei -20°C gelagert.The spot synthesis was carried out according to a modified method according to Frank (Tetrahedron 48 (1992) 9217-9232). Whatman 540 cellulose was dried in a desiccator overnight. The membrane was then functionalized with 0.2 M Fmoc-β-alanine, 0.24 M diisopropyl carbodiimide and 0.4 M 1-methyl-imidazole for 3 hours. After washing with 3 × dimethylfor amide (DMF), the Fmoc group was split off by treatment with 20% piperidine in DMF. The peptides were synthesized using an AutoSpot Robot ASP222 (INTAVIS, Germany). Another ß-alanine spacer was always introduced as the first amino acid. After the first β-alanine coupling, the remaining free amino groups were acetylated with 2% acetic anhydrides. Fmoc-protected Opfp esters of the amino acids (O-pentafluorophenyl, Novabiochem) were dissolved as amino acids in a concentration of 0.3 M in N- Methyl pyrolidone used. Each amino acid was spotted twice per cycle. After each cycle, the Fmoc group was cleaved off with piperidine as described above and the membrane was then washed 6 times with DMF. After the synthesis of the last amino acid, acetylation was carried out again and the side protecting groups were then split off. First the membrane was treated with 90% trifluoroacetic acid, 3% triisobutylsilane, 1% phenol, 2% water and 4% dichloromethane for 30 minutes. The membrane was then washed 5 times with DCM, 3 times with DMF and methanol and dried. DA '■ after there was a further cleavage by treatment with 50% trifluoroacetic acid, 3% Triisobutylsilane, 1% phenol, 2% water and 44% dichloromethane for two hours. The mixture was then washed again with DCM, DMF and methanol and the membrane was stored at −20 ° C.
Um die Aminosäuresequenz des extrazellulären Teils von Ep-CAM abzudecken wurden 77 Decapeptide mit jeweils 6-fach Überlappungen auf Zellulosemembranen über Doppel-ß-Alanin Linker immobilisiert. Die N-terminalen Seitengruppen wurden mittels Fmoc- Strategie geschützt. In order to cover the amino acid sequence of the extracellular part of Ep-CAM, 77 decapeptides, each with 6-fold overlaps, were immobilized on cellulose membranes via double-ß-alanine linkers. The N-terminal side groups were protected by Fmoc strategy.
Tabelle. 1: Aminosäure-Sequenz der für das Epitopmapping von EpCAM synthetisierten DecapeptideTable. 1: Amino acid sequence of the decapeptides synthesized for the epitope mapping of EpCAM
Da die überwiegende Immunantwort in Form von IgG vorliegt, wurde IgG aus den Seren über Protein G Sepharose gereinigt. Nach der sauren Elution mit 0.2 M Essigsäure in Gegenwart von 20% Ethy- lenglykol wurden die einzelnen Fraktionen in IM Natriumcarbonat aufgefangen, um den pH-Wert so rasch wie möglich zu erhöhen. Diese Bedingungen erlauben auch das direkt anschließende Bioti- nylieren des aufgereinigten IgG ohne vorhergehendes Umpuffern. Nach Zugabe des Biotinylierungsreagenz (Biotinamidocaproate N- hydroxysuccinimideester, NHS-LC-Biotin, ECL protein biotinylati- on module, RPN 2202, Fa. Amersham Biosciences) in einem 40fachen molaren Überschuss zur IgG - Lösung und einer einstündigen Inkubation bei Raumtemperatur wurde das überschüssige Biotin über Sephadex G25 Säulchen entfernt und gleichzeitig das biotiny- lierte Material in PBS umgepuffert. Die Konzentration des bioti- nylierten IgG wurde über photometrische Absorptionsmessung bei 280nm bestimmt und das Material nach Zugabe von 0.2% Natriumazid bei 4°C gelagert.Since the predominant immune response is in the form of IgG, IgG was purified from the sera via Protein G Sepharose. After acidic elution with 0.2 M acetic acid in the presence of 20% ethylene glycol, the individual fractions were collected in 1M sodium carbonate in order to increase the pH as quickly as possible. These conditions also allow the purified IgG to be biotinylated immediately afterwards without previous buffering. After adding the biotinylation reagent (Biotinamidocaproate N-hydroxysuccinimideester, NHS-LC-Biotin, ECL protein biotinylation module, RPN 2202, from Amersham Biosciences) in a 40-fold molar excess to the IgG solution and a one-hour incubation at room temperature, the excess biotin became removed via Sephadex G25 columns and at the same time the biotinylated material was buffered in PBS. The concentration of the biotinylated IgG was determined by photometric absorption measurement at 280 nm and the material was stored at 4 ° C. after adding 0.2% sodium azide.
Entwicklung der Membranen:Development of the membranes:
Die Durchführung der Membranentwicklung ist der eines Western Blot sehr ähnlich und ist aus denselben Schritten aufgebaut: die Zellulosemembranen wurden in 20% Methanol konditioniert und mit 3% BSA/PBS-T geblockt. Anschließend erfolgte die Inkubation mit den biotinylierten Seren. Nach einem Waschschritt wurde mit Streptavidin - HRP Konjugat (ECL protein biotinylation module, RPN 2202, Fa. Amersham Biosciences) inkubiert, um dann über Oxi- dation der Peroxidase in Gegenwart einer Verstärkerlösung die detektierbare Lichtentwicklung zu induzieren. Die Membranen wurden sofort nach der Entwicklung und bei konstanter Messdauer vermessen. Die mitgeführten Blindwertmembranen wurden nur mit Verdünnungspuffer anstelle von biotinyliertem IgG und anschließend mit Streptavidin-HRP inkubiert.The membrane development is very similar to that of a Western blot and is built up from the same steps: the cellulose membranes were conditioned in 20% methanol and blocked with 3% BSA / PBS-T. The incubation with the biotinylated sera then took place. After a washing step, incubation was carried out with streptavidin-HRP conjugate (ECL protein biotinylation module, RPN 2202, from Amersham Biosciences), in order then to induce the detectable light development via oxidation of the peroxidase in the presence of an amplifier solution. The membranes were measured immediately after development and with a constant measurement duration. The blank membranes carried were only incubated with dilution buffer instead of biotinylated IgG and then with streptavidin-HRP.
Zusammenfassend ergaben sich nach der Optimierung der Blankmembranen und der Regenerierbarkeit für die folgenden Versuche nachstehenden Bedingungen:In summary, the optimization of the blank membranes and the regenerability for the following tests resulted following conditions:
Tabelle 2: Optimierte Bedingungen für EpitopmappingTable 2: Optimized conditions for epitope mapping
Um die Aussagekraft und Vergleichbarkeit der Methode zu erhöhen wurde zusätzlich zur Analyse der biotinylierten Präimmun- und Immunseren ein Verdrängungstest durchgeführt. Dazu wurde jeweils ein Teil der gereinigten Präimmun- Seren nicht biotinyliert, sondern nur in PBS umgepuffert.In order to increase the informative value and comparability of the method, a displacement test was carried out in addition to the analysis of the biotinylated preimmune and immune sera. For this purpose, part of the purified preimmune sera was not biotinylated, but only buffered in PBS.
Weiters wurden jeweils die Analysen aller Seren eines Individuums sowie der Verdrängungstest an aufeinander folgenden Tagen auf ein und derselben Membran durchgeführt. Zur Überprüfung der erfolgreichen Regenerierung der Membranen, wurden routinemäßig zwischen den einzelnen Untersuchungen Blankmembranen entwickelt.Furthermore, the analyzes of all sera of an individual and the displacement test were carried out on successive days on the same membrane. In order to check the successful regeneration of the membranes, blank membranes were routinely developed between the individual examinations.
Die Ergebnisse sind der Figur 1 zu entnehmen. Das Immunserum stammte aus einem Patienten, der einer aktiven Immuntherapie mit mAbl7-lA, einem EpCAM Antikörper unterzogen wurde. Das Immunserum wurde 71 Tage nach der Immunisierung entnommen. Das Präserum wurde am Tag 1, d.h. kurz vor Beginn der Behandlung entnommen. Wie sich zeigte, bindet Immunserum verstärkt an die lOmer Pepti- de 1-2, 34-36, 42, 45 53, 61, 68-71 während das Präserum diese Bindung nicht oder nur in geringem Maße zeigt.The results can be seen in FIG. 1. The immune serum came from a patient who underwent active immunotherapy with mAbl7-1A, an EpCAM antibody. The immune serum was withdrawn 71 days after the immunization. The preserum was removed on day 1, ie shortly before the start of treatment. It was shown that immune serum binds more strongly to the lomer peptides. de 1-2, 34-36, 42, 45 53, 61, 68-71 while the preserum does not show this bond or shows it only to a small extent.
Eine Erhöhung der Salzkonzentration in auf 0.8M NaCI Endkonzentration im Verdünnungspuffer des Konjugates mit einer gleichzeitigen Reduktion der Konjugatkonzentration auf 0.25μg/ml erlaubte eine optimale Reduzierung der nachweisbaren Spots auf den Blankmembranen unter gleichzeitiger Nachweisbarkeit der gebundenen Serum IgGs.An increase in the salt concentration in 0.8M NaCI final concentration in the dilution buffer of the conjugate with a simultaneous reduction in the conjugate concentration to 0.25μg / ml allowed an optimal reduction of the detectable spots on the blank membranes while at the same time the bound serum IgGs could be detected.
Daher wurden als optimierte Bedingungen für den Detektions- schritt der Methode mit Streptavidin-HRP Konjugat gewählt:Therefore, the optimized conditions for the detection step of the method with streptavidin-HRP conjugate were chosen:
Reagens: 0.25μg/ml Streptavidin-HRP, 1:6400 verdünnt (AmershamReagent: 0.25μg / ml streptavidin-HRP, diluted 1: 6400 (Amersham
Biosiences)Biosciences)
1% BSA/PBS-T mit 0.8M NaCI Endkonzentration1% BSA / PBS-T with 0.8M NaCI final concentration
Inkubationsdauer: lh Incubation period: lh

Claims

Patentansprüche : Claims:
1. Verfahren zur Selektion von Epitopen zur Immuntherapie dadurch gekennzeichnet, dass jeweils ein Immunserum A und ein Präserum mit Peptidsequenzen eines Antigens von mindestens 6 Aminosäuren Länge in Kontakt gebracht werden und die Bindung des Immunserums an die Peptidsequenzen mit der Bindung des Präserums an die Peptidsequenzen verglichen wird, wobei die Epitope aufgrund von unterschiedlichen Bindungsmustern selektiert werden.1. A method for the selection of epitopes for immunotherapy, characterized in that in each case an immune serum A and a preserum are brought into contact with peptide sequences of an antigen of at least 6 amino acids in length and the binding of the immune serum to the peptide sequences is compared with the binding of the preserum to the peptide sequences the epitopes are selected on the basis of different binding patterns.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass die Bindung des Immunserums an die Peptidsequenzen stärker ist als die Bindung des Präserums.2. The method according to claim 1, characterized in that the binding of the immune serum to the peptide sequences is stronger than the binding of the preserum.
3. Verfahren nach Anspruch 1 oder 2, dadurc ' gekennzeichnet, dass das Immunserum aus Patienten stammt, die mit einem gegen das Antigen wirksamen Impfstoff behandelt wurden.3. The method according to claim 1 or 2, dadurc 'characterized in that the immune serum comes from patients who have been treated with a vaccine effective against the antigen.
. Isoliertes Peptid mit mindestens 6 Aminosäuren Länge erhältlich durch ein Verfahren nach einem der Ansprüche 1 bis 3., Isolated peptide with at least 6 amino acids in length obtainable by a method according to one of claims 1 to 3.
5. Isoliertes Peptid nach Anspruch 4, dadurch gekennzeichnet, dass es an die extrazelluläre Domäne eines Selbstadhäsionsproteins bindet.5. Isolated peptide according to claim 4, characterized in that it binds to the extracellular domain of a self-adhesion protein.
6. Peptid nach Anspruch 5, dadurch gekennzeichnet, dass es an die extrazelluläre Domäne eines EpCAM Moleküls bindet.6. Peptide according to claim 5, characterized in that it binds to the extracellular domain of an EpCAM molecule.
7. Peptid nach einem der Ansprüche 4 bis 6, dadurch gekennzeichnet, dass es innerhalb einer Aminosäuresequenz, ausgewählt aus der Gruppe bestehend aus7. Peptide according to one of claims 4 to 6, characterized in that it is within an amino acid sequence selected from the group consisting of
Asn Cys Phe Val Asn Asn (NCFVNN)Asn Cys Phe Val Asn Asn (NCFVNN)
Ala Gin Asn Thr Val Ile Cys Ser Lys Ala Ala Lys Cys (AQNTVICS-Ala Gin Asn Thr Val Ile Cys Ser Lys Ala Ala Lys Cys (AQNTVICS-
KLAAKC)KLAAKC)
Lys Leu Gly Arg Arg Ala (KLGRRA)Lys Leu Gly Arg Arg Ala (KLGRRA)
Glu Ser Gly Leu Phe Lys Ala Lys Gin Cys Asn Gly Thr Ser Thr CysGlu Ser Gly Leu Phe Lys Ala Lys Gin Cys Asn Gly Thr Ser Thr Cys
Trp Cys Val Asn Thr Ala (ESGLFKAKQCNGTSTCWCVNTA)Trp Cys Val Asn Thr Ala (ESGLFKAKQCNGTSTCWCVNTA)
Cys Ser Glu Arg Val Arg (CSERVR)Cys Ser Glu Arg Val Arg (CSERVR)
Leu Phe His Ser Lys Lys (LFHSKK)Leu Phe His Ser Lys Lys (LFHSKK)
Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile (ITCSER- VRTYWI I I )Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr Trp Ile Ile Ile (ITCSER- VRTYWI II)
Thr Tyr Trp Ile Ile Ile Glu Leu Lys His (TYWIIIELKH)Thr Tyr Trp Ile Ile Ile Glu Leu Lys His (TYWIIIELKH)
Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys (IIELKHKAREK)Ile Ile Glu Leu Lys His Lys Ala Arg Glu Lys (IIELKHKAREK)
Ser Leu Arg Thr Ala Leu Gin Lys Glu Ile (SLRTALQKEI)Ser Leu Arg Thr Ala Leu Gin Lys Glu Ile (SLRTALQKEI)
Ala Leu Gin Lys Glu Ile Thr Thr Arg Tyr (ALQKEITTRY)Ala Leu Gin Lys Glu Ile Thr Thr Arg Tyr (ALQINESSTRY)
Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys (IADVAYYFEK)Ile Ala Asp Val Ala Tyr Tyr Phe Glu Lys (IADVAYYFEK)
Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly (AYYFEKDVKG)Ala Tyr Tyr Phe Glu Lys Asp Val Lys Gly (AYYFEKDVKG)
Asp Leu Asp Pro Gly Gin Thr Leu Ile Tyr (DLDPGQTLIY)Asp Leu Asp Pro Gly Gin Thr Leu Ile Tyr (DLDPGQTLIY)
Val Ile Ala Val Ile Val Val Val Val Met Ala (IAVIVVVVMA)Val Ile Ala Val Ile Val Val Val Val Met Ala (IAVIVVVVMA)
Met Ala Val Val Ala Gly Ile Val Val Leu (MAVVAGIVVL)Met Ala Val Val Ala Gly Ile Val Val Leu (MAVVAGIVVL)
Ala Gly Ile Val Val Leu Val Ile Ser Arg (AGIVVLVISR)Ala Gly Ile Val Val Leu Val Ile Ser Arg (AGIVVLVISR)
Met Ala Pro Pro Gin Val Leu Ala Phe Gly Leu Leu Leu Ala (MAPPQV-Met Ala Pro Pro Gin Val Leu Ala Phe Gly Leu Leu Leu Ala (MAPPQV-
LAFGLLLA)LAFGLLLA)
Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly (MNGS-Met Asn Gly Ser Lys Leu Gly Arg Arg Ala Lys Pro Glu Gly (MNGS-
KLGRRAKPEG)KLGRRAKPEG)
Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp (WCVN-Trp Cys Val Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp (WCVN-
TAGVRRTDKD) , liegt.TAGVRRTDKD).
8. Peptid nach einem der Ansprüche 4 bis 7, dadurch gekennzeichnet, dass es an ein Trägermolekül gekoppelt ist.8. Peptide according to one of claims 4 to 7, characterized in that it is coupled to a carrier molecule.
9. Peptid nach Anspruch 8, dadurch gekennzeichnet, dass das Trägermolekül ein Antikörper, Antikörperderivat, KLH oder Serum Albumin ist.9. Peptide according to claim 8, characterized in that the carrier molecule is an antibody, antibody derivative, KLH or serum albumin.
10. Peptid nach einem der Ansprüche 8 oder 9, dadurch gekennzeichnet, dass das Trägermolekül immunogen ist.10. Peptide according to one of claims 8 or 9, characterized in that the carrier molecule is immunogenic.
11. Vakzine umfassend ein Peptid nach einem der Ansprüche 4 bis 10 und ein geeignetes Adjuvans.11. Vaccine comprising a peptide according to any one of claims 4 to 10 and a suitable adjuvant.
12. Verwendung eines Peptids nach einem der Ansprüche 4 bis 7 als Zielantigen für eine passive Immuntherapie.12. Use of a peptide according to any one of claims 4 to 7 as a target antigen for passive immunotherapy.
13. Verwendung eines Peptids nach einem der Ansprüche 4 bis 10 zur Herstellung von Antikörpern zur Erkennung eines Proteins enthaltend das Peptid nach einem der Ansprüche 4 bis 7.13. Use of a peptide according to one of claims 4 to 10 for the production of antibodies for the detection of a protein containing the peptide according to one of claims 4 to 7.
14. Diagnostikum umfassend ein Peptid nach einem der Ansprüche 4 bis 9 und ein Detektionsmittel zur Bestimmung der Bindung eines Immunserums.14. Diagnostic device comprising a peptide according to one of the claims 4 to 9 and a detection means for determining the binding of an immune serum.
15. Verwendung eines Verfahrens nach einem der Ansprüche 1 bis 3 zum Nachweis von zellspezifisch unterschiedlichen Expressionsmustern eines Proteins.15. Use of a method according to one of claims 1 to 3 for the detection of cell-specifically different expression patterns of a protein.
16. Verwendung eines Verfahrens nach einem der Ansprüche 1 bis 3 zum Nachweis von unterschiedlichen Glykosylierungsmustern eines Proteins .16. Use of a method according to one of claims 1 to 3 for the detection of different glycosylation patterns of a protein.
17. Verwendung eines Peptids nach einem der Ansprüche 4 bis 9, dadurch gekennzeichnet, dass das Peptid als antisense-Peptid verwendet wird.17. Use of a peptide according to one of claims 4 to 9, characterized in that the peptide is used as an antisense peptide.
18. Immunserum umfassend Antikörper gegen Epitope, die nach einem Verfahren nach einem der Ansprüche 1 bis 3 selektioniert wurden.18. Immune serum comprising antibodies against epitopes which have been selected by a method according to one of claims 1 to 3.
19. Vakzine gegen EpCAM exprimierende Tumorzellen, die zur Bildung eines Immunserums wie definiert in einem der Ansprüche 1 bis 3 führt. 19. Vaccine against EpCAM-expressing tumor cells, which leads to the formation of an immune serum as defined in one of claims 1 to 3.
EP04735707A 2003-06-02 2004-06-02 Method for the selection of epitopes for immunotherapy Withdrawn EP1629275A2 (en)

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PCT/AT2004/000193 WO2004106917A2 (en) 2003-06-02 2004-06-02 Method for the selection of epitopes for immunotherapy

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