EP1606308A2 - Peptide associe a une tumeur et se liant a des molecules mhc - Google Patents

Peptide associe a une tumeur et se liant a des molecules mhc

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Publication number
EP1606308A2
EP1606308A2 EP04722556A EP04722556A EP1606308A2 EP 1606308 A2 EP1606308 A2 EP 1606308A2 EP 04722556 A EP04722556 A EP 04722556A EP 04722556 A EP04722556 A EP 04722556A EP 1606308 A2 EP1606308 A2 EP 1606308A2
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EP
European Patent Office
Prior art keywords
peptide
seq
peptides
tumor
cells
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.)
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Application number
EP04722556A
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German (de)
English (en)
Inventor
Hans Georg Rammensee
Stefan Stevanovic
Toni Weinschenk
Claudia Lemmel
Jörn DENGJEL
Oliver Schoor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immatics Biotechnologies GmbH
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Immatics Biotechnologies GmbH
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Publication date
Application filed by Immatics Biotechnologies GmbH filed Critical Immatics Biotechnologies GmbH
Priority to EP10009968A priority Critical patent/EP2280023A1/fr
Priority to EP10009967A priority patent/EP2295443A1/fr
Publication of EP1606308A2 publication Critical patent/EP1606308A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • 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
    • 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
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to tumor-associated peptides which have the ability to bind to a molecule of the major human histocompatibility complex (MHC), Class I.
  • MHC major human histocompatibility complex
  • Such peptides are used, for example, in the immunotherapy of tumor diseases.
  • TAA tumor-associated antigens
  • CD8-expressing cytotoxic T-ymphocytes are particularly involved in the rejection of tumors.
  • CTL cytotoxic T-ymphocytes
  • T cells To trigger such an immune reaction by cytotoxic T cells, foreign proteins / peptides must be presented to the T cells.
  • T cells only recognize antigens as peptide fragments if they are presented on the cell surfaces of MHC molecules.
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • Human MHC molecules are also referred to as human leukocyte antigens (HLA).
  • MHC class I molecules which are found on most cells with a nucleus, present peptides that result from the proteolytic degradation of endogenous proteins.
  • MHC class II molecules occur only on professional antigen-presenting cells (APC) and present peptides of exogenous proteins that are absorbed and processed by APC in the course of endocytosis.
  • APC professional antigen-presenting cells
  • CD8-positive cytotoxic T lymphocytes complexes of peptide and MHC class II are recognized by CD4 helper T cells.
  • CD4 helper T cells In order for a peptide to trigger a cellular immune response, it must bind to an MHC molecule.
  • MHC class I binding peptides are usually 8-10 residues long and contain in their sequence two conserved residues (“anchors") that interact with the corresponding binding groove of the MHC molecule.
  • T cell receptor T Cells with specific T cell receptors
  • the main goal in developing a tumor vaccine is to identify and characterize tumor-associated antigens that are recognized by CD8 + CTL.
  • the antigens that are recognized by the tumor-specific cytotoxic T lymphocytes, or their epitopes can be molecules from all protein classes, such as e.g. Enzymes, receptors, transcription factors, etc.
  • Another important class of tumor-associated antigens are tissue-specific structures, such as, for example, CT ("cancer testis”) antigens, which are expressed in various types of tumor and in healthy testicular tissue.
  • Tumor cells are expressed and not by normal tissues or only in smaller amounts than in the tumors. It is also desirable if the antigen in question is present not only in one tumor type, but also in a high concentration in others.
  • epitopes in the amino acid sequence of the antigen is also absolutely essential, since such peptides (“immunogenic peptides”) derived from a tumor-associated antigen are said to lead to a T cell response, whether in vitro or in vivo.
  • TAAs therefore represent a starting point for the development of a tumor vaccine.
  • the methods for identifying and characterizing the TAAs are based on the one hand on the use of CTL that has already been induced in patients or on the creation of differential transcription profiles between tumors and normal tissues.
  • a tumor-associated peptide with an amino acid sequence which is selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 101 from the accompanying sequence listing, wherein the peptide has the ability to bind to a molecule of the major human histocompatibility complex (MHC) class I.
  • MHC major human histocompatibility complex
  • the peptides identified by the tumor are synthesized in order to obtain larger amounts and for use for the purposes listed below, or are expressed in cells.
  • tumor-associated refers to peptides that have been isolated and identified from tumor material. These peptides, which are presented on real (primary) tumors, are therefore subject to antigen processing in a tumor cell.
  • the specific ligands can be used in cancer therapy, for example to induce an immune response against tumor cells that express the corresponding antigens from which the peptides are derived.
  • Such an immune response in the form of an induction of CTL can be achieved in vivo.
  • the peptide is administered to a patient suffering from a tumor disease associated with the TAA, for example in the form of a pharmaceutical composition.
  • a CTL response to a tumor that expresses the antigens from which the peptides are derived can also be triggered ex vivo.
  • the CTL precursor cells are incubated together with the antigen-presenting cells and the peptides.
  • the CTL stimulated thereby is then cultivated and the activated CTL is administered to the patient.
  • APC APC with the peptides ex vivo and to administer these loaded APC to the patient who expresses in the tumor tissue the antigen from which the peptide is derived.
  • the APC can then present the peptide to the CTL in vivo and activate it.
  • the peptides according to the invention can also be used as diagnostic reagents.
  • the peptides can thus be used to find out whether a CTL population has specifically directed against a peptide or has been induced by therapy.
  • the peptides can also be used to test the increase in precursor T cells which are reactive towards the defined peptide.
  • the peptide can be used as a marker to monitor the course of the disease of a tumor that expresses the antigen from which the peptide is derived.
  • the peptides identified are listed in the attached Table 1.
  • the table also shows the proteins from which the peptides are derived and the respective positions of the peptides in the proteins in question.
  • the English names of the proteins have been retained in order to avoid misleading translations.
  • the Acc numbers are given, which are kept in the gene bank of the "National Center for Biotechnology Information" of the National Institute of Health (see http: ⁇ www.ncbi.nl. Ih. Gov).
  • the inventors were able to isolate the peptides (or ligands) from renal cell tumors of two patients, RCC68 and RCC44.
  • 101 ligands could be identified from the tumors of the patients, which bind to the HLA subtypes HLA-A * 02, HLA-A * 29, HLA-B * 15 or HLA-B * 45 (patient RCC68) and to HLA-A * 3201, HLA-A * 1101, HLA-B * 4002, HLA-B * 2705 or HLA-Cw * 0202 (patient RCC44).
  • ligands were derived from highly expressed so-called "housekeeping" genes, which are uniform in most tissues are expressed, but many were characterized by tissue-specific and tumor-specific expression.
  • Some peptides have been identified that derive from proteins that are overexpressed, particularly in tumor tissue. For example, fragments of vimentin (ALRDVRQQY, position 268-276, SEQ ID No. 7; EENFAVEA, position 348-355, SEQ ID No. 15; MEENFAVEA, position 347-355; NYIDKVRFL, position 116-124) be identified. Young et al. , Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers, 2001, Am. J. Pathol., 158: 1639-1651) showed that this protein was overexpressed in tissue from renal cell tumors.
  • the inventors were also able to Identify ligands derived from alpha-catenin (LQHPDVAAY, positions 229-237, SEQ ID No. 43) and beta-catenin (AQNAVRLHY, positions 481-489, SEQ ID No. 8).
  • cytotoxic T-lymphocytes CTL
  • the inventors were able to show in their own experiments that it was possible to generate cytotoxic T-lymphocytes (CTL) in vitro, which were specific for the selected peptides, using peptides selected by way of example.
  • CTLs cytotoxic T-lymphocytes
  • the CTL mentioned also lysed, for example, dendritic cells which had previously been “pulsed” (loaded) with the respective peptides. It could thus be shown that human T cells could be activated in vitro with the peptides according to the invention as epitopes.
  • CTL peripheral blood ononuclear cells
  • peptides can also be used to stimulate an immune response that have the sequence ID no. 1 to 101 and in which at least one amino acid is replaced by another amino acid with similar chemical properties.
  • these are, for example, the anchor amino acids, which can be replaced by amino acids with similar chemical properties.
  • leucine in the case of peptides which are associated with the MHC subtype HLA-A * 02, leucine can be exchanged at position 2 with isoleucine, valine or with methionine and vice versa, and at the C-terminus leucine with valine, isoleucine and alanine, all of which have non-polar side chains.
  • peptides with the sequence ID no. 1 to 101 which have at the N- or / and C-terminal at least one further amino acid or in which at least one amino acid is deleted.
  • peptides with the sequence ID no. 1 to 101 can be used in which at least one amino acid is chemically modified.
  • the varying amino acid (s) is (are) chosen so that the variation does not impair the immunogenicity of the peptide, ie has a similar binding affinity to the MHC molecule and the ability to stimulate T cells.
  • the peptide can be used for the treatment of tumor diseases and / or adenomatous diseases.
  • the tumor diseases to be treated include, for example, kidney, breast, pancreas, stomach, testicular and / or skin cancer.
  • the list of tumor diseases is only an example and is not intended to limit the area of use.
  • the inventors were able to show in their own experiments that the peptides according to the invention are suitable for such a use. It proved that specially generated CTLs, which were specific for certain peptides, could effectively and selectively kill tumor cells.
  • the peptide can be used with the addition of adjuvants, or else on its own.
  • the granulocyte macrophage colony stimulating factor can be used as an adjuvant.
  • adjuvants are aluminum hydroxide, emulsions of mineral oils, such as Freund's adjuvant, saponins or silicon compounds.
  • the use with adjuvants offers the advantage that the immune response triggered by the peptide can be strengthened and / or that the peptide is stabilized.
  • the peptide is used bound on an antigen-presenting cell.
  • This measure has the advantage that the peptides can be presented to the immune system, in particular the cytotoxic T-lymphocytes (CTL).
  • CTL cytotoxic T-lymphocytes
  • antigen-presenting cells for example, dendritic cells, monocytes or B-lyitiphoocytes are suitable for such an application.
  • the cells will be loaded with the peptides ex vivo, for example.
  • the cells with the DNA coding for the peptides or the corresponding Transfect RNA to then express the peptides on the cells.
  • the inventors were able to show in their own experiments that it is possible to load dendritic cells (DC) with specific peptides and that these loaded dendritic cells activate peptide-specific CTL. This means that the immune system can be stimulated to develop CTL against the tumors that express the corresponding peptides.
  • DC dendritic cells
  • the antigen-presenting cells carrying the peptide can either be used directly, or they can be activated, for example, with the heat shock protein gp96 before use.
  • This heat shock protein induces the expression of MHC class I molecules and of costimulatory molecules such as B7 and also stimulates the production of cytokines. Overall, this triggers the triggering of immune responses.
  • the peptides are used for labeling leukocytes, in particular T-lymphocytes.
  • the peptide can be used as a marker for assessing a course of therapy for a tumor disease.
  • the peptide can also be used for monitoring the therapy in other immunizations or therapies.
  • the peptide can therefore not only be used therapeutically but also diagnostically.
  • the peptides are used to produce an antibody.
  • Polyclonal antibodies can be obtained in a conventional manner by immunizing animals by injection of the peptides and subsequent purification of the immunoglobulin.
  • Monoclonal antibodies can be produced according to standard protocols, for example in Methods Enzymol. (1986), 121, Hybridoma technology and monoclonal antibodies.
  • the invention also relates to a pharmaceutical composition which contains one or more of the peptides.
  • composition is used, for example, for parenteral administration, for example subcutaneously, intradermally or intramuscularly, or for oral administration.
  • the peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier.
  • the composition can contain auxiliary substances such as buffers, binders, diluents, etc. contain.
  • the peptides can also be administered together with immunostimulating substances, for example cytokines.
  • immunostimulating substances for example cytokines.
  • auxiliaries such as can be used in such a composition, is presented, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients, 3rd Ed., 2000, American Pharmaceutical Association and pharmaceutical press.
  • the agent can be used for the prevention, prophylaxis and / or therapy of tumor diseases and / or adenomatous diseases.
  • the pharmaceutical agent which contains at least one of the peptides with the sequence ID no. 1 to 101 is administered to a patient suffering from a tumor disease with which the peptide or antigen in question is associated. This can trigger a tumor-specific immune response based on tumor-specific CTL.
  • the amount of the peptide or peptides present in the pharmaceutical composition is present in a therapeutically effective amount.
  • the peptides contained in the composition can also bind to at least two different HLA types.
  • the present invention relates to nucleic acid molecules which code for the peptides with the sequence ID numbers 1 to 101, and to the use of at least one of the nucleic acid molecules for the production of a medicament for the treatment of tumor diseases and / or adenomatous diseases.
  • the nucleic acid molecules can be DNA or RNA molecules and can also be used for the immunotherapy of cancer.
  • the peptide expressed by the nucleic acid molecule induces an immune response against tumor cells that express the peptide.
  • the nucleic acid molecules can also be present in a vector.
  • the invention relates to cells which are generated with the aid of the nucleic acid molecule which codes for the peptides. was changed in such a way that it contains a peptide with the sequence ID no. 1 to 101 produced.
  • the cells are transfected with the DNA encoding the peptides or the corresponding RNA, as a result of which the peptides are expressed on the cells.
  • suitable antigen-presenting cells are, for example, dendritic cells, monocytes or other human cells which express molecules which are suitable for co-stimulation, such as, for example, B7.1 or B7.2.
  • the invention further relates to a diagnostic method in which the presence of one of the new peptides is used as a diagnostic marker, and to a method for treating a pathological condition in which an immune response against a protein of interest is triggered, a therapeutically effective amount of at least one of the new peptides is administered.
  • the new peptides can also be used as markers for a pathological condition, so that a corresponding diagnostic method in which a blood sample is taken from the patient and examined in the usual way for the presence of lymphocytes directed against one of the new peptides is used as early detection or for the targeted selection of a suitable treatment.
  • the invention further relates to an electronic storage medium which contains the amino acid sequence of at least one of the new peptides and / or the nucleic acid sequence of nucleic acid molecules coding for the new peptides.
  • RCC68 had the following HLA typing: HLA-A * 02 A * 29 B * 15 B * 45; patient No. 2 (hereinafter referred to as RCC44) HLA-A * 3201 A * 1101 B * 4002 B * 2705 Cw * 0202.
  • the shock-frozen tumor samples were processed as already described in Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach, 2000, European Journal of Immunology, 30: 2216-2225.
  • the peptides were isolated according to standard protocols using the W6 / 32 monoclonal antibody specific for HLA class I molecules or the BB7.2 monoclonal antibody specific for HLA-A2.
  • Barnstable, CJ et al. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis, 1978, Cell, 14: 9-20 and Parham, P. & Brodsky, FM, Partial purification and some properties of BB7.2.
  • the peptides were separated by "reversed phase HPLC" (SMART system, ⁇ RPC C2 / C18 SC 2.1 / 19, Amersham Pharmacia Biotech) and the fractions obtained were analyzed by nanoESI MS.
  • SMART system ⁇ RPC C2 / C18 SC 2.1 / 19, Amersham Pharmacia Biotech
  • nanoESI MS nanoESI MS.
  • the procedure was as described in Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach, 2000, European Journal of Immunolgy, 30: 2216-2225.
  • the peptides obtained from tumor tissue were identified by capillary LC-MS as just mentioned, but with minor changes: 100 ⁇ l of the samples were each loaded, desalted and on a 300 ⁇ m * 5 mm C18 ⁇ precolumn (LC packings ) pre-concentrated.
  • the solvent and sample were delivered using a syringe pump (PHD 2000, Harvard Apparatur, Inc.) with a sealed 100 ul syringe (1710 RNR, Hamilton) at a rate of 2 ul / min.
  • PLD 2000 Harvard Apparatur, Inc.
  • the preconcentration column was placed in front of a 75 ⁇ m * 250 mm C-18 column (LC Packings).
  • a binary gradient with 25-60% B was then run within 70 min, the flow rate being reduced from 12 ⁇ l / min to approximately 300 nl / min, using a TEE connection (ZT1C, Valco) and a 300 ⁇ m * 150 mm C-18 column.
  • HLA-A * 02 had the allele-specific peptide motif: Leucine, valine, isoleucine, alanine or methionine were found at position 2 and leucine, valine, isoleucine, or alanine was found at the C-terminus .
  • Most of the ligands were derived from so-called "housekeeping" proteins, but ligands of proteins associated with tumors could also be identified. For example, fragments of Vi entin (ALRDVRQQY, position 268-276, SEQ ID No.
  • HLA-A * subtype tetramers in question were treated with the HLA-A * subtype tetramers in question, which were constituted with peptides in question:
  • HLA-A * subtype molecules were constituted in vitro with the peptides, purified by gel filtration, biotinylated and mixed with streptavidin to link the monomers ,
  • results of the double staining are evaluated by analysis using FACS and the specific binding of the peptide tetramers is verified.
  • DC dendritic cells
  • PBMNC peripheral blood mononuclear cells
  • the DC were isolated from heparinized blood by Ficoll / Paque (Biochrom, Berlin, Germany) density gradient centrifugation from PBMNC.
  • the heparinized blood was obtained from "buffy coat" preparations from healthy donors of the blood bank of the University of Tübingen.
  • the cells were placed on 6-well plates (Falcon, Heidelberg, Germany) (1 x 10 7 cells / 3 ml per well) in RP10 Medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and with antibiotics) sows.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Leukomax Novartis
  • lOOng / ml Interleukin IL-4
  • TNF- ⁇ tumor necrosis factor ⁇
  • peptides selected by way of example were synthesized using F-moc (9-fluorenylmethyloxycarbonyl) protective groups on a peptide synthesizer (432A, Applied Biosystems, Rothstadt, Germany) and analyzed by “reversed phase” HPLC and mass spectroscopy Amounts of the identified peptides are made.
  • Tumor cells peptide-pulsed cells from different cell lines and autologous DC were used as target cells for the CTL assays.
  • Peptide-pulsed cells were pulsed with 50 ug / ml peptide for 2 hours. All target cells were labeled in RPIO medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and with antibiotics) for 1 hour at 37 ° C. with [ 51 Cr] sodium chromate ( 51 Cr). Then 10 4 cells / per well were placed on a 96-well plate with a rounded bottom. Different amounts of CTL were added to reach a final volume of 200 ul, followed by incubation for 4 hours at 37 ° C.
  • the supernatants (50 ⁇ l / well) were then harvested and counted in a beta plate counter.
  • the specific lysis was calculated in percent as follows: 100 x (experimental release - spontaneous release / maximum release - spontaneous release). The spontaneous and maximum release were determined in the presence of either medium or 2% Triton X-100.
  • T2 cell line is HLA-A * 02- positive and TAP (Transporter associated with Antigen processing) —deficient; (TAP peptide transporters transport peptide fragments of a protein antigen from the cytosol into the endoplasmic reticulum, where they associate with MHC molecules.)
  • a 51 Cr release assay was used to test whether the CTL, which were specific for the selected peptides, recognize and lyse tumor cells which endogenously express the selected peptides.
  • HCT 116 colonal cancer; obtained from Prof. G. Pawelec, Tübingen, Germany
  • a 498, MZ 1257 and MZ 1774 renal cell carcinoma; obtained from Prof. A. Knuth , Frankfurt, Germany
  • MCF-7 breast cancer; purchased from the ATCC, American Type Culture Collection
  • Mel 1479 melanoma; received from Prof. G. Pawelec, Tübingen, Germany
  • U 266 multiple myeloma; obtained from Prof. G. Pawelec, Tübingen, Germany.
  • These cell lines express certain proteins as target structures.
  • the B cell line Croft (EBV (Epstein-Barr virus) immortalized; HLA-A * 02 positive; obtained from OJ Finn, Pittsburgh, USA) and the cell line SK-OV-3 (ovarian tumor; HLA-A * 03 -positive; obtained from OJ Finn, Pittsburgh, USA) were included in the studies as negative controls.
  • K 562 cells (for example available from the German Collection of Microorganisms and Cell Cultures, DSMZ; ACC 10) were used to determine the activity of natural killer cells (NK), since this cell line is highly sensitive to these killer cells.
  • All cell lines were cultivated in RP10 medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and with antibiotics).
  • the CTLs which are specific for the selected peptides, efficiently lysed tumor cells that express both the corresponding HLA molecule and the selected peptides.
  • the specific lysis was - as in 2.4 above. indicated - measured by the 51 Cr release.
  • the control cell line SK-OV-3 (HLA-A- * 02 negative), however, was not lysed by the CTL induced by the peptides bound by HLA-A * 02. This showed that the peptides in connection with the corresponding HLA molecules had to be presented on the tumor cells in order to efficiently lyse the target cells. This also confirms the antigen specificity and the MHC restriction of the CTL.
  • the CTL cells induced in vitro by the peptides also did not recognize the cell line K562, which shows that the cytotoxic activity was not mediated by natural killer cells (NK) cells.
  • NK natural killer cells
  • the ability of peptide-pulsed cell lines to inhibit or compete for the lysis of tumor cells was analyzed. An excess of inhibitor (ie pulsed, unlabeled cells) was used for this. The ratio of the inhibitor (peptide-pulsed cells) to the target (tumor cells) was 20: 1. When the inhibitor cell lines were lysed, 51 Cr could not be released because the inhibitor cell lines were unlabeled.
  • Cell line T2 (HLA-A * 02; TAP deficient; see under 2.5.a)) was used as an inhibitor. This cell line T2 was pulsed with the relevant peptides or an irrelevant control peptide before the assays.
  • the MHC restriction and the antigen specificity of the cytotoxic activity mediated by the HLA-A * 02 peptide-induced CTL could be determined using an HLA-A * 02-specific monoclonal antibody and in an inhibition assay with unlabelled ("cold") inhibitor:
  • the A 498 tumor cells were blocked by adding the HLA-A * 02-specific antibody (monoclonal antibody BB7.2, IgG2b, obtained from S. Stefanovic, Tübingen), so that they were not lysed by adding the CTL and no 51 Cr was released.
  • a non-specific antibody which did not block the HLA-A * 02 molecule was used as a control.
  • the cells were used for these inhibition experiments Sowing on the 96-well plates incubated for 30 min with 10 ⁇ g / ml antibody.
  • RNA was isolated from the tumor cells using the QIAGEN Rneasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The amount and purity of the RNA was determined photometrically and stored in aliquots at -80 ° C.
  • CTL was generated from the PBMNC of a patient with chronic lymphatic leukemia (CLL), which were specific for selected peptides.
  • CLL chronic lymphatic leukemia
  • autologous primary CLL cells and DC of this patient were used as 51 Cr-labeled targets in an assay in which sl Cr release was mediated by the peptide-induced CTL.
  • both the autologous DC of this patient which was pulsed with the selected peptides, and the autologous CLL cells were lysed by the peptide-induced CTL.
  • DC which were pulsed with an irrelevant peptide, however, were not lysed.
  • Non-malignant B cells and the cell line K 562 were also not lysed by the CTL.
  • the specificity of the CTL response was confirmed in a target inhibition assay, the cell line T2 (see above) being used as the inhibitor cells, each of which was pulsed with the selected peptides or with an irrelevant peptide.
  • the CTL induced by the use of the peptides also lysed the excess inhibitor cell lines which were pulsed with the relevant peptides that the 51 Cr-labeled tumor cells were not lysed in this case.
  • the inventors were able to show that the identified peptides are promising substances in the context of immunotherapy for a large number of (tumor) diseases.
  • AVCEVALDY 2260-2268 NM_003128 SEQ ID no. 10 spectrin, beta, non-erythrocytic 1
  • transglutaminase 2 C polypeptides, protein-glutamine-gamma-gluta yl transferase
  • CD49C alpha 3 subunit of VLA-3 re ⁇ eptor
  • HSPC038 protein 73 VEHPSLTSP 170-178 M15374 SEQ ID no. 73
  • VEPDHFKVA 204-212 NM 002306 SEQ ID no. 74 le ⁇ tin, gala ⁇ toside-binding, soluble, 3 (galectin 3)
  • VMDSKIVQV 432-440 NM 012316 SEQ ID no. 80 karyopherin alpha 6 (importin alpha 7)

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  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un peptide associé à une tumeur, présentant une séquence aminoacide choisie à partir du groupe formé par SEQ ID-Nr. 1 à SEQ ID-Nr. 101 à partir d'un protocole de séquence associé, ledit peptide étant capable de se lier à une molécule du complexe majeur d'histocompatibilité humain (MHC) classe I. L'invention concerne en outre l'utilisation des peptides précités et des acides nucléiques codant pour ces peptides, pour la fabrication d'un médicament, et pour le traitement de maladies tumorales et/ou adénomateuses. L'invention concerne également une composition pharmaceutique comprenant au moins l'un desdits peptides.
EP04722556A 2003-03-24 2004-03-23 Peptide associe a une tumeur et se liant a des molecules mhc Withdrawn EP1606308A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP10009968A EP2280023A1 (fr) 2003-03-24 2004-03-23 Peptides spécifiques aux tumeurs se fixant sur des molécules CMH
EP10009967A EP2295443A1 (fr) 2003-03-24 2004-03-23 Peptides spécifiques aux tumeurs se fixant sur des molécules CMH

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10313819A DE10313819A1 (de) 2003-03-24 2003-03-24 An MHC-Moleküle bindende Tumor-assoziierte Peptide
DE10313819 2003-03-24
PCT/EP2004/003077 WO2004085461A2 (fr) 2003-03-24 2004-03-23 Peptide associe a une tumeur et se liant a des molecules mhc

Publications (1)

Publication Number Publication Date
EP1606308A2 true EP1606308A2 (fr) 2005-12-21

Family

ID=32946241

Family Applications (3)

Application Number Title Priority Date Filing Date
EP04722556A Withdrawn EP1606308A2 (fr) 2003-03-24 2004-03-23 Peptide associe a une tumeur et se liant a des molecules mhc
EP10009968A Withdrawn EP2280023A1 (fr) 2003-03-24 2004-03-23 Peptides spécifiques aux tumeurs se fixant sur des molécules CMH
EP10009967A Withdrawn EP2295443A1 (fr) 2003-03-24 2004-03-23 Peptides spécifiques aux tumeurs se fixant sur des molécules CMH

Family Applications After (2)

Application Number Title Priority Date Filing Date
EP10009968A Withdrawn EP2280023A1 (fr) 2003-03-24 2004-03-23 Peptides spécifiques aux tumeurs se fixant sur des molécules CMH
EP10009967A Withdrawn EP2295443A1 (fr) 2003-03-24 2004-03-23 Peptides spécifiques aux tumeurs se fixant sur des molécules CMH

Country Status (9)

Country Link
US (2) US20090221509A1 (fr)
EP (3) EP1606308A2 (fr)
JP (1) JP4365405B2 (fr)
AU (1) AU2004224159B2 (fr)
CA (2) CA2731275A1 (fr)
DE (1) DE10313819A1 (fr)
HR (1) HRP20050808A2 (fr)
PL (1) PL378679A1 (fr)
WO (1) WO2004085461A2 (fr)

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US20090004213A1 (en) * 2007-03-26 2009-01-01 Immatics Biotechnologies Gmbh Combination therapy using active immunotherapy
PT2105501E (pt) * 2008-03-27 2015-12-21 Immatics Biotechnologies Gmbh Nova imunoterapia para tumores neuronais e cerebrais
DE102010026063A1 (de) 2010-06-30 2012-01-05 Siemens Aktiengesellschaft 11C-markiertes Peptid zur Detektion eines krankhaften Gewebes
GB201214007D0 (en) * 2012-08-07 2012-09-19 Scancell Ltd Anti-tumour immune responses to modified self-epitopes
GB201505585D0 (en) 2015-03-31 2015-05-13 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides and scaffolds for use in immunotherapy against renal cell carinoma (RCC) and other cancers
NL2014935B1 (en) 2015-06-08 2017-02-03 Applied Immune Tech Ltd T cell receptor like antibodies having fine specificity.
CN106645677B (zh) * 2016-11-15 2019-08-09 恒瑞源正（上海）生物科技有限公司 体外检测肿瘤新生抗原特异性t细胞的方法、试剂盒以及肿瘤疫苗
EP4317432A3 (fr) 2016-12-08 2024-04-17 Immatics Biotechnologies GmbH Récepteurs de lymphocytes t à appariement amélioré
DE102016123893A1 (de) 2016-12-08 2018-06-14 Immatics Biotechnologies Gmbh T-Zellrezeptoren mit verbesserter Bindung
JP6841782B2 (ja) 2018-03-28 2021-03-10 ヤンマーパワーテクノロジー株式会社 自律走行システム

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Also Published As

Publication number Publication date
JP4365405B2 (ja) 2009-11-18
US20110070253A1 (en) 2011-03-24
AU2004224159B2 (en) 2011-06-02
PL378679A1 (pl) 2006-05-15
HRP20050808A2 (en) 2005-12-31
WO2004085461A2 (fr) 2004-10-07
JP2006521093A (ja) 2006-09-21
CA2731275A1 (fr) 2004-10-07
EP2280023A1 (fr) 2011-02-02
DE10313819A1 (de) 2004-10-07
WO2004085461A3 (fr) 2005-09-15
CA2520497A1 (fr) 2004-10-07
AU2004224159A1 (en) 2004-10-07
EP2295443A1 (fr) 2011-03-16
US20090221509A1 (en) 2009-09-03

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