EP1363942A2 - Polypeptides d'un recepteur de lymphocytes t alpha/beta des murides specifique de la proteine hdm2, acides nucleiques les codant et leur utilisation - Google Patents

Polypeptides d'un recepteur de lymphocytes t alpha/beta des murides specifique de la proteine hdm2, acides nucleiques les codant et leur utilisation

Info

Publication number
EP1363942A2
EP1363942A2 EP02722160A EP02722160A EP1363942A2 EP 1363942 A2 EP1363942 A2 EP 1363942A2 EP 02722160 A EP02722160 A EP 02722160A EP 02722160 A EP02722160 A EP 02722160A EP 1363942 A2 EP1363942 A2 EP 1363942A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
cell
hdm2
seq
protein
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.)
Withdrawn
Application number
EP02722160A
Other languages
German (de)
English (en)
Inventor
Thomas Stanislawski
Matthias Theobald
Holger Voss
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.)
Immugenics AG
Original Assignee
Immugenics AG
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Immugenics AG filed Critical Immugenics AG
Publication of EP1363942A2 publication Critical patent/EP1363942A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to polypeptides of the murine ⁇ / ⁇ T cell receptor which mediates an hdm2 protein-specific T-cell response, these nucleic acids coding therefor and their use in the therapy, diagnosis and / or prevention of diseases associated with the hdm2 protein.
  • Oligopeptides of the hdm2 protein can be presented in the context of MHC class I molecules on the cell surface and represent attractive target structures for CD8-positive T cells.
  • the extent of the T cell response runs in a defined kinetic window (Kersh et al., 1998).
  • WO 97/32603 generally describes a method for the production of recombinant T lymphocytes that express specific T cell receptors directed against tumor tissue, whereby an HLA transgenic mouse (in this case HLA-A2) is immunized with tumor-associated antigen, so as to cause the production of cytotoxic T lymphocytes, the specific Express T-cell receptors on their surface:
  • Tumor-associated antigens are peptides of various genes, such as Her-2 / neu, Ras, p53, tyrosinase, MART, gplOO, MAGE, BAGE and MUC-1 b esch rubbed.
  • the present invention is therefore based on the object of making available the murine genes of the T cell receptors ⁇ -TZR and ⁇ -TZR directed against the hdm2 protein. These have the effect that cells expressing hdm2 protein are recognized by CD8-positive T cells, cytokines are released, and T cell-induced lysis and / or apoptosis of tumor or leukemia cells is brought about.
  • the genes were introduced as wild type (WT) or modified constructs retrovirally into human peripheral blood lymphocytes (PBLs) and the HLA-restricted antigen recognition was functionally checked by CD8-mediated cytotoxic lysis of various cell lines in the 51 chromium release test.
  • the genes of the hdm2-specific TZR according to the invention do not belong to the hitherto known suitable targets for the diagnosis - such as the indication - and / or the treatment - such as the modulation - of diseases related to hdm2 protein or for the identification of pharmacologically active substances, so that completely new therapeutic approaches result from this invention.
  • Another aspect of the invention relates to a fusion protein comprising the polypeptide according to the invention or functional variants or parts thereof or nucleic acids encoding it, functional variants or parts thereof.
  • the fusion protein can be characterized in that it comprises the ⁇ region of CD3 or CD8 or CD 16 or parts thereof, in particular the ⁇ region of human CD3 or CD8 or CD 16 or parts thereof.
  • a fusion protein according to the invention which comprises a flexible linker (Whitlow et al., "An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability", Prot. Engin. 6 (8), pp. 989-995, 1993), in particular a linker of the amino acid sequence (GGGGS) 3.
  • the fusion protein according to the invention can comprise the ⁇ chain of the CD3 complex or ITAM motifs of the ⁇ chain or parts thereof, in particular the ⁇ chain of human CD3 or parts thereof
  • the fusion protein can further be characterized in that it comprises CD8 ⁇ or the Lck binding motif of CD8 ⁇ or parts thereof, in particular human CD8 ⁇ .
  • Fusion proteins are produced here which contain the polypeptides according to the invention described above, the fusion proteins themselves already having the function of a polypeptide of the invention or the specific one only after the fusion portion has been split off Function is active. Above all, this includes fusion proteins with a proportion of approximately 1-200, preferably approximately 1-150, in particular approximately 1-100, especially approximately 1-50 foreign amino acids. Examples of such peptide sequences are prokaryotic peptide sequences which e.g. B. can be derived from the galactosidase of E. coli. Furthermore, viral peptide sequences, such as, for example, from bacteriophage Ml 3, can also be used in order to generate fusion proteins for the "phage display" method known to the person skilled in the art.
  • a further polypeptide can be added to purify the proteins according to the invention.
  • Protein tags according to the invention allow, for example, high affinity absorption to a matrix, stringent washing with suitable buffers without eluting the complex to any appreciable extent, and then targeted elution of the absorbed complex.
  • Examples of the protein tags known to the person skilled in the art are a (His) 6 tag, a Myc tag, a FLAG tag, a Strep tag, a Strep tag II, a hemagglutinin tag, glutathione transferase (GST) - tag, intein with an affinity chitin binding tag or maltose binding protein (MBP) tag.
  • These protein tags can be located at the N-, C-terminal and / or internally.
  • all of the polypeptides according to the invention or parts thereof may have been produced under cell-free conditions, e.g. B. by synthesis or by in v tro translation.
  • all or part of the polypeptide can be synthesized using classic synthesis (Merrifield technique).
  • Parts of the polypeptides according to the invention are particularly suitable for obtaining antisera, with the aid of which suitable gene expression banks can be searched in order to arrive at further functional variants of the polypeptide according to the invention.
  • nucleic acid according to the invention can be chemically determined using the protein sequences described in SEQ ID No. 1 to SEQ ID No. 8 using the genetic code z. B. can be synthesized by the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Revievs, 90, 543-584).
  • oligonucleotides are rapidly degraded by endo- or exonucleases, in particular by DNases and RNases occurring in the cell. It is therefore advantageous to modify the nucleic acid in order to stabilize it against degradation so that a high concentration of the nucleic acid is maintained in the cell over a long period of time (WO 95/11910; Macadam et al., 1998, WO 98 / 37240; Reese et al., 1997, WO 97/29116). Typically, such stabilization can be obtained by introducing one or more internucleotide phosphor groups or by introducing one or more non-phosphor internucleotides.
  • Suitable modified internucleotides are summarized in Uhlmann and Peymann (1990 Chem. Rev. 90, 544) (WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116).
  • Another aspect of the present invention relates to a vector, preferably in the form of a plasmid, shuttle vector, phagemid, cosmid, expression vector, adenoviral vector, retroviral vector (Miller, et al. "Improved retroviral vectors for gene transfer and expression", BioTechniques Vol. 7, No. 9, p 980, 1989) and / or gene therapy-effective vector which contains a nucleic acid according to the invention.
  • a vector preferably in the form of a plasmid, shuttle vector, phagemid, cosmid, expression vector, adenoviral vector, retroviral vector (Miller, et al. "Improved retroviral vectors for gene transfer and expression", BioTechniques Vol. 7, No. 9, p 980, 1989) and / or gene therapy-effective vector which contains a nucleic acid according to the invention.
  • the nucleic acid according to the invention can preferably be contained in a vector in an expression vector or a gene therapy vector.
  • the gene-therapeutic vector T cell preferably contains specific regulatory sequences which are functionally linked to the nucleic acid according to the invention.
  • the expression vectors can be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors for expression in E. coli are, for example, the vectors pGEM or pUC derivatives and for eukaryotic expression vectors for expression in Saccharomyces cerevisiae z. B. the vectors p426Met25 or p426GALl (Mumberg et al. (1994) Nucl.
  • Bac lovirus vectors as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, and for expression in mammalian cells z.
  • the nucleic acid can be present as a plasmid, as part of a viral or non-viral vector or particle.
  • viral vectors or particles baculoviruses, vaccinia viruses, retroviruses, adenoviruses, adenoas-associated viruses and herpes viruses.
  • non-viral carriers virosomes, liposomes, cationic lipids or poly-lysine-conjugated DNA.
  • virus vectors for example adevirus vectors or retroviral vectors (Lindemann et al., 1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell. 2: 549-58; Weijtens et al. "A retroviral vector system, 'STITCH'; in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T-lymphocytes", Gene Therapy (1998) 5,1995-1203) ,
  • Retroviral vector systems create the prerequisites for long-term expression of the transgene through the stable but non-directional integration into the host genome. Younger generation vectors have no irrelevant and potentially immunogenic proteins, furthermore there is no pre-existing immunity of the recipient against the vector.
  • Retroviruses contain an RNA genome that is packaged in a lipid shell that consists of parts of the host cell membrane and virus proteins. To express viral genes, the RNA genome is reverse transcribed and integrated with the enzyme integrase in the target cell DNA. This can then be transcribed and translated by the infected cell, creating viral components that combine to form retroviruses. RNS is only then inserted into the newly created viruses.
  • New, non-viral vectors consist of autonomous, self-integrating DNA sequences, the transposons, which are e.g. liposomal transfection were introduced into the host cell and were successfully used for the first time to express human transgenes in mammalian cells (Yant et al., 2000).
  • Vectors with gene therapy effects can also be obtained by complexing the nucleic acid according to the invention with liposomes, since a very high transfection efficiency, in particular of skin cells, can be achieved with this (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 2279 -85).
  • Excipients that increase the transfer of nucleic acids into the cell can be, for example, proteins or peptides that are bound to DNA or synthetic peptide-DNA molecules that enable the transport of the nucleic acid into the nucleus of the cell (Schwartz et al. ( 1999) Gene Therapy 6, 282; Branden et al. (1999) Nature Biotech. 17, 784).
  • Auxiliaries also include molecules that enable the release of nucleic acids into the cytoplasm of the cell (Planck et al. (1994) J. Biol. Chem. 269, 12918; Kichler et al. (1997) Bioconj. Chem. 8, 213) or for example liposomes (Uhlmann and Peymann (1990) supra).
  • Another particularly suitable form of gene therapy vectors can be obtained by applying the nucleic acid according to the invention to gold particles and shooting them into tissue, preferably into the skin, or cells using the so-called “gene gun” (Wang et al., 1999, J. Invest. Dermatol., 112: 775-81).
  • the part of the nucleic acid which codes for the polypeptide has one or more non-coding sequences including intron sequences, preferably between the promoter and the start codon of the polypeptide, and / or a polyA sequence , in particular the naturally occurring polyA sequence or an SV40 virus polyA sequence, especially at the 3 'end of the gene, since this can stabilize the mRNA (Jackson, RJ (1993) Cell 74, 9-14 and Palmiter, RD et al. (1991) Proc. Natl. Acad. Sci. USA 88, 478-482).
  • Another object of the present invention is a host cell, in particular a T cell, which is transformed with a vector according to the invention or another gene construct according to the invention.
  • Host cells can be both prokaryotic and eukaryotic cells, examples of prokaryotic host cells are E. coli and Saccharomyces cerevisiae or insect cells for eukaryotic cells.
  • a particularly preferred transformed host cell is a transgenic T precursor cell or a stem cell, which is characterized in that it comprises a gene construct according to the invention or an expression cassette according to the invention.
  • Methods for transforming or transducing host cells and / or stem cells are well known to those skilled in the art and include, for example, electroporation or microinjection.
  • a particularly preferred transformed host cell is a patient's own T cell which, after removal, is transfected with a gene construct according to the invention.
  • Host cells according to the invention can in particular be obtained by removing one or more cells, preferably T cells, in particular CD8 + T cells, from the patient, which are then transfected or transduced ex vivo with one or more genetic constructs according to the invention in order to to obtain host cells according to the invention.
  • the specific T cells generated ex vivo can then be re-implanted in the patient.
  • the process is thus similar to that in Darcy et al. ("Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL" J. Immunol., 2000. 164: 3705-3712) described methods using scFv anti-CEA receptor transduced ZTL, perforin and ⁇ -IFN.
  • the method is carried out according to methods generally known to the person skilled in the art by immunizing a mammal, for example a rabbit, with the polypeptide according to the invention or the parts thereof, optionally in the presence of e.g. B. incomplete Freund's adjuvant and / or aluminum hydroxide gels (see e.g. Diamond, B.A. et al. (1981) The New England Journal of Medicine, 1344-1349).
  • B. incomplete Freund's adjuvant and / or aluminum hydroxide gels see e.g. Diamond, B.A. et al. (1981) The New England Journal of Medicine, 1344-1349.
  • the polyclonal antibodies produced in the animal due to an immunological reaction can then be easily isolated from the blood by generally known methods and z. B. clean over column chromatography.
  • Monoclonal antibodies can be produced, for example, by the known method from Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299
  • Another object of the present invention is an antibody for the diagnosis and / or treatment of diseases associated with hdm2 protein or for the identification of pharmacologically active substances, which is directed against a polypeptide according to the invention and reacts specifically with the polypeptides according to the invention, the above-mentioned parts of the polypeptide are either themselves immunogenic or by coupling to suitable carriers, such as. B. bovine serum albumin can be increased in their immunogenicity.
  • This antibody is either polyclonal or monoclonal, a monoclonal antibody is preferred.
  • the present invention further relates to a medicament produced by this method for the treatment of diseases associated with hdm2 protein which contains at least one nucleic acid, at least one polypeptide or at least one antibody according to the present invention, optionally together with suitable additives and auxiliaries.
  • the invention further relates to the use of this medicament for the treatment of diseases associated with hdm2 protein.
  • the therapy of diseases associated with hdm2 protein can be carried out in a conventional manner, for example by infusions or injections, which contain the medicaments according to the invention.
  • the medicaments according to the invention can furthermore optionally be administered in the form of liposome complexes or gold particle complexes.
  • Treatment by means of the medicaments according to the invention can, however, also take place via oral dosage forms, such as tablets or capsules, via the mucous membranes, for example the nose or the oral cavity, or in the form of disposers implanted under the skin.
  • TTS are known for example from EP 0 944 398 AI, EP 0 916 336 AI, EP 0 889 723 AI or EP 0 852 493 AI.
  • the present invention further relates to a method for producing a test for finding functional interactors in connection with diseases associated with hdm2 protein, which is characterized in that at least one nucleic acid, at least one polypeptide or at least one antibody according to the invention together with suitable additives and Auxiliary materials are combined.
  • the term "functional interactors" within the meaning of the present invention is to be understood as meaning all those molecules, compounds and / or compositions and substance mixtures which are suitable for use with the nucleic acids, polypeptides or antibodies according to the invention, optionally together with suitable additives and auxiliaries Can interact.
  • Possible interactors are simple chemical organic or inorganic molecules or compounds, but can also include peptides, proteins or complexes thereof.
  • the functional interactors can influence the function (s) of the nucleic acids, polypeptides or antibodies in vivo or in vitro or can only bind to the nucleic acids, polypeptides or antibodies according to the invention or have other interactions with them covalently or non-covalently.
  • the present invention also relates to a medicament for the indication and therapy of diseases associated with hdm2 protein, which contains a nucleic acid or a polypeptide according to the invention and, if appropriate, suitable additives or auxiliaries, and a method for producing such a medicament for the treatment of hdm2 protein-associated diseases, in which a nucleic acid or a polypeptide according to the invention is formulated with a pharmaceutically acceptable carrier.
  • Suitable therapeutic agents and / or prophylactic agents are in particular vaccines, recombinant particles or injections or infusion solutions which contain as active ingredient (a) the TCR receptor according to the invention polypeptide and / or its derivatives and / or (b) a nucleic acid according to the invention and / or ( c) T lymphocytes produced in vitro or ex vivo, which contain a TZR specifically directed against hdm2.
  • a drug and / or recombinant particle which contains the nucleic acid according to the invention in naked form or in the form of one of the gene therapy-active vectors described above or in a form complexed with liposomes or gold particles is particularly suitable for gene therapy use in humans.
  • the pharmaceutical carrier is, for example, a physiological buffer solution, preferably with a pH of approximately 6.0-8.0, preferably approximately 6.8-7.8. In particular of approximately 7.4 and / or an osmolarity of approximately 200-400 milliosmol / liter, preferably of approximately 290-310 milliosmol / liter.
  • the pharmaceutical carrier can contain suitable stabilizers, such as e.g. B. nuclease inhibitors, preferably complexing agents such as EDTA and / or other auxiliaries known to those skilled in the art.
  • the invention further relates to a method for producing a polypeptide for the diagnosis and / or treatment of diseases associated with hdm2 protein or for the identification of pharmacologically active substances in a suitable host cell, which is characterized in that a nucleic acid according to the invention is appropriately expressed.
  • the polypeptide is thus produced, for example, by expression of the nucleic acid according to the invention in a suitable expression system, as already described above, using methods which are generally known to the person skilled in the art.
  • Suitable host cells are, for example, the E. coli strains DHS, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, insect cell lines, eg. B. from Spodoptera frugiperda, or the animal cells COS, Vero, 293, HaCaT, and HeLa, all of which are commonly available.
  • a diagnostic agent according to the invention for monitoring therapy contains the polypeptide according to the invention or the immunologically active parts thereof described in more detail above.
  • the polypeptide or parts thereof which are preferably attached to a solid phase, e.g. B. from nitrocellulose or nylon, can for example with the body fluid to be examined, for. As blood, are brought into contact in vitro in order to be able to react with autoimmune antibodies, for example.
  • the antibody-peptide complex can then be detected, for example, using labeled anti-human IgG or anti-human IgM antibodies.
  • the label is, for example, an enzyme, such as peroxidase, that catalyzes a color reaction. The presence and the amount of autoimmune antibodies present can thus be easily and quickly detected via the color reaction.
  • Another diagnostic agent for therapy monitoring contains the antibodies according to the invention themselves.
  • a tissue sample can be easily and quickly examined to determine whether the relevant polypeptide is present in an increased amount, thereby indicating that there is an hdm2 protein to get related diseases.
  • the antibodies according to the invention are labeled, for example, with an enzyme, as already described above. The specific antibody-peptide complex can thus be detected easily and just as quickly via an enzymatic color reaction.
  • the derived nucleic acid sequences can be used to synthesize oligonucleotides which are suitable as primers for a polymerase chain reaction.
  • Suitable fragments are, for example, DNA fragments with a length of approx. 10-100 nucleotides, preferably with a length of approx. 15 to 50 nucleotides, in particular with a length of 20-30 nucleotides, whose sequence from the polypeptides according to SEQ ID No. 1 to SEQ ID No. 8 of the sequence listing.
  • coding nucleic acid refers to a DNA sequence that codes for an isolatable bioactive polypeptide according to the invention or a precursor.
  • the polypeptide can be encoded by a full-length sequence or any part of the coding sequence, as long as the specific, for example enzymatic, activity is retained.
  • this also includes polypeptides which have a sequence homology, in particular a sequence identity, of approximately 70%, preferably approximately 80%, in particular approximately 90%, in particular approximately 95% of the polypeptide with the amino acid sequence according to one of the SEQ ID No. 1 to SEQ ID No. 8 and / or to DNA sequences derived from the peptide sequences.
  • sequence homology in particular a sequence identity
  • these also include additions, inversions, substitutions, deletions, insertions or chemical / physical modifications and / or exchanges or parts of the polypeptide in the range from about 1-60, preferably from about 1-30, in particular from about 1-15, especially from about 1-5 amino acids.
  • the first amino acid methionine may be absent without significantly changing the function of the polypeptide.
  • SEQ ID No. 1 polypeptide of the wild-type mouse mouse ⁇ TZR (muv ⁇ -muc ⁇ )
  • SEQ ID No. 2 polypeptide of the wild-type mouse mouse ßTZR (muvß-mucß)
  • SEQ. ID No. 20 Primer for_NcoI_aTCR_5b; phosphorylated at the 5 'end,
  • SEQ. ID No. 22 Primer for_NcoI_bTCR_5b; phosphorylated at the 5 'end,
  • SEQ. ID no. 39 polynucleotide of the wild-type mouse mouse ßTZR (muvß-mucß) from SEQ ID no.
  • SEQ. ID No. 40 Polynucleotide of the chimeric partially humanized TZR gene (muv ⁇ -huc ⁇ ) from SEQ ID No. 3
  • SEQ. ID No. 41 Polynucleotide of the chimeric partially humanized TZR gene (muvß-hucß) from SEQ ID No. 4
  • SEQ. ID No. 42 polynucleotide of the single-chain TZR muv ⁇ -Li-muvß-mucß from SEQ ID No.
  • SEQ. ID no. 43 polynucleotide of the single chain TCR muvß-Li-muv ⁇ -muc ⁇ from SEQ ID no.
  • SEQ. ID No. 44 polynucleotide of the double chain TZR in fusion with human CD3 ⁇ chain muv ⁇ -huc ⁇ -huCD3 ⁇ from SEQ ID No. 7; and SEQ. ID No. 45: polynucleotide of the double chain TZR in fusion with human CD3 ⁇ chain muvß-hucß-huCD3 ⁇ from SEQ ID No. 8.
  • FIG. 2 shows a v6-specific antibody blockade of the murine T cell clone 3 in the cytotoxic 51 chromium release test (see 1.4).
  • the blockade was evaluated depending on the dose.
  • An anti-V ⁇ 3.2 antibody (BD) and, on the other hand, the p53 - 264-272-specific T cell clone 46 which served as a negative control served as negative control recognizing p53 peptide was cross-checked.
  • the target cell was the TAP-deficient cell line T2, which was loaded exogenously with 10 "8 M peptide.
  • FIG. 5 shows the polylinker of the construct muv ⁇ -Li-muvß-mucß (see 2.3).
  • the polylinker was ultimately inserted into the resulting construct via Kpn I and Dra III.
  • Figure 6 shows the polylinker of the construct muvß-Li-muv ⁇ -muc ⁇ (see 2.3).
  • the polylinker was ultimately inserted into the resulting construct via Sca I and Bsg I.
  • Figure 7 shows the fusion site of the chimeric double chain TZR in fusion to the human CD3 ⁇ chain muv ⁇ -huc ⁇ -huCD3 ⁇ (see 2.4).
  • the extracytosolic cysteine which dimerizes via a disulfide bridge, is given with a position relative to the starting methionine.
  • Figure 8 shows the fusion site of the chimeric double chain TZR in fusion to the human CD3 ⁇ chain muvß-hucß-huCD3 ⁇ (see 2.4).
  • the extracytosolic cysteine which dimerizes via a disulfide bridge, is given with a position relative to the starting methionine.
  • FIG. 9 shows the "fluorescence activated cell sorting" (FACS) analysis of the constructs described under 2.) after retroviral transduction. Examples of average transduction efficiencies for OKT3-activated PBL cultures are shown 6 days after transduction. on. These are in the range of 10-25% of the inserted CD3 + lymphocytes. A transfection / transduction approach with the empty retroviral vector pBullet served as a negative control.
  • FACS fluorescence activated cell sorting
  • FIG. 10 to 12 show peptide titrations (see 3.4.1) wherein T2 target cells were loaded exogenously with the hdm2-81-88 peptide in a concentration series of 1 * 10 " M to 1 * 10 " M.
  • the murine T cell clone 3 hdm2-81-88 (synonym: P2) of the huCD8 x A2K b - transgenic mouse served as a reference.
  • the 51 chromium release tests shown were carried out with transduced PBLs, which were enriched for vß6-directed magnetic cell sorting (Miltenyi-Biotec) to over 90% vß6 + T cells.
  • nfTCR non-functional single chain T cell receptor
  • FIGS. 13 to 15 show the detection of malignantly transformed Zeil lines.
  • the target cells chosen were HLA-A2 + .
  • the murine T cell clone 3 hdm2- 81-88 (synonym: P2) of the huCD8 x A2K b - transgenic mouse served as reference.
  • the 51 chromium release tests shown were carried out with transduced PBLs which were enriched for vß6-directed magnetic cell sorting (Miltenyi-Biotec) to over 90% vß6 + T cells.
  • nfTCR non-functional single chain T cell receptor
  • the mildly solubilized and aliquoted cells were centrifuged at 300 g / 2 '(minutes) / 4 ° C and the supernatant was removed. 600 ⁇ l of a ⁇ -mercaptoethanol (MSH) -containing (1.45 mM) “RLT” buffer was added, mixed well and 430 ⁇ l of 96% ETOH were added with mixing. The solution per aliquot was placed on a silica minispin column and centrifuged for 15 "(seconds) at 10,000 rpm (revolutions per minute) in an Eppendorf table joint.
  • MSH ⁇ -mercaptoethanol
  • the TZR-encoding RNA had to be specifically rewritten and amplified in DNA before it could be cloned.
  • the RT-PCR was used methodically, which is implemented in the "Titan One Tube RT-PCR System” kit from Röche (1888 382).
  • the "one-step protocol” was chosen, in which the reverse transcription is followed by the polymerase chain reaction without further intervention.
  • the enzymes used were AMV - reverse transcriptase and a mixture of Thermus Aquaticus / Pwo - thermostable polymerase ("Expand TM High fidelity" enzyme mix), which were used in a universal reaction buffer.
  • An artificial gene segment that codes for a murine TCR signal peptide should originally be connected upstream of synthetic oligonucleotides to ensure that the protein synthesized in vivo is directed into the lumen of the endoplasmic reticulum.
  • a primer (rev_bTCR_c4; SeqlD No. 10) which reverses within the constant gene segment was chosen in order to account for the additional variance of the two possible c-gene segments (c-ßl or c-ß2) of the ß-chain, which have some base differences at their 3rd Have 'end to escape.
  • the oligonucleotides of the "nested PCR” also had sequences of the restriction sites for EcoRI or Ascl at its 5 'end.
  • the primer hybridizing in the forward direction (for_ ⁇ co_bTCR_v5; SeqlD No. 11) of the nested "PCR” was congruent in the sequence of the v gene segment with the degenerate RT-PCR primer, whereas the primer hybridizing in the reverse direction complementary to the c gene segment (rev_Asc_bTCR_c6; No. 12) was more than 120 bases offset inwards relative to the reverse 1st primer of the TZR reading frame, so that it is exactly a "3'- located nested PCR ".
  • the complete c gene segment was determined using a reverse degenerate primer (rev_Asc_bTCR_c2; SeqlD No. 14 ) to detect the two potentially possible cßl / cß2 gene segments and a primer (for_Eco_bTCR_c2; SeqlD No.
  • the pipetting scheme and the PCR protocol of the RT-PCR basically corresponded to the method described by the Röche company.
  • the subsequent "nested PCR” was carried out analogously, with the components RNase inhibitor and DTT being replaced by autoclaved water. Instead of the 2 ⁇ l R ⁇ A (0.1-1 ⁇ g) 2 ⁇ l of the amplificate of the RT-PCR was used.
  • the technical requirements of the PCR device made it possible to simultaneously test different hybridization temperatures in the second step of the classic PCR cycle in a temperature gradient from 35 ° C - 55 ° C at intervals for 5 selected temperatures of 36 ° C, 39 ° C, 44 ° C, 52 ° C and 56 ° C. For the higher temperatures of 44-56 ° C singular bands of the desired length could be achieved, the intensity of which was increased by the 2nd PCR and were sufficient to be inserted in vectors.
  • the sequencing of 8 bacterial clones resulted in some randomized base changes based on the error rate of the Expand TM enzyme mix, which was comparable to other polymerases.
  • the selected clones also all carried the sequence information for the signal peptide and a short region of the non-codogenic, 5'-untranslated region of less than 100 bases, so that it could be assumed that the v gene segment-specific degenerate primer was one recognized similar sequence upstream and provided the additional sequence of the wild-type signal peptide.
  • the RACE technology is established in the system of the same name from Röche (1 734 792) and must be supplemented with ⁇ TZR gene-specific oligonucleotides: for this purpose, the 5'-localized variance is part of the "5'-RACE-PCR" of the codogenic sequence by generating a known, short adenine oligonucleotide sequence at the 3 'end of the reverse transcribed cDNA sequence by means of the terminal transferase, which subsequently acts as a recognition sequence for a 5' localized PCR primer ,
  • the reverse PCR primers lie within the invariant sequence that codes for the constant domain.
  • the primer rev_Asc_aTCR_cl (SeqlD No.
  • the multi-step protocol is largely based on the commercial protocol with the aid of a programmable PCR device: the reverse transcription, the transferase reaction and the subsequent 2-step "nested PCR” were carried out in the "MasterCycler” PCR device from Eppendorf.
  • the gradient technology was used in the hybridization step for the temperatures 39 ° C, 44 ° C, 52 ° C and 56 ° C.
  • the R / w polymerase (Stratagene) was chosen because at this point it was considered to be the enzyme with the lowest error incorporation rate.
  • the 1st PCR already showed a weak band of the expected size of 0.9 kB in the Entire temperature spectrum, the intensities of which could be amplified by the subsequent PCR and, in addition, "primer dimers" which occurred could be thinned out.
  • the amplificate was shortened by 70 bp according to the theoretical position of the offset hybridizing primers.
  • a double band of 100 bp in length difference appeared in the agarose gel, which indicated the presence of the functional and the non-functional ⁇ chain.
  • the bands were cut out together in the preparative gel in order to record both amplificates in the subsequent cloning. 7 clones were found which encoded a functional reading frame and had identical sequences.
  • An influenza matrix protein (FluMl 58-66) specific T cell clone served as a negative control.
  • FACS analysis Figure 1 using a Vß6-specific FITC-labeled antibody (Pharmingen). Both the murine T cell line and the resulting clone 3 were checked.
  • FITC-labeled antibody was used as a positive control, which recognizes the ß-chain portion independently of the subfamily.
  • the retroviral vector pBullet a functional derivative of pStitch (Weijtens et al., 1998) belongs to the so-called splice vectors, in which the transgene clones in the same sequence context relative to the splice acceptor and donor elements SA and SD must be like the env gene to ensure maximum expression.
  • an Ncol interface within the start codon "ccATGg" is designed using appropriate primers. This changed the 2nd amino acid of Lys for both wild-type chains ⁇ TZR and ßTZR Glu or Asn according to Asp.
  • the singular BamRI interface was selected at the 3 'end of the reading frame.
  • the restriction enzymes and modifying enzymes were obtained from New England Biolabs (NEB).
  • thermostable polymerase was mostly the native or cloned R / w DNA polymerase (Pyrococcus furiosus, Stratagene) or the R / 5c DNA polymerase (Pyrococcus sp., Life Technologies)
  • the gene segments could be amplified and cloned directly via primers which contained the sequences for Ncol (for_ ⁇ coI_aTCR_4, SeqlD No. 19; for_NcoI_bTCR_4, SeqlD No. 21) ,
  • the murine constant domains should be exchanged for the corresponding human domains in such a way that these domains are completely recorded in their immunoglobulin-like folding structure.
  • the complete variable domains were also to be recorded in order not to cause any impairment of the CDR3 loop which was almost carboxyterminal in this domain.
  • the fusion had to take place in such a way that the transition generated as little as possible an anteoantigen that could lead to an unwanted immune reaction.
  • the positions are relative in a range of up to 8 amino acids behind the last consensus motif "GxG", which closes the CDR3 loop heavily preserved. This area is completed by a highly conserved sequence of up to 11 amino acids.
  • the murine sequences then begin to deviate from the human sequences, highly conserved for each species.
  • the central area lies in the extended loop that connects the variable domain with the constant one (see criterion a)).
  • the chimerization should be carried out within a highly conserved amino acid (AS) triplet sequence.
  • the highly conserved triplet sequence consisted of "QNP" (base 400-408 relative to the start codon of the murine ⁇ TZR). Position 407-415 was followed by a singular / wNI interface that could be used for the fusion: the 3'-offset position of the AlwNl meant that instead of the aspartate conserved in human sequences, the glutamate conserved in murine sequences was present. This conservative amino acid exchange was considered to be less immunogenic and therefore tolerable.
  • the chimeric, highly conserved region was I 133 -QNPEPAVYQLR 144 in which R 144 represents the first human-specific amino acid. I is at the ninth position behind the consensus motif "GxG" that closes the CDR3 loop.
  • the cloning strategy was to ligate the LMP-agarose-isolated EcoRI / / wNI-cut v ⁇ segments with the AlwNl / BamHl-digested c ⁇ -PCR amplicons, to recut the mixed ligation products with EcoRI and BamRl and to prepare the prepared ligation product to clone potentially correct fragment length into the expression vector pcDNA3.1 (-) (Invitrogen).
  • An asymmetric test digest resulted in the clones with the desired sequence of the ligation starting materials.
  • the human constant gene segments c ⁇ and cß were isolated from the human leukemia cell line Jurkat by classic RT-PCR, as implemented as part of the Roche kit described above, with subsequent "nested” PCR.
  • the .4 / wNI interface (T to A; see table below) had to be generated with the aid of mutagenic PCR primers at 2 positions secondly, compatibility with the murine sequence is established (c according to a; see table below), since the middle 3 positions of the / wNI interface may be degenerate:
  • the -4 / wNI cleavage site that occurs naturally in the murine sequence is shown in bold, the 3 ′ offset cutting position is underlined.
  • the degenerate triplet is in lower case.
  • the base exchanges required for the human “primer” to maintain a mouse sequence-compatible / wNI interface are shown in bold.
  • the 5 ′ initial base of the mutagenic primer is in italics.
  • the resulting chimeric AS sequence is shown in bold (see text); the highly preserved AS triplet is also underlined.
  • vß fragment was cut with EcoRI and isolated from an LMP gel, the cß-PCR amplificate was blunt-ended as an additionally enzymatically phosphorylated (T4 polynucleotide kinase, N ⁇ B) final BamHl fragment used in the ligation.
  • the mixed ligation products were cut with EcoRI / BamHl and the fragment with the potentially correct fragment length was cloned in pcDNA3.1 (-).
  • An asymmetric test digest gave the clones with the desired sequence and orientation of the ligation starting materials.
  • the est YI interface that occurs naturally in the murine sequence is shown in bold, the 5 'offset cutting position which generates 5' overhangs is underlined.
  • the 5 'initial and phosphorylated base "T" of the human "nested" primer, which supplements the last base of BstYl in the chimeric construct, is in italics.
  • the resulting chimeric AS sequence is shown in bold (see text); the highly preserved AS triplet is also underlined.
  • Single chain TCRs consist of the fusion of the variable domains v ⁇ with vß and an added constant domain c ⁇ or cß (Eshar et al., 1993).
  • the advantage of such constructs lies in the defined 1: 1 stoichiometry of the two variable domains in one construct and the defined reactivity due to the heterogeneous pairing of endogenous and exogenous double chain TZRs that can be excluded.
  • the variable domains were connected via a flexible polylinker of a redundant (GGGGS) 3 motif.
  • the ends of the synthetic oligonucleotide harbored unique interfaces with which the carboxy-terminal end of one variable domain, located behind the essential CDR3 loop, could be linked to the beginning of the other variable domain, behind the sequence encoding the signal peptide.
  • five criteria were considered: a) The polylinker had to be long enough to connect the ends of the variable ends. For this purpose, murine TZR crystal structures with an MHC-H-2K b specificity were consulted. b) the polylinker had to connect the ends in such a way that on the one hand it did not impair the specificity of the CDR3 loop, on the other hand it sufficiently eliminated the signal peptide of the downstream variable domain while maintaining its functional structure.
  • the chimeric intermediate was cut open with DrllllBamHl and the ßTZR coding sequence, which had lost its signal peptide coding sequence via a DrallllBamHl digest, was cloned into it.
  • the first glycine of the polylinker was at position 6 relative to the amino-terminal gly of the "GxG" consensus motif in v ⁇ , the last serine of the polylinker, connected by arginine due to an intermediate blunt-ended ligation, before position Asp 22 of the vß domain.
  • muvß-Li-muv ⁇ -muc ⁇ (SeqlD No. 43; FIG.
  • a modified ligation-PCR-based technique was established, with the help of which one could generate any fusions for the production of chimeric proteins, regardless of singular interfaces, and the specificity of the PCR amplificates obtained was significantly increased in comparison to a conventional ligation-PCR.
  • the genetic fusion is defined via a reverse, chimeric primer (rev_huca-hu_tm_Zeta, SeqlD No. 36, FIG. 7; rev_hucb-hu_tm_Zeta; SeqlD No. 37, FIG. 8), which is linked to a forward primer (T7_for; SeqlD No.
  • a functional derivative of the pStitch system (Weijtens et al., 1999) was used for transduction of human peripheral T lymphocytes.
  • the retroviral genes required for packaging are encoded via individual plasmids by cotransfection of the packaging cell line 293T (Soneoka et al., 1995): pHit60 encodes the gag-pol structure and polymerase genes from the Moloney murine leukemia virus ( MoMuLV), pColt-Galv for the env - envelope protein of the "gibbon ape leukemia virus", which is able to bind to the Na + - / phosphate synporter pit of human cells and thus to transduce the latter.
  • the chimeric virus particles thus have an amphotropic pseudotype and can transduce various mammalian cells, except for the mouse.
  • the isolated bacterial clones of the T cell receptor genes cloned into the pStitch derivative were purified via plasmid preparations which promise removal of residual endotoxins (Qiagen, product 12362) and adjusted to 1 ⁇ g / ⁇ l.
  • the DNA was transiently introduced into the packaging cell line 293 T via the calcium phosphate precipitation (GiboBRL-Life Technologies, product 18306-019). Up to 80 ⁇ g DNA are used in the context of the WT or modified double chain T cell receptors ⁇ TZR and ßTZR: 20 ⁇ g ⁇ TZR construct 20 ⁇ g ßTZR - construct 20 ⁇ g pColt-Galv 20 ⁇ g pHit 60
  • DMEM / H modified DMEM medium

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des polypeptides d'un récepteur de lymphocytes T α/β des muridés induisant une réponse spécifique de la protéine hdm2 ou de variantes fonctionnelles ou de leurs parties ou des acides nucléiques les codant, des variantes fonctionnelles ou leurs parties. Ils ont pour effet de permettre d'identifier des cellules de lymphocytes T exprimant la protéine hdm2, qui ont été pourvues de ces gènes, d'exprimer de la cytokine et d'obtenir une lyse induite par lymphocyte T et/ou l'apoptose de cellules tumorales ou leucémiques.
EP02722160A 2001-03-01 2002-02-28 Polypeptides d'un recepteur de lymphocytes t alpha/beta des murides specifique de la proteine hdm2, acides nucleiques les codant et leur utilisation Withdrawn EP1363942A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10109854A DE10109854A1 (de) 2001-03-01 2001-03-01 Polypeptide eines hdm2-Protein spezifischen murinen alpha/beta T-Zell Rezeptors, diese kodierende Nukleinsäuren und deren Verwendung
DE10109854 2001-03-01
PCT/EP2002/002187 WO2002070552A2 (fr) 2001-03-01 2002-02-28 POLYPEPTIDES D'UN RECEPTEUR DE LYMPHOCYTES T Α/β DES MURIDES SPECIFIQUE DE LA PROTEINE HDM2, ACIDES NUCLEIQUES LES CODANT ET LEUR UTILISATION

Publications (1)

Publication Number Publication Date
EP1363942A2 true EP1363942A2 (fr) 2003-11-26

Family

ID=7675927

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02722160A Withdrawn EP1363942A2 (fr) 2001-03-01 2002-02-28 Polypeptides d'un recepteur de lymphocytes t alpha/beta des murides specifique de la proteine hdm2, acides nucleiques les codant et leur utilisation

Country Status (5)

Country Link
EP (1) EP1363942A2 (fr)
JP (1) JP2004535778A (fr)
CA (1) CA2445013A1 (fr)
DE (1) DE10109854A1 (fr)
WO (1) WO2002070552A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE386306T1 (de) 2002-09-11 2008-03-15 Nxp Bv Verfahren zum lesen einer vielzahl von nicht- kontakt-datenträgern, einschliesslich eines antikollisionsschemas
DE10244457A1 (de) * 2002-09-24 2004-04-01 Johannes-Gutenberg-Universität Mainz Verfahren zur rationalen Mutagenese von alpha/beta T-Zell Rezeptoren und entsprechend mutierte MDM2-Protein spezifische alpha/beta T-Zell Rezeptoren
DE10259713A1 (de) * 2002-12-19 2004-07-08 Johannes-Gutenberg-Universität Mainz Verfahren zur Expressionsstabilisierung und Verbesserung der spezifischen Effektorfunktion von Einzelketten-Antigenerkennenden genetischen Konstrukten (scARC) und entsprechend mutierten MDM2-Protein spezifischen scT-Zell Rezeptoren
GB0427585D0 (en) * 2004-12-16 2005-01-19 Avidex Ltd Assay
US20110070191A1 (en) * 2008-03-19 2011-03-24 Wong Hing C T cell receptor fusions and conjugates and methods of use there of
DK2618835T3 (en) 2010-09-20 2017-08-28 Biontech Cell & Gene Therapies Gmbh ANTIGEN-SPECIFIC T-CELL RECEPTORS AND T-CELL EPITOPES
ES2861901T3 (es) * 2014-10-03 2021-10-06 Univ Oxford Innovation Ltd Análisis de monotipia de células T
EA034306B1 (ru) * 2014-10-20 2020-01-27 Зе Скриппс Рисёрч Инститьют Основанные на близости расположения способы выбора партнеров для связывания
WO2016070061A1 (fr) * 2014-10-31 2016-05-06 The Trustees Of The University Of Pennsylvania Procédés et compositions permettant l'obtention de lymphocytes t modifiés

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0910409A4 (fr) * 1996-03-05 2003-03-19 Scripps Research Inst Elements recombinants codant pour des recepteurs de lymphocytes t specifiques des antigenes de tumeurs limites par hla humains

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02070552A2 *

Also Published As

Publication number Publication date
CA2445013A1 (fr) 2002-09-12
WO2002070552A2 (fr) 2002-09-12
JP2004535778A (ja) 2004-12-02
WO2002070552A8 (fr) 2002-10-10
DE10109854A1 (de) 2002-09-12
WO2002070552A3 (fr) 2002-12-12

Similar Documents

Publication Publication Date Title
US11339199B2 (en) Chimeric NK receptor and methods for treating cancer
US11325962B2 (en) Therapeutic and diagnostic cloned MHC-unrestricted receptor specific for the MUC1 tumor associated antigen
DE102006041455B4 (de) Verfahren zur Herstellung einer einen stabilisierten funktionellen humanen Einzelketten-Antigen-erkennenden-TCR (scTCR) exprimierenden Zelllinie, damit hergestellt Zelllinie, stabilisierter TAA-spezifischer scTCR, deren Verwendungen und diese enthaltende pharmazeutische Zusammmensetzungen
EP1543032B1 (fr) Procede de mutagenese rationnelle de recepteurs de lymphocytes t a/beta et recepteurs de lymphocytes t a/beta specifiques de la proteine mdm2 ayant subi ladite mutagenese rationnelle
WO2017076308A1 (fr) Tcr pour identifier un oligopeptide d'antigènes ny-eso-1
US11472860B2 (en) Chimeric antigen receptors
KR20210141573A (ko) 항-bcma 키메라 항원 수용체
CN113166228A (zh) 用于治疗b细胞恶性肿瘤的cd22特异性t细胞受体和过继性t细胞疗法
JP2008541695A (ja) 炎症障害の阻害に有用な可溶性ｂｔｎｌ２タンパク質
EP1363942A2 (fr) Polypeptides d'un recepteur de lymphocytes t alpha/beta des murides specifique de la proteine hdm2, acides nucleiques les codant et leur utilisation
DE10259713A1 (de) Verfahren zur Expressionsstabilisierung und Verbesserung der spezifischen Effektorfunktion von Einzelketten-Antigenerkennenden genetischen Konstrukten (scARC) und entsprechend mutierten MDM2-Protein spezifischen scT-Zell Rezeptoren
WO1997016557A1 (fr) TRAITEMENT DE TUMEURS PAR TRANSFERT ADOPTIF DE LYMPHOCYTES T CYTOTOXIQUES SPECIFIQUES DU CD44v
US6699972B1 (en) Chimeric protein and method of controlling tumor growth using the protein
WO2002070556A9 (fr) POLYPEPTIDES D'UN RECEPTEUR DE LYMPHOCYTE T Α/β MURIN SPECIFIQUE A LA PROTEINE P53, LES ACIDES NUCLEIQUES CODANTS CORRESPONDANTS ET LEUR UTILISATION
CN108929378B (zh) 一种识别prame抗原的t细胞受体及编码该受体的核酸
KR20210150993A (ko) Pa63 도메인 4 변이체를 세포외 결합 도메인으로 포함하는 키메라 항원 수용체 및 이의 용도
CN116103239A (zh) 工程化免疫细胞及其用途

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20030926

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20051125