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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• MSI + microsatellite-unstable tumors
• the present invention relates to genes relevant for MSI + tumors and their gene products.
• the invention further relates to a method for identifying such genes and the use of the genes or their gene products for the prevention, diagnosis and / or therapy of MSI + tumors.
• mutations Tumor cells accumulate instabilities (mutations) in genes that are essential for the maintenance of normal growth and normal differentiation.
• CIN chromosomal instability
• MSI microsatellite instability
• the type and spectrum of mutated genes differs greatly between CIN and MSI + tumors, which indicates different, but not mutually exclusive, ways of developing cancer.
• MSI occurs in about 90% of hereditary non-polypous colorectal tumors (HNPCC) and in about 15% of sporadic tumors of the colon and other organs and is caused by a mutation-related inactivation of different DNA mismatch repair genes.
• MSI + tumors have special histopathological features. MSI + tumors are also classified, for which microsatellites in non-coding areas or intron sequences are generally used. However, there are indications that microsatellites in coding gene regions are also subject to instability. This could be of great importance for the tumorigenesis of MSI + -Tu o- have.
• the present invention is therefore based on the technical problem of providing a means with which MSI + tumors can be investigated at the molecular level and which may be suitable for the diagnosis and / or therapy of MSI + tumors.
• the present invention is based on the knowledge of the applicant that genes encoding MSI + tumors containing mononucleotide microsatellites (cMNR) often have instabilities (mutations) in their cMNR. For this purpose, using a computer algorithm-supported database analysis, he identified approx. 17000 coding mononucleotide microsatellites (cMNR) and approx.
• the present invention thus relates to genes with coding mononucleotide microsatellites (cMNR) or dinucleotide microsatellites (cDNR), wherein the genes can be isolated from MSI + tumor cells and differ from the corresponding genes from non-MSI + (tumor) cells by mutations in the cells Differentiate between cMNR and cDNR and encode comprehensive gene products for neo-peptides.
• cMNR mononucleotide microsatellites
• cDNR dinucleotide microsatellites
• coding mononucleotide microsatellites encompasses repeat units of at least three identical mononucleotides A, T, G or C (n 3 3), the repeat units being present in coding gene regions.
• coding dinucleotide microsatellites includes repeat units of at least three identical dinucleotides (AC, AG, AT, CA, CG, CT, GA, GC, GT, TA, TC, TG, n> 3), preferably at least six (n ⁇ 6) and very particularly preferably at least nine (n ⁇ 9), the repeat units being in coding gene regions.
• genes with mutated cMNR or cDNR which can be isolated from MSI + tumor cells includes such genes in full length, as well as the mutations and the parts thereof containing the sequences encoding the neo-peptides.
• MSI + tumor cells encompasses any tumor cells that have microsatellite instability. Such tumor cells can be in any form, e.g. in a cluster of cells, in particular in a tumor, or kept as such in culture.
• Preferred MSI + tumor cells include the cell lines LoVo, KM12, HCT116, LS174 and SW48.
• non-MSI + (tumor) cells includes any cells that do not have microsatellite instability. Such cells can be of any type and lineage, for example the cells can come from healthy individuals or from tumors, or Tumor cell lines.
• mutants and “gene products comprising neo-peptides” indicate that mutations in the coding microsatellites (cMNR or cDNR) of genes isolatable from MSI + tumor cells compared to the cMNR or cDNR of the corresponding genes from non-MSI + ( Tumor) cells are present, the mutations being such that the genes encode gene products comprising neo-peptides.
• the mutations are insertions and / or deletions of one or more mono- or dinucleotides.
• the mutations lead to reading frame shifts such that the gene products in the form of neo-peptides, i.e. newly generated peptides, comprehensive gene products are available.
• the genes according to the invention are those which differ from the genes from non-MSI + (tumor) cells indicated in FIG. 1 by mutations in the cMNR or cDNR and which encode gene products comprising neo-peptides.
• the genes according to the invention very particularly have the mutations indicated in FIG. 2 in the cMNR or cDNR and code for the gene products comprising the specified neopeptides.
• Genes according to the invention can be identified and provided by various methods.
• a cheap method is one in which one searches in databases of non-MSI + (tumor) cells for coding mononucleotide microsatellites (cMNR) or dinucleotide microsatellites (cDNR) containing gene sequences, uses these to find the same genes in MSI + tumor cells and the latter genes selected in such a way that they have mutations in the cMNR or cDNR compared to the gene sequences from the non-MSI + (tumor) cells and encode gene products comprising neo-peptides.
• cMNR mononucleotide microsatellites
• cDNR dinucleotide microsatellites
• DNA is subjected to a PCR reaction with primers which are derived from the cMNR or gene sequences comprising cDNR are developed.
• the primers preferably comprise the sequences given in Table 1.
• Tumor cells use the appropriate gene sequences from the database to create suitable primers and use them to amplify the genes in the MSI + tumor cells.
• amplified genes can then be cloned and their expressions carried out by customary methods.
• vectors for amplified genes can then be cloned and their expressions carried out by customary methods.
• Expression in E. coli are e.g. To name pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8. Furthermore, for expression in animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 as well as for expression in insect cells e.g. the bacculovirus
• Expression vector pAcSGHisNT-A The person skilled in the art knows suitable cells in order to express the genes present in the expression vectors. Examples of such cells include the E. coli strains HB101, DH 1, xl776, JM101, JM109,
• yeast strain Saccharomyces cerevisiae and the animal cells L, NIH 3T3, FM3A, CHO, C05, VERO and HeLa and the insect cells sfg.
• the expert also knows
• the present invention further relates to gene products which are encoded by the above genes.
• Ins ⁇ special is such gene products that are characterized by mutations in the proteins encoded by the CMNR or CDN r areas differ from those gene products of the genes shown in Fig. 1 and include neo-peptides.
• the gene products very particularly comprise the mutations caused by the cMNR or cDNR indicated in FIG. 2 and have the specified neopeptides.
• Conventional methods can be used to provide the above gene products. Reference is made to the above explanations. It can also be advantageous to provide the neopeptides as such, in particular by means of peptide synthesis.
• the present invention furthermore relates to antibodies which are directed against the above gene products.
• These antibodies are preferably monoclonal, polyclonal or synthetic antibodies or fragments thereof.
• fragment means all parts of the monoclonal antibody (e.g. Fab, Fv or "single chain Fv” fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art.
• the antibodies according to the invention are preferably monoclonal antibodies.
• the antibodies according to the invention can be produced according to standard methods, the above gene products preferably serving as an immunogen. Methods for obtaining monoclonal antibodies are known to the person skilled in the art.
• kits which are suitable for the investigation at the molecular level of MSI + tumors and for their diagnosis.
• the kits are also suitable for identifying genes relevant for MSI + tumors.
• Such kits comprise one or more members of an OF INVENTION ⁇ to the invention the gene, gene product, antibody and / or primer ⁇ pair. It will refer to the above Genes, gene products and antibodies according to the invention are referenced.
• kits can also contain other substances, such as reverse
• Transcriptase DNA polymerase, ligase, buffer and reagents, e.g. Tags, dNTPs, included.
• reagents e.g. Tags, dNTPs.
• Primer pairs must be marked. They can also be free or attached to a solid support, e.g. a test tube, one
• Microtiter plate, a test stick, etc. can be immobilized.
• kits can also contain suitable reagents for the detection of
• the present invention further provides methods for immunizing an individual against MSI + tumors and their precursors, in which the individual is administered an above gene in expressible form or a gene product encoded by it. Reference is made to the above statements regarding genes and gene products according to the invention.
• RNA or DNA preferably as DNA.
• elements suitable for its expression or in conjunction with a vector.
• elements suitable for its expression or in conjunction with a vector.
• elements suitable for its expression or in conjunction with a vector.
• elements suitable for its expression or in conjunction with a vector.
• promoters and enhancers such as CMV, SV40, RSV, metallothionein I and polyhedrin promoters or CMV and SV40 enhancers.
• Further sequences suitable for expression can be found in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
• any vectors suitable for expression in mammalian cells can be used as vectors.
• pcDNA3, pMSX, pKCR, pEFBOS, cDM8 and pCEV4 as well as from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSGy, pSVTo, and pSVT7 vectors.
• Recombinant viruses for example adenovirus, vaccinia virus or adeno-associated, can also be used as vectors Virus.
• this can be present as such or in conjunction with carriers. It is favorable if the carriers in the individual do not act as immunogenic. Such carriers can be individual's own or foreign proteins or fragments thereof. Carriers such as serum albumin, fibrinogen or transferrin or a fragment thereof are preferred.
• an individual can be immunized who may or may already be suffering from an MSI + tumor. Examples of such an individual are humans and animals as well as cells from them.
• the immunization can be carried out under customary conditions, the amount of the gene to be administered or of the gene product encoded by this being easy to determine. It depends, among other things, on whether the immunization of the individual aims more at induction of antibodies directed against the gene product or at stimulation of cytotoxic T cells directed against the gene product, eg CD8 + T cells. Both possibilities of immunization can be achieved by the present invention. Furthermore, the amount depends on whether the immunization is intended as a prophylactic or therapeutic treatment.
• the injection can take place intramuscularly, subcutaneously, intradermally or in any other form of application in several places of the individual. It may also be advantageous to use one or more "booster Injections "with approximately the same amount.
• the present invention thus makes it possible to diagnose MSI + tumors diagnostically. These tumors can also be treated prophylactically and therapeutically.
• 14 human colon cancer cell lines were examined for microsatellite changes in each gene above.
• Five of the 14 colon cancer cell lines are classified as MSI + (LoVo, KM12L4, HCT116, LS174T and SW48), while nine cell lines are classified as MSI-low or MSI-negative (CXF94, SW948, LS180, SW707, CaCo-2, HT29, Colo320DM, SW480 and CX-2).
• the cell lines SW48 and HCT 116 were obtained from the ECACC [http://www.camr.org.uk/frame.htm].
• LoVo, LS174T, and LS180 were obtained through the tumor bank of the German Cancer Research Center. KM12L4 cells were developed by Dr. IJ Fidler, MD Anderson Cancer Center, Houston, USA. In addition, 10 MSI + CRC tumors were analyzed, one MSI + ovarian tumor (B190 TU) and two MSI-low and MSI-negative CRC tumors (B215 TU and B245 TU2). The paraffin-embedded tumors were taken from the archived material of the Surgical University Clinic in Heidelberg or made available by the Institute for Pathology in Mannheim. Genomic DNA of the tumor and the corresponding mucosa samples obtained by microdissection using standard methods were provided by Ch. Sutter (Sutter et al., Mol. Cell Probes. 13 (1999), 157-165). The MSI status was determined using the “NCI ICG-HNPCC ⁇ N - microsatellite marker panel (Boland et al., Cancer Res. 58_
• sequence homologues to other human sequences by a "FASTA U analysis [HUSAR program package]. checked. The primer positions were selected so that they were as close as possible to the repeat region in order to obtain a short amplimer with a length of approximately 100 bp. This was optimal for accurate fragment analysis of the DNA obtained from tissue samples embedded in paraffin. This also turned out to be necessary for the analysis of candidates with unknown genomic structure. All primers used are listed in Table 1 below.
• PCR reactions were carried out in a total volume of 25 ⁇ l (50 ng genomic DNA, 2.5 ⁇ l 10 ⁇ reaction buffer (Life Technologies, Eggenstein-Leopoldshafen, Germany), 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 0.25 ⁇ M from each primer and 0.5 U Taq DNA polymerase (Life Technologies)).
• a primer was fluorescein labeled at the 5 'end.
• PCR products were 'analyzed on a 2% agarose gel prior to fragment analysis the amplification products were diluted 1:. 2 diluted to 1:10 and 1 ul of the diluted product were mixed with 5 ul loading buffer (0.6% "blue dextran” , 100% formamide). The samples were 3 min.
• Microsatellite peaks were analyzed using the “AlleleLinks ⁇ software (Amersham Pharmacia Biotech).
• PCR reactions were carried out as described above. PCR products were purified using the “QIAquick” PCR cleaning kit (Qiagen, Hilden, Germany) and with the appropriate primers using the "Big Dye terminator cycle sequencing kit (Perkin Eimer, Darmstadt, Germany) sequenced.
• RNA from 14 colon cancer cell lines was extracted using the oligo (dT) cellulose method (Vennstrom and Bishop, Cell 2_8 (1982), 135-143). The quality of the RNA preparation and the reverse transcription was checked by means of GAPDH amplification (Hsu et al., Int. J. Cancer, 55: 397-401, 1993). Primer pairs which allowed size differentiation between cDNA and possibly genomic DNA amplimers contained as contamination were considered suitable for expression analysis by semi-quantitative RT-PCR. In the case of an unknown exon structure, primers were designed that were located on the cDNA and it was checked whether genomic PCR gave either the same amplification product as the RT-PCR, a longer one or none at all.
• RNA 100 ng of poly (A +) RNA were analyzed by 0.5 ug oligo (dT) ⁇ 2 - ⁇ in a final volume of 20 ul with 200 units of M-MLV reverse transcriptase (Superscript, Life Technologies) for 1 hour at 37 ° C "reverse transcribed To check the RNA integrity and the synthesis of the first strand of cDNA, control PCR reactions were carried out using GAPDH-specific primers (Hsu et al., Int. J. Cancer 55 (1993), 397-401).
• Reactions were carried out in a total volume of 50 ⁇ l (1 ⁇ l cDNA, 5 ⁇ l 10 ⁇ reaction buffer (Life Technologies), 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 0.25 ⁇ M from each primer and 0.5 units Taq DNA polymerase (Life Technologies) using the above for amplification genomic DNA described amplification protocol performed.
• the PCR products were separated on 2% agarose gels and visualized by means of ethidium bromide staining.
• PCR reactions for cDNA-MSI analyzes were carried out as described for the expression analyzes, except that a primer labeled with fluorescein at the 5 'end was used.
• the fragment analysis was carried out as described for the genomic analysis.
• MSI + cell lines and MSI + tumors were found for the two cMNR markers (HPDMPK (T14) and U79260 (T14)) high mutation rates detected: All 5 MSI + cell lines and 10 of 11 MSI + tumors showed a sequence change with regard to HPDMPK. Analogous results were found for the mono-cMNR in the U79260 gene, which is found in all 5 MSI + Cell lines and 9 out of 11 MSI + tumors was mutated (see Table 2 below).
• Table 2 Frequency of mutations in cMNR in MSI + tumor cell lines and MSI + tumors.
• the expression levels of the above 15 cMNR-containing genes differed greatly and fluctuated between undetectable expression and - ⁇ constant strong transcription activity in all 14 colon cancer cell lines tested.
• the SYCPI gene involved in meiosis and the gene coding for the hematopoietic growth factor FLT3LG were not expressed in colon cancer cell lines.
• the HPDMPK gene which is located downstream of the gene locus for two genes associated with myotonic dystrophy (Dystrophia myotonica) and codes for a hypothetical protein, and the gene coding for the ER membrane protein SEC63 were found in all cell lines not very strong, but constantly expressed.
• the aCl mRNA and the splice variant 3 of the PTHrP gene were expressed to different extents in colon cancer cell lines. Both genes were expressed in approximately 50% of the cell lines examined.
• the trifunctional ribonucleotide synthetase-encoding gene GART, the DNA-dependent protein kinase-encoding gene PRKDC and the cell cycle-related gene ATR were highly expressed in colon cell lines.
• MAC30X is also highly expressed in colon cancer cell lines (see FIG. 3). In summary, however, it can be stated that the expression levels of the genes in question do not correlate with the MSI status of the cell lines concerned.
• Example 8 Stimulation of CD8 + T cells against an above gene product and yse of MSI + tumor cells expressing this gene product.
• Peripheral blood lymphocytes were purified from an HLA-A0201 positive healthy subject by density centrifugation using a Ficoll Paque® gradient. T-lymphocytes were obtained by separating the B-lymphocytes or the monocytes using antibody-coupled magnetobeads (CDU, CD16, CD19, CD36 and CD56) (Pan T-cell isolation Kit®, Milteny, Bergisch Gladbach, Germany). 30 ml of blood became about 2 x 10 7 T cells obtained. Of these, about 2 ⁇ 10 6 T cells with autologous CD40-activated B cells (about 5 ⁇ 10 5 ) that were restricted with one of the HLA-A0201 neo-peptides of Table 3 below (see also FIG. 2) had been loaded, stimulated, ie co-cultivated in 24 perforated plates. This stimulation was repeated weekly for five to six weeks.
• Table 3 Examples of HLA-A0201 restricted neo-peptides encoded by mutant cMNR.
• the reactivity to the neo-peptides was determined weekly, starting on day 0. On day 28 a reactivity of 1760 specific cells / 1,000,000 cells against peptide # 16 (SLYKFSPFPL), on day 35 of 1123 specific cells / 1,000,000 cells against peptide # 15 (FLSASHFLL) and of 733 specific cells / 1,000,000 cells against the Peptide # 21 (TLSPGWSAV) observed.
• the strength of the reaction was thus in ranges which can normally only be achieved with viral antigens, the value for the peptide GILGFVFTL, which was derived from a matrix protein of the influenza virus, was 1170 specific cells / 1,000,000 cells on day 35. It is thus clear that CD8 + T cells activated against the neo-peptides according to the invention can be stimulated.
• the cytotoxic The potential of the activated CD8 + T cells was tested against the HLA-A2.1 + colon carcinoma cell lines SW 480 and HCT 116, as well as T2 cells, which were loaded with the neopeptides. Unloaded cells served as controls. Each lxlO 6 cells were radioactively labeled with 51 Cr (100 ⁇ Ci) for 1 h at 37 ° C. and co-cultivated for 4 h with increasing amounts of activated CD8 + T cells. The specific lysis of the respective cell line was determined by measuring the released radioactivity in the supernatant. It was shown that the HLA-A0201 expressing cell lines can be lysed when they are loaded with neo-peptides, unloaded cells are not lysed.

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