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WO2002004508A1 - Antigene specifique de tumeur (b345) se caracterisant par une sequence d'acides amines telle que dans seq id no 4 - Google Patents

Antigene specifique de tumeur (b345) se caracterisant par une sequence d'acides amines telle que dans seq id no 4 Download PDF

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Publication number
WO2002004508A1
WO2002004508A1 PCT/EP2001/007705 EP0107705W WO0204508A1 WO 2002004508 A1 WO2002004508 A1 WO 2002004508A1 EP 0107705 W EP0107705 W EP 0107705W WO 0204508 A1 WO0204508 A1 WO 0204508A1
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Prior art keywords
seq
tumor
cdna
sequence
pcr
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German (de)
English (en)
Inventor
Norbert Schweifer
Marwa Scherl-Mostageer
Wolfgang Sommergruber
Roger Abseher
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Boehringer Ingelheim International GmbH
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Boehringer Ingelheim International GmbH
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Priority to EP01960465A priority Critical patent/EP1301533A1/fr
Publication of WO2002004508A1 publication Critical patent/WO2002004508A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the chemotherapy of tumor diseases.
  • Normal body cells are subject to a strictly ordered system that controls the growth, cell division and death of certain cells. So share
  • Cancer cells 10 body cells of an adult only if they have to replace dead cells or heal an injury. Cancer cells, on the other hand, continue to grow uncontrollably, they accumulate and form a tumor. If the tumor reaches a critical size, can
  • cancer cells can also be transported to other areas of the body via the bloodstream or the lymphatic system and form colonies there (metastases). Not all tumors are carcinogenic, because benign tumors do not metastasize and are therefore usually not life-threatening because they
  • Cancer cells can be triggered by a number of factors, such as environmental influences, radiation, viruses or chemical reagents.
  • epigenetic methylation, Acetylations and changed chromatin structure
  • genetic modifications point mutation, deletion, amplification, translocation
  • Mutations in coding regions of genes that are involved in the regulation of cell proliferation can contribute to the transfer of a normal cell into a tumor cell, since the transformed cell has growth advantages over its healthy neighboring cell.
  • Cancer therefore arises from an accumulation of inherited or acquired mutations in critical proto-oncogenes or tumor suppressor genes.
  • Cell proliferation is under the control of various gene systems, while products of oncogenes are involved in signaling from the cell surface to the nucleus, cyclin-dependent protein kinase and its inhibitors guide the cell through the cell cycle. Disturbances in the synthesis of these proteins are often found in tumor cells.
  • the p53 protein plays a central role in this.
  • RB protein-type proteins regulate the availability of crucial transcription factors.
  • transcription level the level of gene expression
  • Chemotherapy is the administration of substances that interfere with metabolism, signal transduction and
  • Chemotherapeutic agents can be classified based on the influence of specific targets in the tumor cell, the type of cellular interaction and the Interaction with a specific cell cycle phase, divided into different categories.
  • the type of cancer treatment depends on the tumor stage, the decisive factor here is whether metastases are already present and how widespread they are in the body.
  • the application of cell poisons for cancer treatment is, in addition to operative measures and radiation therapy, an integral part of today's therapy concepts in oncology.
  • Chemotherapy First and foremost, it is curing cancer; this means that the tumor disappears and no longer occurs. If a cure is no longer possible for various reasons, one tries to restrict or control the growth and spread of the tumor.
  • Each tissue has a characteristic growth behavior, which includes cell division, growth arrest, differentiation and aging and is influenced and regulated by internal and external factors.
  • Chemotherapeutic agents are divided into different categories depending on how they affect specific substances within the tumor cell, the cellular processes with which the drugs interact and the cell cycle phase they influence.
  • the genes upregulated in tumor tissues thus represent potential new target structures and since proteins of various functions are highly expressed, the approach for therapeutic interventions is very versatile.
  • Targets Genes upregulated in tumor tissues represent points of attack and thus potential target structures ("targets”) for chemotherapy.
  • the object of the present invention was to provide a new protein, preferably expressed by tumor cells, which is a target molecule for the Intervention using chemotherapy methods.
  • Normal lung tissue was created using a cDNA subtraction library.
  • the cDNA clones obtained were then sequenced and compared with sequences available in databases.
  • the genes that were annotated were 321 unknowns, most of which had ESTs (expressed sequence tags) in the database.
  • the number of candidate clones was restricted to 59, whose ESTs do not originate from critical normal tissues.
  • the length of the transcripts was determined by means of Northern blot analysis and the expression pattern in different cell systems was precisely characterized by quantitative PCR. Only unknown genes or ESTs with tumor-specific expression profiles were found followed up and subjected to a "full length cloning". Potential ORFs ("open reading frames") are converted into the corresponding amino acid sequence and analyzed for possible function prediction using in silico strategies.
  • the human B345 cDNA was cloned, the sequence obtained in a first cloning approach is shown in SEQ ID NO: 1. Sequence analysis of the human B345 CDNA cloned in this approach showed a continuous open reading frame from position 215 to position 2461 (excluding stop codon) which, at the nucleotide and protein levels, has no homology or identity in the known sequences of the databases. It can be concluded from the data obtained from Northern blot experiments that the B345 transcript has a length of approximately 6.5 kb.
  • a B345 CDNA with 5897 bp (exclusive polyA region) was obtained as the cloned region, the presence of a polyadenylation signal and the polyA tail at the 3 'end of the sequence indicating the completeness of the cDNA in this region , Due to the fact that there was no continuous reading frame in the 5 'region of the cloned cDNA from position 1 to 214, it was initially assumed that the ATG at position 215, which is also 75% a Kozak translation initiation site (ACCATGT) (Kozak , 1987) corresponds to the start codon of B345.
  • ACCATGT Kozak translation initiation site
  • primer extension analysis can be located precisely and is at position 201 (SEQ ID NO: 3).
  • SEQ ID NO: 3 Through repeated sequencing also in the 3 ⁇ region, in comparison to the sequence shown in SEQ ID NO: 1, an additional base at position 2430 was found, which leads to a reading frame shift and thus shifts the stop codon from position 2729 to 2791.
  • the cDNA obtained (SEQ ID NO: 3) has an open reading frame which codes for a potential protein with a length of 836 amino acids (SEQ ID NO: 4).
  • the translation initiation site at position 283 corresponds approximately to 70% of a Kozak consensus sequence.
  • the promoter region 200bp upstream of the putative transcription start site contains neither a TATA nor a CCAAT box, but a unique GC box, which represents a binding site of the SP1 protein.
  • the fact that the GC content in the 5 "region is over 60% indicates a CpG Island (Bird, 1986).
  • the resulting primary amino acid sequence of B345 is shown in SEQ ID NO: 4.
  • Analysis of the hydrophilicity plot of the amino acid sequence shows that the B345 protein has two characteristic hydrophobic domains which represent a signal peptide and a helical transmembrane domain (FIG. 6). This polarized structure indicates indicates that B345 is an integral membrane protein.
  • the extracellular domain suggests the existence of definitely one, possibly three, CUB domains.
  • CUB domains are found in various proteins, most of which are regulated during embryonic development.
  • a recent publication (Gerstein et al., 2000) demonstrated that proteins containing CUB domains are the most pronounced differentially regulated proteins in C. elegans. Since genes that play a key role in embryonic development have corresponding functions, e.g. in cell division, cell proliferation or signal transmission in cancer, it can be assumed that overexpression of B345 in cells causes a change in the properties of substrate adhesion or the extracellular matrix.
  • the B345 protein has 12 potential N-glycosylation sites found in the putative extracellular domain.
  • B345 protein forms a ß-sheet secondary structure, since CUB domains are known to fold as a ß-sandwich.
  • the intracellular domain (range 691-836) has no significant homologies. However, the entire C-terminus shows an identity (82%) over 124 amino acids with an EST (Acc No. AW063026) from human ovarian cancer cells.
  • B345 transmembrane protein plays a role in communication, interaction and / or signal transduction with extracellular components or ligands. Furthermore, the data from the expression analysis are a strong indication that B345 is metastatic
  • cell lines preferably human cell lines
  • the cells are transfected with a plasmid containing the B345 sequence and B345 expressed. Changes in the
  • Morphology and / or migration behavior e.g. Using a soft agar assay (Hamburger and Salmon, 1977) or a migration assay (Liaw et al; 1995) of the cells expressing B345 versus the non-transfected cells indicate a role of B345 in the biological process responsible for this. This is a clear indication of the involvement of B345 in the interaction of tumor cells with one another and / or with the extracellular matrix and thus for a function in metastasis.
  • B345 in cells which endogenously express this protein is suppressed in a complementary approach in order to also determine any changes in morphology and / or migration behavior.
  • it may be investigated whether protein components exist that interact with B345 inter- or extracellularly eg using the Yeast Two Hybrid System (Fields and Song, 1989).
  • the invention thus relates in a first aspect to a tumor-specific polypeptide with the designation B345, with the amino acid sequence given in SEQ ID NO: 4, or to a polypeptide which is encoded by a polynucleotide which, under stringent conditions, is linked to a polynucleotide of the type shown in SEQ ID NO: 3 shown sequence or a partial sequence thereof hybridized, as well as derived protein fragments or peptides.
  • the present invention relates to an isolated DNA molecule coding for the tumor-specific polypeptide of the designation B345.
  • the DNA molecule according to the invention is preferably a polynucleotide of the sequence shown in SEQ ID NO: 3 or a fragment thereof or a DNA molecule which is linked to a DNA molecule of the sequence shown in SEQ ID NO: 3 or a partial sequence thereof under stringent conditions hybridized, or a fragment thereof.
  • the DNA molecules according to the invention code for (poly) peptides of the designation B345 with the amino acid sequence shown in SEQ ID NO: or for protein fragments or peptides derived therefrom; this also includes DNA molecules or fragments which, due to the degeneration of the genetic code, have deviations from the sequence shown in SEQ ID NO: 3
  • the invention relates to an isolated DNA molecule of the sequence shown in SEQ ID NO: 3 or a fragment thereof, or a DNA molecule which is associated with a DNA molecule of the sequence shown in SEQ ID NO: 3 or with a Partial sequence hybridized, coding for the natural B345 polypeptide or for a fragment thereof.
  • the B345 DNA molecules can be used in a so-called DNA vaccine for the immunotherapy of tumors.
  • the B345 DNA molecules of the invention can be administered, preferably in recombinant form as plasmids, directly or as part of a recombinant virus or bacterium.
  • any gene therapy method for the immunotherapy of cancer based on DNA (“DNA vaccine”) on B345-DNA can be used, both in vivo and ex vivo.
  • Examples of in vivo administration are the direct injection of "naked" DNA, either intramuscularly or by means of a gene gun, which has been shown to lead to the formation of CTLs against tumor antigens.
  • Examples of recombinant organisms are vaccinia virus, adenovirus or Listeria monocytogenes (an overview was given by Coulie, 1997).
  • synthetic carriers for nucleic acids such as cationic lipids, microspheres, microspheres or liposomes, can be used for the in vivo administration of nucleic acid molecules, coding for B345 peptide.
  • cytokines either in the form of proteins or plasmids encoding them.
  • the application can optionally be combined with physical methods, eg electroporation.
  • ex vivo administration is the transfection of dendritic cells as described by Tuting, 1997, or other APCs . which are used as cellular cancer vaccines.
  • the present invention thus relates to the use of cells which express B345, either on their own or, in optionally modified form, after transfection with the corresponding coding sequence, for the production of a cancer vaccine.
  • modified derivatives can be used. These include sequences with modifications for a protein (fragment) or peptides with stronger ones
  • Coding immunogenicity with the same considerations for the modifications at the DNA level as for the peptides described above.
  • Another type of modification is the stringing together of numerous sequences, coding for immunologically relevant peptides, according to Art a string of beads ("string-of-beads"; Toes et al., 1997).
  • the sequences can also be modified by adding auxiliary elements, for example functions which ensure more efficient delivery and processing of the immunogen (Wu et al., 1995). For example, by adding a
  • ER targeting sequence Localization sequence in the endoplasmic reticulum
  • the present invention relates to a recombinant DNA molecule containing B345 DNA, e.g. linked to a regulatory DNA sequence, especially a heterologous regulatory DNA sequence, e.g. a promoter or enhancer.
  • the invention relates to antibodies against B345 or fragments thereof.
  • Polyclonal antibodies can be obtained in a conventional manner by immunizing animals, in particular rabbits, by injection of the B 345 antigen or fragments thereof, and subsequent purification of the immunoglobulin.
  • Monoclonal anti-B345 antibodies can be obtained according to standard protocols according to the principle described by Köhler and Milstein, 1975, by immunizing animals, in particular mice, then immortalizing antibody-producing cells of the immunized animals, for example by fusion with myeloma cells, and the supernatant hybridomas obtained is screened for monoclonal anti-B345 antibodies by means of standard immunological assays.
  • these animal antibodies can optionally be chimerized in a conventional manner (Neuberger et al., 1984; Boulianne et al., 1984) or humanized (Riechmann et al., 1988; Graziano et al., 1995) ,
  • Human monoclonal anti-B345 antibodies fragments can also be obtained from so-called "phage display libraries” (Winter et al., 1994; Griffiths et al., 1994; Kruif et al.,
  • the anti-B345 antibodies according to the invention can be used in immunohistochemical analyzes for diagnostic purposes, or as a therapeutic agent in cancer therapy.
  • a monoclonal antibody in cancer therapy is Herceptin; an antibody against the proto-oncogene HER2.
  • Herceptin can be used in breast cancer patients who overexpress HER2.
  • the invention relates to the use of B345-specific antibodies in order to selectively bring any substances into or into a tumor which expresses B345.
  • examples of such substances are cytotoxic agents or radioactive nuclides, the effect of which is to damage the tumor on site. Due to the relatively tumor-specific expression of B345, only a few are involved Expected side effects.
  • B345 antibodies can be used to visualize tumors that express B345. This is useful for the diagnosis and evaluation of the course of therapy.
  • the protein of the designation B345 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom can be used in cancer therapy, e.g. B. to induce an immune response against tumor cells that express the corresponding antigen determinants. They are preferably used for the therapy of
  • B345-positive tumors are used, especially in lung and colon carcinoma.
  • B345 or peptides, peptide equivalents and peptidomimetics can be used for the immunotherapy of cancer, e.g. in WO 00/73438, the disclosure of which is hereby incorporated by reference.
  • tumor-associated antigens can have tumor-specific mutations that contribute to: an immunological differentiation between tumor and normal tissue (Mandruzzato et al., 1997; Hogan et al., 1998; Gaudi et al., 1999; Wölfel et al., 1994).
  • the B345 cDNA is expediently determined using probes from the isolated cDNA according to the invention cloned one or more different tumors and compared the sequences obtained with normal tissue B345 CDNA.
  • the present invention thus relates to B345 peptides derived from regions of a tumor-expressed B345 which have tumor-specific mutations.
  • B345 Due to the preferred expression of B345 in tumor cells, it can be assumed that this protein has an important function for the tumor, e.g. for emergence, infiltration and growth and thus a target for chemotherapeutic intervention.
  • B345 is characterized in more detail in order to develop the appropriate strategy for the intervention with this function.
  • a bioinformatic analysis is expediently carried out in a first step, which is trend-setting for the experimental validation of B345 as a target.
  • the bioinformatics concepts based on similarity and modular structure represent an essential basis for this analysis.
  • Established bioinformatics tools for determining similarities are BLAST
  • B345 has a helical transmembrane domain, with both the N-terminal and the C-terminal region being hydrohpil, which suggests that this protein is a transmembrane protein.
  • the N-terminal, The extracellular region has some CUB domains which tend to form disulfide bridges and are therefore involved in dimerization or in protein-protein interactions (Bork et al., 1993).
  • the C-terminal, intracellular end shows homologies to a receptor kinase and to a C-kinase substrate.
  • B345 is subjected to a biochemical and biological analysis.
  • Proliferation assays in vitro or in animal models that overexpress the B345 gene to be examined (constitutive or inducible) and express it as a control either in deleted (inactive) form or downregulate via antisense (see e.g. Grosveld and Kollias, 1992).
  • B345 can be used in screening assays to identify substances that modulate, especially inhibit, the activity of this protein.
  • an assay may e.g. B. consist of introducing the B345 protein, or an active fragment thereof, into cells which react to the activity of B345 with proliferation or to express the corresponding B345 cDNA in the cell, and the proliferation of the cells in the presence and to determine in the absence of a test substance.
  • test cells are cells with a low division rate, for example primary cells that have no endogenous B345.
  • Substances with an anti-proliferative effect can be used for the treatment of tumors with strong B345 expression, particularly in the case of lung and colon carcinoma.
  • FIG. 1A Expression profile of B345, B452 and B540 in individual lung carcinomas and lung tumor cell lines.
  • Fig. IC Graphical representation of the alignment of B345, B452 and B540.
  • Fig. 2A Northern blot analysis of tumor cell line A549 with a 490bp long B345 PCR product
  • Fig. 2B Northern blot analysis of various
  • Fig. 4 mRNA expression analysis of B345 by real-time PCR of laser microscope-prepared colon tumors (LCM) as well as normal colon tissue and tumor cell lines.
  • Fig. 5 Graphical representation of the gene structure of B345.
  • Fig. 6 Hydrophilicity and transmembrane blot of the B345 protein
  • RDA Representative Difference Analysis
  • the human lung adenocarcinoma cell line A549 obtained from ATCC was grown up in T150 cell culture flasks. MEM served with 10% heat-inactivated, fetal calf serum and 2 mM L-glutamine as the nutrient medium. Every 3 to 4 days the cells were cleaved by trypsinization 1: 5 to 1:10 for propagation. After approximately 80% confluence had been reached, 4 ml of a trypsin solution (data per liter: 8 g NaCl, 0.2 g KC1, 1.13 g Na 2 HP0 4 - water-free, 0.2 g KH 2 PO 4 ,
  • the poly-A (+) RNA of the lung adenocarcinoma cell line A549 was described as "solid", that of normal lung tissue (1 mg / ml; Clontech, Palo Alto; # 6524-1) used as a "driver”.
  • the RDA was carried out using the PCR-select TM kit (Clontech, Palo Alto) according to the manufacturer's protocol, except that a modified primer / adapter-2 oligonucleotide system was used: adapter-2-alt-l (SEQ ID NO: 31) and nested PCR primer-2-alt (SEQ ID NO: 32) and adapter-2-alt-2 (SEQ ID NO: 33).
  • the newly generated primer / adapter sequences enable the presence of three new restriction enzyme interfaces (Kpn I, Sac I and Xho I) in the sequence of the nested PCR primer-2-alt after cloning the subtracted cDNA fragments in the pPCRII- Vector a subsequent cutting out of the respective cDNA fragments.
  • Restriction enzyme interfaces were necessary because point mutations were often observed, particularly in the primer sequences due to the PCR amplification steps.
  • the cDNA obtained was digested by "tester” and "driver” with Rsal (Rsal is a 4-base-recognizing restriction enzyme and provides a statistical average of 256 bp long cDNA fragments).
  • Identical parts of "tester cDNA” were ligated either with adapters 1 or 2 and then hybridized separately with an excess of "driver cDNA” at 65 ° C. The two approaches were then combined and subjected to a second hybridization with freshly denatured "driver cDNA”.
  • the enriched "tester” -specific cDNAs were then exponentially amplified by PCR, with primers 1 and 2 specific for the adapters.
  • 712 positive transformants (blue-white selection) were obtained and cultured in 96-well blocks in LB-Amp medium (1.3 ml per well) for 48 h at 37 ° C.
  • 750 ⁇ l of the E. coli suspensions were used per well for the preparation of the plasmid DNA (96-well mini-preparation method from QIAgen according to the manufacturer's instructions).
  • the remaining bacterial cultures were stored as glycerol stocks at -80 ° C.
  • a cDNA subtraction library consisting of 712 individual clones was obtained, which was available both in the form of E. coli glycerol stock cultures and in the form of purified plasmids.
  • the isolated plasmid DNA of all 712 clones was sequenced according to the Sanger method on an ABI-377 Prism device. The sequences obtained were annotated using the BioScout software (LION, Heidelberg) and subjected to database comparisons (Genbank). From 712 clones, 678 could be sequenced and annotated. The rest (34) had either only poly (A) sequences as an insert or corresponded to a religious vector or could not be sequenced. Of the 678 annotable sequences, 357 proved to be genes with a known function. The remaining 321 represented clones encoding genes with unknown function; 59 of them did not even have entries in the human EST database. Known genes were not further treated. For those unknown
  • oligonucleotide primer pairs were designed and synthesized from the sequences determined from the 200 selected clones.
  • 8 different human tissue-derived cDNA libraries (GibcoBRL "SUPERSCRIPT TM"), which are directionally cloned into pCMV-SPORT, were tested for the presence of the respective candidates by means of qualitative PCR.
  • the cDNA libraries used here came from tissue from the heart (# 10419-018), liver (# 10422-012), leukocytes (# 10421-022), kidney (# 10420-016), lung (# 10424-018), Testis (# 10426-013), brain (# 10418-010) and fetal brain (# 10662-013).
  • PCR conditions were like follows: 20 ⁇ l total volume per PCR mixture contained 1 ⁇ TaqPol buffer (50 mM KC1, 10 mM Tris-HCl pH9, 0.1% Triton X-100), 1.5 mM MgCl 2 , 0.2 mM dNTPs (Promega ), 0.025 U / ⁇ l Taq DNA polymerase (Promega), each 5 pM at specific oligonucleotide primers for B345 (B345-D,
  • Primer pairs of oligonucleotide primers (SEQ ID NO: 34) and (SEQ ID NO: 35) were also tested in parallel for the isolated plasmid with the B345 "original fragment” (originally isolated cDNA fragment from B345).
  • a number of clones were selected from the dBE ⁇ T database via nucleotide sequence search, the most diverse
  • IMAGE clones (of which 1024 already known genes) were ordered and sequenced for control. Microtiter plates with bacteria containing sequences of approximately 800 bp from the 3 "end of the gene in the vector were sent to Incyte Pharmaceuticals, Inc. (USA) and spotted there on 60 chips. In addition to these clones, 120 EST identified by RDA were also found The DNA chips produced in this way were then hybridized with Cy3-labeled cDNA from normal tissue, tumor tissue and cell lines together with Cy5-labeled cDNA from a mixture of nine different normal tissues and the two signals for normalizing the expression values were compared.
  • B345 is a gene which, according to DNA chip analyzes, is up-regulated in tumor tissues (see FIGS. 1A and 1B, Tab. La and Tab. 1B).
  • a Northern blot analysis was carried out for B345 using human cell lines and the "Human Multiple Tissue Northern Blot" (Clontech and Invitrogen) , The 490 bp and 318 bp long PCR products labeled with [ ⁇ - 32 P] dCTP (NEN, Boston) (primers (SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 8)). The hybridization took place at 68 ° for 2 h; visualization using standard autoradiography (Hyperfilm, Amersham). 2A, 2B and 2C and Tab.
  • Fig. 2A from cell line A549
  • Fig. 2B from 12 normal tissues (peripheral blood lymphocytes (PBL), lungs, placenta, small intestine, liver, kidney, spleen , Thymus, colon, skeletal muscle, heart and brain) and FIG.
  • PBL peripheral blood lymphocytes
  • the B345 transcript is 6.5 kb in length.
  • RNA was isolated from frozen tissue using trizole in accordance with Gibco's manufacturer's protocol. To remove any contaminating DNA, the prepared RNA was digested with DNAase I as follows: 3 ⁇ g of total RNA were mixed with 20 ⁇ l of 5 ⁇ AMV buffer (Promega), 1 ⁇ l of RNasin (Promega) and 2 ⁇ l of DNase I (Boehringer Mannheim) incubated for a total of 80 ⁇ l at 37 ° C. for 15 minutes. 120 ⁇ l phenol: chloroform: Isoamyl alcohol (25: 24: 1) was added, mixed on a vortex mixer and centrifuged briefly. The aqueous phase was removed, mixed with 120 ul chloroform: isoamyl alcohol (24: 1) and centrifuged as before. The purified RNA was ethanol-precipitated and dissolved in water.
  • DNAase I 3 ⁇ g of total RNA were mixed with 20 ⁇ l of 5 ⁇ AMV
  • RNA was then transcribed with reverse transcriptase (Superscript, Gibco, BRL) into cDNA: 1 ⁇ l of oligo dT primer (Promega) was added to 3 ⁇ g of total RNA and water was added to it
  • a TaqMan PCR Run included samples of ⁇ -actin control sequence with 10 2 , 10 3 , 10 4 , 10 5 and 10 6 copies / ⁇ l (Perkin Elmer) to determine the standard curve, a negative control without DNA and the cDNA pools to be quantified. All samples were analyzed in triplicate. For a 25 ⁇ l
  • the reaction mixture was 1 ul cDNA, 2.5 ul lOx buffer A (Perkin Elmer), 4 ul MgCl 2 (25 mM, (Perkin Elmer)), 0.5 ul per nucleotide (10 mM dATP, dCTP, dGTP; 20 mM dUTP ), 0.125 ⁇ l TaqMan probe (20 ⁇ M; TaqMan probe for ß-actin (SEQ ID NO: 20 fluorescence-labeled at the 5 'end with 6-carboxyfluorescein and with
  • the PCR was carried out as follows: a cycle with 2 minutes 50 ° C for the UNG reaction, a cycle 10 minutes 95 ° C to activate the AmpliTaq, 40 cycles with 15 seconds each 95 ° and 1 minute 60 ° C. The samples were then kept at 25 ° C. The data are evaluated with the program "Sequence Detection System 1.5bl" * (PE Applied Biosystems), the fluorescence signals of the cDNA samples to be quantified being compared in principle with the signals of the control plasmid dilutions of known concentration.
  • GAPDH-TaqMan PCR The following primers or probes were used for the quantification of GAPDH, which was used to normalize the RNAs used, such as ⁇ -actin or tubulin.
  • a TaqMan probe for GAPDH (SEQ ID NO: 23) was used at the 5 'end
  • SybrGreen PCR see manufacturer information (Perkin Elmer).
  • a SybrGreen PCR run contained samples of tubulin control plasmid with 10 2 , 10 3 , 10 4 , 10 5 and 10 e copies / ⁇ l (Perkin. Elmer) to determine the standard curve, a negative control without DNA and the cDNA pools to be quantified , All samples were analyzed in triplicate.
  • B345 specific primers SEQ ID NO: 28 and SEQ ID NO: 29
  • SEQ ID NO: 30, 20 ⁇ M B345 specific probe
  • Fig. 3 illustrates the TaqMan expression analysis (Fig. 3A: ⁇ -actin; Fig. 3B: tubulin). It was shown that B345 is expressed higher in colon cancer tissue than in normal tissue (see Table 2a). Now, however, both the normal tissue and the tumor tissue represent a very heterogeneous mixture of different cell types. Furthermore, the proportion of tumor cells in the tumor tissue varies greatly from about 30 to 80%. In order to minimize this biological heterogeneity, the epithelial cells of the large intestine, which are the original cells of the adenocarcinoma, and the cancer cells or cancer areas were specifically prepared by laser microdissection.
  • Tissue sections of 10 ⁇ m thickness were made with the Kyromikrotome from Leica, Jung CM1800 and onto one with polyethylene coated slides applied (Böhm et al., 1997). The sections, dried at room temperature for about 30 minutes, were incubated with Mayer's hematoxylin (SIGMA DIAGNOSTICS) and then washed under running water for five minutes to remove non-specifically bound dye. After drying for five minutes at 37 ° C, the laser microdissection was carried out. The laser microscope from PALM (PALM GmbH, Bernried, Germany) was used for this and about 2000 to 5000 cells were prepared. The cDNA obtained by reverse transcription was also analyzed here by real time PCR. The result shows that the B345 expression in colon carcinoma cell lines and in patient material is many times higher than that of the normal colon tissue. The expression level of GAPDH was determined for normalization (see FIG. 4 and Table 2B).
  • RNA from the lung carcinoma cell line Calu 6 (AACC No.HTB56) was reverse transcribed using the primer (SEQ ID NO: 9) and the resulting single-stranded cDNA was PCR by means of the gene-specific primer SEQ ID NO: 5 and the adapter primer SEQ ID NO: 10 amplified.
  • PCR For a 25 ul PCR approach, 1 ul of the cDNA pool with 2.5 ul lOxTaq buffer (Promega), 1.5 ul MgCl 2 (25 mM, Promega), 0.5 ul dNTPs (10 mM each, Boehringer Mannheim) , 1 ⁇ l primer mixture (20 ⁇ M each), 0.15 ⁇ l Taq polymerase (Promega) mixed in water.
  • the PCR was carried out as follows: 1 ⁇ 94 ° C. for 3 minutes; 30x 94 ° C 30 seconds,
  • the PCR was analyzed on a 1.2% agarose gel.
  • the two primers were then used to sequence the purified PCR product.
  • the determined Sequences showed high homology with the "in silico" cloned DNA section (including the PolyA tract).
  • Calu 6 was also used as the starting cell line.
  • a linker consisting of the two oligos SEQ ID NO: 11 and SEQ ID NO: 12 was ligated to the double-strand cDNA (Abe et al., 1992) .
  • the resulting LoneLinker cDNA library was then linearly amplified with the gene-specific primer SEQ ID NO: 6 over 35 cycles.
  • An aliquot of the B345-enriched cDNA could then be further amplified with the primers SEQ ID NO: 13 and LLEcoRIA SEQ ID NO: 11.
  • the cDNA mentioned has an open reading frame (ORF) which codes for a potential protein with a length of 749 amino acids.
  • ORF open reading frame
  • the translation initiation site at position 215 corresponds to approximately 75% of a Kozak consensus sequence.
  • the 5 ⁇ region and the promoter region as well as the accurate transcription initiation site were determined.
  • the 5 'region was performed using a genomic DNA library from Clontech with B345 specific primers ( SEQ ID NO: 38 or nested SEQ ID NO: 39) and adapter primer in the kit amplified.
  • the primer extension reaction was carried out to determine the exact start of transcription.
  • the primer SEQ ID NO: 40 at the 5 "end was labeled using 10 U of the T4 polynucleotide kinase (Promega) and 3 ⁇ l [ ⁇ - 32 P] ATP, (3000 Ci / mmol) according to standard protocols (Sa brook et al
  • the labeled oligonucleotide was purified by precipitation and 10,000 cpm oligonucleotide in a total volume of 20 ⁇ l to 25 ⁇ g total RNA of the Colo 205 cell line (ATCC: CCL-222) were used for the primer extension reaction.
  • RNA of the cell line was reverse transcribed with the radioactively labeled primer and applied to a 10% polyacryamide gel.
  • a PCR fragment of nt 1000 - nt 1362 with 35 S-labeled nucleotides was sequenced and also applied.
  • the fragment of 209 nucleotides resulting from the elongation of the reverse primer specifies the start of transcription exactly at position 201 B345 sequence obtained in Example 6a expanded in the 5 "region and a new start codon at position 283 determined.
  • the resulting primary amino acid sequence of B345 is shown in SEQ ID NO: 4.
  • Analysis of the hydrophilicity plot of the amino acid sequence by the method of Kyte and Doolittle (1982) shows that the B345 protein has two characteristic hydrophobic domains (amino acids pos. 1 - 29 and 666 - 691), which represent a signal peptide and a helical transmembrane domain ( Fig. 6).
  • This polarized structure indicates that B345 is an integral membrane protein.
  • the transmembrane helix connects an approximately 666 amino acid long extracellular and a short (145 amino acid) intracellular part (see FIG. 7).
  • the extracellular domain also has clear indications for the existence of a CUB domain at positions 220-350 as well as indications for two possible further CUB domains in the region of amino acids 425-660.
  • CUB domains are found in various proteins, most of which are regulated during embryonic development. In addition, CUB domains are sometimes found in EGF (epidermal growth factor) -like domains.
  • EGF epidermal growth factor
  • a recent publication (Gerstein et al., 2000) demonstrated that proteins containing CUB domains are the most pronounced differentially regulated proteins in C. elegans. Since genes that have a key role in embryonic development also perform analogous functions in cancer, it can be assumed overexpression of B345 in cells causes a change in the properties of the substrate adhesion or the extracellular matrix.
  • the protein also has 12 potential N-glycosylation sites found in the predicted extracellular domain, which is consistent with the predicted orientation of the protein.
  • a BLAST hit (E-value: 5.8 x 10 ⁇ 2 ) for the amino acid range from 235 to 282 of B345 identified a complement-activating component of the _RA-reactive factor (RARF) from mus musculus.
  • the alignment is located within CUB Domain 1 of B345.
  • the CUB domains 2 and 3 (range 425-535 and 545-660) have marginal homologies to the human and fugu
  • PCOLCE Procollagen C-Proteinase Enhancer Protein
  • the B345 protein presumably forms a ß-sheet secondary structure, since CUB domains are known to fold as a ß-sandwich.
  • the intracellular domain (range 691-836) has no significant homologies. However, the entire C-terminus was aligned with an EST (AW063026) of human ovarian cancer cells (82% identity over 124 amino acids).
  • Bac clones were first searched in public databases (BLAST search) which contained the B345 gene. Bac clones Ac068625 and Ac010170 contained one
  • the chromosomal location of the gene was determined using fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • the human, digoxigenin-labeled B345 probe was used together with the biotin-labeled probe from B47a2 (Knight et al., 1997), which is located on the sub-telomeric region of the chromosome arm 3p, with metaphase chromosomes from two "normal" individuals hybridizes (Lichter et al., 1988).
  • the hybridized digoxygenin probe was detected using anti-sheep dig (Boehringer Mannheim FRG) and rabbit anti-sheep FITC-labeled antibodies.
  • the sample labeled with biotin was visualized with mouse anti-biotin and rabbit anti-mouse (TRITC) and then stained with DAPI.
  • TRITC mouse anti-biotin and rabbit anti-mouse
  • the FISH results show that a majority of the metaphases have unique signals at one or have two chromatids of chromosome 3 in the region p21-p23.
  • the co-localization of the B47a2 (TRITC) probe on the same chromosomal arm served as confirmation of the position.

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Abstract

La présente invention concerne un antigène spécifique de tumeur (B345) et la molécule d'ADN codant pour ledit antigène.
PCT/EP2001/007705 2000-07-07 2001-07-05 Antigene specifique de tumeur (b345) se caracterisant par une sequence d'acides amines telle que dans seq id no 4 Ceased WO2002004508A1 (fr)

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EP1396501A1 (fr) * 2002-09-04 2004-03-10 Eberhard-Karls-Universität Tübingen Universitätsklinikum Anticorps pour l'identification et/ou l'isolement d'au moins une population de cellules selectionnée de cellules hématopoietiques souches, de cellules neuronales souches ou progenitrices, de cellules du mesenchyme souches ou progenitrices
WO2004074481A1 (fr) * 2003-02-19 2004-09-02 Novartis Ag Antigene glycoproteine sima135 exprime dans des cellules tumorales humaines metastatiques
WO2011023389A1 (fr) 2009-08-28 2011-03-03 Roche Glycart Ag Anticorps anti-cdcp1 humanisés
WO2011023390A1 (fr) 2009-08-28 2011-03-03 F. Hoffmann-La Roche Ag Anticorps contre cdcp1 destinés au traitement du cancer
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US8263071B2 (en) 2003-10-22 2012-09-11 Ucb Pharma S.A. Protein involved in ovarian cancer
WO2015082446A1 (fr) 2013-12-02 2015-06-11 F. Hoffmann-La Roche Ag Traitement du cancer à l'aide d'un anticorps anti-cdcp1 et d'un taxane

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Cited By (17)

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Publication number Priority date Publication date Assignee Title
DE10242146A1 (de) * 2002-09-04 2004-03-18 Eberhard-Karls-Universität Tübingen Universitätsklinikum Antikörper zur Identifizierung und/oder Isolierung von hämatopoetischen Stammzellen
US7541030B2 (en) 2002-09-04 2009-06-02 Hoffmann-La Roche Inc. Antibodies for identifying and/or isolating at least one cell population which is selected from the group comprising haematopoietic stem cells, neuronal stem cells, neuronal precursor cells, mesenchymal stem cells and mesenchymal precursor cells
US7741114B2 (en) 2002-09-04 2010-06-22 Hoffmann-La Roche, Inc. Antibodies for identifying and/or isolating at least one cell population
EP2206729A1 (fr) * 2002-09-04 2010-07-14 F. Hoffmann-La Roche AG Anticorps pour l'identification et/ou l'isolement de cellules hématopoiétiques souches, de cellules neuronales souches ou progénitrices, de cellules souches ou progénitrices du mésenchyme
EP1396501A1 (fr) * 2002-09-04 2004-03-10 Eberhard-Karls-Universität Tübingen Universitätsklinikum Anticorps pour l'identification et/ou l'isolement d'au moins une population de cellules selectionnée de cellules hématopoietiques souches, de cellules neuronales souches ou progenitrices, de cellules du mesenchyme souches ou progenitrices
WO2004074481A1 (fr) * 2003-02-19 2004-09-02 Novartis Ag Antigene glycoproteine sima135 exprime dans des cellules tumorales humaines metastatiques
CN100422328C (zh) * 2003-02-19 2008-10-01 诺瓦提斯公司 在人转移性肿瘤细胞中表达的糖蛋白抗原sima135
US7589173B2 (en) 2003-02-19 2009-09-15 The Scripps Research Institute Methods for diagnosing cancer and decreasing metastasis by cancer cells
AU2008201545B2 (en) * 2003-02-19 2012-08-02 Novartis Ag Glycoprotein antigen SIMA135 expressed in metastatic human tumor cells
US8263071B2 (en) 2003-10-22 2012-09-11 Ucb Pharma S.A. Protein involved in ovarian cancer
US9120860B2 (en) 2003-10-22 2015-09-01 Ucb Pharma S.A. Protein involved in ovarian cancer
WO2011023389A1 (fr) 2009-08-28 2011-03-03 Roche Glycart Ag Anticorps anti-cdcp1 humanisés
US8394928B2 (en) 2009-08-28 2013-03-12 Roche Glycart Ag Humanized anti-CDCP1 antibodies
US8883159B2 (en) 2009-08-28 2014-11-11 Hoffmann-La Roche, Inc. Antibodies against CDCP1 for the treatment of cancer
WO2011023390A1 (fr) 2009-08-28 2011-03-03 F. Hoffmann-La Roche Ag Anticorps contre cdcp1 destinés au traitement du cancer
US9346886B2 (en) 2009-08-28 2016-05-24 Roche Glycart Ag Humanized anti-CDCP1 antibodies
WO2015082446A1 (fr) 2013-12-02 2015-06-11 F. Hoffmann-La Roche Ag Traitement du cancer à l'aide d'un anticorps anti-cdcp1 et d'un taxane

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