WO2004050870A2 - Genetic switches for the detection of fusion proteins - Google Patents

Genetic switches for the detection of fusion proteins Download PDF

Info

Publication number
WO2004050870A2
WO2004050870A2 PCT/EP2003/013323 EP0313323W WO2004050870A2 WO 2004050870 A2 WO2004050870 A2 WO 2004050870A2 EP 0313323 W EP0313323 W EP 0313323W WO 2004050870 A2 WO2004050870 A2 WO 2004050870A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
nucleic acid
complex
acid molecule
cell
Prior art date
Application number
PCT/EP2003/013323
Other languages
French (fr)
Other versions
WO2004050870A3 (en
Inventor
Stefan Bohlander
Nicole FRÖHLICH
Original Assignee
Ludwig-Maximilians-Uni Versität
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 Ludwig-Maximilians-Uni Versität filed Critical Ludwig-Maximilians-Uni Versität
Priority to AU2003289899A priority Critical patent/AU2003289899A1/en
Publication of WO2004050870A2 publication Critical patent/WO2004050870A2/en
Publication of WO2004050870A3 publication Critical patent/WO2004050870A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/72—Fusion polypeptide containing domain for protein-protein interaction containing SH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present invention relates to a complex comprising (a) a fusion protein comprising at least two epitopes; (b) protein A comprising an interaction domain capable of interacting with said first epitope of the protein of (a) and comprising a first effector domain; and c) protein B comprising an interaction domain capable of interacting with said second epitope of the protein of (a) and comprising a second effector domain whereby said interaction domains of protein A and protein B are not capable of directly interacting with each other.
  • specific nucleic acid molecules encoding said protein A and/or said protein B are provided as well as expressed protein A/B molecules.
  • compositions, in particular pharmaceutical and diagnostic compositions are described which comprise the members of the complex of the present invention.
  • the invention provides for in vivo and/or in vitro methods for the detection of a protein, preferably a fusion protein, more preferably an oncogenic fusion protein.
  • fusion proteins found associated with specific types of cancer especially leukemias and certain entities of sarcomas.
  • BCR/ABL fusion protein which is found in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL).
  • CML chronic myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • the BCR/ABL fusion protein is able to cause leukemia in a transgenic mouse model. More recently, using an inducible BCR ABL transgenic mouse model, it could be demonstrated that the expression of the BCR/ABL fusion protein is necessary to maintain the malignant status of the transformed cells. Switching off the fusion gene reverted the malignant phenotype of the cells. Since the fusion proteins represent the fundamental difference between a normal and a transformed cells it seems logical to use these fusion protein as the preferred targets for specific therapeutic strategies. Several strategies have been used up to now in the case of the BCR/ABL fusion to achieve specficity for cells harboring the leukemic fusion.
  • RNA binding domain that interacts with its RNA binding site in a bifunctional RNA molecule
  • the other part of the RNA molecule interacts with a second hybrid protein consisting of another RNA binding domain linked to a transcription activation domain.
  • This system is limited to the use in yeast cells and relates to the detection or confirmation of RNA-protein interactions.
  • WO 02/070662 provides for compositions and methods for isolating ligand binding polypeptides for a user-specified ligand and for isolating small molecule ligands for a user-specified target polypeptide.
  • the used hybrid ligand in WO 02/070662 is characterized by a R1-Y-R2 structure, whereby R1 represents a first ligand, Y represents a polyethylene linker and R2 a user-specified second ligand.
  • R1 represents a first ligand
  • Y represents a polyethylene linker
  • R2 a user-specified second ligand.
  • WO 02/070662 describes a method whereby the interacting between a polypeptide and a small molecule ligand can be discovered.
  • the "middle" compound in this three hybrid system is a complex small molecule consisting of a known small interactor and a variable other small molecule for which the interaction with a given other polypeptide or protein can be detected.
  • This system is specifically designed to discover pharmacologically relevant small molecule-protein interactions in a screening method.
  • WO 02/070662 relates to a method to identify cellular targets of pharmacological agents, in particular pharmacological agents displaying a given activity.
  • WO 02/070662 also discusses further prior art relating to "two- and/or three hybrid systems". In particular to Fields, Nature 340 (1989), 245-246; Gyuris, Cell 75 (1993), 791-803; US 5,468,614; Yang, Nuc. Acid Res. 23 (1995), 1152- 1156; WO 94/23025, WO 95/30012 or WO 97/41255. Most of this cited art is related to the elucidation of the interaction of proteins, since the elucidation of particular interacting protein partners has been advanced by the development of in vivo "two-hybrid” or "interaction trap” methods for detecting and selecting interacting protein partners (see Fields & Song (1989), loc.
  • WO 94/23025 and WO 95/30012 describe a screening assay for identifying molecules capable of binding cell surface receptor so as to activate a selected signal transduction pathway. These references describe the modification of selected yeast signalling pathways so as to mimic steps in the mammalian signalling pathway.
  • US Patent Nos. 5,585,245 and 5,503,977 describe the "split ubiquitin" methods, which can detect protein-protein interactions by use of a ubiquitin specific protease to cleave a reporter polypeptide from a fusion protein.
  • Two fusion proteins are constructed, one consisting of the N-terminal half of ubiquitin and a prey protein (Nub-prey or prey-Nub), and the other consisting of the C-terminal half of ubiquitin, a bait protein and the report (bait-Cub-reporter).
  • Association of prey and bait reconstitutes a ubiquitin structure recognized by the ubiquitin specific protease, whereby the reporter is cleaved from the fusion protein.
  • the cleavage of the reporter from the fusion protein can be detected by several techniques, e.g. cleavage or destabilizing the reporter or allow for its translocation.
  • WO 97/41255 relates to a screening methods for small molecules that bind cellular targets so as to identify new drugs that are capable of specific therapeutic effects, whereby a ligand-hybrid (similar to WO 02/070662) is detected. Again, this system does not provide for detection means in mammalian cells and is limited to the detection of hybrid ligands.
  • US 5,503,977 relates to specific fusion proteins comprising an mutationally altered N-terminal subdomain of ubiquitin, whereby said fusion proteins or nucleic acid molecules encoding the same may be employed in methods for identifying interacting proteins or peptides.
  • US 5,503,977 essentially describes a two hybrid system in which the effect of the interaction between two proteins is the reconstitution of ubiquitin as the target of the ubiquitin-specific protease. This protease (which seems to be present in all cells or has to be externally provided) cleaves off a reporter moiety. This proteolytic cleavage can be detected by immunoprecipitation, gel electrophoresis etc.
  • US 5,585,245 also describes the use ubiquitin subdomain fusion proteins which are useful for studying the interaction of two members of a specific binding pair or the determination of a predetermined ligand in a sample.
  • a further variant of the mammalian two hybrid system is described for the detection the indirect in vivo association of two proteins whereby said association is mediated by a third (or fourth) protein acting as a bridge between the two assayed proteins.
  • a third (or fourth) protein acting as a bridge between the two assayed proteins.
  • fusion proteins which are the result of chromosomal translocations play a pivotal role in the malignant transformation process in numerous types of leukemias and solid tumors. In some cases it could be shown that switching off the production of such a fusion protein in the cell will revert the malignant phenotype. However, there are only very few therapies available today that use these fusion proteins as a therapeutic targets.
  • the technical problem underlying the present invention is the provision of means and methods for the elimination of cells comprising detrimental, e.g. oncogenic fusion proteins.
  • the present invention relates to a complex comprising
  • a protein B comprising an interaction domain capable of interacting with said second epitope of the protein of (a) and comprising a second effector domain whereby said interaction domains of protein A and protein B are not capable of directly interacting with each other.
  • the term "complex" as used above relates to a specific protein interaction between the defined protein comprising at least two epitopes, the protein A and the protein B as defined herein.
  • Said complex is preferably a complex formed in vivo in a mammalian cell, whereby most preferably said protein comprising said at least two epitopes is produced/expressed by said mammalian cell. Yet, said complex may also be formed ex vivo as well as in vitro.
  • the complex as defined herein may be detected by specific means disclosed herein and may be useful for the elimination of a cell expressing said protein comprising two epitopes as defined herein.
  • the invention relates in a preferred embodiment to a complex, wherein a first of said epitopes of the protein comprising at least two epitopes is encoded by a first gene (or a fragment thereof) and wherein a second of said epitopes is encoded by a second gene (or a fragment thereof).
  • said protein comprising at least two epitopes is a fusion protein, even more preferably said fusion protein is a fusion protein derived from a chromosomal translocation.
  • said fusion protein is comprised/expressed in a blood cell or a bone marrow cell.
  • Said protein comprising two epitopes and composed in the complex of the invention may be related to or may be associated with malignancy.
  • said protein comprising two epitopes is oncogenic, even more preferred said protein is an oncogenic fusion protein.
  • a complex wherein the first epitope of said protein comprising at least two epitopes is encoded by a gene (or a fragment thereof) selected from the group consisting of RNM15 (OTT), AF1 p (eps15), PAX7, MLL, AF4 (MLLT2), MSF (homologous to PNUTL1), GMPS, AF10 (MLLT10), PAX3, PBX1 , TFE3, ALK, HOXD13, HOXA9, RAP1GDS1 , NSD1 , PMX1, MLF1 , EAP, EVI1 , MDS1 , HMGIC, BTL (CHIC2), ACS2, PDGFRB, NPM, CAN, AF6 (MLLT4), FOP, JAZF1, HLXB9, MOZ, FGFR1 , ETO, LEDGF, CHN, CSMF, ABL, AF10, RET, PDGFRB
  • MKL1 MAL
  • MLL FOX01A
  • PNUTL1 PNUTL1
  • FBP17 LPP
  • AF9 MLLT3, GPHN
  • ENL MLLT1, ABU (SSH3BP1)
  • AFX1 MLLT7
  • FOXA1A E2A
  • PRCC NUP98
  • NPM ETV6
  • FGFR1 ETV6
  • FGFR1 FGFR1
  • JJAZ1 JJAZ1
  • TIF2 AML1
  • CBP ZNF198
  • FIM RAMP
  • BCR P300
  • TEL/Abl ETV6/Abl
  • PML/LT3-ITD fusion PML/LT3-ITD fusion
  • APL-like disease Kelly, PNAS 99 (2002), 8283-8288.Yet, it is of note that these examples relate to transgenic mice or mice that received retrovirally transduced bone marrow cells.
  • epitope as employed herein above in context with said protein comprising at least two epitopes relates to any potential, but specific interaction site for the interaction domain of protein A or protein B as defined herein.
  • Said “epitope” comprises preferably a stretch of at least 5 amino acids, more preferably of at least 7 amino acids.
  • epitopes are envisaged which comprise less than 5 amino acids.
  • epitopes which comprise secondary modifications, like, e.g. phophorylations.
  • said first epitope comprised in said protein comprising at least two epitopes has to be distinct and different from said second epitope. Accordingly, the interaction of the interaction domain of protein A with said first epitope is distinct from the interaction of the interaction domain of protein B with said second epitope.
  • Said first effector domain in said protein A of the complex of the invention is, preferably, selected from:
  • DBD DNA-binding domain
  • said DNA-binding domain is or is derived from a bacterial DBD, from a fungal DBD, from a plant DBD or a DBD from a transcription factor.
  • said bacterial DBD of protein A is or is derived from lexA or Tet repressor
  • said fungal DBD is or is derived from GAL4 DBD (this is a fungal ie.
  • said plant DBD is or is derived from the DBD of TIZZ or the DBD of PHR1 and wherein said DBD from a transcription factors is the DBD of a mammalian (like Hox-Proteins, ETS-Proteins, GATA-1 , GATA-2, ATF2, c-Jun) a yeast transcription factor.
  • a mammalian like Hox-Proteins, ETS-Proteins, GATA-1 , GATA-2, ATF2, c-Jun
  • DNA binding domains for a DNA binding domain to be useful in the context of this invention the precise sequence of the the DNA sequence that is recognizes must be known. DNA binding domains derived from bacterial, fungal or plant proteins are best suited because there should be less cross recoginition of mammalian promoter sequences. Particular designed transcription factors as tools for therapeutics and functional genomics are described in Urnov (Biochem Pharmacol. 64 (2002), 919).
  • Protein A as employed in accordance with this invention is also illustrated in the appended examples and further described herein below.
  • the invention also provides for a complex as desribed above, wherein said second effector domain in said protein B is
  • AD transcriptional activation domain
  • said transcriptional activation domain is selected from the group consisting of GAL4 activation domain, VP16 activation domain, c-Jun, c- Fos, ELK1 , CREB, ATF2 and CHOP.
  • GAL4 activation domain is employed in yeast systems, whereas, e.g. VPI6 activation domain may be employed in mammalian systems.
  • further transcriptional activation domains may be employed, e.g. domains rich in glutamine, serine and threonine, as, inter alia, described in DE-A1 198 31 420. consulting the appended figures and examples, the skilled artisan is readily in the position to employ further transcriptional activation domains (AD) known in the art.
  • the recognition complex could direct a specific proteolytic cleavage event.
  • protein A consists of interactor X fused to a site specific protease and protein B consists of interactor Y linked by a cleavable linker to a transcription factor or an effector protein.
  • protein A cleaves protein B and releases either a transcription factor or an effector protein, e.g. an active caspase or other effectors described herein.
  • the transcription factor then translocates into the nucleus and initiates transcription of effector genes.
  • the transcription factor would contain a DNA binding domain with known specificity, a nuclear localization signal and a transcriptional activation domain.
  • interactor Y would be designed in such a way that it not only interacts with one part of the fusion protein but that it contains additional interaction surfaces that would interact intramolecularly with both the NLS, the DNA binding domain and the activation domain of the transcription factor.
  • interactor Y would be designed in such a way that it not only interacts with one part of the fusion protein but that it contains additional interaction surfaces that would interact intramolecularly with both the NLS, the DNA binding domain and the activation domain of the transcription factor.
  • Every protein-protein interaction has certain interaction kinetics, i.e. on and off rates.
  • the time of binding is very high since the interacting surfaces cannot move away very far from each other.
  • the interaction is converted from an intra- to an inter-molecular interaction and the interacting surfaces can move away from each other easily.
  • the transcription factor is free to move away from the interactor Y part of protein B and to interact with the components (e.g. transportin oc) of the nuclear translocation machinery.
  • the cleaved portion of protein B would be an activated caspase the uncleaved form would similarly be held in an inactive state by intramolecular interactions (procaspase: here the active site is covered by intramolecular interaction).
  • the polypeptide comprised in protein B of the complex of the invention as activated by said proteolytic cleavage may be a caspase, a transcription factor or a protein involved in a signal transduction cascade or another effector gene.
  • said caspase is selected from the polypeptide consisting of caspase-9, caspase-3, caspase-6, caspase-8 and/or wherein said transcription factor or said protein involved is a signal transduction cascade in NF-kappa B or Notch.
  • NFKB translocates into the nucleus and activates transcription after proteolytic cleavage.
  • Notch signaling is initiated by proteolytic cleavage at the membrane and the intracellular portion of NOTCH then complexes with another protein, translocates into the nucleus, and activates transcription.
  • said polypeptide comprised in protein B of the complex of the invention may also be an "engineered transcription factor", comprised an engineered DNA bidning domain (DBD) and an activation domain (AD) which is liberated and/or activated upon cleavage from "protein B" by a protease comprised in "protein A”.
  • proteolytic cleavage mediated by "protein A” is mediated by thrombin, TEV protease, secretase, enterokinase.
  • proteases are preferred in this context that are involved in viral protein processing since these often comprise a very specific protease activity.
  • proteases to be employed in this context also comprise HIV-1 retropepsin (human immunodeficiency virus type 1), nodavirus endopeptidase (flock house virus), ubiquitin C-terminal hydrolase UCH-L1 (Homo sapiens), foot-and-mouth disease virus L-proteinase (foot-and- mouth disease virus), caspase-1 (Rattus norvegicus), hedgehog protein (Drosophila melanogaster), poliovirus-type picornain 3C (poliovirus type 1), procine transmissible gastroenteritis virus main protease (porcine transmissible gastroenteritis virus), togavirin (Sindbis virus), signal peptidase I (Escherichia coli), or C-terminal processing protease-1 (Escherichia coli). Said protease is comprised (at least in part) in said protein A. Accordingly, only the proteolytically active fragment of such a protease described
  • Protein A as well as protein B may further comprise a "nuclear localization signal" (NLS).
  • NLS nuclear localization signal
  • the invention also relates to a complex as described herein when said complex further comprises a nucleic acid molecule comprising a binding site for said
  • Said effector gene preferably encodes for a polypeptide (or a fragment thereof) which is selected from the group consisting of
  • a marker gene (a) a marker gene; (b) a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug;
  • effector genes as defined herein may be employed in combination. These combined effector genes may be linked, e.g., by "internal ribosome entry sites” (IRES).
  • IRS internal ribosome entry sites
  • the effector gene which is a marker gene may be selected from group consisting of a fluorescent protein, a cell surface marker, ⁇ -Gal, luciferase,
  • a desired fluorescent protein may be "green fluorescent protein", fluorescent proteins (green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, red fluorescent protein and the like), a cell surface marker may be neurotrophin receptor.
  • said prodrug converting enzyme may be selected from the group consisting of purine nucleoside phosphorylase, thymidine kinase, DeoD (PNP) cytosine deaminase, carboxyl esterase, carboxypeptidase A, carboxypeptidase G2, cytochrome P450, D-amino-acid oxidase, deoxycytidine kinase, DT- diaphorase, ⁇ -galactosidase, ⁇ -glucuronidase, ⁇ -lactamase, methionine-liase, nitroreductase, thymidine phosphorylase, and xanthine-guanine phosphoribosyl transferase.
  • the prodrug converting enzymes described here may also be capable of sensitising a cell for a drug.
  • thymidine kinase as "effector gene" in the complex of the invention, i.e. in a cell comprising a detrimental protein (like an oncogenic fusion protein) makes said cell sensitive for medicaments, like gancyclovin.
  • the expression DeoD encoding purine nucleoside phosphorylase (PNP) sensitizes cells for G-methylpurine desoryribose.
  • the effector gene to be expressed in accordance with this invention may also be an immunomodulatory molecule like interleukin 2, costimulatory molecules (B7), and MHC molecules.
  • said effector gene encodes an antigen which may be a bacterial, viral or plant antigen and/or an antigen which is capable, when expressed, to elucidate immune response in a subject.
  • a cell expressing a protein comprising at least two epitopes as defined above, e.g. an oncogenic fusion protein may be driven (by the complex of the invention) to the expression of a specific antigen on the surface of said cell.
  • This antigen is capable of eliciting an immune response in a subject, said cell is eliminated by the subjects own immune system. Accordingly, such an approach is in particular useful in a gene therapeutic setting.
  • the molecule capable of activating a senescence program and to expressed by the effector gene may be CDKNIb, CDKN2b or CDKN2a.
  • said effector gene encodes a molecule that is capable of activating a differentiation program, whereby said molecule may be selected from the group consisting of CEBPA (drives granulocytic differentiation), PU.1 (drives monocytic differentiation), and GATA-1 (drives erythroid differentiation).
  • CEBPA drives granulocytic differentiation
  • PU.1 drives monocytic differentiation
  • GATA-1 drives erythroid differentiation
  • the effector gene encodes a molecule that is capable of activating apoptosis, whereby said molecule may be a caspase, a cytochrom-C and Fas receptor.
  • said fusion protein comprising at least two epitopes is the oncogenic fusion protein BCR/ABL.
  • Said fusion protein comprises two known forms (p210 and p190). The uses described herein are envisaged to be employed in the detection/in the screening for the p190 version as well as the p210 protein. Also the pharmaceutical and medical uses are to be employed on both version BCR/ABL (ABL/BCR).
  • the interaction domain capable of interacting with said first epitope derived from BCR may be selected from the group consisting of a specific antibody part, an antibody construct in intracellular antibodies, an aptamer, BAP1 , GRB2, xeroderma pigmentosum group B protein, GRB10 (only to BCR/ABL), c-Fes, Ras GTPase activating protein, phospholipase C-gamma, 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, Abl (-fragments), Gads. RhoA GTP, RAC1, BCR and ARG or (a) fragment(s) of said interacting molecules.
  • said fragment of BAP-1 capable of interacting with said first epitope of BCR is cBAP-1 or BAP ⁇ 7-9.
  • BAP1 Pigments, Cell Signal 11 (1999), 507-14
  • GRB2 Million, Blood 15 (2000), 664-70
  • xeroderma pigmentosum group B protein Maru, Biochem. Biophys. Res. Commun. 260 (1999), 309-12
  • GRB10 only to BCR/ABL
  • Oncogene 17 1998, 941-8
  • c-Fes cters, (1999) loc.
  • Ras GTPase activating protein (Liu, Oncogene 17 (1998), 3073-82); phospholipase C-gamma (Peters, (1999), loc. cit.); 85,000 M(r) subunit of phosphatidylinositol 3'-kinase (Peters, (1999), loc. cit.); Abl (Muller, Mol. Cell Biol. 12 (1992), 5087-93; Pendergast, Cell 66 (1991), 161-71); Gads (Liu (1998) loc. cit.); RhoA GTP (Zhang, Biochemistry 14 (1998), 5249-57); RAC1 (Zhang, J. Biol. Chem. 273 (1998), 8776-82); BCR (Lu, Blood 82 (1993), 1257-63); ARG (Muller (1992), loc. cit).
  • the interaction domain capable of interacting with said second epitope derived from ABL may be selected from the group consisting of a specific antibody part, an antibody construct, an intracellular antibody, an aptamer, N-terminal 80 amino acids of ABL, Vav, Ikappa alpha which is directed against the to c-ABL SH2 domain, DDB1 , VASP, BRCA1 , RAD9 c-terminus which is directed against the c-ABL SH3 domain, ik3-1 , ik3-2, caveolinl , RIN1 , Abi-1 , EphB2 receptor, phospholipid scramblase 1 , CRKL, SORBS1 , Amyloid precusor protein, Fe65, HPK1 , p21-activated protein kinase (PAK) family, gamma-PAK, Ggrgb4, hTERT, Cbl, N-methyl-D-aspartic acid receptor NR2D subunit, protein kinas
  • TrkA TrkA, p53, p73, ATM, p62(dok), Ku70, SHPTP1 , JAK1 , ST5, RFXI, E2F- 1 , RB, receptor tyrosine kinase p145c-kit, She, cyclic AMP response element (CRE)-binding protein (CREB), CRK, actin, EGFR, HCK, and SYP or (a) fragment(s) of said interacting molecules.
  • said fragment of ABL to be used as interactor with an epitope an BCR/ABL comprises the N-terminal part of ABL and the fragment of CRKL interacting with ABL in BCR/ABL comprises the SH3 domain (CRKL-SH3n).
  • N-terminal 80 amino acids of ABL (Pluk, Cell 108 (2002), 247-59); Intracellular antibodies (Tse, J. Mol. Biol. 317 (2002), 85-94); Vav (Bassermann, J. Biol. Chem. 277 (2002), 12437-45); Ikappa alpha (to c-ABL SH2 domain) (Kawai, Mol. Cell Biol. 22 (2002), 6079-88); DDB1 (Cong, J. Biol. Chem. (2002) [epub ahead of print]); VASP (Howe, J. Biol. Chem. (2002) [epub ahead of print]); BRCA1 (Foray, Mol. Cell Biol.
  • RAD9 c-terminus (c-ABL SH3 domain) (Yoshida, Mol. Cell Biol. 22 (2002), 3292-300); ik3-1 , ik3-2 (Sato, Biochim. Biophys Acta 1574 (2002), 157-63); caveolinl (is substrate of c-ABL) (Sato (2002), loc. cit.); RIN1 (Tall, Dev. Cell 1 (2001), 73-82); Abi-1 (Ikeguchi, Oncogene 20 (2001), 4926-34); EphB2 receptor (Yu, Oncogene 20 (2001), 3995-4006); phospholipid scramblase 1 (Sun, J. Biol. Chem.
  • TrkA Koch, FEBS Lett. 469 (2000), 72-6
  • p53 Nie, Mol. Cell Biol. 20 (2000), 741-8
  • p73 Agami, Nature 399 (1999), 809-13
  • ATM Choen, J. Biol. Chem. 274 (1999), 12748-52; Shafman, Nature 387 (1997), 520- 3
  • p62(dok) Bhat, J. Biol. Chem. 273 (1998), 32360-8
  • Ku70 Kumaravel, Int. J. Radiat Biol.
  • a further, illustrative protein comprising at least two epitopes and being comprised in the complex of the invention is AML1/ETO.
  • Corresponding interaction domain capable of interacting with said first epitope derived from AML1 may, inter alia, be selected from the group consisting of a specific antibody part (or a fragment thereof), an antibody construct, an intracellular antibody, an aptamer, CEBPA, PU.1 , MORF, CBFB, HES-1 , BSAP, MITF, MEF, TLE and P300, or (a) fragment(s) of said interacting molecules.
  • a functional fragment of an interactor must be capable of specifically interacting with at least one epitope. For example a functional fragment of PU.1 (an interactor for AML1 PU.1 ( ⁇ 3- ⁇ 4)).
  • AML1 interactors are known in the art, see, e.g., CEBPA (Petrovick, Mol Cell Biol. 18 (1998), 3915-25; Zhang, Mol Cell Biol. 16 (1996), 1231-40), PU.1 (Petrovick, Mol Cell Biol. 18 (1998), 3915-25; Zhang, Mol Cell Biol. 16 (1996), 1231-40), Aptamers, MORF (Pelletier, Oncogene 21 (2002), 2729-40), CBFB (Warren, EMBO J. 19 (2000), 3004-15), HES-1 (McLarren, J Biol Chem.;275 (2000), 530-8), BSAP (Libermann, J Biol Chem.
  • the interaction domain capable of interacting with said first epitope derived from ETO may be selected from the group consisting of a specific antibody part (or a fragment thereof), an antibody construct, an intracellular antibody, an aptamer, N-CoR, importin ⁇ , importin ⁇ , ETO, mSin3A and SMRT, or (a) fragment(s) of said interacting molecules.
  • the unique complex described herein provides for the first time for means and methods to detect and/or eliminate cells which express a detrimental fusion protein, e.g. an oncogenic fusion protein.
  • a detrimental fusion protein e.g. an oncogenic fusion protein.
  • the introduction of a "protein A” and a "protein B” as defined above into a cell comprising and/or expressing such a fusion protein can drive the expression of a specific marker gene (e.g. for detecting said cell) or an effector gene which makes said cell susceptible to certain drugs (e.g.
  • said effector gene may be a gene which is activated by the binding of the complex described herein to the corresponding gene activation domains, e.g. to a specific promoter.
  • the DNA-binding domain of the herein described “protein A” may be specifically engineered to bind to a specific, intracellular promoter which drives the expression of an endogenous "effector gene” as described herein.
  • the "proteins A and B" as used in the complex of the invention may be introduced via methods known in the art which are described herein. Said introduction may be the introduction of the protein compounds themselves, as well as the introduction of one or more vectors expressing said protein A and/or said protein B. As detailed herein, it is also feasible to introduce a further nucleic acid molecule which comprises a specific domain/motif on which the DNA binding domain of the A protein may bind as well as the nucleic acid sequence coding for a specific marker or effector gene.
  • an vector is introduced in the cell which is suspected to express a detrimental fusion proteins which comprises the coding sequences for both protein A and B, as well as the corresponding sequences for an effector gene, i.e. a vector or a nucleic acid molecule comprising a nucleic acid molecule comprising a binding site for said DNA-binding domain and an effector gene as well as the nucleic acid molecules coding for protein A/B or (a) functional fragment(s) thereof.
  • the present invention provides for unique genetic switches which are particularly useful in diagnostic as well as pharmaceutical settings. Accordingly, the present invention particularly provides for methods for the elimination of a cell comprising a fusion protein comprising at least two epitopes, preferably an oncogenic fusion protein, comprising the steps of: (a) contacting, introducing and/or expressing (into) a cell suspected of comprising such a fusion protein (with) a protein A and a protein B as defined herein or contacting, introducing and/or expressing (into) said cell (with) at least one nucleic acid molecule coding for a protein A and a protein B as defined herein and; (b) eliciting in said cell the expression of an effector gene or an cellular effector which leads to the elimination of said cell.
  • the methods described herein may be carried out in vivo, in vitro as well as ex vivo. It is, inter alia, envisaged to contact introduce or express ex vivo blood cells or bone marrow cells of a patient, preferably a human patient (with) the above described protein A and protein B (and, optionally, to contact said cell with (and/or) express in said cell) a nucleic acid molecule comprising a specific domain/motif on which the DNA binding domain of the A protein may bind as well as the nucleic acid sequence coding for a specific marker or effector gene).
  • a cell expressing a detrimental fusion protein as defined herein will be detected by the corresponding expression of a marker gene (e.g.
  • a luciferase, GFP and the like may easily detected by methods known in the art, like, e.g. FACS-analysis.
  • a corresponding embodiment is illustrated in the appended example and in particular in figure 29.
  • said cell, expressing said fusion protein as well as a protein A/B as defined herein will elicit the expression of an endogenous effector gene as defined herein, a gene which elicits a senescence or adipose program or will elicit the expression of an effector gene comprised on a further nucleic acid molecule introduced which comprises a specific domain/motif on which the DNA binding domain of the A protein may bind as well as the nucleic acid sequence coding for said effector gene.
  • the invention also and particularly provides for a "protein A” or a "protein B" as defined herein above.
  • Said “protein A” and/or said “protein B” is(are) useful in the methods and described herein. It is in particular preferred said "protein A” and/or said “protein B” is expressed in a cell that expresses a protein comprising two epitopes as defined above, preferably in a cell expression a fusion protein, more preferably a fusion protein which is oncogenic and/or leads to a malignant state of the cell.
  • a protein A and a protein B as defined herein in said cell the cell comprising said detrimental protein/fusion protein may be distinguished from those cells that do not express that protein/fusion protein in vivo. Corresponding methods are illustrated below as well as in the appended examples and figures.
  • the present invention relates to a nucleic acid molecule encoding a protein A or a nucleic acid molecule encoding a protein B as described and defined herein above and in the appended examples.
  • said nucleic acid molecule is selected from the group consisting of
  • nucleic acid molecule comprising a nucleic acid sequence as shown in any one of SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 or 35;
  • nucleic acid molecule encoding a polypeptide as shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36;
  • nucleic acid molecule encoding a functional fragment or a functional domain of the polypeptide as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36;
  • nucleic acid molecule which is at least 60% identical to the nucleic acid molecule as defined in (a), (b) or (c) and which encodes a protein A or a protein B of claim 34 or 35;
  • nucleic acid molecule which hybridises under stringent conditions with the complementary strand of the nucleic acid molecule as defined in (a) to (d).
  • the nucleic acid molecule encoding a "protein A” or a “protein B” may be a DNA or a RNA.
  • nucleic acid sequence means the sequence of bases comprising purine- and pyrimidine bases which are comprised by nucleic acid molecules, whereby said bases represent the primary structure of a nucleic acid molecule.
  • Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • hybridizes as used in accordance with the present invention may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent.
  • Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds) "Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington DC, (1985).
  • the setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art.
  • the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O.lxSSC, 0.1% SDS at 65°.
  • Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1 % SDS at 65°C.
  • the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.
  • variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hydridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid sequences which code for a functional protein A or protein B fragment and which have a length of at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably of at least 21 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 40 nucleotides and most preferably at least 60 nucleotides. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules.
  • a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g. C 0 t or R 0 t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).
  • complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • Complementarity between two single- stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single- stranded molecules.
  • the degree of complementartity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.
  • hybridizing sequences preferably refers to sequences which display a sequence identity of at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with a nucleic acid sequence as described above encoding a "protein A” and/or a "protein B” having a described mutation.
  • hybridizing sequences preferably refers to sequences encoding a "protein A” and/or a "protein B” having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, particularly preferred at least 90%, more particularly preferred at least 95%, even more particularly preferred at least 97% and most preferably at least 99% identity with an amino acid sequence of a "protein A” and/or a "protein B" sequence as described herein above.
  • the term "identical” or “percent identity” in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence.
  • the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length.
  • Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson (1994), Nucl. Acids Res. 2, 4673-4680) or FASTDB (Brutlag (1990), Comp. App. Biosci. 6, 237-245), as known in the art.
  • the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above- described hybridzing molecule.
  • the term "being degenerate as a result of the genetic code” means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.
  • a vector comprising the nucleic acid molecule encoding for a "protein A” and/or a "protein B" as defined herein is described.
  • Said vector is particularly useful for the diagnostic and pharmaceutic methods described herein.
  • Such a vector may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the nucleic acid molecules of the present invention may be inserted into several commercially available vectors.
  • Nonlimiting examples include plasmid vectors compatible with mammalian cells, such as pUC, pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1 neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag , plZD35, pLXIN and pSIR (Clontech) and plRES-EGFP (Clontech).
  • plasmid vectors compatible with mammalian cells such as pUC
  • vectors as described in the appended examples and figures are useful in the context of the present invention. These vectors are, e.g. pM1(-3), pVP, pcDNA3, pVP-FLAG5 or pVP- HA1(-3), etc.
  • Baculovirus vectors such as pBIueBac, BacPacz Baculovirus Expression System (CLONTECH), and MaxBacTM Baculovirus Expression System, insect cells and protocols (Invitrogen) are available commercially and may also be used to produce high yields of biologically active protein, (see also, Miller (1993), Curr. Op. Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: A Laboratory Manual, p. 127).
  • prokaryotic vectors such as pcDNA2; and yeast vectors such as pYes2, pACT2, pGBT9, pGBKT7, pGAD424, or pGAD-GH are nonlimiting examples of other vectors suitable for use with the present invention.
  • vector modification techniques see Sambrook and Russel (2001), loc. cit.
  • Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, e. g. antibiotic resistance, and one or more expression cassettes.
  • the coding sequences inserted in the vector can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e. g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods.
  • transcriptional regulatory elements e. g., promoters, enhancers, and/or insulators
  • the vectors may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts.
  • control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site or internal ribosomal entry sites (IRES) (Owens (2001), Proc Natl Acad Sci USA 98,1471-1476) for introducing an insert into the vector.
  • the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Particularly preferred are in this context control sequences which allow for correct expression in blood cells or bone marrow cells.
  • Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art and also described in the appended examples. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymidine kinase promoter, SV40, RSV- promoter (Rous sarcome virus), human elongation factor 1 ⁇ -promoter, CMV enhancer, CaM-kinase promoter or SV40-enhancer.
  • CD34 promoter which is active in stem cells (Okuno et al, (Blood. 2002) ;100(13):4420-6; Radomska et al., Blood. (2002) 100(13):4410-9), MRP8 promoter, which is active in early myeloid cells (Yuan et al., PNAS USA (2001) 98(18): 10398-403) and many other promoters/enhancers that are specifically acitve in the hematopoietic compartment can readily be found described in the literature
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI, pcDNA3 (In-Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, such as lambda gt11.
  • An expression vector according to this invention is at least capable of directing the replication, and preferably the expression, of the nucleic acids and protein of this invention.
  • Suitable origins of replication include, for example, the Col E1 , the SV40 viral and the M 13 origins of replication.
  • Suitable promoters include, for example, the cytomegalovirus (CMV) promoter, the lacZ promoter, the gal 10 promoter and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter.
  • Suitable termination sequences include, for example, the bovine growth hormone, SV40, lacZ and AcMNPV polyhedral polyadenylation signals.
  • selectable markers include neomycin, ampicillin, and hygromycin resistance and the like.
  • Specifically-designed vectors allow the shuttling of DNA between different host cells, such as bacteria-yeast, or bacteria-animal cells, or bacteria-fungal cells, or bacteria invertebrate cells.
  • said vector may also be, besides an expression vector, a gene transfer and/or gene targeting vector.
  • Gene therapy is one of the pharmaceutical methods envisaged in context of this invention and provides for means and methods for the selection and/or elimination of cells comprising a detrimental protein comprising at least two epitopes as defined above.
  • Gene therapy which is based on introducing therapeutic genes (for example for vaccination) into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • nucleic acid molecules of the invention and vectors as described herein above may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.
  • viral vectors e.g. adenoviral, retroviral
  • baculoviral systems or systems based on vaccinia virus or Semliki Forest Virus can be used as eukaryotic expression system for the nucleic acid molecules of the invention.
  • fragments of the protein, the fusion protein or antigenic fragments of the invention may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) Solid Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc.
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer. Various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • the present invention in addition relates to a host transformed with a vector of the present invention or to a host comprising the nucleic acid molecule of the invention.
  • Said host may be produced by introducing said vector or nucleotide sequence into a host cell which upon its presence in the cell mediates the expression of a protein encoded by the nucleotide sequence of the invention or comprising a nucleotide sequence or a vector according to the invention wherein the nucleotide sequence and/or the encoded polypeptide is foreign to the host cell.
  • nucleotide sequence and/or the encoded polypeptide is either heterologous with respect to the host, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host but located in a different genomic environment than the naturally occurring counterpart of said nucleotide sequence. This means that, if the nucleotide sequence is homologous with respect to the host, it is not located in its natural location in the genome of said host, in particular it is surrounded by different genes. In this case the nucleotide sequence may be either under the control of its own promoter or under the control of a heterologous promoter.
  • the location of the introduced nucleic acid molecule or the vector can be determined by the skilled person by using methods well-known to the person skilled in the art, e.g., Southern Blotting.
  • the vector or nucleotide sequence according to the invention which is present in the host may either be integrated into the genome of the host or it may be maintained in some form extrachromosomally. In this respect, it is also to be understood that the nucleotide sequence of the invention can be used to restore or create a mutant gene via homologous recombination.
  • Said host may be any prokaryotic or eukaryotic cell. Suitable prokaryotic/bacterial cells are those generally used for cloning like E. coli, Salmonella typhimurium, Serratia marcescens or Bacillus subtilis. Said eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell, a plant cell or a bacterial cell (e.g., E coli strains HB101 , DH5a, XL1 Blue, Y1090 and JM101). Eukaryotic recombinant host cells are preferred.
  • eukaryotic host cells include, but are not limited to, yeast, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichia pastoris cells, cell lines of human, bovine, porcine, monkey, and rodent origin, as well as insect cells, including but not limited to, Spodoptera frugiperda insect cells and Drosophila-derived insect cells as well as zebra fish cells.
  • yeast e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichia pastoris cells
  • insect cells including but not limited to, Spodoptera frugiperda insect cells and Drosophila-derived insect cells as well as zebra fish cells.
  • Mammalian species-derived cell lines suitable for use and commercially available include, but are not limited to, L cells, CV-1 cells, COS-1 cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HeLa cells (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • said mammalian cell is a leukocyte, a bone marrow cell, blood cell and/or a cultured cell like, inter alia, a HEK 293 (human embryonic kidney) cell, a CHO, HeLa, NIH3T3, BHK, PC12 cell or a cultured blood cell, like 32D cells (murine myeloid cells), BaF3 cells (murine pre B cells, FDCP-mix (murine stem cell / progenitor cells, Jurkat (human T-ALL cell line or U937 (human myeloid/monocytic cell line) Most preferably said cells are derived from a mammal and more preferably from a human.
  • said amphibian cell is an oocyte.
  • said oocyte is a frog oocyte, particularly preferred a Xenopus laevis oocyte.
  • said host according to the invention may also be a non-human transgenic organism.
  • Said non-human organism may be a mammal, amphibian, a fish, an insect, a fungus or a plant.
  • Particularly preferred non-human transgenic animals are Drosophila species, Caenorhabditis elegans, Xenopus species, zebra fish, Spodoptera frugiperda, Autographa califomica, mice and rats.
  • Transgenic plants comprise, but are not limited to, wheat, tobacco, parsley and Arabidopsis.
  • Transgenic fungi are also well known in the art and comprise, inter alia, yeasts, like S. pombe or S. cerevisae, or Aspergillus, Neurospora or Ustilago species.
  • the present invention relates to a method for producing the polypeptide encoded by a nucleic acid molecule of the invention comprising culturing/raising the host of the invention and isolating the produced polypeptide.
  • the invention also relates to a method for the preparation of a protein A and/or a protein B as defined herein comprising culturing the host described above under conditions that allow synthesis of said protein A and/or protein B or (a) fragment(s) thereof and recovering said said protein A and/or protein B or (a) fragment(s) from said culture.
  • the host is a unicellular organism or a mammalian or insect cell, the person skilled in the art can revert to a variety of culture conditions that can be further optimized without an undue burden of work.
  • the produced protein is harvested from the culture medium or from isolated (biological) membranes by established techniques.
  • the produced polypeptide may be directly isolated from the host cell. Said host cell may be part of or derived from a part of a host organism, for example said host cell may be part of the blood system of an animal. Additionally, the produced polypeptide may be isolated from fluids derived from said host, like blood, milk or cerebrospinal fluid.
  • the present invention relates to a polypeptide that is encoded by a nucleic acid molecule of the invention or produced by the method of the invention.
  • the polypeptide of the invention may accordingly be produced by microbiological methods or by transgenic mammals. It is also envisaged that the polypeptide of the invention is recovered from transgenic plants. Alternatively, the polypeptide of the invention may be produced synthetically or semi-synthetically.
  • nucleotide acid sequences comprising all or a portion of any one of the nucleotide sequences according to the invention can be synthesized by PCR, inserted into an expression vector, and a host cell transformed with the expression vector. Thereafter, the host cell is cultured to produce the desired polypeptide, which is isolated and purified.
  • Protein isolation and purification can be achieved by any one of several known techniques; for example and without limitation, ion exchange chromatography, gel filtration chromatography and affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, preparative disc gel electrophoresis.
  • cell-free translation systems can be used to produce the polypeptides of the present invention. Suitable cell-free expression systems for use in accordance with the present invention include rabbit reticulocyte lysate, wheat germ extract, canine pancreatic microsomal membranes, E. coli S30 extract, and coupled transcription/translation systems such as the TNT-system (Promega).
  • protein isolation/purification techniques may require modification of the proteins of the present invention using conventional methods. For example, a histidine tag can be added to the protein to allow purification on a nickel column. Other modifications may cause higher or lower activity, permit higher levels of protein production, or simplify purification of the protein.
  • nucleic acid sequences/molecules described above e.g. the nucleic acid molecules coding for a "protein A” and/or a “protein B” as defined herein may be comprised on a single nucleic acid molecule and/or a single vector.
  • said vector may, in addition, comprise a nucleic acid molecule coding for an effector gene as defined above.
  • a method to delivery the components of the protein detection system of the present invention and the effector components to the cells is in the form of DNA vectors encoding the corresponding proteins.
  • the whole range of gene delivery systems can be used (naked DNA, gene guns, Iiposomal transfection reagents, electroporation and viral vectors).
  • One advantage of the present invention is that the three components of the system (protein A, protein B, and effector gene) do not have to be very large and could be coded for in as little as 3 kilobase pairs (kbp) or less. Proteins A and B can be made very small if one were to use oligopeptides (aptamers) as specific interaction partners for the parts of the fusion protein.
  • the DNA binding domain in protein A is encoded in less than 500 bp of DNA and the, e.g. VP16 activation domain in less than 200 bp.
  • Each aptamer would not need more than 50 bp of coding region.
  • the largest portion of the coding region would be used for the effector gene.
  • the system is thus sufficiently compact to be even delivered by e.g. adeno associated virus gene delivery systems which have a carrying capacity of about 4 kbp (or by other delivery systems described above).
  • a cellular effector gene is to be activated or an activated effector protein is to be generated by proteolytic cleavage
  • all the components of the system could be delivered as proteins.
  • the interacting regions in a protein comprising at least two epitopes as defined herein that are recognized by the interactor molecules should be close together on the fusion protein. If such close proximity can be achieved it might be possible to attach a molecule to interactor X that would activate another molecules that is attached to interactor Y. The activated molecule of interactor Y could then act, for example, like a cytotoxic drug.
  • the invention also provides for a composition
  • a composition comprising a complex, a protein A, a protein B, a nucleic acid molecule, a vector, a host cell and/or nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined herein above and as illustrated in the appended examples.
  • said composition is a pharmaceutical or a diagnostic composition.
  • said composition is particularly useful in detecting a cell which comprises a detrimental protein comprising at least two epitopes, e.g., a fusion protein, most preferably an oncogenic fusion protein, like BCR/ABL.
  • a detrimental protein comprising at least two epitopes, e.g., a fusion protein, most preferably an oncogenic fusion protein, like BCR/ABL.
  • Leukemias are often caused by fusion proteins.
  • the present invention it is envisaged to, inter alia, use the described detection system of the invention to purge patients' bone marrow extracorporally or to apply the inventive system in a systemic fashion to eliminate malignant cells in the patient or to eliminate such cells ex vivo.
  • the (fusion) protein detection system of the invention will first be used extracorporally to purge the, e.g. bone marrow of patients with e.g. chronic myeloid leukemia of all malignant cells.
  • Bone marrow would be harvested from the patients prior to an intensive myeloablative chemotherapy regimen.
  • the chemotherapy regimen would be so intensive that the patients would die without receiving a bone marrow transplant.
  • the patients will receive their own purged bone marrow.
  • one would be free to use a variety of different techniques (see above) to introduce the components of the detection/complex system of the invention into the bone marrow cells and use a variety of different ways to exploit the activation of effector genes.
  • the technologies to achieve the required gene transfer efficiencies in such a setting are well within the capabilities of today's technologies.
  • the detection system/complex system of the invention In order to employ the detection system/complex system of the invention directly in vivo, in the patient, one would have to be able to efficiently introduce the components of the detection system into the majority of, e.g. haematopoietic cells and especially into stem cells and progenitor cells. Ways of such an introduction are provided herein above.
  • One problem that may arise in the course of detecting a (fusion) protein or another protein comprising two epitopes as defined above is that the interactor proteins that are part of proteins A and B may interact with other cellular proteins and that this interaction either disturbs the interaction between the interactors and the fusion protein or that it prevents the trimeric recognition complex to efficiently translocate into the nucleus and initiate transcription of the effector genes.
  • This problem can be solved in, e.g. the following ways: (1) The interactor proteins can be reduced to the actual interacting protein domain. As documented in the appended examples, this strategy has been successfully employed in the case of CRKL, from which only the N-terminal SH3 domain was used. (2) Furthermore, the need for nuclear translocation of the trimeric recognition complex of the invention can be circumvented if a proteolytic cleavage event is used to initiate the action step.
  • IRS internal ribosome entry site
  • GFP green fluorescence protein
  • neomycin resistance gene all the cells containing the resistance gene could be selected for with the antibiotic G418. In this way a positive negative selection could be employed.
  • BCR/ABL positive cells will be sensitive to gancyclovir whereas BCR/ABL negative cells will survive gancyclovir treatment.
  • positive and negative selection systems e.g. Fluorescence activated cell sorting when using GFP as a positive transfection maker
  • the invention also provides for the use of a composition comprising a complex, a protein A, a protein B, a nucleic acid molecule, a vector, a host cell and/or nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined herein for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of a proliferative disorder.
  • Said proliferative disorder may be cancer or a tumorous disease. Most preferably, said proliferative disorder is a carcinoma disease, a sarcoma- disease, a lymphoma disease, a lipoma disease, or a leukemia.
  • Said proliferative disorder may be AML (acute myeloid leukemia), mixed lineage leukemia, J, T-ALL (acute T cell Iymphoblastic leukemia), B-ALL (actue B cell lymphoblastic leukemia), CMMoL (chronic myelomonocytic leukemia), AL (acute leukemia), MPD (myedodysplastic syndrome), CML (chronic myeloid leukemia), MPS (Myelo proliferative syndrome), CLL (chronic lymphocytic leukemia), MALT-lymphoma, adenocarcinoma, alveolar (soft part) sarcoma, fibrosarcoma, nephroma, RAEB-T (refractory anemia with excess blasts in transformation), clear cell sarcoma, rhabdomyosarcoma, papillary renal cell carcinoma.
  • AML acute myeloid leukemia
  • mixed lineage leukemia J
  • T-ALL
  • the invention also provides for the use of a protein A, a protein B, a nucleic acid molecule encoding a "protein A” and/or a “protein B” and/or a nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined above, for the preparation of a diagnostic composition for the detection of a protein comprising at least two epitopes, in particular a (fusion) protein, most preferably an oncogenic fusion protein like BCR/ABL.
  • these compounds are employed in in vivo and/or in vitro methods for the detection of a protein comprising at least two epitopes as defined above or for the detection of a cell comprising such a protein, said method comprising the steps of: (a) introducing and/or expressing, a protein A and a protein B as defined herein in cell suspected to comprise a protein comprising at least two epitopes, e.g. a fusion protein;
  • This method(s) may also comprise, as step (aa) the additional introduction of a nucleic acid molecules which comprise a binding site for a DNA-binding domain of "protein A” and an effector gene as defined above,
  • (a) method(s) is (are) provided which are useful in the detection/recognition of the in vivo presence of detrimental (fusion) proteins in a cell.
  • detrimental (fusion) proteins in a cell.
  • the complex of detrimental fusion protein, protein A and protein B leads to the activation of certain effector genes as defined herein and as illustrated in the figures and examples.
  • these effector genes may then produce proteins that could act as surface antigens to mark the malignant cell, that could convert a prodrug into an active metabolite or that could initiate the cell death program. This would either facilitate the physical removal of the malignant cells, direct an immune response against the malignant cells, to sensitize the malignant cells against a chemotherapeutic agent or cause the malignant cell to self-destruct.
  • the recognition of the fusion protein may be achieved through protein-protein interactions.
  • the effector genes may be transcriptionally activated through a protein complex consisting of three proteins with the fusion protein being the central bridge mediating between a DNA recognition domain and a transcription activation domain.
  • BCR/ABL fusion protein (p210), which is found in chronic myeloid leukemia, as an example, it could be demonstrated in the appended examples that this strategy is capable of metabolically marking yeast cells distinguishing cells which express the BCR/ABL fusion protein from cells that do not express the fusion protein. It could also be shown that mammalian cells expressing the BCR/ABL fusion protein may be distinguishable from cells that do not express the fusion protein using a reporter gene (luciferase) in transient transfection assays.
  • reporter gene luciferase
  • said effector gene may encode for
  • the methods described herein is employed in the detection and/or elimination of cells which comprise an oncogenic fusion protein, like BCR/ABL. Accordingly, said method(s) is (are) preferably used on blood cells or on bone marrow cells.
  • the present invention provides for novel methods to determine the absence or presence of detrimental (fusion) proteins in a cell.
  • the method may also be employed in the selection of stem cells which do not comprise such a detrimental protein, e.g. an oncogenic fusion protein. This is illustrated, but not limited, to an example for the BCR ABL fusion protein:
  • the expression of the BCR/ABL protein can be specifically inhibited by interfering with the BCR/ABL mRNA using anti-sense oligonucleotide or ribozymes.
  • the expression of BCR/ABL could be specifically inhibited by inducing DNA tripelhelix formation.
  • the function of the ABL tyrosine kinase in the BCR/ABL fusion protein could be inhibited using substances like STI571.
  • This invention also provides for a kit comprising
  • kits a nucleic acid molecule comprising a binding site for a DNA-binding protein (comprised in protein A) and an effector gene.
  • Said kit may also comprise the vectors described herein above. Said kit is particularly useful in practicing the methods of the invention.
  • the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific, medical or diagnostic assays or the like.
  • parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
  • the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
  • the invention also provides for a pharmaceutical composition
  • a pharmaceutical composition comprising
  • nucleic acid molecule comprising a binding site for a DNA-binding protein (comprised in protein A) and an effector gene.
  • Said pharmaceutical composition may also comprise (a) vector(s) comprising the nucleic acid molecules described herein.
  • said pharmaceutical composition comprises, optionally, a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein.
  • pharmaceutically acceptable means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the. like.
  • composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the aforementioned compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the pharmaceutical composition of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • In vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems.
  • the pharmaceutical composition is administered directly or in combination with an adjuvant.
  • the pharmaceutical composition is preferably designed for the application in gene therapy.
  • the technique of gene therapy has already been described above in connection with the nucleic acid molecules of the invention and all what has been said there also applies in connection with the pharmaceutical composition.
  • the nucleic acid molecule in the pharmaceutical composition is preferably in a form which allows its introduction, expression and/or stable integration into cells of an individual to be treated.
  • the present invention relates to a method of treating a oncological disease comprising administering a therapeutically effective amount of the pharmaceutical composition described herein above to a subject suffering from said disease. Yet, also the treatment of further disorders caused by a detrimental (fusion) protein is envisaged.
  • the term "subject” means an individual in need of a treatment of a neurological disease.
  • the subject is a vertebrate, even more preferred a mammal, particularly preferred a human.
  • administered means administration of a therapeutically effective dose of the aforementioned nucleic acid molecule encoding a functional protein A and/or protein B and, optionally, a nucleic acid molecule encoding for an effector gene as defined above and comprising a binding site for the DBD of protein A, to an individual.
  • therapeutically effective amount is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.
  • the methods are applicable to both human therapy and veterinary applications.
  • the compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein.
  • the compounds may be formulated in a variety of ways as discussed below.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %.
  • the agents maybe administered alone or in combination with other treatments.
  • the administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-arterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermally, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the candidate agents may be directly applied as a solution dry spray.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the dosages are preferably given once a week, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily.
  • the immune response is monitored using herein described methods and further methods known to those skilled in the art and dosages are optimized, e.g., in time, amount and/or composition. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 12 copies of the DNA molecule. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute. Progress can be monitored by periodic assessment.
  • the pharmaceutical composition of the invention may be administered locally or systemically.
  • Administration will preferably be parenterally, e.g., intravenously.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. It is also envisaged that the pharmaceutical compositions are employed in co- therapy approaches, i.e. in co-administration with other medicaments or drugs, for example other drugs for preventing, treating or amelioration an malignancy, like cancer, in particular a leukemia.
  • the invention also provides for a transgenic, non-human animal
  • Fig. 1 Schematic representation of the complex of the invention.
  • the abbreviations have the following meaning: FP: Fusion protein; X: one part of the FP, Y: second part of FP; A: Protein A; B: Protein B, J: Interactor X of Protein A; I: Interactor Y of Protein B; DBD: DNA Binding Domain of Protein A or a specific protease; AD: transcriptional activation Domain of Protein B or a polypeptide activated by proteolytic cleavage.
  • Fig. 2 Exemplified transcriptional activation of effector genes.
  • FP Fusion protein
  • X one part of the FP
  • Y the second part of FP
  • A Protein A
  • B Protein B
  • J Interactor X of Protein A
  • I Interactor Y of Protein B
  • DBD Binding Domain of Protein A
  • AD transcriptional activation Domain of Protein B
  • b binding site
  • EG Effector genes.
  • Fig. 3 Exemplified further activation modus.
  • the trimeric (recognition) complex directs proteolytic cleavage of protein B. this liberates/activates a polypeptide or a fragment thereof capable of eliciting a (cellular) response.
  • the proteolytic cleavage of protein B liberates, e.g., a transcription factor (left side) or may, as a further example, activate caspase(s) which can initiate, inter alia, apoptosis (right side).
  • FP Fusion protein
  • X one part of the FP
  • Y the second part of FP
  • A Protein A
  • B Protein B
  • J Interactor X of Protein A
  • I Interactor Y of Protein B
  • P Protease
  • L linker
  • AD Activation Domain
  • b binding site
  • N Nucleus
  • EG Effector gene
  • C i Caspase inactive
  • C a Caspase active.
  • FIG. 4 Schematic representation of BCR/ABL as fusion protein, BAP-
  • HIS3/lacZ As "read-out” or "effector gene” in this example HIS3/lacZ is chosen.
  • the abbreviations have the following meaning: FP: BCR/ABL; X: BCR, Y: ABL; A: Protein A; B: Protein B, J: BAP-1; I: CRKL-SH3n; DBD: GAL4-BDB; AD: GAL4-AD; , b: UAS GAL4 binding site; EG: HIS3, LacZ.
  • FIG. 5 Schematic representation of BCR/ABL fusion protein and CRKL-
  • SH3n/GAL4-BDB as "protein A” and BAP-1 /GAL4-AD as "protein B”.
  • the abbreviations have the following meaning: FP: BCR/ABL; X: ABL, Y: BCR; A: Protein A; B: Protein B, JI: CRKL-SH3n; I: BAP-1 ; DBD: GAL4-BDB; AD: GAL4-AD; b: UAS, GAL4 binding site; EG: HIS3, LacZ.
  • Fig. 8 Graphical representation of average normalized luciferase values of BCR/ABL detection experiments as well as negative and posisitve controls in N1H3T3 cells.
  • the plasmids that were used for the transfections are indicated below the columns with a "+" sign. 0.5 ⁇ g of each plasmid were employed. In addition, 0.1 ⁇ g of the plasmid pCMV- ⁇ GAL was included in every transfection as a control.
  • Fig. 9 Schematic representation of successful detection of BCR/ABL in mammalian cells.
  • the abbreviations have the following meaning: FP: BCR/ABL; X: ABL, Y: BCR; A: Protein A; B: Protein B, J: CRKL-SH3n; I: BAP-1; DBD: GAL4-BDB; AD: VP16; b: UAS, GAL4 binding site; EG: Luciferase.
  • Fig. 10 to 25 show sequences related to "protein A” and/or “protein B” employed in accordance with this invention in the complex formation with BCR/ABL.
  • pVP-FLAG5/BAP-1 Insert from: pVP-FLAG5/BAP-1 (nt 126-879); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); BAP-1 origin: Homo sapiens
  • pVP-FLAG5/CRKL Insert from: pVP-FLAG5/CRKL (nt 510-1422); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); CRKL origin: Homo sapiens
  • pVP-FLAG5/CRKL-SH3n Insert from: pVP-FLAG5/CRKL-SH3n (nt 867-1051 [HindUI site]); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); CRKL origin: Homo sapiens Fig. 20 Sequence ID No: 21 and 22 (nucleic acid and amino acid)
  • pVP-FLAG5/cBAP-1 Insert from: pVP-FLAG5/cBAP-1 (nt 441-879); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain); origin: Viral (herpes simplex virus); BAP-1 origin: Homo sapiens
  • pVP-FLAG5/ BAP 7-9 Insert from: pVP-FLAG5/ BAP 7-9 (nt 617-879); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); BAP-1 origin: Homo sapiens Fig. 26 Normalized relative luciferase values obtained with the BCR/ABL detection system after transient transfection in 293 cells (mammalian cells). Columns 1 and 2, 3 and 4, 5 and 6, 7 and 8 correspond to experiments No 1 , 2, 3, and 4 in Table 1.
  • Fig. 27 and 28 Sequences related to "protein A” and/or “protein B”employed in accordance with this invention in the complex formation with AML1/ETO Fig. 27 Sequence ID No: 33 and 34 (nucleic acid and amino acid)
  • Fig. 29 Use of inventive genetic switch complex in the murine myeloid cell line 32D (a hematopoietic cell line) and a 32D derived cell line (32DP210) that is constitutively expressing the BCR/ABL (P210) fusion protein.
  • 32D a hematopoietic cell line
  • 32DP210 a 32D derived cell line
  • effector gene luciferase is employed.
  • Example I General principle of the inventive protein detection system
  • the detection system works in two distinct steps: (1) the protein to be detected is recognized; and (2) an action is initiated.
  • the recognition of the protein of choice is accomplished by forming a trimeric protein complex in which the (fusion) protein ("X") is contacted by protein A which consists of a protein domain (interactor X) that can interact with one part of the fusion protein fused to a DNA binding domain and by protein B which consists of a protein domain (interactor Y) that can interact with the other portion of the (fusion) protein (“Y”) fused to a transcriptional activation domain.
  • This recognition complex is shown in appended figure 1.
  • step 2 After the recognition complex is formed an action is initiated (step 2). If Protein A contains a DNA binding domain and protein B a transcriptional activation domain the recognition complex will form a transcriptional activation complex that is able to activate the transcription of (an) effector gene(s) that is/are under the control of the appropriate DNA binding site. This is shown in appended figure 2.
  • the recognition complex may also direct a specific proteolytic cleavage.
  • protein A consists of interactor X fused to a site specific protease and protein B consists of interactor Y linked to, e.g., a transcription factor by a (protease-) cleavable linker, an effector protein or a protein which is activated by proteolytic cleavage.
  • protein A cleaves protein B and releases either a transcription factor or an effector protein (e.g. an active caspase). This situation is shown in appended figure 3.
  • Example II Detection of the oncogenic fusion protein BCR/ABL
  • a strategy was developed by which cells that express the BCR/ABL fusion protein can be distinguished from those cells that do not express BCR/ABL in vivo.
  • an inventive genetic switch was designed which was first tested in yeast cells and then employed in mammalian cells according to the detection system illustrated in Example I.
  • BCR/ABL The recognition of BCR/ABL was achieved through protein-protein interaction.
  • BCR/ABL was contacted by two proteins.
  • One protein (protein A) consists of a protein that interacts with BCR, for example BAP-1.
  • BAP-1 was fused to the DNA binding domain of the yeast transcription factor GAL4 (other DNA binding proteins may be used, as illustrated in this invention).
  • the other protein (protein B) consists of a protein that interacts with ABL, e.g. CRKL (other ABL interactors may be employed), which was fused to the transcriptional activation domain of the yeast transcription factor GAL4 or VP16 in mammalian cells(other transcriptional activation domains can be used).
  • BCR/ABL Only in cells expression BCR/ABL a trimeric recognition complex as illustrated in this invention is formed consisting of protein A, protein B and the protein to be detected, BCR/ABL.
  • the recognition complex can also be formed if BCR contacting protein is fused to a transcriptional activation domain and the ABL contacting protein is fused to a specific DNA binding domain. It is of note that the "protein A” and “protein B” function may also be reversed (see appended Fig 5).
  • This recognition complex is then able to activate the transcription of a reporter/effector gene or reporter/effector genes that have an appropriate transcription factor binding site in their promoter region. It is of note that preferably multimeric binding sites on the DNA level are employed.
  • the HIS3 and the LACZ gene as reporter genes/effector genes were used. Transcription of HIS3 allows the yeast strain CG1945 to grow on plates that lack the amino acid histidine. If the HIS3 gene in CG1945 is not activated the cells are not able to grow on plates lacking histidine. The activation of the LACZ gene leads to the production of the enyzme beta-galactosidase which can be visualized by a special-, known staining technique.
  • Example III Material and Methods for the illustrative example BCR/ABL
  • the BAP-1 coding region was amplified with primers BAT.T125.Eco and
  • the CRKL coding region was amplified with primers CRKL.T509Sma and
  • the plasmid pM1/CRKL-SH3n (see below) was cut with HindUI blunt ended and then cut with EcoRI. The fragment containing the CRKL-SH3n (N-terminal SH3 domain of CRKL) was then ligated with EcoRI and Smal cut pGBT9.
  • the BAP-1 coding region was amplified with primers BAP.T125.Eco and
  • PGAD424/ CRKL The CRKL coding region was amplified with primers CRKL.T509Sma and CRKLB1412Sal from the plasmid E1.7K15-6SK (ten Hoeve, Oncogene 8 (1993), 2469-2474) restricted with Smal and Sail and ligated with Smal and Sail restricted pGAD424.
  • the plasmid pM1/CRKL-SH3n (see below) was cut with Hindlll blunt ended and then cut with EcoRI. The fragment containing the CRKL-SH3n (N-terminal SH3 domain of CRKL) was then ligated with EcoRI and Smal cut pGAD424.
  • pES1 yeast expression vector
  • pES1 was constructed on the basis of pGAD424 by removing the GAL4 activation domain and the multiple cloning site through a Hindlll digest and replacing it with a modified (restricted with Clal and EcoRV, blunt ended and religated) polylinker from pBluescript-SK II (Clontech) which was inserted into the blunted Hindlll sites of pGAD424 after BssHII excision and blunt ending from pBluescript-SK II (modified).
  • the LEU2 gene was partially removed from the modified pGAD424 through a Clal and EcoRV restriction and it was replaced by the LYS2 gene (from plasmid pDP6 (Dieter Gallwitz, MPI- Biophysikalische Chemie, G ⁇ ttingen; Eibel Mol. Gen. Genet. 191 (1983), 66-73) with modified restriction sites after it was cloned into pBluescript-SK II to obtain Smal and Clal sites flanking the gene.
  • BCR/ABL was cloned into the EcoRI site of pES1 from pcDNA3/BCR/ABL (Warmuth, J. Biol. Chem. 272 (1997), 33260-33270) by cutting the insert with EcoRI.
  • Yeast strain CG1945 (Clontech) already contains the reporter genes HIS3 and LACZ under the control of a GAL4 DNA binding site.
  • This yeast strain has the following genotype: MATa, trp1-901 , leu2-3, his3-200, gal4-542, LYS2::GAL1- HIS3, URA3::(GAL4 17-mere) 3 -CYC1-lacZ.
  • BAP-1 was excised from pGBT9-BAP-1 with EcoRI and Sail and cloned into
  • CRKL was excised from pGBT9-CRKL with Smal and Sail and cloned into Smal and Sail linearized pM1.
  • the CRKL-SH3n region was amplified with primers CRKL-SH3.T867 and
  • BAP-1 was excised from pGBT9-BAP-1 with EcoRI and Sail and cloned into
  • CRKL was excised from pGBT9-CRKL with Smal and Sail blunt ended and cloned into Hindlll cut, blunt ended pVP-FLAG5.
  • the CRKL-SH3n region was amplified with primers CRKL-SH3.T867 and
  • the pcDNA3/BCR-ABL expression vector was obtained from Michael Hallek, LMU, Kunststoff.
  • the plasmid pCMV- ⁇ GAL was used as a transfection control and the plasmid pBluescript-SKII(+) (pBSK) was used as filler to bring the total amount of plasmid in the transfection assays to a constant amount.
  • the plasmids pM1/BR-304 and pVP-HA/B202-NB were used as a positive mammalian two hybrid control (Wu (1996), loc. cit.)
  • Example IV Detection of BCR/ABL is a yeast system
  • SD -T, -L, -K, -H plates were used without 3-amino triazole (3AT) and SD -T, -L, -K, -H plates with 20 mM 3AT.
  • 3AT inhibits residual HIS3 gene acitivity so that a strong activation of the HIS3 reporter gene is required.
  • Figure 6 and 7 show the results of the assays described in table 2.
  • Example V Detection of BCR/ABL in a mammalian cellular system
  • plasmids described in Materials and Methods were employed. Said NIH 3T3 cells do not normally express the BCR/ABL fusion protein. Accordingly, a further plasmid (pcDNA3/BCR-ABL) has introduced which (artificially) drives the expression of BCR/ABL in said test system. About 1.4 x 10 5 cells in 35 mm plates were transfected with a total of 2.1 ⁇ g of plasmid DNA using Superfect transfection reagent. The luciferase measurements were corrected for transfection efficiency by using dividing the raw luciferase values by the measurements of the ⁇ - galactosidase from the same culture plate. The following combinations were employed:
  • reporter gene sequence As “reporter gene sequence” the above described G5E1bLUC was employed.
  • the strategy presented here is useful for the detection of fusion proteins (demonstrated for the BCR/ABL fusion protein) in vivo.
  • GFP as an effector gene it is possible to detect the presence of the BCR/ABL fusion protein in individual living cells.
  • Cells expressing BCR/ABL may be detected by greenfluorescence in UV light and could be sorted according to their BCR/ABL status. All other detection methods of BCR/ABL that are available today will only work on isolated DNA, RNA or proteins or on fixed cells, which means that the presence or absence of BCR/ABL can only be determined in cells that are non- viable.
  • Example V Minimal interaction domains for protein A and/or protein B
  • the N-terminal SH3 domain of CRKL was used in protein A (fused to the DBD of GAL4) and the full length BAP-1 protein in protein B (fused to the VP16 transcriptional activation domain) was it possible to detect BCR/ABL in NIH3T3 cells.
  • c-BAP-1 and BAPo.7-9 were then cloned into the mammalian expression vectors pM1 (to be expressed with the DNA binding domain of GAL4) and into pVP-FLAG5 (to be expressed with the VP16 transcriptional activation domain). Corresponding sequences are shown in Figures 23 to 25. Both c-BAP-1 and BAP 7-9 could be used successfully (either fused to the DNA binding domain or to the VP16 transactivation domain) to detect the presence of BCR/ABL in transfected cells (see table 4).
  • Example VII The use of additional nuclear localization signals
  • sequence coding for a nuclear localization signal can improve the performance of the detection system.
  • Example VIII The detection of a further fusion protein AML1/ETO
  • the fusion gene detection system is capable of detecting the presence of the BCR/ABL fusion protein in transiently transfected cells. Since the interactor portions of protein A and protein B are specific for the the two components of a fusion gene, specific proteins A and B have to be designed if a different fusion gene is to be detected. As an example the development of an AML1/ETO fusion gene detection system is described.
  • AML1 is a transcription factor and ETO is a transcriptional cofactor it was also necessary to test whether any of the components of or the complete AML1/ETO fusion protein would have transcriptional activation potential. To this end the AML1 and the ETO portions of AML1/ETO as well as the complete AML1/ETO were cloned in frame into the GAL4 DBD fusion expression vector pGBT9. These experiments showed that the pGBT9-AML1 contruct was able to transactivate the yeast reporter genes.
  • the interactor portions of the system (N-CoR for Protein A) and PU.1( ⁇ 3- ⁇ ) for Protein B) were cloned into the mammalian expression vectors pM1 and pVP-FLAG5, respectively.
  • the components and AML1/ETO were transiently expressed in mammalian cells (NIH3T3).
  • the expression of the reporter gene luciferase did not appreciably change between the presence and the absence of the AML1/ETO fusion gene.
  • EXAMPLE IX Exemplified use of the inventive genetic switch in cells expressing an oncogenic fusion protein.
  • u the murine myeloid cell line 32D (a hematopoietic cell line) and a 32D derived cell line (32DP210) that is constitutively expressing the BCR/ABL (P210) fusion protein were employed.
  • Protein A pM1-BAPD7-9 or pM1-CRKL-SH3n
  • protein B pVP-FLAG5- BAPD7-9 or pVP-FLAG5-CRKL-SH3n
  • the luciferase reporter G5E1 bLUC the luciferase reporter G5E1 bLUC
  • a plasmid expressing Renilla luciferase pRL-nulI, Promega
  • Two combinations of protein A and protein B were used: 1) pM1-BAPD7-9 and pVP-FLAG5-CRKL- SH3n (left two columns); 2) pM1-CRKL-SH3n and pVP-FLAG5-BAPD7-9 (right two columns).
  • a genetic switch as described herein can be employed to successfully drive the expression of effector genes capable of eliciting a cellular response which leads to either a specific signal in the cell comprising said fusion protein (e.g., the expression of a marker gene, like GFP or luciferase) or which may activate a cellular response, like the specific activation of, e.g., a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug, an immunomodulating molecule; an antigen, as well as a molecule capable of activating a senescence program, a differentiation program or apoptosis,

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a complex comprising a fusion protein (a) comprising at least two epitopes; (b) protein A comprising an interaction domain capable of interacting with said first epitope of the protein of (a) and comprising a first effector domain; and (c) protein B comprising an interaction domain capable of interacting with said second epitope of the protein of (a) and comprising a second effector domain whereby said interaction domains of protein A and protein B are not capable of directly interacting with each other. Furthermore, specific nucleic acid molecules encoding said protein A and/or said protein B are provided as well as expressed protein A/B molecules. In addition, compositions, in particular pharmaceutical and diagnostic compositions are described which comprise the members of the complex of the present invention. Finally, the invention provides for in vivo and/or in vitro methods for the detection or elimination of a fusion protein, more preferably an oncogenic fusion protein.

Description

Genetic switches for the detection of fusion proteins
The present invention relates to a complex comprising (a) a fusion protein comprising at least two epitopes; (b) protein A comprising an interaction domain capable of interacting with said first epitope of the protein of (a) and comprising a first effector domain; and c) protein B comprising an interaction domain capable of interacting with said second epitope of the protein of (a) and comprising a second effector domain whereby said interaction domains of protein A and protein B are not capable of directly interacting with each other. Furthermore, specific nucleic acid molecules encoding said protein A and/or said protein B are provided as well as expressed protein A/B molecules. In addition, compositions, in particular pharmaceutical and diagnostic compositions are described which comprise the members of the complex of the present invention. Finally, the invention provides for in vivo and/or in vitro methods for the detection of a protein, preferably a fusion protein, more preferably an oncogenic fusion protein.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions etc.) are hereby incorporated by reference.
There are many examples of fusion proteins found associated with specific types of cancer especially leukemias and certain entities of sarcomas.
Beginning in the early 80ties a growing number of translocations that result in fusion proteins has been characterized at the molecular level.
One example is the BCR/ABL fusion protein which is found in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). There are at the present time no cures for CML except for allogeneic bone marrow transplant and the situation is quite similar for ALL caused by the BCR/ABL fusion protein which is associated with a very poor prognosis.
More than 10 years ago it could be shown that the BCR/ABL fusion protein is able to cause leukemia in a transgenic mouse model. More recently, using an inducible BCR ABL transgenic mouse model, it could be demonstrated that the expression of the BCR/ABL fusion protein is necessary to maintain the malignant status of the transformed cells. Switching off the fusion gene reverted the malignant phenotype of the cells. Since the fusion proteins represent the fundamental difference between a normal and a transformed cells it seems logical to use these fusion protein as the preferred targets for specific therapeutic strategies. Several strategies have been used up to now in the case of the BCR/ABL fusion to achieve specficity for cells harboring the leukemic fusion. These strategies are based on targeting the BCR/ABL mRNA (anti sense oligonuceotides or BCR/ABL specific ribozymes), inhibiting the transcription of BCR/ABL (triple helix approach) or inhibiting the tyrosine kinase acitivity of the ABL moiety of BCR/ABL (tyrosine kinase inhibitors like STI571). However, all these strategies have in common that they will only lead to a reduction of BCR/ABL expression or an inhibition of the function of BCR/ABL but not to a complete elimination of BCR/ABL positive cells. The BCR/ABL antisense, ribozyme or triple helix approaches have not yet been used in clinical trials (ref Verfaillie et al., Mol Med Today 5:359-366 1999). Efforts to generate a specific immune response against the BCR/ABL fusion protein which would be sufficient for clinical applications have not been successful yet. The majority of intracellular processes are governed by protein-protein interactions. In PNAS 93:4192-4196, Da Caosta et al. 1996 showed that it is possible use a system which is based on protein-protein interaction to express the enzyme purine nucleoside phosphroylase (PNP) in cells that overexpress a mutant form of the tumor suppressor gene TP53. PNP converts the non-toxic 6- methylpurine desoxyribose into the toxic 6-methylpurine. Using this approach it was possible to selectively kill those cells that express the mutated form of TP53.
The prior art has described screening methods or detection systems which may be summarized as "two- or three hybrid systems". Most of these systems have the disadvantage that they only work in a limited number of eukaryotic cells, e.g. yeast cells, and that these methods and techniques are restricted to the detection of protein-protein complexes or to the detection of RNA-protein interactions.
For example, Bernstein and colleagues (Methods 26 (2002), 123-141) have developed a yeast-based three-hybrid system for the analysis of mRNA-protein complexes. Yet, the system is based on a trimeric complex which comprises a RNA binding domain that interacts with its RNA binding site in a bifunctional RNA molecule, whereas the other part of the RNA molecule interacts with a second hybrid protein consisting of another RNA binding domain linked to a transcription activation domain. This system is limited to the use in yeast cells and relates to the detection or confirmation of RNA-protein interactions.
WO 02/070662 provides for compositions and methods for isolating ligand binding polypeptides for a user-specified ligand and for isolating small molecule ligands for a user-specified target polypeptide. The used hybrid ligand in WO 02/070662 is characterized by a R1-Y-R2 structure, whereby R1 represents a first ligand, Y represents a polyethylene linker and R2 a user-specified second ligand. In particular WO 02/070662 describes a method whereby the interacting between a polypeptide and a small molecule ligand can be discovered. The "middle" compound in this three hybrid system is a complex small molecule consisting of a known small interactor and a variable other small molecule for which the interaction with a given other polypeptide or protein can be detected. This system is specifically designed to discover pharmacologically relevant small molecule-protein interactions in a screening method. Accordingly, WO 02/070662 relates to a method to identify cellular targets of pharmacological agents, in particular pharmacological agents displaying a given activity.
WO 02/070662 also discusses further prior art relating to "two- and/or three hybrid systems". In particular to Fields, Nature 340 (1989), 245-246; Gyuris, Cell 75 (1993), 791-803; US 5,468,614; Yang, Nuc. Acid Res. 23 (1995), 1152- 1156; WO 94/23025, WO 95/30012 or WO 97/41255. Most of this cited art is related to the elucidation of the interaction of proteins, since the elucidation of particular interacting protein partners has been advanced by the development of in vivo "two-hybrid" or "interaction trap" methods for detecting and selecting interacting protein partners (see Fields & Song (1989), loc. cit; Gyuris (1993), loc. cit., US 5,468,614 and Yang (1995), loc. cit.). These methods rely upon the reconstitution of a nuclear transcriptional activator via the interaction of two binding partner polypeptides - i.e. a first polypeptide fused to a DNA binding domain (BD) and a second polypeptide fused to a transcriptional activation domain (AD) and are limited to the use in yeast systems.
WO 94/23025 and WO 95/30012 describe a screening assay for identifying molecules capable of binding cell surface receptor so as to activate a selected signal transduction pathway. These references describe the modification of selected yeast signalling pathways so as to mimic steps in the mammalian signalling pathway.
US Patent Nos. 5,585,245 and 5,503,977 describe the "split ubiquitin" methods, which can detect protein-protein interactions by use of a ubiquitin specific protease to cleave a reporter polypeptide from a fusion protein. Two fusion proteins are constructed, one consisting of the N-terminal half of ubiquitin and a prey protein (Nub-prey or prey-Nub), and the other consisting of the C-terminal half of ubiquitin, a bait protein and the report (bait-Cub-reporter). Association of prey and bait reconstitutes a ubiquitin structure recognized by the ubiquitin specific protease, whereby the reporter is cleaved from the fusion protein. The cleavage of the reporter from the fusion protein can be detected by several techniques, e.g. cleavage or destabilizing the reporter or allow for its translocation.
Similarly, WO 97/41255 relates to a screening methods for small molecules that bind cellular targets so as to identify new drugs that are capable of specific therapeutic effects, whereby a ligand-hybrid (similar to WO 02/070662) is detected. Again, this system does not provide for detection means in mammalian cells and is limited to the detection of hybrid ligands.
US 5,503,977 relates to specific fusion proteins comprising an mutationally altered N-terminal subdomain of ubiquitin, whereby said fusion proteins or nucleic acid molecules encoding the same may be employed in methods for identifying interacting proteins or peptides. US 5,503,977 essentially describes a two hybrid system in which the effect of the interaction between two proteins is the reconstitution of ubiquitin as the target of the ubiquitin-specific protease. This protease (which seems to be present in all cells or has to be externally provided) cleaves off a reporter moiety. This proteolytic cleavage can be detected by immunoprecipitation, gel electrophoresis etc.
US 5,585,245 also describes the use ubiquitin subdomain fusion proteins which are useful for studying the interaction of two members of a specific binding pair or the determination of a predetermined ligand in a sample.
A further variant of the mammalian two hybrid system is described for the detection the indirect in vivo association of two proteins whereby said association is mediated by a third (or fourth) protein acting as a bridge between the two assayed proteins. (Osada et al. Cell 48:777-783 (1 995); Wadman et al. EMBO J 1 3:4831 -4839 (1 994)). Accordingly, Osada (1995) loc. cit. and Wadman (1994) loc. cit. use the mammalian two hybrid method for the analysis of multi protein complexes. All the proteins that are suspected to form a complex are transiently expressed in the assay system. The use of the bridge mammalian two-hybrid assay for studies of protein-protein interactions was also described in the review by Tsan "The yeast two hybrid system" Eds Bartel PL, Fields S: Oxford University Press, New York, Oxford, 1997:217-232).
As mentioned above, fusion proteins which are the result of chromosomal translocations play a pivotal role in the malignant transformation process in numerous types of leukemias and solid tumors. In some cases it could be shown that switching off the production of such a fusion protein in the cell will revert the malignant phenotype. However, there are only very few therapies available today that use these fusion proteins as a therapeutic targets.
Furthermore, there is a need for in vivo and/or ex vivo detection and elimination of cells comprising detrimental, e.g. oncogenic proteins (for example oncogenic fusion proteins).
Taken together there are, at the present time, no strategies described achieve a therapeutic effect in malignancies that are caused by (oncogenic) fusion proteins
The only examples where the prior art has tried to eliminate cells expressing oncogenic fusion proteins are some immunological approaches. Yet, as pointed out above, these approaches do not eliminate cells comprising (oncogenic) fusion proteins.
Accordingly, the technical problem underlying the present invention is the provision of means and methods for the elimination of cells comprising detrimental, e.g. oncogenic fusion proteins.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Thus, the present invention relates to a complex comprising
(a) a protein comprising at least two epitopes;
(b) a protein A comprising an interaction domain capable of interacting with said first epitope of the protein of (a) and comprising a first effector domain; and
(c) a protein B comprising an interaction domain capable of interacting with said second epitope of the protein of (a) and comprising a second effector domain whereby said interaction domains of protein A and protein B are not capable of directly interacting with each other.
In accordance with this invention, the term "complex" as used above relates to a specific protein interaction between the defined protein comprising at least two epitopes, the protein A and the protein B as defined herein. Said complex is preferably a complex formed in vivo in a mammalian cell, whereby most preferably said protein comprising said at least two epitopes is produced/expressed by said mammalian cell. Yet, said complex may also be formed ex vivo as well as in vitro. As will be detailed herein below, the complex as defined herein may be detected by specific means disclosed herein and may be useful for the elimination of a cell expressing said protein comprising two epitopes as defined herein. Accordingly, the invention relates in a preferred embodiment to a complex, wherein a first of said epitopes of the protein comprising at least two epitopes is encoded by a first gene (or a fragment thereof) and wherein a second of said epitopes is encoded by a second gene (or a fragment thereof). More preferably, said protein comprising at least two epitopes is a fusion protein, even more preferably said fusion protein is a fusion protein derived from a chromosomal translocation. In a most preferred embodiment, said fusion protein is comprised/expressed in a blood cell or a bone marrow cell.
Said protein comprising two epitopes and composed in the complex of the invention may be related to or may be associated with malignancy.
In a most preferred embodiment, said protein comprising two epitopes is oncogenic, even more preferred said protein is an oncogenic fusion protein.
In a specific aspect of the invention, a complex is described, wherein the first epitope of said protein comprising at least two epitopes is encoded by a gene (or a fragment thereof) selected from the group consisting of RNM15 (OTT), AF1 p (eps15), PAX7, MLL, AF4 (MLLT2), MSF (homologous to PNUTL1), GMPS, AF10 (MLLT10), PAX3, PBX1 , TFE3, ALK, HOXD13, HOXA9, RAP1GDS1 , NSD1 , PMX1, MLF1 , EAP, EVI1 , MDS1 , HMGIC, BTL (CHIC2), ACS2, PDGFRB, NPM, CAN, AF6 (MLLT4), FOP, JAZF1, HLXB9, MOZ, FGFR1 , ETO, LEDGF, CHN, CSMF, ABL, AF10, RET, PDGFRB, WT1 , NUP98, HOXD11 , FN1, API2, ETV6, EVI, CHOP, ATF1, PML, FUS, MYH11, MTG16, PLZF, TFE3, SSX1 , SSX2, HLF, LCX, SEPTIN6, JAK2, ABL2 and ARG; and wherein the second epitope of said protein comprising at least two epitopes is encoded by a gene (or a fragment thereof) selected from the group consisting of
MKL1 (MAL), MLL, FOX01A (FKHR), PNUTL1 (CDCrel), FBP17, LPP, AF9, MLLT3, GPHN (gephyrin), ENL, MLLT1, ABU (SSH3BP1), AFX1 (MLLT7), FOXA1A, E2A, PRCC, NUP98, NPM, ETV6, AML1 , RARA, DEK, FGFR1, JJAZ1 , TIF2, CBP, ZNF198, FIM, RAMP, BCR, P300, AML1, NUP98, TAF2N, RBP56, CALM, H4, EWS, DDX10, MALT1 , CBFA2, NTRK3, MN1 , FUS (TLS), EWS, CDX2, ERG, CBFB, CBFA2, RCC17, SYT, ELL, E2A, CDCREL1 , PSF, SYT, SH3GL1 (EEN) and NonO (p54nrb).
The person skilled in the art is aware of the fat that these proteins comprising at least two epitopes (wherein said epitopes are encoded by specific genes) often relate to proteins which are encoded by (oncogenic) fusion genes. A selection of corresponding fusion genes encoding for such (fusion-) proteins is given in Table 1 herein below. These fusion genes do not only relate to oncological disorders but also to further disorders as mentioned herein below.
Table I: Exemplified fusion genes encoding for fusion proteins
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
The cited references in the Table comprise:
1 Ma, Nature Genet. 28 (2001), 220-221
2 Mercher, Proc. Nat. Acad. Sci. 98 (2001), 5776-5779,
3 Bernard, Oncogene 9 (1994), 1039-1045
4 Barr, Hum. Molec. Genet. 5 (1996), 15-21
5 Osaka, Proc. Nat. Acad. Sci. 96 (1999), 6428-6433
6 Eguchi, Genes Chromosomes Cancer 32 (2001), 212-221 Pegram, Blood 96 (2000), 4360-4362 Gu, Cell 71 (1992), 701-708 Chaplin, Blood 85 (1995), 1435-1441 Taki, Blood 92 (1998), 1125-1130 Borkhardt, Oncogene 14 (1997), 195-202 Parry, Genes Chromosomes Cancer 11 (1994), 79-84 Nourse, Cell 60 (1990), 535-545 Sidhar, Hum. Molec. Genet. 5 (1996), 1333-1338 Morris, Science 263 (1994), 1281-1284 Borrow, Nature Genet. 12 (1996), 159-167 Nakamura, Nature Genet. 12 (1996), 154-158 Hussey, Blood 94 (1999), 2072-2079 Jaju, Blood 98 (2001), 1264-1267 Nakamura, Blood 94 (1999), 741-747 Kas, Nature Genet. 15 (1997), 170-174 Yoneda-Kato, Oncogene 12 (1996), 265-275 Cools, Blood 94 (1999), 1820-1824 Yagasaki, Genes Chromosomes Cancer 26 (1999), 192-202 Golub, Cell 77 (1994), 307-316 Redner, Blood 87 (1996), 882-886 von Lindern, Molec. Cell. Biol. 12 (1992), 1687-1697 Prasad, Cancer Res. 53 (1993), 5624-5628 Popovici, Blood 93 (1999), 1381-1389 Koontz, Proc. Nat. Acad. Sci. 98 (2001), 6348-6353 Beverloo, Cancer Res. 61(14) (2001), 5374-7 Carapeti, Blood 91(9) (1998), 3127-33 Borrow, Nature Genet. 14 (1996), 33-41 Popovici, Proc. Nat. Acad. Sci. 95 (1998), 5712-5717 Reiter, Blood 92 (1998), 1735-1742
Smedley, Hum. Molec. Genet. 7 (1998), 637-642 Xiao, Nature Genet. 18 (1998), 84-87 Fioretos, Genes Chromosomes Cancer. 32(4) (2001), 302-10 Demiroglu, Blood98(13) (2001), 3778-83
36 Chaffanet, Genes Chromosomes Cancer. 28(2) (2000), 138-44 Kitabayashi, Leukemia 15 (2001), 89-94
37 Miyoshi, Proc. Nat. Acad. Sci. 88 (1991), 10431-10434 Erickson, Blood 80 (1992); 1825-1831
38 Nakamura, Proc. Nat. Acad. Sci. 90 (1993), 4631-4635
39 Ahuja, Cancer Res. 60 (2000), 6227-9 Hussey, BMC Genet. 2 (2001); 20
40 Panagopoulos, Oncogene 18 (1999), 7594-7598
41 Dreyling, Proceedings Of The National Academy Of Science, U.S.A. 93 (1996), 4804-4809
42 Grieco, Cell 60 (1990), 557-563
43 Schwaller, Blood 97 (2001), 3910-8 Kulkami, Cancer Res. 60 (2000), 3592-8
44 Ladanyi, Cancer Res. 54 (1994), 2837-40
45 Arai, Blood 89 (1997), 3936-44
46 Raza-Egilmez, Cancer Res. 58 (1998), 4269-73
47 Taketani, Cancer Res. 62 (2000), 33-7
48 Arai, Leukemia 14 (2000), 1621-9
49 Dierlamm, Blood 93 (1999), 3601-3609
50 Golub, Proceedings Of The National Academy Of Science, U.S.A., 92 (1995), 4917-4921
51 Knezevich, Nature Genetics 18 (1998), 184-187
52 Buijs, Oncogene 10 (1995), 1511-1519
53 Peeters, Cancer Research 57 (1997), 564-569
54 Rabbitts, Nature Genet. 4 (1993), 175-180
55 Zucman, Nature Genet. 4 (1993), 341-345
56 Chase, Blood 93 (1999), 1025-1031
57 de The, Nature 347 (1990), 558-561
58 lchikawa, Cancer Res. 54 (1994), 2865-2868
59 Liu, Science 261 (1993), 1041-1044
60 Gamou, Blood 91 (1998), 4028-4037 Chen, EMBO J. 12 (1993), 1161-1167 Ladanyi, Oncogene 20 (2001), 48-57 Clark, Nature Genet. 7 (1994), 502-508 Crew, EMBO J. 14 (1995), 2333-2340 Tkachuk, Cell 71 (1992), 691-700 Thirman, Proc. Nat. Acad. Sci. 91 (1994), 12110-12114 Inaba, Science 257 (1992), 531-534
Smith, Molec. Cell. Biol. 19 (1999), 4443-4451 Ono, Cancer Res. 62 (2002), 4075-80 Ono, Cancer Res. 62 (2002), 333-7 Tatsumi, Genes Chromosomes Cancer 30 (2002), 230-5 Cazzaniga, Blood 94 (1999), 4370-4373. lijima, Blood 95 (2000), 2126-2131 Golub, Molecular and Cellular Biology 16 (1996), 4107-4116 Papadopoulos, TEL. Cancer Research 55 (1995), 34-38 Griesinger, Blood 96 (2000), 353a Lacronique, Science 278 (1997), 1309-1312 Peeters, Blood 90 (1997), 2535-2540 Fuchs, Proc. Nat. Acad. Sci. 98 (2001), 8756-8761 Daheron, Genes Chromosomes Cancer 31 (2001), 382-389 Galili, Nature Genet. 5 (1993), 230-235 Sorensen, J. Clin. Oncol. 20 (2002), 2672-2679 Clark, Oncogene 15 (1997), 2233-9 Peeters, Cancer Research 57 (1997), 564-569 Nucifora, Proc Natl Acad Sci U S A. 90 (1993),7784-8 Nucifora, Proc Natl Acad Sci U S A. 91 (1994), 4004-8 Baffa, Proc. Nat. Acad. Sci. 92 (1995), 4922-4926 Heisterkamp, Nature 306 (1983), 239-242 Shtivelman, Nature 315 (1985), 550-554 Schoenmakers, Nature Genet. 10 (1995), 436-444 Yet, not only translocations in human cells lead to detrimental fusion proteins, but similar events are described in other (mammalian) organisms which provided interesting model systems. Examples of such fusion proteins found in the mouse system are TEL/Abl (ETV6/Abl) causing a myeloproliferative disorder (Million, Blood 99 (2002), 4568-4577) or PML/LT3-ITD fusion, which causes an APL-like disease (Kelly, PNAS 99 (2002), 8283-8288).Yet, it is of note that these examples relate to transgenic mice or mice that received retrovirally transduced bone marrow cells.
The term "epitope" as employed herein above in context with said protein comprising at least two epitopes relates to any potential, but specific interaction site for the interaction domain of protein A or protein B as defined herein. Said "epitope" comprises preferably a stretch of at least 5 amino acids, more preferably of at least 7 amino acids. Yet, also epitopes are envisaged which comprise less than 5 amino acids. Also envisaged are epitopes which comprise secondary modifications, like, e.g. phophorylations. However, in accordance with this invention said first epitope comprised in said protein comprising at least two epitopes has to be distinct and different from said second epitope. Accordingly, the interaction of the interaction domain of protein A with said first epitope is distinct from the interaction of the interaction domain of protein B with said second epitope.
Said first effector domain in said protein A of the complex of the invention is, preferably, selected from:
(a) a DNA-binding domain (DBD); or
(b) a specific protease.
Most preferably, said DNA-binding domain (DBD) is or is derived from a bacterial DBD, from a fungal DBD, from a plant DBD or a DBD from a transcription factor. Even more preferred is a complex, wherein said bacterial DBD of protein A is or is derived from lexA or Tet repressor, said fungal DBD is or is derived from GAL4 DBD (this is a fungal ie. yeast transcription factor), said plant DBD is or is derived from the DBD of TIZZ or the DBD of PHR1 and wherein said DBD from a transcription factors is the DBD of a mammalian (like Hox-Proteins, ETS-Proteins, GATA-1 , GATA-2, ATF2, c-Jun) a yeast transcription factor.
The above mentioned DBD are known in the art. For example, for a DNA binding domain to be useful in the context of this invention the precise sequence of the the DNA sequence that is recognizes must be known. DNA binding domains derived from bacterial, fungal or plant proteins are best suited because there should be less cross recoginition of mammalian promoter sequences. Particular designed transcription factors as tools for therapeutics and functional genomics are described in Urnov (Biochem Pharmacol. 64 (2002), 919).
Protein A as employed in accordance with this invention is also illustrated in the appended examples and further described herein below.
Yet, the invention also provides for a complex as desribed above, wherein said second effector domain in said protein B is
(a) a transcriptional activation domain (AD); or
(b) a polypeptide activated by proteolytic cleavage.
Most preferably, said transcriptional activation domain (AD) is selected from the group consisting of GAL4 activation domain, VP16 activation domain, c-Jun, c- Fos, ELK1 , CREB, ATF2 and CHOP. It is understood that the GAL4 activation domain is employed in yeast systems, whereas, e.g. VPI6 activation domain may be employed in mammalian systems. Yet, further transcriptional activation domains may be employed, e.g. domains rich in glutamine, serine and threonine, as, inter alia, described in DE-A1 198 31 420. Consulting the appended figures and examples, the skilled artisan is readily in the position to employ further transcriptional activation domains (AD) known in the art. To circumvent the requirement for the trimeric recognition complex to move into or assemble in the nucleus, the recognition complex could direct a specific proteolytic cleavage event. In this case, protein A consists of interactor X fused to a site specific protease and protein B consists of interactor Y linked by a cleavable linker to a transcription factor or an effector protein. Upon forming the trimeric recognition complex, protein A cleaves protein B and releases either a transcription factor or an effector protein, e.g. an active caspase or other effectors described herein. The transcription factor then translocates into the nucleus and initiates transcription of effector genes. The transcription factor would contain a DNA binding domain with known specificity, a nuclear localization signal and a transcriptional activation domain.
In order to prevent spurious activation of the effector gene by the transcription factor when it is still in the intact protein B (i.e. fused via the cleavable linker to interactor Y) the following strategy can be used: interactor Y would be designed in such a way that it not only interacts with one part of the fusion protein but that it contains additional interaction surfaces that would interact intramolecularly with both the NLS, the DNA binding domain and the activation domain of the transcription factor. Thus as long as the transcription factor is covalently bound to interactor Y in protein B it could not translocate into the nucleus since its NLS is hidden and even if it reached the nucleus it could not bind to its DNA recognition site or activate transcription because these domain would be masked by intramolecular interaction.
Every protein-protein interaction has certain interaction kinetics, i.e. on and off rates. In the case of an intramolecular interaction the time of binding is very high since the interacting surfaces cannot move away very far from each other. However, after cleavage of the linker in the example described herein, the interaction is converted from an intra- to an inter-molecular interaction and the interacting surfaces can move away from each other easily. This means that as soon as the linker is cleaved the transcription factor is free to move away from the interactor Y part of protein B and to interact with the components (e.g. transportin oc) of the nuclear translocation machinery. In those cases in which the cleaved portion of protein B would be an activated caspase the uncleaved form would similarly be held in an inactive state by intramolecular interactions (procaspase: here the active site is covered by intramolecular interaction).
Accordingly, the polypeptide comprised in protein B of the complex of the invention as activated by said proteolytic cleavage may be a caspase, a transcription factor or a protein involved in a signal transduction cascade or another effector gene. Preferably, said caspase is selected from the polypeptide consisting of caspase-9, caspase-3, caspase-6, caspase-8 and/or wherein said transcription factor or said protein involved is a signal transduction cascade in NF-kappa B or Notch. There are many distinct examples in the prior art wherein a specific signal is initiated through proteolytic cleavage. For example, NFKB translocates into the nucleus and activates transcription after proteolytic cleavage. Notch signaling is initiated by proteolytic cleavage at the membrane and the intracellular portion of NOTCH then complexes with another protein, translocates into the nucleus, and activates transcription. It is also of note that, in accordance with the present invention, said polypeptide comprised in protein B of the complex of the invention may also be an "engineered transcription factor", comprised an engineered DNA bidning domain (DBD) and an activation domain (AD) which is liberated and/or activated upon cleavage from "protein B" by a protease comprised in "protein A".
Most preferably, in the complex of the invention, wherein said proteolytic cleavage mediated by "protein A" is mediated by thrombin, TEV protease, secretase, enterokinase. Especially those proteases are preferred in this context that are involved in viral protein processing since these often comprise a very specific protease activity. Accordingly, proteases to be employed in this context also comprise HIV-1 retropepsin (human immunodeficiency virus type 1), nodavirus endopeptidase (flock house virus), ubiquitin C-terminal hydrolase UCH-L1 (Homo sapiens), foot-and-mouth disease virus L-proteinase (foot-and- mouth disease virus), caspase-1 (Rattus norvegicus), hedgehog protein (Drosophila melanogaster), poliovirus-type picornain 3C (poliovirus type 1), procine transmissible gastroenteritis virus main protease (porcine transmissible gastroenteritis virus), togavirin (Sindbis virus), signal peptidase I (Escherichia coli), or C-terminal processing protease-1 (Escherichia coli). Said protease is comprised (at least in part) in said protein A. Accordingly, only the proteolytically active fragment of such a protease described herein has to be comprised in protein A.
Protein A as well as protein B may further comprise a "nuclear localization signal" (NLS). The introduction of sequence coding for a nuclear localization signal (NLS) into the sequences coding for protein A and/or protein B can improve the performance of the system described herein.
In experiments shown in the appended examples, only protein B (comprising the transcriptional activation domain of VP16) had also a nuclear localization signal (from the SV40 large T antigen). There was no obvious NLS in protein A which contains the DNA binding domain of the yeast transcription factor GAL4. However, in order to check whether GAL4DBD is translocated into the nucleus, GAL4DBD (1-147) was fused to green fluorescence protein (GFP) and transient transfections in NIH3T3 cells were transformed. The GFP-GAL4DBD fusion protein was evenly distributed both in the cytoplasm and the nucleus. There was no preferential nuclear staining visible. This proved that there are no sequences in GAL4DBD that would promote a preferential nuclear localization of protein A suggesting that the addition of a NLS may improve the performance of the system described herein, in particular the detection system/screening systems described below.
The invention also relates to a complex as described herein when said complex further comprises a nucleic acid molecule comprising a binding site for said
DNA-binding domain and an effector gene.
Said effector gene preferably encodes for a polypeptide (or a fragment thereof) which is selected from the group consisting of
(a) a marker gene; (b) a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug;
(c) an immunomodulating molecule;
(d) an antigen;
(e) a molecule capable of activating a senescene program, a differentiation program or apoptosis.
It is of note that several effector genes as defined herein may be employed in combination. These combined effector genes may be linked, e.g., by "internal ribosome entry sites" (IRES).
The effector gene which is a marker gene may be selected from group consisting of a fluorescent protein, a cell surface marker, β-Gal, luciferase,
SEAP.
For example, a desired fluorescent protein may be "green fluorescent protein", fluorescent proteins (green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, red fluorescent protein and the like), a cell surface marker may be neurotrophin receptor.
Furthermore, said prodrug converting enzyme may be selected from the group consisting of purine nucleoside phosphorylase, thymidine kinase, DeoD (PNP) cytosine deaminase, carboxyl esterase, carboxypeptidase A, carboxypeptidase G2, cytochrome P450, D-amino-acid oxidase, deoxycytidine kinase, DT- diaphorase, β-galactosidase, β-glucuronidase, β-lactamase, methionine-liase, nitroreductase, thymidine phosphorylase, and xanthine-guanine phosphoribosyl transferase. The prodrug converting enzymes described here may also be capable of sensitising a cell for a drug.
For example, the expression of thymidine kinase as "effector gene" in the complex of the invention, i.e. in a cell comprising a detrimental protein (like an oncogenic fusion protein) makes said cell sensitive for medicaments, like gancyclovin. Similarly, the expression DeoD, encoding purine nucleoside phosphorylase (PNP) sensitizes cells for G-methylpurine desoryribose. The effector gene to be expressed in accordance with this invention may also be an immunomodulatory molecule like interleukin 2, costimulatory molecules (B7), and MHC molecules.
Furthermore, it is envisaged that said effector gene encodes an antigen which may be a bacterial, viral or plant antigen and/or an antigen which is capable, when expressed, to elucidate immune response in a subject. Accordingly, a cell expressing a protein comprising at least two epitopes as defined above, e.g. an oncogenic fusion protein may be driven (by the complex of the invention) to the expression of a specific antigen on the surface of said cell. This antigen is capable of eliciting an immune response in a subject, said cell is eliminated by the subjects own immune system. Accordingly, such an approach is in particular useful in a gene therapeutic setting.
The molecule capable of activating a senescence program and to expressed by the effector gene may be CDKNIb, CDKN2b or CDKN2a.
Furthermore, it is expected that said effector gene encodes a molecule that is capable of activating a differentiation program, whereby said molecule may be selected from the group consisting of CEBPA (drives granulocytic differentiation), PU.1 (drives monocytic differentiation), and GATA-1 (drives erythroid differentiation).
It is also envisaged in this invention that the effector gene encodes a molecule that is capable of activating apoptosis, whereby said molecule may be a caspase, a cytochrom-C and Fas receptor.
In a particular preferred embodiment of the complex of the invention, said fusion protein comprising at least two epitopes is the oncogenic fusion protein BCR/ABL. Said fusion protein comprises two known forms (p210 and p190). The uses described herein are envisaged to be employed in the detection/in the screening for the p190 version as well as the p210 protein. Also the pharmaceutical and medical uses are to be employed on both version BCR/ABL (ABL/BCR).
In case the protein comprising at least two epitopes is BCR/ABL the interaction domain capable of interacting with said first epitope derived from BCR may be selected from the group consisting of a specific antibody part, an antibody construct in intracellular antibodies, an aptamer, BAP1 , GRB2, xeroderma pigmentosum group B protein, GRB10 (only to BCR/ABL), c-Fes, Ras GTPase activating protein, phospholipase C-gamma, 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, Abl (-fragments), Gads. RhoA GTP, RAC1, BCR and ARG or (a) fragment(s) of said interacting molecules.
Preferably, said fragment of BAP-1 capable of interacting with said first epitope of BCR is cBAP-1 or BAPα7-9.
Corresponding examples of a "protein A" comprising such an interaction domain are shown in the appended experimental part.
Most of the "interactors" for a first epitope on BCR are known in the art, see, e.g. BAP1 (Peters, Cell Signal 11 (1999), 507-14); GRB2 (Million, Blood 15 (2000), 664-70); xeroderma pigmentosum group B protein (Maru, Biochem. Biophys. Res. Commun. 260 (1999), 309-12; Takeda, Proc. Natl. Acad. Sci. USA 96 (1999), 203-7); GRB10 (only to BCR/ABL) (Bai, Oncogene 17 (1998), 941-8); c-Fes (Peters, (1999) loc. cit.); Ras GTPase activating protein (Liu, Oncogene 17 (1998), 3073-82); phospholipase C-gamma (Peters, (1999), loc. cit.); 85,000 M(r) subunit of phosphatidylinositol 3'-kinase (Peters, (1999), loc. cit.); Abl (Muller, Mol. Cell Biol. 12 (1992), 5087-93; Pendergast, Cell 66 (1991), 161-71); Gads (Liu (1998) loc. cit.); RhoA GTP (Zhang, Biochemistry 14 (1998), 5249-57); RAC1 (Zhang, J. Biol. Chem. 273 (1998), 8776-82); BCR (Lu, Blood 82 (1993), 1257-63); ARG (Muller (1992), loc. cit).
The interaction domain capable of interacting with said second epitope derived from ABL may be selected from the group consisting of a specific antibody part, an antibody construct, an intracellular antibody, an aptamer, N-terminal 80 amino acids of ABL, Vav, Ikappa alpha which is directed against the to c-ABL SH2 domain, DDB1 , VASP, BRCA1 , RAD9 c-terminus which is directed against the c-ABL SH3 domain, ik3-1 , ik3-2, caveolinl , RIN1 , Abi-1 , EphB2 receptor, phospholipid scramblase 1 , CRKL, SORBS1 , Amyloid precusor protein, Fe65, HPK1 , p21-activated protein kinase (PAK) family, gamma-PAK, Ggrgb4, hTERT, Cbl, N-methyl-D-aspartic acid receptor NR2D subunit, protein kinase C delta. TrkA, p53, p73, ATM, p62(dok), Ku70, SHPTP1 , JAK1 , ST5, RFXI, E2F- 1 , RB, receptor tyrosine kinase p145c-kit, She, cyclic AMP response element (CRE)-binding protein (CREB), CRK, actin, EGFR, HCK, and SYP or (a) fragment(s) of said interacting molecules. Most preferably, said fragment of ABL to be used as interactor with an epitope an BCR/ABL comprises the N-terminal part of ABL and the fragment of CRKL interacting with ABL in BCR/ABL comprises the SH3 domain (CRKL-SH3n).
These interactors are also demonstrated in the appended examples. It is of note that corresponding interactors are known in the art, for example the following interaction partners for ABL are described:
N-terminal 80 amino acids of ABL (Pluk, Cell 108 (2002), 247-59); Intracellular antibodies (Tse, J. Mol. Biol. 317 (2002), 85-94); Vav (Bassermann, J. Biol. Chem. 277 (2002), 12437-45); Ikappa alpha (to c-ABL SH2 domain) (Kawai, Mol. Cell Biol. 22 (2002), 6079-88); DDB1 (Cong, J. Biol. Chem. (2002) [epub ahead of print]); VASP (Howe, J. Biol. Chem. (2002) [epub ahead of print]); BRCA1 (Foray, Mol. Cell Biol. 22 (2002), 4020-32); RAD9 c-terminus (c-ABL SH3 domain) (Yoshida, Mol. Cell Biol. 22 (2002), 3292-300); ik3-1 , ik3-2 (Sato, Biochim. Biophys Acta 1574 (2002), 157-63); caveolinl (is substrate of c-ABL) (Sato (2002), loc. cit.); RIN1 (Tall, Dev. Cell 1 (2001), 73-82); Abi-1 (Ikeguchi, Oncogene 20 (2001), 4926-34); EphB2 receptor (Yu, Oncogene 20 (2001), 3995-4006); phospholipid scramblase 1 (Sun, J. Biol. Chem. 276 (2001), 28984-90); CRKL (ten Hoeve, Cancer Res. 54 (1994), 2563-7); SORBS 1 (Lin, Genomics 74 (2001), 12-20); Amyloid precusor protein, Fe65 (Zambrano, J. Biol. Chem. 276 (2001), 19787-92); HPK1 (Ito, J. Biol. Chem. 276 (2001), 18130-8); p21 -activated protein kinase (PAK) family, gamma-PAK (Roig, Proc. Natl. Acad. Sci. USA 97 (2000), 14346-51); Ggrgb4 (Coutinho, Blood 96 (2000), 618-24); hTERT (Kharbanda, Curr. Biol. 10 (2000), 568-75); Cbl (Shishido, Proc. Natl. Acad. Sci. USA 97 (2000), 6439-44; Salgia, Exp. Hematol. 24 (1996), 310-3); N-methyl-D-aspartic acid receptor NR2D subunit (Glover, J. Biol. Chem. 275 (2000), 12725-9); protein kinase C delta (Sun, J. Biol. Chem. 275 (2000), 7470-3); TrkA (Koch, FEBS Lett. 469 (2000), 72-6); p53 (Nie, Mol. Cell Biol. 20 (2000), 741-8); p73 (Agami, Nature 399 (1999), 809-13); ATM (Chen, J. Biol. Chem. 274 (1999), 12748-52; Shafman, Nature 387 (1997), 520- 3); p62(dok) (Bhat, J. Biol. Chem. 273 (1998), 32360-8); Ku70 (Kumaravel, Int. J. Radiat Biol. 74 (1998), 481-9); SHPTP1 (Liedtke, Oncogene 17 (1998), 1889-92); JAK1 (Danial, Mol. Cell Biol. 18 (1998), 6795-804); ST5 (Majidi, J. Biol. Chem. 273 (1998), 16608-14); RFXI (Agami, Oncogene 16 (1998), 1779- 88); E2F-1 (Birchenall-Roberts, J. Biol. Chem. 272 (1997), 8905-11); RB (Miyamura, Int. J. Hematol. 65 (1997), 115-21 ; Welch, Cell 75 (1993), 779-90); receptor tyrosine kinase p145c-kit (Hallek, Br. J. Haematol. 94 (1996), 5-16); She (Raffel, J. Biol. Chem. 271 (1996), 4640-5); cyclic AMP response element (CRE)-binding protein (CREB) (Birchenall-Roberts, Mol. Cell Biol. 15 (1995), 6088-99); CRK (Ren, Genes Dev. 8 (1994), 783-95); actin (Van Etten, J. Cell Biol. 124 (1994), 325-40); EGFR (Zhu, J. Biol. Chem. 268 (1993), 1775-9); HCK (Warmuth, J. Biol. Chem. 272 (1997), 33260-70); SYP (Tauchi, J. Biol. Chem. 269 (1994), 15381-7).
A further, illustrative protein comprising at least two epitopes and being comprised in the complex of the invention is AML1/ETO.
Corresponding interaction domain capable of interacting with said first epitope derived from AML1 may, inter alia, be selected from the group consisting of a specific antibody part (or a fragment thereof), an antibody construct, an intracellular antibody, an aptamer, CEBPA, PU.1 , MORF, CBFB, HES-1 , BSAP, MITF, MEF, TLE and P300, or (a) fragment(s) of said interacting molecules. A functional fragment of an interactor must be capable of specifically interacting with at least one epitope. For example a functional fragment of PU.1 (an interactor for AML1 PU.1 (β3-β4)).
AML1 interactors are known in the art, see, e.g., CEBPA (Petrovick, Mol Cell Biol. 18 (1998), 3915-25; Zhang, Mol Cell Biol. 16 (1996), 1231-40), PU.1 (Petrovick, Mol Cell Biol. 18 (1998), 3915-25; Zhang, Mol Cell Biol. 16 (1996), 1231-40), Aptamers, MORF (Pelletier, Oncogene 21 (2002), 2729-40), CBFB (Warren, EMBO J. 19 (2000), 3004-15), HES-1 (McLarren, J Biol Chem.;275 (2000), 530-8), BSAP (Libermann, J Biol Chem. 274 (1999), 24671-6), MITF (Morii, Biochem Biophys Res Commun. 261 (1999), 53-7), MEF (Mao, Mol Cell Biol. 19 (1999), 3635-44), TLE (Imai, Biochem Biophys Res Commun. 252 (1998), 582-9), P300 (Kitabayashi, EMBO J. 17 (1998), 2994-3004).
The interaction domain capable of interacting with said first epitope derived from ETO may be selected from the group consisting of a specific antibody part (or a fragment thereof), an antibody construct, an intracellular antibody, an aptamer, N-CoR, importin α, importin β, ETO, mSin3A and SMRT, or (a) fragment(s) of said interacting molecules.
Also interactors for ETO are known and, inter alia, described in N-CoR (Wang, Proc Natl Acad Sci U S A. 95 (1998), 10860-5), Aptamers, Importin α, importin β (Odaka, Oncogene 19 (2000), 3584-97), ETO (Minucci, Mol Cell. 5 (2000), 811-20), mSin3A (Hildebrand, J Biol Chem. 276 (2001), 9889-95), SMRT (Zhang, Mol Cell Biol. 21 (2001), 156-63).
The unique complex described herein provides for the first time for means and methods to detect and/or eliminate cells which express a detrimental fusion protein, e.g. an oncogenic fusion protein. Here, it was surprisingly found (as documented in the appended examples) that the introduction of a "protein A" and a "protein B" as defined above into a cell comprising and/or expressing such a fusion protein can drive the expression of a specific marker gene (e.g. for detecting said cell) or an effector gene which makes said cell susceptible to certain drugs (e.g. by expressing a prodrug converting enzyme and/or a polypeptide capable of sensitizing a cell for a drug), which drives said cell in a "suicid" program, which drives said cell into apoptosis, which elicits in said cell a senescence program or a specific differentiation program, which leads said cell to the expression of an immunomodulating molecule or a specific antigen (e.g. for the elicitation of an individuals immune response to said cell), or which elicits further cellular responses. As described herein, said effector gene may be a gene which is activated by the binding of the complex described herein to the corresponding gene activation domains, e.g. to a specific promoter. Accordingly, in particular the DNA-binding domain of the herein described "protein A" may be specifically engineered to bind to a specific, intracellular promoter which drives the expression of an endogenous "effector gene" as described herein. The "proteins A and B" as used in the complex of the invention may be introduced via methods known in the art which are described herein. Said introduction may be the introduction of the protein compounds themselves, as well as the introduction of one or more vectors expressing said protein A and/or said protein B. As detailed herein, it is also feasible to introduce a further nucleic acid molecule which comprises a specific domain/motif on which the DNA binding domain of the A protein may bind as well as the nucleic acid sequence coding for a specific marker or effector gene. Such a setting is also illustrated in the appended examples. Yet, it is also possible that an vector is introduced in the cell which is suspected to express a detrimental fusion proteins which comprises the coding sequences for both protein A and B, as well as the corresponding sequences for an effector gene, i.e. a vector or a nucleic acid molecule comprising a nucleic acid molecule comprising a binding site for said DNA-binding domain and an effector gene as well as the nucleic acid molecules coding for protein A/B or (a) functional fragment(s) thereof.
As detailed herein, the present invention provides for unique genetic switches which are particularly useful in diagnostic as well as pharmaceutical settings. Accordingly, the present invention particularly provides for methods for the elimination of a cell comprising a fusion protein comprising at least two epitopes, preferably an oncogenic fusion protein, comprising the steps of: (a) contacting, introducing and/or expressing (into) a cell suspected of comprising such a fusion protein (with) a protein A and a protein B as defined herein or contacting, introducing and/or expressing (into) said cell (with) at least one nucleic acid molecule coding for a protein A and a protein B as defined herein and; (b) eliciting in said cell the expression of an effector gene or an cellular effector which leads to the elimination of said cell. The methods described herein may be carried out in vivo, in vitro as well as ex vivo. It is, inter alia, envisaged to contact introduce or express ex vivo blood cells or bone marrow cells of a patient, preferably a human patient (with) the above described protein A and protein B (and, optionally, to contact said cell with (and/or) express in said cell) a nucleic acid molecule comprising a specific domain/motif on which the DNA binding domain of the A protein may bind as well as the nucleic acid sequence coding for a specific marker or effector gene). A cell expressing a detrimental fusion protein as defined herein will be detected by the corresponding expression of a marker gene (e.g. a luciferase, GFP and the like) and may easily detected by methods known in the art, like, e.g. FACS-analysis. A corresponding embodiment is illustrated in the appended example and in particular in figure 29. In an other approach, said cell, expressing said fusion protein as well as a protein A/B as defined herein will elicit the expression of an endogenous effector gene as defined herein, a gene which elicits a senescence or adipose program or will elicit the expression of an effector gene comprised on a further nucleic acid molecule introduced which comprises a specific domain/motif on which the DNA binding domain of the A protein may bind as well as the nucleic acid sequence coding for said effector gene. The invention also and particularly provides for a "protein A" or a "protein B" as defined herein above.
Said "protein A" and/or said "protein B" is(are) useful in the methods and described herein. It is in particular preferred said "protein A" and/or said "protein B" is expressed in a cell that expresses a protein comprising two epitopes as defined above, preferably in a cell expression a fusion protein, more preferably a fusion protein which is oncogenic and/or leads to a malignant state of the cell. By expressing a protein A and a protein B as defined herein in said cell, the cell comprising said detrimental protein/fusion protein may be distinguished from those cells that do not express that protein/fusion protein in vivo. Corresponding methods are illustrated below as well as in the appended examples and figures.
In another embodiment, the present invention relates to a nucleic acid molecule encoding a protein A or a nucleic acid molecule encoding a protein B as described and defined herein above and in the appended examples.
Preferably, said nucleic acid molecule is selected from the group consisting of
(a) a nucleic acid molecule comprising a nucleic acid sequence as shown in any one of SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 or 35;
(b) a nucleic acid molecule encoding a polypeptide as shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36;
(c) a nucleic acid molecule encoding a functional fragment or a functional domain of the polypeptide as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36;
(d) a nucleic acid molecule which is at least 60% identical to the nucleic acid molecule as defined in (a), (b) or (c) and which encodes a protein A or a protein B of claim 34 or 35; and
(e) a nucleic acid molecule which hybridises under stringent conditions with the complementary strand of the nucleic acid molecule as defined in (a) to (d).
The nucleic acid molecule encoding a "protein A" or a "protein B" may be a DNA or a RNA.
In accordance with the present invention, the term "nucleic acid sequence" means the sequence of bases comprising purine- and pyrimidine bases which are comprised by nucleic acid molecules, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. The term "hybridizes" as used in accordance with the present invention may relate to hybridizations under stringent or non-stringent conditions. If not further specified, the conditions are preferably non-stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as O.lxSSC, 0.1% SDS at 65°. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6xSSC, 1 % SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hydridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid sequences which code for a functional protein A or protein B fragment and which have a length of at least 12 nucleotides, preferably at least 15, more preferably at least 18, more preferably of at least 21 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 40 nucleotides and most preferably at least 60 nucleotides. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g. C0t or R0t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). The terms complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single- stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single- stranded molecules. The degree of complementartity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands. The term "hybridizing sequences" preferably refers to sequences which display a sequence identity of at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, particularly preferred at least 80%, more particularly preferred at least 90%, even more particularly preferred at least 95% and most preferably at least 97% identity with a nucleic acid sequence as described above encoding a "protein A" and/or a "protein B" having a described mutation. Moreover, the term "hybridizing sequences" preferably refers to sequences encoding a "protein A" and/or a "protein B" having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, particularly preferred at least 90%, more particularly preferred at least 95%, even more particularly preferred at least 97% and most preferably at least 99% identity with an amino acid sequence of a "protein A" and/or a "protein B" sequence as described herein above.
In accordance with the present invention, the term "identical" or "percent identity" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson (1994), Nucl. Acids Res. 2, 4673-4680) or FASTDB (Brutlag (1990), Comp. App. Biosci. 6, 237-245), as known in the art.
Moreover, the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above- described hybridzing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid. In a most preferred embodiment of the present invention, a vector comprising the nucleic acid molecule encoding for a "protein A" and/or a "protein B" as defined herein is described.
Said vector is particularly useful for the diagnostic and pharmaceutic methods described herein.
Such a vector may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
The nucleic acid molecules of the present invention may be inserted into several commercially available vectors. Nonlimiting examples include plasmid vectors compatible with mammalian cells, such as pUC, pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1 neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag , plZD35, pLXIN and pSIR (Clontech) and plRES-EGFP (Clontech). Furthermore, vectors as described in the appended examples and figures are useful in the context of the present invention. These vectors are, e.g. pM1(-3), pVP, pcDNA3, pVP-FLAG5 or pVP- HA1(-3), etc. Baculovirus vectors such as pBIueBac, BacPacz Baculovirus Expression System (CLONTECH), and MaxBacTM Baculovirus Expression System, insect cells and protocols (Invitrogen) are available commercially and may also be used to produce high yields of biologically active protein, (see also, Miller (1993), Curr. Op. Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: A Laboratory Manual, p. 127). In addition, prokaryotic vectors such as pcDNA2; and yeast vectors such as pYes2, pACT2, pGBT9, pGBKT7, pGAD424, or pGAD-GH are nonlimiting examples of other vectors suitable for use with the present invention. For vector modification techniques, see Sambrook and Russel (2001), loc. cit. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, e. g. antibiotic resistance, and one or more expression cassettes.
The coding sequences inserted in the vector can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e. g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods.
Furthermore, the vectors may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site or internal ribosomal entry sites (IRES) (Owens (2001), Proc Natl Acad Sci USA 98,1471-1476) for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Particularly preferred are in this context control sequences which allow for correct expression in blood cells or bone marrow cells.
Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art and also described in the appended examples. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymidine kinase promoter, SV40, RSV- promoter (Rous sarcome virus), human elongation factor 1 α-promoter, CMV enhancer, CaM-kinase promoter or SV40-enhancer. CD34 promoter which is active in stem cells (Okuno et al, (Blood. 2002) ;100(13):4420-6; Radomska et al., Blood. (2002) 100(13):4410-9), MRP8 promoter, which is active in early myeloid cells (Yuan et al., PNAS USA (2001) 98(18): 10398-403) and many other promoters/enhancers that are specifically acitve in the hematopoietic compartment can readily be found described in the literature
For the expression for example in blood cells/ leucocytes and/or cells derived therefrom, several regulatory sequences are well known in the art, like CD34 promoter which is active in stem cells (Okuno et al, (Blood. 2002) ;100(13):4420-6; Radomska et al., Blood. (2002) 100(13):4410-9), MRP8 promoter, which is active in early myeloid cells (Yuan et al., PNAS USA (2001) 98(18):10398-403). For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter, the lacUVδ or the trp promoter, has been described. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI, pcDNA3 (In-Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, such as lambda gt11.
An expression vector according to this invention is at least capable of directing the replication, and preferably the expression, of the nucleic acids and protein of this invention. Suitable origins of replication include, for example, the Col E1 , the SV40 viral and the M 13 origins of replication. Suitable promoters include, for example, the cytomegalovirus (CMV) promoter, the lacZ promoter, the gal 10 promoter and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter. Suitable termination sequences include, for example, the bovine growth hormone, SV40, lacZ and AcMNPV polyhedral polyadenylation signals. Examples of selectable markers include neomycin, ampicillin, and hygromycin resistance and the like. Specifically-designed vectors allow the shuttling of DNA between different host cells, such as bacteria-yeast, or bacteria-animal cells, or bacteria-fungal cells, or bacteria invertebrate cells. Furthermore, said vector may also be, besides an expression vector, a gene transfer and/or gene targeting vector. Gene therapy is one of the pharmaceutical methods envisaged in context of this invention and provides for means and methods for the selection and/or elimination of cells comprising a detrimental protein comprising at least two epitopes as defined above. Gene therapy, which is based on introducing therapeutic genes (for example for vaccination) into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, vector systems and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, Current Opinion in Biotechnology 7 (1996), 635-640 or Verma, Nature 389 (1997), 239-242 VandenDriessche et al., Curr Gene Ther. (2001) 1 (3):301-15; Woods et al., Leukemia. (2002)16(4):563- 9; Pfeifer et al.,. Annu Rev Genomics Hum Genet. (2001) 2:177-211; Chang et al., Curr Opin Mol Ther. (2001) 3(5):468-75] and references cited therein. The nucleic acid molecules of the invention and vectors as described herein above may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, baculoviral systems or systems based on vaccinia virus or Semliki Forest Virus can be used as eukaryotic expression system for the nucleic acid molecules of the invention. In addition to recombinant production, fragments of the protein, the fusion protein or antigenic fragments of the invention may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) Solid Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer. Various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
The present invention in addition relates to a host transformed with a vector of the present invention or to a host comprising the nucleic acid molecule of the invention. Said host may be produced by introducing said vector or nucleotide sequence into a host cell which upon its presence in the cell mediates the expression of a protein encoded by the nucleotide sequence of the invention or comprising a nucleotide sequence or a vector according to the invention wherein the nucleotide sequence and/or the encoded polypeptide is foreign to the host cell.
By "foreign" it is meant that the nucleotide sequence and/or the encoded polypeptide is either heterologous with respect to the host, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host but located in a different genomic environment than the naturally occurring counterpart of said nucleotide sequence. This means that, if the nucleotide sequence is homologous with respect to the host, it is not located in its natural location in the genome of said host, in particular it is surrounded by different genes. In this case the nucleotide sequence may be either under the control of its own promoter or under the control of a heterologous promoter. The location of the introduced nucleic acid molecule or the vector can be determined by the skilled person by using methods well-known to the person skilled in the art, e.g., Southern Blotting. The vector or nucleotide sequence according to the invention which is present in the host may either be integrated into the genome of the host or it may be maintained in some form extrachromosomally. In this respect, it is also to be understood that the nucleotide sequence of the invention can be used to restore or create a mutant gene via homologous recombination.
Said host may be any prokaryotic or eukaryotic cell. Suitable prokaryotic/bacterial cells are those generally used for cloning like E. coli, Salmonella typhimurium, Serratia marcescens or Bacillus subtilis. Said eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell, a plant cell or a bacterial cell (e.g., E coli strains HB101 , DH5a, XL1 Blue, Y1090 and JM101). Eukaryotic recombinant host cells are preferred. Examples of eukaryotic host cells include, but are not limited to, yeast, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis or Pichia pastoris cells, cell lines of human, bovine, porcine, monkey, and rodent origin, as well as insect cells, including but not limited to, Spodoptera frugiperda insect cells and Drosophila-derived insect cells as well as zebra fish cells. Mammalian species-derived cell lines suitable for use and commercially available include, but are not limited to, L cells, CV-1 cells, COS-1 cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HeLa cells (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
In a particularly preferred embodiment said mammalian cell is a leukocyte, a bone marrow cell, blood cell and/or a cultured cell like, inter alia, a HEK 293 (human embryonic kidney) cell, a CHO, HeLa, NIH3T3, BHK, PC12 cell or a cultured blood cell, like 32D cells (murine myeloid cells), BaF3 cells (murine pre B cells, FDCP-mix (murine stem cell / progenitor cells, Jurkat (human T-ALL cell line or U937 (human myeloid/monocytic cell line) Most preferably said cells are derived from a mammal and more preferably from a human. In another more preferred embodiment said amphibian cell is an oocyte. In an even more preferred embodiment said oocyte is a frog oocyte, particularly preferred a Xenopus laevis oocyte.
As will be discussed below said host according to the invention may also be a non-human transgenic organism. Said non-human organism may be a mammal, amphibian, a fish, an insect, a fungus or a plant. Particularly preferred non-human transgenic animals are Drosophila species, Caenorhabditis elegans, Xenopus species, zebra fish, Spodoptera frugiperda, Autographa califomica, mice and rats. Transgenic plants comprise, but are not limited to, wheat, tobacco, parsley and Arabidopsis. Transgenic fungi are also well known in the art and comprise, inter alia, yeasts, like S. pombe or S. cerevisae, or Aspergillus, Neurospora or Ustilago species.
In another embodiment, the present invention relates to a method for producing the polypeptide encoded by a nucleic acid molecule of the invention comprising culturing/raising the host of the invention and isolating the produced polypeptide.
Therefore, the invention also relates to a method for the preparation of a protein A and/or a protein B as defined herein comprising culturing the host described above under conditions that allow synthesis of said protein A and/or protein B or (a) fragment(s) thereof and recovering said said protein A and/or protein B or (a) fragment(s) from said culture.
A large number of suitable methods exist in the art to produce polypeptides in appropriate hosts. If the host is a unicellular organism or a mammalian or insect cell, the person skilled in the art can revert to a variety of culture conditions that can be further optimized without an undue burden of work. Conveniently, the produced protein is harvested from the culture medium or from isolated (biological) membranes by established techniques. Furthermore, the produced polypeptide may be directly isolated from the host cell. Said host cell may be part of or derived from a part of a host organism, for example said host cell may be part of the blood system of an animal. Additionally, the produced polypeptide may be isolated from fluids derived from said host, like blood, milk or cerebrospinal fluid.
Additionally the present invention relates to a polypeptide that is encoded by a nucleic acid molecule of the invention or produced by the method of the invention. The polypeptide of the invention may accordingly be produced by microbiological methods or by transgenic mammals. It is also envisaged that the polypeptide of the invention is recovered from transgenic plants. Alternatively, the polypeptide of the invention may be produced synthetically or semi-synthetically.
For example, chemical synthesis, such as the solid phase procedure described by Houghton (1985), Proc. Natl. Acad. Sci. USA (82), 5131-5135, can be used. Another method is in vitro translation of mRNA. A preferred method involves the recombinant production of protein in host cells as described above. For example, nucleotide acid sequences comprising all or a portion of any one of the nucleotide sequences according to the invention can be synthesized by PCR, inserted into an expression vector, and a host cell transformed with the expression vector. Thereafter, the host cell is cultured to produce the desired polypeptide, which is isolated and purified. Protein isolation and purification can be achieved by any one of several known techniques; for example and without limitation, ion exchange chromatography, gel filtration chromatography and affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, preparative disc gel electrophoresis. In addition, cell-free translation systems can be used to produce the polypeptides of the present invention. Suitable cell-free expression systems for use in accordance with the present invention include rabbit reticulocyte lysate, wheat germ extract, canine pancreatic microsomal membranes, E. coli S30 extract, and coupled transcription/translation systems such as the TNT-system (Promega). These systems allow the expression of recombinant polypeptides or peptides upon the addition of cloning vectors, DNA fragments, or RNA sequences containing coding regions and appropriate promoter elements. As mentioned supra, protein isolation/purification techniques may require modification of the proteins of the present invention using conventional methods. For example, a histidine tag can be added to the protein to allow purification on a nickel column. Other modifications may cause higher or lower activity, permit higher levels of protein production, or simplify purification of the protein.
It is of particular note that, in accordance with this invention, the nucleic acid sequences/molecules described above, e.g. the nucleic acid molecules coding for a "protein A" and/or a "protein B" as defined herein may be comprised on a single nucleic acid molecule and/or a single vector.
Furthermore, said vector may, in addition, comprise a nucleic acid molecule coding for an effector gene as defined above.
As mentioned above, a method to delivery the components of the protein detection system of the present invention and the effector components to the cells is in the form of DNA vectors encoding the corresponding proteins. Here the whole range of gene delivery systems can be used (naked DNA, gene guns, Iiposomal transfection reagents, electroporation and viral vectors). One advantage of the present invention is that the three components of the system (protein A, protein B, and effector gene) do not have to be very large and could be coded for in as little as 3 kilobase pairs (kbp) or less. Proteins A and B can be made very small if one were to use oligopeptides (aptamers) as specific interaction partners for the parts of the fusion protein. Small "fragments" of protein A or protein B are also illustrated in the appended examples The DNA binding domain in protein A is encoded in less than 500 bp of DNA and the, e.g. VP16 activation domain in less than 200 bp. Each aptamer would not need more than 50 bp of coding region. The largest portion of the coding region would be used for the effector gene. The system is thus sufficiently compact to be even delivered by e.g. adeno associated virus gene delivery systems which have a carrying capacity of about 4 kbp (or by other delivery systems described above).
If a cellular effector gene is to be activated or an activated effector protein is to be generated by proteolytic cleavage, all the components of the system could be delivered as proteins. There are various signal peptides available that will facilitate cellular up-take of external proteins at high efficiency. This type of delivery will be most practical, e.g. in an extracorporal bone marrow purging setting but might also be achievable in a patient (in particular a human patient) through the use of, e.g. liposomally encapsulated proteins. It might also be feasible to reduce the size of interactor X and Y so much that they are no longer oligopeptides but are some other molecules that have been found to interact with protein X and Y of the fusion protein. The interacting regions in a protein comprising at least two epitopes as defined herein that are recognized by the interactor molecules should be close together on the fusion protein. If such close proximity can be achieved it might be possible to attach a molecule to interactor X that would activate another molecules that is attached to interactor Y. The activated molecule of interactor Y could then act, for example, like a cytotoxic drug.
Accordingly, the invention also provides for a composition comprising a complex, a protein A, a protein B, a nucleic acid molecule, a vector, a host cell and/or nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined herein above and as illustrated in the appended examples.
Most preferably, said composition is a pharmaceutical or a diagnostic composition.
As illustrated herein, said composition is particularly useful in detecting a cell which comprises a detrimental protein comprising at least two epitopes, e.g., a fusion protein, most preferably an oncogenic fusion protein, like BCR/ABL. In the following, the intended medical and diagnostic use is illustrated by example of the treatment of leukemias.
Leukemias are often caused by fusion proteins. With the present invention, it is envisaged to, inter alia, use the described detection system of the invention to purge patients' bone marrow extracorporally or to apply the inventive system in a systemic fashion to eliminate malignant cells in the patient or to eliminate such cells ex vivo.
It is envisaged that the (fusion) protein detection system of the invention will first be used extracorporally to purge the, e.g. bone marrow of patients with e.g. chronic myeloid leukemia of all malignant cells. Bone marrow would be harvested from the patients prior to an intensive myeloablative chemotherapy regimen. The chemotherapy regimen would be so intensive that the patients would die without receiving a bone marrow transplant. After the chemotherapy the patients will receive their own purged bone marrow. In such an extracorporal setting one would be free to use a variety of different techniques (see above) to introduce the components of the detection/complex system of the invention into the bone marrow cells and use a variety of different ways to exploit the activation of effector genes. The technologies to achieve the required gene transfer efficiencies in such a setting are well within the capabilities of today's technologies.
In order to employ the detection system/complex system of the invention directly in vivo, in the patient, one would have to be able to efficiently introduce the components of the detection system into the majority of, e.g. haematopoietic cells and especially into stem cells and progenitor cells. Ways of such an introduction are provided herein above.
One problem that may arise in the course of detecting a (fusion) protein or another protein comprising two epitopes as defined above is that the interactor proteins that are part of proteins A and B may interact with other cellular proteins and that this interaction either disturbs the interaction between the interactors and the fusion protein or that it prevents the trimeric recognition complex to efficiently translocate into the nucleus and initiate transcription of the effector genes. This problem can be solved in, e.g. the following ways: (1) The interactor proteins can be reduced to the actual interacting protein domain. As documented in the appended examples, this strategy has been successfully employed in the case of CRKL, from which only the N-terminal SH3 domain was used. (2) Furthermore, the need for nuclear translocation of the trimeric recognition complex of the invention can be circumvented if a proteolytic cleavage event is used to initiate the action step.
In the practical setting which will be the extracorporal purging of leukemic bone marrow one challenging problem will be to ensure that all cells receive the components of the detection system. This is critical because those cells that do not receive the detector system and that do harbor the fusion protein would not be detected and could not be eliminated from the bone marrow sample. Since it is practically impossible to supply all cells with the components of the detection system (either through transfection or through direct introduction of the proteins) it will be necessary to incorporate a marker for the presence of the detection system in the cells. This can be conveniently be achieved by adding for example an internal ribosome entry site (IRES) sequence to the sequence that codes for protein A or protein B followed the sequences coding for a marker like green fluorescence protein (GFP) or a neomycin resistance gene. In case of the GFP, cells that have received the components of the detection system could be distinguished from those that did not receive the system by their green fluoresence. In case of the neomycin resistance gene, all the cells containing the resistance gene could be selected for with the antibiotic G418. In this way a positive negative selection could be employed. For clarification, this positive negative selection is illustrated in the appended examples and below using the "BCR/ABL fusion protein" as an example, the tk-gene as an effector gene and the neomycin resistance gene as a "transfection" marker: Cells that have not received the detection system (either BCR/ABL positive or negative) will die in G418 selection.
Cells that have received the detection system (either BCR/ABL positive or negative) will survive G418 selection. Accordingly, BCR/ABL positive cells will be sensitive to gancyclovir whereas BCR/ABL negative cells will survive gancyclovir treatment. The same results could be achieved by using other combinations of positive and negative selection systems (e.g. Fluorescence activated cell sorting when using GFP as a positive transfection maker) as illustrated above.
The invention also provides for the use of a composition comprising a complex, a protein A, a protein B, a nucleic acid molecule, a vector, a host cell and/or nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined herein for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of a proliferative disorder. Said proliferative disorder may be cancer or a tumorous disease. Most preferably, said proliferative disorder is a carcinoma disease, a sarcoma- disease, a lymphoma disease, a lipoma disease, or a leukemia. Said proliferative disorder may be AML (acute myeloid leukemia), mixed lineage leukemia, J, T-ALL (acute T cell Iymphoblastic leukemia), B-ALL (actue B cell lymphoblastic leukemia), CMMoL (chronic myelomonocytic leukemia), AL (acute leukemia), MPD (myedodysplastic syndrome), CML (chronic myeloid leukemia), MPS (Myelo proliferative syndrome), CLL (chronic lymphocytic leukemia), MALT-lymphoma, adenocarcinoma, alveolar (soft part) sarcoma, fibrosarcoma, nephroma, RAEB-T (refractory anemia with excess blasts in transformation), clear cell sarcoma, rhabdomyosarcoma, papillary renal cell carcinoma.
As illustrated in Table I herein above, certain chromosomal translocations lead to distinct disorders due to the expression of a detrimental (fusion) protein comprising at least two distinct epitopes derived from different genes. Accordingly, in particular in the treatment of these disorders, the complex system described herein is useful.
Yet, also diagnostic purposes are envisaged. Therefore, the invention also provides for the use of a protein A, a protein B, a nucleic acid molecule encoding a "protein A" and/or a "protein B" and/or a nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined above, for the preparation of a diagnostic composition for the detection of a protein comprising at least two epitopes, in particular a (fusion) protein, most preferably an oncogenic fusion protein like BCR/ABL.
Due to the usefulness of the compounds of the complex of the invention, it is also envisaged that these compounds are employed in in vivo and/or in vitro methods for the detection of a protein comprising at least two epitopes as defined above or for the detection of a cell comprising such a protein, said method comprising the steps of: (a) introducing and/or expressing, a protein A and a protein B as defined herein in cell suspected to comprise a protein comprising at least two epitopes, e.g. a fusion protein;
(b) detecting the increase or the decrease of the expression of an effector gene, detecting the increase or decrease of a specific signal elucidated by said effector gene, and/or detecting the increase or decrease of the activity of an cellular effector.
This method(s) may also comprise, as step (aa) the additional introduction of a nucleic acid molecules which comprise a binding site for a DNA-binding domain of "protein A" and an effector gene as defined above,
Accordingly, (a) method(s) is (are) provided which are useful in the detection/recognition of the in vivo presence of detrimental (fusion) proteins in a cell. It is, e.g. envisaged that the complex of detrimental fusion protein, protein A and protein B leads to the activation of certain effector genes as defined herein and as illustrated in the figures and examples. For example, these effector genes may then produce proteins that could act as surface antigens to mark the malignant cell, that could convert a prodrug into an active metabolite or that could initiate the cell death program. This would either facilitate the physical removal of the malignant cells, direct an immune response against the malignant cells, to sensitize the malignant cells against a chemotherapeutic agent or cause the malignant cell to self-destruct.
The recognition of the fusion protein may be achieved through protein-protein interactions. The effector genes may be transcriptionally activated through a protein complex consisting of three proteins with the fusion protein being the central bridge mediating between a DNA recognition domain and a transcription activation domain.
Using the BCR/ABL fusion protein (p210), which is found in chronic myeloid leukemia, as an example, it could be demonstrated in the appended examples that this strategy is capable of metabolically marking yeast cells distinguishing cells which express the BCR/ABL fusion protein from cells that do not express the fusion protein. It could also be shown that mammalian cells expressing the BCR/ABL fusion protein may be distinguishable from cells that do not express the fusion protein using a reporter gene (luciferase) in transient transfection assays.
In the herein described methods, which may also be employed ex vivo (e.g. on blood cells or bone marrow cells) said effector gene may encode for
(a) a marker gene;
(b) a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug;
(c) an immunomodulating molecule;
(d) an antigen;
(e) a molecule capable of activating a senescence program, a differentiation program or apoptosis.
The corresponding cellular effector genes, marker genes, prodrug converts enzymes and the like have been described above.
Most preferably, the methods described herein is employed in the detection and/or elimination of cells which comprise an oncogenic fusion protein, like BCR/ABL. Accordingly, said method(s) is (are) preferably used on blood cells or on bone marrow cells.
Thus, the present invention provides for novel methods to determine the absence or presence of detrimental (fusion) proteins in a cell. The method may also be employed in the selection of stem cells which do not comprise such a detrimental protein, e.g. an oncogenic fusion protein. This is illustrated, but not limited, to an example for the BCR ABL fusion protein:
In the case of the BCR/ABL fusion protein, previous strategies habe been mainly aimed at downregulating the expression or inhibiting the function of
BCR/ABL.
The expression of the BCR/ABL protein can be specifically inhibited by interfering with the BCR/ABL mRNA using anti-sense oligonucleotide or ribozymes. The expression of BCR/ABL could be specifically inhibited by inducing DNA tripelhelix formation. Finally the function of the ABL tyrosine kinase in the BCR/ABL fusion protein could be inhibited using substances like STI571.
While repression of BCR/ABL expression or inhibition of its function will cause apoptosis in some cells these treatments are not affecting all malignant cells. More undifferentiated (stem cells) and dormant cells that harbor the Philadelphia chromosome will not be driven into apoptosis. It is for this reason that the very effective ABL inhibitor STI571 can induce disease remissions but it is not able to cure patients.
Attempts to induce a specific immune response against the BCR/ABL fusion protein are hampered by the fact that both BCR and ABL are normal cellular proteins and that only the very short protein sequence where both protein are joined can be used as a BCR/ABL unique epitope. The same difficulties are encountered when other fusion proteins are used as immunological targets. The strategy to detect the fusion protein in vivo and then to initiate a specific action is fundamentally different from techniques of the prior art and offers many advantages over the strategies described above. Since the detection of the fusion protein and the resulting action are two distinct and individually designable entities great flexibility is achieved. Thus one is not dependent on the state of the malignant cell which might or might not allow the induction of apoptosis after inhibition of a fusion protein as stated above. Rather one can tailor the action which is triggered after the fusion protein is detected in such a way that all cells harboring the fusion protein can be marked or made sensitive to a chemotherapeutic agent while normal cells are spared.
This invention also provides for a kit comprising
(a) a protein A or a nucleic acid molecule encoding the same;
(b) a protein B or a nucleic acid molecule encoding the same; and/or
(c) a nucleic acid molecule comprising a binding site for a DNA-binding protein (comprised in protein A) and an effector gene. Said kit may also comprise the vectors described herein above. Said kit is particularly useful in practicing the methods of the invention.
Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific, medical or diagnostic assays or the like. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
As mentioned above, the invention also provides for a pharmaceutical composition comprising
(a) a protein A or a nucleic acid molecule encoding the same;
(b) a protein B or a nucleic acid molecule encoding the same; and/or
(c) a nucleic acid molecule comprising a binding site for a DNA-binding protein (comprised in protein A) and an effector gene.
Said pharmaceutical composition may also comprise (a) vector(s) comprising the nucleic acid molecules described herein.
Preferably, said pharmaceutical composition comprises, optionally, a pharmaceutically acceptable carrier.
The pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the. like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the aforementioned compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The pharmaceutical composition of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. In vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. Preferably, the pharmaceutical composition is administered directly or in combination with an adjuvant.
The pharmaceutical composition is preferably designed for the application in gene therapy. The technique of gene therapy has already been described above in connection with the nucleic acid molecules of the invention and all what has been said there also applies in connection with the pharmaceutical composition. For example, the nucleic acid molecule in the pharmaceutical composition is preferably in a form which allows its introduction, expression and/or stable integration into cells of an individual to be treated.
In another aspect the present invention relates to a method of treating a oncological disease comprising administering a therapeutically effective amount of the pharmaceutical composition described herein above to a subject suffering from said disease. Yet, also the treatment of further disorders caused by a detrimental (fusion) protein is envisaged.
In the context of the present invention the term "subject" means an individual in need of a treatment of a neurological disease. Preferably, the subject is a vertebrate, even more preferred a mammal, particularly preferred a human. The term "administered" means administration of a therapeutically effective dose of the aforementioned nucleic acid molecule encoding a functional protein A and/or protein B and, optionally, a nucleic acid molecule encoding for an effector gene as defined above and comprising a binding site for the DBD of protein A, to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents maybe administered alone or in combination with other treatments.
The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-arterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermally, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the candidate agents may be directly applied as a solution dry spray.
The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
The dosages are preferably given once a week, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily. In a preferred case the immune response is monitored using herein described methods and further methods known to those skilled in the art and dosages are optimized, e.g., in time, amount and/or composition. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 1012 copies of the DNA molecule. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute. Progress can be monitored by periodic assessment. The pharmaceutical composition of the invention may be administered locally or systemically. Administration will preferably be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. It is also envisaged that the pharmaceutical compositions are employed in co- therapy approaches, i.e. in co-administration with other medicaments or drugs, for example other drugs for preventing, treating or amelioration an malignancy, like cancer, in particular a leukemia. The invention also provides for a transgenic, non-human animal
(a) expressing a protein A and/or a protein B; or
(b) comprising an inventive nucleic acid molecule described herein.
The Figures show:
Fig. 1 Schematic representation of the complex of the invention. The abbreviations have the following meaning: FP: Fusion protein; X: one part of the FP, Y: second part of FP; A: Protein A; B: Protein B, J: Interactor X of Protein A; I: Interactor Y of Protein B; DBD: DNA Binding Domain of Protein A or a specific protease; AD: transcriptional activation Domain of Protein B or a polypeptide activated by proteolytic cleavage.
Fig. 2 Exemplified transcriptional activation of effector genes.
The abbreviations have the following meaning: FP: Fusion protein; X: one part of the FP, Y: the second part of FP; A: Protein A; B: Protein B, J: Interactor X of Protein A; I: Interactor Y of Protein B; DBD: Binding Domain of Protein A; AD: transcriptional activation Domain of Protein B; b: binding site; EG: Effector genes.
Fig. 3 Exemplified further activation modus. The trimeric (recognition) complex directs proteolytic cleavage of protein B. this liberates/activates a polypeptide or a fragment thereof capable of eliciting a (cellular) response. As shown in this figure the proteolytic cleavage of protein B liberates, e.g., a transcription factor (left side) or may, as a further example, activate caspase(s) which can initiate, inter alia, apoptosis (right side). The abbreviations have the following meaning: FP: Fusion protein; X: one part of the FP, Y: the second part of FP; A: Protein A; B: Protein B, J: Interactor X of Protein A; I: Interactor Y of Protein B; P: Protease; L: linker; AD: Activation Domain; b: binding site; N: Nucleus; EG: Effector gene; C i: Caspase inactive; C a: Caspase active.
Fig. 4 Schematic representation of BCR/ABL as fusion protein, BAP-
1/GAL4-BDB as an example for "protein A" and CRKL- SH3n/GAL4-AD as an example for "protein B". As "read-out" or "effector gene" in this example HIS3/lacZ is chosen. The abbreviations have the following meaning: FP: BCR/ABL; X: BCR, Y: ABL; A: Protein A; B: Protein B, J: BAP-1; I: CRKL-SH3n; DBD: GAL4-BDB; AD: GAL4-AD; , b: UAS GAL4 binding site; EG: HIS3, LacZ.
Fig. 5 Schematic representation of BCR/ABL fusion protein and CRKL-
SH3n/GAL4-BDB as "protein A" and BAP-1 /GAL4-AD as "protein B". The abbreviations have the following meaning: FP: BCR/ABL; X: ABL, Y: BCR; A: Protein A; B: Protein B, JI: CRKL-SH3n; I: BAP-1 ; DBD: GAL4-BDB; AD: GAL4-AD; b: UAS, GAL4 binding site; EG: HIS3, LacZ.
Fig. 6 and 7 Results as illustrated in appended table 2. Figure 6 a:
Growth of triple transformed yeast strain CG1945 on medium lacking leucine, lysine and tryptophane. This plate illustrates that all strains were successfully transformed with all three plasmids.
The following plasmids were used:
Number 1 through 4 are negative controls that do not express
BCR/ABL.
1) pGBT9/CRKL + pGAD424/BAP-1 + pES1
2) pGBT9/CRKL-SH3n + pGAD424/BAP-1 + pES1
3) pGBT9/BAP-1 + pGAD424/CRKL + pES1
4) pGBT9/BAP-1 + pGAD424/CRKL-SH3n + pES1
5) pGBT9/CRKL + pGAD424/BAP-1 + pES1/BCR-ABL 6) pGBT9/CRKL-SH3n + pGAD424/BAP-1 + pES1/BCR-ABL
7) pGBT9/BAP-1 + pGAD424/CRKL + pES1/BCR-ABL
8) pGBT9/BAP-1 + pGAD424/CRKL-SH3n + pES1/BCR-ABL Figure 6 b:
The same triple fransformants as shown in figure 6, left side, were streaked out on plates that also lacked histidine (the plate did not contain 3 amino-triazole). Only those transformants that acitvate the reporter gene HIS3 would be able to grow. Growth was readily observed for the transformants No. 5, 6 and 8. The weak growth of transformants 1 and 2 (negative controls) was absent when these transformants were streaked out on a plate containing 20 mM 3 amino-triazole which is an inhibitor of the HIS3 encoded enzyme (see Figure 7, left). The experiments for the plasmid combination of transformant 7 was repeated at a later time point and showed the expected growth on plates lacking histidine (not shown).
Figure 7 a:
Growth of triple transformed yeast strain CG1945 on medium lacking leucine, lysine, tryptophane and histidine with the addition of 3-AT to a final concentration of 20 mM. The combination of plasmids was the same as those used in figure 6. Only transformants 5, 6, and 8 show growth. Figure 7 b: β-galactosidase filter assay of plate from figure 6, a. Only transformants 5, 6 and 8 show a positive β-galactosidase reaction. Transformants 1 through 4 and 7 are negative.
Fig. 8 Graphical representation of average normalized luciferase values of BCR/ABL detection experiments as well as negative and posisitve controls in N1H3T3 cells. The plasmids that were used for the transfections are indicated below the columns with a "+" sign. 0.5 μg of each plasmid were employed. In addition, 0.1 μg of the plasmid pCMV-βGAL was included in every transfection as a control.
Fig. 9 Schematic representation of successful detection of BCR/ABL in mammalian cells. The abbreviations have the following meaning: FP: BCR/ABL; X: ABL, Y: BCR; A: Protein A; B: Protein B, J: CRKL-SH3n; I: BAP-1; DBD: GAL4-BDB; AD: VP16; b: UAS, GAL4 binding site; EG: Luciferase.
Fig. 10 to 25 show sequences related to "protein A" and/or "protein B" employed in accordance with this invention in the complex formation with BCR/ABL.
Fig. 10 Sequence ID No: 1 and 2 (nucleic acid and amino acid)
Insert from: pGBT9/BAP1 (nt 126-879) or pM1 /BAP-1 (nt 126- 879); numbering according to pGBT9 plasmid; GAL4-DBD1-147 origin: Saccharomyces cerevisiae; BAP-1 origin: Homo sapiens.
Fig. 11 Sequence ID No: 3 and 4 (nucleic acid and amino acid)
Insert from: pGBT9/CRKL (nt 510-1422); numbering according to pGBT9 plasmid; GAL4-DBD1-147 origin: Saccharomyces cerevisiae; CRKL origin: Homo sapiens
Fig. 12 Sequence ID No: 5 and 6 (nucleic acid and amino acid)
Insert from: pGBT9/CRKLSH3n (nt 867-1051 [HindUI site]); numbering according to pGBT9 plasmid; GAL4-DBD1-147 origin: Saccharomyces cerevisiae; CRKL origin: Homo sapiens
Fig. 13 Sequence ID No: 7 and 8 (nucleic acid and amino acid)
Insert from: pM1/CRKLSH3n (nt 867-1051 [HindUI site]); numbering according to pGBT9 plasmid (sequence of pM1 except for MCS not available); GAL4-DBD 1-147 origin: Saccharomyces cerevisiae; CRKL origin: Homo sapiens Fig. 14 Sequence ID No: 9 and 10 (nucleic acid and amino acid)
Insert from: pGAD424/BAP-1 (nt 126-879); numbering according to pGAD424 plasmid; GAL4-AD origin: Saccharomyces cerevisiae CRKL origin: Homo sapiens
Fig. 15 Sequence ID No: 11 and 12 (nucleic acid and amino acid)
Insert from: pGAD424/CRKL (nt 510-1422); numbering according to pGAD424 plasmid; GAL4-AD origin: Saccharomyces cerevisiae CRKL origin: Homo sapiens
Fig. 16 Sequence ID No: 13 and 14 (nucleic acid and amino acid)
Insert from: pGAD424/CRKL-SH3n (nt 867-1051[Hindlll site]); numbering according to pGAD424 plasmid; GAL4-AD origin: Saccharomyces cerevisiae; CRKL origin: Homo sapiens
Fig. 17 Sequence ID No: 15 and 16 (nucleic acid and amino acid)
Insert from: pVP-FLAG5/BAP-1 (nt 126-879); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); BAP-1 origin: Homo sapiens
Fig. 18 Sequence ID No: 17 and 18 (nucleic acid and amino acid)
Insert from: pVP-FLAG5/CRKL (nt 510-1422); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); CRKL origin: Homo sapiens
Fig. 19 Sequence ID No: 19 and 20 (nucleic acid and amino acid)
Insert from: pVP-FLAG5/CRKL-SH3n (nt 867-1051 [HindUI site]); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); CRKL origin: Homo sapiens Fig. 20 Sequence ID No: 21 and 22 (nucleic acid and amino acid)
Insert from: pGBT9/c-BAP-1 (nt 441-879) or pM1/c-BAP-1 (nt 441- 879); numbering according to pGBT9 plasmid; GAL4-DBD1-147 origin: Saccharomyces cerevisiae; BAP-1 origin: Homo sapiens
Fig. 21 Sequence ID No: 23 and 24 (nucleic acid and amino acid)
Insert from: pGAD424/c-BAP-1 (nt 441-879); numbering according to pGAD424 plasmid; GAL4-AD origin: Saccharomyces cerevisiae BAP-1 origin: Homo sapiens
Fig. 22 Sequence ID No: 25 and 26 (nucleic acid and amino acid)
Insert from: pVP-FLAG5/cBAP-1 (nt 441-879); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain); origin: Viral (herpes simplex virus); BAP-1 origin: Homo sapiens
Fig. 23 Sequence ID No: 27 and 28 (nucleic acid and amino acid)
Insert from: pGBT9/BAPα7-9 (nt 617-879) or pM1/BAP 7-9 (nt 617-879); numbering according to pGBT9 plasmid; GAL4-DBD1- 147 origin: Saccharomyces cerevisiae; BAP-1 origin: Homo sapiens
Fig. 24 Sequence ID No: 29 and 30 (nucleic acid and amino acid)
Insert from: pGAD424/BAPα7-9 (nt 617-879); numbering according to pGAD424 plasmid; GAL4-AD origin: Saccharomyces cerevisiae; BAP-1 origin: Homo sapiens
Fig. 25 Sequence ID No: 31 and 32 (nucleic acid and amino acid)
Insert from: pVP-FLAG5/ BAP 7-9 (nt 617-879); numbering according to pVP-FLAG5(VP16&MCS); VP16 AD (VP 16 transactivation domain) origin: Viral (herpes simplex virus); BAP-1 origin: Homo sapiens Fig. 26 Normalized relative luciferase values obtained with the BCR/ABL detection system after transient transfection in 293 cells (mammalian cells). Columns 1 and 2, 3 and 4, 5 and 6, 7 and 8 correspond to experiments No 1 , 2, 3, and 4 in Table 1. Odd columns give relative luciferase activity in the presence of the BCR/ABL fusion protein (pcDNA3.1 -BCR/ABL), even columns in the absence of the BCR ABL fusion protein (pcDNA3.1 empty). Columns 9 and 10 use a mammalian two hybrid interaction as positive control: Column 9 pM1/BR304 and pVP-HA-B202-NB; column 10 pM1/BR304 and pVP-FLAG5
Fig. 27 and 28: Sequences related to "protein A" and/or "protein B"employed in accordance with this invention in the complex formation with AML1/ETO Fig. 27 Sequence ID No: 33 and 34 (nucleic acid and amino acid)
Insert from: pGBT9/N-CoR (nt3734-5791); numbering according to pGBT9 plasmid; GAL4-DBD1-147 origin: Saccharomyces cerevisiae; N-CoR origin: Homo sapiens (AF044209)
Fig. 28 Sequence ID No: 35 and 36 (nucleic acid and amino acid)
Insert from: pGAD424/PU.1 b3-b4 (nt 892-1006); numbering according to pGAD424 plasmid; GAL4-AD origin: Saccharomyces cerevisiae; PU.1 b3-b4 origin: Homo sapiens (NM_003120)
Fig. 29: Use of inventive genetic switch complex in the murine myeloid cell line 32D (a hematopoietic cell line) and a 32D derived cell line (32DP210) that is constitutively expressing the BCR/ABL (P210) fusion protein. As exemplified "effector gene", luciferase is employed.
The following examples illustrate the invention. Example I: General principle of the inventive protein detection system
The detection system works in two distinct steps: (1) the protein to be detected is recognized; and (2) an action is initiated.
The recognition of the protein of choice (step 1), e.g. a fusion protein or protein comprising at least two epitopes "X" and "Y", is accomplished by forming a trimeric protein complex in which the (fusion) protein ("X") is contacted by protein A which consists of a protein domain (interactor X) that can interact with one part of the fusion protein fused to a DNA binding domain and by protein B which consists of a protein domain (interactor Y) that can interact with the other portion of the (fusion) protein ("Y") fused to a transcriptional activation domain. This recognition complex is shown in appended figure 1.
After the recognition complex is formed an action is initiated (step 2). If Protein A contains a DNA binding domain and protein B a transcriptional activation domain the recognition complex will form a transcriptional activation complex that is able to activate the transcription of (an) effector gene(s) that is/are under the control of the appropriate DNA binding site. This is shown in appended figure 2.
Instead of activating transcription by forming a transcriptional activation complex the recognition complex may also direct a specific proteolytic cleavage. In this case protein A consists of interactor X fused to a site specific protease and protein B consists of interactor Y linked to, e.g., a transcription factor by a (protease-) cleavable linker, an effector protein or a protein which is activated by proteolytic cleavage. Upon forming the trimeric recognition complex protein A cleaves protein B and releases either a transcription factor or an effector protein (e.g. an active caspase). This situation is shown in appended figure 3. Example II: Detection of the oncogenic fusion protein BCR/ABL
A strategy was developed by which cells that express the BCR/ABL fusion protein can be distinguished from those cells that do not express BCR/ABL in vivo. In detail, an inventive genetic switch was designed which was first tested in yeast cells and then employed in mammalian cells according to the detection system illustrated in Example I.
The recognition of BCR/ABL was achieved through protein-protein interaction. As shown in appended figure 4 BCR/ABL was contacted by two proteins. One protein (protein A) consists of a protein that interacts with BCR, for example BAP-1. BAP-1 was fused to the DNA binding domain of the yeast transcription factor GAL4 (other DNA binding proteins may be used, as illustrated in this invention). The other protein (protein B) consists of a protein that interacts with ABL, e.g. CRKL (other ABL interactors may be employed), which was fused to the transcriptional activation domain of the yeast transcription factor GAL4 or VP16 in mammalian cells(other transcriptional activation domains can be used).
Only in cells expression BCR/ABL a trimeric recognition complex as illustrated in this invention is formed consisting of protein A, protein B and the protein to be detected, BCR/ABL.
The recognition complex can also be formed if BCR contacting protein is fused to a transcriptional activation domain and the ABL contacting protein is fused to a specific DNA binding domain. It is of note that the "protein A" and "protein B" function may also be reversed (see appended Fig 5).
This recognition complex is then able to activate the transcription of a reporter/effector gene or reporter/effector genes that have an appropriate transcription factor binding site in their promoter region. It is of note that preferably multimeric binding sites on the DNA level are employed. As an illustrative example the HIS3 and the LACZ gene as reporter genes/effector genes (figure 4 and 5) were used. Transcription of HIS3 allows the yeast strain CG1945 to grow on plates that lack the amino acid histidine. If the HIS3 gene in CG1945 is not activated the cells are not able to grow on plates lacking histidine. The activation of the LACZ gene leads to the production of the enyzme beta-galactosidase which can be visualized by a special-, known staining technique.
Example III: Material and Methods for the illustrative example BCR/ABL
Yeast system Construction of protein A
PGBT9/BAP-1
The BAP-1 coding region was amplified with primers BAT.T125.Eco and
BAP.B879Sal from the plasmid plK9.9 (Nielsen, Biochim Biohpys Acta
1088:425-428 (1991)) restricted with EcoRI and Sail and ligated with EcoRI and
Sail restricted pGBT9 (Clontech).
PGBT9/CRKL
The CRKL coding region was amplified with primers CRKL.T509Sma and
CRKL.B1412Sal from the plasmid E1.7K15-6SK (ten Hoeve et al., Oncogene
8:2469-2474 (1993)) restricted with Smal and Sail and ligated with Sma1 and
Sail restricted pGBT9 (Clontech). pGBT9/CRKL-SH3n
The plasmid pM1/CRKL-SH3n (see below) was cut with HindUI blunt ended and then cut with EcoRI. The fragment containing the CRKL-SH3n (N-terminal SH3 domain of CRKL) was then ligated with EcoRI and Smal cut pGBT9.
Construction of protein B
PGAD424/BAP-1
The BAP-1 coding region was amplified with primers BAP.T125.Eco and
BAP.B879Sal from the plasmid plK9.9 (Nielsen, Bioch. Bioph. Acta 1088
(1991), 425-428) restricted with EcoRI and Sail and ligated with EcoRI and Sail restricted pGAD424 (Clontech).
PGAD424/ CRKL The CRKL coding region was amplified with primers CRKL.T509Sma and CRKLB1412Sal from the plasmid E1.7K15-6SK (ten Hoeve, Oncogene 8 (1993), 2469-2474) restricted with Smal and Sail and ligated with Smal and Sail restricted pGAD424. PGAD424/ CRKL-SH3n
The plasmid pM1/CRKL-SH3n (see below) was cut with Hindlll blunt ended and then cut with EcoRI. The fragment containing the CRKL-SH3n (N-terminal SH3 domain of CRKL) was then ligated with EcoRI and Smal cut pGAD424.
BCR ABL
Construction of a yeast BCR/ABL expression vector
In order to express the BCR/ABL fusion protein in yeast, we constructed a yeast expression vector (pES1) that had the LYS2 gene as a metabolic marker. pES1 was constructed on the basis of pGAD424 by removing the GAL4 activation domain and the multiple cloning site through a Hindlll digest and replacing it with a modified (restricted with Clal and EcoRV, blunt ended and religated) polylinker from pBluescript-SK II (Clontech) which was inserted into the blunted Hindlll sites of pGAD424 after BssHII excision and blunt ending from pBluescript-SK II (modified). Subsequently the LEU2 gene was partially removed from the modified pGAD424 through a Clal and EcoRV restriction and it was replaced by the LYS2 gene (from plasmid pDP6 (Dieter Gallwitz, MPI- Biophysikalische Chemie, Gδttingen; Eibel Mol. Gen. Genet. 191 (1983), 66-73) with modified restriction sites after it was cloned into pBluescript-SK II to obtain Smal and Clal sites flanking the gene.
BCR/ABL was cloned into the EcoRI site of pES1 from pcDNA3/BCR/ABL (Warmuth, J. Biol. Chem. 272 (1997), 33260-33270) by cutting the insert with EcoRI.
Reporter genes
Yeast strain CG1945 (Clontech) already contains the reporter genes HIS3 and LACZ under the control of a GAL4 DNA binding site. This yeast strain has the following genotype: MATa, trp1-901 , leu2-3, his3-200, gal4-542, LYS2::GAL1- HIS3, URA3::(GAL4 17-mere)3-CYC1-lacZ.
Mammalian system Construction of protein A pM1 /BAP-1
BAP-1 was excised from pGBT9-BAP-1 with EcoRI and Sail and cloned into
EcoRI and Sail linearized pM1 (Wu, Nat. Genet. 14 (1996), 430-440).
PM1/CRKL
CRKL was excised from pGBT9-CRKL with Smal and Sail and cloned into Smal and Sail linearized pM1. pM1/CRKL-SH3n
The CRKL-SH3n region was amplified with primers CRKL-SH3.T867 and
CRKL.B1412Sal by PCR, cut with EcoRI and Hindlll and ligated into EcoRI and
Hindlll cut pM1.
Construction of protein B
PVP-FLAG5/BAP-1
BAP-1 was excised from pGBT9-BAP-1 with EcoRI and Sail and cloned into
EcoRI and Sail linearized pVP-FLAG5 (Wu, Nat. Genet. 14 (1996), 430-440).
To correct the reading frame the resulting contruct was cut with Bglll, blunt ended and religated.
PVP-FLAG5/ CRKL
CRKL was excised from pGBT9-CRKL with Smal and Sail blunt ended and cloned into Hindlll cut, blunt ended pVP-FLAG5.
PVP-FLAG5/ CRKL-SH3n
The CRKL-SH3n region was amplified with primers CRKL-SH3.T867 and
CRKL.B1412Sal by PCR, cut with EcoRI and Hindlll and ligated into EcoRI and
Hindlll cut pVP-FLAG5. To correct the reading frame the resulting contruct was cut with Bglll, blunt ended and religated. BCR ABL
The pcDNA3/BCR-ABL expression vector was obtained from Michael Hallek, LMU, Munich.
Reporter genes
As a reporter gene the contruct G5E1bLUC was used (PNAS 91 :3181-3185,
1994)
Additional plasmids
The plasmid pCMV-βGAL was used as a transfection control and the plasmid pBluescript-SKII(+) (pBSK) was used as filler to bring the total amount of plasmid in the transfection assays to a constant amount.
The plasmids pM1/BR-304 and pVP-HA/B202-NB were used as a positive mammalian two hybrid control (Wu (1996), loc. cit.)
Corresponding sequences are depicted in appended Figures 10 to 25.
Example IV: Detection of BCR/ABL is a yeast system
Yeast System
Four combination of plasmids (1 to 4; see below) were transformed into CG1945 (Clontech) and the resulting transformants assayed for growth on Synthetic Dropout (SD) plates lacking tryptophane, leucine and lysine (SD -T, - L, -K). This assay ensured that all three plasmids were transformed into the yeast cells. Subsequently the transformants were assayed for the activation of the reporter gene HIS3 by plating them on SD -T, -L, -K plates that also lacked histidine (SD -T, -L, -K, -H). SD -T, -L, -K, -H plates were used without 3-amino triazole (3AT) and SD -T, -L, -K, -H plates with 20 mM 3AT. 3AT inhibits residual HIS3 gene acitivity so that a strong activation of the HIS3 reporter gene is required.
1) pGBT9/CRKL + pGAD424/BAP-1 + pES1/BCR-ABL
2) pGBT9/CRKL-SH3n + pGAD424/BAP-1 + pES1/BCR-ABL
3) pGBT9/BAP-1 + pGAD424/CRKL + pES1/BCR-ABL 4) pGBT9/BAP-1 + pGAD424/CRKL-SH3n + pES1/BCR-ABL
As negative controls (5 to 8; see below) the same combination of Protein A and Protein B expressing plasmids were used, however the pES1 expression plasmid did not contain the BCR/ABL fusion gene.
5) pGBT9/CRKL + pGAD424/BAP-1 + pES1
6) pGBT9/CRKL-SH3n + pGAD424/BAP-1 + pES1
7) pGBT9/BAP-1 + pGAD424/CRKL + pES1
8) pGBT9/BAP-1 + pGAD424/CRKL-SH3n + pES1
The following results were obtained:
Table 2
Figure imgf000067_0001
Assay number 2 was subsequently repeated and showed the expected behaviour (these results are shown in parenthesis).
Figure 6 and 7 show the results of the assays described in table 2.
In addition, the negative controls listed in appended table 3 were performed. None of these negative controls showed activation of the HIS3 reporter gene as assayed by growth on the corresponding SD plate lacking hisitidine either in the presence or the absence of 3AT. Table 3
Figure imgf000068_0001
Figure imgf000069_0001
These negative results taken together with the positive detection of BCR/ABL as show in figure 6 and 7 convincingly demonstrate that the detection of BCR/ABL is indeed dependent on the presence of proteins A and B and is not an artefact.
Example V: Detection of BCR/ABL in a mammalian cellular system
To test the BCR/ABL detection system in mammalian cells, transient cotransfection of the plasmids described in Materials and Methods (Example III) into NIH3T3 mouse fibroblast cells were employed. Said NIH 3T3 cells do not normally express the BCR/ABL fusion protein. Accordingly, a further plasmid (pcDNA3/BCR-ABL) has introduced which (artificially) drives the expression of BCR/ABL in said test system. About 1.4 x 105 cells in 35 mm plates were transfected with a total of 2.1 μg of plasmid DNA using Superfect transfection reagent. The luciferase measurements were corrected for transfection efficiency by using dividing the raw luciferase values by the measurements of the β- galactosidase from the same culture plate. The following combinations were employed:
1) pM1/CRKL + pVP-FLAG5/BAP-1 + pcDNA3/BCR-ABL (test)
2) pM1 /BAP-1 + pVP-FLAG5/CRKL + pcDNA3/BCR-ABL (test)
As "reporter gene sequence" the above described G5E1bLUC was employed.
With unmodified, i.e. using the full length coding region for CRKL and BAP-1 as interactors in proteins A and B, the normalized luciferase values of the detection experiments were not significantly different from those of the negative control experiments (data not shown). These negative results were thought to be due to interference with the function of protein A or protein B through interaction by other cellular proteins. As a first step to minimize unwanted interactions of proteins A or B with cellular proteins, only the N terminal SH3 domain of CRKL was used for the construction of protein A (pM1/CRKL-SH3n) and protein B (pVP-FLAG5/ CRKL-SH3n). When the modified protein A (pM1/CRKL-SH3n) in combination with an unmodified protein B (pVP-FLAG5/ BAP-1) was used, a positive detection of BCR/ABL was achieved in the mammalian system (figure 8, column 2 arrow, Figure 9). The luciferase values for this combination of protein A and protein B were on average about twice the values of the highest negative control (pcDNA3 empty; no BCR/ABL expression, figure 8, column 4 arrow) and reached a third of the strongly positive mammalian two hybrid control experiment (figure 8, column 6).
The strategy presented here is useful for the detection of fusion proteins (demonstrated for the BCR/ABL fusion protein) in vivo. Using, for example, GFP as an effector gene it is possible to detect the presence of the BCR/ABL fusion protein in individual living cells. Cells expressing BCR/ABL may be detected by greenfluorescence in UV light and could be sorted according to their BCR/ABL status. All other detection methods of BCR/ABL that are available today will only work on isolated DNA, RNA or proteins or on fixed cells, which means that the presence or absence of BCR/ABL can only be determined in cells that are non- viable.
Example V: Minimal interaction domains for protein A and/or protein B
Using only those domains of the interactor proteins that actually interact with the fusion proteins will improve the performance of the system especially in mammalian cells and may even be necessary to obtain positive results in the mammalian system (see Example V). These experiments describe the use of only the N-terminal SH3 domain of CRKL (the interactor of ABL) for the construction of protein A (pM1/ CRKL-SH3n) or protein B (pVP-FLAG5/CRKL- SH3n). In the case of CRKL, the N-terminal SH3 domain is known to interact with ABL. However, in practice it may be necessary to confirm that the domain which is supposed to interact actually does so. This test for interaction can conveniently be performed in the yeast two hybrid system (Fields, Nature 340 (1989), 245-247).
As described above, the N-terminal SH3 domain of CRKL was used in protein A (fused to the DBD of GAL4) and the full length BAP-1 protein in protein B (fused to the VP16 transcriptional activation domain) was it possible to detect BCR/ABL in NIH3T3 cells.
However, the system did not function when CRKL-SH3n was fused to the VP16 activation domain and BAP-1 was fused to the GAL4 DBD in the mammalian system. Even though the same combination of proteins A and B (pGBT9/BAP-1 and pGAD424/CRKL-SH3n) in the appropriate vectors (and CRKL-SH3n fused correspondingly to the GAL4 activation domain) did function in the yeast system.
Accordingly, it might be necessary to further improve the system by only using the interaction domain of BAP-1 with BCR (in the same manner that the N- terminal SH3 domain of CRKL had been used). Since it was not known which portion of BAP-1 would constitute the interaction domain with BCR, several deletion mutants of BAP-1 were constructed and tested for their interaction with BCR in the yeast two hybrid system (data not shown). Yet, the corresponding deletion mutants were prepared by standard methods and the two-hybrid system followed standard methods (Fields (1989), loc. cit.; Clontech Product Protocoll PT/265-1). It could be shown that the c-terminal half of BAP-1 (c-BAP- 1) and the c-terminal quarter (comprising alpha helices 7, 8, and 9: BAPα7-9) are sufficient for interaction with BCR. c-BAP-1 and BAPo.7-9 were then cloned into the mammalian expression vectors pM1 (to be expressed with the DNA binding domain of GAL4) and into pVP-FLAG5 (to be expressed with the VP16 transcriptional activation domain). Corresponding sequences are shown in Figures 23 to 25. Both c-BAP-1 and BAP 7-9 could be used successfully (either fused to the DNA binding domain or to the VP16 transactivation domain) to detect the presence of BCR/ABL in transfected cells (see table 4).
Table 4
Figure imgf000072_0001
Especially noteworthy are the results of experiments 1 and 3 in table 4: the full length BAP-1 fused to the DBD in combination with CRKL-SH3n fused to the transactivation domain had previously not been able to detect the presence of BCR/ABL. These results clearly show that even short, "minimally" interaction domains are efficient in the system described herein.
Example VII: The use of additional nuclear localization signals
The introduction of sequence coding for a nuclear localization signal (NLS) into the sequences coding for Protein A and/or Protein B can improve the performance of the detection system.
In the above described experiments in (mammalian system), only protein B (containing the transcriptional activation domain of VP16) comprised a nuclear localization signal (from the SV40 large T antigen). There was no obvious NLS in protein A which comprises the DNA binding domain of the yeast transcription factor GAL4. In order to check whether GAL4DBD is translocated into the nucleus, GAL4DBD (1-147) was fused to green fluorescence protein (GFP) and transient transfections in NIH3T3 cells were performed. The GFP-GAL4DBD fusion protein was evenly distributed both in the cytoplasm and the nucleus. There was no preferential nuclear staining visible. This proved that there are no sequences in GAL4DBD that would promote a preferential nuclear localization of protein A. This suggests that the addition of a NLS can further improve the performance of the system, in particular when employed in detection assays.
Example VIII: The detection of a further fusion protein AML1/ETO
It could be shown that the fusion gene detection system is capable of detecting the presence of the BCR/ABL fusion protein in transiently transfected cells. Since the interactor portions of protein A and protein B are specific for the the two components of a fusion gene, specific proteins A and B have to be designed if a different fusion gene is to be detected. As an example the development of an AML1/ETO fusion gene detection system is described.
Selection of interactors:
Several interacting partners for both AML1 and ETO are described (like CEBPA (Petrovick, Mol Cell Biol. 18 (1998), 3915-25; Zhang, Mol Cell Biol. 16 (1996), 1231-40), PU.1 (Petrovick, Mol Cell Biol. 18 (1998), 3915-25; Zhang, Mol Cell Biol. 16 (1996), 1231-40), Aptamers, MORF (Pelletier, Oncogene 21 (2002), 2729-40), CBFB (Warren, EMBO J. 19 (2000), 3004-15), HES-1 (McLarren, J Biol Chem.;275 (2000), 530-8), BSAP (Libermann, J Biol Chem. 274 (1999), 24671-6), MITF (Morii, Biochem Biophys Res Commun. 261 (1999), 53-7), MEF (Mao, Mol Cell Biol. 19 (1999), 3635-44), TLE (Imai, Biochem Biophys Res Commun. 252 (1998), 582-9), P300 (Kitabayashi, EMBO J. 17 (1998), 2994- 3004). N-CoR (Wang, Proc Natl Acad Sci U S A. 95 (1998), 10860-5), Aptamers, Importin α, importin β (Odaka, Oncogene 19 (2000), 3584-97), ETO (Minucci, Mol Cell. 5 (2000), 811-20), mSin3A (Hildebrand, J Biol Chem. 276 (2001), 9889-95), SMRT (Zhang, Mol Cell Biol. 21 (2001), 156-63)). Initially, as interacting partner for AML1 the transcription factor PU.1 was chosen. As an interaction partner for ETO the nuclear corepressor N-CoR was employed.
Testing of interactors in the yeast system :
To verify the interaction of PU.1 and N-CoR with AML1 and ETO, respectively, the coding sequences for PU.1 , N-CoR and the fusion protein AML1/ETO were cloned in frame both into the GAL4-DBD fusion vector pGBT9 and into the GAL4 AD fusion vector pGAD424. Since N-CoR is a very big gene only the middle domain, which had been described as interacting with ETO (Amann, Mol. Cell Biol. 19 (2001), 6470-6483), was used in these experiments. The first test was to investigate whether any of the GAL4-DBD domain fusions had transactivation potential. To do this the pGBT9-PU.1 and pGBT9-N- CoR(middle fragment) were transformed into yeast strain CG1945 and the cells were plated on to drop-out plates lacking tryptophane and histidine. These experiments showed that pGBT9-PU.1 had transactivation potential. With this experiments it was determined that the full length PU.1 protein should not be used in the detection system because it may constitutively activate the effector genes. To circumvent this problems only the β3-β4 region of the PU.1 ets domain which had been shown to be necessary for the interaction with AML1 was used in the subsequent experiments. The β3-β4 region of PU.1 and N-CoR did not exhibit transactivation potential in the yeast system. Since AML1 is a transcription factor and ETO is a transcriptional cofactor it was also necessary to test whether any of the components of or the complete AML1/ETO fusion protein would have transcriptional activation potential. To this end the AML1 and the ETO portions of AML1/ETO as well as the complete AML1/ETO were cloned in frame into the GAL4 DBD fusion expression vector pGBT9. These experiments showed that the pGBT9-AML1 contruct was able to transactivate the yeast reporter genes.
Next the interaction between pGBT9-PU.1(β3-β4) and pGAD424-AML1/ETO and between pGBT9-N-CoR and pGAD424-AML1/ETO was tested in the yeast system by assaying growth on the appropriate selection plates. Interaction between PU.1(β3-β4) and AML1/ETO and between N-CoR and AML1/ETO could be confirmed. If these initial interaction assays are negative it may be necessary to confirm the expression of the proteins that are tested for interaction by Western blotting using commercially available antibodies against the proteins or against the GAL4 DBD or the GAL4 AD. The corresponding sequences as used in this assay are depicted in SEQ ID NOs: 33 to 36.
Testing of the AML1/ETO detection system in yeast:
Once the individual interactions were confirmed and verified, all the components of the system as well as the fusion gene are expressed in the appropriate yeast strain and assayed for growth on drop-out plates and LACZ gene expression. Since it had been shown in the preliminary experiments that the AML1 portion of AML1/ETO and consequently also AML1/ETO act as transcriptional activators only the combination Protein A (pGBT9-N-CoR) and Protein B (pGAD424-PU.1(β3-β4)) were tested. This combination gave a positive read-out only in the presence of AML1/ETO. The combination Protein A (pGAD424- PU.1(β3-β4)) and Protein B (pGBT9-N-CoR) was not tested because - on theoretical grounds - this combination should result in the activation of the reporter genes in the presence of AML1 alone and should not require the fusion protein for activation. If one of proteins of the fusion protein contains a transcriptional acitivation domain, then protein A (the DNA-binding domain interactor X fusion) should not contact the protein that contains the transactivation domain because then the presence of the wild type (not-fused) protein could be sufficient to activate the reporter gene(s). This precaution is not necessary if the proteolytic cleavage route of effector activation is used.
Testing of the AML1/ETO detection system in mammalian cells: After successfully testing the system in yeast, the interactor portions of the system (N-CoR for Protein A) and PU.1(β3-β) for Protein B) were cloned into the mammalian expression vectors pM1 and pVP-FLAG5, respectively. The components and AML1/ETO were transiently expressed in mammalian cells (NIH3T3). However, the expression of the reporter gene luciferase did not appreciably change between the presence and the absence of the AML1/ETO fusion gene. Since Protein B (VP16AD- PU.1(β3-β 4)) already had a very small interaction domain, the interaction domain of the middle fragment of N-CoR (Protein A) with ETO was mapped in more detail. After using a subfragment of the middle portion of N-CoR in Protein A, that could be shown to strongly interact with ETO, the experiment was repeated and showed the expected result. The luciferase reporter gene was strongly activated only in the presence of Protein A, Protein B and AML1/ETO.
It could thus be shown that following certain rules (i.e. using the minimal interactor domains and avoiding interference by the wild type proteins contained in the fusion protein) that another fusion protein is amenable to this in vivo detection method.
EXAMPLE IX: Exemplified use of the inventive genetic switch in cells expressing an oncogenic fusion protein.
In order to investigate the utility of the fusion protein detection system (the genetic switch as described herein), u the murine myeloid cell line 32D (a hematopoietic cell line) and a 32D derived cell line (32DP210) that is constitutively expressing the BCR/ABL (P210) fusion protein were employed. Protein A (pM1-BAPD7-9 or pM1-CRKL-SH3n) and protein B (pVP-FLAG5- BAPD7-9 or pVP-FLAG5-CRKL-SH3n), the luciferase reporter G5E1 bLUC, and a plasmid expressing Renilla luciferase (pRL-nulI, Promega) as a transfection control were introduced into these cells by electroporation. Two combinations of protein A and protein B were used: 1) pM1-BAPD7-9 and pVP-FLAG5-CRKL- SH3n (left two columns); 2) pM1-CRKL-SH3n and pVP-FLAG5-BAPD7-9 (right two columns). As illustrated in appended figure 29, in the 32DP210 cell line the ratio of luciferase to Renilla luciferase activity was 81 and 35 times higher than in the 32D cell line for the two protein A and protein B combinations, respectively. Accordingly, a genetic switch as described herein can be employed to successfully drive the expression of effector genes capable of eliciting a cellular response which leads to either a specific signal in the cell comprising said fusion protein (e.g., the expression of a marker gene, like GFP or luciferase) or which may activate a cellular response, like the specific activation of, e.g., a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug, an immunomodulating molecule; an antigen, as well as a molecule capable of activating a senescence program, a differentiation program or apoptosis,
Thus, these results illustrate and document that the "genetic switch system" described herein is useful and can readily be employed for detecting and/or eliminating cells comprising an oncogenic fusion protein.

Claims

Claims
1. A complex comprising
(a) a protein comprising at least two epitopes;
(b) a protein A comprising an interaction domain capable of interacting with said first epitope of the protein of (a) and comprising a first effector domain; and
(c) a protein B comprising an interaction domain capable of interacting with said second epitope of the protein of (a) and comprising a second effector domain whereby said interaction domains of protein A and protein B are not capable of directly interacting with each other.
2. The complex of claim 1, wherein a first of said epitopes of the fusion protein of (a) is encoded by a first gene (or a fragment thereof) and wherein a second of said epitopes is encoded by a second gene (or a fragment thereof).
3. The complex of claim 1 or 2, wherein said protein of (a) is an oncogenicfusion protein.
4. The complex of any one of claims 1 to 3, wherein said fusion protein of (a) is a fusion protein derived from a chromosomal translocation.
5. The complex of any one of claims 1 to 4, wherein said fusion protein of (a) is related to or associated with malignancy.
6. The complex of any one of claims 1 to 5, wherein said fusion protein is expressed in a blood cell or a bone marrow cell..
7. The complex of claim 5 or 6, wherein the first epitope of said fusion protein is encoded by a gene (or a fragment thereof) selected from the group consisting of RNM15 (OTT), AF1p (eps15), PAX7, MLL, AF4 (MLLT2), MSF (homologous to PNUTL1), GMPS, AF10 (MLLT10), PAX3, PBX1 , TFE3, ALK, HOXD13, HOXA9, RAP1GDS1 , NSD1, PMX1 , MLF1, EAP, EVI1, MDS1 , HMGIC, BTL (CHIC2), ACS2, PDGFRB, NPM, CAN, AF6 (MLLT4), FOP, JAZF1 , HLXB9, MOZ, FGFR1 , ETO, LEDGF, CHN, CSMF, ABL, AF10, RET, PDGFRB, WT1 , NUP98, HOXD11 , FN1, API2, ETV6, EVI, CHOP, ATF1 , PML, FUS, MYH11, MTG16, PLZF, TFE3, SSX1 , SSX2, HLF, LCX, SEPTIN6, JAK2, ABL2 and ARG; and wherein the second epitope of said protein is encoded by a gene (or a fragment thereof) selected from the group consisting of MKL1 (MAL), MLL, FOX01A (FKHR), PNUTL1 (CDCrel), FBP17, LPP, AF9, MLLT3, GPHN (gephyrin), ENL, MLLT1 , ABU (SSH3BP1), AFX1 (MLLT7), FOXA1A, E2A, PRCC, NUP98, NPM, ETV6, AML1 , RARA, DEK, FGFR1 , JJAZ1 , TIF2, CBP, ZNF198, FIM, RAMP, BCR, P300, AML1, NUP98, TAF2N, RBP56, CALM, H4, EWS, DDX10, MALT1, CBFA2, NTRK3, MN1 , FUS (TLS), EWS, CDX2, ERG, CBFB, CBFA2, RCC17, SYT, ELL, E2A, CDCREL1 , PSF, SYT, SH3GL1 (EEN) and NonO (p54nrb).
8. The complex of any one of claims 1 to 7, wherein said first effector domain in said protein A is selected from:
(a) a DNA-binding domain (DBD); or
(b) a specific protease.
9. The complex of claim 8, wherein said DNA-binding domain (DBD) is or is derived from a bacterial DBD, from a fungal DBD, from a plant DBD or a DBD from a transcription factor.
10. The complex of claim 9, wherein said bacterial DBD is or is derived from lexA or Tet repressor, said fungal DBD is or is derived from GAL4 DBD, said plant DBD is or is derived from the DBD of TIZZ or the DBD of PHR1.
11. The complex of claim 9, wherein said DBD from a transcription factors is the DBD of a mammalian or a yeast transcription factor.
12. The complex of any one of claims 1 to 11 , wherein said second effector domain in said protein B is
(a) a transcriptional activation domain (AD); or
(b) a polypeptide activated by proteolytic cleavage.
13. The complex of claim 12, wherein said transcriptional activation domain (AD) is selected from the group consisting of GAL4 activation domain, VP16 activation domain, c-Jun, c-Fos, ELK1, CREB, ATF2 and CHOP.
14. The complex of claim 12, wherein said polypeptide activated by said proteolytic cleavage is a caspase, a transcription factor or a protein involved in a signal transduction cascade.
15. The complex of claim 14, wherein said caspase is selected from the polypeptide consisting of caspase-9, caspase-3, caspase-6, caspase-8 and/or wherein said transcription factor or said protein involved is a signal transduction cascade in NF-kappa B or Notch.
16. The complex of claim 12, 14 or 15, wherein said proteolytic cleavage is mediated by thrombin, TEV protease, secretase, enterokinase, HIV-1 retropepsin (human immunodeficiency virus type 1), nodavirus endopeptidase (flock house virus), ubiquitin C-terminai hydrolase UCH- L1 (Homo sapiens), foot-and-mouth disease virus L-proteinase (foot-and- mouth disease virus), caspase-1 (Rattus norvegicus), hedgehog protein (Drosophila melanogaster), poliovirus-type picornain 3C (poliovirus type 1), porcine transmissible gastroenteritis virus main protease (porcine transmissible gastroenteritis virus), togavirin (Sindbis virus), signal peptidase I (Escherichia coli), C-terminal processing protease-1 (Escherichia coli).
17. The complex of any one of claims 1 to 12 further comprising a nucleic acid molecule comprising a binding site for said DNA-binding domain and an effector gene.
18. The complex of claim 17, wherein said effector gene encodes for a polypeptide (or a fragment thereof) which is selected from the group consisting of
(a) a marker gene;
(b) a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug;
(c) an immunomodulating molecule;
(d) an antigen;
(e) a molecule capable of activating a senescence program, a differentiation program or apoptosis.
19. The complex of claim 18, wherein said marker gene is selected from group consisting of a fluorescent protein, a cell surface marker, β-Gal, luciferase and SEAP.
20. The complex of claim 18, wherein said prodrug converting enzyme is selected from the group consisting of purine nucleoside phosphorylase, thymidine kinase, cytosine deaminase, carboxyl esterase, carboxypeptidase A, carboxypeptidase G2, cytochrome P450, D-amino- acid oxidase, deoxycytidine kinase, DT-diaphorase, β-galactosidase, β- glucuronidase, β-lactamase, methionine-liase, nitroreductase, thymidine phosphorylase, and xanthine-guanine phosphoribosyl transferase.
21. The complex of claim 18, wherein said immunomodulatory molecule is selected from the group consisting of interleukin 2, costimulatory molecules (B7), and MHC molecules.
22. The complex of claim 18, wherein said antigen is a bacterial, viral or plant antigen and/or wherein said antigen is capable, when expressed, to elucidate immune response in a subject.
23. The complex of claim 18, wherein said molecule capable of activating a senescence program is CDKNI b, CDKN2b and/or CDKN2a.
24. The complex of claim 18, wherein said molecule capable of activating a differentiation program is selected from the group consisting of CEBPA (drives granulocytic differentiation), PU.1 (drives monocytic differentiation), and GATA-1 (drives erythroid differentiation).
25. The complex of claim 18, wherein said molecule capable of activating apoptosis is a caspase, a cytochrom-C and Fas receptor.
26. The complex of any one of claims 1 to 25, wherein said fusion protein comprising at least two epitopes is BCR/ABL (ABL/BCR).
27. The complex of claim 26, wherein the interaction domain capable of interacting with said first epitope derived from BCR is selected from the group consisting of a specific antibody part, an antibody construct in intracellular antibodies, an aptamer, BAP1 , GRB2, xeroderma pigmentosum group B protein, GRB10 (only to BCR/ABL), c-Fes, Ras GTPase activating protein, phospholipase C-gamma, 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, Abl (-fragments), Gads. RhoA GTP, RAC1 , BCR and ARG or (a) fragment(s) of said interacting molecules.
28. The complex of claim 27, wherein said fragment of BAP-1 is cBAP-1 or BAPα7-9.
29. The complex of any one of claims 26 to 28, wherein the interaction domain capable of interacting with said second epitope derived from ABL is selected from the group consisting of a specific antibody part, an antibody construct, an intracellular antibody, an aptamer, N-terminal 80 amino acids of ABL, Vav, Ikappa alpha, DDB1 , VASP, BRCA1 , RAD9 c- terminus, ik3-1, ik3-2, caveolinl, RIN1, Abi-1 , EphB2 receptor, phospholipid scramblase 1 , CRKL, SORBS1 , Amyloid precusor protein, Fe65, HPK1 , p21-activated protein kinase (PAK) family, gamma-PAK, Ggrgb4, hTERT, Cbl, N-methyl-D-aspartic acid receptor NR2D subunit, protein kinase C delta. TrkA, p53, p73, ATM, p62(dok), Ku70, SHPTP1 , JAK1 , ST5, RFXI, E2F-1 , RB, receptor tyrosine kinase p145c-kit, She, cyclic AMP response element (CRE)-binding protein (CREB), CRK, actin, EGFR, HCK, and SYP or (a) fragment(s) of said interacting molecules.
30. The complex of claim 29, wherein said fragment of ABL comprises the N- terminal part of ABL and herein the fragment of CRKL comprises the SH3 domain (CRKL-SH3n).
31. The complex of any one of claims 1 to 25, wherein said protein comprising at least two epitopes is AML1/ETO.
32. The complex of claim 31 , wherein the interaction domain capable of interacting with said first epitope derived from AML1 is selected from the group consisting of a specific antibody part (or a fragment thereof), an antibody construct, an intracellular antibody, an aptamer, CEBPA, PU.1, MORF, CBFB, HES-1 , BSAP, MITF, MEF, TLE and P300, or (a) fragment(s) of said interacting molecules.
33. The complex of claim 32, wherein said fragment of PU.1 is PU.1 (β3-β4).
34. The complex of any one of claims 31 to 33, wherein the interaction domain capable of interacting with said first epitope derived from ETO is selected from the group consisting of a specific antibody part (or a fragment thereof), an antibody construct, an intracellular antibody, an aptamer, N-CoR, importin , importin β, ETO, mSin3A and SMRT, or (a) fragment(s) of said interacting molecules.
35. A protein A as defined in any one of claims 1 to 34.
36. A protein B as defined in any one of claims 1 to 34.
37. A nucleic acid molecule encoding a protein A as defined in any one of claims 1 to 35.
38. A nucleic acid molecule encoding a protein B as defined in any one of claims 1 to 34 and claim 36.
39. The nucleic acid molecule of claim 37 and 38, whereby said nucleic acid molecule is selected from the group consisting of
(a) a nucleic acid molecule comprising a nucleic acid sequence as shown in any one of SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33 or 35;
(b) a nucleic acid molecule encoding a polypeptide as shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36;
(c) a nucleic acid molecule encoding a functional fragment or a functional domain of the polypeptide as shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36;
(d) a nucleic acid molecule which is at least 60% identical to the nucleic acid molecule as defined in (a), (b) or (c) and which encodes a protein A or a protein B of claim 34 or 35; and (e) a nucleic acid molecule which hybridises under stringent conditions with the complementary strand of the nucleic acid molecule as defined in (a) to (d).
40. A vector comprising the nucleic acid molecule of any one of claims 37 to
39 or comprising a nucleic acid molecule as defined in any one of claim 17 to 25.
41. A host cell comprising the vector of claim 40.
42. A method for the preparation of a protein A and/or a protein B as defined in any one of claims 1 to 36 comprising culturing the host of claim 41 under conditions that allow synthesis of said protein A and/or protein B or (a) fragment(s) thereof and recovering said said protein A and/or protein B or (a) fragment(s) from said culture.
43. A composition comprising a complex of any one of claims 1 to 35, a protein A of claim 35, a protein B of claim 36, a nucleic acid molecule of claims 37 to 39, a vector of claim 40, a host cell of claim 41 and/or nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined in any one of claims 17 to 25.
44. The composition of claim 43, which is a pharmaceutical or a diagnostic composition.
45. Use of a composition comprising a complex of any one of claims 1 to 35, a protein A of claim 35, a protein B of claim 36, a nucleic acid molecule of claims 37 to 39, a vector of claim 40, a host cell of claim 41 and/or nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined in any one of claims 17 to 25 for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of a proliferative disorder.
46. The use of claim 45, wherein said proliferative disorder is cancer or a tumorous disease.
47. The use of claims 45 or 46, wherein said proliferative disorder is a carcinoma disease, a sarcoma-disease, a lymphoma disease, a lipoma disease, or a leukemia.
48. The use of claims 45 to 47, wherein said proliferative disorder is AML (acute myeloid leukemia), mixed lineage leukemia.J, T-ALL (acute T cell lymphoblastic leukemia), B-ALL (actue B cell lymphoblastic leukemia), CMMoL (chronic myelomonocytic leukemia), AL (acute leukemia), MPD (myedodysplastic syndrome), CML (chronic myeloid leukemia), MPS(myelo proliferative syndrome), CLL (chronic lymphocytic leukemia), MALT-lymphoma, adenocarcinoma, alveolar (soft part) sarcoma, fibrosarcoma, nephroma, RAEB-T (refractory anemia with excess blasts in transformation), clear cell sarcoma, rhabdomyosarcoma, papillary renal cell carcinoma.
49. Use of a protein A of claim 35, a protein B of claim 36, a nucleic acid molecule of any one of claims 37 to 39 and/or a nucleic acid molecule comprising a binding site for a DNA-binding domain and an effector gene as defined in any one of claims 17 to 25, for the preparation of a diagnostic composition for the detection of a protein comprising at least two epitopes as defined in any one of claims 1 to 7.
50. The use of claim 49, wherein said diagnostic composition is employed for the detection of a fusion protein.
51. The use of claim 50, wherein said fusion protein is an oncogenic fusion protein.
52. An in vivo and/or in vitro method for the detection of a protein comprising at least two epitopes as defined in any one of claims 1 to 7 or for the detection of a cell comprising such a protein, said method comprising the steps of:
(a) introducing and/or expressing in cell suspected to comprise a protein comprising at least two epitopes as defined in any one of claims 1 to 7, a protein A and a protein B as defined in any one of claims 1 to 36;
(b) detecting the increase or the decrease of the expression of an effector gene, detecting the increase or decrease of a specific signal elucidated by said effector gene, and/or detecting the increase or decrease of the activity of an cellular effector.
53. A method for the elimination of a cell comprising a fusion protein comprising at least two epitopes as defined in any one of claims 1 to 7 comprising the steps of:
(a) introducing and/or expressing in a cell suspected of comprising a fusion protein comprising at least two epitopes as defined in any one of claims 1 to 7 a protein A and a protein B as defined in any one of claims 1 to 36 or introducing and/or expressing in said cell at least one nucleic acid molecule coding for a protein A and a protein B as defined in any one of claims 1 to 36; and
(b) eliciting in said cell the expression of an effector gene or an cellular effector which leads to the elimination of said cell.
54. The method of claim 52 or 53, further comprising as step (aa) the introduction of a nucleic acid molecule as defined in any one of claims 17 to 25.
55. The method of any one of claims 52 to 54, wherein said effector gene encodes for
(a) a marker gene; (b) a prodrug converting enzyme and/or a polypeptide capable of sensitising a cell for a drug;
(c) an immunomodulating molecule;
(d) an antigen;
(e) a molecule capable of activating a senescence program, a differentiation program or apoptosis.
56. The method of claim 52 or 54, wherein cellular effector is selected from the group consisting of caspase, NF-kappa B and NOTCH.
57. The method of any one of claims 52 to 56, wherein said method is carried out ex vivo.
58. The method of any one of claims 52 to 57, wherein said method comprises the detection of a fusion protein comprising two epitopes in a blood cell/a bone marrow cell or wherein said elimination comprising the elimination of a blood cell/a bone marrow cell comprising a fusion protein comprising two epitopes as defined in any one of claims 1 to 7..
59. A kit comprising
(a) a protein A of claim 35 or a nucleic acid molecule of claim 37 or 39;
(b) a protein B of claim 36 or a nucleic acid molecule of claim 34 or 38;
(c) a nucleic acid molecule as defined in any one of claims 17 to 25; and/or
(d) a vector as defined in claim 40 or a vector comprising a nucleic acid molecule as defined in any one of claim 17 to 25.
60. A pharmaceutical composition comprising
(a) a protein A of claim 35 or a nucleic acid molecule of claim 37 or 39; (b) a protein B of claim 36 or a nucleic acid molecule of claim 34 or 38; and/or
(c) a nucleic acid molecule as defined in any one of claims 17 to 25; and/or
(d) a vector as defined in claim 40 or a vector comprising a nucleic acid molecule as defined in any one of claim 17 to 25.
61. A transgenic, non-human animal
(a) expressing a protein A of claim 35 and/or a protein B of claim 36; or
(b) comprising a nucleic acid molecule of claim 37 or 39 and/or a nucleic acid molecule of claim 38 or 39.
62. The transgenic, non human animal further comprising a nucleic acid molecule as defined in any one of claims 17 to 25.
PCT/EP2003/013323 2002-12-05 2003-11-26 Genetic switches for the detection of fusion proteins WO2004050870A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003289899A AU2003289899A1 (en) 2002-12-05 2003-11-26 Genetic switches for the detection of fusion proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP02027501.2 2002-12-05
EP02027501 2002-12-05

Publications (2)

Publication Number Publication Date
WO2004050870A2 true WO2004050870A2 (en) 2004-06-17
WO2004050870A3 WO2004050870A3 (en) 2004-09-23

Family

ID=32405696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/013323 WO2004050870A2 (en) 2002-12-05 2003-11-26 Genetic switches for the detection of fusion proteins

Country Status (2)

Country Link
AU (1) AU2003289899A1 (en)
WO (1) WO2004050870A2 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150152160A1 (en) * 2005-05-12 2015-06-04 Zymogenetics, Inc. Compositions and methods for modulating immune responses
CN107189986A (en) * 2017-07-25 2017-09-22 南京大学 A kind of reporter gene cell line construction method and its application based on er stress
CN107365783A (en) * 2017-07-26 2017-11-21 中国人民解放军南京军区南京总医院 A kind of new fusion of MiT families transposition clear-cell carcinoma and its detection primer and application
WO2021026601A1 (en) * 2019-08-12 2021-02-18 Macquarie University Compositions and methods for treatment
US10946095B2 (en) 2015-09-02 2021-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human T-cell immunoglobulin and ITIM domain (TIGIT)
WO2024026465A1 (en) * 2022-07-29 2024-02-01 The Broad Institute, Inc. Programmable pattern recognition compositions
US12397035B2 (en) 2019-08-12 2025-08-26 Celosia Therapeutics Pty Ltd Compositions and methods for treatment

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117867032B (en) * 2024-01-22 2024-07-02 湛江中心人民医院 Construction method and application of aplastic anemia zebra fish model

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6686165B2 (en) * 1997-05-20 2004-02-03 Erasmus Universiteit Rotterdam Recognition of tumor-specific gene products in cancer

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150152160A1 (en) * 2005-05-12 2015-06-04 Zymogenetics, Inc. Compositions and methods for modulating immune responses
US9695238B2 (en) 2005-05-12 2017-07-04 Zymogenetics, Inc. Screening method for inhibitors of binding of zBR7R1 to CD155
US9994637B2 (en) 2005-05-12 2018-06-12 Bristol-Myers Squibb Company Method of treating CD155 expressing cancers
US10946095B2 (en) 2015-09-02 2021-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human T-cell immunoglobulin and ITIM domain (TIGIT)
US12121579B2 (en) 2015-09-02 2024-10-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Antibodies specific to human t-cell immunoglobulin and ITIM domain (TIGIT)
CN107189986A (en) * 2017-07-25 2017-09-22 南京大学 A kind of reporter gene cell line construction method and its application based on er stress
CN107365783A (en) * 2017-07-26 2017-11-21 中国人民解放军南京军区南京总医院 A kind of new fusion of MiT families transposition clear-cell carcinoma and its detection primer and application
WO2021026601A1 (en) * 2019-08-12 2021-02-18 Macquarie University Compositions and methods for treatment
JP2022544532A (en) * 2019-08-12 2022-10-19 マッコーリー ユニバーシティー Compositions and methods for treatment
US12397035B2 (en) 2019-08-12 2025-08-26 Celosia Therapeutics Pty Ltd Compositions and methods for treatment
WO2024026465A1 (en) * 2022-07-29 2024-02-01 The Broad Institute, Inc. Programmable pattern recognition compositions

Also Published As

Publication number Publication date
WO2004050870A3 (en) 2004-09-23
AU2003289899A1 (en) 2004-06-23

Similar Documents

Publication Publication Date Title
KR100404737B1 (en) Tumor Necrosis Factor / Nerve Growth Factor (TNF / NGF) Superfamily Receptor and Water-soluble Oligomeric Tumor Necrosis Factor / Nerve Growth Factor
Cosentino et al. Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo-and heterodimers in vivo.
KR101476458B1 (en) Protease screening methods and proteases identified thereby
US6429298B1 (en) Assays for identifying functional alterations in the p53 tumor suppressor
JP2002526108A (en) Fusion of scaffold protein with random peptide library
SK287889B6 (en) Isolated polypeptide that binds to TRAF2 and modulates activity of NF-B, DNA sequence encoding its, pharmaceutical composition containing them and their use
CN101517074A (en) Protease screening methods and proteases indentified thererby
EP2185707B1 (en) Cell permeable nm23 recombinant proteins, polynucleotides encoding the same, and anti-metastatic composition comprising the same
AU9720598A (en) Binding partners for inhibitors of cyclin-dependent kinases and their use for searching for inhibitors and for the diagnosis or therapy of a disease
WO2004050870A2 (en) Genetic switches for the detection of fusion proteins
JPWO2002099104A1 (en) Polypeptides that destabilize proteins in cells under aerobic conditions and DNA encoding them
RU2297241C2 (en) Chimeric protein for malignant lymphoma treatment
KR100820526B1 (en) IREN protein, its manufacture and uses
JP4927546B2 (en) Interactive polypeptides containing heptapeptide patterns and cell permeation domains
US5770720A (en) Ubiquitin conjugating enzymes having transcriptional repressor activity
AU720755B2 (en) Ubiquitin conjugating enzymes having transcriptional repressor activity
AU4284799A (en) Screening system
KR100552547B1 (en) TNF Receptor Associated Factor (TRAF) Modulators, Preparations and Their Uses
WO2001018036A2 (en) Methods and reagents for regulating gene expression
Lee et al. Tra2α and hnRNP K might be functional partners of Rbm for regulation of RNA processes during spermatogenesis
Podack et al. Perforin and the mechanism of lymphocyte-mediated cytolysis
Botha A yeast-based assay for detection of mutations in the human p16 gene
Thomas The interaction of FADD with death receptors
AU2006202361A1 (en) Human P53 mutations and a genetic system in yeast for functional identification of human P53 mutations
Skepu Identification and characterisation of a novel gene, DWNN, isolated from promoter-trapped Chinese hamster ovary cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP