WO1999025736A1 - Proteines et nucleotides de la leucemie lymphocytaire chronique - Google Patents

Proteines et nucleotides de la leucemie lymphocytaire chronique Download PDF

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Publication number
WO1999025736A1
WO1999025736A1 PCT/SE1998/002052 SE9802052W WO9925736A1 WO 1999025736 A1 WO1999025736 A1 WO 1999025736A1 SE 9802052 W SE9802052 W SE 9802052W WO 9925736 A1 WO9925736 A1 WO 9925736A1
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sequence
seq
nucleic acid
leukemia associated
base pairs
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PCT/SE1998/002052
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English (en)
Inventor
Stefan Einhorn
Yie Liu
Omid Rasool
Dan GRANDÉR
Eugene Zabarovsky
David Oscier
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Karolinska Innovations Ab
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Priority to AU12672/99A priority Critical patent/AU1267299A/en
Publication of WO1999025736A1 publication Critical patent/WO1999025736A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the present invention relates to nucleotides and proteins associated with chronic lympho- cytic leukemia as well as a process for the manufacture of proteins and peptides from such nucleotides.
  • Leukemia is a generic term relating to a group of blood diseases with disturbed matura- tion and an increased development of white blood cells, which was first described by
  • B-cell chronic lymphocytic leukemia (B-CLL) is the most common haematological ma- lignancy in the Western world and accounts for approximately 30% of all leukemia cases. It is recognized by a slowly growing increase of relatively mature lymphocytes, which are enriched in the blood, bone marrow, lymph nodes and spleen. The affected cells are primary B-lymphocytes, which are relatively similar to the normal lymphocytes in the blood. The illness usually has a quiet course with few or no symtoms during several years, even though the course may be more rapid in some patients. The condition is usually not treated until symtoms have occured, when irradiation and chemotherapy may have a suppressing effect.
  • D13S25 locus approximately 1.6 cM telomeric of the RBI gene
  • D13S319 located between the RBI gene and D13S25
  • BRCA2 breast cancer susceptibility gene 2
  • Kalachikov et al (Genomics 42, 369-377 (1997)) have defined a region of about 300 kb, which is derived from patients with relatively limited deletions. However, only parts of said defined region have been sequenced. As there are no complete sequences or suggestions regarding the exon/intron structure of the many expressed sequences in the area, it is not possible to produce any cDNA thereof and consequently, it is not possible to produce any peptides or proteins based on this reference. Regardless of the above, Kalachikov et al have not performed any detailed mutational analysis of the region disclosed therein, which leaves the question of whereabout the regions essential in B-CLL are located unanswered. Accordingly, it is not possible to produce any peptides or proteins relevant in B- CLL on the basis of the information provided by this reference. Conclusively, this refer- ence does not constitute any sufficient basis for the development of diagnostic tools or treatment schemes for the disease.
  • the present inventors constructed and analyzed a YAC, PAC and cosmid contig covering the deleted region of chromosome 13ql4.
  • the results showed that the minimally deleted region, defining the location of the putative tumor suppressor gene(s), spans an area of less than 130 kb centromeric to the locus D13S272 (Corcoran MM, Rasool O, Liu Y, Iyengar A, Grander D, Ibbotson RE, Merup M, Wu X, Brodyansky V, Gardiner AC, Juliusson G, Chapman RM, Tiller M, Gahrton G, Yankonsky N, Zabarovsky E, Oscier DG and Einhorn S. Blood, in press).
  • the object of the present invention is to fulfill the needs defined above.
  • the minimally deleted region has been narrowed to a 10 kb area and two neighbouring genes that span this region have been identified. Consequently, the invention relates to such leukemia associated deoxyribonucleic acids (DNAs) as well as to complementary deoxyribonucleotides (cDNAs) derived from within said region.
  • the invention also relates to peptides, proteins and processes for the manufacture thereof as well as to antibodies towards such peptides and proteins useful inter alia in the diagnosis, treatment and prevention of B-CLL.
  • Figure 1 is a general physical and transcriptional map of the region on 13ql4.3 frequently deleted in B-CLL.
  • Figure 2 A discloses a detailed restriction map of the cosmids covering the minimally deleted region
  • Figure 2B discloses a detailed deletion map of CLL patients 2, 4, 5, 6 and the CLL cell line 183-E95, in the 8.5 kb EcoRI/EcoRI fragment of cosmid c30a and the areas surrounding this fragment.
  • Figure 3 discloses a Southern blot analysis of Hindlll-digested DNA hybridized to DNA markers overlapping or surrounding the minimally deleted area. DNA from CLL patients is compared with DNA from healthy donors acting as controls.
  • Figure 4 discloses a Southern blot hybridization of EcoRI-digested DNA with DNA markers overlapping or surrounding the minimally deleted region.
  • Figure 5 discloses the general characteristics of cDNA clones in the present region.
  • Figure 6 discloses an expression analysis of Leul and Leu2 genes.
  • Figure 7 discloses the sequence of forward (F) and reverse (R primers used in mutation screening and/or in amplification of exons of the Leul and Leu2 genes.
  • Figure 8 discloses EcoRl fragments in the minimal deleted area as well as location of ESTs (expressed sequence tags) mapped to the introns of the Leul and Leu2 gene area according to the invention.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • a “label” is a composition detectable by spectroscopic, photochemical, biochemical, im- munochemical, or chemical means.
  • useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in a ELISA), biotin, di- oxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (for example, the peptide encoded by SEQ ID NO 1 can be made detectable, e.g., by incorporating a radio-label into the peptide).
  • nucleic acid probe is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond for- mation.
  • a probe may include natural (i.e. A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.)
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather that phosphodiester linkages.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labeled as with isotopes, chromo- phores, lumiphore, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind.
  • biotin to which a streptavidin complex may later bind.
  • a "labeled nucleic acid probe” is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
  • target nucleic acid refers to a nucleic acid (often derived from a biological sample), to which a nucleic acid probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified.
  • the target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target.
  • target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the ovarall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from context.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • a “peptide mimetic” is a molecule that mimics the biological activity of a peptide, but is no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.
  • peptidomimetic refers to a compound exhibiting a spatial arrangement of reactive chemical entities that closely resembles the three dimen- sional arrangement of active groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems which are similar to the biological activity of the peptide.
  • Subsequence refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide), respectively.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are gen- erally present in excess, at Tm, 50% of the probes are occupies at equilibrium).
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
  • Stringent condi- tions may also be achieved with the addition of destabilizing agents such as formamide.
  • nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • Bind(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accomodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
  • polypeptide or nucleotide sequence indicates that a polypeptide or nucleotide comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues, or the corresponding number of bases.
  • An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. In this context, see e.g.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody as used herein, also includes antibody fragments either produced by the modifi- cation of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
  • a “chimeric antibody” is an antibody molecule in which (a) the constant region, or a por- tion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • immunoassay is an assay that utilizes an antibody to specifically bind an analyte.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the analyte.
  • isolated purified or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.
  • recombinant when used with reference to a cell, or nucleic acid, or vector, indicates that the cell, or nucleic acid, or vector, has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • nucleic acids or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, GESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by inspection.
  • BLAST refers in the present context to the BLAST algorithm, which in addition to e.g. FASTA is an algorithm that is suitable for determining sequence similarity and which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology In- formation (http://www.ncbi.nlm.nih.gov/). See also Henikoff and Henikoff ( 1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919); Karlin and Altschul (1993) Proc. Nat 'I Acad. Sci. USA 90: 5873-5787.
  • the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbour Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a “gene product”, as used herein, refers to a nucleic acid whose presence, absence, quantity, or nucleic acid sequence is indicative of a presence, absence, quantity, or nucleic acid composition of the gene.
  • Gene products thus include, but are not limited to, a mRNA transcript, a cDNA reverse transcribed from a mRNA, and RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA or subsequences of any of these nucleic acids.
  • Polypeptides expressed by the gene or subsequences thereof are also gene products. The particular type of gene product will be evi- dent from the context of the usage of the term.
  • the present invention relates to leukemia associated nucleic acids, such as cDNAs and DNAs, involved in B-cell chronic lymphocytic leukemia for use in diagno- sis and/or treatment of said condition as well as in other conditions related to defects or malignacies in the same genetic region.
  • the nucleic acids according to the invention may inter alia be used in the development of diagnostic tools and may also be used in the production of peptides or proteins encoded by such sequences, as is described in more detail below.
  • An additional advantageous use is in research, such as in various biochemical studies.
  • a second aspect of the present invention is a leukemia associated protein, which comprises an amino acid sequence encoded by a nucleotide as defined above or any variant thereof.
  • one aspect of the invention is a leukemia associated nucleic acid, such as a cDNA molecule, which comprises at least about 300, preferably about 380-390 and most preferred about 385 base pairs of the sequence according to SEQ ID NO 1 , such as the region defined as the first exon of said sequence.
  • a leukemia associated nucleic acid such as a cDNA molecule comprising at least about 600, preferably about 610-620 and most preferred 616 base pairs of the sequence according to SEQ ID NO 1, such as the region defined as the second exon of said sequence.
  • the leukemia associated cDNA molecule as defined above comprises essentially all of the sequence according to SEQ ID NO 1.
  • the nucleic acid of this embodiment of the invention is substantially identical to said sequence. This embodiment also encompasses any variants of such a sequence.
  • the leukemia associated cDNA molecule comprises at least about 100, preferably about 150-160 and most preferred about 156 base pairs of the sequence according to SEQ ID NO 6, such as the region defined as the first exon of said sequence.
  • a leukemia associated nucleic acid such as a cDNA mole- cule, which comprises at least about 50, preferably about 90-100 and most preferred about 96 base pairs of the sequence according to SEQ ID NO 6, such as the region defined as the second exon of said sequence.
  • a leukemia associated nucleic acid such as a cDNA molecule, which comprises at least about 100, preferably about 125-135 and most preferred about 131 base pairs of the sequence according to SEQ ID NO 6, such as the region defined as the third exon of said sequence.
  • a leukemia associated nucleic acid such as a cDNA molecule, which comprises at least about 1300, preferably about 1370-1380 and most preferred about 1376 base pairs of the sequence according to SEQ ID NO 6, such as the region defined as the fourth exon of said sequence.
  • the leukemia associated cDNA molecule as defined above comprises essentially all of the sequence according to SEQ ID NO 6.
  • the nucleic acid of this embodiment of the invention is substantially identical to said sequence. This embodiment also encompasses any variants of such a sequence.
  • the leukemia associated nucleic acid is of mouse origin. More specifically, the mouse nucleic acid according to the invention comprises one or more of the sequences selected from the group consisting of SEQ. ID NO. 9, SEQ. ID NO. 10, SEQ. ID NO. 11, SEQ. ID NO. 12 and 13, originating from mouse Leu2 gene, wherein SEQ ID NOS. 9-12 shows clones of said sequence and SEQ ID NO. 13 discloses the mouse homologue of Leu2.
  • the homology between human and mouse Leu2 is about 75-80%, some mouse exons being longer than the human correspondence.
  • the novel mouse Leu2 gene according to the invention was identified and located by first searching available EST databases using the BLAST and FASTA programs, see reference above. Three homologous ESTs were found. Partial sequencing of these ESTs on an ABI PRISM 310 Genetic Analyzer further established the homology to the human Leu2 gene.
  • the invention also encompasses any variants of the present sequences.
  • the present invention also relates to an isolated leukemia associated nucleic acid having a sequence substantially identical with a sequence selected from the group comprised of SEQ. ID NO. 14, SEQ. ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18. The locations of said sequences are described in more detail in Figure 8, from which it appears that they are located between exon 1 and exon 3 of Leul .
  • the nucleic acids according to the present invention are cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS) and the self-sustained sequence repli- cation system (SSR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • SSR self-sustained sequence repli- cation system
  • nucleic acids according to the invention may also be isolated by routine cloning methods, e.g by using a cDNA, or any other gene product, according to the invention as a probe that specifically hybridize to all, or parts, of the leukemia associated gene according to the invention in a genomic sample, see e.g the references above.
  • the nucleic acids according to the invention may be synthesized by conventional means, for example on commercially automated DNA synthesizers, such as an Applied Biosystems (Foster City, CA), Model 380B, 392 or 394 DNA/RNA synthesizer, or by methods such as the phosphotriester method (Narang et al., Methods Enzymol.
  • the present nucleic acids are used as probes.
  • probes are preferably labeled with detectable labels.
  • the labels may be incorporated by any one of a number of means well known to those of skill in this field. The label is pref- erably incorporated simultaneously during the amplification step in the preparation of the sample nucleic acids.
  • PCR polymerase chain reaction
  • a label is incorporated into the transcribed nucleic acid by transcription amplification using a labeled nucleotide, such as a fluorescein-labeled UTP and/or CTP.
  • a label is added directly to an original nucleic acid sample (e.g. mRNA, polyA mRNA, cDNA etc.) or to the amplification product after the amplification is completed.
  • Means of attaching labels to nucleic acids are well known to those skilled in the art and include for example nick-translation and end-labelling (e.g.
  • Detectable labels for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful examples include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes (e.g. fluorescein, texas red, rhodamine, green fluorescent protein etc.), radiolabels (e.g. 3 H, 125 1, 35 S, 14 C and 32 P), enzymes (e.g. horse radish peroxidase, alkaline phosphatase and others commonly used in ELIZA) and col- orimetric labels.
  • biotin for staining with labeled streptavidin conjugate magnetic beads, fluorescent dyes (e.g. fluorescein, texas red, rhodamine, green fluorescent protein etc.), radiolabels (e.g. 3 H, 125 1, 35 S, 14 C and 32 P), enzymes (e.g. horse radish peroxidase,
  • a further aspect of the present invention is a leukemia associated DNA molecule, which comprises at least about 45, preferably about 50, base pairs of the sequence according to SEQ. ID NO. 3.
  • the first 90 bases are the ones preceeding the Leu2 gene in the chromosome and may comprise the promotor sequence.
  • Bases nos 91- 575 corresponds to the first exon of Leul and the Leul coding sequence ends at base no 575.
  • said DNA molecule comprises essentially all of the se- quence as defined in SEQ. ID NO. 4.
  • the nucleic acid of this embodiment of the invention is substantially identical to said sequence. This embodiment also encompasses any variants of such a sequence.
  • leukemia associated DNA molecule defined above comprises essentially all of the sequence according to SEQ. ID NO. 3, or any variant thereof.
  • the leukemia associated DNA molecule comprises essentially all of the sequence according to SEQ ID NO 8, or any variant thereof.
  • bp nos 747-1902 represent DNA encoding Leu4
  • bp nos 4392-6022 represents the DNA encoding Leu3, both of which are desribed more detailed below.
  • the above defined DNA molecule also includes parts, or all, of the sequence according to SEQ ID NO 5. Preferably, such parts exhibits regulatory elements, such as promotors, terminators etc.
  • Leu3 it has been shown that one exon, comprising about 80% of the gene, is located within intron 1 of Leu2. It has also been shown that another exon of Leu3 is located within intron 2 of Leu2. Presumably, a small Leu3 exon is located closer to exon 1 of Leu2.
  • the present invention also relates to cDNA sequences other than those specifically mentioned above, such as nucleic acids that hybridize specifically under stringent conditions to the nucleic acids according to the invention, which are easily produced by someone skilled in this field with reference to the present specification as well as to the references cited herein.
  • cDNA sequences other than those specifically mentioned above, such as nucleic acids that hybridize specifically under stringent conditions to the nucleic acids according to the invention, which are easily produced by someone skilled in this field with reference to the present specification as well as to the references cited herein.
  • a variety of hybridization formats are known in the art. See, for example, ' “Nucleic Acid Hybridization, A Practical Approach", ed. Hames, B.D. and Higgins, S.J., IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci., USA, 63:378-383 (1969); and John et al, Nature 223:582-587 (1969).
  • the present invention provides the necessary information concerning the region suggested to be the most essential one in B-CLL to enable the development of diagnostic and prognos- tic tools as well as treatments and possibly schemes for preventing the disease.
  • RT-PCR genomic and reverse transcription polymerase chain reaction
  • SSCP single strand conformation polymorphism
  • the present invention also relates to various assays, wherein the nucleic acids described above are used.
  • assays are for example nucleic acid assays, e.g. wherein human expression of the gene according to the invention is detected and/or quantified.
  • they are hybridization assays, wherein the presence, absence or quantity of DNA encoding proteins or polypeptides according to the invention is evaluated.
  • they may be amplification based assays, wherein the gene according to the invention is de- tected using an amplification based assay.
  • the nucleic acids according to the invention may also advantageously be used as primers in PCR.
  • the present invention also relates to the use of the nucleic acids disclosed above in methods for diagnosis, such as diagnosis of patients suffering from chronic lymphocytic leukemia and/or individuals with a predisposition for developping chronic lymphocytic leukemia, as well as to kits for performing such analyses per se.
  • a kit according to the invention will comprise one or more reagents for determining the presence or absence of the gene according the invention or mutations thereof, or for quantifying the expression thereof.
  • reagents may e.g. be nucleic acid probes binding to the normal gene according to the invention or probes that specifically bind to an abnormal such gene, which for example may include premature truncations, insertions or deletions.
  • the kit may also include antibodies with the desired binding properties.
  • the antibody or hybridization probe may be free or immobilized on a solid support, such as a test tube, a microtiter plate, a dipstick or the like.
  • a kit according to the invention may contain appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers etc.
  • the kits according to the invention may e.g. be PCR based kits.
  • the present invention relates to various gene products originating from the novel leukemia associated genes according to the invention as well as the uses thereof, such as the uses disclosed above. More specifically, the present invention also relates to genomic DNA sequences encoding allelic variant forms of the present proteins useful in therapy and/or prophylaxis relating to leukemia, preferably B-CLL, but also in other malignacies of the same genetic area.
  • the invention is understood to encompass any se- quence which, but for the degeneracy of the genetic code, would hybridize to the above defined sequences.
  • the genomic sequences are e.g. useful in gene activation techniques (supra), as well as in biochemical studies.
  • yet a further aspect of the present invention is a leukemia associated protein en- coded by the amino acid sequence disclosed in SEQ. ID. NO. 2 or any analogue orvariant thereof.
  • This protein has a molecular weight of approximately 8 kb.
  • all embodiments relating to proteins also relates to any analogues or functional fragments thereof.
  • the leukemia associated protein is encoded by the amino acid sequence disclosed in SEQ. ID. NO. 7 or any variant thereof, and has a molecular weight of about 10 kb.
  • the invention also encompasses all proteins that may be obtained by the use of the above defined cDNA molecules, such as the ones defined as exons in the sequence listings herein.
  • the proteins and polypeptides according to the invention may be produced by general laboratory procedures as disclosed in the literature. See e.g. Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2 nd ed.), vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, as well as other references cited herein. Synthetic methods, such as solid phase synthesis, may also be used for the preparation of the pro- teins and polypeptides disclosed herein, see e.g. Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology, vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield et al., J. Am. Chem. Soc, 85:219-2156 (1963) and Stewart et al., Solid Phase Synthesis, 2n d ed., Pierce Chem. Co., Rockford, 111. (1984)).
  • the present invention also relates to peptidomimetic compositions, which are analogues that mimic the activity of biologically active peptides according to the invention, i.e., compositions, which may function in leukemia patients to replace the protein expressed by the gene according to the invention in a healthy individual.
  • peptidomimetic compositions which are analogues that mimic the activity of biologically active peptides according to the invention, i.e., compositions, which may function in leukemia patients to replace the protein expressed by the gene according to the invention in a healthy individual.
  • the techniques of developing peptidomimetics are conventional and the development of peptidomimetics may be aided by determining the tertiary structure of the original peptide, e.g. by NMR spectros- copy, crystallography and/or, preferably, computer-aided molecular modelling etc.
  • the peptidomimetic compositions according to the invention are advantageously of higher potency, of greater stability and/or of greater bio
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • the present invention encompasses use of the peptides, polypeptides and proteins according to the invention as lead compounds in such methods.
  • another aspect of the present invention is a process for the manufacture of a leukemia associated protein or peptide.
  • Such a process will comprise the following steps: a) the introduction of a nucleic acid as defined above in a suitable vector; b) the transformation of a suitable host by said vector; c) the expression thereof in said host; and d) the recovery of the desired protein or peptide.
  • the product protein or polypeptide is purified by any suitable conventional method, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like, see e.g R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutcher, Methods in Enzy- mology, vol. 182: Guide to Protein Purification, Academic Press, Inc.
  • the specific nucleic acid used in the process may be chosen in such a way as to fit a correct open reading frame for the expression of a protein or peptide which is useful for any of the advantageous purposes defined elsewhere in this application.
  • the present proteins may possess a conformation substantially different from the native conformations of the constituent polypeptides. Thus, it may be necessary to denature and reduce the polypeptide and then cause it to re-fold into the preferred conformation, see e.g. Debinski et al. (1993) J. Biol. Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconj.
  • the present invention also relates to a vector which comprises a nucleic acid according to the invention as well as to a recombinant cell harbouring such a vector.
  • the vector may be any suitable vector, such as a plasmid, a virus, an oligonucleotide etc. and may be used for the transformation of cells in vitro or in vivo to express the proteins ac- cording to the invention. They are also useful for expressing those proteins to provide immunogens for antibody production, which will be disclosed in detail below.
  • the proteins may be provided in order to provoke a T-cell response for immunisation purposes, such as in a DNA based vaccine.
  • the cell may be procaryotic or eucaryotic, such as a bacterial cell, e.g. E.coli, a yeast cell, a mammalian cell, e.g. a human cell, etc. Gen- erally, the use of a eucaryotic cell is preferred, as the present gene is of eucaryotic origin.
  • the culture of cells used in the present invention including cell lines and cultured cells from tissue and blood samples, is well known in the art.
  • Freshney (Culture of Animal Cells, A Manual of Basic Training, 3 rd ed., New York, NY (1994)) and the references cited therein provide a general guide to the culture of cells.
  • the invention also relates to the use of tissue for the herein described purposes.
  • the invention encompasses an isolated human cell, wherein the gene according to the invention is present and wherein the expression thereof has been modified by the inclusion and/or depletion, preferably by homologous recombination, of regulatory elements by gene activation technique (see e.g. US Patent No. 5 641 670; and Genetic Engineering News, April 15, 1994).
  • the present invention relates to an antibody directed towards a leukemia associated protein or peptide as defined above.
  • the antibody may be monoclonal or poly- clonal, while the preferred embodiment is a monoclonal antibody.
  • the antibodies according to the invention are raised to the present proteins and/or polypeptides, including individual, allelic, strain or species variants, and fragments thereof, both in their naturallly occuring (full-length) and in recombinant forms.
  • the antibodies are raised in either their native configurations or in non-native configurations. Anti-idiotypic antibodies are also included.
  • the antibodies according to the invention are also useful in immunohistochemical methods for the diagnosis of tu- mor material or in other functional studies of these proteins.
  • the antibodies according to the present invention are easily produced by someone skilled in this field with reference to the background given herein concerning the various sequences.
  • the invention relates to the use of the present antibodies in screening methods, wherein the antibodies are used as target to screen for pharmaceutically active compounds.
  • the present invention will provide a useful basis for the development of a plurality of advantageous applications, which have not been possible before.
  • diagnosis of patients with chronic lymphocytic leukemia, and possibly other malignancies, such as various lymphomas and leukemias will be greatly improved by the aid of the nucleotides, proteins and antibodies according to the invention.
  • Suitable kits for such diagnosis are now easily developped and also fall within the scope of the present invention.
  • the gene according to the invention may be cloned with a tag, preferably a conventionally used tag, such as His, Flag or Myc, in order to provide easier purification, to study protein interaction etc. Methods for such an inclusion of a tag are known to those of skill in the field.
  • the invention also relates to different applications of the peptides and proteins disclosed herein, such as the use thereof for the identification of substances and molecules useful as medicaments and/or prophylaxis of B-CLL as well as the use thereof for various research purposes.
  • some of said proteins and peptides may be used pharmaceutically er se, which use is also encompassed in the scope of the present invention.
  • the term "protein” may also be in- terpreted as a fusion protein, which in some instances advantageously may be used as an alternative or substitute to the protein, as it may render further desired properties to the protein. Additionally, or alternatively, it may enhance the production of protein.
  • fusion proteins produced in mammalian cells include cytokine im- munoconjugates, immunodhesins and immunotoxins. Based on the present description, a fusion protein is easily created by someone skilled within this area with reference to conventional techniques and methods, see e.g. Smith and Johnson (1988), Gene 67:31 ; Hopp et al. (1988) Biotechnology 6: 1204; La Value et al. (1993) Biotechnology 11 :187.
  • the invention also relates to methods for the treatment of patients suffering from B-CLL or any other malinancy of the same gene and/or the prevention thereof by aid of the above defined products in states where such claim categories are allowed.
  • the invention also relates to the use of peptides, proteins, fusion proteins, antibodies etc. in therapy.
  • the DNA sequences according to the invention may be used in therapy in order to correct deficiences present in B-CLL patients and/or as a prevention.
  • the invention also relates to the use of such molecules as mentioned above as medicaments and/or in the manufacture of medicaments for the treatment of leukemia, such as B-CLL.
  • medicaments or pharmaceutical preparations may be in a form suitable for parental, topical, oral or local administration and may be administered in a variety of dosage forms, depending on the mode of administration.
  • the pharmaceutical prepa- ration will commonly comprise a solution of a polypeptide, antibody or fusion protein dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier, e.g. buffered saline.
  • a pharmaceutically acceptable carrier such as an aqueous carrier, e.g. buffered saline.
  • a pharmaceutically acceptable carrier such as an aqueous carrier, e.g. buffered saline.
  • a pharmaceutically acceptable carrier such as an aqueous carrier, e.g. buffered saline.
  • auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents etc., e.g. sodium acetate, sodium chloride, potassium chloride, sodium lactate and the like.
  • the pharmaceutical preparation according to the in- vention comprises one or more of the above described mimetics and such mimetics are used in methods of treatment and/or prophylaxis.
  • the pharmaceutical preparation according to the invention is a DNA vaccine composition, comprising one or more of the nucleic acids according to the invention.
  • the invention also encompasses methods aimed at vaccination of subjects, such as humans, for protection against one or more of the malignacies related to the herein disclosed genetic area, e.g. against B-CLL, as well as the use of the present nucleic acids in prophylactic compounds and the use thereof in the manufacture of a DNA vaccine.
  • the present invention relates to transgenic animals, wherein a molecule according to the invention has been introduced.
  • Such animals may be produced by methods, which are used conventionally for this purpose.
  • Transgenic animals such as mammals, e.g. mice, rats, etc, comprising the molecules within their genomes may be used as models for studies of leukemia, such as B-CLL, and other malignacies of the same ge- netic area, as well as in the development of drugs, therapies etc.
  • the invention also encompasses knock-out animals, such as knock-out mice, wherein the function of the present genes have been knocked out through recombination by standard methods and by the aid of products developped from the invention.
  • knock-out animals according to the invention may also provide additional information, as the knock-out of one gene some- times give rise to additional, totally unexpected symptoms.
  • a knock-out animal, such as a knock-out mouse, according to the invention may also be used as a model for the study of other conditions, for which it has been made suitable by the knock-out of the present gene or parts thereof.
  • the present invention relates to use of the present nucleic acids in methods of gene and/or cell therapy as well as such methods er se. Accordingly, the invention also relates to packageable nucleic acids according to the invention for the transformation of cells in vitro and in vivo. These can be inserted in any number of well known vectors for the transection or transformation of target cells and organisms.
  • the nucleic acid will, under the control of a promoter, then express the encoded protein thereby mitigating the effects of absent or defect genes or partial inactivation of the gene or abnormal expression thereof.
  • the methods according to the invention can involve transfecting cells of a mammal with a vector expressing a polypeptide or antibody according to the invention.
  • the transfection can be in vivo or ex vivo. Ex vivo transfection is suitably followed by re- infusing the cells into the organism. Other methods involve administering to the mammal, e.g. a human, of a therapeutically effective dose of a composition comprising a polypeptide according to the invention and a pharmacological excipient and/or carrier.
  • Delivery of the gene or genetic material into the cell is the first critical step in gene therapy treatment of disease.
  • a large number of delivery methods are well known to those of skill in the art. Such methods include, for example liposome-based gene delivery (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691 ; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Feigner et al. (1987) Proc. Natl. Acad. Sci.
  • retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof.
  • Figure 1 is a general physical and transcriptional map of the region on 13ql4.3 frequently deleted in B-CLL. Six overlapping cosmids are shown (c6a, c25a, cla, c32a, c30a and
  • Figure 2(A) is a detailed restriction map of the cosmids covering the minimally deleted region.
  • the cosmids were digested with either EcoRI or Hindlll. The location of the different exons of Leul and Leu2 is also indicated.
  • Figure 2(B) is a detailed deletion map of CLL patients 2, 4, 5, 6 and the CLL cell line
  • Figure 3 is a Southern blot analysis of Hindlll-digested DNA hybridized to DNA markers overlapping or surrounding the minimally deleted area.
  • CLL 1 was used as a control of specificity, as this sample was known to have a large homozygous deletion in the area. The residual signals in this sample represent contaminating normal cells.
  • Cl represents DNA from a healthy donor.
  • CLL2 homozygous deletion was found with the markers p30.4, p30.2 and p30.1, whereas the markers pi 8 (a fragment of cosmid cl8), p32.3 as well as Leu2 exon 5 were found to be hemizygously deleted in this patient.
  • Bcl-1 was used as a control probe (this probe gives rise to three different bands of which only one is shown).
  • Figure 4 is a Southern blot hybridization of EcoRI-digested DNA with DNA markers overlapping or surrounding the minimally deleted region.
  • CLL 3 is a sample with a large deletion in the area and serves as a negative control. The samples on this figure are taken from three different filters and therefore three different control DNAs from healthy donors, Cl , C2, and C3, are shown.
  • (A) Bcl-1 was used as a control probe; (B) p30.5 (19 kb) and p30.4 (8.5 kb); the former is hemizygously deleted in 183-E95, CLL 4, and CLL 5 and the latter is homozygously deleted in 183-E95 and CLL 5 and with a reduced signal intensity in CLL 4; (C), p30.3, 183-E95 and CLL 5 showed no signals with this probe, whereas CLL 4 showed a reduced signal intensity.
  • a rearranged band (R) of about 17 kb in size was seen in CLL 4 and CLL 5; (D) the p32.3 marker was homozygously deleted in 183-E95, whereas CLL 4 showed no deletion and CLL 5 showed a hemizygously deleted signal with this probe.
  • Figure 5 shows the general characteristics of cDNA clones of the present region.
  • Figure 6 is an expression analysis of Leul and Leu2 genes.
  • the figure shows multiple tissue northern blots with poly (A)+ RNA, probed with the whole Leul cDNA (A) or the last exon (exon 5) of the Leu2 gene (B).
  • a 1.1 kb band was detected when Le l cDNA was used as probe.
  • the Leu2 probe gives rise to 1.4 and 1.8 kb bands.
  • the lanes contain the following RNAs: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon (mucosal lining); and 8, peripheral blood lymphocytes.
  • Figure 7 is the sequence of forward (F) and reverse (R primers used in mutation screening and/or in amplification of exons of the Leul and Leu2 genes.
  • Figures 8 shows EcoRI fragments in the minimal deleted area as well as location of ESTs (expressed sequence tags) mapped to the introns of the Leul and Leu2 genes.
  • the minimal deleted region, as well as a 150 bp deletion found in one patient alone, is also assigend as well as the location of a CpG-island covering a Notl site in the 8.5 kb Ecorl fragment.
  • Blood was obtained with informed consent from 206 patients with B-CLL, whose diagnosis was based on lymphocyte morphology, CD 19, CD23 and CD5 phenotype and the weak expression of monotypic immunoglobulin.
  • Peripheral blood samples were subjected to ficoll gradient centrifugation.
  • the mononuclear cells were either T-cell depleted using sheep red blood cell rosetting or positively selected for B-cells using CD 19 positive magnetic beads (Dynal, Norway).
  • the resulting mononuclear cells consisted of > 95% leukemic cells as deter- mined by immunophenotyping studies.
  • Polymorphonuclear cells or T-lymphocytes obtained with CD3 positive magnetic beads were used as control fractions.
  • DNA was extracted from the separated leukemic and control cell fractions using a standard proteinase K phenol-chloroform extraction (Liu et al., 1992, supra).
  • the following CLL cell lines were also included in this study: 183-E95, WaC3CD5+ and 232 B4-CLL (Wendel- Hansen V, Wendel-Hansen V, Sallstrom J, De Campos-Lima PO, Kjellstr ⁇ m G, Sand- lund A, Siegbahn A, Carlsson M, Nilsson K and Rosen A. (1994). Leukemia, 8, 476- 484).
  • ICRF 61C1 ICRF 126A9 and CEPH 922A8 YACs were used in a subcloning procedure into the cosmid vector Supercosl .
  • the isolated human specific cosmids were subsequently used as the basis for a cosmid contig encompassing the critical region.
  • Single copy non-repetitive subclones from several of the cosmids were then used to screen an arrayed human PAC library (RPCIl) and the isolated PAC clones were then integrated into the detailed map.
  • Cosmid libraries prepared from genomic DNA and specific for human chromosome 13 were also used for verification of the cosmid contig (Kapanadze BI, Brodianskii VM, Baranova AV, Sevat'ianov S, Fedorova ND, Kurskov MM, Kostina MA, Mironov AA, Sineokii SP, Zakhar'ev VM, Grafodatskii AS, Modianov NN and Iankovskii NK. (1996).
  • Probes generated from the cosmids overlapping the minimally deleted region including different subcloned fragments of the YAC ICRF 61C1 derived cosmids cla, cl ⁇ a, c29a, c30a, c32a and the LANL cosmid 71al 1 (largely overlapping with the ICRF 61 Cl YAC derived cosmid c9a) as well as cDNA clones, were used for hybridization.
  • Cosmid or plasmid DNAs were digested with appropriate restriction enzymes and the fragments were purified by electrophoresis in agarose gels.
  • DNA probes were labelled with 32P-dCTP by the random oligonucleotide priming method, according to the protocols of the manufacturer (Pharmacia, Uppsala, Sweden).
  • DNA probes were labelled with 32P-dCTP by the random oligonucleotide priming method, according to the protocols of the manufacturer (Pharmacia, Uppsala, Sweden).
  • To remove repetitive DNA sequences of the fragments we used a preliminary reassociation step as previously described (Sealey PG, Whittaker PA and Southern EM. (1985). Nucl. Acids Res., 13, 1905-1922). Colony and Southern hybridizations were performed according to standard protocols (Sambrook et al., 1989, supra).
  • Samples were defined as hemizygous loss if the leukemic signal was between 40% to 50% less intense compared to the control signal or as homozygous loss if the tumor signal was below 10% of the control signal.
  • the specificity of the Southern hybridization signals was determined by the use of the B-CLL cell line WaC3CD5+, which has homozygous deletion at 13ql4, including the markers RBkpt, D13S272, D13S319 and D13S25 and the microcell hybrid cell line MCH 240.3.
  • MCH 240.3 is a mouse-human microcell hybrid containing 1-3 copies of cytogenetically intact human chromosome 13 (Imreh S, Kost-Alimova M, Kholodnyuk I, Ying Y, Szeles A, Kiss H, Liu Y, Foster K, Zabarovsky E, Stanbridge E and G Klein. Genes Chrom. Cancer , in press).
  • Control probes used to help in the quantification of loss were the following: a genomic clone from the renin gene located on chromosome 1 , the ⁇ -interferon gene on chromosome 9, pB16 (bcl-2 cDNA clone corresponding to the 1.6 kb 3' part of the first exon) on chromosome 18, pl05-153A (5ql l .2-5ql3.3; D5S39) and pel 17.3, a genomic fragment from the bcl-1 gene on chromosome 1 1 (Tsujimoto Y and Croce CM. (1986). Proc. Natl. Acad. Sci.
  • PCR conditions were designed using the PRIMER program http://www.bmb.psu.edu/597A/stdnts96/ducker/primer.htm).
  • PCR conditions for amplification of sequences were 1-2.5 mM MgCl 2 ; 10 mM Tris-HCl, pH 8.8; 50-100 mM KC1; 0.2 mM of each dNTP; 100 nM of each primer, 96° C for 5 min, 30 cycles with denaturation at 96° C for 1 min, annealing between 53 and 65 C for 1 min, extension at 72° C for 1 min and final extension at 72° C for 10 min.
  • primers used are listed in the table shown in Figure 7. Primers derived from either the exons or the introns of Leul and Leu2 were used to perform RT-PCR and genomic PCR-SSCP as described previously (Liu et al., 1993, supra). In some cases the resulting PCR products were restriction enzyme digested before SSCP analysis. Sequencing and computer analysis
  • Sequencing was done either manually (Sanger F, Nicklen S and Coulson AR. (1977). Proc. Natl. Acad. Sci. USA, 74, 5463-5467) or using the A.L.F. sequenator (Pharmacia, Uppsala, Sweden) according to manufacturer's protocols. DNA sequences were compiled and compared using the DNASIS sequence analyzing program (Hitachi Software Engineering Europe, Ardon, France). To detect human cDNA sequences a search of the Gen- Bank and dbEST nucleotide database was performed using the FASTA and the BLAST programs.
  • Metaphase spreads were obtained by standard techniques from phytohemagglutinin- stimulated human lymphocytes. In situ hybridization and detection were performed as described previously (Wu X, Merup M, Juliusson G, Jansson M, Stellan B, Grander D, Zabarovsky E, Spasokoukotskaja T, Gahrton G and Einhorn S. Genes Chrom. Cancer, in press). Briefly, we used 80-160 ng labelled DNA probe and 4-10 mg Cotl DNA (BRL) per 11 ml hybridization mixture (50% formamide, 2xSSC, 10% dextran sulphate, 0.1% Tween 20).
  • the signals were visualized with a Zeiss Axiophot fluorescence microscope equipped with a cooled CCD camera (Photometries or Hamamatsu), and analyzed using the Smartcapture software (Digital Scientific, Cambridge). At least twenty metaphase spreads with paired signals on each copy of chromosome 13 were analyzed for each probe. Two or three colour FISH on metaphase chromosomes using biotin and digoxi- genin labelled probes was used to confirm the correct order of the cosmid clones along the chromosome.
  • Fiber-FISH was done as previously described (Heiskanen M, Karhu R, Hellsten E, Pel- tonen L, Kallioniemi OP and Palotie A. (1994). Biotechniques, 17, 928-929, 932-933).
  • the 5' and 3' sequence of cDNA transcripts were extended using a standard RACE proto- col with oligonucleotide primers derived from the known sequence (Frohman MA, Dush MK and Martin GR. (1988). Proc. Natl. Acad. Sci. USA, 85, 8898-9002).
  • the cloned product was isolated using the Marathon cDNA amplification kit (Clontech) using cDNA prepared from a normal lymph node as template.
  • a second approach of selective genomic sequencing was applied to the genomic regions within the critical area containing clusters of rare cutting restriction enzyme sites, indicating possible CpG islands which may be associated with up to 60% of genes (Allikmets RL, Kashuba VI, Pettersson B, Gizatullin R, Lebedeva T, Kholodnyuk ID, Bannikov VM, Petrov N, Zakharyev VM, Winberg G, Dean M, Uhlen M, Kisselev LL, Klein G and Za- barovsky ER. (1994). Genomics, 19, 303-309.) A NotI site was identified within c30a.
  • restriction fragments from six cosmids covering the 130 kb minimally deleted region were used as probes to analyze our patient material by Southern hybridization.
  • telomeric border of the deletion could be mapped to the region centromeric of the marker p30.5, as it has retained one unrearranged copy of this marker.
  • the telomeric border of this patient is less than 10 kb away from the cen- tromeric breakpoint in fragment p30.3, and is similar to the telomeric border detected in the 183-E95 cell line (See Figure 2B).
  • the minimally deleted region in our CLL material covers an area of less than 10 kb, extending from fragment p30.3 to the 6 kb area centromeric to fragment p30.5.
  • 14% (28/206) of leukemic DNA samples show homozygous loss and 36% (67/206) of cases show hemizygous loss in the minimally deleted area.
  • Leu3/Leu4 are not part of the Leul/Leu2 genes.
  • Northern blot analysis of Leu3 and Leu4 does not give rise to any bands overlapping with the ones obtained using Leul or Leu2 as probes (see below).
  • repeated RT-PCR using one primer from Leu3 or Leu4 and one primer from Leul or Leu2 did not result in any product, strengthening the con- elusion that they are not part of the same genes.
  • the fact that these ESTs are located within the intron of another gene and consist of uninterrupted genomic sequences casts doubt on the possibility that they are true genes and suggests that they may be derived from intronic sequences, which have previously been shown to occur in publicly accessible cDNA libraries (Hillier L. (1996). Genome Res. 6, 807-828).
  • both ESTs are larger in size than commonly observed for human exons.
  • the Leu3 sequence contains a polyA stretch of a genomic ALU tail at its 3' end, indicating the possibility of inappropriate cDNA synthesis priming by the oligo(dT) primer during library construction.
  • the previously defined 10 kb minimally deleted region was compared to the genomic organization of the Leul and Leu2 genes.
  • the minimally deleted region contains Leul exon 1 and Leu2 exon 1 and the intervening region, whereas large parts of the first introns as well as the remaining exons lie outside the critical area of loss.
  • the only possible alternative candidate tumor suppressor genes to be disrupted by this deletion would be the rare occurrence of intronic genes located within the minimally deleted area.
  • Southern analysis revealed that Leu3 is outside the minimally deleted region, whereas Leu4 is within the area of interest, however, it is unclear if these latter ESTs relate to real genes, as discussed elsewhere.
  • Leu3 and Leu4 hybridization did not reveal any bands corresponding to Leul or Leu2.
  • Leu3 gives rise to a band of approximately 2.3 kb in size in pancreatic tissue alone
  • Leu4 hybridization gives rise to two weak bands corresponding to an 8 kb and a 5.5 kb transcript in skeletal muscle and a 5.5 kb transcript in heart tissue only (data not shown).
  • the promoter region between the Leul and Leu2 genes lacks a canonical TATA box. Sequence analysis of the region upstream of both genes demonstrates several potential binding sites for cis-acting DNA elements including Spl, ETF, API, AP2, GCF, PEA3, CP1 and CTF/NF1, located within 300 bps 5' of the predicted major transcription start sites. The occurrence of several potential binding sites for transcription factors, common to both genes, in addition to their physical co- localisation, within a head to head arrangement, suggests that their expression may be co- regulated.
  • PCR based SSCP analysis was performed on DNA and cDNA from 170 CLL patients. A few different SSCP band shifts were detected in some of the samples which were were further analyzed by nucleotide sequencing. In all cases analyzed, the data suggests that the shifts were due to polymorphisms. PCR based SSCP analysis was also performed for the genomic area between Leul and Leu2 in addition to the regions covered by the Leu3 and Leu4 sequences. No small aberrations were observed in these regions.
  • Leul and Leu2 are one or both of two adjacent genes, termed Leul and Leu2.
  • Leu3 and Leu4 are located within the first intron of Leu2.
  • the Leu3 sequence has the same transcriptional orientation as Leu2, whereas the Leu4 sequence has the same transcriptional orientation as Leul .
  • the Leu3 sequence is, however, located around 5 kb centromeric of the minimally deleted region.
  • cDNA data base searches revealed that Leu3 and Leu4 are only represented by one homologous EST respectively in dbEST, and genomic data show that the proposed genes would be intron- less and consist of unusually large exons.
  • the pl6INK4 gene was defined as a tumor suppressor gene in 1994 (Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS3rd, Johnson BE and Skolnick MH. (1994). Science, 264, 436-440). Located adjacent to the pl6INK4 gene is another CDK-inhibitor, the pl5INK4B gene. Furthermore, an alternative pi 6 exon 1, causing an alternative open reading frame has been described; the pl9ARF gene (Quelle DE, Zindy F, Ashmun RA and Sherr CJ. (1995).Cell, 83, 993-1000). It is still an open question whether the latter two genes act as tumor suppressor genes in malignant disease (Quelle et al, supra; Heyman M and Einhorn S. (1996). Leuk. Lymph., 23, 235- 245).
  • the B-CLL minimally deleted region in 13ql4 can be defined within an area not larger than 10 kb.
  • these deletions include parts of the coding sequence of two adjacent genes termed Leul and Leu2, suggesting that these genes are strong candidates as tumor suppressors in B-CLL.
  • B-CLL B-cell chronic lymphocytic leukemia
  • Leul and Leu2 leukemia-associated gene 1 and 2
  • the Leul and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leul and 2 genes in 170 CLL samples revealed no small in- tragenic mutations or pointmutations. However, in all cases of 13ql4 loss examined, the first exon of both genes, which are only 300 bps apart, were deleted. Thus, the present invention shows that the Leul and Leu2 genes are strong candidates tumor suppressor gene(s) involved in B-CLL leukemogenesis.

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Abstract

La présente invention concerne les séquences d'acides aminés et nucléotidiques associées à la leucémie lymphocytaire chronique telles que présentées dans la liste de séquences annexe. Les séquences nucléotidiques sont isolées de la région du chromosome 13q4. L'invention concerne également l'utilisation desdits acides nucléiques dans la production de protéines, qui, entre autres, sont utiles pour l'élaboration de médicaments contre la leucémie et des proliférations malignes de la même zone génétique ainsi que des kits de diagnostic. La présente invention concerne, en outre, des anticorps produits par les séquences définies ci-dessus.
PCT/SE1998/002052 1997-11-13 1998-11-13 Proteines et nucleotides de la leucemie lymphocytaire chronique WO1999025736A1 (fr)

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BLOOD, Volume 88, No. 10, 1996, M. CORCORAN et al., "Characterisation of the Minimal Area of Homozygous 13q14.3 Deletion in B-CLL and Isolation of Candidate Tumour Suppressor Genes", page 358a, Suppl. 1, Part 1-2. *
BLOOD, Volume 88, No. 8, October 1996, FLORENCIA BULLRICH et al., "Minimal Region of Loss at 13q14 in B-Cell Chronic Lymphocytic Leukemia", pages 3109-3115. *
DATABASE MEDLINE, Accession No. 84223943, TSUJIMOTO Y. et al., "Molecular Cloning of the Chromosomal Breakpoint of B-Cell Lymphomas and Leukemias with the t(11;14) Chromosome Translocation"; & SCIENCE, 29 June 1984, 224(4656), 1403-6. *
DIALOG INFORMATION SERVICES, File 154, MEDLINE, Dialog Accession No. 09348451, Medline Accession No. 98055620, LIU Y. et al., "Cloning of Two Candidate Tumor Suppressor Genes within a 10kb Region on Chromosome 13q14, Frequently Deleted in Chronic Lymphocytic Leukemia"; & ONCOGENE, 13 Nov. 1997, 15(20), p. 2463-2473, *
EMBL, Database/Genbank/DDBJ, Accession No. AA641505, NCI-CGAP, "National Cancer Institute, Cancer Genome Anatomy Project (CGAP), Tumor Gene Index http://www.ncbi.nlm.nih.gov/ncicgap", 20-02-1998. *
EMBL, Database/Genbank/DDBJ, Accession No. AI046364, MARRA M. et al., "The WashU-HHMI Mouse EST Project", 09-07-1998. *
GENOMICS, Volume 42, 1997, S. KALACHIKOV et al., "Cloning and Gene Mapping of the Chromosome 13q14 Region Deleted in Chronic Lymphocytic Leukemia", pages 369-377. *

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