USOO8603740B2

(12) United States Patent (10) Patent No.: US 8,603,740 B2 Ma (45) Date of Patent: Dec. 10, 2013

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ABL fusion protein isoform X8 (BCRABL fusion) mRNA, com 1?report gbwithparts&logS-seqview, printed Aug. 8, 2012 1 page. plete cds; http://www.ncbi.nlm.nih.gov/nuccore/Eu216065, printed NCBIGenBankAccession No. AY789 120, Homo sapiens BCR/ABL Aug. 8, 2012 2 pages. fusion mRNA sequence; http://www.ncbi.nlm.nih.gov/nuccore? NCBI GenBank Accession No. EU216066. Homo sapiens BCR/ AY789 120.1?report=gbwithparts&logS=seqview, printed Aug. 8, ABL fusion protein isoform X9 (BCRABL fusion) mRNA, com 2012 1 page. plete cds; http://www.ncbi.nlm.nih.gov/nuccore/Eu216066, printed NCBI GenBank Accession No. DQ898313, Homo sapiens isolate Aug. 8, 2012 3 pages. ela4 BCR/ABL fusion protein (BCRABL fusion) mRNA, partial NCBI GenBank Accession No. EU216067, Homo sapiens BCR/ cds, alternatively spliced; http://www.ncbi.nlm.nih.gov/nuccore? ABL fusion protein isoformY1 (BCRABL fusion) mRNA, complete dq898313, printed Aug. 8, 2012 2 pages. cds; http://www.ncbi.nlm.nih.gov/nuccore/Eu216067, printed Aug. 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NCBI GenBank Accession No. M15025, Human BCR/ABL mRNA NCBIGenBankAccession No. EF158045, Homo sapiens BCR/ABL (product of translocation tC22q11:9q34)), 5'end; www.ncbi.nlm.nih. p210 fusion protein (BCRABL fusion) mRNA, partial cds; http:// gov/nuccore/m 15025, printed Aug. 8, 2012, 2 pages. www.ncbi.nlm.nih.gov/nuccore/EF158045.1?report=gbwithparts NCBI GenBank Accession No. M17541, Human bcr/abl fusion pro &logS-seqview, printed Aug. 8, 2012 1 page. tein (product of translocation tC22d 11; 9q34)), exons 1 and 2: http:// NCBIGenBankAccession No. EF423615, Homo sapiens BCR/ABL www.ncbi.nlm.nih.gov/nuccore/m 17541, printed Aug. 8, 2012 1 fusion protein (BCRABL fusion) mRNA, partial cds; http://www. page. incbi.nlm.nih.gov/nuccore/EF423615?report-gbwithparts NCBIGenBankAccession No. M17542, Humanbcr/ablprotein gene &logS-seqview, printed Aug. 8, 2012 1 page. (product of tranlocation tC22q11; 9q34)), exons 1 and 2. http://www. 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Pharmacogenomics: Translating Functional Genomics positive cell lines with differential sensitivity to the tyrosine kinase into Rational Therapeutics Science vol. 286:487-491 (1999). inhibitor STI571: diverse mechanisms of resistance, 96:1070-1079 Kievits, et al., NASBATM isothermal enzymatic nucleic acid (2000). amplification optimized for the diagnosis of HIV-1 infection. JViro Manley, P.W., Imatinib: a selective tyrosine kinase inhibitor, Eur. J. logical Methods 35:273-286, (1991). Cancer, 38: S19-S27 (2002). Quintas-Cardama et al. Phase I/II study of subcutaneous homohar Melo, J.V. & Chuah, C., Resistance to imatinib mesylate in chronic ringtonine inpatients with chronic myeloid leukemia who have failed myeloid leukaemia, Cancer Lett. 249:121-132 (2007). prior therapy, Cancer, 109(2):248-255 (2006). Mensink et al., Quantitation of minimal residual diseases in Phila Tokarski et al. The structure of dasatinib (BMS-354825) bound to delphia chromosome positive chronic myeloid leukemia patients activated ABL kinase domain elucidates its inhibitory activity against using real time quantitative PCR. British J. Haematology 102:768 imatinib-resistant ABL mutants, Cancer Research, 66(11):5790 774 (1998). 5797 (2006). Nagar et al., Crystal Structures of the Kinase Domain of c-Abl in Urdea, et al., Direct and quantitative detection of HIV-1 RNA in Complex with the Small Molecule Inhibitors PD173955 and Imatinib human plasma with a branched DNA signal amplification assay. (STI-571), Cancer Res., 62:4236-4243 (2002). AIDS 7 (suppl 2):S11-S 14. (1993). Nagar, B., Structural Basis for the Autoinhibition of C-Abl Tyrosine US Office Action dated Mar. 14, 2012 in U.S. Appl. No. 12472.319. Kinase, Cell, 1.12: 859-871 (2003). US Office Action dated Jul. 6, 2011 in U.S. Appl. No. 1 1/301.272.

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US 8,603,740 B2 1. 2 BCR-ABL1 SPLCE VARIANTS AND USES rence) occurs varies dependent on the stage of diseases. Pri THEREOF mary resistance has been reported in chronic-, accelerated or blast-phase at rates of 3%, 9%, and 51%, respectively FIELD OF THE INVENTION (Melo, J. V. & Chuah, C. Cancer Lett. 2007:249:121-132: Hughes. T. Blood. 2006: 108:28-37). Secondary resistance The present invention relates to BCR-ABL1 variants and has been reported in these patients at rates of 22%, 32%, and resistance to kinase inhibitor therapy. 41%, respectively. The complete mechanism of kinase inhibitor resistance in BACKGROUND OF THE INVENTION CML patients is unclear and a significant number of patients 10 resistant to imatinib have no mutation in the bcr-abl1 gene. The following description is provided to assist the under However, 35-45% of patients with imatinib resistance have standing of the reader. None of the information provided or mutations in the kinase domain of the BCR-ABL1 protein references cited is admitted to be prior art to the present (Mahon, F. X. Blood. 2000; 96: 1070-1079). Most of the invention. reported mutations disrupt critical contact points between Chronic Myelogenous Leukemia (“CML:) is a cancer of 15 imatinib and the tyrosine kinase receptor or induce a transi bone marrow and blood cells. In CML, healthy bone marrow tion from the inactive to the active protein configuration, cells are replaced with leukemic cells: myeloid, erythroid, preventing imatinib binding (Nagar, B. Cell. 2003; 112: 859 megakaryocytic and B lymphoid cells are among the blood 871; Nagaret al. Cancer Res. 2002: 62:4236-4243; Branford cells which become leukemic due to the effects of a charac S. Blood. 2002; 99: 3472-3475; Branford et al. Blood. 2003: teristic chromosomal translocation. 102: 276-283; O'Hare et al., Blood 2007 110: 2242-2249 CML is associated with a specific chromosomal abnormal (2007)). ity called Philadelphia chromosome. The genetic defect is The T315I mutation (Gorre et al. Science. 2001; 293: 876 caused by the reciprocal translocation designated tC9:22) 880; Hochhaus et al. Leukemia. 2002; 16: 2190-2196) and (q34:q11), which refers to an exchange of genetic material some mutations affecting the P-loop of BCR-ABL1 confer a between region q34 of chromosome 9 and region q11 of 25 greater level of resistance to imatinib (Branford et al. Blood. chromosome 22 (Rowley, J. D. Nature. 1973: 243: 290-3: 2002; 99: 3472-3475; Branford et al. Blood. 2003: 102: 276 Kurzrock et al. N. Engl. J. Med. 1988:319:990-998). This 283; and Gorre etal. Blood. 2002: 100: 3041-3044) as well as translocation results in a portion of the her (“breakpoint clus other tyrosine kinase inhibitors that are currently used and ter region') gene from chromosome 22 (region q11) becom tested in these patients (Hughes et al. Blood. 2006; 108: ing fused with a portion of the abl1 gene on chromosome 9 30 28-37; Hochhaus, et al. Blood. 2006: 108: 225a). The role of (region q34). (Wong & Witte, Annu. Rev. Imunol. 2004; 22: Src family kinases has received particular interest as possible 247-306). mechanism for imatinib resistance (Levinson et al. PLoS Biol. The fused “bcr-abl gene is located on chromosome 22, 2006: 4: e144). Overexpression and activation of the Lyn has which is shortened as a result of the translocation. The fused been implicated in imatinib-resistance (Donato, N.J. Blood. gene retains the tyrosine kinase domain of the abl gene, which 35 2003: 101: 690–698). is constitutively active (Elefanty et al. EMBO J. 1990; 9: Furthermore. Chu et al. (N. Engl. J. Med. 2006; 355: 10) 1069-1078). This kinase activity activates various signal describe an insertion/truncation mutant of BCR-ABL1 in a transduction pathways leading to uncontrolled cell growth CML patient resistant to imatinib. Chu et al. report that the and division (e.g., by promoting cell proliferation and inhib mutant results from a 35 base insertion of abl1 intron 8 into iting apoptosis). For example. BCR-ABL may cause undif 40 the junction between exons 8 and 9, resulting in a new C-ter ferentiated blood cells to proliferate and fail to mature. minus and truncation of the normal C-terminus of the ABL1 Alternative bcr-abl 1 splice variants in Philadelphia chro portion of the fusion protein. Laudadio et al. (J. Mol. Diag. mosome-positive CML patient have been reported. Specifi 2008; 10(2): 177-180) and Leeet al. (Mol. Cancer Ther: 2008: cally, alternative splice variants between BCR exon 1, 13 and 7(12): 3834-41) also report a similar splice variant in CML ABL exon 4 or 5 were reported by Volpe et al., (Cancer Res. 45 patients that had undergone imatinib therapy. An additional 67:53.00-07 (2007). splice variant without c-ABL exon 7 has also been reported in Treatment of CML may involve drug therapy (e.g., chemo Imatinib-resistant patients. Curvo et al., Leuk. Res. 2007; therapy), bone marrow transplants, or combinations of both. 32:508-51O. One class of drugs that may be used for treating CML is kinase inhibitors. For example, “imatinib mesylate (also 50 SUMMARY OF THE INVENTION known as ST1571 or 2-phenylaminopyrimidine or “Ima tinib) has proven effective for treating CML (Deininger et The present inventions are based on bcr-abl 1 splice vari al., Blood. 1997: 90:3691-3698: Manley. P. W. Eur: J. Cancer. ants which result from insertion and/or truncation of the bcr 2002:38: S19 S27). Imatinib is marketed as a drug under abl1 transcript and the finding that these variants provide the trade name “Gleevec” or “Glivec.” Other examples of 55 resistance to kinase domain inhibitors such as imatinib, nilo kinase inhibitor drugs for treating CML include nilotinib, tinib and dasatinib. dasatinib, Bosutinib (SKI-606) and Aurora kinase inhibitor In one aspect, the invention provides a method for predict VX-680. ing likelihood for resistance of a patient with a bcr-abl1 Imatinib is an ATP competitive inhibitor of BCR-ABL1 translocation to treatment with one or more BCR-ABL1 kinase activity and functions by binding to the kinase domain 60 kinase inhibitors, comprising: (a) assessing the bcr-abll of BCR-ABL1 and stabilizing the protein in its closed, inac mRNA in a sample obtained from the patient for the presence tive conformation. Monotherapy with imatinib has been or absence of a polynucleotide sequence encoding the shown to be effective for all stages of CML. 195INSbcr-abl1 splice variant or the 243INSbcr-abl1 splice Resistance to imatinib, and other kinase inhibitors, remains variant; and (b) identifying the patient as having an increased a major problem in the management of patients with CML. 65 likelihood of being resistant to treatment with one or more Rates at which primary (failure to achieve any hematologic BCR-ABL1 kinase inhibitors when a polynucleotide encod response) and secondary resistance (i.e., hematologic recur ing at least one of said splice variants is detected. US 8,603,740 B2 3 4 In some embodiments, the presence or absence of the (w/w) whole cellular material. Plasma or serum are examples 195INSbcr-abl 1 splice variant is determined by detecting the of acellular bodily fluids. A sample may include a specimen presence or absence of abcr-abl 1 nucleic acid comprising the of natural or synthetic origin. sequence of SEQID NO:3. “Nucleic acid' or “nucleic acid sequence' as used herein In another aspect, the invention provides a method for refers to an oligonucleotide, nucleotide or polynucleotide, predicting likelihood for resistance of a patient with a bcr and fragments or portions thereof, which may be single or abl1 translocation to treatment with one or more BCR-ABL1 double stranded, and represent the sense or antisense strand. kinase inhibitors, comprising: (a) assessing the BCR-ABL1 A nucleic acid may include DNA or RNA, and may be of protein in a sample obtained from a patient for the presence or natural or synthetic origin and may contain deoxyribonucle absence of a truncated Abl protein encoded by the 195INS 10 otides, ribonucleotides, or nucleotide analogs in any combi bcr-abl 1 splice variant or the protein encoded by the 243INS nation. bcr-abl1 splice variant; and (b) identifying the patient as Non-limiting examples of polynucleotides include a gene having an increased likelihood of being resistant to treatment or gene fragment, genomic DNA, exons, introns, mRNA, with one or more BCR-ABL1 kinase inhibitors when at least tRNA, rRNA, ribozymes, cDNA, recombinant polynucle one of said truncated proteins is detected. 15 otides, branched polynucleotides, , vectors, isolated In some embodiments, the presence or absence of the DNA of any sequence, isolated RNA of any sequence; syn BCR-ABL1 protein encoded by the bcr-abl 1 splice variants is thetic nucleic acid, nucleic acid probes and primers. Poly determined by detecting the size of the BCR-ABL1 protein(s) may be natural or synthetic. Polynucleotide may in the patient sample, or by using an antibody that specifically comprise modified nucleotides, such as methylated nucle binds to the BCR-ABL1 protein encoded by the 195INS otides and nucleotide analogs, uracyl, other Sugars and link bcr-abl 1 splice variant or the 243INSbcr-abl 1 splice variant, ing groups such as fluororibose and thiolate, and nucleotide or by sequencing the C-terminus of the BCR-Abl protein. In branches. A nucleic acid may be modified Such as by conju some embodiments, the C-terminus of the Abl protein gation, with a labeling component. Other types of modifica encoded the 195INS bcr-abl 1 splice variant comprises the tions included in this definition are caps, Substitution of one or sequence of SEQID NO: 5 and/or the Abl protein 25 more of the naturally occurring nucleotides with an analog, encoded by the 243INS bcr-abl splice variant comprising the and introduction of chemical entities for attaching the poly amino acid sequence of SEQID NO: 6. nucleotide to other molecules Such as proteins, metal ions, In some embodiments of either of the foregoing aspects, labeling components, other polynucleotides or a solid Sup the patient is diagnosed as having a myeloproliferative dis port. Nucleic acid may include a nucleic acid that has been ease (e.g., chronic myelogenous leukemia (CML)). In other 30 amplified (e.g. using polymerase chain reaction). embodiments, the kinase inhibitors include one or more of A fragment of a nucleic acid generally contains at least imatinib, nilotinib, bosutinib, and dasatinib. In other embodi about 15, 20, 25, 30, 35, 40.45, 50, 75, 100, 200, 300, 400, ments, the sample is a blood or bone marrow. Preferably, the 500, 1000 nucleotides or more. Larger fragments are possible sample contains blood cells (e.g., peripheral mononuclear and may include about 2,000, 2,500, 3,000, 3.500, 4,000, cells) and/or platelets. 35 5,000 7,500, or 10,000 bases. In another aspect, the invention provides a recombinant “Gene' as used herein refers to a DNA sequence that com polynucleotide which encodes the 195INS bcr-abl 1 splice prises control and coding sequences necessary for the pro variant or the 243INS bcr-abl 1 splice variant. In some duction of an RNA, which may have a non-coding function embodiment, the recombinant polynucleotide is operably (e.g. a ribosomal or transfer RNA) or which may include a linked to an expression regulatory element capable of modu 40 polypeptide or a polypeptide precursor. The RNA or polypep lating the expression of said recombinant polynucleotide. The tide may be encoded by a full length coding sequence or by regulatory element optionally contains a promoter, an any portion of the coding sequence so long as the desired enhancer, and/or a poly-adenylation signal. The vectors activity or function is retained. include expression vectors and may be eukaryotic, prokary Although a sequence of the nucleic acids may be shown in otic, or viral. 45 the form of DNA, a person of ordinary skill in the art recog The term “myeloproliferative disease as used herein nizes that the corresponding RNA sequence will have a simi means a disorder of a bone marrow-derived cell type, Such as lar sequence with the thymine being replaced by uracil, i.e. a white blood cell. A myeloproliferative disease is generally “t with “u. manifest by abnormal cell division resulting in an abnormal “Identity” and “identical as used herein refer to a degree level of aparticular hematological cell population. The abnor 50 of identity between sequences. There may be partial identity mal cell division underlying a myeloproliferative disease is or complete identity. A partially identical sequence is one that typically inherent in the cells and not a normal physiological is less than 100% identical to another sequence. Preferably, response to infection or inflammation. A leukemia is a type of partially identical sequences have an overall identity of at myeloproliferative disease. Exemplary myeloproliferative least 70% or at least 75%, more preferably at least 80% or at disease include, but are not limited to, acute myeloid leuke 55 least 85%, most preferably at least 90% or at least 95% or at mia, acute lymphoblastic leukemia, chronic lymphocytic leu least 99%. Sequence identity determinations may be made for kemia, myelodysplastic syndrome, chronic myeloid leuke sequences which are not fully aligned. In Such instances, the mia, hairy cell leukemia, leukemic manifestations of most related segments may be aligned for optimal sequence lymphomas, and multiple myeloma. identity by and the overall sequence identity reduced by a As used herein, the term “sample' or “biological sample' 60 penalty for gaps in the alignment. refers to any liquid or solid material obtained from a biologi “Hybridize” or “hybridization” as used herein refers to the cal source, such a cell or tissue sample or bodily fluids. pairing of substantially complementary nucleotide sequences “Bodily fluids' may include, but are not limited to, blood, (strands of nucleic acid) to form a duplex or heteroduplex serum, plasma, saliva, cerebrospinal fluid, pleural fluid, tears, through formation of hydrogen bonds between complemen lactal duct fluid, lymph, sputum, urine, saliva, amniotic fluid, 65 tary base pairs. Hybridization and the strength of hybridiza and semen. A sample may include a bodily fluid that is “acel tion (i.e., the strength of the association between the nucleic lular.” An “acellular bodily fluid includes less than about 1% acids) is influenced by Such factors as the degree of comple US 8,603,740 B2 5 6 mentary between the nucleic acids, stringency of the condi example, the oligonucleotide may be labeled with an agent tions involved, and the thermal melting point (T) of the that produces a detectable signal (e.g., a fluorophore). formed hybrid. An oligonucleotide or polynucleotide (e.g., a “Primeras used herein refers to an oligonucleotide that is probe or a primer) that is specific for a target nucleic acid will capable of acting as a point of initiation of synthesis when “hybridize' to the target nucleic acid under suitable condi placed under conditions in which primer extension is initiated tions. (e.g., primer extension associated with an application Such as “Specific hybridization” as used herein refers to an indica PCR). The primer is complementary to a target nucleotide tion that two nucleic acid sequences share a high degree of sequence and it hybridizes to a Substantially complementary complementarity. Specific hybridization complexes form sequence in the target and leads to addition of nucleotides to under permissive annealing conditions and remain hybrid 10 the 3'-end of the primer in the presence of a DNA or RNA ized after any Subsequent washing steps. Permissive condi polymerase. The 3'-nucleotide of the primer should generally tions for annealing of nucleic acid sequences are routinely be complementary to the target sequence at a corresponding determinable by one of ordinary skill in the art and may occur, nucleotide position for optimal expression and amplification. for example, at 65°C. in the presence of about 6xSSC. Strin An oligonucleotide “primer may occur naturally, as in a gency of hybridization may be expressed, in part, with refer 15 purified restriction digest or may be produced synthetically. ence to the temperature under which the wash steps are car The term “primer' as used herein includes all forms of prim ried out. Such temperatures are typically selected to be about ers that may be synthesized including nucleic acid 5°C. to 20° C. lower than the T for the specific sequence at primers, locked nucleic acid primers, phosphorothioate a defined ionic strength and pH. The T is the temperature modified primers, labeled primers, and the like. (under defined ionic strength and pH) at which 50% of the Primers are typically between about 10 and about 100 target sequence hybridizes to a perfectly matched probe. nucleotides in length, preferably between about 15 and about Equations for calculating T and conditions for nucleic acid 60 nucleotides in length, more preferably between about 20 hybridization are known in the art. and about 50 nucleotides in length, and most preferably “Stringent hybridization conditions” as used herein refers between about 25 and about 40 nucleotides in length. In some to hybridization conditions at least as stringent as the follow 25 embodiments, primers can be at least 8, at least 12, at least 16, ing: hybridization in 50% formamide, 5xSSC, 50 mM at least 20, at least 25, at least 30, at least 35, at least 40, at least NaH2PO, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon 45, at least 50, at least 55, at least 60 nucleotides in length. An sperm DNA, and 5xDenhart's solution at 42°C. overnight; optimal length for a particular primer application may be washing with 2xSSC, 0.1% SDS at 45° C.; and washing with readily determined in the manner described in H. Erlich, PCR 0.2xSSC, 0.1% SDS at 45°C. In another example, stringent 30 Technology. Principles and Application for DNA Amplifica hybridization conditions should not allow for hybridization tion (1989). of two nucleic acids which differ over a stretch of 20 contigu “Probe' as used herein refers to nucleic acid that interacts ous nucleotides by more than two bases. with a target nucleic acid via hybridization. A probe may be “Substantially complementary” as used herein refers to fully complementary to a target nucleic acid sequence or two sequences that hybridize under Stringent hybridization 35 partially complementary. The level of complementarity will conditions. The skilled artisan will understand that substan depend on many factors based, in general, on the function of tially complementary sequences need not hybridize along the probe. A probe or probes can be used, for example to their entire length. detect the presence or absence of a mutation in a nucleic acid Oligonucleotides can be used as primers or probes for sequence by virtue of the sequence characteristics of the specifically amplifying (i.e. amplifying a particular target 40 target. Probes can be labeled or unlabeled, or modified in any nucleic acid sequence) or specifically detecting (i.e., detect of a number of ways well known in the art. A probe may ing a particular target nucleic acid sequence) a target nucleic specifically hybridize to a target nucleic acid. acid generally are capable of specifically hybridizing to the Probes may be DNA, RNA or a RNA/DNA hybrid. Probes target nucleic acid. may be oligonucleotides, artificial chromosomes, fragmented “Oligonucleotide' as used herein refers to a molecule that 45 artificial chromosome, genomic nucleic acid, fragmented has a sequence of nucleic acid bases on a backbone comprised genomic nucleic acid, RNA, recombinant nucleic acid, frag mainly of identical monomer units at defined intervals. The mented recombinant nucleic acid, peptide nucleic acid bases are arranged on the backbone in Such a way that they (PNA), locked nucleic acid, oligomer of cyclic heterocycles, can enter into a bond with a nucleic acid having a sequence of or conjugates of nucleic acid. Probes may comprise modified bases that are complementary to the bases of the oligonucle 50 nucleobases, modified Sugar moieties, and modified inter otide. The most common oligonucleotides have a backbone of nucleotide linkages. A probe may be fully complementary to Sugar phosphate units. A distinction may be made between a target nucleic acid sequence or partially complementary. A oligodeoxyribonucleotides that do not have a hydroxyl group probe may be used to detect the presence or absence of a target at the 2' position and oligoribonucleotides that have a nucleic acid. Probes are typically at least about 10, 15, 21, 25, hydroxyl group in this position. Oligonucleotides also may 55 30, 35, 40, 50, 60, 75, 100 nucleotides or more in length. include derivatives, in which the hydrogen of the hydroxyl “Detecting as used herein refers to determining the pres group is replaced with organic groups, e.g., an allyl group. ence of a nucleic acid of interest in a sample or the presence Oligonucleotides of the method which function as primers or of a protein of interest in a sample. Detection does not require probes are generally at least about 10-15 nucleotides long and the method to provide 100% sensitivity and/or 100% speci more preferably at least about 15 to 25 nucleotides long, 60 ficity. although shorter or longer oligonucleotides may be used in "Detectable label' as used herein refers to a molecule or a the method. The exact size will depend on many factors, compound or a group of molecules or a group of compounds which in turn depend on the ultimate function or use of the used to identify a nucleic acid or protein of interest. In some oligonucleotide. The oligonucleotide may be generated in cases, the detectable label may be detected directly. In other any manner, including, for example, chemical synthesis, 65 cases, the detectable label may be a part of a binding pair, DNA replication, reverse transcription, PCR, or a combina which can then be subsequently detected. Signals from the tion thereof. The oligonucleotide may be modified. For detectable label may be detected by various means and will US 8,603,740 B2 7 8 depend on the nature of the detectable label. Detectable labels the mouse mammary tumor (MMTV) steroid-inducible may be isotopes, fluorescent moieties, colored Substances, promoter, Moloney murine leukemia virus (MMLV) pro and the like. Examples of means to detect detectable label moter. Exemplary promoters suitable for use with prokaryotic include but are not limited to spectroscopic, photochemical, hosts include T7 promoter, beta-lactamase promoter, lactose biochemical, immunochemical, electromagnetic, radio 5 promoter systems, alkaline phosphatase promoter, a tryp chemical, or chemical means, such as , chemif tophan (trp) promoter system, and hybrid promoters such as luorescence, or , or any other appropriate the tac promoter. CaS. Antibody” as used herein refers to a polypeptide, at least “TaqMan(R) PCR detection system as used herein refers to a portion of which is encoded by at least one immunoglobulin a method for real time PCR. In this method, a TaqMan(R) probe 10 which hybridizes to the nucleic acid segment amplified is gene, or fragment thereof, and that can bind specifically to a included in the PCR reaction mix. The TaqMan(R) probe com desired target molecule. The term includes naturally-occur prises a donor and a quencher fluorophore on either end of the ring forms, as well as fragments and derivatives. Fragments probe and in close enough proximity to each other so that the within the scope of the term “antibody' include those pro fluorescence of the donor is taken up by the quencher. How 15 duced by digestion with various , those produced by ever, when the probe hybridizes to the amplified segment, the chemical cleavage and/or chemical dissociation, and those 5'-exonuclease activity of the Taq polymerase cleaves the produced recombinantly, so long as the fragment remains probe thereby allowing the donor fluorophore to emit fluo capable of specific binding to a target molecule. Among Such rescence which can be detected. fragments are Fab, Fab'. Fv, F(ab)', and single chain Fv “Vector” as used herein refers to a recombinant DNA or (scFv) fragments. Derivatives within the scope of the term RNA plasmid or virus that comprises a heterologous poly include antibodies (or fragments thereof) that have been nucleotide capable of being delivered to a target cell, either in modified in sequence, but remain capable of specific binding vitro, in vivo or ex-vivo. The polynucleotide can comprise a to a target molecule, including: interspecies chimeric and sequence of interest and can be operably linked to another humanized antibodies; antibody fusions; heteromeric anti nucleic acid sequence Such as promoter or enhancer and may 25 body complexes and antibody fusions, such as diabodies control the transcription of the nucleic acid sequence of inter (bispecific antibodies), single-chain diabodies, and intrabod est. As used herein, a vector need not be capable of replication ies (see, e.g., Marasco (ed.). Intracellular Antibodies. in the ultimate target cell or subject. The term vector may Research and Disease Applications, Springer-Verlag New include expression vector and cloning vector. York, Inc. (1998) (ISBN: 3540641513). As used herein, anti Suitable expression vectors are well-known in the art, and 30 include vectors capable of expressing a polynucleotide opera bodies can be produced by any known technique, including tively linked to a regulatory sequence, such as a promoter harvest from cell culture of native B lymphocytes, harvest region that is capable of regulating expression of such DNA. from culture of hybridomas, recombinant expression sys Thus, an “expression vector” refers to a recombinant DNA or tems, and . RNA construct, Such as a plasmid, a phage, recombinant virus 35 “Specifically binds to a polypeptide' as used herein in the or other vector that, upon introduction into an appropriate context of an antibody refers to binding of an antibody spe host cell, results in expression of the inserted DNA. Appro cifically to certain of a polypeptide Such that the priate expression vectors include those that are replicable in antibody can distinguish between two proteins with and with eukaryotic cells and/or prokaryotic cells and those that out Such epitope. remain episomal or those which integrate into the host cell 40 genome. BRIEF DESCRIPTION OF THE DRAWINGS The term “vector or “expression vector is used herein to mean vectors used in accordance with the present invention as FIG. 1 shows the mRNA sequence of the human abl1 gene a vehicle for introducing into and expressing a desired gene in as provided in GenBank Accession No.: NM 005157.3 a host cell. As known to those skilled in the art, such vectors 45 (SEQID NO: 1). may easily be selected from the group consisting of plasmids, FIG. 2 shows the amino acid sequence for human ABL1 phages, and retroviruses. In general, vectors compat protein (SEQ ID NO: 2) as provided in GenBank Accession ible with the instant invention will comprise a selection No: NM 005157.3. marker, appropriate restriction sites to facilitate cloning of the FIG. 3 shows the mRNA sequence of the 195INS abl1 desired gene, and the ability to enter and/or replicate in 50 splice variant (SEQID NO:3). The 195bp insertion is under eukaryotic or prokaryotic cells. Additionally elements may lined. also be included in the vector Such as signal sequences, splice FIG.4 show the mRNA sequence of the 243INS abl1 splice signals, as well as transcriptional promoters, enhancers, and variant (SEQID NO: 4). The 243 bp insertion is underlined. termination signals. FIG. 5A shows the amino acid sequence encoded by the Examples of suitable vectors include, but are not limited to 55 195INS abl1 splice variant (SEQID NO. 5): FIG. 5B shows plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF 1/His, the amino acid sequence encoded by the 243INS abl1 splice pEMD/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, variant (SEQID NO: 6). The amino acids that differ from the pUB6/V5-His, pVAX1, and pZeoSV2 (available from invit non-variant protein are underlined. rogen, San Diego, Calif.), and plasmid pCl (available from FIG. 6 shows the relative frequency of individual BCR Promega, Madison, Wis.). 60 ABL kinase domain mutations detected in a group of 245 “Promoter as used herein refers to a segment of DNA that patients, 219 of which have CML and 26 of which have controls transcription of polynucleotide to which it is opera Ph-positive acute lymphoblastic leukemia. The numbering of tively linked. Promoters, depending upon the nature of the amino acids is based on the Abl protein variant B (which regulation, may be constitutive or regulated. Exemplary includes ABL exon 1b but not exon 1a). At some positions, 2 eukaryotic promoters contemplated for use in the practice of 65 or 3 possible mutants with different amino acids are possible. the present invention include the SV40 early promoter, the The percentage of patients with each mutation specified is cytomegalovirus (CMV) major immediate-early promoter, shown in the right most column. US 8,603,740 B2 9 10 DETAILED DESCRIPTION OF THE INVENTION cation mutant of BCR-ABL1 in a CML patient resistant to imatinib has been reported which results from a 35 base BCR-ABL1 insertion of abl intron 8 into the junction between exons 8 and The inventions described herein include polynucleotides 9 of the bcr-abl mRNA due to alternate splicing (see, for which encode all or portions of the splice variants in the example, Laudadio et al. J. Molec. Diag. 10: 177-180, 2008). bcr-abl1 gene product, cells that express all or portions of 195INS Splice Variant those splice variants, and proteins encoded by those splice This splice mutation resulted from the insertion of 195 variants. Recombinant cells expressing the truncated BCR nucleotides from abl1 intron 4 into the abl1 exon 4-5 junction, ABL protein with an active kinase domain are useful for and causes a frameshift and protein truncation (FIGS. 3 and identifying drug candidates for treating CML. Methods for 10 5A). The intron 4 nucleotide sequence inserted by the alter predicting likelihood for responsiveness to kinase inhibitor nate splicing is:

(SEO ID NO: 7) gggagctgct ggtgaggatt attittagact gtgagtaatt gacctgacag acagtgatga

ctgctt catt aagagcc.cac gaccacgtgc cagaatagitt cago atcc tic togttgctact gtactittgag acatcqttct tctttgttgat goaatacctic tittcttgtca togagggtctic

tt CCCttaala to agg therapy are included along with methods, compositions and The inserted sequence results in a non-native ABL1 protein reagents for detecting the splice variants. The genomic C-terminus having the following amino acid sequence: sequence of the abl1 gene is known, and may be found in GenBank Accession No: NT 035014, from nucleotides 487, 25 774 to 538,010. The genomic sequence of the bcr gene is also (SEQ ID NO: 8) known and may be found at NG 009244.1. - - TASDGKGSCW Variants of the bcr-abl1 mRNA Because the insertion only affects the abl1 portion of bcr-abl1 Several splice variants of bcr-abl1 mRNA have been translocations, FIGS. 1-5 only show the sequence of abl1 reported. Many of the known sequences are full length cDNA 30 mRNA and resulting protein, and not the entire bcr-abl1 sequences and some are partial cDNA sequences. For translocation and resulting fusion protein sequences. example bcr-abl1 mRNA sequences include: NCBIGenBank Included in the invention are oligonucleotides, primers or accession numbers: EU216072, EU216071, EU216070, probes which are designed to be complementary to Some or all of the above sequence or to be complementary to Some or EU216069, EU216068, EU216067, EU216066, EU216065, 35 all of the above sequence and to some adjoining sequence (not EU2 16064, EU216063, EU216062, EU216061, EU216060, shown) in the mRNA (i.e., a junction sequence). Such oligo EU216059. EU216058, EU236680, DQ912590, DQ912589, nucleotides primers or probes can be readily designed so as to DQ912588, DQ898315, DQ898314, DQ898313, EF423615, hybridize under stringent conditions to 195INS splice vari EF158045. S72479, S72478, AY789 120, AB069693, ants but not hybridize to the regular bcr-abl1 mRNA or to any AF487522, AF113911, AF251769, M30829, M30832. 40 other known bcr-abl 1 insertion splice variants. M17542. M15025, and M17541. 243INS Splice Variant Additionally. BCR-ABL1 variant protein sequences have This splice mutation comprised an 243-nucleotide been reported, for example NCB1 protein database accession sequence from intron 6 inserted into the abl1 exon 6-7 junc numbers: ABX82708. ABX82702, AAA35594. tion, causing an insertion of 85 amino acids (FIGS. 4 and 5B). CML patients undergoing BCR-ABL1 tyrosine kinase 45 The intron 6 nucleotide sequence inserted by the alternate inhibitor therapy may develop resistance to the therapy. The splicing is:

(SEQ ID NO: 9) gtaggggcct ggcCaggcag Cctg.cgc.cat ggagt cacag gg.cgtggagc cgggcagc ct

tttacaaaaa. gccc.ca.gc.ct aggaggtot C agggcgcagc titct aacctic agtgctggca

acacattgga Cctcggalaca aaggcaaac a ct aggctic ct ggcaaa.gc.ca gCtttgggca

tgcatcCagg gctaaattica gcc aggcct a gactctggac cagtggagca gctaatcc cc

gga resistance in patients has been associated with mutant splice The amino acid sequence that is inserted in the 243INS splice variant forms of BCR-ABL1. For example, an insertion/trun variant protein is:

(SEQ ID NO: 10) RGLAROPAPW SHRAWSRAAF YKKPOPRRSO GAASNLSAGN TLDLGTKANT RLLAKPALGM

HPGLNSARPR LWTSGAANPR. R. US 8,603,740 B2 11 12 Because the insertion only affects the abl1 portion ofbcr-abl1 labeled probe, detection by size, allele specific PCR, ligation translocations. FIGS. 1-5 only show the sequence of abl1 amplification reaction (LAR), detection by oligonucleotide mRNA and resulting protein, and not the entire bcr-abl1 arrays. translocation and resulting fusion protein sequences. Biological Sample Collection and Preparation Included in the invention are oligonucleotides, primers or 5 Sample: Samples, for use in the methods of the present probes which are designed to be complementary to some or invention, may be obtained from an individual who is Sus all of the 243 nucleotides from intron 6 that are present in the pected of having a disease, e.g. CML, or a genetic abnormal mRNA (or cDNA) or which are designed to be complemen ity, or who has been diagnosed with CML. Samples may also tary to Some or all of the 243 nucleotides and to Some adjoin be obtained from a healthy individual who is assumed of ing sequence in the mRNA (i.e., a junction sequence). Such 10 having no disease, e.g. CML, or genetic abnormality. Addi probes can be readily designed so as to hybridize under strin tionally, the sample may be obtained from CML patients gent conditions to 243INS splice variants but not hybridize to undergoing kinase inhibitor therapy or from CML patients the regular bcr-abl1 mRNA or any other known bcr-abl1 not undergoing kinase inhibitor therapy. insertion splice variants. Sample Collection: Methods of obtaining samples are well Mutations in the ABL Kinase Domain: 15 known to those of skill in the art and include, but are not CML patients undergoing tyrosine kinase inhibitor therapy limited to, aspirations, tissue sections, drawing of blood or (such as, imatinib, nilotinib, dasatinib, Bosutinib (SK1-606) other fluids, Surgical or needle biopsies, collection of paraffin and Aurora kinase inhibitor VX-680) may develop resistance embedded tissue, collection of body fluids, collection of to such inhibitors. Several underlying mechanisms of resis stool, urine, buccal Swab and the like. tance to kinase inhibitors have been identified. One major Methods of plasma and serum preparation are well known cause is the presence of point mutations within the ABL in the art. Either “fresh' blood plasma or serum, or frozen kinase domain of BCR-ABL1. In one embodiment, such (stored) and Subsequently thawed plasma or serum may be mutations inhibit the ability of imatinib to bind to BCR used. Frozen (stored) plasma or serum should optimally be ABL1 by altering the binding sites or preventing the kinase maintained at storage conditions of -20 to -70 degrees cen domain from assuming the inactive conformation required for 25 tigrade until thawed and used. “Fresh' plasma or serum imatinib binding (O'Hare et al. Blood, 2007: 110: 2242 should be refrigerated or maintained on ice until used. Exem 2249). Point mutations develop inapproximately 35% to 70% plary methods of preparation are described below. of patients displaying resistance to imatinib, either spontane DNA/RNA/ ously or through the evolutionary pressure of imatinib (Bran Polynucleotides (e.g., DNA or RNA) or polypeptides may ford et al. Blood. 2003: 102: 276-283). 30 be isolated from the sample according to any methods well More than 40 distinct resistance-conferring mutations have known to those of skill in the art. If necessary, the sample may been detected; the majority fall within four regions of the be collected or concentrated by centrifugation and the like. kinase domain: the ATP-binding loop (P-loop) of the ABL The sample may be subjected to , such as by treatments kinase domain, the contact site, the SHY binding site (acti with , heat, Surfactants, ultrasonication, or a combi Vation loop), and the catalytic domain (Hughes et al. Blood. 35 nation thereof. The lysis treatment is performed in order to 2006: 108:28-37). A list of such mutations are shown in FIG. obtain a Sufficient amount of nucleic acid or polypeptide. The 6 and incorporated herein by reference. Approximately 85% sample may be subjected to liquid chromatography to par of all imatinib-resistant mutations are associated with amino tially purity the nucleic acid or polypeptide. In some embodi acid substitutions at just seven residues (P-loop: M244V, ments, the whole cell lysates or tissue homogenate may used G250E, Y253F/H and E255K/V; contact site: T315I; and 40 as source of nucleic acid or polypeptide without further iso catalytic domain: M351T and F359V). The most frequently lation and purification. mutated region of BCR-ABL is the P-loop, accounting for Suitable DNA isolation methods include phenol and chlo 36% to 48% of all mutations. roform extraction, see Sambrook, et al., Molecular Cloning: The importance of P-loop mutations is further underlined A Laboratory Manual (1989). Second Edition, Cold Spring by in vitro evidence Suggesting that these mutations are more 45 Harbor Press, Plainview, N.Y. oncogenic with respect to un-mutated BCR-ABL as well as Numerous commercial kits also yield suitable DNA other mutated variants. In various biological assays, P-loop including, but not limited to, QIAampTM mini blood kit. mutants Y253F and E255K exhibited an increased transfor Agencourt GenfindTM, Roche Cobas(R Roche MagNA Pure(R) mation potency relative to un-mutated BCR-ABL. Overall, or phenol: chloroform extraction using Eppendorf Phase the relative transformation potencies of various mutations 50 Lock Gels(R). Total DNA (e.g. genomic, mitochondrial, were found to be as follows: Y253F>E255K>native BCR microbial, viral.) can be purified from any biological sample ABL>T315IDH396P>M351T. Transformation potency also Such as whole blood, plasma, serum, buffy coat, bone mar correlated with intrinsic BCR-ABL kinase activity in this row, other body fluids, lymphocytes, cultured cells, tissue, study. and forensic specimens using commercially available kits In some embodiments, CML, patients undergoing kinase 55 e.g., QIAamp DNA and QIAamp DNA Blood mini kits from inhibitor therapy may develop two kinds of mutations: a) an Qiagen. insertion/truncation mutant of BCR-ABL due to alternate In another embodiment, the polynucleotide may be mRNA splicing and b) one or more point mutations in the kinase or cDNA generated from mRNA or total RNA. RNA is iso domain of Abl. lated from cells or tissue samples using standard techniques, In preferred embodiments, the alternate splice variant of 60 see, e.g. Sambrook, et al. Molecular Cloning. A Laboratory bcr-abl1 mRNA can be detected simultaneously with the Manual, Second Edition (1989), Cold Spring Harbor Press, detection of mutations in abl portion of bcr-abl 1 mRNA. In Plainview, N.Y. In addition, reagents and kits for isolating another embodiment, the mutations in the abl portion of bcr RNA from any biological sample such as whole blood, abl1 mRNA can be detected separately. Several methods are plasma, serum, buffy coat, bone marrow, other body fluids, known in the art for detection of the presence or absence of 65 lymphocytes, cultured cells, tissue, and forensic specimens Such mutations. Non-limiting examples include, DNA are commercially available e.g., RNeasy Protect Mini kit, sequencing, detection by hybridization of a detectably RNeasy Protect Cell Mini kit, QIAamp RNA Blood Mini kit, US 8,603,740 B2 13 14 RNeasy Protect Saliva Minikit, Paxgene Blood RNAkit from art as described herein, as well as with specific requirements Qiagen; MELTTM, RNaqueous(R, ToTALLY RNATM, as described herein for each step of the particular methods RiboPurerTM-Blood, Poly(A)PuristTM from Applied Biosys described. tems; TRIZOL(R) reagent, Dynabeads(R mRNA direct kit In some embodiments, oligonucleotide primers for cDNA from . synthesis and PCR are 10 to 100 nucleotides in length, pref In some embodiments, the nucleic acid is isolated from erably between about 15 and about 60 nucleotides in length, paraffin embedded tissue. Methods of extracting nucleic acid more preferably 25 and about 50 nucleotides in length, and from paraffin embedded tissue are well known in the art e.g., most preferably between about 25 and about 40 nucleotides in paraffin blocks containing the tissue are collected, de-waxed length. There is no standard length for optimal hybridization 10 or polymerase chain reaction amplification. by treatment with xylene, treated with proteinase to remove Methods of designing primers have been described in U.S. protein contaminants, and then finally extracted with phenol patent application Ser. No. 10/921,482. Primers useful in the and chloroform, followed by ethanol precipitation. Alterna methods described herein are also designed to have a particu tively, nucleic acid from a paraffin embedded tissue can be lar melting temperature (T) by the method of melting tem isolated by commercially available kits e.g., EZI DNA kit. 15 perature estimation. Commercial programs, including QIAamp DINA Mini Kit from Qiagen; Paraffin Block RNA OligoTM, Primer Design and programs available on the inter Isolation Kit, Recover AllTMTotal Nucleic Acid Isolation Kit net, including Primer3 and Oligo Calculator can be used to from Ambion. calculate a T of a polynucleotide sequence useful according Nucleic acid need not be extracted, but may be made avail to the invention. able by suitable treatment of cells or tissue such as described T of a polynucleotide affects its hybridization to another in U.S. patent application Ser. No. 11/566,169, which is polynucleotide (e.g., the annealing of an oligonucleotide incorporated herein by reference. primer to a template polynucleotide). In the Subject methods, Polypeptides detected in the methods of the present inven it is preferred that the oligonucleotide primer used in various tion may be detected directly from a biological sample or may steps selectively hybridizes to a target template or polynucle be further purified for detection. Any known method for 25 otides derived from the target template (i.e., first and second polypeptide purification may be used including, but not lim strand cDNAs and amplified products). Typically, selective ited to Sucrose gradient purification, size exclusion chroma hybridization occurs when two polynucleotide sequences are tography, ion exchange chromatography, affinity chromatog substantially complementary (at least about 65% comple raphy, immunoaffinity chromatography, HPLC, gel mentary over a stretch of at least 14 to 25 nucleotides, pref 30 erably at least about 75%, more preferably at least about 90% electrophoresis (e.g. SDS-PAGE or QPNC-PAGE), and complementary). See Kanehisa, M., Polynucleolides Res. with a BCR-ABL1 specific antibody. (1984), 12:203, incorporated herein by reference. As a result, Detection it is expected that a certain degree of mismatch at the priming The bcr-abl 1 polynucleotides or BCR-ABL1 polypeptides site is tolerated. Such mismatch may be small, such as a may be detected by a variety of methods known in the art. 35 mono-, di- or tri-nucleotide. In preferred embodiments, 100% Non-limiting examples of detection methods are described complementarity is preferred. below. The detection assays in the methods of the present Probes: invention may include purified or isolated DNA, RNA or Probes are capable of hybridizing to at least a portion of the protein or the detection step may be performed directly from nucleic acid of interest or a reference nucleic acid. Probes a biological sample without the need for further DNA, RNA 40 may be an oligonucleotide, artificial chromosome, frag or protein purification/isolation. mented artificial chromosome, genomic nucleic acid, frag Nucleic Acid Amplification mented genomic nucleic acid. RNA, recombinant nucleic Polynucleotides encoding bcr-abl 1 can be detected by the acid, fragmented recombinant nucleic acid, peptide nucleic use of nucleic acid amplification techniques which are well acid (PNA), locked nucleic acid, oligomer of cyclic hetero known in the art. The starting material may be genomic DNA, 45 cycles, or conjugates of nucleic acid. Probes may be used for cDNA, RNA mRNA. Nucleic acid amplification can be linear detecting and/or capturing/purifying a nucleic acid of inter or exponential. Specific variants or mutations may be eSt. detected by the use of amplification methods with the aid of Typically, probes can be about 10 nucleotides, about 20 oligonucleotide primers or probes designed to interact with or nucleotides, about 25 nucleotides, about 30 nucleotides, hybridize to a particular target sequence in a specific manner, 50 about 35 nucleotides, about 40 nucleotides, about 50 nucle thus amplifying only the target variant. otides, about 60 nucleotides, about 75 nucleotides, about 100 Non-limiting examples of nucleic acid amplification tech nucleotides long. niques include the polymerase chain reaction (PCR), reverse However, longer probes are possible. Longer probes can be transcriptase polymerase chain reaction (RT-PCR), nested about 200 nucleotides, about 300 nucleotides, about 400 PCR, ligase chain reaction (see Abravaya, K., et al., Nucleic 55 nucleotides about 500 nucleotides, about 750 nucleotides, Acids Res. (1995), 23:675-682), branched DNA signal ampli about 1.000 nucleotides, about 1,500 nucleotides, about fication (see Urdea, M. S., et al., AIDS (1993), 7(suppl 2.000 nucleotides, about 2,500 nucleotides, about 3,000 2):S11-S14, amplifiable RNA reporters, Q-beta replication, nucleotides, about 3,500 nucleotides, about 4,000 nucle transcription-based amplification, boomerang DNA amplifi otides, about 5,000 nucleotides, about 7,500 nucleotides, cation, strand displacement activation, cycling probe technol 60 about 10,000 nucleotides long. ogy, isothermal nucleic acid sequence based amplification Probes may also include a detectable label or a plurality of (NASBA) (see Kievits, T. et al., J. Virological Methods detectable labels. The detectable label associated with the (1991), 35:273-286), Invader Technology, or other sequence probe can generate a detectable signal directly. Additionally, replication assays or signal amplification assays. the detectable label associated with the probe can be detected Primers: 65 indirectly using a reagent, wherein the reagent includes a Oligonucleotide primers for use amplification methods can detectable label, and binds to the label associated with the be designed according to general guidance well known in the probe. For example, a detectable label includes a labeled US 8,603,740 B2 15 16 antibody or a primary antibody/secondary antibody pair, amount of final amplified product. Real-time PCR does not wherein the detectable label may be in the primary antibody, detect the size of the amplicon. The probes employed in or in the secondary antibody or in both. ScorpionTM and TaqMan(R) technologies are based on the Primers or probes may be prepared that hybridize under principle of fluorescence quenching and involve a donor fluo stringent conditions to the insert sequence or to a junction 5 rophore and a quenching moiety. sequence that includes some normal bcr-abl1 mRNA TaqMan(R) probes (Heid, et al., Genomme Res 6: 986-994, sequence and some of the adjoining insertion sequence. Such 1996) use the fluorogenic 5' exonuclease activity of Taq poly primers or probes can be designed so that they hybridize merase to measure the amount of target sequences in cDNA under stringent conditions to the specific splice variant tran samples. TaqManR probes are oligonucleotides that contain a 10 donor fluorophore usually at or near the 5' base, and a quench script but not to normal bcr-abl 1 transcript. Primers or probes ing moiety typically at or near the 3' base. The quencher also can be prepared that are complementary and specific for moiety may be a dye such as TAMRA or may be a non the normal bcr-abl 1 splice junction. Such primers or probes fluorescent molecule Such as 4-(4-dimethylaminophenylazo) can be used to detect the normal bcr-abl1 mRNA and not the benzoic acid (DABCYL). See Tyagi, et al., 16 Nature Bio corresponding insertion mutation Such as is described herein. 15 technology 49-53 (1998). When irradiated, the excited fluo Primers and/or probes specific for the inserted arising from rescent donor transfers energy to the nearby quenching moi the bcr-abl 1 splice variants described herein are designed to ety by FRET rather than fluorescing. Thus, the close specifically hybridize to a diagnostic portion of the inserted proximity of the donor and quencher prevents emission of sequence of the 195INS and 243INS variants including, for donor fluorescence while the probe is intact. example, SEQ ID NOs: 7 and 9, respectively. Alternatively, Taq ManR probes are designed to anneal to an internal the 195INS and 234 INS variants may be identified using region of a PCR product. When the polymerase (e.g., reverse primers and/or probes that are directed to the novel junctions transcriptase) replicates a template on which a TaqMan(R) created by the inserted sequences. Suitable primers and probe is bound, its 5' exonuclease activity cleaves the probe. probes include, for example, those which contain the follow This ends the activity of the quencher (no FRET) and the ing nucleotide sequences (the junction is indicated by a 25 donor fluorophore starts to emit fluorescence which increases colon): in each cycle proportional to the rate of probe cleavage. Accumulation of PCR product is detected by monitoring the increase in fluorescence of the reporter dye (note that primers are not labeled). If the quencher is an acceptor fluorophore, Junction D 30 then accumulation of PCR product can be detected by moni Sequence Description NO toring the decrease in fluorescence of the acceptor fluoro ggcaa.g:gggag 5' junction between exon 4 1. phore. and the intron 4 insertion In a preferred embodiment, the detectable label is a fluo in the 195INS variant rophore. The term “fluorophore” as used herein refers to a 35 molecule that absorbs light at a particular wavelength (exci atcagg: ct ctac 3' junction between the 2 intron 4 insertion and exon tation frequency) and Subsequently emits light of a longer 5 in the 195INS variant wavelength (emission frequency). The term “donor fluoro phore' as used herein means a fluorophore that, when in close t ccttg: gtaggg 5' junction between exon 6 3 proximity to a quencher moiety, donates or transfers emission and the intron 6 insertion 40 energy to the quencher. As a result of donating energy to the in the 231INS variant quencher moiety, the donor fluorophore will itself emit less cc.cgga: gggtot 3' junction between the 4. light at a particular emission frequency that it would have in intron 6 insertion and exon the absence of a closely positioned quencher moiety. 7 in the 231INS variant The term "quencher moiety' as used herein means a mol 45 ecule that, in close proximity to a donor fluorophore, takes up Detectable Label emission energy generated by the donor and either dissipates The term “detectable label” as used herein refers to a mol the energy as heat or emits light of a longer wavelength than ecule or a compound or a group of molecules or a group of the emission wavelength of the donor. In the latter case, the compounds associated with an oligonucleotide (e.g., a probe quencher is considered to be an acceptor fluorophore. The or primer) and is used to identify the probe hybridized to a 50 quenching moiety can act via proximal (i.e., collisional) genomic nucleic acid or reference nucleic acid. quenching or by Förster or fluorescence resonance energy Detectable labels include but are not limited to fluoro transfer (“FRET). Quenching by FRET is generally used in phores, isotopes (e.g., P. P. S., H, C, "'I, ''I), elec Taq ManR probes while proximal quenching is used in tron-dense reagents (e.g. gold, silver), nanoparticles, molecular beacon and ScorpionTM type probes. enzymes commonly used in an ELISA (e.g., horseradish per 55 Suitable fluorescent moieties include but are not limited to oxidase, beta-galactosidase, luciferase, alkaline phos the following fluorophores working individually or in com phatase), chemiluminescent compound, colorimetric labels bination: 4-acetamido-4-isothiocyanatostilbene-2.2"disul (e.g., colloidal gold), magnetic labels (e.g., Dynabeads.TM), fonic acid; acridine and derivatives: acridine, acridine biotin, digoxigenin, haptens, proteins for which antisera or isothiocyanate; Alexa Fluors: Alexa Fluor R 350. Alexa monoclonal antibodies are available, ligands, hormones, oli 60 Fluorr 488, Alexa Fluorr 546, Alexa Fluorr 555, Alexa gonucleotides capable of forming a complex with the corre Fluor R. 568, Alexa Fluor R 594. Alexa Fluor R 647 (Molecu sponding oligonucleotide complement. lar Probes); 5-(2-aminoethyl)aminonaphthalene-1-sulfonic One general method for real time PCR uses fluorescent acid (EDANS): 4-amino-N-3-vinylsulfonyl)phenylnaph probes such as the TaqMan(R) probes, molecular beacons, and thalimide-3.5 disulfonate (Lucifer YellowVS); N-(4-anilino Scorpions. Real-time PCR quantifies the initial amount of the 65 1-naphthyl)maleimide; anthranilamide: Black Hole template with more specificity, sensitivity and reproducibil QuencherTM (BHQTM) dyes (biosearch Technologies): ity, than other forms of quantitative PCR, which detect the BODIPY dyes: BODIPYR R-6G, BOPIPYR 530/550, US 8,603,740 B2 17 18 BODIPYR FL: Brilliant Yellow; coumarin and derivatives: analyzer (Applied Biosystems, Foster City, Calif.). Near the coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin end of the CE column, in these devices the amplified DNA 120), 7-amino-4-trifluoromethylcouluarin (Coumarin 151); fragments pass a fluorescent detector which measures signals Cy2(R), Cy3(R), Cy3.5(R, Cy5(R, Cy5.5(R); cyanosine; 4,6-di of fluorescent labels. These apparatuses provide automated aminidino-2-phenylindole (DAPI); 5'5"-dibromopyrogallol high throughput for the detection of fluorescence-labeled sulfonephthalein (Bromopyrogallol Red): 7-diethylamino-3- PCR products. (4-isothiocyanatophenyl)-4-methylcoumarin; In some embodiments, nucleic acid may be analyzed and diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihy detected by size using . Methods dro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostil of performing agarose gel electrophoresis are well known in bene-2,2'-disulfonic acid; 5-dimethylaminonaphthalene-1- 10 the art. See Sambrook et al., Molecular Cloning: A Labora sulfonyl chloride (DNS, dansyl chloride); 4-(4- tory Manual (2nd Ed.) (1989), Cold Spring Harbor Press. dimethylaminophenylazo)benzoic acid (DABCYL): N.Y. 4-dimethylaminophenylaZophenyl-4-isothiocyanate DNA Sequencing: (DABITC); EclipseTM (Epoch Biosciences Inc.); eosin and In some embodiments, detection of nucleic acid is by DNA derivatives: eosin, eosin isothiocyanate; erythrosin and 15 sequencing. Sequencing may be carried out by the dideoxy derivatives: erythrosin 13, erythrosin isothiocyanate: chain termination method of Sanger et al. (PNAS USA (1977), ethidium; fluorescein and derivatives: 5-carboxytluorescein 74, 5463-5467) with modifications by Zimmermann et al. (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (Nucleic Acids Res. (1990), 18:1067). Sequencing by dideoxy (DTAF), 2,7-dimethoxy-4'5'-dichloro-6-carboxyfluorescein chain termination method can be performed using Thermo (JOE), fluorescein, fluorescein isothiocyanate (FITC), Sequenase (Amersham Pharmacia, Piscataway, N.J.), Seque hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC), nase reagents from US Biochemicals or Sequatherm sequenc tetrachlorofluorescein (TET); fluorescamine: IR 144: ing kit (Epicenter Technologies, Madison. Wis.). Sequencing IR 1446; lanthamide phosphors; Malachite Green isothiocy may also be carried out by the “RR dRhodamine Terminator anate: 4-methylumbelliferone; ortho cresolphthalein: nitroty Cycle Sequencing Kit' from PE Applied Biosystems (prod rosine; pararosaniline; Phenol Red: B-phycoerythrin, R-phy 25 uct no. 403044, Weiterstadt, Germany), Taq DyeDeoxyTM coerythrin; allophycocyanin; o-phthaldialdehyde; Oregon Terminator Cycle Sequencing kit (Perkin-Elmer/Applied Green R; propidium iodide, pyrene and derivatives: pyrene, Biosystems) using an Applied Biosystems Model 373 ADNA pyrene butyrate, succinimidyl 1-pyrene butyrate; QSYR 7: or in the presence of dye terminators CEQTM Dye Terminator QSYR 9; QSYR) 21: QSYR35 (Molecular Probes); Reactive Cycle Sequencing Kit, (Beckman 608000). Alternatively, Red 4 (Cibacron(RBrilliant Red 3B-A): rhodamine and 30 sequencing can be performed by a method known as Pyrose derivatives: 6-carboxy-X-rhodamine (ROX), 6-carbox quencing (Pyrosequencing, Westborough, Mass.). Detailed yrhodamine (R6C), lissamine rhodamine B sulfonyl chloride, protocols for Pyrosequencing can be found in: Alderborn et rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine al., Genomne Res. (2000), 10:1249-1265. green, rhodamine X isothiocyanate, riboflavin, rosolic acid, Detection of Polypeptide by Size: sulforhodamine B, sulforhodamine 101, sulfonyl chloride 35 Methods for detecting the presence or amount of polypep derivative of sulforhodamine 101 (Texas Red); terbium che tides are well known in the art and any of them can be used in late derivatives: N.N.N',N'-tetramethyl-6-carboxyrhodamine the methods described herein so long as they are capable of (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine separating polypeptides by a difference in size. The separa isothiocyanate (TRITC). tion can be performed under denaturing or under non-dena Detection of Nucleic Acid by Size: 40 turing or native conditions. Useful methods for the separation Methods for detecting the presence or amount of poly and analysis of polypeptides include, but are not limited to, nucleotides are well known in the art and any of them can be electrophoresis (e.g., SDS-PAGE electrophoresis, capillary used in the methods described herein so long as they are electrophoresis (CE)), immunoblot analysis, size exclusion capable of separating individual polynucleotides by a differ chromatography, chromatography (HPLC), and mass spec ence in size. The separation technique used should permit 45 trometry. resolution of nucleic acid as long as they differ from one Antibody Production and Screening another by at least one nucleotide or more. The separation can Various procedures known in the art may be used for the be performed under denaturing or under non-denaturing or production of antibodies which bind to variants of the BCR native conditions—i.e., separation can be performed on ABL1 protein. Such antibodies include but are not limited to single- or double-stranded nucleic acids. Useful methods for 50 polyclonal, monoclonal, chimeric, single chain, Fab frag the separation and analysis of polynucleotides include, but ments and fragments produced by a Fab expression library. are not limited to, electrophoresis (e.g., agarose gel electro Antibodies that specifically bind to a diagnostic epitope of phoresis, capillary electrophoresis (CE)), chromatography SEQID NOs: 5 and 6 are useful for detection and diagnostic (HPLC), and . purposes. Suitable include, for example the polypep In one embodiment, CE is a preferred separation means 55 tides encoded by SEQID NOs: 8 and 10. because it provides exceptional separation of the polynucle Antibodies that differentially bind to the polypeptide of otides in the range of at least 10-1,000 base pairs with a SEQ ID NOS 5 and/or 6 relative to the native BCR-ABL1 resolution of a single base pair. CE can be performed by protein may also specifically detect and distinguish insertion/ methods well known in the art, for example, as disclosed in truncation variants of the BCR-ABL1 protein from other U.S. Pat. Nos. 6,217,731; 6,001.230; and 5.963,456, which 60 BCR-ABL1 proteins without such insertion/truncation muta are incorporated herein by reference. High-throughput CE tions. apparatuses are available commercially, for example, the For the production of antibodies, various host animals, HTS9610 High throughput analysis system and SCE 9610 including but not limited to rabbits, mice, rats, etc., may be fully automated 96-capillary electrophoresis genetic analysis immunized by injection with the full length or fragment of system from Spectrumedix Corporation (State College, Pa.); 65 variants of the BCR-ABL1 protein. Various adjuvants may be P/ACE 5000 series and CEQ series from Beckman Instru used to increase the immunological response, depending on ments Inc (Fullerton, Calif.); and ABI PRISM 3100 genetic the host species, including but not limited to Freund's (com US 8,603,740 B2 19 20 plete and incomplete), mineral gels such as aluminum moter. T7 terminator, the inducible E. coli lac operator, and hydroxide, Surface active Substances Such as lysolecithin, the lac repressor gene; and plT 12a-c, which contain the T7 pluronic polyols, polyanions, , oil emulsions, key promoter. T7 terminator, and the E. coliompT secretion sig hole limpet hemocyanin, dinitrophenol, and potentially use nal. Another suitable vector is the plN-IIIomp A2 (see Duf ful human adjuvants such as BCG (bacilli Calmette-Guerin) faudet al., Meth, in Enzymology, 153:492-507, 1987), which and Corynebacterium parvum. contains the Ipp promoter, the lacUV5 promoter operator, the Monoclonal antibodies to BCR-ABL1 protein variants ompA secretion signal, and the lac repressor gene. may be prepared by using any technique which provides for Eukaryotic Vectors: the production of antibody molecules by continuous cell lines Exemplary, eukaryotic transformation vectors, include the in culture. These include but are not limited to the hybridoma 10 cloned bovine papilloma virus genome, the cloned technique originally described by Kohler and Milstein, (Na of the murine retroviruses, and eukaryotic cassettes, such as ture (1975), 256:495-497), the human B-cell hybridoma tech the pSV-2 gpt system described by Mulligan and Berg, nique (Kosboret al., Immunology Today (1983), 4:72: Cote et Nature Vol. 277:108-114 (1979) the Okayvama-Berg clon al. Proc. Natl. Acad. Sci. (1983),80:2026-2030) and the EBV ing system Mol. Cell. Biol. Vol. 2:161-170 (1982), and the hybridoma technique (Cole et al., Monoclonal Antibodies 15 expression cloning vector described by Genetics Institute and Cancer Therapy (1985), Alan R. Liss, Inc., pp. 77-96). In (Science. 1985; 228: 810-815), pCMV Sport, pcDNATM3.3 addition, techniques developed for the production of “chi TOPOR, BaculodirectTM Baculovirus Expression System mericantibodies' (Morrison et al., Proc. Nail. Acad. Sci. USA (Invitrogen Corp., Carlsbad, Calif., USA), StrataCloneTM (1984). 81:6851-6855; Neuberger et al. Nature (1984), 312: (Stratagene, California, USA), pBAC vectors (EMD Chemi 604-608: Takeda et al., Nature (1985),314:452-454) by splic cals Inc. New Jersey, USA). ing the genes from a mouse antibody molecule of appropriate Vector Components: specificity together with genes from a human anti Vector components generally include, but are not limited body molecule of appropriate biological activity can be used. to, one or more of a regulatory elements such as an enhancer Alternatively, techniques described for the production of element, a promoter, and a transcription termination single chain antibodies (U.S. Pat. No. 4,946,778) can be 25 sequence, an origin of replication, one or more selection adapted to produce specific single chain antibodies which marker genes, and a cloning site. bind to variants of the BCR-ABL1 protein. Origin of Replication: Antibody fragments which recognize variants of the BCR Both expression and cloning vectors contain a nucleic acid ABL1 protein may be generated by known techniques. For sequence that enables the vector to replicate in one or more example, such fragments include but are not limited to: the 30 selected host cells. Such sequences are well known for a F(ab')2 fragments which can be produced by pepsin digestion variety of , yeast, and viruses. Non-limiting examples of the antibody molecule and the Fab fragments which can be include the origin of replication from the plasmidpBR322 for generated by reducing the disulfide bridges of the F(ab'), most Gram-negative bacteria, the plasmid origin is Suitable fragments. Alternatively. Fab expression libraries may be for yeast, and various viral origins (SV40, cytomegalovirus, constructed (Huse et al., Science. 1989: 246: 1275-1281) to 35 polyoma, adenovirus, VSV or BPV) useful for cloning vec allow rapid and easy identification of monoclonal Fab frag tors in mammalian cells. ments with the desired specificity to variants of the BCR Selection Marker: ABL1 protein. Expression and cloning vectors will typically contain a Antibodies to BCR-ABL1 variants can be used in a variety selection gene, also termed a selectable marker. Typical selec of techniques for detecting BCR-ABL1 polypeptides in the 40 tion genes encode proteins that (a) confer resistance to anti methods of the present invention including, but non limited biotics or other toxins, e.g., amplicillin, neomycin, methotr to, immunoprecipitation, -linked immunosorbent exate, or tetracycline, (b) complement auxotrophic assay (ELISA), protein immunoblotting techniques such as deficiencies, or (c) Supply critical nutrients not available from Westerns, etc. complex media. e.g., the gene encoding D-alanine racemase Cloning 45 for Bacilli. The nucleic acid (e.g. cDNA or genomic DNA) encoding at An example of suitable selectable markers for mammalian least a portion of bcr-abl or its variants may be inserted into a cells is those that enable the identification of cells competent replicable vector for cloning (amplification of the DNA) or to take up the bcr-abl-encoding nucleic acid, such as DHFR or for expression. Various vectors are publicly available. The thymidine kinase. An appropriate host cell when wild-type vector may, for example, be in the form of a plasmid, cosmid, 50 DHFR is employed is the CHO cell line deficient in DHFR viral particle, or phage. The appropriate nucleic acid activity, prepared and propagated as described by Uirlaub et sequence may be inserted into the vector by a variety of al., Proc. Natl. Acad. Sci. USA (1980), 77:4216. A suitable procedures. In general, DNA is inserted into an appropriate selection gene for use in yeast is the trp 1 gene present in the restriction endonuclease site(s) using techniques known in yeast plasmid YRp7 (Stinchcomb et al. Nature (1979), 282: the art, see Sambrook, et al., Molecular Cloning: A Labora 55 39-43; Kingsman et al., Gene (1979). 7:141-152). The trp 1 tory Manual (1989), Second Edition, Cold Spring Harbor gene provides a selection marker for a mutant strain of yeast Press, Plainview, N.Y. lacking the ability to grow in tryptophan, for example. ATCC Prokaryotic Vectors: No. 44076 or PEP4-1 (Jones, Genetics (1977). 86:85-102). Prokaryotic transformation vectors are well-known in the Regulatory Elements: art and include plBluescript and phage Lambda ZAP vectors 60 Expression and cloning vectors usually contain a promoter (Stratagene, La Jolla, Calif.), and the like. Other suitable and/or enhancer operably linked to the bcr-abl 1 encoding vectors and promoters are disclosed in detail in U.S. Pat. No. nucleic acid sequence to direct mRNA synthesis. Promoters 4,798,885, issued Jan. 17, 1989, the disclosure of which is recognized by a variety of potential host cells are well known. incorporated herein by reference in its entirety. Promoters suitable for use with prokaryotic hosts include the Other suitable vectors for transformation of E. coli cells 65 beta-lactamase and lactose promoter systems (Chang et al., include the pET expression vectors (Novagen, see U.S. Pat. Nature (1978), 275:617-624; Goeddelet al., Nature (1979), No. 4,952,496), e.g., pET11a, which contains the T7 pro 281:544–548), alkaline phosphatase, a tryptophan (trp) pro US 8,603,740 B2 21 22 moter system (EP36,776), T7 promoter, and hybrid promot Expression vectors used in eukaryotic host cells (yeast, ers such as the tac promoter (deboer et al., Proc. Natl. Acad. fungi, insect, plant, animal, human, or nucleated cells from Sci. USA (1983), 80:21-25). Promoters for use in bacterial other multicellular organisms) will also contain sequences systems also will contain a Shine-Dalgarno (S.D) sequence necessary for the termination of transcription and for stabi operably linked to the DNA encoding bcr-abl1. lizing the mRNA. Such sequences are commonly available Examples of Suitable promoting sequences for use with from the 5' and, occasionally 3', untranslated regions of yeast hosts include the promoters for 3-phosphoglycerate eukaryotic or viral DNA or cDNA. These regions contain kinase (Hitzeman et al., J. Biol. Chem. (1980), 255:12073 nucleotide segments transcribed as polyadenylated fragments 12080) or other glvycolytic enzymes (Holland and Holland, in the untranslated portion of the mRNA encoding bcr-abl1. Biochemistry (1978). 17:4900-4907), such as enolase, glyc 10 Still other methods, vectors, and host cells suitable for eraldehyde-3-phosphate dehydrogenase, hexokinase, pyru adaptation to the synthesis of bcr-abl 1 in recombinant verte vate decarboxylase, phosphofructokinase, glucose-6-phos brate cell culture are described in Gething et al., Nature (1981), 293:620-625; Mantei et al., Nature. 1979; 281:40-46; phate isomerase, 3-phosphoglycerate mutase, pyruvate EP 117,060; and EP 117,058. kinase, triosephosphate isomerase, phosphoglucose 15 Genetically Modifying Host Cells by Introducing Recombi isomerase, and glucokinase. nant Nucleic Acid Other yeast promoters, which are inducible promoters hav The recombinant nucleic acid (e.g., cDNA or genomic ing the additional advantage of transcription controlled by DNA) encoding at least a portion of bcr-abl 1 or its variants growth conditions, are the promoter regions for alcoholdehy may be introduced into host cells thereby genetically modi drogenase 2, isocytochrome C, acid phosphatase, degradative fying the host cell. Host cells may be used for cloning and/or enzymes associated with nitrogen metabolism, metallothio for expression of the recombinant nucleic acid. Host cells can nein, glyceraldehyde-3-phosphate dehydrogenase, and be prokaryotic, for example bacteria. Host cell can be also be enzymes responsible for maltose and galactose utilization. eukaryotic which includes but not limited to yeast, fungal Suitable vectors and promoters for use in yeast expression are cell, insect cell, plant cell and animal cell. In preferred further described in EP 73,657. 25 embodiment, the host cell can be a mammalian cell. In bcr-abl 1 transcription from vectors in eukaryotic host cells another preferred embodiment host cell can be human cell. In may be controlled, for example, by promoters obtained from one preferred embodiment, the eukaryotic host cell may be the genomes of viruses such as polyomavirus, fowlpox virus K562 cell. K562 cells were the first human immortalized (UK 2.211,504 published 5 Jul. 1989), adenovirus (such as myelogenous leukemia line to be established and are abcr-abl Adenovirus 2), bovine papilloma virus, avian sarcoma virus, 30 positive erythroleukemia line derived from a CML patient in cytomegalovirtis, a retrovirus, hepatitis-B Virus and Simian blast crisis (Lozzio & Lozzio, Blood. 1975; 45(3): 321-334; Virus 40 (SV40), from heterologous mammalian promoters. Drexler, H. G. The Leukemia-Lymphoma Cell Line Facts e.g., the promoter or an immunoglobulin promoter, and book. (2000), Academic Press. from heat-shock promoters, provided Such promoters are Host cells may comprise wild-type genetic information. compatible with the host cell systems. 35 The genetic information of the host cells may be altered on Transcription of a DNA encoding the bcr-abl1 gene by purpose to allow it to be a permissive host for the recombinant higher eukaryotes may be increased by inserting an enhancer DNA. Examples of such alterations include mutations, partial sequence into the vector. Enhancers are cis-acting elements of or total deletion of certain genes, or introduction of non-host DNA, usually about from 10 to 300 bp that act on a promoter nucleic acid into host cell. Host cells may also comprise to increase its transcription. Many enhancer sequences are 40 mutations which are not introduced on purpose. now known from mammalian genes (globin, elastase, albu Several methods are known in the art to introduce recom min, alpha.-fetoprotein, and insulin). Typically, however, binant DNA in bacterial cells that include but are not limited one will use an enhancer from a eukaryotic cell virus. to transformation, transduction, and electroporation, see Examples include the SV40 enhancer on the late side of the Sambrook, et al., Molecular Cloning: A Laboratory Manual replication origin (bp 100-270), the cytomegalovirus early 45 (1989), Second Edition, Cold Spring Harbor Press, Plain promoter enhancer, the polyoma enhancer on the late side of view, N.Y. Non limiting examples of commercial kits and the replication origin, and adenovirus enhancers. The bacterial host cells for transformation include NovaBlue enhancer may be spliced into the vector at a position 5' or 3' to SinglesTM (EMD Chemicals Inc., New Jersey, USA). Max the bcr-abll coding sequence, but is preferably located at a Efficiency(R DH5TM. One Shot(R) BL21 (DE3) E. coli cells, site 5' from the promoter. 50 One ShotR BL21 (DE3) plys E. coli cells (Invitrogen Corp., Vectors encoding bcr-abll nucleic acid sequence and its Carlsbad, Calif., USA), XL1-Blue competent cells (Strat variants may further comprise non-bcr-abll nucleic acid agene, California, USA). Non limiting examples of commer sequence which may be co-expressed with bcr-abl1 and its cial kits and bacterial host cells for electroporation include variants either as a fusion product or as a co-transcript. Non ZappersTM electrocompetent cells (EMD Chemicals Inc., New limiting examples of Such non-bcr-abll nucleic acid 55 Jersey, USA), XL1-Blue Electroporation-competent cells sequence includes His-tag (a stretch of poly histidines), (Stratagene, California, USA), ElectroMAXTM A. tumefa FLAG-tag, and Green Fluorescent Protein (GFP). His-tag and ciens LBA4404 Cells (Invitrogen Corp., Carlsbad, Calif., FLAG-tag can be used to in many different methods, such as USA). purification of BCR-ABL1 protein and or insertion/trunca Several methods are known in the art to introduce recom tion mutant of BCR-ABL1 protein fused to such tags. The 60 binant nucleic acid in eukaryotic cells. Exemplary methods tags can also serve as an important site for antibody recogni include transfection, electroporation, liposome mediated tion. This is particularly important in detecting BCR-ABL1 delivery of nucleic acid, microinjection into to the host cell, proteins and or insertion/truncation mutant of BCR-ABL1 see Sambrook, et al., Molecular Cloning: A Laboratory protein fused to such tags. GFP may be used as a reporter of Manual (1989), Second Edition, Cold Spring Harbor Press, expression (Phillips G. J. FEMS Microbiol. Lett. 2001; 204 65 Plainview, N.Y. Non limiting examples of commercial kits (1): 9-18), such as the expression of bcr-abl1 and the splice and reagents for transfection of recombinant nucleic acid to variant of bcr-abl1. eukaryotic cell include LipofectamineTM2000, OptifectTM US 8,603,740 B2 23 24 Reagent, Calcium Phosphate Transfection Kit (Invitrogen sequence of any one of SEQ ID NOs: 5 and 6. Methods for Corp., Carlsbad, Calif., USA), Gene Jammer R Transfection detecting the absence or presence of Such polypeptides are Reagent, LipoTAXIR. Trasfection Reagent (Stratagene, Cali discussed above. The presence of the polypeptide sequence fornia, USA). Alternatively, recombinant nucleic acid may be indicates that the patient has an increased likelihood of being introduced into insect cells (e.g. Sf9. Sf21, High FiveTM) by resistant to treatment with one or more BCR-ABL1 kinase using baculo viral vectors. inhibitors relative to a patient not having the polynucleotide In one preferred embodiment, an exemplary vector com sequence. The presence of one or more of these BCR-ABL1 prising the cDNA sequence of bcr-abl splice variant (pCMV/ fusion proteins indicates that the patient has an increased GFP/195INS bcr-abl) may be transfected into K562 cells. likelihood of being resistant to treatment with one or more Stable transfected K562 cells may be developed by transfect 10 BCR-ABL1 kinase inhibitors relative to a patient not having ing the cells with varying amounts of the pCMV/GFP/ the polynucleotide sequence. 195INS bcr-abl vector (Ong-500 ng) using various methods In another embodiment, a sample from a CML patient, or a known in the art. In one exemplary method. The ProFection(R) Subject Suspected of having CML, is assessed for the presence Mammalian Transfection System—Calcium Phosphate or absence of a polypeptide having an amino acid sequence of (Promega Corporation, Wisconsin, USA) may be used. This 15 SEQID NO: 5 or 6. is a simple system containing two buftfers: CaCl and Identifying a Compound for Treating Leukemic Patients HEPES-buffered saline. A precipitate containing calcium In one preferred embodiment, cell lines expressing BCR phosphate and DNA is formed by slowly mixing a HEPES ABL1 (both wild-type and/or mutant) proteins may be uti buffered phosphate Solution with a solution containing cal lized to screen compounds for treating CML patients. In cium chloride and DNA. These DNA precipitates are then preferred embodiments, the compounds may be targeting distributed onto eukaryotic cells and enter the cells through an BCR-ABL1 protein. In some embodiments, the compounds endocytic-type mechanism. This transfection method has may be inhibitor of ABL kinase activity. Non-limiting been successfully used by others (Hay et al. J. Biol. Chem. examples of kinase inhibitors include but not limited to ima 2004; 279: 1650-58). The transfected K562 cells can be tinib, dasatinib, nilotinib, Bosutinib (SKI-606) and Aurora selected from the non-transfected cells by using the antibiot 25 kinase inhibitor VX-680. In other embodiments, the com ics Neomycin and Ampicillin. Expression of the spliced vari pounds may not be an inhibitor of ABL kinase activity. ant of bcr-abl can assessed from the co-expression of the The effect of the compounds on the cells may be assessed. reporter gene GFP. Several parameters may be assessed for identifying the com Alternatively, in a 24-well format complexes are prepared pounds that may be beneficial for treatment of CML patients. using a DNA (ug) to LipofectamineTM 2000 (Invitrogen Cor 30 Non-limiting examples of the parameters that may be poration. Carlsbad, Calif., USA) (ul) ratio of 1:2 to 1:3. Cells assessed includes cell viability, cell proliferation, apoptosis, are transfected at high cell density for high efficiency, high kinase activity of BCR-ABL1 protein, additional mutations expression levels, and to minimize cytotoxicity. Prior to pre in BCR-ABL1 protein, additional mutation in ABL protein. paring complexes, 4-8x10 cells are plated in 500 ul of In one embodiment, human chronic myeloid leukemia growth medium without antibiotics. For each transfection 35 (CML) cell lines expressing BCR-ABL1 (both wild-type and/ sample, complexes are prepared as follows: a. DNA is diluted or mutant) proteins may be used to study the effect of such in 50 ul of Opti-MEMR I Reduced Serum Medium without compounds on their effect on the cells. Non-limiting serum (Invitrogen Corporation, Carlsbad, Calif., USA) or examples of human chronic myeloid leukemia (CML) cell other medium without serum and mixed gently. b. Lipo lines include BV 173, K562, KCL-22, and KYO-1, LAMA84, fectamineTM 2000 is mixed gently before use and the mixture 40 EM2, EM3, BV 173, AR230, and KU812 (Mahon, F. X., is diluted to appropriate amount in 50 ul of Opti-MEMR I Blood. 2000; 96: 1070-1079; Lerma et al. Mol. Cancer Ther. Medium. The mixture is incubated for 5 minutes at room 2007; 6(2): 655-66). temperature. c. After 5 minute incubation, the diluted DNA is In other embodiments, non-CML cells may be transfected combined with diluted LipofectamineTM2000 (total vol with expression vectors comprising the bcr-abl1 gene or vari ume=100 ul) and is mixed gently. The mixture is incubated 45 ants of the bcr-abl1 gene including splice variants of the for 20 minutes at room temperature. 100 ul of complexes is bcr-abl1 gene resulting in genetically modified cells compris added to each well containing cells and medium. The contents ing the recombinant polynucleotide. Thus, the transfected are mixed gently by rocking the plate back and forth. Cells are cells will be able to express BCR-ABL1 protein or its vari incubated at 37°C. in a CO, incubator for 18–48 hours prior ants. The genetically modified cells can be used to Screen to testing for transgene expression. Medium may be changed 50 compounds for treating CML patients. after 4-6 hours. Cells are passaged at a 1:10 (or higher dilu In yet other embodiments. CML cell lines, for example tion) into fresh growth medium 24 hours after transfection. BV 173, K562, KCL-22, and KYO-1, LAMA84, EM2, EM3, Selective medium (containing Neomycin and Ampicillin) is BV 173, AR230, and KU812 may be transfected with expres added the following day. sion vectors comprising splice variants of bcr-abl1 gene Prediction of the Likelihood of Drug Resistance in CML 55 resulting in genetically modified cells comprising the recom Patients or Subjects Suspected of having CML binant polynucleotide. The gene product of the splice variants Methods of the invention can be used for predicting the of the bcr-abl1 gene and the insertion/truncation mutant of likelihood that a CML patient or a subject suspected of having BCR-ABL1 may impart partial or total resistance to ABL CML with a BCR-ABL1 translocation will be resistant to kinase inhibitors to these genetically modified cells. The treatment with one or more BCR-ABL1 kinase inhibitors. A 60 genetically modified cells may be used to screen compounds sample from a CML patient, or a Subject Suspected of having for treating CML. The compounds may be inhibitors of ABL CML, is assessed for the presence or absence of a polynucle kinase activity or these compounds may have other mecha otide sequence encoding the 195INS and/or the 243INS bcr nism of action. abl1 splice variants described herein, or a complement The CML cell lines and the genetically modified cell lines thereof. Optionally, a sample from a CML patient, or a subject 65 as discussed above may be grown in appropriate growth Suspected of having CML, is assessed for the presence or medium and using appropriate selective antibiotics. Methods absence of a BCR-ABL1 fusion protein having an amino acid for cell culture is well known in the art (Sambrook, et al., US 8,603,740 B2 25 26 Molecular Cloning: A Laboratory Manual (1989), Second pound. In one embodiment, the detectable label can be FITC. Edition, Cold Spring Harbor Press, Plainview, N.Y.). Several The amount of signal from FITC-labeled anti-BrdU bound to growth media for cell culture are commercially available. the DNA can be measured by . Commercially Non-limiting examples include GIBCORRPMI Media 1640, available kits for flow cytometry based cell proliferation Dulbecco's Modified Eagle Medium (DMEM), DMEM: assays are available. Such as, Click-iTR, EdU (Invitrogen Nutrient Mixture F-12 (DMEM/F12), Minimum Essential Corp., Carlsbad, Calif., USA). ELISA based assays for mea Media (Invitrogen Corp., Carlsbad, Calif., USA), RF-10 Suring BrdU incorporation by proliferating cells care com medium. Non-limiting examples of selective antibiotics mercially available examples include BrdUCell Proliferation include amplicillin, neomycin, Geneticin R, Hygromycin B. Assay kit (Calbiochem. EMD Chemicals Inc. New Jersey, In one preferred embodiment, K562 cells (ATCC catalog 10 USA). no: CCL-243) may be genetically modified by transfecting In another method, proliferation of cells treated with vari with different amounts of the expression vector pCMV/GFP/ ous amounts of the compound can be measured by monitor 195INS bcr-abl or pCMV/GFP/243INS bcr-abl. In one ing the incorporation of radioactively labeled deoxynucle embodiment, the amount the expression vector used for trans otides (Sun et al. Cancer Res. 1999:59:940-946). fection can be 0 ng, or can be at least about: 1 ng, 2ng, 5 ng, 15 Kinase Activity of BCR-ABL1: 7.5 ng, 10 ng, 12.5 ng, 15 ng, 20 ng, 25 ng, 30 ng, 40 ng, 50 The effect of a compound on the kinase activity of the ng, 60 ng, 75 ng, 100 ng, 125 ng, 200 ng, 500 ng, 750 ng, or BCR-ABL1 protein is assessed by monitoring tyrosine phos 1 ug. The transfected cells may be grown in RF-10 medium phorylation profile of the cellular proteins. CrlkL is a sub with neomycin/and or amplicillin. strate of BCR-ABL1 tyrosine kinase (Ren et al. Genes Dev. Assessing the Effect of a Compound for Treatment of Leu 1994; 8(7):783-95). Genetically modified cells comprising kemia on Genetically Modified Cells recombinant bcr-abl or variant so of bcr-abl including the Several parameters may be assessed for identifying the splice variant are grown in presence of various amounts of a compounds that may be beneficial for treatment of CML compound for treating CML patients. In a preferred embodi patients. Non-limiting examples of the parameters that may ment, the compounds are ABL tyrosine kinase inhibitors. be assessed includes cell viability, cell proliferation, apopto 25 Non-limiting examples of kinase inhibitors include imatinib, sis, kinase activity of BCR-ABL1 protein, additional muta nilotinib, dasatinib, Bosutinib (SKI-606) and Aurora kinase tions in BCR-ABL1 protein, and additional mutation in the inhibitor VX-680. Amount of phosphorylated CrkL protein ABL protein. can be measured by using detectably labeled anti-phospho Cell Viability: CrkL antibody. In one embodiment, the detectable label is Cells can be plated at a density of 2-2.5x10 cells/mL in 30 phycoerythrin. The signal Canbe detected by flow cytometer. RF-10 with varying amounts of the compound or without the Alternatively, the signal can be detected by Fluorescent compound. Aliquots are taken out at 24-hour intervals for Microtiter plate reader. assessment of cell viability by trypan blue exclusion. Sequencing of the ABL Kinase Domain: Alternatively, cell viability can be measured by colorimet To further investigate the reason for some cells that do not ric assay Such as MTT assay (Mosman et al. J. Immunol. 35 overexpress BCR-ABL1 but that have higher resistance to a Meth. 1983; 65: 55-63). Commercial kits for MTT assay are compound that target the ATP-binding site of the ABL kinase available. For example, CelTiter 96(R) Non-Radioactive Cell domain (such as imatinib, nilotinib, dasatinib, and Aurora Proliferation Assay (MTT) (Promega Corporation, Wiscon kinase inhibitor VX-680) than their sensitive counterparts, sin, USA), Vybrant(R) MTT Cell Proliferation Assay Kit (In the entire kinase domain of K562-sensitive and -resistant vitrogen Corp. Carlsbad, Calif., USA). 40 cells can be sequenced. Sequencing can be performed using Cell Proliferation: ABI prism 377 automated DNA sequencer (PE Applied Bio Proliferation of the genetically modified cells in presence systems; USA). Sequence analysis can performed using the of a compound for treatment of CML patient can be measured GCG version 10 software. in several ways. The proliferation of the cells can be indica In summary, 195INS BCR-Abland 243INSbcr-abl1 vari tive of the effectiveness of the compound for CML therapy. 45 ants described herein are created by an exonic insertion of a In one Such method, cell proliferation assay can performed sequence from the adjacent preceding intron and produce an using MTS tetrazolium such as Cell Titer96 Aqueous exclusively-expressed splicing variant in the absence of wild (Promega corporation, Wisconsin, USA), which measures type bcr-abl 1 transcript. The 195INS variant causes early numbers of viable cells. Between 2x10 and 2x10 cells are translational termination and truncation of the BCR-ABL1 washed twice in RF-1-10 and plated in quadruplicate into 50 protein missing a significant portion of the C-terminal regu microtiter-plate wells in 100 uL, RF-10 plus various doses of latory region and is associated with significant drug resis the compound. Controls using the same concentrations of tance not only to imatinib, but also to one or more of the newer compound without cells are set up in parallel. Twenty micro tyrosine kinase inhibitors—nilotinib and dasatinib. The 243 liters MTS is added to the wells at daily intervals. Two hours INS variant results in a non-native 81 amino acid insertion after MTS is added, the plates are read in a microplate auto 55 which may result in resistance to tyrosine kinase inhibitors. reader (Dynex Technologies, Billingshurst, UK) at 490-nm The invention will now be described in greater detail by wavelength. Results are expressed as the mean optical density reference to the following non-limiting examples. for each dose of the compound. All experiments are repeated at least 3 times. EXAMPLE 1. In another method, cell proliferation assays can be per 60 formed by monitoring the incorporation bromo-deoxyuracil bcr-abl1 Mutation Detection and Analysis (BrdU) into newly synthesized DNA. The Amount of BrdU incorporated into the DNA will be proportional to the amount Venous blood was collected from a CML patient who was of DNA synthesis and will be indicative of the proliferating resistant to more than one of the three kinase inhibitors: cells. In one such method, detectably labeled anti-BrdU anti 65 imatinib, nilotinib and dasatinib. The bcr-abl1 allele was body can be used to measure the amount of BrdU incorpo amplified from the blood sample in a first round one step rated into the cells treated with various amounts of the com RT-PCR. A forward primer that anneals at bcr exon b2 (BCR US 8,603,740 B2 27 28 F: SEQID NO: 15) and a reverse primer (ABL-R2: SEQ ID ABL1 sequence shown in FIG. 2. The altered ABL1 protein NO: 16) that anneals at the junction of ablexons 9 and 10 were does not truncate early. The amino acid sequence of the used in first round PCR to ensure that the normal, non-trans altered ABL1 portion of the BCR-ABL1 protein is shown in located abl gene would not be analyzed. FIG. 5B, with the differing amino acids underlined. This particular patient shows no additional mutations in the ABL1 The ABL kinase domain was then amplified in semi-nested kinase domain. PCR followed by direct sequencing using ABI/prism Big Approximately 40-60% of human genes undergo alterna Dye terminator cycle sequencing kit on automated capillary tive splicing, and alterations in alternative splicing have been DNA sequencer (ABI Prism R 3100 Genetic Analyzer). The manifested by its clinical connections to many human dis nested PCR amplified the region encoding the entire BCR eases, including cancers (Caceres & Kornblihtt, Trends in ABL tyrosine kinase domain and the activation loop using a 10 Genetics 2002 18:186-93: Stoilov et al., DNA and Cell Biol. forward primer that anneals to exon 4 (ABL-F1; SEQID NO: 2002 21:803-18: Wu et al., “Alternatively Spliced Genes.” In 17) and a reverse primer that anneals to the junction of abl Encyclopedia of Molecular and Cell Biology and Molecular exon 9 and 10 (ABL-R2: SEQ ID NO: 16). The resulting Medicine, Vol.1, 2" ed., 125-177 (2004)). Alternative splic fragment was gel extracted, purified and sequenced in both ing mutations in patients with CML being treated with 15 tyrosine kinase inhibitors could be an overlooked mechanism forward and reverse directions using SEQID NOs: 16, 17, 18, for the resistance to therapy. Two alternative splicing muta and 19. Sequencing data were base-called by Sequencing tions, 195INS and 234INS, were detected in multidrug resis Analysis software and assembled and analyzed by ABI tant CML patients, in which they are the only isoform of Prism(R) SeqScape Software using GenBank accession num bcr-abl 1 transcript to be detected. ber M14752 as a reference. Primer sequences for the first and In Summary, each of the two splicing variants described second rounds of PCR are listed below. herein 1) are created by an exonic insertion of a sequence from the adjacent preceding intron; and 2) are associated with significant drug resistance not only to imatinib, but also to one SEO ID NO: 15 or more of the newer tyrosine kinase inhibitors—nilotinib and TGA, CCA ACT CGT GTG. TGA AAC TC dasatinib. These 2 identified mutations, along with the previ SEO ID NO: 16 25 ously reported 35INS (Lee et al.), appear to be a part of a new TCC ACT TCG TCT GAG ATA CTG GAT T class of alternative splicing mutations. SEO ID NO: 17 Unless otherwise defined, all technical and scientific terms CGC AAC AAG CCC ACT GTC T used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention SEO ID NO: 18 30 belongs. All nucleotide sequences provided herein are pre CAA. GTG GTT CTC CCC TAC CA sented in the 5' to 3' direction. SEO ID NO: 19 Thus, it should be understood that although the present TGG TAG GGG AGA ACC ACT TOG invention has been specifically disclosed by preferred embodiments and optional features, modification, improve 35 ment and variation of the inventions embodied therein herein EXAMPLE 2 disclosed may be resorted to by those skilled in the art, and that Such modifications, improvements and variations are bcr-abl1 195INS and 243INS Splice Variants considered to be within the scope of this invention. The mate rials, methods, and examples provided here are representative Two mutations, resulting from alternative splicing in the 40 ABL1 kinase domain, were detected in kinase resistant CML of preferred embodiments, are exemplary, and are not patients. The two splice variants, 195INS and 243INS both intended as limitations on the scope of the invention. resulted in frameshift mutations. FIG. 1 shows the mRNA The invention has been described broadly and generically sequence for the human abl1 gene, which is the gene region in herein. Each of the narrower species and Subgeneric group the bcr-abl 1 translocation affected by these mutations. FIG. 2 ings falling within the generic disclosure also form part of the shows the amino acid sequence for human ABL1 protein, 45 invention. This includes the generic description of the inven which is the part of the BCR-ABL1 fusion protein that is tion with a proviso or negative limitation removing any Sub changed in these mutations. ject matter from the genus, regardless of whether or not the The 195INS splice variant carries an insertion of a 195 excised material is specifically recited herein. nucleotide sequence in the abll exon 4-5 junction at position In addition, where features or aspects of the invention are 553 in FIG. 1. The 195 nucleotide sequence is derived from 50 described in terms of Markush groups, those skilled in the art intron 4 of the abl1 gene. FIG.3 shows the altered nucleotide will recognize that the invention is also thereby described in sequence of the abl1 mRNA with the insertion underlined. terms of any individual member or subgroup of members of The resulting amino acid sequence of the ABL1 portion of the the Markush group. BCR-ABL1 fusion protein changes at amino acid 184 and All publications, patent applications, patents, and other truncates at amino acid 187 in the ABL1 sequence shown in 55 references mentioned herein are expressly incorporated by FIG. 2: FIG. 5A shows the amino acid sequence of the ABL1 reference in their entirety, to the same extent as if each were portion of the BCR-ABL1 protein with the differing amino incorporated by reference individually. In case of conflict, the acids underlined. This particular patient shows no additional present specification, including definitions, will control. mutations in the ABL1 kinase domain. Other embodiments are set forth within the following The 243INS splice variant carries an insertion of a 243 60 claims. nucleotide sequence in the abll exon 6-7 junction at position 911 in FIG. 1. The 243 nucleotide sequence is derived from SEQUENCE LISTING intron 6 of the abl1 gene. The altered nucleotide sequence of the abl1 mRNA is shown in FIG.4, with the 243 bp insertion The instant application contains a Sequence Listing which underlined. The resulting amino acid sequence of the ABL1 65 has been submitted via EFS-Web and is hereby incorporated portion of the BCR-ABL1 fusion protein acquires an addi by reference in its entirety. Said ASCII copy, created on Feb. tional 81 amino acids starting at amino acid 304 of in the 8, 2011 is named 09527602.txt and is 48,391 bytes in size.

US 8,603,740 B2 33 34 - Continued acagcc titca cittittctgag ttcttgaagc atttcaaag.c cctgcct citg tdtagcc.gc.c 4500

Ctgaga.gaga atagagctgc Cactgggcac Ctgcgcacag gtgggaggaa agggcCtggc 456 O

Cagt cctggit Cctggctgca citc.ttgaact ggg.cgaatgt Cttatttaat taccgtgagt gacatagoct catgttctgt ggggg.tcatC agggagggitt aggaaaacca caaacggagc 468O

CCCtgaaagc citcacg tatt t cacagagca cgc.ctgc cat cittct coccg aggctgc.ccc 474. O aggc.cggagc ccagatacgg gggctgttgac tctgggCagg gacCCggggt ctic ct ggacc 48OO ttgacagagc agctaactico gaga.gcagtg ggCaggtggc cgc.ccct gag gct tcacgc.c 486 O gggagaa.gc.c acct tccCaC CCCtt Catac cgc.ct cqtgc cagcago ct c gcacaggccC 492 O tagctttacg CtcatCacct aaacttgtac titt attitt to tgatagaaat ggttt cotct 498O ggat.cgttitt atgcggttct tacagcacat cacct ctittg CCC cc.gacgg Ctgtgacgca 5040 gcc.ggaggga ggc act agtic accgacagcg gcc ttgaaga cagagcaaag cgc.ccaccca ggtc.ccc.cga ctgcctgtct c catgaggta ctggit coctit c ctitttgtta acgtgatgtg 516 O

CCactatatt ttacacgitat citc.ttggitat gcatcttitta tagacgctict tittctaagtg 522 O gcgtgtgcat agcgt.cctgc cctg.cccc ct cgggggcctg tggtggct cc ccctctgctt 528 O

Ctcggggit Co agtgcattitt gtttctgtat atgattctict gtggttttitt ttgaatccaa 534 O atctgtcc to tgtag tattt tittaaataaa. t cagtgttta Catt 53.84

<210s, SEQ ID NO 2 &211s LENGTH: 113 O 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2

Met Lieu. Glu Ile Cys Lieu Lys Lieu. Val Gly Cys Ser Lys Gly 1. 1O 15

Lell Ser Ser Ser Ser Ser Cys Tyr Lieu. Glu Glu Lell Glin Arg Pro 25

Wall Ala Ser Asp Phe Glu Pro Glin Gly Lieu. Ser Ala Ala Arg Trp 35 4 O 45

Asn Ser Glu Asn Lieu. Lieu. Ala Gly Pro Ser Asn Asp Pro Asn SO 55

Lell Phe Wall Ala Lieu. Tyr Asp Phe Wall Ala Ser Asp Asn Thir Lieu. 65 70 7s

Ser Ile Thir Lys Gly Glu Lys Lieu Arg Val Lieu. Asn His Asn 85 90 95

Gly Glu Trp Cys Glu Ala Glin Thr Lys Asn Gly Gly Trp Wall Pro 105 11 O

Ser Asn Tyr Ile Thr Pro Wall Asn Ser Lieu. Glu His Ser Trp Tyr 115 12 O 125

His Gly Pro Val Ser Arg Asn Ala Ala Glu Tyr Lell Lell Ser Ser Gly 13 O 135 14 O

Ile Asn Gly Ser Phe Lieu Val Arg Glu Ser Glu Ser Ser Pro Gly Glin 145 150 155 160

Arg Ser Ile Ser Lieu. Arg Tyr Glu Gly Arg Val His Arg Ile 1.65 17O 17s

Asn Thir Ala Ser Asp Gly Llys Lieu Tyr Val Ser Ser Glu Ser Arg Phe 18O 185 19 O

Asn Thir Luell Ala Glu Lieu Val His His His Ser Thir Wall Ala Asp Gly 195 2O5

Lell Ile Thir Thr Lieu. His Tyr Pro Ala Pro Llys Arg Asn Pro Thir US 8,603,740 B2 35 36 - Continued

21 O 215 22O

Wall Gly Wall Ser Pro Asn Asp Trp Glu Met Glu Arg Thir 225 23 O 235 24 O

Asp Ile Thir Met Lys His Luell Gly Gly Gly Glin Gly Glu Wall 245 250 255

Glu Gly Wall Trp Ser Luell Thir Wall Ala Wall Thir 26 O 265 27 O

Lell Glu Asp Thir Met Glu Wall Glu Glu Phe Lell Lys Glu Ala Ala 28O 285

Wall Met Glu Ile His Pro Asn Luell Wall Glin Lell Luell Gly Wall 29 O 295 3 OO

Cys Thir Arg Glu Pro Pro Phe Ile Ile Thir Glu Phe Met Thir Tyr 3. OS 310 315

Gly Asn Luell Luell Asp Lell Arg Glu Cys ASn Arg Glin Glu Wall Asn 3.25 330 335

Ala Wall Wall Luell Lell Met Ala Thir Glin Ile Ser Ser Ala Met Glu 34 O 345 35. O

Luell Glu Lys Asn Phe Ile His Arg Asp Lell Ala Ala Arg Asn 355 360 365

Luell Wall Gly Glu Asn His Luell Wall Wall Ala Asp Phe Luell 37 O 375

Ser Arg Luell Met Thir Gly Asp Thir Thir Ala His Ala Gly Lys 385 390 395 4 OO

Phe Pro Ile Trp Thir Ala Pro Glu Ser Luell Ala Asn Phe 4 OS

Ser Ile Ser Asp Wall Trp Ala Phe Gly Wall Lell Lell Trp Ile 425 43 O

Ala Thir Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Lell Ser Wall 435 44 O 445

Glu Luell Luell Glu Asp Arg Met Glu Arg Pro Glu 450 45.5 460

Pro Glu Wall Tyr Glu Lell Met Arg Ala Cys Trp Glin Trp Asn Pro 465 470 48O

Ser Asp Arg Pro Ser Phe Ala Glu Ile His Glin Ala Phe Glu Thir Met 485 490 495

Phe Glin Glu Ser Ser Ile Ser Asp Glu Wall Glu Glu Luell Gly SOO 505

Glin Gly Wall Arg Gly Ala Wall Ser Thir Luell Luell Glin Ala Pro Glu Luell 515 525

Pro Thir Thir Thir Ser Arg Arg Ala Ala Glu His Arg Asp Thir 53 O 535 54 O

Thir Asp Wall Glu Met Pro His Ser Gly Glin Gly Glu Ser Asp 5.45 550 555 560

Pro Luell Asp His Glu Pro Ala Wall Ser Pro Luell Lell Pro Arg Lys Glu 565 st O sts

Arg Gly Pro Glu Gly Gly Luell Asn Glu Asp Glu Arg Luell Luell Pro 585 59 O

Asp Lys Thir Asn Lell Phe Ser Ala Luell Ile Lys 595 605

Thir Ala Thir Pro Pro Arg Ser Ser Ser Phe Arg Glu Met 610 615

Asp Gly Glin Glu Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp 625 630 635 64 O US 8,603,740 B2 37 38 - Continued Ile Ser Asn Gly Ala Lell Ala Phe Thr Pro Lieu. Asp Thr Ala Asp Pro 645 650 655

Ala Llys Ser Pro Llys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly Ala 660 665 67 O

Lell Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser Pro His Leu Trp 675 685

Ser Ser Thir Lieu. Thir Ser Ser Arg Lieu Ala Thr Gly Glu Glu Glu 69 O. 695 7 OO

Gly Gly Gly Ser Ser Ser Lys Arg Phe Lieu. Arg Ser Cys Ser Ala Ser 7 Os

Wall Pro His Gly Ala Lys Asp Thr Glu Trp Arg Ser Val Thr Luell 72 73 O 73

Pro Arg Asp Lieu. Glin Ser Thr Gly Arg Glin Phe Asp Ser Ser Thr Phe 740 74. 7 O

Gly Gly His Llys Ser Glu Llys Pro Ala Lieu Pro Arg Lys Arg Ala Gly 760 765

Glu Asn Arg Ser Asp Glin Val Thr Arg Gly Thr Val Thr Pro Pro Pro 770 775 78O

Arg Lieu Wall Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp 79 O 79.

Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro Asn Lieu. Thr Pro 805 810 815

Pro Lieu. Arg Arg Glin Wall Thr Val Ala Pro Ala Ser Gly Lieu Pro His 82O 825 83 O

Glu Glu Ala Gly Gly Ser Ala Lieu. Gly Thr Pro Ala Ala Ala 835 84 O 845

Glu Pro Wall Thir Pro Thir Ser Lys Ala Gly Ser Gly Ala Pro Gly Gly 850 855 860

Thir Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His Lys His 865 88O

Ser Ser Glu Ser Pro Gly Arg Asp Llys Gly Llys Lieu. Ser Arg Lieu Lys 885 890 895

Pro Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 9 OO 905 91 O

Pro Ser Glin Ser Pro Ser Glin Glu Ala Ala Gly Glu Ala Val Luell 915 92 O 925

Gly Ala Lys Thr Lys Ala Thir Ser Lieu Val Asp Ala Val Asn. Ser Asp 93 O 935 94 O

Ala Ala Lys Pro Ser Glin Pro Gly Glu Gly Lieu Lys Llys Pro Val Luell 945 950 955 96.O

Pro Ala Thr Pro Llys Pro Gln Ser Ala Lys Pro Ser Gly Thr Pro Ile 965 97O 97.

Ser Pro Ala Pro Wall Pro Ser Thir Lieu Pro Ser Ala Ser Ser Ala Luell 98O 985 99 O

Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Ile Pro Leu Ile Ser Thr 995 1OOO 1005

Arg Wall Ser Lieu. Arg Llys Thir Arg Glin Pro Pro Glu Arg Ile Ala 1010 1015 1 O2O

Ser Gly Ala Ile Thr Lys Gly Val Val Lieu. Asp Ser Thr Glu Ala 1025 1O3 O 1035

Lell Cys Lieu. Ala Ile Ser Arg Asn. Ser Glu Glin Met Ala Ser His 104 O 1045 1 OSO

Ser Ala Wall Lieu. Glu Ala Gly Lys Asn Leu Tyr Thir Phe Cys Wall 105.5 106 O 1065

US 8,603,740 B2 49 - Continued gCag.ccggag gaggcacta gtcaccgaca gcggccttga agacagagca aag.cgcc cac 534 O cCaggit cocc cactgcctg t ct coatgag gtactggtc.c citt cottttg ttaacgtgat 54 OO gtgccact at attittacacg tat ct cittgg tatgcatctt ttatagacgc tottt totala 546 O gtggcgtgtg catagogt CC tgc cctdcc.c Cct C9ggggc tocccctctg 552O

Cttcticgggg. tccagtgcat tttgtttctg tatatgattic tctgtggittt tttittgaatc 558 O caaatctgtc. citctgtag ta tttitt taaat aaatcagtgt ttacatt 5 627

<210s, SEQ ID NO 5 &211s LENGTH: 1.87 212. TYPE: PRT ORGANISM: Artificial Sequence 22 Os. FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 5

Met Lieu. Glu Ile Cys Lieu Lys Lieu. Val Gly Cys Lys Ser Lys Gly 1. 5 1O 15

Lell Ser Ser Ser Ser Ser Cys Tyr Lieu. Glu Glu Lell Glin Arg Pro 25

Wall Ala Ser Asp Phe Glu Pro Glin Gly Lieu. Ser Ala Ala Arg Trp 35 4 O 45

Asn Ser Lys Glu Asn Lieu. Lieu Ala Gly Pro Ser Asn Asp Pro Asn SO 55

Lell Phe Val Ala Lieu. Tyr Asp Phe Wall Ala Ser Asp Asn Thir Lieu. 65 70 7s

Ser Ile Thir Lys Gly Glu Lys Lieu. Arg Val Lieu. Asn His Asn 85 90 95

Gly Glu Trp Cys Glu Ala Glin Thr Lys Asn Gly Gly Trp Wall Pro 105 11 O

Ser Asn Tyr Ile Thr Pro Val Asn Ser Lieu. Glu His Ser Trp Tyr 115 12 O 125

His Gly Pro Val Ser Arg Asn Ala Ala Glu Tyr Lell Lell Ser Ser Gly 13 O 135 14 O

Ile Asin Gly Ser Phe Lieu Val Arg Glu Ser Glu Ser Ser Pro Gly Glin 145 150 155 160

Arg Ser Ile Ser Lieu. Arg Tyr Glu Gly Arg Val His Arg Ile 1.65 17O 17s

Asn Thir Ala Ser Asp Gly Lys Gly Ser Cys Trp 18O 185

<210s, SEQ ID NO 6 &211s LENGTH: 1211 212. TYPE: PRT ORGANISM: Artificial Sequence 22 Os. FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 6

Met Lieu. Glu Ile Cys Lieu Lys Lieu. Val Gly Cys Ser Lys Gly 1. 5 1O 15

Lell Ser Ser Ser Ser Ser Cys Tyr Lieu. Glu Glu Ala Lell Glin Arg Pro 25 3O

Wall Ala Ser Asp Phe Glu Pro Glin Gly Lieu. Ser Glu Ala Ala Arg Trp 35 4 O 45

Asn Ser Lys Glu Asn Lieu. Lieu Ala Gly Pro Ser Glu Asn Asp Pro Asn US 8,603,740 B2 51 52 - Continued

SO 55 6 O

Lell Phe Wall Ala Lell Tyr Asp Phe Wall Ala Ser Gly Asp Asn Thir Luell 65 70

Ser Ile Thir Gly Glu Luell Arg Wall Luell Gly Tyr Asn His Asn 85 90 95

Gly Glu Trp Cys Glu Ala Glin Thir Lys Asn Gly Glin Gly Trp Wall Pro 105 11 O

Ser Asn Tyr Ile Thir Pro Wall Asn Ser Luell Glu His Ser Trp Tyr 115 12 O 125

His Gly Pro Wall Ser Arg Asn Ala Ala Glu Tyr Lell Lell Ser Ser Gly 13 O 135 14 O

Ile Asn Gly Ser Phe Lell Wall Arg Glu Ser Glu Ser Ser Pro Gly Glin 145 150 155 160

Arg Ser Ile Ser Lell Arg Glu Gly Arg Wall His Arg Ile 1.65

Asn Thir Ala Ser Asp Gly Luell Tyr Wall Ser Ser Glu Ser Arg Phe 18O 185 19 O

Asn Thir Luell Ala Glu Lell Wall His His His Ser Thir Wall Ala Asp Gly 195

Lell Ile Thir Thir Lell His Tyr Pro Ala Pro Arg Asn Pro Thir 21 O 215

Wall Gly Wall Ser Pro Asn Asp Trp Glu Met Glu Arg Thir 225 23 O 235 24 O

Asp Ile Thir Met Lys His Luell Gly Gly Gly Glin Tyr Gly Glu Wall 245 250 255

Glu Gly Wall Trp Ser Luell Thir Wall Ala Wall Thir 26 O 265 27 O

Lell Glu Asp Thir Met Glu Wall Glu Glu Phe Lell Lys Glu Ala Ala 285

Wall Met Glu Ile His Pro Asn Luell Wall Glin Lell Luell Gly Arg 29 O 295 3 OO

Gly Luell Ala Arg Glin Pro Ala Pro Trp Ser His Arg Ala Trp Ser Arg 3. OS 310 315

Ala Ala Phe Lys Pro Glin Pro Arg Arg Ser Glin Gly Ala Ala 3.25 330 335

Ser Asn Luell Ser Ala Asn Thir Luell Asp Luell Gly Thir Lys Ala Asn 34 O 345 35. O

Thir Arg Luell Luell Ala Pro Ala Luell Gly Met His Pro Gly Luell Asn 355 360 365

Ser Ala Arg Pro Arg Lell Trp Thir Ser Gly Ala Ala Asn Pro Arg Arg 37 O 375

Wall Thir Arg Glu Pro Pro Phe Ile Ile Thir Glu Phe Met Thir 385 390 395 4 OO

Gly Asn Luell Lell Asp Luell Arg Glu Cys Asn Arg Glin Glu Wall 4 OS 415

Asn Ala Wall Wall Lell Lell Tyr Met Ala Thir Glin Ile Ser Ser Ala Met 425 43 O

Glu Tyr Luell Glu Asn Phe Ile His Arg Asp Lell Ala Ala Arg 435 44 O 445

Asn Cys Luell Wall Gly Glu Asn His Luell Wall Wall Ala Asp Phe Gly 450 45.5 460

Lell Ser Arg Luell Met Thir Gly Asp Thir Tyr Thir Ala His Ala Gly Ala 465 470 47s 48O US 8,603,740 B2 53 54 - Continued

Phe Pro Ile Lys Trp Thir Ala Pro Glu Ser Lell Ala Tyr Asn 485 490 495

Phe Ser Ile Lys Ser Asp Wall Trp Ala Phe Gly Wall Lell Luell Trp Glu SOO 505

Ile Ala Thir Gly Met Ser Pro Pro Gly Ile Asp Luell Ser Glin 515 525

Wall Tyr Glu Luell Lell Glu Lys Asp Arg Met Glu Arg Pro Glu Gly 53 O 535 54 O

Cys Pro Glu Wall Tyr Glu Luell Met Arg Ala Trp Glin Trp Asn 5.45 550 555 560

Pro Ser Asp Arg Pro Ser Phe Ala Glu Ile His Glin Ala Phe Glu Thir 565 st O sts

Met Phe Glin Glu Ser Ser Ile Ser Asp Glu Wall Glu Glu Luell Gly 58O 585 59 O

Glin Gly Wall Arg Gly Ala Wall Ser Thir Luell Lell Glin Ala Pro Glu 595 605

Lell Pro Thir Thir Arg Thir Ser Arg Arg Ala Ala Glu His Arg Asp 610 615

Thir Thir Asp Wall Glu Met Pro His Ser Lys Gly Glin Gly Glu Ser 625 630 635 64 O

Asp Pro Luell Asp His Glu Pro Ala Wall Ser Pro Lell Lell Pro Arg Lys 645 650 655

Glu Arg Gly Pro Glu Gly Gly Luell Asn Glu Asp Glu Arg Luell Luell 660 665 67 O

Pro Asp Thir Asn Luell Phe Ser Ala Lell Ile Lys 675 685

Lys Thir Ala Thir Pro Pro Arg Ser Ser Ser Phe Arg Glu 69 O. 695 7 OO

Met Asp Gly Glin Glu Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg 7 Os

Asp Ile Ser Asn Gly Ala Lell Ala Phe Thir Pro Lell Asp Thir Ala Asp 72 73 O 73

Pro Ala Ser Pro Pro Ser Asn Gly Ala Gly Wall Pro Asn Gly 740 74. 7 O

Ala Luell Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser Pro His Luell Trp 760 765

Lys Ser Ser Thir Lell Thir Ser Ser Arg Luell Ala Thir Gly Glu Glu 770 775

Glu Gly Gly Gly Ser Ser Ser Arg Phe Luell Arg Ser Ser Ala 79 O 79.

Ser Wall Pro His Gly Ala Asp Thir Glu Trp Arg Ser Wall Thir 805 810 815

Lell Pro Arg Asp Lell Glin Ser Thir Gly Arg Glin Phe Asp Ser Ser Thir 825 83 O

Phe Gly Gly His Ser Glu Lys Pro Ala Luell Pro Arg Arg Ala 835 84 O 845

Gly Glu Asn Arg Ser Asp Glin Wall Thir Arg Gly Thir Wall Thir Pro Pro 850 855 860

Pro Arg Luell Wall Lys Asn Glu Glu Ala Ala Asp Glu Wall Phe Lys 865

Asp Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro Asn Luell Thir Pro 885 890 895

Pro Luell Arg Arg Glin Wall Thir Wall Ala Pro Ala Ser Gly Luell Pro 9 OO 905 91 O US 8,603,740 B2 55 - Continued

His Lys Glu Glu Ala Gly Lys Gly Ser Ala Lieu. Gly Thr Pro Ala Ala 915 92 O 925 Ala Glu Pro Val Thr Pro Thir Ser Lys Ala Gly Ser Gly Ala Pro Gly 93 O 935 94 O Gly. Thir Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His Lys 945 950 955 96.O His Ser Ser Glu Ser Pro Gly Arg Asp Llys Gly Llys Lieu. Ser Arg Lieu. 965 97O 97. Llys Pro Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly 98O 985 99 O Gly Llys Pro Ser Glin Ser Pro Ser Glin Glu Ala Ala Gly Glu Ala Val 995 1OOO 1005 Lieu. Gly Ala Lys Thir Lys Ala Thir Ser Lieu Val Asp Ala Val Asn O1O O15 O2O Ser Asp Ala Ala Lys Pro Ser Glin Pro Gly Glu Gly Lieu Lys Llys O25 O3 O O35 Pro Val Lieu Pro Ala Thr Pro Llys Pro Glin Ser Ala Lys Pro Ser O4 O O45 OSO Gly Thr Pro Ile Ser Pro Ala Pro Val Pro Ser Thr Lieu Pro Ser O55 O6 O O65 Ala Ser Ser Ala Lieu Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Of O O7 O8O Ile Pro Lieu. Ile Ser Thr Arg Val Ser Lieu. Arg Llys Thr Arg Glin O85 O9 O O95 Pro Pro Glu Arg Ile Ala Ser Gly Ala Ile Thr Lys Gly Val Val OO O5 10 Lieu. Asp Ser Thr Glu Ala Lieu. Cys Lieu Ala Ile Ser Arg Asn. Ser

Glu Gln Met Ala Ser His Ser Ala Val Lieu. Glu Ala Gly Lys Asn

Lieu. Tyr Thr Phe Cys Val Ser Tyr Val Asp Ser e Glin Glin Met

Arg Asn Llys Phe Ala Phe Arg Glu Ala Ile Asn Llys Lieu. Glu Asn

Asn Lieu. Arg Glu Lieu. Glin e Cys Pro Ala Thr Ala Gly Ser Gly

Pro Ala Ala Thr Glin Asp Phe Ser Lys Lieu Lleu Ser Ser Wall Lys

Glu e Ser Asp Ile Val Glin Arg 2O5 21 O

<210s, SEQ ID NO 7 &211s LENGTH: 195 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OO > SEQUENCE: 7 gggagctgct ggtgaggatt attittagact gtgagtaatt gacctgacag acagtgatga 6 O ctgctt catt aagagcc.cac gaccacgtgc cagaatagitt cagcatcct c tdttgctact 12 O gtactittgag acatcgttct tctttgttgat gcaatacctic titt cittgtca togagggit ct c 18O titcc cittaaa totagg 195 US 8,603,740 B2 57 58 - Continued

<210s, SEQ ID NO 8 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 8 Thir Ala Ser Asp Gly Lys Gly Ser Cys Trp 1. 5

<210s, SEQ ID NO 9 &211s LENGTH: 243 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OOs, SEQUENCE: 9 gtaggggcct gcCaggcag cctg.cgc.cat ggagt cacag ggcgtggagc C9ggCagcct 6 O tttacaaaaa gcc cca.gc.ct aggaggtotC agggcgcagc ttctaac ct c agtgctggca 12 O acacattgga ccttggaaca aaggcaaaca c taggct cot ggcaaagcca gCtttgggca 18O tgcatcCagg gctaaattica gcc aggccta gactctggac Cagtggagca gctaatcCCC 24 O gga 243

<210s, SEQ ID NO 10 &211s LENGTH: 81 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 10

Arg Gly Lieu Ala Arg Glin Pro Ala Pro Trp Ser His Arg Ala Trp Ser 1. 5 15

Arg Ala Ala Phe Tyr Llys Llys Pro Glin Pro Arg Arg Ser Glin Gly Ala 25 3O

Ala Ser Asn Lieu. Ser Ala Gly Asn Thir Luell Asp Lieu. Gly. Thir Lys Ala 35 4 O 45

Asn Thr Arg Lieu. Lieu Ala Lys Pro Ala Luell Gly Met His Pro Gly Lieu SO 55 6 O

Asn Ser Ala Arg Pro Arg Lieu. Trp Thir Ser Gly Ala Ala Asn Pro Arg 65 70 7s 8O Arg

<210s, SEQ ID NO 11 &211s LENGTH: 11 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 11 ggcaa.gggga g 11

<210s, SEQ ID NO 12 &211s LENGTH: 12 &212s. TYPE: DNA US 8,603,740 B2 59 60 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 12 atcaggct ct ac 12

<210s, SEQ ID NO 13 &211s LENGTH: 12 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 13 t ccttggtag gg 12

<210s, SEQ ID NO 14 &211s LENGTH: 12 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 14 cc.cggagggit ct 12

<210s, SEQ ID NO 15 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 15 tgacca actic gtgttgttgaaa citc 23

<210s, SEQ ID NO 16 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 16 tccactitcgt ctdagatact ggatt 25

<210s, SEQ ID NO 17 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 17 cgcaacaa.gc ccactgtct 19

<210s, SEQ ID NO 18 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 8,603,740 B2 61 62 - Continued

223 OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 18 caagtggttc. tcc cctacca

SEQ ID NO 19 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 19 tgg tagggga galaccacttg

What is claimed is: 2O 3. The method of claim 2, wherein the myeloproliferative 1. A method for predicting likelihood for resistance of a disease is chronic myelogenous leukemia (CML). patient with a bcr-abl 1 translocation to treatment with one or 4. The method of claim 1, wherein said one or more kinase more BCR-ABL1 kinase inhibitors, comprising: inhibitors are selected from a group consisting of imatinib, (a) detecting in a biological sample from the patient a 25 nilotinib, bosutinib, and dasatinib. polynucleotide sequence encoding a 195INS bcr-abl1 5. The method of claim 4, wherein said one or more kinase splice variant or a 243INS bcr-abl 1 splice variant in inhibitors is imatinib. bcr-abl1 mRNA; and (b) identifying the patient as having an increased likelihood 6. The method of claim 1, wherein the sample comprises of being resistant to treatment with one or more BCR blood cells. ABL1 kinase inhibitors, whereby the presence of a poly 30 7. The method of claim 6, wherein the sample comprises nucleotide encoding at least one of the splice variants peripheral mononuclear cells. indicates that the patient has an increased likelihood of 8. The method of claim 1, wherein the assaying comprises being resistant to treatment with one or more BCR hybridizing an oligonucleotide comprising a nucleotide ABL1 kinase inhibitors. sequence selected from the group consiting of SEQID NOs: 2. The method of claim 1, wherein the patient is diagnosed 11-14 to the bcr-ab11 mRNA or cDNA derived therefrom. as having a myeloproliferative disease. k k k k k