US 20050287530A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0287530 A1 Croce et al. (43) Pub. Date: Dec. 29, 2005

(54) TCL-1B AND AND Publication Classification RELATED METHODS AND COMPOSITIONS (51) Int. Cl." ...... C12O 1/68; CO7H 21/04; (76) Inventors: Carlo M. Croce, Philadelphia, PA A61K 39/395; CO7K 14/82; (US); Yuri Pekarsky, Philadelphia, PA CO7K 16/30 (US) (52) U.S. Cl...... 435/6; 435/69.1; 435/320.1; 435/325; 530/350; 530/388.8; Correspondence Address: 424/155.1; 536/23.5 HAMILTON, BROOK, SMITH & REYNOLDS, P.C. 530 VIRGINA ROAD (57) ABSTRACT P.O. BOX 91.33 CONCORD, MA 01742-9133 (US) The TCL1 gene family, located on the human at the 14q32.1 locus, are implicated in the development of (21) Appl. No.: 10/678,790 T-cell malignancies. The present invention discloses the identification and characterization of new members of this (22) Filed: Oct. 2, 2003 gene family, the TCL-1b, TNG1 and TNG2 . The TCL-1b, TNG1 and TNG2 gene sequences are expressed at Related U.S. Application Data very low levels in normal bone marrow and peripheral lymphocytes, but are activated in T-cell leukemia and lym (63) Continuation of application No. 09/526,329, filed on phoma by rearrangements of the 14q32.1 locus. The present Mar. 15, 2000, now abandoned. invention relates to the identification of these abnormalities, and methods for detecting and treating any (60) Provisional application No. 60/124,714, filed on Mar. T-cell malignancies that develop, as well as preventing the 15, 1999. development of these T-cell malignancies.

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TCL-1B GENE AND PROTEIN AND RELATED in humans (T-CLL) and is activated in these leukemias by, METHODS AND COMPOSITIONS iuxtaposition to the T-cell receptor C/6 locus caused by chromosomal translocations, t(14:14)(q11;32), CROSS REFERENCE TO RELATED t(7,14)(q35;q32), or inversions inv(14)(q11;q32). Normally APPLICATIONS TCL1 expression is observed in early T-cell progenitors (CD4-CD8CD3) and lymphoid cells of the B-cell lineage: 0001. This application claims priority, in part, under 35 pre B-cells and immature IgM expressing B-cells. Introduc USC S 119 based upon U.S. Provisional Patent Application tion of a TCL1 transgene under the control of alck promoter No. 60/124,714 filed Mar. 15, 1999. caused mature T-cell leukemia in mice. (Virgilio et al., 1998, 0002 This invention was made in part with government Proc Natl AcadSci USA, 95:3885-3889). support under Grant numbers CA39880 and CA51083 awarded by the National Institutes of Health. The govern 0008 However, some cases of T-cell malignancies with ment has certain rights in the invention. abnormalities Such as gene amplification at 14q32.1 did not show activation of the TCL1 expression, Suggesting that FIELD OF THE INVENTION perhaps an additional oncogene may be located in 14q32.1. The second member of the TCL1 gene family, MTCP1, is 0003. The present invention relates to the field of molecu located at Xq28 and activated in rare cases of mature T-cell lar biology, more particularly to the isolation and character leukemia with a tX;14)(q28;q11) translocation. The present ization of a third member of the TCL1 gene family, specifi invention involves the isolation and characterization of the cally TCL-1b, also activated by chromosomal third member of the TCL1 gene family, TCL1b, located at rearrangements in T cell leukemias. 14q32.1 and also activated by rearrangements at 14q32.1 in T-cell leukemias. BACKGROUND OF THE INVENTION 0009 Rearrangements of the TCL-1 locus at chromo 0004. There is a cow association between particular chro Some 14q32.1 are unique, in that the other locus involved in mosomal abnormalities, e.g., chromosomal translocations, these rearrangements, namely the TCR C/6 locus, is also on inversions, and deletions, and certain types of malignancy chromosome 14 at Subband q11 (Croce et al., 1985, Science indicating that Such abnormalities may have a causative role 227:1044-1047; Isobe et al., 1988, Proc Natl AcadSci USA, in the cancer process. Chromosomal abnormalities may lead 85:3933-3937). For this reason, the rearrangements to gene fusion resulting in chimeric oncoproteins, Such as is observed cytogenetically are either chromosomal inver observed in the majority of the tumors involving the myeloid Sions, inv(14) (q11;q32), involving only one of the chromo lineage. Alternatively, chromosomal abnormalities may lead Somes 14 or translocations involving both. 14 to deregulation of protooncogenes by their juxtaposition to Such as the t014;14) (q11;q32), or more rarely, the t07:14) a regulatory element active in the hematopoietic cells, Such (q35;q32) involving the TCR Blocus at 7q35 (Isobe et al., as is observed in the translocation occurring in the lympho 1988, Proc Natl AcadSci USA, 85:3933-3937). Several of cytic lineage (Virgilio et al., 1993, Proc. Natl. Acad. Sci. the breakpoints at 14q32.1 involved in these translocations USA, 90:9275-9279). have been cloned and characterized (Russo et al., 1988, Cell, 0005 Non random chromosomal translocations are char 51:137-144; Baer, et al., 1987, Proc Natl AcadSci, 84:9069 acteristic of most human hematopoietic malignancies 9073; Mengle-Gaw et al., 1987, EMBO 1:2273-2280; Bert (Haluska et al., 1987, Ann. Rev. Genet, 21:321-345) and may ness et al., 1990, Cancer Genet Cytogenet, 44:47-54). be involved in some solid tumors (Croce, 1987, Cell, 0010. The TCL-1 locus, a chromosomal region of 49:155-156). In B and T cells, chromosomal translocations approximately 350 kb as determined by placement of trans and inversions often occur as a consequence of mistakes location breakpoints on the long range genomic map, has during the normal process of recombination of the genes for recently been cloned (Virgilio, et al., 1993, Proc Natl Acad immunoglobulins (Ig) or T-cell receptors (TCR). These Sci USA, 90:9275-9279). The involvement of such a large rearrangements juxtapose enhancer elements of the Ig or region in translocation events Suggests that activation of the TCR genes to oncogenes whose expression is then deregu putative TCL-1 gene may occur from a distance of many lated (Croce, 1987, Cell, 41: 155-156). In the majority of the kilobases, as previously observed for the BCL-1/CCNDI cases, the rearrangements observed in lymphoid malignan gene in mantle cell lymphoma (Tsujimoto, et al., 1984, cies occur between two different chromosomes. Science 22.4:1403-1406; Rosenberg, et al., 1991, Proc Natl 0006. The TCL-1 locus on chromosome 14 band q32.1 is AcadSci USA, 88:9638-9642; Withers, et al., 1991, Mol Cell frequently involved in the chromosomal translocations and Biol, 11:4846-4853; Motokura and Arnold, 1993, Genes inversions with the T-cell receptor genes observed in Several Chrom & Cancer, 7:89-95) and the MYC oncogene in post-thymic types of T-cell leukemias and lymphomas, Burkitt lymphoma (Dalla-Favera, et al., 1982, Proc Natl including T-prolymphocytic leukemias (T-PLL) (Brito-Ba AcadSci USA, 79:7824-7827; Nishikura, et al., 1983, Proc bapulle and Catovsky, 1991, Cancer Genet. Cytogenet, Natl Acad Sci USA, 80:4822-4826) and in acute T-cell 55:1-9), acute and chronic leukemias associated with the leukemia (Erikson, et al., 1986, Science, 232:884-886). immunodeficiency Syndrome ataxia-telangiectasia (AT) 0011 Introduction of a TCL1 transgene under the control (Russo et al., 1988, Cell, 53:137-144; Russo et al., 1989, of the T-cell Specific lek promoter into mice causes T-cell Proc. Natl. Acad. Sci. USA, 86:602-606), and adult T-cell proliferative disorder and, at the age of 15 months, T-cell leukemia (Virgilio et al., 1993, Proc. Natl. Acad. Sci. USA, leukemia (Virgilio, L., et al., 1998, Proc Natl AcadSci USA, 90:9275-9279). 95:3885-3889). Another member of the TCL1 gene family is 0007. The TCL1 oncogene on chromosome 14q32.1 is the MTCP1 gene on chromosome Xq28. MTCP1 is also also involved in the development of chronic T-cell leukemia activated in rare cases of T-cell leukemia by a US 2005/0287530 A1 Dec. 29, 2005 t(X;14)(q28;q11) translocation (Soulier, J., et al., 1994, respectively, as well as derivatives and analogs thereof, and Oncogene, 9:3565-3570). In rare cases of mature T-cell antibodies thereto. The present invention further relates to leukemias with chromosomal abnormalities at 14q32.1, acti nucleic acids hybridizable to or complementary to the fore vation of the TCL1 gene was not observed (Sakashita, K., et going nucleotide Sequences, as well as equivalent nucleic al., 1998, Leukemia, 12:970-971; Takizawa, J., et al., 1998, acid Sequences encoding a Tcl1b, Tng 1 or Tng2 protein. Jpn.J Cancer Res, 89,712-718). A second putative oncogene in this region was isolated, as described below, the TCL1b 0017. The present invention relates to expression vectors gene. This gene is located approximately 16 kb centromeric encoding a Tcl1b, Tng1 or Tng2 protein, derivative or analog to TCL1 and shares 60% amino acid sequence similarity thereof, as well as host cells containing the expression with TCL1. vectors encoding the Tcl1b, Tng1 or Tng2 protein, derivative or analog thereof. 0012. The expression profiles of both genes are very similar. TCL1 and TCL1b are expressed at very low levels 0018. The present invention further relates to the use of in normal bone marrow and peripheral blood lymphocytes TCL1b, TNG1 and TNG2 genes and their encoded (Virgilio, L., et al., 1994, Proc Natl Acad Sci USA, as diagnostic and therapeutic tools for the detection and 91:12530-12534; Pekarsky, Y., et al., 1999, Proc Natl Acad treatment of disease States associated with chromosomal Sci USA, 96:2949-2951), but at higher levels in T-cell lines abnormalities, Specifically abnormalities at 14q32.1. In one containing rearrangements of the 14q32.1 region (Virgilio, embodiment of the present invention the use of nucleotide L., et al., 1994, Proc Natl AcadSci USA, 91:12530-12534; sequences of TCL-1b, TNG1 or TNG2 genes and amino acid Pekarsky, Y., et al., 1999, Proc Natl AcadSci USA, 96:2949 Sequences of their encoded Tcl-1b, Tng1, or Tng2 proteins, 2951). Since genes in close proximity to TCL1 and TCL1b respectively, are used as diagnostic reagents or in the prepa may also be activated in leukemias with rearrangements at ration of diagnostic agents useful in the detection of disease 14q32.1, the chromosomal region bracketed by two previ States, Such as T-cell leukemias and lymphomas, associated ously published breakpoint cluster regions observed in T-cell with chromosomal abnormalities, in particular at 14q32.1, neoplasias (Virgilio, L., et al., 1994, Proc Natl Acad Sci and/or increased levels of expression of the Tcl1b, Tng 1 or USA, 91: 12530-12534; Virgilio, L., et al., 1993, Proc Natl Tng2 protein. AcadSci USA, 90:9275-9279) at 14q32.1 was investigated 0019. The invention further relates to the use of nucle for the presence of additional genes. otide sequences of TCL-1b, TNG1 or TNG2 genes and 0013 The murine Tcl1 locus was also examined in order amino acid Sequences of their encoded Tcl1b, Tng 1 or Tng2 to investigate the function of TCL1 and TCL1b. In the protein, respectively, as therapeutic/prophylactic agents in mouse the Syntenic region of human chromosome 14q32 is the treatment/prevention of disease States, Such as T-cell the region of the murine chromosome 12 proximal to the leukemias, associated with chromosomal abnormalities, in immunoglobulin locus. The murine Tcl1 protein shows a particular at 14q32.1, and/or increased levels of expression 50% homology to the human Tcl 1 (Narducci, M. G., et al., of the Tcl1b, Tng 1 or Tng2 protein. 1997, Oncogene, 15:919-926) and is expressed in fetal 0020) The TCL-1b, TNG1 or TNG2 genes and Tel1b, hematopoietic organs and in immature T and B-cells as well Tng1 or Tng2 protein Sequences disclosed herein, and as in adult spleen and thymus (Narducci, M. G., et al., 1997, antibodies thereto, are used in assays to diagnose T-cell Oncogene, 15:919-926). In order to identify other members leukemias and lymphomas associated with chromosomal of the murine Tcl1 family the murine Tcl 1 locus was also abnormalities, and/or increased expression of Tcl1b, Tng 1 or investigated for the presence of homologous genes. Tng2 protein. 0.014. There remains an unfulfilled need to fully isolate and characterize the other member of the TCL-1 gene 0021. The Tcl 1b, Tng1 or Tng2 protein, or derivatives or family, TCL1b, and the genes located very closely to TCL1b analogs thereof, disclosed herein, are used for the production and TCL1; TNG1 and TNG2. The identification of addi of anti-Tcl 1b, anti-Tng1 or anti-Tng2 antibodies, respec tional oncogenes that are associated with chromosomal tively, which antibodies are useful diagnostically in immu abnormalities causing T-cell leukemias and lymphomas fur noassays for the detection or measurement of Tcl1b, Tng 1 or ther expands the efficacy by which a diagnostic and thera Tng2 protein, respectively, in a patient Sample. peutic/prophylactic reagent will detect, treat, and prevent 0022. Another aspect of the present invention relates to Such disease States. The present invention fulfills this need methods of treatment of diseases or conditions associated by the identification and characterization of the TCL1b, with chromosomal abnormalities and/or increased expres TNG1 and TNG2 genes. sion of Tcl 1b, Tng1 or Tng2 proteins. Abnormalities of 0.015 Citation of references herein above shall not be chromosome 14, Such as inversions and translocations, construed as an admission that Such references are prior art particularly at 14q32.1, are associated with T-cell leukemias to the present invention. and lymphomas. TCL-1b, TNG1 or TNG2 gene sequences and their protein products are used therapeutically in the SUMMARY OF THE INVENTION treatment of disease States associated with chromosome 14 abnormalities. Anti-Tcl 1b, anti-Tng 1 or anti-Tng2 antibod 0016. The TCL1 gene family is implicated in the devel ies are used therapeutically, for example, in neutralizing the opment of T-cell malignancies. The present invention dis activity of an overexpressed Tcl1b, Tng1 or Tng2 protein, closes the identification and characterization of new mem respectively, associated with disease. bers of this gene family, the TCL-1b, TNG1 and TNG2 genes. The present invention relates to the nucleotide 0023 Oligonucleotide Sequences, including antisense sequences of TCL1b, TNG1 and TNG2, and amino acid RNA and DNA molecules and ribozymes, designed to Sequences of their encoded Tcl1b, Tng1, and Tng2 proteins, inhibit the transcription or translation of TCL-1b, TNG1 or US 2005/0287530 A1 Dec. 29, 2005

TNG2 mRNA, are used therapeutically in the treatment of Bold lines under the exons show various splicing products of disease States associated with increased expression of Tcl1b, TNG1, TNG2, and TCL1b genes. (C) Murine Tcl 1 locus. Tng1 or Tng2, respectively. Restriction sites and exons are indicated as in (A). 0024 Proteins, peptides and organic molecules capable 0032 FIG. 6. RT-PCR analysis of TNG1 and TNG2 of modulating activity of Tcl 1b, Tng 1 or Tng2 are used genes (A) Leukemia cell lines. Lanes 1-3: T-ALL cell lines: therapeutically in the treatment of disease States associated MOLT3; MOLT4; CEM. Lane 4: pre B-ALL cell line 697. with aberrant expression of Tcl 1b, Tng1 or Tng2. Lane 5: T-ALL cell line SupT11. Lane 6-8: Burkitt's lym phoma cell lines CA-46; Raji; Daudi. Lanes 9-10: bone 0.025 The present invention further relates to therapeutic marrow; peripheral blood lymphocytes (PBL). First panel, compositions comprising Tcl1b, Tng 1 or Tng2 proteins, TCL1 primers, Second panel, TCL1b primers; third panel, derivatives or analogs thereof, antibodies thereto, nucleic TNG1 primers; fourth panel, TNG2 primers; bottom, control acids encoding the Tcl1b, Tng1 or Tng2 proteins, derivatives G3PDH primers (B). Normal human tissues. Lanes I-23: or analogs, and TCL-1b, TNG1 or TNG2 antisense nucleic heart, liver; brain; muscle, placenta, kidney, lung, pancreas, acid. spleen; lymph node; thymus, tonsil; PBL, fetal liver; fetal 0026. The present invention further relates to methods of brain; fetal lung, fetal kidney, fetal heart, fetal skeletal production of the Tcl1b, Tng1 or Tng2 proteins, derivatives muscle; fetal spleen; fetal thymus, negative control. (C) and analogs, Such as, for example, by recombinant means. Lanes 1-4, T cell PLL samples: 3047; 3046; 3050; 3048. Lanes 5-6: bone marrow; PBL. (B-C). Top, TNG1 primers; DESCRIPTION OF THE DRAWINGS middle, TNG2 primers; bottom, control G3PDH primers. 0033 FIG. 7. Northern analysis of TNG1 and TNG2 0027 FIG. 1. Sequence comparison of Tcl 1, Tcl-1b and genes. Lanes 1-3: Burkitt's lymphomas Raji; Daudi; CA-46; Mtcp1. Identities are shown in black boxes, similarities are Lane 4: T-ALL SupT11; Lanes 5-6: bone marrow; placenta. shown in shaded boxes. For Tcl1 and Mctp GenBank Top, TNG1 probe; middle, TNG2 probe; bottom, actin accession numbers are X82240 and Z24459, respectively. probe. Each lane contains 3 lug of polyA+ RNA. 0028 FIG. 2FIG. 2. Genomic organization of the TCL1 0034 FIG. 8. RT-PCR analysis of murine Tcl1b genes. and TCL1b genes. Vertical arrows refer to cloned 14q32.1 (A-B) Nested PCR, except B-actin. The panels are in the breakpoints. Restriction sites are given for BSSHII (B), Cla same order. (A) Normal mouse tissues. Lanes 1-13: heart; (C), Eag (E), Sfil (F), KspI (K), Mlul (M), NotI (N), NruI brain; spleen; lung; liver; Sceletal muscle, kidney; testis, 7 (R) and SalI (S). Solid boxes represent TCL1 and TCL1b day embryo; 11-day embryo; 15-day embryo; 17-day CXOS. embryo, negative control. (B) Lymphoid cell lines. Lanes 0029 FIG. 3. Northern analysis of the TCL1 and TCL1b 1-5: B-cell lines NFS-5; NFS-70; WEHI-279; MOPC-3 1C: genes. (A). Human immune System Northern blot. Lanes MPC-11. Lanes 6-7: T-cell lines S49.1, BW5147. Lane 8-9: 1-6: Spleen, lymph node; thymus, peripheral blood leuko ES cells; negative control. (C) Single round of RCR. Lanes cyte; bone marrow; fetal liver. (B). Human cancer cell line 1-4: ES cells, mouse oocytes; 2-cell embryos, negative Northern blot. Lanes 1-8: promyelocytic leukemia, HL-60; control. Hela cells, chronic myelogenous leukemia, K-562; T-lym phoblastic leukemia, MOLT4; Burkitt's lymphoma Raji; 0035 FIG. 9. Sequence comparison of human and colorectal adenocarcinoma, SW480; lung carcinoma, A549; murine Tcl 1, Tcl 1b and Mtcp1 proteins. Identities are shown melanoma, G361. (C). Lanes 1-6: Burkitt's lymphoma Raji; in black boxes, Similarities are indicated by Shaded boxes. Burkitt's lymphoma Daudi; Burkitt's lymphoma CA-46; * mark the conserved residues of the inner hydrophobic core. SupT11, bone marrow; placenta. (D). Lane 1: bone marrow; 0036 FIG. 10. Location of the insertion in human and lanes 2-7, EBV transformed lymphoblastiod cell lines: Ado murine Tcl 1b proteins: A side view of human Tcl 1 is shown 1471; Ado-1476; Ado-1701; Ado-1727; Ado-2069; Ado in green. The Tcl-1b insert into the C-D loop is shown in 2199; lane 8: CA-46. (A-D). Top, TCL1b probe; middle, blue. Tcl1 probe; bottom, actin probe. 0030 FIG. 4. RT-PCR analysis of the TCL1 and TCL1b DESCRIPTION OF THE INVENTION genes. (A). Normal human tissues. Lanes 1-23: heart, liver; 0037 Methods brain; muscle, placenta, kidney, lung; pancreas, Spleen; lymph node, thymus, tonsil; peripheral blood lymphocytes 0.038 Cell Lines. (PBL); fetal liver; fetal brain; fetal lung; fetal kidney; fetal 0039) Cell lines, except EBV transformed lymphoblas heart, fetal Skeletal muscle, fetal Spleen; fetal thymus, toid cell lines, were obtained from ATCC (Rockville, Md.) negative control. (B) Lanes 1-4, T cell PLL samples: 3047; and grown in RPMI media with 10% fetal bovine serum. 3046; 3050; 3048. Lanes 5-6: bone marrow; PBL (A-B). Lymphoblastoid cell lines were made from peripheral blood Top, TCL1b primers; middle, TCL1 primers; bottom, con lymphocytes of patients with Alzheimer's disease by trans trol G3PDH primers. formation with Epstein-Barr virus (EBV) as previously 0.031 FIG. 5. Genomic organization of human and reported (Ounanian, A., et al., 1992, Mech Ageing Dev, mouse TCL1 loci. (A) Human TCL1 locus. Vertical arrows 63:105-116). refer to cloned 14q32.1 breakpoints (1,7). Restriction sites 0040) Human leukemia cell lines MOLT 3, MOLT 4, are given for BssHII (B), ClaI (C), Eagl (E), Sfil (F), KspI CEM, and SupT11 (T-cell leukemias) and 697 (pre B-cell (K), Mlul (M), Not (N), and Sal (S). Solid boxes represent leukemia) and CA-46, Raji, and Daudi (Burkitt's lympho exons of the four genes. (B) Striped boxes indicate translated mas) were obtained from American Type Culture Collection parts of exons, white boxes indicate untranslated regions. (Manassas, Va.) Mouse lymphatic cell lines NFS-70 C-10 US 2005/0287530 A1 Dec. 29, 2005

(pro B-cells), NFS-5 C-1 and WEHI-279 (pre B-cells), TAGGATGTGCACAGAGCA, (SEQ. ID. NO: 15); and 4A MOPC-31C and MPC-11 (plasma cells), and S49.1 and BW (nested) for 5 RACE and TNG2: 3B and 4B (nested) for 3' 5147 (thymocytes) were also purchased from American RACE and 8B and 9B (nested) for 5 RACE together with Type Culture Collection (Manassas, Va.). All cell lines were primers AP1 and AP2 Supplied by Clontech (Palo Alto, grown in RPMI 1640 medium with 10% fetal bovine serum. Calif.) fitting to the adapters of the cDNA. The murine Tcl1b genes were amplified using the respective R reverse: 1R: 0041) Northern, Rapid Amplification of cDNA Ends GAGAACGGTCAGGACCCAAACC, (SEQ. ID. NO:16); (RACE) and Reverse Transcripton-PCR (RT-PCR) Analysis. 2R: CAGGCTATCAAGACCTTTACTC, (SEQ. ID. NO: 0042. These experiments were carried out as previously 17); 3/5R: TCAACCTCGCATATTACTATGTC, (SEQ. ID. described (Pekarsky, Y, et al., 1998, Proc Natl AcadSci USA, NO: 18); 4R: CAAAGGCACAAAGTGAGCAAGAG, 95:8744-8749) with the following exceptions. Human bone (SEQ. ID. NO: 19); and F forward: 1F. AATGTGGAAACT marrow and placenta mRNAS, human immune System and TCTCACTCAT, (SEQ. ID. NO: 20); 2F: ACTGGAAACT human cancer cell line Northern blots were purchased from TGTTCTCATTCAC, (SEQ. ID. NO: 21); 3/5F: CACTTG Clontech (Pato Alto, Calif.). Each line on FIGS. 3C and D CAGCATATGACCACAAT, (SEQ. ID. NO: 22); 4F: contains 3 mg Poly A+ RNA. PCR shown on FIG. 4A was CCTGGTCTGCACAAGAGATGA, (SEQ. ID. NO. 23); carried out for 25-35 cycles using Multiple Tissue cDNA primers for 28 cycles. Subsequently the respective R and FN Panels (Clontech) and manufacturer's protocol. Primers forward nested: 1FN: CTGTCCACTTGTGGAAGTTAAT, were: top panel, TC1GGCAGCTCTACCCCGGGATGAA, (SEQ. ID. NO: 24); 2FN: CACTTGTGGCAGATGACCA (SEQ. ID. NO: 1); and TC39 ACAGACCTGAGTGGGA GATA, (SEQ. ID. NO:25); 3/5FN: CCAGGAGCCTACTC CAGGA, (SEQ. ID. NO: 2); middle panel, TCLBTCCTC CCCAGCAG, (SEQ. ID. NO: 26); 4FN: GTGGCAGAT CTTGGCAGGAGTGGTA, (SEQ. ID. NO:3); and TCLC GACCACACTCTT, (SEQ. ID. NO: 27); primers were used CAGTTACGGGTGCTCTTGCGT, (SEQ. ID. NO: 4); in a seminested PCR for 25 cycles to amplify 1 ul of the first lower panel, control 3' and 5 RACE G3PDH primers reaction. PCR conditions were the same as described for (Clontech). FIG. 4B, middle and bottom panels, primers human tissues. Due to the Similarity of mouse Tcl 1b genes were the same as above. FIG. 4B, top panel. PCR was it was difficult to find specific primers for each of them. carried out for 22 cycles with primers TC8 ATGGCCTC Subsequently Tcl1b3 and Tcl1b5 were amplified with the CGAAGCTTCTGTG, (SEQ. ID. NO: 5), and TC39. 0.1 ml Same primers and Sequenced to Verify the expressed gene. of the reaction was used for the second PCR with nested However, in the case of embryonic tissue, unique forward primers TC10 TGGTCGTGCGGTTCAATCCCT, (SEQ. ID. primers were used to analyze the expression of Tcl1b3 and NO: 6); and TC5 AATCTGGCCATGGTCTGCTATTTC, Tcl1b5 separately. The expression of both alternative first (SEQ. ID. NO: 7); for 15 cycles. RACE primers were: TC1 exons of Tcl 1b3 was verified using the primers 3F horn (for 3' RACE) and TC5 (for 5 RACE). homologous exon 1: CATTACTATGGCTGATTCAGTTC, (SEQ. ID. NO: 28); and 3F alt alternative exon 1: GGAAT. 0043 Mouse and human tissue cDNAs for RT-PCR and GAGACTCTCAGGGCAC, (SEQ. ID. NO: 29); instead of RACE experiments were purchased from Clontech (Palo 3/5F. RT-PCR for Tcl 1 was carried out similarly with prim Alto, Calif.). Mouse egg and 2 cell embryo cDNA libraries ers Tel1R, CCTGGGCAAGGCAGACAGGAGC, (SEQ. for embryonic expression Studies in mouse were previously ID. NO: 30); and TCL1F, TGCTTCTTGCTCTTATCG described (Rothstein, J. L., et al., 1992, Genes Dev, 6:1190 GATG, (SEQ. ID:NO:31); followed by a nested PCR using 1201). The DNAS from these libraries were diluted to the primers Tel1RN, TTCATCGTTGGACTCCGAGTC, (SEQ. Same concentration of cDNA as in mouse tissue Samples. ID, NO. 32); and Tel1FN, AATTCCAGGTGATCT RNA extractions and reverse transcriptions from human and TGCGCC, (SEQ. ID. NO:33). The quality of the cDNA was mouse cell lines and mouse embyonic Stem cells were verified by 25 cycles of B-actin RT-PCR using primers actR, performed using TrizolTM reagent (Gibco BRL, Grand GTACCACCAGACAGCACTGTG, (SEQ. ID. NO. 34); Island, N.Y.). 2 ug of total RNA were transcribed into cDNA and actF, GACCCAGATCATGTTTGAGACC, (SEQ. ID. in a total volume of 20 ul using SuperScript'TM reverse NO:35); RACE analysis from mouse tissues was performed transcription kit (Gibco BRL, Grand Island, N.Y.) according as described above for human tissues. The Specific primers to the manufacturers instructions. 1 ul of this reaction was were: allR, AAGCCATCTATAAGGTCAGG, (SEQ. ID. used for PCR. RT-PCR for TNG1 was carried out with NO:36); for the first step and the respective R primers for primers 1A TGCATCCCTCCAGCCAAGGAT, (SEQ. ID. the nested step of 5 RACE and the respective F (first) and NO: 8); and 4ATGGCCTGCAGAGGCTCTCAAG, (SEQ. ID, NO:9); for 25-35 cycles. For TNG2 primers 3B GTGC FN (nested) primers for 3' RACE. CTGTCTCATTCGCCTCTG, (SEQ. ID. NO: 10); and 8B 0044 Pulsed-Field Gel Electrophoresis (PFGE) Analysis AGTGGGCACATGTTACAGCATTC, (SEQ. ID. NO: 11); and Chromosomal Localization. were used for the first round of 25 cycles and primers 4B 0045 PFGE analysis was performed as described (Pekar GCATCCAGGACTGTGCCAGCA, (SEQ. ID. NO: 12); sky, Y., et al., 1998, Proc Natl AcadSci USA, 95:8744-8749), and 9B TTCTGTTAGCCTTGCTGTCCGT, (SEQ. ID. NO: except pulse time was 1-6 Second for 11 hours. Chromo 13); were used to amplify 0.1 ul of the first reaction in a Somal localization of the TCL1b gene was carried out using nested PCR of 20 cycles. PCR conditions were 94° C. GeneBridge 4 radiation hybrid mapping panel (Research denaturation for 30 Sec, 54 to 62 C. annealing for 30 sec and Genetics, Huntsville, Ala.) according to the manufacturer's 72 C. extension for 30 sec. TCL1, TCL1b and, as control, protocol. Primers were TC1 and TC4, TGCTAGGAC G3PDH were amplified as described previously (6). RACE CAGCTGCTCCATAGA, (SEQ. ID. NO:37). analysis was carried out in a nested reaction with 30 cycles in the first round and 25 in the second. The primers were: 0046 Sequencing. TNG1: 1A and 2A TTGAACCCAGGTCTCGTCTGAC, 0047 Products from RACE and RT-PCR experiments nested, (SEQ. ID. NO: 14); for 3' RACE and 3A AACG were cut and extracted from agarose gels using a QIAquick US 2005/0287530 A1 Dec. 29, 2005 gel extraction kit (Qiagen, Valencia, Calif.) according to the The TCL1b pseudogene does not have the initiating ATG or manufacturers instructions. Subsequently they were introns and has a stop codon in the middle of the open Sequenced using an automated Sequencer model 377 (Perkin reading frame. Elmer, Foster City, Calif.). A human Bacterial Artificial 0056 TCL1 and TCL1b are both located at 14q32, there Chromosome library (BAC) (277A8) was partially digested fore, a determination was made as to whether TCL1 and with Sau3A and TSP509I and cloned into a pUC18 vector TCL1b are physically linked. The human bacterial artificial using Standard methods. 100 random clones were isolated chromosome (BAC) library and found several BAC clones and Sequenced from both ends using a 377 automated containing TCL1 and TCL1b. The TCL1b gene (SEQ. ID. Sequencer. The DNA sequences were compared to the NO: 40) is 6.5 kb in size and contains 4 exons of 189, 171, expressed sequence tag (EST) database. The mouse BAC 69 and 697 bp respectively (FIG. 2), but only the first three 452-I24 was Sequenced and analyzed as described previ exons are coding. Pulsed field analysis of the positive BAC ously (Inoue, H., et al., 1997, Proc Natl Acad Sci USA, clone with both probes revealed that the TCL1 and TCL1b 94:14584-14589). EST clones were purchased from genes have opposite directions of transcription and are Research Genetics (Huntsville, Ala.) and Sequenced. separated only by 16 kb (FIG. 2). Both genes are located in 0048 Northern Blot and Pulse-Field Gel Electrophoresis the ~160 kb region between previously published two sets of (PFGE) for TNG Gene breakpoints observed in T-cell acute lymphoblastic leukemia (ALL) cases with translocations or inversions at 14q32.1 0049 Total RNA for Northern blot experiments was (Virgilio, L., et al., 1994, Proc Natl Acad Sci USA, isolated as described above. Poly A+ RNA isolation, North 91:12530-12534; Virgilio, L., et al., 1993, Proc Natl Acad ern blotting and hybridization was performed as previously Sci USA, 90:9275-9279). described (Hallas, C., et al., 1999, Clin Cancer Res, In press). TNG1 and TNG2 probes were generated by RT-PCR. 0057 Expression of TCL1b Gene and its Activation in PFGE analysis was performed as described" using BAC T-Cell Malignancies. (277A8) DNA and TNG1, TNG2, TCL1, and TCL1b probes. 0.058 Because of the similarities between the TCL1 and TCL1b genes in their structure, Sequence, and location, it 0050 Protein Structure. Seemed possible that they would exhibit Similar expression 0051 A computer model was created for the human and patterns. To verify this, we carried out a series of Northern murine Tcl 1b proteins based on their similarity to Tcl 1. The and RT-PCR experiments (FIGS. 3 and 4). Northern analy atomic coordinates for human TCL1 are derived from the sis in normal tissues was mostly negative for TCL1b (FIG. crystal structure (Hoh, F., et al., 1998, Structure, 6:147-155). 3A), except that the 1.2 kb transcript was detected after The initial Sequence alignment was generated by maximiz several days exposure in testis and placenta (FIG. 3C). The ing the correlation between the Sequences. Modeling and TCL1 gene expression, however, was detected in most analysis were done using Insight I (BioSym, San Diego, hematopoietic tissues after several days exposure (FIG.3A). Semiquantitative RT-PCR analysis (FIG. 4A) revealed that Calif.). both TCL1 and TCL1b genes are expressed in spleen, tonsil, 0.052 Results fetal liver, fetal kidney, and fetal thymus. However, the TCL1b gene is expressed in wider variety of tissues includ 0053) Identification of the TCL1b Gene. ing placenta, kidney and fetal spleen (FIG. 4A). Northern 0054. In some mature T-cell leukemias with chromo analysis of commercial human cancer cell lines showed that Somal abnormalities at 14q32.1, activation of the TCL1 gene TCL1 and TCL1b are expressed in only the Raji Burkitt at 14q32.1 was not observed (Takizawa, J., et al., 1998, Jpn. lymphoma cell line (FIG. 3B), although TCL1 was J. Cancer Res, 89:712-718, Sakashita, et al., 1998, Leukemia, expressed at a much higher level (FIG. 3B). 12:970-971). To investigate the possibility that other, 0059) The TCL1 and TCL1b genes have similar tran unknown TCL1 family member(s) may be involved, we Scription patterns and are physically linked. Therefore, a Searched the EST database for Sequences homologous to the determination as whether the TCL1b gene could also be TCL1 and MTCP1 gene products. A single EST (accession activated by rearrangements in 14q32 was made. FIGS. 3C number AA689513) was found to be homologous, but not an and 3D show the activation of the TCL1b gene in a exact match to both genes. Thus, a ~1.2 kb full length cDNA T-leukemia cell line with a translocation at 14q32.1 (SEQ. ID. NO: 38) was isolated using 5' and 3' RACE (SupT11) compared with the normal bone marrow and with procedure and human testis mRNA as a cDNA source. The EBV transformed lymphoblastiod B cell lines expressing 1.2 kb TCL1b cDNA encodes a 14 kDa protein of 128 amino TCL1. (FIGS. 3C and 3D, middle panels). Since TCL1 and acids (SEQ. ID. NO:39) (FIG. 1). It contains a starting ATG TCL1b are normally not expressed in post-thymic T-cells codon at position 28 within a perfect Kozak consensus and post-thymic T-cell leukemias lacking 14q32.1 abnor Sequence. The Tcl 1b protein has a 14 amino acid insertion malities (for example, in T-ALL MOLT4 with no abnormali compared to the Tcl1 and Mtcp1 proteins (FIG. 1); it is 30% ties at 14q32.1, FIG. 3B, lane 4), the expression of TCL1 identical and 60% similar to Tcl1, and 36% identical and and TCL1b in SupT11 cells carrying a to 14;14)(q11;q32,1) 63% similar to Mtcp1 (FIG. 1). translocation indicates that juxtaposition of TCL1 and 0055 A radiation hybrid mapping panel (GeneBridge 4) TCL1b to the C/6 locus of the T-cell receptor deregulates was used to determine the chromosomal localization of the both genes. human TCL1b gene. By analysis of PCR data at the MIT 0060. To further investigate TCL1b expression, four database (http://www-genome.wi.mit.edu), the TCL1b gene T-cell leukemias and six EBV transformed lymphoblastoid was localized to 3.05 cR from the marker D14S265, at cell lines with elevated levels of TCL1 were analyzed. FIG. 14q32. ATCL1b pseudogene and localized it to 5q12-5q13. 4B shows the activation of the TCL1b expression in one US 2005/0287530 A1 Dec. 29, 2005 leukemic Sample from a patient with T-cell prolymphocycic tionally, a bacterial artificial chromosome (BAC) library was leukemia. Human T-cell prolymphocytic leukemias carry the Screened and three clones containing murine Tcl1 were 14q32.1 translocation or inversion and overexpress TCL1 obtained. PCR analysis of these BAC clones confirmed the (Virgilio, L., et al., 1994, Proc Natl Acad Sci USA, presence of all three EST Sequences. By a combination of 91:12530-12534; Narducci, M. G., et al., 1997, Cancer Res, RACE and RT-PCR experiments, database analysis of EST 57:5452-5456). The TCL1b gene was also expressed in two Sequences and Sequencing of Selected EST clones, full out of six EBV transformed lymphoblastoid B cell lines length cDNAS corresponding to these Sequences were iso (FIG. 3D, upper panel, lanes 2-7). lated. 0065. Because of the shared similarity among the cDNAS 0061 The Human TCL1 Locus. it was not possible to obtain unique probes for each. Thus, 0062) The TCL1 and TCL1b genes are both located on the genomic structure of the region by conventional methods chromosome 14q32.1 within a ~160 kb region between two Such as Southern hybridization and pulse field gel analysis previously published breakpoint cluster regions observed in could not be determined. Subsequently, the BAC (452-124) T-cell neoplasms (Virgilio, L., et al., 1994, Proc Natl Acad was Sequenced and the position and the exon-intron bound Sci USA, 91.12530-12534; Virgilio, L., et al., 1993, Proc aries of the three cDNAS was determined. Natl AcadSci USA, 90:9275-9279). Both genes are activated 0066 Further analysis of the region also revealed that it by translocations and inversions involving 14q32.1 (Pekar contains two other Sequence related genes. RT-PCR experi sky, Y., et al., 1999, Proc Natl AcadSci USA,96:2949-2951). ments with Specific primers for these two genes confirmed To investigate whether other, unknown genes within this that they are transcribed. Altogether, five full length cDNAS region are also activated by the Same rearrangements a (SEQ. ID. NO: 47-51) were isolated located on murine previously isolated bacterial artificial chromosome library chromosome 12 centromeric to the Igh locus homologous to (BAC) of 110 kb (277A8, ref. 6) covering the majority of human TCL1b. Murine Tcl 1b1-Tcl1b5 cDNAs had a length this region was analyzed. This BAC was partially digested of ~1 kb (SEQ. ID. NO: 47-51, respectively) encoding for with the restriction enzymes Sau3A and TSP509I and cloned proteins ranging in size from 117-123 amino acids (SEQ. ID. into a puC18 vector. 100 clones (the equivalent of the length NO: 57-63, respectively). They share 70-90% nucleic acid of the BAC) were picked randomly and sequenced from homology and 55-75% amino acid identity and 65-80% both Sides. These Sequences were compared to the expressed amino acid similarity. The murine Tcl 1b family members sequence tag (EST) database and two different sets of ESTs show ~25% identity and ~35% similarity to murine Tcl1 and homologous to the BAC Sequences were found. Two full are 25-30% identical and 30-40% similar to human TCL1b. length cDNAs using 3' and 5 RACE and RT-PCR of cDNA from human testis, peripheral blood lymphocytes, and the 0067. The five genes are aligned on murine chromosome Burkitt's lymphoma cell line Raji were isolated using prim 12 (FIG.5C) in tire order Tel1b2, Tel1b1, Tcl 1b5, Tcl 1b3, ers made from the different ESTs. The 1.5 kb cDNA of the and Tcl 1b4 with distances of 4.5 kb, 9.7 kb, 9.9 kb, and 6.8 TCL1 neighboring gene 1 (TNG1) (SEQ. ID. NO: 41) kb, respectively, from each other and 9.8 kb between Tcl1b4 contains an open reading frame coding for a protein of 141 and Tcl 1. The total sizes of the genes are: Tcl1b1: 6.9 kb, amino acids (SEQ. ID. NO: 42) with the start codon ATG at (SEQ. ID. NO: 52); Tel1b2: 8.2 kb, (SEQ. ID. NO: 53); position 161. The 2 kb cDNA of TCL1 neighboring gene 2 Tel1b3 (SEQ. ID, NO. 54); and Tel1b4: 4.6 kb, (SEQ. ID. (TNG2) (SEQ. ID. NO: 43) encodes a shorter protein of 110 NO: 55); Tel1b5: 4.8 kb (SEQ. ID. NO:56). The direction amino acids (SEQ. ID. NO: 44) with the start codon at of transcription of Tcl1b1-Tcl1b5 is opposite to that of Tcl1. position 36. Both genes do not show homology to any Each of the murine Tcl 1b genes contains four exons of known genes found in the database. Relative positions of the approximately 200, 170, 70, and 590 bp in size. The only genes and their distances from each other were determined exceptions are the exons 3 of Tcl 1b2 and Tcl1b4, in which by Southern hybridization and pulse field Southern analysis. a different Splicing Site leads to a transcript 29bp shorter. In TNG2 is located 8 kb centromeric of TCL1b and TNG1 is addition, sequences of RT-PCR and RACE products and only 118 bp centromeric of TNG2. TNG1, TNG2 and ESTs derived from Genebank showed alternatively spliced TCL1b have the same transcriptional orientation, opposite to cDNAs for Tcl 1b1 and Tcl1b3. Tcl 1b1 may have a deletion TCL1 (FIG. 5A). The TNG1 gene is 4.5 kb (SEQ. ID. NO: of 73 bp consisting of nearly the complete exon 3 and the 45) in size and contains only two exons of 215 and 1239 bp. first 6 bp of exon 4. Because this deletion includes the stop The TNG2 gene has a size of 8.6 kb (SEQ. ID. NO: 46) codon the deduced protein Sequence is slightly longer containing four exons of 134, 136, 157, and 1651 bp, all of (Tcl1b1a, SEQ. ID. NO:58). For Tcl1b3 an alternative exon which are coding. RT-PCR and RACE experiments revealed 1 was found leading to a shorter protein (Tcl1b3a, SEQ. ID. Several alternatively spliced RNAS linking various exons of NO: 61) with an alternative N-terminal end without homol TNG1 and TNG2 to exon 2 of TCL1b (FIG. 5B). Only one ogy to other Tcl 1b proteins. of these RNAS, linking the exon 1 of TNG1 in frame to the 0068 Although the homology of murine Tcl 1b proteins Second eXon of TCL1b, contains a new open reading frame (SEQ. ID. NO: 57-63) to human Tel-1b (SEQ. ID. NO:39) encoding a TCL1b protein with an alternative N-terminal is lower than typically observed between mouse and human end. homologues (70-100%), the position of the genes on the map, their direction of transcription and their exon-intron 0063) The Murine Tcl 1 Locus. structure are similar to the human TCL1b locus and indicate 0064. In order to identify the murine Tcl1b gene the that these genes are authentic homologues to the human murine expressed sequence tag (EST) database was searched TCL1b gene (SEQ. ID. NO: 40). for sequences homologous to human TCL1b. Three sets of 0069 Expression of Human TNG1 and TNG2. ESTs were found that were very similar, but not identical, to 0070 ?? Because TNG1 and TNG2 are located at the each other and showed homology to human TCL1b. Addi same locus as TCL1 and TCL1b, it seemed possible that they US 2005/0287530 A1 Dec. 29, 2005 would exhibit Similar expression patterns. To investigate low level of expression of Tcl 1b2 in all lymphoid cell lines this, a series of Northern blot and RT-PCR experiments were (FIG. 8B) and nearly all normal tissues Further, Tcl1b2 performed. TNG1 and TNG2 are both transcribed in a wide expression increased during embryonic developement (7-17 variety of normal tissues (FIG. 6B). The results demonstrate days old embryos, FIG. 8A). A low level of expression was a low level of expression in most tissues examined including also found for Tcl 1b1 in nearly all lymphoid cell lines, but placenta, kidney, fetal kidney, fetal lung, and fetal heart and not in any other tissues. Tcl 1b4 only showed a low level of all lymphoid tissues including fetal liver and fetal Spleen. expression in testis and in the pro B-cell line NFS 5, which The only exception is thymus, which only showed tran also expressed Tcl1b3, as confirmed by sequencing (FIG. 8). scripts of TNG2, whereas fetal thymus only expressed Tcl1b5 expression was not detected in any tisuue or cell line TNG1 (FIG. 6B). TCL1b was expressed in the same tissues examined. In comparison to the Tcl1b genes, Tcl1 was as TNG1 except thymus, fetal lung, and fetal heart???(Vir expressed at low levels in testis, 11 and 15 days old embryos gilio, L., et al., 1998, Proc Natl Acad Sci USA, 95:3885 and in the thymocyte cell line S49-1. Interestingly, murine 3889). Northern blot analysis of normal adult and embryonic Tcl 1 was not detected in any of the early B-cell lines, tissues was negative for TNG1 and TNG2, probably due to although early B-cells show expression of TCL1 in humans the low level of expression. (Virgilio, L., et al., 1994, Proc Natl AcadSci USA, 95:3885 0071. Because of the similarity of transcription patterns 3889; Virgilio, L., et al., 1998, Proc Natl Acad Sci USA, of TNG1 and TNG2 to those of TCL1 and especially TCL1b, 95:3885-3889). and the physical linkage of these genes, the activation of the 0075 Since the original three sets of ESTs all derived TNG genes by rearrangements at 14q32.1 was investigated. from a 2 cell embryonic cDNA library where they make up FIG. 6A demonstrates that all four genes show an identical ~0.5% of the total ESTs, cDNA from mouse embryonic stem expression pattern in lymphoid tumor cell lines. They are all (ES) cells, oocytes, and 2 cell embryos was investigated for expressed in early B-tumor cell lines (697, Raji, Daudi, and the expression of Tcl1b genes. After a single round of CA-46), but not in postthymic T-cell lines without 14q32.1 RT-PCR, expression of all five Tcl1b genes and Tcl 1 was rearrangements. Nevertheless, TCL1, TCL1b, TNG1, and found in mouse oocytes and 2 cell embryoS at a level TNG2 are all transcribed in the T-ALL cell line SupT11 comparable to that of B-actin expression (FIG.8C). In 2 cell carrying a t14,14)(q11;q32) translocation. Northern blot embryos, both splicing variants of Tcl 1b1 were amplified. experiments confirmed these transcription patterns (FIG. 7). Interestingly, in the mouse oocyte cDNA library only a The 1.5 kb transcript of TNG1 was found in Burkitt's Shorter transcript of Tcl1 was detected, missing a part of lymphoma cell lines Daudi and CA-46 and to a lesser extent exon 2. Only Tcl1 showed expression in ES cells after a also in the Raji cell line and in the T-cell acute lymphocytic single round of PCR, but nested PCR revealed a low level leukemia cell line SupT11 (T-ALL) that carries a 14q32.1 of expression also of Tcl1b1, Tcl 1b2, and Tcl1b4. The high translocation. The Second band, a -2.3 kb transcript is likely expression of all five Tcl 1b genes and Tcl1 in mouse oocytes to be a product of alternative Splicing or incompletly pro and 2 cell embryos implies that an important function of cessed hnRNA. However, activation of TNG2 in the SupT11 these genes occurs in the early embryogenesis of the mouse. cell line was not confirmed by Northern blotting, due to a 0076) Protein Structure of TCL1 Family. lower expression level. The 2 kb TNG2 transcript was 0077 Tcl1 and Mtcp1 proteins both consist of an eight detected in all three Burkitt's lymphoma cell lines, but not Stranded antiparallel B-barrel with a hydrophobic core and in the pre B-cell line 697 or in any of the T-cell lines are predicted to bind Small hydrophobic ligands (Fu, Z. Q., investigated. The diffuse signal around the bands is due to et al., 1998, Proc Natl AcadSci USA, 95:3413-3418). Amino the various alternative splicing products known to involve acid Sequence alignment of these proteins with human and these gene. mouse Tcl1b (FIG. 9) shows that, despite only an overall 0072) To further study the activation of TNG1 and TNG2 30-40% homology, all 14 amino acids forming the hydro by rearrangements at 14q32.1 the expression of TNG1 and phobic core are conserved except Pro36. 10 of these 14 TNG2 was investigated in four T-cell prolymphocytic leu amino acids are identical in all 10 members of the Tcl1 kemias (T-PLL) overexpressing TCL1. FIG. 6C shows the family, whereas three residues show conservative Substitu activation of both genes in 2 out of 4 cases. The transcripts tions in some of the proteins (Leu49->Val, Leu92->Ile, of TNG1 and TNG2 were detected after 27 cycles of PCR Met104->Leu). Therefore, those residues have an important in these two cases even though at these conditions bone function in all Tcl1 family members. marrow and peripheral blood lymphocytes were negative. 0078 Human Tel1b (SEQ. ID. NO: 39) shows a 14 Interestingly, activation of TCL1b in one of the two cases residue insertion (Arg44-Glu58) relative to human TC11 not expressing the TNG genes???(Pekarsky, Y., et al., 1999, (FIG. 9). Mouse Tcl1b has a smaller, 10-11 residue insertion Proc Nail Acad Sci USA, 96:2949-2951) was previously in the same position. A molecular model was built for human found. These results indicate that juxtaposition of the TCL1 and murine Tcl1b based on the 35% similarity in amino acid locus at 14q32.1 to the C/6 locus of the T-cell receptor Sequence to Tcl1. In this model, the Tcl 1b insertion aligns activates TNG1 and TNG2, as well as TCL1 and TCL1b. with a non-canonical, 5 residue turn (Lys42-Gln46) observed in the crystal structure of human Tcl 1 (Hoh, F., et 0073 Expression of Murine Tcl 1b Genes. al., 1998, Structure, 6:147-155). The additional residues in 0.074 To investigate the expression pattern of the murine human and mouse Tcl1b may form a Surface accessible Tcl1b genes a series of RT-PCR experiments was carried out beta-sheet extension or a flexible loop with conserved for each of the five genes. After a single round of PCR no charged amino acids (FIG. 10). mRNA expression was found in a Series of normal tissue, 0079 Discussion embryonic cDNA libraries, and lymphoid cell lines for any 0080. The present invention discloses the cloning, map of the five genes. However, nested PCR analysis revealed a ping and expression analysis to of a novel member of the US 2005/0287530 A1 Dec. 29, 2005

TCL1 gene family, TCL1b. The TCL1 and TCL1b genes are but it remains to be determined whether they all code for physically linked, show Structural Similarity, Similar expres active proteins or whether Some of them might be pseudo Sion patterns and involvement in T-cell malignancies. genes. Their genomic Structure, though, is untypical for Because the remaining two members of the TCL1 family are pseudogenes, Since it includes introns. oncogenes (Virgilio, L., et al., 1998, Proc Natl Acad Sci USA, 95:3885-3889; Gritti, C., et al., 1998, Blood, 92:368 0086 The five Tcl1b genes show different expression 373), it seems likely that TCL1b is also an oncogene. It is patterns, Suggesting different regulatory elements for each of also likely that TCL1b activation would explain cases of them, but Since the expression of all of the genes in all adult T-cell leukemia with amplification at 14q32 without activa tissues and cell lines is very low, the Significance of this is tion of TCL1. not clear. Interestingly, the expression of murine Tcl1 and Tcl1b genes in lymphoid tissues and cell lines is much lower 0081. It is possible that two TCL1 genes are the result of than the expression of their human homologues. The most duplication, although the TCL1b gene is slightly more striking feature of murine Tcl 1 and Tcl 1b genes is their very homologous to the MTCP1 gene at Xq28 than to the TCL1 high expression level (up to 0.5% of all mRNA) in mouse gene. oocytes and 2-cell embryos. This finding is consistent with 0082 Neither the in vivo function of Tcl 1, nor the mecha the presence of human TCL1b in a syncytiotrophoblast nism(s) of its oncogenic potential is known, although its subtracted cDNA library (genebank accession # AF137027), crystal structure (Fu, Z. Q., et al., 1998, Proc Natl Acad Sci implying a function of murine and human TCL1b genes in USA, 95:3413-3418) suggests, it may function as a trans the early embryogenesis. porter of Small molecules, Such as retinoids, nucleosides or 0087. The identification of five more murine members of fatty acids. The same study (Fu, Z. Q., et al., 1998, Proc Natl the Tcl1 family provides a better understanding of the Acad Sci USA, 95:3413-3418). Suggested that Tcl1 might structural differences and similarities between the Tcl1 fam function as dimer, implying the possibility that Tcl1 and ily of proteins. A comparison of the protein Sequences of all Tcl1b might form heterodimers. members of the family including murine and human MTCP1 0083. Since TCL1 and MTCP1 transgenic mice develop shows that, although overall homologies between the genes mature T-cell leukemia only after 15 months (Virgilio, L., et are low, the hydrophobic core region as described by Fu et al., 1998, Proc Natl AcadSci USA, 95:3885-3889; Gritti, C., al. (14) is preserved. This indicates a similar function for all et al., 1998, Blood, 92:368-373), it will be of considerable of these proteins as transporters of Small molecules Such as interest to determine whether TCL1b transgenic mice also retinoids, nucleosides, or fatty acids as Suggested previously develop mature T-cell leukemia late and whether TCL1 and for Tcl 1 and Mtcp1 (Hoh, F., et al., 1998, Structure, 6:147 TCL1b double transgenic mice develop leukemia faster. 155; Fu, Z. O., et al., 1998, Proc Natl Acad Sci USA, Thus, is Seems possible that translocations and inversions at 95:3413-3418). However, compared to Tcl1 and Mtcp1 14q32.1 contribute to malignant transformation by activat mouse and human Tcl 1b proteins show an insertion which ing two oncogenes at the same time. form a Surface accessible flexible loop or beta-sheet eXten Sion. The conserved charged residues in the insert loop play 0084. The present invention discloses the cloning, map a significant role in mediating interactions with other pro ping, and expression analysis of the human and murine teins or ligands and also influence the quaternary Structure of TCL1/Tcl1 locus. Human TCL1 and TCL1b genes (SEQ. mouse Tcl 1b (Hoh, F, et al., 1998, Structure, 6:147-155). ID. NO: 40); are located between two clusters of chromo Somal breakpoints and are activated by translocations and 0088 Altogether, murine and human TCL1 loci show inversions at 14q32.1 juxtaposing them to regulatory ele Significant differences: There are five murine Tcl 1b genes ments of T-cell receptor genes (Pekarsky, Y., et al., 1999, compared to one human TCL1b. The homology of human Proc Natl AcadSci USA, 96:2949-2951). Between these two and mouse TCL1b is low and the expression levels of Sets of breakpoints two new genes were found and charac murine Tcl 1 and Tcl 1b in lymphoid tissues and cell lines is terized, TNG1 and TNG2 (SEQ. ID. NO: 45 and 46, much lower than the expression levels of their human respectively). Both show no homology to any known genes, equivalents. Moreover, murine homologues of TNG1 and but similar expression patterns to that of TCL1 and TCL1b. TNG2 were not found. This implies that there are also be Both TNG genes are also activated in the T-cell leukemia Significant differences in the function of human and mouse cell line SupT11 carrying at 14,14) translocation, and in two TCL1 loci. Further investigation should lead to a better out of four T-PLL samples. Therefore, like TCL1 and understanding of the role of Tcl 1 and Tcl1b in normal TCL1b, these two genes are also activated by rearrange development and T cell leukemia. ments at 14q32.1 involved in T-cell malignancies. Thus, 0089. The present invention relates to nucleotide T-cell leukemias, in Some cases, are induced by the activa sequences of TCL-1b (SEQ. ID. NO: 40); TNG1 (SEQ. ID. tion of a Single gene or in others by the cumulative activation NO: 45); and TNG2 (SEQ. ID. NO: 46); genes and amino of two or more of these four genes, although the oncogenic acid sequences of their encoded Tcl-1b (SEQ. ID. NO:39);, potential of TNG1 and TNG2 remains to be determined. TNG1 (SEQ. ID. NO: 42); and TNG2 (SEQ. ID. NO: 44), 0085 To assist in the structural and functional analysis of respectively, proteins, as well as derivatives and analogs the human TCL1 gene activation, the murine Tcl 1 locus was thereof, and antibodies thereto. The present invention further searched for homologues to human TCL1b and TNG genes. relates to the use of TCL-1b, TNG1 and TNG2 genes and Five genes homologous to human TCL1b were found. The their encoded proteins or derivatives or analogs thereof, and high shared similarity between the five murine Tcl 1b genes antibodies thereto, in assays for the detection and in treat (SEQ. ID. NO: 52-56), not only in the exons but also in ment/prevention of disease States associated with chromo intronic Sequences, implies that they are most likely a result Somal abnormalities and/or increased expression of TCL1b, of duplications. All five genes are transcribed into mRNA TNG1 and TNG2. The present invention also relates to US 2005/0287530 A1 Dec. 29, 2005 therapeutic compositions comprising Tcl-1b, TNG1 and expression, however, was detected in most hematopoietic TNG2, proteins, derivatives or analogs thereof, antibodies tissues and both TCL1 and TCL1b are expressed in spleen, thereto, nucleic acids encoding these proteins, derivatives or tonsil, fetal liver, fetal kidney and fetal thymus. The TCL1b analogs, and antisense nucleic acids. gene (SEQ. ID. NO:39) is expressed in a wider variety of 0090 The TCL-1b, TNG1 and TNG2 gene sequences are tissues including placenta, kidney and fetal Spleen, as shown from one of many different species, including but not limited in FIG. 8A. Low levels of expression of TNG1 and TNG2 to, mammalian, bovine, Ovine, porcine, equine, rodent and were present in most tissues examined, in addition to those human, in naturally occurring Sequence or in variant form, for TCL1b, expression in fetal lung, fetal heart and fetal or from any Source, whether natural, Synthetic, or recombi liver. The only exception is the thymus, which showed nant. In a specific embodiment described herein, the TCL transcripts of TNG2, whereas fetal thymus only expressed 1b, TNG1 and TNG2 gene Sequence are human Sequences. TNG1 (FIG. 6B). The detection of TCL-1b, TNG1 and The Tcl-1b, Tng1 and Tng1 proteins are those present in one TNG2 mRNA in patient samples, such as biopsied cells and of many different species, including but not limited to, tissues, is used as an indicator of the presence of T-cell mammalian, bovine, Ovine, porcine, equine, rodent and leukemias and lymphomas associated with certain chromo human, in naturally occurring or variant form, or from any Some 14 abnormalities and/or increased expression of Tcl Source, whether natural, Synthetic, or recombinant. In the 1b, Tng1 or Tng2 proteins. Also, the Tcl-1b, Tng 1 or Tng2 Specific embodiment described herein, the above proteins amino acid Sequences of the present invention (SEQ. ID. are human proteins. NO: 39, 42, and 44, respectively), are used to generate 0.091 AS defined herein, a Tcl-1b, Tng1 and Tng2 deriva antibodies useful in immunoassays for the detection or tive is a fragment or amino acid variant of the Tcl-1b, Tng 1 measurement of Tcl-1b, Tng 1 or Tng2 proteins in patient and Tng2 sequence (SEQ. ID. NO:39, 42, and 44), respec Samples, respectively. Such antibodies are used in diagnostic tively, as long as the fragment or amino acid variant is immunoassays, for the detection or measurement of capable of displaying one or more biological activities increased levels of Tcl-1b, Tng1 or Tng2 proteins, respec asSociated with the full-length proteins. Such biological tively, associated with T-cell leukemias and lymphomas. activities include, but are not limited to, antigenicity, i.e., the ability to bind to an their respective antibodies, and immu 0094. In accordance with the present invention, poly nogenicity, i.e., the ability to generate an antibody which is nucleotide Sequences coding for a Tcl-1b, Tng1 or Tng2 capable able of binding a Tcl-1b, Tng1 or Tng2 protein, proteins (SEQ. ID. NO: 38, 41, and 43), derivatives, e.g. respectively. fragment, or analog thereof, are inserted into an appropriate expression vector, i.e., a vector which contains the necessary 0092. The invention provides fragments of a Tcl-1b, elements for the transcription and translation of the inserted Tng1 or Tng2 protein consisting of at least 10 amino acids, protein-coding Sequence, for the generation of recombinant or of at least 25 amino acids, or of at least 50 amino acids, DNA molecules that direct the expression of a Tcl-1b, Tng 1 or of at least 114 amino acids. Nucleic acids encoding Such or Tng2 proteins. Such Tcl-1b, Tng 1 or Tng2 polynucleotide derivatives or analogs are also within the Scope of the Sequences, as well as other polynucleotides or their comple invention. A preferred Tcl-1b, Tng1 or Tng2 protein variant ments, are also used in nucleic acid hybridization assays, is one sharing at least 70% amino acid Sequence homology, Southern and Northern blot analysis, etc. In a specific a particularly preferred Tcl-1b, Tng 1 or Tng2 protein variant embodiment, a human TCL-1b, TNG1 or TNG2 gene (SEQ. is one Sharing at least 80% amino acid Sequence homology ID. NO: 40, 45, and 46, respectively), or a sequence encod and another particularly preferred Tcl-1b, Tng1 or Tng2 ing a functionally active portion of a human TCL-1b, TNG1 protein variant is one sharing at least 90% amino acid or TNG2 gene, is expressed. In yet another embodiment, a Sequence homology to the naturally occurring Tcl-b, Tng1 or derivative or fragment of a human TCL-1b, TNG1 or TNG2 Tng2 protein over at least 25, at least 50, at least 75 or at gene is expressed. least 100 contiguous amino acids of the Tcl-1b, Tng 1 or Tng2 amino acid Sequence, respectively. AS used herein, 0.095 The TCL-1b, TNG1 and TNG2 Coding Sequences amino acid Sequence homology refers to amino acid Sequences having identical amino acid residues or amino 0096. In a specific embodiment disclosed herein the acid Sequences containing conservative changes in amino invention relates to the nucleic acid Sequence of the human acid residues. In another embodiment, a Tcl-1b, Tng 1 or TCL-1b, TNG1 and TNG2 genes (SEQ. ID. NO:40, 45, and Tng2 homologous protein is one that shares the foregoing 46, respectively). In a preferred, but not limiting, aspect of percentages of Sequences identical with the naturally occur the invention, a human TCL-1b cDNA sequence (SEQ. ID. ring Tcl-1b, Tng 1 or Tng2 protein, respectively, over the NO: 38) was identified in the expressed sequence tag cited lengths of amino acids. database (accession no. AA689513) that was homologous to TCL-1 and MTCP1, the other members of the TCL1 gene 0093. The TCL-b1, TNG1 and TNG2 genes (SEQ. ID. family. Such a Sequence was isolated and cloned as a 1.2 NO: 40, 45, and 46, respectively), are located in the region kilobase full-length cDNA, as described, Supra. The TNG1 of chromosome 14q32.1 that is located in a region banded by and TNG2 genes were also isolated and identified and their two clusters of breakpoints. Due to the similarities between Sequences compared to the expressed Sequence database the TCL1 and TCL-1b gene Structure, Sequence and loca (EST). The 1.5 kilobase cDNA of TNG1 (SEQ. ID. NO:41) tion, their expression patterns were compared. In addition contains an open reading frame encoding a protein of 141 the expression patterns of TNG1 and TNG2, which are amino acids and the 2 kilobase TNG2 gene (SEQ. ID. NO: located at the same locus as that of TCL1 and TCL-1b, were 43) encodes a protein of 110 amino acids, as described, investigated (FIG. 7). Expression in normal tissue was Supra. The invention also relates to nucleic acid Sequences mostly negative for TCL1b, FIG. 7A. The TCL1 gene hybridizable or complementary to the foregoing Sequences, US 2005/0287530 A1 Dec. 29, 2005

of equivalent to the foregoing Sequences, in that the equiva encode the desired gene. The DNA is cleaved at Specific Sites lent nucleic acid Sequences also encode a Tcl-1b, Tng 1 or using various restriction enzymes. Alternatively, DNASe in Tng2 protein product. the presence of manganese is used to fragment the DNA, or the DNA is physically sheared, as for example, by Sonica 0097. In a preferred aspect, polymerase chain reaction tion. The linear DNA fragments is then Separated according (PCR) is used to amplify the desired nucleic acid sequence to Size by Standard techniques, including but not limited to, in the library by using oligonucleotide primerS representing agarose and polyacrylamide gel electrophoresis and column known TCL-1b, TNG1 or TNG2 sequences (SEQ. ID. NO: chromatography. 38, 41, and 43, respectively). Such primers are used to amplify sequences of interest from an RNA or DNA source, 0101. Once the DNA fragments are generated, identifi preferably a cDNA library. PCR is carried out by use of a cation of the Specific DNA fragment containing the desired Perkin-Elmer Cetus thermal cycler and Taq polymerase, as gene is accomplished in a number of ways. For example, a is well known by those skilled in the art. The DNA being TCL-1b, TNG1 or TNG2 gene (SEQ. ID. NO: 40, 45, and amplified is mRNA or cDNA or genomic DNA from any 46, respectively) of the present invention or its specific eukaryotic Species. Several different degenerate primers are RNA, or a fragment thereof, Such as a probe or primer, is synthesized for use in PCR amplification reactions. The isolated and labeled and then used in hybridization assays to Stringency of hybridization conditions used in priming the detect a generated TCL-1, TNG1 or TNG2 gene (Benton, W. PCR reactions are also varied in order to allow for greater or and Davis, R., 1977, Science, 196:180; Grunstein, M. And lesser degrees of nucleotide Sequence homology between the Hogness, D., 1975, Proc Natl Acad Sci USA, 72:3961). TCL-1b, TNG1 or TNG2 gene being cloned and that of the Those DNA fragments sharing Substantial Sequence homol TCL-1b, TNG1 or TNG2 genes (SEQ. ID. NO: 40, 45, and ogy to the probe will hybridize under Stringent conditions. 46, respectively) of the present invention. The phrase “stringent conditions' as used herein refers to those hybridizing conditions that (Virgilio, L., et al., 1994, 0.098 After successful amplification of a segment of the Proc Natl Acad Sci USA, 91:12530-12534) employ low TCL-1b, TNG1 or TNG2 gene, an allelic, a polymorphic ionic Strength and high temperature for Washing, for variant, or a species homology of the TCL-1b, TNG1 or example, 0.015 M NaCl/0.0015M sodium citrate/0.1% SDS TNG2 gene, that Segment is molecularly cloned and at 50...degree. C., (Narducci, M. G., et al., 1997, Cancer Res, Sequenced, and utilized as a probe to isolate a complete 57:5452-5456) employ, during hybridization, a denaturing cDNA or genomic clone. This will permit the determination agent such as formamide, for example, 50% (vol/vol) for of the gene's complete nucleotide Sequence, the analysis of mamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% its expression, and the production of its protein product for polyvinylpyrrolidone/50 mMSodium phosphate buffer at pH functional analysis. This allows for the identification of 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42.degree. additional genes encoding the Tcl-1b, Tng1 or Tng2, respec C.; or (Virgilio, L., et al., 1998, Proc Natl AcadSci USA, tively, proteins. 95:3885-3889) employ 50% formamide, 5.times.SSC (0.75 0099 Potentially, any eukaryotic cell can serve as the M NaCl, 0.075 M Sodium pyrophosphate, 5.times. Den nucleic acid Source for the molecular cloning of the TCL-1b, hardt’s solution, Sonicated salmon sperm DNA (50 g/ml), TNG1 or TNG2 gene. The nucleic acid sequences encoding 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with TCL-1b, TNG1 or TNG2 gene are isolated from, for washes at 42.degree. C. in 0.2. times.SSC and 0.1% SDS. example, human, porcine, boVine, feline, avian, equine, 0102) The appropriate fragment is also identified by canine, rodent, as well as additional primate Sources. The restriction enzyme digestion(s) and comparison of fragment DNA is obtained by standard procedures known in the art sizes with those expected according to a known restriction from, for example, cloned DNA (e.g., a DNA “library”), by map. Further Selection is carried out on the basis of the chemical Synthesis, by cDNA cloning, or by the cloning of properties of the gene. Alternatively, the presence of the genomic DNA, or fragments thereof, purified from a desired gene is detected by assays based on the physical, chemical, cell. (See, for example, Sambrook et al., 1989, Molecular or immunological properties of its expressed product. For Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor example, cDNA clones, or genomic DNA clones which Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. hybrid-select the proper mRNAS, are selected which pro (ed.), 1985, DNA Cloning: A Practical Approach, MRL duce a protein that has similar or identical electrophoretic Press, Ltd., Oxford, U.K. Vol. I, II.) A preferred source is migration, isolectric focusing behavior, proteolytic digestion cDNA of leukemic cells in which the leukemia is associated maps, binding activity or antigenic properties as known for with a 14q32.1 chromosomal abnormality. Clones derived Tcl-1b. Alternatively, the Tcl-1b protein may be identified by from genomic DNA contain regulatory and intron DNA binding of labeled antibody to the putatively Tcl-1b express regions in addition to coding regions, while clones derived ing clones, e.g., in an ELISA (enzyme-linked immunosor from cDNA will contain only TCL-1b exon sequences. In a bent assay)-type procedure. particular embodiment of the present invention, a genomic Sequence is one that is not more than 10 kilobases (kb), or 0103) The TCL-1b, TNG1 or TNG2 gene is also identi not more than 20 kb, or not more than 50 kb or not more than fied by mRNA selection by nucleic acid hybridization fol 70 kb. Whatever the source, the gene should be molecularly lowed by in vitro translation. In this procedure, fragments cloned into a Suitable vector for propagation of the gene. In are used to isolate complementary mRNAS by hybridization. a particular embodiment, a preferred Source of nucleic acid Such DNA fragments may represent available, purified for the isolation of TCL-1b, TNG1 or TNG2 gene sequences TCL-1b, TNG1 or TNG2 DNA of another TCL-1b, TNG1 or is from pre B-cells. TNG2 gene, respectively. Immunoprecipitation analysis, or functional assays, of the in vitro translation products of the 0100. In the molecular cloning of the gene from genomic isolated products of the isolated mRNAS identifies the DNA, DNA fragments are generated, some of which will mRNA and, therefore, the complementary DNA fragments US 2005/0287530 A1 Dec. 29, 2005 that contain the desired Sequences. In addition, Specific Synthesized by Standard methods commonly known in the mRNAS are selected by adsorption of polysomes isolated art. Such oligonucleotides preferably have a size in the range from cells to immobilized antibodies specifically directed of 8 to 25 nucleotides. In a particular embodiment herein, against Tcl-1b, Tng 1 or Tng2 protein. A radiolabelled TCL Such oligonucleotides have a size in the range of 15 to 25 1b, TNG1 or TNG2 cDNA is synthesized using the selected nucleotides or 18 to 25 nucleotides. mRNA (from the adsorbed polysomes) as a template. The radiolabeled mRNA or cDNA is then used as a probe to 0109 Expression of the TCL-1b, TNG1 or TNG2 Gene identify the TCL-1b, TNG1 or TNG2 DNA fragments, 0110. In accordance with the present invention, poly respectively, from among other genomic DNA fragments. nucleotide Sequences coding for a Tcl-1b, Tng1 or Tng2 0104. Alternatives to isolating the TCL-1b, TNG1 or protein, derivative, e.g. fragment, or analog thereof, can be TNG2 genomic DNA include, but are not limited to, chemi inserted into an appropriate expression vector, i.e., a vector cally Synthesizing the gene Sequence itself from a known which contains the necessary elements for the transcription sequence or making cDNA to the mRNA which encodes the and translation of the inserted protein-coding Sequence, for Tcl-1b, Tng1 or Tng2, respectively, protein. For example, the generation of recombinant DNA molecules that direct RNA useful in cDNA cloning of the TCL-1b, TNG1 or the expression of a Tcl-1b, Tng1 or Tng2 protein. Such TNG2 gene is isolated from cells which express Tcl-1b, TCL-1b, TNG1 or TNG2, respectively, polynucleotide Tng1 or Tng2, respectively, e.g., pre-B acute lymphoblastic Sequences, as well as other polynucleotides or their comple leukemia cells or endemic Burkitt's lymphoma cells which ments, may also be used in nucleic acid hybridization express cell Surface IgM and do not Secrete immunoglobu assays, Southern and Northern blot analysis, etc. In a spe lin. Other methods are known to those of skill in the art and cific embodiment, a human TCL-1b, TNG1 or TNG2 gene, are within the Scope of the invention. or a Sequence encoding a functionally active portion of a human TCL-1b, TNG1 or TNG2 gene is expressed. In yet 0105 The identified and isolated gene is then inserted another embodiment, a derivative or fragment of a human into an appropriate cloning vector. A large number of TCL-1b, TNG1 or TNG2 gene is expressed. vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified 0111. Due to the inherent degeneracy of the genetic code, viruses, but the vector system must be compatible with the other DNA sequences which encode Substantially the same host cell used. Such vectors include, but are not limited to, or a functionally equivalent Tcl-1b amino acid Sequence, is bacteriophages Such as lambda derivatives, or plasmids Such within the scope of the invention. Such DNA sequences as PBR322 or puC plasmid derivatives. The insertion into a include those which are capable of hybridizing to the human cloning vector can, for example, be accomplished by ligat TCL-1b, TNG1 or TNG2 sequence under stringent condi ing the DNA fragment into a cloning vector which has tions. complementary cohesive termini. However, if the comple 0112 Altered DNA sequences which are used in accor mentary restriction sites used to fragment the DNA are not dance with the invention include deletions, additions or present in the cloning vector, the ends of the DNA molecules Substitutions of different nucleotide residues resulting in a may be enzymatically modified. Alternatively, any site Sequence that encodes the same or a functionally equivalent desired may be produced by ligating nucleotide Sequences gene product. The gene product itself may contain deletions, (linkers) onto the DNA termini; these ligated linkers com additions or Substitutions of amino acid residues within a prise Specific chemically Synthesized oligonucleotides TCL-1b, TNG1 or TNG2 sequence, which result in a silent encoding restriction endonuclease recognition Sequences. In change, thus producing a functionally equivalent Tcl-1b, an alternative method, the cleaved vector and TCL-1b, Tng1 or Tng2, respectively, protein. Such amino acid Sub TNG1 or TNG2 gene is modified by homopolymeric tailing. Stitutions are made on the basis of Similarity in polarity, Recombinant molecules are introduced into host cells via charge, Solubility, hydrophobicity, hydrophilicity, and/or the transformation, transfection, infection, electroporation, or amphipathic nature of the residues involved. For example, other methods known to those of skill in the art, So that many negatively charged amino acids include aspartic acid and copies of the gene Sequence are generated. glutamic acid; positively charged amino acids include lysine 0106. In an alternative method, the desired gene is iden and arginine; amino acids with uncharged polar head groups tified and isolated after insertion into a Suitable cloning having Similar hydrophilicity values include the following: vector in a “shotgun' approach. Enrichment for the desired leucine, isoleucine, Valine, glycine, alanine; asparagine, gene, for example, by Size fractionization, is done before glutamine, Serine, threonine, phenylalanine, tyrosine. insertion into the cloning vector. 0113. The DNA sequences of the invention are engi 0107. In specific embodiments, transformation of host neered in order to alter a TCL-1b, TNG1 or TNG2 coding cells with recombinant DNA molecules that incorporate the Sequence for a variety of ends, including but not limited to isolated TCL-1b, TNG1 or TNG2 gene, cDNA, or synthe alterations which modify processing and expression of the sized DNA sequence enables generation of multiple copies gene product. For example, mutations introduced using of the gene. Thus, the gene is obtained in large quantities by techniques which are well known in the art, e.g., Site growing transformants, isolating the recombinant DNA mol directed mutagenesis, to insert new restriction sites, to alter ecules from the transformants and, when necessary, retriev phosphorylation, etc. ing the inserted gene from the isolated recombinant DNA. 0114. In another embodiment of the invention, a TCL-1b, 0108 Oligonucleotides containing a portion of the TCL TNG1 or TNG2 gene sequence or a derivative thereof is 1b, TNG1 or TNG2 coding or non-coding sequences, or ligated to a non-TCL-1b, non-TNG1 or non-TNG2 gene which encode a portion of the Tcl-1b, Tng 1 or Tng2, Sequence to encode a chimeric fusion protein. A fusion respectively, protein (e.g., primers for use in PCR) are protein is engineered to contain a cleavage Site located US 2005/0287530 A1 Dec. 29, 2005

between a Tcl-1b, Tng 1 or Tng2, respectively, Sequence and isms. Such as bacteria transformed with recombinant bacte the non-Tcl-1b, non-Tng1 or non-Tng2, respectively, protein riophage DNA, plasmid DNA or cosmid DNA expression Sequence, So that the Tcl-1b, Tng1 or Tng2 protein, respec vectors containing a TCL-1b, TNG1 or TNG2 coding tively may be cleaved away from the non-Tcl-1b, non-Tng 1 Sequence; yeast transformed with recombinant yeast expres or non-Tng2, respectively, moiety. In a specific embodiment, sion vectors containing a TCL-1b, TNG1 or TNG2 coding the Tcl-1b, non-Tng1 or non-Tng2, respectively, amino acid Sequence; insect cell Systems infected with recombinant Sequence present in the fusion protein consists of at least 10 virus expression vectors (e.g., baculovirus) containing an contiguous amino acids, at least 25 contiguous amino acids, TCL-1b, TNG1 or TNG2 coding sequence; plant cell sys at least 50 contiguous amino acids, at least 75 contiguous tems infected with recombinant virus expression vectors amino acids, at least 100 contiguous amino acids, or at least (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic 114 amino acids of the Tcl-1b, non-Tng1 or non-Tng2, virus, TMV) or transformed with recombinant plasmid protein Sequence. expression vectors (e.g., Tiplasmid) containing a TCL-1b, TNG1 or TNG2 coding sequence; or animal cell systems. 0115) In an alternate embodiment of the invention, the The expression elements of these Systems vary in their coding Sequence of a Tcl-1b, Tng 1 or Tng2, is Synthesized Strength and Specificities. Depending on the host/vector in whole or in part, using chemical methods well known in System utilized, any of a number of Suitable transcription the art. See, for example, Caruthers et al., 1980, Nuc. Acids and translation elements, including constitutive and induc Res. Symp. Ser. 7:215-233; Crea and Horn, 1980, Nuc. ible promoters, are used in the expression vector. For Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, example, when cloning in bacterial Systems, inducible pro Tetrahedron Letters 21:719; and Chow and Kempe, 1981, moterS Such as pil of bacteriophage lambda, plac, ptrp, ptac Nuc. Acids Res. 9(12):2807-2817. Alternatively, the protein (ptrp-lac hybrid promoter) and the like are used; when itself is produced using chemical methods to Synthesize a cloning in insect cell Systems, promoterS Such as the bacu Tcl-1b, Tng1 or Tng2 amino acid Sequence in whole or in lovirus polyhedrin promoter are used; when cloning in plant part. For example, peptides are Synthesized by Solid phase cell Systems, promoters derived from the genome of plant techniques, cleaved from the resin, and purified by prepara cells (e.g., heat shock promoters; the promoter for the Small tive high performance liquid chromatography. (e.g., See subunit of RUBISCO; the promoter for the chlorophyll a?b Creighton, 1983, Proteins Structures And Molecular Prin binding protein) or from plant viruses (e.g., the 355 RNA ciples, W. H. Freeman and Co., N.Y. pp. 50-60). The promoter of CaMV; the coat protein promoter of TMV) are composition of the Synthetic peptides may be confirmed by used; when cloning in mammalian cell Systems, promoters amino acid analysis or sequencing (e.g., the Edman degra derived from the genome of mammalian cells (e.g., metal dation procedure; see Creighton, 1983, Proteins, Structures lothionein promoter) or from mammalian viruses (e.g., the and Molecular Principles, W. H. Freeman and Co., N.Y., pp. adenovirus late promoter; the vaccinia virus 7.5 K promoter) 34-49. are used; when generating cell lines that contain multiple 0116. In order to express a biologically active Tcl-1b, copies of a TCL-1b, TNG1 or TNG2 DNA, SV40-, BPV Tng1 or Tng2 protein or derivative thereof, a polynucleotide and EBV-based vectors are used with an appropriate Select Sequence encoding a Tcl-1b, Tng 1 or Tng2, resepectively, able marker. protein, or a derivative thereof, is inserted into an appropri 0119). In bacterial systems, a number of expression vec ate expression vector, i.e., a vector which contains the tors are advantageously Selected depending upon the use necessary elements for the transcription and translation of intended for the Tcl-1b, Tng1 or Tng2 protein expressed. For the inserted coding sequence. The TCL-1b, TNG1 or TNG2 example, when large quantities of Tcl-1b, Tng1 or Tng2 gene products, as well as host cells or cell lines transfected protein are produced for the generation of antibodies, vec or transformed with recombinant TCL-1b, TNG1 or TNG2, tors which direct the expression of high levels of fusion respecitvely, expression vectors, are used for a variety of protein products that are readily purified are desirable. Such purposes. These include, but are not limited to, generating vectors include, but are not limited to, the E. coli expression antibodies (i.e., monoclonal or polyclonal) that immunospe vector pUR278 (Ruther, et al., 1983, EMBOJ, 2:1791), in cifically bind a Tcl-1b protein. Anti-Tcl-1b, antig-Ting1 or which the TCL-1b, TNG1 or TNG2 coding sequence are anti-Tng2 antibodies are used in detecting or measuring ligated into the vector in frame with the lac Z coding region levels of a Tcl-1b, Tng 1 or Tng2, respectively, protein in so that a hybrid AS-lac Z protein is produced; plN vectors patient Samples. (Inouye & Inouye, 1985, Nucleic Acids Res, 13:3101-3109; Van Heeke & Schuster, 1989, J Biol Chem, 264:5503–5509); 0117 Methods which are well known to those skilled in and the like. pCEX vectors are also used to express foreign the art are used to construct expression vectors containing a polypeptides as fusion proteins with glutathione S-trans TCL-1b, TNG1 or TNG2 coding sequence and appropriate ferase (GST). In general, Such fusion proteins are soluble transcriptional/translational control Signals. These methods and easily purified from lysed cells by adsorption to glu include in vitro recombinant DNA techniques, synthetic tathione-agarose beads followed by elution in the presence techniques and in Vivo recombination/genetic recombina of free glutathione. The PGEX vectors are designed to tion. See, for example, the techniques described in Sam include thrombin or factor Xa protease cleavage Sites So that brook et al., 1989, Molecular Cloning, A Laboratory Manual the cloned polypeptide of interest is released from the GST 2d ed., Cold Spring Harbor Laboratory, N.Y. and Ausubel et moiety. al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. 0120 In yeast, a number of vectors containing constitu tive or inducible promoters are used. For a review See, 0118) A variety of host-expression vector systems are Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. utilized to express a TCL-1b, TNG1 or TNG2 coding Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Sequence. These include, but are not limited to, microorgan Ch. 13; Grant et al., 1987, Expression and Secretion Vectors US 2005/0287530 A1 Dec. 29, 2005

for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, coding Sequences. These signals include the ATG initiation 1987, Acad. Press, N.Y. 153:516-544; Glover, 1986, DNA codon and adjacent Sequences. In cases where an entire Cloning. Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, TCL-1b, TNG1 or TNG2 gene, including its own initiation 1987, Heterologous Gene Expression in Yeast, Methods in codon and adjacent Sequences, is inserted into the appropri Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. ate expression vector, no additional translational control 152:673-684; and The Molecular Biology of the Yeast Signals may be needed. However, in cases where only a Saccharomyces, 1982, Eds. Strathern et al., Cold Spring portion of a TCL-1b, TNG1 or TNG2 coding sequence is Harbor Press, Vols. I and II. inserted, lacking the 5' end, exogenous translational control 0121. In cases where plant expression vectors are used, Signals, including the ATG initiation codon, must be pro the expression of a TCL-1b, TNG1 or TNG2 coding Vided. Furthermore, the initiation codon must be in phase Sequence is driven by any of a number of promoters. For with the reading frame of a TCL-1b, TNG1 or TNG2 coding example, viral promoters such as the 35S RNA and 19S Sequence to ensure translation of the entire insert. These RNA promoters of CaMV (Brisson, et al., 1984, Nature, exogenous translational control Signals and initiation codons 310:511-514), or the coat protein promoter of TMV (Taka of are of a variety of origins, both natural and Synthetic. The matsu, et al., 1987, EMBOJ, 6:307-311) are used; alterna efficiency of expression are enhanced by the inclusion of tively, plant promoterS Such as the Small Subunit of appropriate transcription enhancer elements, transcription RUBISCO (Coruzzi, et al., 1984, EMBOJ, 3:1671-1680; terminators, etc. (see Bittner, et al., 1987, Methods in Broglie, et al., 1984, Science, 224:838-843); or heat shock Enzymol, 153:516-544). promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley, et 0.125. In addition, a host cell strain is chosen which al., 1986, Mol Cell Biol, 6:559-565) are used. These con modulates the expression of the inserted Sequences, or Structs are introduced into plant cells using Ti plasmids, Ri modifies and processes the gene product in the Specific plasmids, plant virus vectors, direct DNA transformation, fashion desired. Such modifications (e.g., phosphorylation) microinjection, electroporation, etc. For reviews of Such and processing (e.g., cleavage) of protein products may be techniquessee, for example, Weissbach & Weissbach, 1988, important for the function of the protein. Different host cells Methods for Plant Molecular Biology, Academic Press, have characteristic and Specific mechanisms for the post N.Y., Section VIII, pp. 421-463; and Grierson & Corey, translational processing and modification of proteins. 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Appropriate cells lines or host Systems are chosen to ensure Ch. 7-9. the correct modification and processing of the foreign pry 0122) An alternative expression system which could be expressed. To this end, eukaryotic host cells which possess used to express a TCL-1b, TNG1 or TNG2 gene is an insect the cellular machinery for proper processing of the primary ystem. In one Such System, Autographa Californica nuclear transcript, and phosphorylation of the gene product are used. polyhedrosis virus (AcNPV) is used as a vector to express Such mammalian host cells include but are not limited to foreign genes. The virus grows in Spodoptera frugiperda CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc. cells. ATCL-1b, TNG1 or TNG2 coding sequence is cloned 0.126 For long-term, high-yield production of recombi into non-essential regions (for example the polyhedrin gene) nant proteins, Stable expression is preferred. For example, of the virus and placed under control of an AcNPV promoter cell lines which stably express a Tcl-1b, Tng 1 or Tng2 (for example, the polyhedrin promoter). Successful insertion protein are engineered. Rather than using expression vectors of a TCL-1b, TNG1 or TNG2 coding sequence will result in which contain Viral origins of replication, host cells are inactivation of the polyhedrin gene and production of non transformed with TCL-1b, TNG1 or TNG2 DNA, respec occluded recombinant virus (i.e., virus lacking the proteina tively, controlled by appropriate expression control elements ceous coat coded for by the polyhedrin gene). These recom (e.g., promoter, enhancer, Sequences, transcription termina binant viruses are then used to infect Spodoptera frugiperda tors, polyadenylation sites, etc.), and a selectable marker. cells in which the inserted gene is expressed. (e.g., see Following the introduction of foreign DNA, engineered cells Smith, et al., 1983, J Virol, 46:584; Smith, U.S. Pat. No. are allowed to grow for 1-2 days in an enriched media, and 4,215,051). are then Switched to a selective media. The selectable 0123. In mammalian host cells, a number of viral based marker in the recombinant plasmid conferS resistance to the expression Systems are utilized. In cases where an adenovi Selection and allows cells to stably integrate the plasmid into rus is used as an expression vector, a TCL-1b, TNG1 or their chromosomes and grow to form foci which in turn are TNG2 coding Sequence is ligated to an adenovirus transcrip cloned and expanded into cell lines. This method is advan tion/translation control complex, e.g., the late promoter and tageously used to engineer cell lines which express a Tcl-1b, tripartite leader Sequence. This chimeric gene is then Tng1 or Tng2, respectively, protein. The present invention inserted in the adenovirus genome by in vitro or in vivo provides a method for producing a recombinant Tcl-1b, recombination. Insertion in a non-essential region of the Tng1 or Tng2 protein comprising culturing a host cell Viral genome (e.g., region E1 or E3) will result in a recom transformed with a recombinant expression vector encoding binant virus that is viable and capable for expressing a a Tcl-1b, Tng1 or Tng2, respectively, protein Such that the TCL-1b in infected hosts. (e.g., see Logan & Shenk, 1984, Tcl-1b, Tng1 or Tng2 protein is expressed by the cell and Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659). Alternatively, recovering the expressed Tcl-1b, Tng1 or Tng2 protein. the vaccinia 7.5 K promoter are used. (See, e.g., Mackett, et 0127. A number of selection systems are used, including, al., 1982, Proc Natl AcadSci USA, 79:7415-7419; Mackett, but not limited to, the herpes simplex virus thymidine kinase et al., 1984, J Virol, 49:857-864; Panicali, et al., 1982, Proc (Wigler, et al., 1977, Cell, 11:223), hypoxanthine-guanine Natl AcadSci USA, 79:4927-4931). phosphoribosyltransferase (Szybalska & Szybalski, 1962, 0.124 Specific initiation signals may also be required for Proc Natl AcadSci USA, 48:2026), and adenine phospho efficient translation of an inserted TCL-1b, TNG1 or TNG2 ribosyltransferase (Lowy, et al., 1980, Cell, 22:817) genes US 2005/0287530 A1 Dec. 29, 2005 can be employed in th:-, hgprt- or aprt-cells, respectively. 0133. In the fourth approach, the levels of a Tcl-1b, Tng 1 Also, antimetabolite resistance is used as the basis of Selec or Tng2 protein product is assessed immunologically, for tion for dhfr, which conferS resistance to methotrexate example by Western blots, immunoassayS. Such as radioim (Wigler, et al., 1980, Natl AcadSci USA, 77:3567; O'Hare, muno-precipitation, enzyme-linked immunoassays and the et al., 1981, Proc Natl AcadSci USA, 78:1527); gpt, which like. conferS resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc Natl AcadSci USA, 78.2072), neo, which confers 0.134 Purification of the Expressed Gene Product resistance to the aminoglycoside G-418 (Colberre-Garapin, 0135). Once a recombinant which expresses the TCL-1b, et al., 1981, J Mol Biol, 150: 1); and hygro, which confers TNG1 or TNG2 gene sequence is identified, the gene resistance to hygromycin (Santerre, et al., 1984, Gene, product is analyzed. This is achieved by assays based on the 30:147). Recently, additional selectable genes have been physical or functional properties of the product, including described, namely trp B, which allows cells to utilize indole radioactive labelling of the product followed by analysis by in place of tryptophan; hisD, which allows cells to utilize gel electrophoresis, immunoassay, or other detection meth histinol in place of histidine (Hartman & Mulligan, 1988, ods known to those of skill in the art. Proc Natl Aca. Sci USA, 85:8047); and ODC (ornithine decarboxylase) which conferS resistance to the ornithine 0.136. Once the Tcl-1b, Tng1 or Tng2 protein is identified, decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, it is isolated and purified by Standard methods including DFMO (McConlogue, L., 1987, In: Current Communica chromatography (e.g., ion exchange, affinity, and sizing tions in Molecular Biology, Cold Spring Harbor Laboratory, column chromatography), centrifugation, differential Solu Ed.). bility, or by any other Standard technique for the purification 0128 Identification of Transfectants or Transformants of proteins. The functional properties are evaluated using that Express Tcl-1b, Tng1 or Tng2 any Suitable assay. 0129. The host cells which contain the coding sequence 0.137 Alternatively, once a Tcl-1b, Tng 1 or Tng2 protein and which express the biologically active gene product are produced by a recombinant is identified, the amino acid identified by at least four general approaches; (a) DNA Sequence of the protein is deduced from the nucleotide DNA or DNA-RNA hybridization; (b) the presence or Sequence of the chimeric gene contained in the recombinant. absence of “marker gene functions; (c) assessing the level AS a result, the protein is Synthesized by Standard chemical of transcription as measured by the expression of TCL-1b, methods known in the art (e.g., see Hunkapiller, et al., 1984, TNG1 or TNG2 mRNA transcripts in the host cell; and (d) Nature, 310:105-111). detection of the gene product as measured by immunoassay or by its biological activity. 0.138. In a specific embodiment of the present invention, such Tcl-1b, Tng 1 or Tng2 proteins, whether produced by 0130. In the first approach, the presence of the TCL-1b, recombinant DNA techniques or by chemical synthetic TNG1 or TNG2 coding sequence inserted in the expression methods, include, but are not limited to, those containing, as vector is detected by DNA-DNA or DNA-RNA hybridiza a primary amino acid Sequence, all or part of the amino acid tion using probes comprising nucleotide Sequences that are sequence, substantially, as in SEQ. ID. NO:39, 42, and 44, homologous to the TCL-1b, TNG1 or TNG2 coding respectively, as well as fragments and other derivatives, and Sequence, respectively, or portions or derivatives thereof. analogs thereof. 0131. In the second approach, the recombinant expres Sion vector/host System is identified and Selected based upon 0.139 Generation of Antibodies to Tcl-1b, Tng1 or Tng2 the presence or absence of certain “marker gene functions (e.g., thymidine kinase activity, resistance to antibiotics, 0140. According to the invention, Tcl-1b, Tng 1 or Tng2 resistance to methotrexate, transformation phenotype, occlu protein, its fragments or other derivatives, or analogs Sion body formation in baculovirus, etc.). For example, if the thereof, are used as an immunogen to generate antibodies human TCL-1b, TNG1 or TNG2 coding sequence is inserted which recognize Such an immunogen. Such antibodies within a marker gene Sequence of the vector, recombinant include but are not limited to polyclonal, monoclonal, chi cells containing the TCL-1b, TNG1 or TNG2 coding meric, Single chain, Fab fragments, and an Fab expression Sequence are identified by the absence of the marker gene library. In a specific embodiment, antibodies to a human function. Alternatively, a marker gene is placed in tandem Tcl-1b, Tng1 or Tng2, respectively, protein are produced. with a TCL-1b, TNG1 or TNG2 sequence under the control 0141 Various procedures known in the art are used for of the same or different promoter used to control the the production of polyclonal antibodies to a Tcl-1b, Tng 1 or expression of the TCL-1b, TNG1 or TNG2 coding sequence. Tng2 protein or derivative or analog. For the production of Expression of the marker in response to induction or Selec antibody, various host animals are immunized by injection tion indicates expression of the TCL-1b, TNG1 or TNG2 with the native Tcl-1b, Tng 1 or Tng2 protein, or a synthetic coding Sequence. version, or derivative (e.g., fragment) thereof, including but 0.132. In the third approach, transcriptional activity of a not limited to rabbits, mice, rats, etc. Various adjuvants are TCL-1b, TNG1 or TNG2 gene is assessed by hybridization used to increase the immunological response, depending on assayS. For example, RNA is isolated and analyzed by the host species, and including but not limited to Freund's Northern blot using a probe having Sequence homology to a (complete and incomplete), mineral gels Such as aluminum TCL-1b, TNG1 or TNG, respectively, coding sequence or hydroxide, Surface active Substances Such as lySolecithin, transcribed noncoding Sequence or particular portions pluronic polyols, polyanions, peptides, oil emulsions, key thereof. Alternatively, total nucleic acid of the host cell are hole limpet hemocyanins, dinitrophenol, and potentially extracted and quantitatively assayed for hybridization to useful human adjuvants such as BCG (bacille Calmette Such probes. Guerin) and corynebacterium parvum. US 2005/0287530 A1 Dec. 29, 2005

0142. In a specific example, the entire protein product of domain. For Selection of an antibody Specific to human the TCL-1b, TNG1 or TNG2 gene expressed in bacteria was Tcl-1b, Tng1 or Tng2, one Selects on the basis of positive used to immunize rabbits against Tcl-1b, Tng 1 or Tng2, binding to human Tcl-1b, Tng1 or Tng2, respectively, and a respectivley. Such antibodies recognized the Tcl-1b, Tng1 or lack of binding to, for example, mouse Tcl-1b, Tng 1 or Tng2 protein, respectively, in a variety of leukemia and Tng2. lymphoma cells by Western Blot and by immunoprecipita tion. 0147 The foregoing antibodies are used in methods known in the art relating to the localization and activity of 0143 For preparation of monoclonal antibodies directed the protein Sequences of the invention, e.g., for imaging toward a Tcl-1b, Tng1 or Tng2 protein sequence (SEQ. ID. these proteins, measuring levels thereof in appropriate NO:39, 42, 44, respectively) or analog thereof, any tech physiological Samples, etc. nique which provides for the production of antibody mol ecules by continuous cell lines in culture are used. For 0.148 Structure of the Tcl-1b, Tng1 and Tng2 Gene and example, the hybridoma technique originally developed by Protein Kohler and Milstein (1975, Nature, 256:495-497), as well as 014.9 The structure of the Tcl-1b, Tng1 and Tng2 gene the trioma technique, the human B-cell hybridoma technique and protein is analyzed by to various methods known in the (Kozbor et al., 1983, Immunology Today, 4:72), an the EBV art. hybridoma technique to produce human monoclonal anti bodies (Cole, et al., 1985, in Monoclonal Antibodies and 0150 Genetic Analysis Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal anti 0151. The cloned DNA or cDNA corresponding to the bodies are produced in germ-free animals utilizing recent TCL-1b, TNG1 or TNG2 gene is analyzed by methods technology (PCT/US90/02545). According to the invention, including but not limited to Southern hybridization (South human antibodies are used and are obtained by using human ern, E. M., 1975, J Mol Biol, 98:503–517), Northern hybrid hybridomas (Cote, et al., 1983, Proc Natl AcadSci USA, ization (see, e.g., Freeman, et al., 1983, Proc Natl AcadSci 80:2026-2030) or by transforming human B cells with EBV USA, 80:4094-4098), restriction endonuclease mapping virus in vitro (Cole et al., 1985, in Monoclonal Antibodies (Maniatis, T., 1982, Molecular Cloning, A Laboratory, Cold and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, Spring Harbor, N.Y.), and DNA sequence analysis. Poly according to the invention, techniques developed for the merase chain reaction (PCR; U.S. Pat. Nos. 4,683,202, production of "chimericantibodies” (Morrison, et al., 1984, 4,683,195, and 4,889,818, Proc Natl AcadSci USA 85:7652 Proc Natl AcadSci USA, 81:6851-6855; Neuberger, et al., 7656; Ochman, et al., 1988, Genetics, 120:621-623; Loh, et 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, al., 1989, Science, 243:217-220) followed by Southern 314:452-454) by splicing the genes from a mouse antibody hybridization with a TCL-1b, TNG1 or TNG2 specific probe molecule Specific for Tcl-1b, Tng 1 or Tng2 proteins together allows the detection of the TCL-1b, TNG1 or TNG2 gene, with genes from a human antibody molecule of appropriate respectively, in DNA from various cell types. In one embodi ment, Southern hybridization is used to determine the biological activity is used; Such antibodies are within the genetic linkage of TCL-1b, TNG1 or TNG2, respectively. Scope of this invention. PCR followed by hybridization assay is also used to detect 0144. According to the invention, techniques described or measure TCL-1b, TNG1 or TNG2 RNA, respectively, or for the production of single chain antibodies (U.S. Pat. No. 14q32.1 chromosomal abnormalities. Northern hybridiza 4.946,778) are adapted to produce Tcl-1b, Tng1 or Tng2 tion analysis is used to determine the expression levels of the Specific Single chain antibodies. An additional embodiment TCL-1b, TNG1 or TNG2 gene. Various cell types, at various of the invention utilizes the techniques described for the states of development or activity are tested for TCL-1b construction of Fab expression libraries (Huse, et al., 1989, expression. The Stringency of the hybridization conditions Science, 246:1275-1281) to allow rapid and easy identifi for both Southern and Northern hybridization, or dot blots, cation of monoclonal Fab fragments with the desired Speci are manipulated to ensure detection of nucleic acids with the ficity for Tcl-1b, Tng 1 or Tng2 proteins, derivatives, or desired degree of relatedness to the specific TCL-1b, TNG1 analogs. or TNG2 probe respectively, used. 0145 Antibody fragments which contain the idiotype of 0152 Restriction endonuclease mapping is used to the molecule are generated by known techniques. For roughly determine the genetic Structure of the TCL-1b, example, Such fragments include, but are not limited to... the TNG1 or TNG2 gene. Restriction maps derived by restric F(ab'). Sub.2 fragment which is produced by pepsin digestion tion endonuclease cleavage are confirmed by DNA sequence of the antibody molecule; the Fab' fragments which are analysis. generated by reducing the disulfide bridges of the F(ab'). Sub.2fragment, and the Fab fragments which are 0153 DNA sequence analysis is performed by any tech generated by treating the antibody molecule with papain and niques known in the art, including, but not limited to, the a reducing agent. method of Maxam and Gilbert (1980, Meth Enzymol, 65:499-560), the Sanger dideoxy method (Sanger, et al., 0146 In the production of antibodies, screening for the 1977, Proc Natl AcadSci USA, 74:5463), the use of T7 DNA desired antibody is accomplished by techniques known in polymerase (Tabor and Richardson, U.S. Pat. No. 4,795, the art, e.g. ELISA (enzyme-linked immunosorbent assay). 699), or use of an automated DNA sequenator (e.g., Applied For example, to Select antibodies which recognize a specific Biosystems, Foster City, Calif.). The cDNA sequence of a domain of a Tcl-1b, Tng 1 or Tng2 protein, one assays the representative TCL-1b, TNG1 or TNG2 gene comprises the generated hybridomas for a product which binds to a Tcl-1b, sequence substantially as disclosed herein (SEQ. ID. NO: Tng1 or Tng2, respectively, fragment containing Such 38, 41 and 43, respectively). US 2005/0287530 A1 Dec. 29, 2005

0154) Protein Analysis TNG1 or TNG2 polynucleotide, its encoded protein product O155 The amino acid sequence of the Tcl-1b, Tng1 and and antibodies thereto are used for therapeutic/prophylactic Tng2 protein are derived by deduction from the DNA purposes alone or in combination with other therapeutics Sequence, or alternatively, by direct Sequencing of the pro useful in the treatment of T-cell leukemias. Such molecules tein, e.g., with an automated amino acid Sequencer. The are also used in diagnostic assays, Such as immunoassays, to amino acid Sequence of a representative Tcl-1b, Tng1 and detect, prognose, diagnose, or monitor various conditions, Tng2 protein comprises the Sequence Substantially as diseases, and disorders associated with TCL-1b, TNG1 or TNG2 gene expression or monitor the treatment thereof. depicted in SEQ ID NO:39, 42, and 44, respectively, with Accordingly, in Specific embodiments, T-cell malignancies the representative mature protein that is shown by amino or premalignant changes in Such tissues is diagnosed by acid numbers 1-128, 1-141, and 1-110, respectively. detecting increased TCL-1b, TNG1 or TNG2 gene expres 0156 The Tcl-1b, Tng1 and Tng2 protein sequence are sion in patient samples relative to the level of TCL-1b, further characterized by a hydrophilicity analysis (Hopp, T. TNG1 or TNG2 gene expression in an analogous non and Woods, K., 1981, Proc Natl AcadSci USA, 78:3824). A malignant sample (from the patient or another person, as hydrophilicity profile is used to identify the hydrophobic and determined experimentally or as is known as a Standard level hydrophilic regions of the Tcl-1b, Tng1 or Tng2 protein and in such samples). For diagnostic purposes, a TCL-1b, TNG1 the corresponding regions of the gene Sequence which or TNG2 polynucleotide is used to detect TCL-1b, TNG1 or encode Such regions. TNG2 gene, respectivley, expression or increased TCL-1b, TNG1 or TNG2 gene expression in disease States, Such as, 0157 Secondary structural analysis (Chou, P and Fas T-cell leukemias and lymphomas. For therapeutic purposes, man, G., 1974, Biochemistry, 13:222) is also done, to a Tcl-1b, Tng 1 or Tng2 protein is used to make anti-Tcl 1b, identify regions of the Tcl-1b, Tng 1 or Tng2 protein that anti-Tng1 or anti-Tng2 antibodies that neutralize the activity assume specific Secondary Structures. of Tcl-1b, Tng1 or Tng2, respectively. Included within the 0158 Manipulation, translation, and secondary structure Scope of the present invention are oligonucleotide prediction, as well as open reading frame prediction and Sequences, that include antisense RNA and DNA molecules plotting, is also accomplished using computer Software and ribozymes, that function to inhibit expression of a programs available in the art. TCL-1b, TNG1 or TNG2 RNA or protein. 0159) Other methods of structural analysis are also 0162 Diagnostic Uses employed. These include, but are not limited to, X-ray 0163 The TCL-1b, TNG1 or TNG2 gene sequence is crystallography (Engstom, A., 1974, Biochem Exp Biol, asSociated with disease States associated with chromosome 11:7-13) and computer modeling (Fletterick, R. and Zoller, 14 translocations and inversions around the TCL-1b, TNG1 M. (eds.), 1986, Computer Graphics and Molecular Model or TNG2 gene locus, is preferentially expressed early in T ing, in Current Communications in Molecular Biology, Cold and B lymphocyte differentiation and demonstrates a high Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). level of expression in cells from patients diagnosed with T-PLL carrying an inversion of chromosome 14, 0160 Uses of TCL-1b, TNG1 or TNG2 and its Tcl-1b inv(14)(q11;q32) or patients carrying a to 14:14)(q11;q32) Tng1 or Tng2, Respectively, Protein Product and Antibodies chromosome translocation. Accordingly, TCL-1b, TNG1 or Thereto TNG2 gene sequences (SEQ. ID. NO: 40, 45, and 46, 0.161 Chromosomal translocations and inversions asso respectively) are used diagnostically for the detection of ciated with the TCL-1b, TNG1 or TNG2 locus on chromo diseases States resulting from chromosomal abnormalities, Some 14, e.g., tC14:14)(q11;q32) chromosome translocation, e.g., translocations, inversions and deletions, involving the inv(14)(q11;q32) chromosome inversion, and TCL-1b, TNG1 or TNG2 gene locus of chromosome 14. t(7:14)(q35:cq32) chromosome translocation, are associated Nucleic acids comprising TCL-1b, TNG1 or TNG2 nucle with Several post-thymic types of T-cell leukemias, includ otide Sequences of at least 8 nucleotides, at least 15 nucle ing, but not limited to, T-prolymphocytic leukemias (T-PLL) otides, at least 25 nucleotides, at least 50 nucleotides, at least (Brito-Babapulle and Catovsky, 1991, Cancer Genet Cyto 100 nucleotides, at least 200 nucleotides, at least 300 genet, 55:1-9), acute and chronic leukemias associated with nucleotides, or at least 387 nucleotides up to 1324 nucle the immunodeficiency Syndrome ataxia-telangiectasia (AT) otides of SEQID NO:38,41 and 43 cDNA, respectivley, are (Russo et al., 1988, Cell, 53:137-144; Russo et al., 1989, used as probes in hybridization assays for the detection and Proc. Natl. Acad. Sci. U.S.A. 86:602-606), and adult T-cell measurement of TCL-1b, TNG1 or TNG2 gene (SEQ. ID. leukemia (Virgilio et al., 1993, PNAS, 90:9275-9279). In NO:40, 45, and 46, respectively). Nucleic acids of not more Some cases of AT-associated translocations, in T-cell leuke than 5 kilobases, of not more than 10 kilobases, not more mia and lymphoma involving the 14q32.1 band, clonal than 25 kilobases, not more than 50 kilobases or not more expansion of cells carrying abnormalities in 14q32.1 have than 70 kilobases which are hybridizable to a TCL-1b, been documented in Some cases prior to the development of TNG1 or TNG2 gene, cDNA, or complementary strand is overt malignancy (Russo, et al., 1988 Cell, 53:137-144). used as probes in hybridization assays for the detection and Therefore, a TCL-1b, TNG1 or TNG2 polynucleotide, its measurement of TCL-1b, TNG1 or TNG2 nucleotide Tcl-1b, Tng 1 or Tng2, respectively, protein product and sequences. As an example, the TCL-1b, TNG1 or TNG2 antibodies thereto are used for diagnostic and/or therapeutic/ DNA sequence is used in hybridization assays, e.g., South prophylactic purposes for the above described diseases, as ern or Northern analysis, including in Situ hybridization well as other disorders associated with chromosomal trans assays, of patient's Samples to diagnose abnormalities of locations and inversions associated with to TCL-1, TNG1 or, TCL-1b, TNG1 or TNG2 gene expression, respectively. TNG2 gene locus and/or, increased expression of TCL-1b, Hybridization assays are used to detect, prognose, diagnose, TNG1 or TNG2 RNA or protein, respectively. A TCL-1b, or monitor conditions, disorders, or disease States, Such as US 2005/0287530 A1 Dec. 29, 2005

T-cell malignancies, associated with aberrant changes in tinguished from amplified cDNA nucleotide Sequences due TCL-1b, TNG1 or TNG2 expression and/or activity as to the Size difference of the resulting amplified Sequences. described Supra. In particular, Such a hybridization assay is Resulting amplified genomic nucleotide Sequences will con carried out by a method comprising contacting a Sample tain amplified intron Sequences and will be of a larger size containing nucleic acid with a nucleic acid probe capable of than amplified cDNA nucleotide sequences that will not hybridizing to TCL-1b, TNG1 or TNG2 DNA or RNA, contain amplified intron Sequences. For amplification of under conditions Such that hybridization can occur, and cDNA nucleotide Sequences, the primer Sequences should be detecting or measuring any resulting hybridization. In par Selected from exons Sequences that are Sufficiently far ticular, hybridization assays are used to detect the presence enough apart to provide a detectable amplified nucleotide of abnormalities associated with increased expression of Sequence. TCL-1b, TNG1 or TNG2 mRNA, by hybridizing mRNA or cDNA from a patient sample to a TCL-1b, TNG1 or TNG2, 0167. The TCL-1b, TNG1 or TNG2 gene sequences of respectively, probe, and measuring the amount of resulting the present invention (SEQ. ID. NO: 40, 45, and 46, respec hybridization. For example, assays which are used include, tively) are used diagnostically for the detection of chromo but are not limited to Northern blots, Dot blots, reverse Some 14 abnormalities, in particular translocations transcriptase PCR, etc. A preferred hybridization assay is t(14:14)(q11:Q32) and inv(14)(q11;q32) inversion at Northern blot analysis of a patient sample using TCL-1b, 14q32.1. Accordingly, the present invention provides a pro TNG1 or TNG2 gene probes of at least 15 polynucleotides ceSS for detecting a target Sequence indicative of or includ up to the full length cDNA sequence of each respective gene ing a chromosome 14 abnormality in a Sample, comprising (SEQ. ID. NO: 38, 41 and 43, respectively). Another pre the Steps of amplifying the target Sequence in the Sample ferred hybridization assay is in Situ hybridization analysis of using a first primer of 8 to 25 nucleotides, preferably 18-25 a patient Sample using anti-Tcl-1b, anti-Tng1 or anti-Tng2 nucleotides, complementary to the nucleotide Sequence of antibodies or TCL-1b, TNG1 or TNG2 nucleotide hybrid SEQID NO:40 (TCL-1b), 45 (TNG1) or 46 (TNG2) or SEQ ization probes. Such techniques are well known in the art, ID NO: 38 (TCL-1b), 41 (TNG1), or 44 (TNG2) and a and are in fact the basis of many commercially available Second primer complementary to a region teleomeric or centromeric to the TCL-1b, TNG1 or TNG2 gene, respec diagnostic kits. tively, and detecting any resulting amplified target Sequence 0164. As used herein, patient samples which are used in which the presence of the amplified target Sequence is include, but are not limited to, fresh or frozen tissue Samples, indicative of the abnormality. The present invention also which are used in in situ hybridization assays, cell or tissue provides a method of diagnosing a T-cell malignancy asso Samples containing T-lymphocytes and, in general, patient ciated with chromosome 14 abnormalities in a patient by Samples containing nucleic acid, Such as peripheral blood detecting a chromosome 14 abnormality according to the lymphocytes (PBL) and T-lymphocytes which are used in method above in which the presence of the amplified target assays that measure or quantitate TCL-1b, TNG1 or TNG2 Sequence indicates the presence of a T-cell malignancy in the nucleic acid. patient. The resultant amplified target Sequence is detected 0165 Polynucleotide sequences of TCL-1b, TNG1 or on gel electrophoresis and compared with a normal Sample TNG2 consisting of at least 8 to 25 nucleotides that are or Standard that does not contain a chromosome 14 abnor useful as primers in primer dependent nucleic acid ampli mality. Virgilio et al., Supra, disclose polynucleotide fication methods are used for the detection of TCL-1b, Sequences useful as Second primers. Other polynucleotide TNG1 or TNG2, resepectively, gene Sequences in patient Sequences useful as Second primers are Selected from the Samples. Primer dependent nucleic acid amplification meth T-cell receptor C/6 locus, the T-cell receptor B. chain, or if the chromosome 14 abnormality involves aninversion, a ods useful in the present invention include, but are not polynucleotide sequence 5' to exon 1 of the TCL-1b, TNG1 limited to, polymerase chain reaction (PCR), competitive or TNG2 gene, or if the chromosome abnormality involves PCR, cyclic probe reaction, and ligase chain reaction. Such a translocation, a polynucleotide Sequence 3' to the 3' intron techniques are well known by those of skill in the art. A of the TCL-1b, TNG1 or TNG2 gene. The amplification of preferred nucleic acid amplification method of the present genomic DNA target Sequences may require generating long invention is reverse transcriptase PCR (RT-PCR) (Siebert, et PCR products. PCR techniques for generating long PCR al., 1992, Nature, 359:557-558). products are described in Science (1994) 263: 1564-1565; 0166 In a particular embodiment of the present inven PCR kits for generating long PCR products are available tion, each primer of a pair of primerS for use in a primer from Perkin Elmer and Takara Shuzo Co., Ltd. The present dependent nucleic aid amplification method is Selected from invention also provides a method for detecting a target a different exon of the genomic TCL-1b, TNG1 or TNG2 nucleotide Sequence indicative of or including at least a nucleotide Sequences. For example, if one primer of a pair portion of a chromosome 14 abnormality in a nucleic acid or primers is selected from exon 1 of the TCL-1b, TNG1 or Sample, comprising the Steps of hybridizing the Sample with TNG2 genomic Sequence, the Second primer will be Selected a nucleic acid probe of not more than 10 kilobases, com from exon 2, 3 or 4 of the TCL-1b or TNG2, respectively, prising in the range of 15-1324 nucleotides complementary or exon 2 of the TNG1 genomic Sequence. AS another to at least a portion of the nucleotide sequence of SEQ ID example, if one primer of a pair of primerS is Selected from NO: 40 (TCL-1b), 45 (TNG1) or 46 (TNG2) and detecting exon 2 of the TCL-1b or TNG2 genomic sequence, the or measuring the amount of any resulting hybridization second primer will be selected from exon 1, 3, or 4 of the between the probe and the target Sequence within the TCL-1b TNG2 genomic sequence, respectively. By select sample. The resultant hybridization between the probe and ing each primer of a pair of primers for use in a primer the target Sequence within the Sample is detected using gel dependent nucleic acid amplification method from a differ electrophoresis and is compared to a target Sequence from a ent exon, amplified genomic nucleotide Sequences are dis normal Sample or Standard that does not contain a chromo US 2005/0287530 A1 Dec. 29, 2005

Some 14 abnormality. The present invention also provides a 0171 Anti-Tcl-1b, anti-Tng1 or anti-Tng2 antibodies are method of diagnosing a T-cell malignancy associated with generated and used diagnostically to detect the presence of chromosome 14 abnormalities in a patient comprising, Tcl-1b, Tng 1 or Tng2 protein product, respectively, in detecting Said chromosome 14 abnormality according to the patient Samples thereby identifying disease States associated method above in which the presence of the amplified target with chromosome 14 abnormalities. For detection of Tcl-1b, Sequence indicates the presence of a T-cell malignancy in the Tng1 or Tng2 protein sequences (SEQ. ID. NO:39, 42, or patient. Absolute complementarity between a hybridization 44, respectively), a diagnostic kit of the present invention probe and a target Sequence, although preferred, is not comprises, in one or more containers, an anti-Tcl-1b, anti required. A sequence “complementary to at least a portion Tng1 or anti-Tng2 antibody which optionally is detectably of, as referred to herein, means a Sequence having Sufficient labeled. In a different embodiment, the kit can comprise in complementarity to be able to hybridize with the nucleic a container, a labeled Specific binding portion of an antibody. acid, forming a stable hybridization complex. The ability to As used herein, the term detectable label refers to any label hybridize will depend on both the degree of complementa which provides directly or indirectly a detectable signal and rity and the length of the nucleic acid. Generally, the longer includes, for example, enzymes, radiolabelled molecules, the hybridizing nucleic acid, the more base mismatches with fluorescent molecules, particles, chemilumineSors, enzyme a TCL-1b, TNG1 or TNG2 RNA it may contain and still Substrates or cofactors, enzyme inhibitors, or magnetic par form a stable duplex (or triplex, as the case is). One skilled ticles. Examples of enzymes useful as detectable labels in in the art can ascertain a tolerable degree of mismatch by use the present invention include alkaline phosphatase and horse of Standard procedures to determine the melting point of the radish peroxidase. A variety of methods are available for hybridized complex. linking the detectable labels to proteins of interest and includee, for example, the use of a bifunctional agent, Such 0168 An additional aspect of the present invention as, 4,4'-difluoro-3,3'-dinitro-phenylsulfone, for attaching an relates to diagnostic kits for the detection or measurement of enzyme, for example, horse radish peroxidase, to a protein TCL-1b, TNG1 or TNG2 gene sequences and Tcl-1b, Tng 1 of interest. The attached enzyme is then allowed to react or Tng2, respectively, protein. Accordingly, the present with a Substrate yielding a reaction product which is detect invention provides a diagnostic kit comprising, in a con able. The present invention provides a method for detecting tainer a compound comprising a probe of not more than 10 a Tcl-1b, Tng1 or Tng2 protein in a patient Sample, com kilobases and comprising in the range of 15-1324 nucle prising, contacting the patient Sample with an anti-Tcl-1b, otides of the nucleotide sequence of SEQ ID NO. 38 anti-Tng1 or anti-Tng2 antibody, respectively, under condi (TCL-1b), 41 (TNG1) or 43 (TNG2) or its complement. tions Such that immunospecific binding occurs, and detect Alternatively, the present invention provides a diagnostic kit ing or measuring the amount of any immunospecific binding comprising, in one or more containers, a pair of primers of by the antibody. at least 8-25 nucleotides in which at least one of the primers is hybridizable to SEQ ID NO:38 (TCL-1b), 41 (TNG1) or 0172 Samples are any Sample from a patient containing 43 (TNG2) or its complement and wherein the primers are Tcl-1b, Tng1 or Tng2 protein, e.g., tissue Sections, periph capable of priming cDNA Synthesis in an amplification eral blood lymphocytes, etc. In diagnosing disease States, the reaction. The present invention also provides a diagnostic kit functional activity of Tcl-1b, Tng1 or Tng2 proteins, deriva in which at least one of the primers is hybridizable to SEQ tives and analogs are assayed by various methods. Accord ID NO: 38 (TCL-1b), 41 (TNG1) or 43 (TNG2) or its ingly, the present invention also provides a method of complement and in which one of the primers is hybridizable diagnosing a T-cell malignancy associated with chromo to a DNA sequence located telomeric or centromeric to the Some 14 abnormalities in a patient comprising, detecting TCL-1b, TNG1 or TNG2 gene. In a specific embodiment, increased expression of Tcl-1b, Tng1 or Tng2 protein in a one of the foregoing compounds of the container is detect Sample from the patient, in which an increase in Tcl-1b, ably labeled. Tng1 or Tng2, respectivley, protein relative to the level found in Such an analogous Sample from a normal indi 0169. The amplification reaction of the present invention vidual, indicates the presence of a T-cell malignancy in the are a polymerase chain reaction, competitive PCR and patient. competitive reverse-transcriptase PCR (Clementi, et al., 0173 For example, in one embodiment, where one is 1994, Genet Anal Tech Appl, 11(1):1-6; Siebert et al., 1992, detecting or measuring Tcl-1b, Tng1 or Tng2 protein by Nature, 359:557-558); cyclic probe reaction, which allows assaying for binding to anti-Tcl-1b, anti-Tng1 or anti-Tng2 for amplification of a target Sequence using a hybrid RNA/ antibody, respectively, various immunoassays known in the DNA probe and RNase (ID Biomedical); ligase chain reac art are used, including, but not limited to, competitive and tion (Wu, et al., 1989, Genomics, 4:560-569). In a particular non-competitive assay Systems using techniqueS Such as embodiment, the chromosomal abnormality associated with radioimmunoassays, ELISA (enzyme linked immunosorbent a TCL-1b, TNG1 or TNG2 locus is detected as described in assay), "Sandwich’ immunoassays, immunoradiometric PCT Publication No. WO/92/19775, dated Nov. 12, 1992. In assays, gel diffusion precipitin reactions, immunodiffusion a specific embodiment, the TCL-1b, TNG1 or TNG2 probe assays, in Situ immunoassays (using colloidal gold, enzyme used in a hybridization assay is detectably labeled. Such a or radioisotope abels, for example), western blots, in Situ label is any known in the art including, but not limited to, hybridizations, precipitation reactions, agglutination SSayS radioactive labels, fluorescent labels, biotin, chemilumines (e.g., gel agglutination assays, hemagglutination assays), cent labels, etc. complement fixation assays, mmunofluorescence assays, 0170 In a specific embodiment in which the assay used protein A assays, and immunoelectrophoresis assays, etc. In employs primers, at least one primer is detectably labeled. In one mbodiment, antibody binding is detected by detecting a another embodiment, one of a primer pair is attached to a label on the primary antibody. In nother embodiment, the moiety providing for capture, e.g., a magnetic bead. primary antibody is detected by detecting binding of a US 2005/0287530 A1 Dec. 29, 2005

Secondary antibody or reagent to the primary antibody. In a on disease States is monitored. For example, the expression further embodiment, the secondary antibody is labelled. of TCL-1b, TNG1 or TNG2 gene sequences (SEQ. ID. NO: Many means are known in the art for detecting binding in an 40, 45, or 46, respectively) or Tcl-1b, Tng1 or Tng2 protein immunoassay and are within the Scope of the present inven sequences (SEQ. ID. NO:38, 42, or 44, respectively) are tion. In particular, Such an immunoassay is carried out by a detected as described, Supra, both before and after admin method comprising contacting a Sample derived from a istration of a therapeutic composition comprising a TCL-1b, patient with an anti-Tcl-1b, anti-Tng1 or anti-Tng2 antibody TNG1 or TNG2 nucleotide sequence, Tcl-1b, Tng 1 or Tng2 under conditions Such that immunospecific binding occurs, protein Sequence, derivative or analog thereof, or antibody and detecting or measuring the amount of any immunospe cific binding by the antibody. In a specific embodiment, thereto, respectively, of the present invention. antibody to a Tcl-1b, Tng1 or Tng2 protein is used to assay 0179 ATCL-1b, TNG1 or TNG2 polynucleotide is use a patient tissue or Serum Sample for the presence of a Tcl-1b, ful in the treatment of various disease States associated with Tng1 or Tng2 protein, respectively, where an increased level chromosome 14 abnormalities, Such as T-cell leukemias and of Tcl-1b, Tng 1 or Tng2 protein is an indication of a diseased lymphomas, and/or increased expression of Tcl-1b, Tng 1 or condition. In one embodiment of the present invention, the Tng2 protein. By introducing TCL-1b, TNG1 or TNG2 Tcl-1b, Tng1 or Tng2 protein is detected or measured by antisense gene Sequences into cells, gene therapy is used to immunocytochemistry of a patient Sample. In another treat conditions associated with Over-expression of TCL-1b, embodiment, assays to measure the levels of Tcl-1b, Tng1 or TNG1 or TNG2 genes, respectively. Accordingly, the Tng2 protein or RNA is used to moniter therapy of disease present invention provides a method for treating a disease associated with increased expression of Tcl-1b. For State associated with a chromosome 14 abnormality in example, a decrease in levels of TCL-1b, TNG1 or TNG2 mammal Suffering from a disease State associated with a RNA or protein after therapy, relative to the level found chromosome 14 abnormality comprising, administering a before therapy, are indicative of a favorable response to therapeutically effective amount of a TCL-1b, TNG1 or therapy. An increase in Such levels after therapy are indica TNG2 antisense molecule to a mammal suffering from a tive of a poor response to therapy. disease State associated with a chromosome 14 abnormality. 0.174. In another embodiment, the levels of Tcl-1b, Tng 1 or Tng2 protein or RNA expression are used to Stage disease, 0180 Oligonucleotide sequences, that include antisense with an increase in Tcl-1b, Tng1 or Tng2 protein or RNA, RNA and DNA molecules and ribozymes that function to respectively, eXpression indicating disease progression. inhibit the translation of a TCL-1b, TNG1 or TNG2 mRNA are within the Scope of the invention. “AntiSense' as used 0175 Other methods will be known to the skilled artisan herein refers to a nucleic acid capable of hybridizing to a and are within the Scope of the invention. portion of a TCL-1b, TNG1 or TNG2 RNA (preferably 0176) Therapeutic/Prophylactic Uses mRNA) by virtue of Some sequence complementarity. Anti 0177. Inhibitors of Tcl-1b, Tng 1 or Tng2 are used thera sense RNA and DNA molecules act to directly block the peutically for the treatment of disease States associated with translation of mRNA by binding to targeted mRNA and chromosome 14 abnormalities, in particular at 14q32.1, preventing protein translation. In regard to antisense DNA, and/or increased expression of Tcl-1b, Tng 1 or Tng2 protein, oligodeoxyribonucleotides derived from the translation ini respectively. In an embodiment of the present invention, a tiation site, e.g., between -10 and +10 regions of a TCL-1b, Tcl-1b, Tng1 or Tng2 protein and/or cell line that expresses TNG1 or TNG2 nucleotide sequence, are preferred. The a Tcl-1b, Tng 1 or Tng2 protein, respectively, is used to present invention provides for an antisense molecule com Screen for antibodies, peptides, or other molecules that bind prising a nucleotide Sequence complementary to at least a to the Tcl-1b, Tng 1 or Tng2 protein and thus may act as part of the coding Sequence of a Tcl-1b, Tng1 or Tng2 agonists or antagonists of Tcl-1b, Tng1 or Tng2 protein. For protein which is hybridizable to a TCL-1b, TNG1 or TNG2 example, anti-Tcl-1b, anti-Tng1 or anti-Tng2 antibodies mRNA, respectively. The present invention also provides for capable of neutralizing the activity of a Tcl-1b, Tng 1 or Tng2 an antisense molecule with a nucleotide Sequence comple protein, respectively, are used to inhibit or prevent a disease mentary to at least a part of the non-coding sequence (SEQ State associated with chromosome 14 abnormalities and/or ID NO: 40, 45, or 46, respectively) which hybridizes to the expression of Tcl-1b, Tng1 or Tng2 protein, Such as T-cell TCL-1b, TNG1 or TNG2 coding sequence (SEQ ID NO:40, leukemia and lymphoma. Accordingly, the present invention 45, or 46, respectively). In a preferred embodiment of the provides a method for treating a disease State associated with present invention, the antisense gene Sequence is derived a chromosome 14 abnormality in mammal Suffering from a from the 5' noncoding sequence of a TCL-1b, TNG1 or disease State associated with a chromosome 14 abnormality TNG2 gene. In a particularly preferred embodiment of the comprising, administering a therapeutically effective present invention, the antisense gene Sequence is derived amount of an anti-Tcl-1b, anti-Tng1 or anti-Tng2 antibody from TCL-1b, TNG1 or TNG2 gene (SEQ ID NO:38, 41, to a mammal Suffering from a disease State associated with or 43, respectively). a chromosome 14 abnormality. Alternatively, Screening of 0181 Ribozymes are enzymatic RNA molecules capable organic or peptide libraries with recombinantly expressed of catalyzing the Specific cleavage of RNA. The mechanism Tcl-1b, Tng 1 or Tng2 protein are useful for identification of of ribozyme action involves Sequence Specific hybridization therapeutic molecules that function to inhibit the activity of of the ribozyme molecule to complementary target RNA, Tcl-1b, Tng1 or Tng2 protein, respectively. Synthetic and followed by a endonucleolytic cleavage. Within the scope of naturally occurring products are Screened in a number of the invention are engineered hammerhead motif ribozyme ways deemed routine to those of skill in the art. molecules that specifically and efficiently catalyze endo 0.178 The ability of antibodies, peptides or other mol nucleolytic cleavage of TCL-1b, TNG1 or TNG2 RNA ecules to modulate the effect of Tcl-1b, Tng 1 or Tng2 protein Sequences. US 2005/0287530 A1 Dec. 29, 2005 2O

0182 Specific ribozyme cleavage sites within any poten present invention. In a preferred aspect, the Therapeutic is tial RNA target are initially identified by Scanning the target Substantially purified. The Subject is preferably an animal, molecule for ribozyme cleavage Sites which include the including but not limited to animals Such as cows, pigs, following sequences, GUA, GUU and GUC. Once identi chickens, etc., and is preferably a mammal, and most fied, short RNA sequences of between 15 and 20 ribonucle preferably human. otides corresponding to the region of the target gene con taining the cleavage Site are evaluated for predicted 0191 Various delivery systems are known and used to Structural features, Such as Secondary Structure that may administer a Therapeutic of the invention, e.g., encapsula render the oligonucleotide Sequence unsuitable. The Suit tion in liposomes, microparticles, microcapsules, expression ability of candidate targets may also be evaluated by testing by recombinant cells, receptor-mediated endocytosis (see, their accessibility to hybridization with complementary oli e.g., Wu and Wu, 1987, J Biol Chem, 262:4429-4432), gonucleotides, using ribonuclease protection assayS. construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction 0183 Both anti-sense RNA and DNA molecules and include but are not limited to intradermal, intramuscular, ribozymes of the invention are prepared by any method intraperitoneal, intravenous, Subcutaneous, intranasal, and known in the art for the synthesis of RNA molecules. These oral routes. The compounds are administered by any con include techniques for chemically Synthesizing oligodeox Venient route, for example by infusion or bolus injection, by yribonucleotides well known in the art Such as for example absorption through epithelial or mucocutaneous linings Solid phase phosphoramidite chemical Synthesis. Alterna (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and are tively, RNA molecules are generated by in vitro and in vivo administered together with other biologically active agents. transcription of DNA sequences encoding the antisense Administration is Systemic or local. In addition, it are RNA molecule. Such DNA sequences are incorporated into desirable to introduce the pharmaceutical compositions of a wide variety of vectors which incorporate suitable RNA the invention into the central nervous System by any Suitable polymerase promoters such as the T7 or SP6 polymerase route, including intraventricular and intrathecal injection; promoters. Alternatively, antisense cDNA constructs that intraventricular injection are facilitated by an intraventricu synthesize antisense RNA constitutively or inducibly, lar catheter, for example, attached to a reservoir, Such as an depending on the promoter used, is introduced Stably into Ommaya reservoir. cell lines. 0.192 In a specific embodiment, it are desirable to admin 0184 Various modifications to the DNA molecules are ister the pharmaceutical compositions of the invention introduced as a means of increasing intracellular Stability locally to the area in need of treatment; this are achieved by, and half-life. Examples of modifications include, but are not for example, and not by way of limitation, local infusion limited to, the addition of flanking Sequences of ribo- or during Surgery, topical application, e.g., in conjunction with deoxy-nucleotides to the 5' and/or 3' ends of the molecule or a wound dressing after Surgery, by injection, by means of a the use of phosphorothioate or 2 O-methyl rather than catheter, by means of a Suppository, or by means of an phosphodiesterase linkages within the oligodeoxyribonucle implant, Said implant being of a porous, non-porous, or otide backbone. gelatinous material, including membranes, Such as Sialastic membranes, or fibers. In one embodiment, administration is 0185. Methods for introducing nucleic acid into cells or by direct injection at the site (or former site) of a malignant tissue include methods for in vitro introduction of nucleic tumor or neoplastic or pre-neoplastic tissue. acid Such as the insertion of naked nucleic acid, i.e., by injection into tissue, the introduction of a nucleic acid in a 0193 In a specific embodiment where the Therapeutic is cell eX Vivo, the use of a vector Such as a virus, retrovirus, a nucleic acid encoding a protein therapeutic, the nucleic phage or plasmic, etc. or techniques Such as electroporation acid is administered in Vivo to promote expression of its which are used in vivo or ex vivo. encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it So that it 0186. Other methods will be known to the skilled artisan becomes intracellular, e.g., by use of a retroviral vector (see and are within the Scope of the invention. U.S. Pat. No. 4,980,286), or by direct injection, or by use of 0187 Demonstration of Therapeutic or Prophylactic Util microparticle bombardment (e.g., a gene gun; Biolistic, ity Dupont), or coating with lipids or cell-Surface receptors or transfecting, agents, or by administering it in linkage to a 0188 The TCL-1b, TNG1 or TNG2 polynucleotides, homeobox-like peptide which is known to enter the nucleus Tcl-1b, Tng 1 or Tng2 protein products, respectivley, deriva (see e.g., Joliot, et al., 1991, Proc Natl Acad Sci USA, tives and analogs thereof, and antibodies thereto, of the 88:1864-1868), etc. Alternatively, a nucleic acid therapeutic invention are tested in Vivo for the desired therapeutic or is introduced intracellularly and incorporated within host prophylactic activity. For example, Such compounds are cell DNA for expression, by homologous recombination. tested in Suitable animal model Systems prior to testing in humans, including but not limited to rats, mice, chicken, 0194 The present invention also provides pharmaceuti cows, monkeys, rabbits, etc. For in Vivo testing, prior to cal compositions. Such compositions comprise a therapeu administration to humans, any animal model System known tically effective amount of a therapeutic, and a pharmaceu in the art are used. tically acceptable carrier or excipient. Such a carrier includes but is not limited to Saline, buffered Saline, dex 0189 Therapeutic/Prophylactic Methods and Composi trose, water, glycerol, ethanol, and combinations thereof. tions The carrier and composition are Sterile. The formulation should Suit the mode of administration. 0190. The invention provides methods of treatment and prophylaxis by administration to a Subject of an effective 0.195 The composition, if desired, can also contain minor amount of a Therapeutic, i.e., a TCL-1b, TNG1 or TNG2 amounts of wetting or emulsifying agents, or pH buffering polynucleotide, Tcl-1b, Tng1 or Tng2 protein, respectively, agents. The composition is a liquid Solution, Suspension, derivative or analog thereof, or antibody thereto of the emulsion, tablet, pill, capsule, Sustained release formulation, US 2005/0287530 A1 Dec. 29, 2005 or powder. The composition is formulated as a Suppository, that are antisense to a TCL-1b, TNG1 or TNG2 gene (SEQ. with traditional binders and carrierS Such as triglycerides. ID. NO: 40, 45, or 46, respectively) or TCL-1b, TNG1 or Oral formulation includes Standard carrierS Such as pharma TNG2 cDNA (SEQ. ID. NO: 38, 41, or 43, respectively) ceutical grades of mannitol, lactose, Starch, magnesium encoding Tcl-1b, Tng1 or Tng2 (SEQ. ID. NO 39, 42 or 44), Stearate, Sodium Saccharine, cellulose, magnesium carbon respectively, or a portion thereof. Such antisense nucleic ate, etc. acids have utility as Antagonist Therapeutics of the inven 0196. In a preferred embodiment, the composition is tion, and is used in the treatment or prevention of disorders, formulated in accordance with routine procedures as a e.g., T-cell malignancies as described Supra. pharmaceutical composition adapted for intravenous admin 0203 The antisense nucleic acids of the invention are istration to human beings. Typically, compositions for intra oligonucleotides that are double-Stranded or Single-Stranded, venous administration are Solutions in Sterile isotonic aque RNA or DNA or a modification or derivative thereof, which ous buffer. Where necessary, the composition may also can be directly administered to a cell, or which are produced include a Solubilizing agent and a local anesthetic Such as intracellularly by transcription of exogenous, introduced lignocaine to ease pain at the Site of the injection. Generally, Sequences. the ingredients are Supplied either Separately or mixed together in unit dosage form, for example, as a dry lyo 0204. In a specific embodiment, the TCL-1b, TNG1 or philized powder or water free concentrate in a hermetically TNG2 antisense polynucleotides provided by the instant Sealed container Such as an ampoule or Sachette indicating invention can be used for the treatment of disease States the quantity of active agent. Where the composition is to be asSociated with chromosome 14 abnormalities, in particular administered by infusion, it is dispensed with an infusion at 14q32.1, wherein the disease State can be demonstrated bottle containing Sterile pharmaceutical grade water or (in vitro or in vivo) to express the TCL-1b, TNG1 or TNG2 Saline. Where the composition is administered by injection, gene, respectively. Such demonstration can be by detection an ampoule of Sterile water for injection or Saline is provided of TCL-1b, TNG1 or TNG2 RNA or of Tcl-1b, Tng 1 or Tng2 So that the ingredients are mixed prior to administration. protein, respectively. 0197) The Therapeutics of the invention are formulated 0205 The invention further provides pharmaceutical as neutral or Salt forms. Pharmaceutically acceptable Salts compositions comprising an effective amount of the TCL include those formed with free amino groupS. Such as those 1b, TNG1 or TNG2 antisense nucleic acids of the invention derived from hydrochloric, phosphoric, acetic, oxalic, tar in a pharmaceutically acceptable carrier, as described Supra. taric acids, etc., and those formed with free carboxyl groups Methods for treatment and prevention of disease States Such as those derived from Sodium, potassium, ammonium, associated with chromosome 14, Such as to T-cell malig calcium, ferric hydroxides, isopropylamine, triethylamine, nancies comprising administering the pharmaceutical com 2-ethylamino ethanol, histidine, procaine, etc. positions of the invention are also provided. 0198 The amount of the Therapeutic of the invention 0206. In another embodiment, the invention is directed to which will be effective in the treatment of a particular methods for inhibiting the expression of a TCL-1b, TNG1 or disorder or condition will depend on the nature of the TNG2 nucleic acid Sequence in a prokaryotic or eukaryotic disorder or condition, and can be determined by Standard cell comprising providing the cell with an effective amount clinical techniques. In addition, in vitro assays are employed of a composition comprising an antisense TCL-1b, TNG1 or to help identify optimal dosage ranges. The precise dose to TNG2 nucleic acid, respectively, of the invention. be employed in the formulation will also depend on the route 0207. The TCL-1b, TNG1 or TNG2 antisense polynucle of administration, and the Seriousness of the disease or otides are of at least Six nucleotides and are preferably disorder, and should be decided according to the judgment oligonucleotides (ranging from 6 to about 50 oligonucle of the practitioner and each patient's circumstances. How otides). In specific aspects, the oligonucleotide is at least 10 ever, Suitable dosage ranges for intravenous administration nucleotides, at least 20 nucleotides, at least 30 nucleotides, are generally about 20-500 micrograms of active compound or at least 40 nucleotides. The oligonucleotides are DNA or per kilogram body weight. Suitable dosage ranges for intra RNA or chimeric mixtures or derivatives or modified ver nasal administration are generally about 0.01 pg/kg body Sions thereof, Single-Stranded or double-Stranded. The oli weight to 1 mg/kg body weight. Effective doses are extrapo gonucleotide is modified at the base moiety, Sugar moiety, or lated from dose-response curves derived from in vitro or phosphate backbone. The oligonucleotide may include other animal model test Systems. appending groups Such as peptides, or agents facilitating 0199 Suppositories generally contain active ingredient in transport across the cell membrane (see, e.g., Letsinger, et the range of 0.5% to 10 k by weight; oral formulations al., 1989, Proc Natl AcadSci USA, 86:6553-6556; Lemaitre, preferably contain 10% to 95% active ingredient. et al., 1987, Proc Natl. Acad Sci USA, 84:648-652; PCT 0200. The invention also provides a pharmaceutical pack Publication No. WO 88/09810, published Dec. 15, 1988) or or kit comprising one or more containers filled with one or blood-brain barrier (see, e.g., PCT Publication No. WO more of the ingredients of the pharmaceutical compositions 89/10134, published Apr. 25, 1988), hybridization-triggered of the invention. Optionally associated with Such contain cleavage agents (see, e.g., Krol, et al., 1988, BioTechniques, er(s) is a notice in the form prescribed by a governmental 6:958-976) or intercalating agents (see, e.g., Zon, 1998, agency regulating the manufacture, use or Sale of pharma Pharm Res 5:539-549). ceuticals or biological products, which notice reflects 0208. The oligonucleotide are conjugated to another mol approval by the agency of manufacture, use or Sale for ecule, e.g., a peptide, hybridization triggered croSS-linking human administration. agent, transport agent, hybridization-triggered cleavage 0201 Antisense Regulation of TCL-1b, TNG1 and TNG2 agent, etc. Gene Expression 0209 Oligonucleotides of the invention are synthesized 0202) The present invention provides the therapeutic or by Standard methods known in the art, e.g., by use of an prophylactic use of nucleic acids of at least Six nucleotides automated DNA synthesizer (Such as are commercially US 2005/0287530 A1 Dec. 29, 2005 22 available from BioSearch, Applied BioSystems, etc.). AS The ability to hybridize will depend on both the degree of examples, phosphorothioate oligos are Synthesized by the complementarity and the length of the antisense nucleic method of Stein et al. (1988, Nucl. Acids Res. 16:3209), acid. Generally, the longer the hybridizing nucleic acid, the methylphosphonate oligos are prepared by use of controlled more base mismatches with a TCL-1b, TNG1 or TNG2 RNA pore glass polymer supports (Sarin, et al., 1988, Proc Natl it may contain and still form a stable duplex (or triplex, as AcadSci USA, 85:7448-7451), etc. the case are). One skilled in the art can ascertain a tolerable 0210. In a specific embodiment, the TCL-1b, TNG1 or degree of mismatch by use of Standard procedures to deter TNG2 antisense oligonucleotide comprises catalytic RNA, mine the melting point of the hybridized complex. or a ribozyme (see, e.g., PCT International Publication WO 0213 The TCL-1b antisense nucleic acids are used to 90/11364, published Oct. 4, 1990; Sarver, et al., 1990, treat (or prevent) T-cell malignancies, of a cell type which Science, 247: 1222-1225). In another embodiment, the oli has been shown to express TCL-1b, TNG1 or TNG2 RNA. gonucleotide is a 2'-O-methylribonucleotide (Inoue, et al., Malignant, neoplastic, and pre-neoplastic cells which are 1987, Nucl Acids Res, 15:6131-6148), or a chimeric RNA tested for Such expression include, but are not limited, to DNA analogue (Inoue, et al., 1987, FEBS Lett, 215:327 those described Supra. In a preferred embodiment, a single 330). stranded DNA antisense TCL-1b, TNG1 or TNG2 oligo 0211). In an alternative embodiment, the TCL-1b, TNG1 nucleotide is used, respectively. or TNG2 antisense nucleic acid of the invention is produced 0214 Malignant (particularly, tumor) cell types which intracellularly by transcription from an exogenous Sequence. express TCL-1b, TNG1 or TNG2 RNA is identified by For example, a vector is introduced in Vivo Such that it is various methods known in the art. Such methods include but taken up by a cell, within which cell the vector or a portion are not limited to hybridization with a TCL-1b, TNG1 or thereof is transcribed, producing an antisense nucleic acid TNG2-specific nucleic acid (e.g., by Northern hybridization, (RNA) of the invention. Such a vector would contain a dot blot hybridization, in situ hybridization), observing the sequence encoding the TCL-1b, TNG1 or TNG2 antisense ability of RNA from the cell type to be translated in vitro into nucleic acid, respectively. Such a vector can remain episo Tcl-1b, Tng1 or Tng2, respectively. In a preferred aspect, mal or become chromosomally integrated, as long as it is primary tumor tissue from a patient is assayed for TCL-1b, transcribed to produce the desired antisense RNA. Such TNG1 or TNG2 expression prior to treatment. vectors are constructed by recombinant DNA technology methods Standard in the art. Vectors are plasmid, Viral, or 0215 Pharmaceutical compositions of the invention, others known in the art, used for replication and expression comprising an effective amount of a TCL-1b, TNG1 or in mammalian cells. Expression of the Sequence encoding TNG2 antisense nucleic acid in a pharmaceutically accept the TCL-1b, TNG1 or TNG2 antisense RNA is by any able carrier, is administered to a patient having a malignancy promoter known in the art to act in mammalian, preferably which is of a type that expresses TCL-1b, TNG1 or TNG2 human, cells. Such promoters are inducible or constitutive. RNA. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, 0216) The amount of TCL-1b, TNG1 or TNG2 antisense Nature, 290:304-310), the promoter contained in the 3' long nucleic acid which will be effective in the treatment of a terminal repeat of Rous sarcoma virus (Yamamoto, et al., particular disease State or condition will depend on the 1980, Cell, 22:787-797), the herpes thymidine kinase pro nature of the disease State or condition, and is determined by moter (Wagner, et al., 1981, Proc Natl Acad Sci USA, Standard clinical techniques. Where possible, it is desirable 78:1441-1445), the regulatory sequences of the metallothio to determine the antisense cytotoxicity of the tumor type to nein gene (Brinster, et al., 1982, Nature, 296:3942), etc. be treated in vitro, and then in useful animal model Systems prior to testing and use in humans. 0212. The antisense nucleic acids of the invention com prise a Sequence complementary to at least a portion of an 0217. In a specific embodiment, pharmaceutical compo RNA transcript of a TCL-1b, TNG1 or TNG2 gene, prefer sitions comprising TCL-1b, TNG1 or TNG2 antisense ably a human TCL-1b, TNG1 or TNG2 gene (SEQ. ID. NO: nucleic acids are administered via liposomes, micropar 40, 45, or 46, respectively). However, absolute complemen ticles, or microcapsules. In various embodiments of the tarity, although preferred, is not required. A sequence invention, it are useful to use Such compositions to achieve “complementary to at least a portion of an RNA,” as referred Sustained release of the TCL-1b, TNG1 or TNG2 antisense to herein, means a Sequence having Sufficient complemen nucleic acids. In a Specific embodiment, it are desirable to tarity to be able to hybridize with the RNA, forming a stable utilize liposomes targeted via antibodies to Specific identi duplex; in the case of double-stranded TCL-1b, TNG1 or fiable tumor antigens (Leonetti, et al., 1990, Proc Natl Acad TNG2 antisense nucleic acids, a Single Strand of the duplex Sci USA, 87:2448-2451; Renneisen, et al., 1990, J Biol DNA may thus be tested, or triplex formation are assayed. Chem, 265:16337-16342).

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 63

<210> SEQ ID NO 1 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &22O > FEATURE US 2005/0287530 A1 Dec. 29, 2005 23

-continued <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer <400 SEQUENCE: 1 ggcagotcta ccc.cgg gatgaa 22

<210> SEQ ID NO 2 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer <400 SEQUENCE: 2 acagacct ga gtgggaCagg a 21

<210> SEQ ID NO 3 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer <400 SEQUENCE: 3 to citcc ttgg caggagtggit a 21

<210> SEQ ID NO 4 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer <400 SEQUENCE: 4 cagttacggg togctottgcg t 21

<210 SEQ ID NO 5 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer <400 SEQUENCE: 5 atggccitc.cg aagcttctgt g 21

<210> SEQ ID NO 6 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: PCR primer <400 SEQUENCE: 6 tggtogtgcg gttcaatc.cc t 21

<210 SEQ ID NO 7 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: TC5 <400 SEQUENCE: 7 US 2005/0287530 A1 Dec. 29, 2005 24

-continued aatctggcca togtotgcta tttc 24

<210 SEQ ID NO 8 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: RT-PCR primer for TNG1 <400 SEQUENCE: 8 tgcatc.cc to cagocaagga t 21

<210 SEQ ID NO 9 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: RT-PCR primer for TNG1 <400 SEQUENCE: 9 tggcctgcag aggctotcaa g 21

<210> SEQ ID NO 10 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: RT-PCR primer for TNG2 <400 SEQUENCE: 10 gtgcctgtct cattc.gc.citc td 22

EQ ID NO 11 ENGTH 23 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: RT-PCR rimer for TNG2

EQUENCE: 11 agtggg caca tottacagoa titc 23

EQ ID NO 12 ENGTH 21 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: RT-PCR Lille

<400 SEQUENCE: 12 gcatcCagga citgtgc.cago a 21

<210> SEQ ID NO 13 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: RT-PCR primer US 2005/0287530 A1 Dec. 29, 2005 25

-continued <400 SEQUENCE: 13 ttctgttago cittgctgtcc git 22

<210> SEQ ID NO 14 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: 3 "RACE primer for TNG1 <400 SEQUENCE: 14 ttgaac coag gtc.tc.gtotg ac 22

<210 SEQ ID NO 15 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: 5' RACE primer for TNG1 <400 SEQUENCE: 15 aacgtaggat gtgcacagag ca 22

<210> SEQ ID NO 16 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 16 gagaacgg to agg accoa aa cc 22

<210 SEQ ID NO 17 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 17 caggctatoa agacctttac to 22

<210> SEQ ID NO 18 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 18 tdaaccitc.gc atattacitat gito 23

<210 SEQ ID NO 19 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b US 2005/0287530 A1 Dec. 29, 2005 26

-continued primer

<400 SEQUENCE: 19 Calaagg caca aagtgagcaa gag 23

<210> SEQ ID NO 20 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 20 aatgttggaa acttctoact cat 23

<210> SEQ ID NO 21 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 21 actggaaact tattotcatt cac 23

<210> SEQ ID NO 22 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 22 cacttgcago atatgaccac aat 23

<210> SEQ ID NO 23 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 23 cctggtotgc acaagagatg a 21

<210> SEQ ID NO 24 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 24 citgtcc actt gtggaagtta at 22

<210> SEQ ID NO 25 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence US 2005/0287530 A1 Dec. 29, 2005 27

-continued

&220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b primer

<400 SEQUENCE: 25 cacttgttggc agatgaccag ata 23

EQ ID NO 26 ENGTH 22 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: murine Tcl 1b Lille

EQUENCE: 26 ccaggagcct acticcc.cago ag 22

EQ ID NO 27 ENGTH 21 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: murine Tcl 1b Lille

<400 SEQUENCE: 27 gtgg Cagatg accacactct t 21

<210> SEQ ID NO 28 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: muring Tcl 1b3 primer

<400 SEQUENCE: 28 cattactatg gotgattcag titc 23

<210 SEQ ID NO 29 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl 1b3 primer

<400 SEQUENCE: 29 ggaatgagac totcagggca c 21

<210 SEQ ID NO 30 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murien Tcl1 primer

<400 SEQUENCE: 30 CCtggg Caag gCalgacagga gC 22

<210> SEQ ID NO 31 <211& LENGTH 22 US 2005/0287530 A1 Dec. 29, 2005 28

-continued

&212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl1 primer

<400 SEQUENCE: 31 tgcttcttgc ticittatcgga tig 22

<210> SEQ ID NO 32 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine Tcl1 primer

<400 SEQUENCE: 32 ttcatcgttg gacitcc gagt c 21

<210 SEQ ID NO 33 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murien Tcl1 primer

<400> SEQUENCE: 33 aatticciaggit gatcttgcgc c 21

<210> SEQ ID NO 34 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: actin RT-PCR primer

<400 SEQUENCE: 34 gtaccaccag acago actgt g 21

<210 SEQ ID NO 35 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: actin RT-PCR primer

<400 SEQUENCE: 35 gacccagatc atgtttgaga cc 22

<210 SEQ ID NO 36 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: murine RACE primer

<400 SEQUENCE: 36 aa.gc.catcta talaggtoagg 20

US 2005/0287530 A1 Dec. 29, 2005 31

-continued agaatggcgt galaccc.ggga gg.cgaagctt gtagtgagcc gagat.cgcac cactgcactic 1620 cagoctoggt gacagagtga gacitc.cgtcc caaaaaaaaa aaaaaaaaaa aatgcc.gttt 1680 aggtottcgt aaacaattca citgcctgttt gtttgtttitt to agaaagttc ttgctctgtt 1740 gc ggctggag to cactggtg tdatgttggc ticactgcaac citccaccitcc caggotcaag 1800 tgattctoat gccitcago ct cocgagtagc titggattaca gg.cgatttitt ttttacagtt 1860 aattittttitt gttattittca ggagagacaa aagtttaatc atgttgggcca ggctggttitt 1920 gaactcct ga cctoaagtga totg.cccacc ttggc citccc aaagtgctgg gattacaggt 1980 gagccaccitc gcc cago cag titcactgaca ctittaaacaa tatalacacat titcctaaaaa 20 40 aagttcaaat aggttatttcaaaaaatgtt got agagaac atggaaaggc titttctgtac 2100 atacactaaa taaag catgc aaaaattgtg gag caaatat tittaagttitt toaaaag.cct 216 O gaaaaagtgt taatggaggg cactgtaaaa togtgcagoc act at ggaaa acaggatgag 2220 gatttctoaa aaaaagaatt acggcataat coagcaatgc cacttctgga tatataccca 228O caag actotg aagcc.ggaac ttaag catgt attcatacat coatgttcac agcagtatca 234. O ttcatactag ccaaaagg to gtggcagocc cc.gtgtc.cat tdatagatga atgggtaaac 24 OO aacacaaacc atgaagtatt caccottaaa agt cagacac acggatgaaa cittggagcca 2460 ttatactaaa taaatatgc cagtcacgga agg acagatt citcttgtatg agg tacticag 252O agtggtctica ttcataaagt ggaatggtag citgcc agggg citggagggag to gaggatgg 258O gaagttaatg ttagta acag gtacggagtc. tcagtttggg aagataaaaa gttctggagg 264 O tggatagtgc cqacggttcc acatgtcaat gcacttaatg ccaccaaact gtact cittaa 27 OO aaac agttga ccgggcacgg togcticacgc ctdaatc.cca gcactittgg g g gacc gaggc 276 O. ggg.cggat.ca caaggtoagg agatc gagac catcc togct aac acggtga aacco cqtct 282O citactaaaaa tacaaaagaa ttago.cgggit gcggtgg.cgg gcgtotgtag toccago tac 2880 toggggggct gaggcaggag aatggcttga acctgggagg cqgagcttgc agtgagctga 2.940 gatccago.ca citgcactcca gcctdggcga cagagcaaga citcc.gtotca aacaaaacaa 3OOO agcaaaacaa aaaaaa.cagt taagatttitt tttittttitta aatgatticag toggaaataga 3060 atggattctt caaataactt agccacgggt gggataaggg accitact tag talagtattitt 312 O titcc ccttct ttcttaaaaa tagat.cgatg tottagggtg g gaattaggc titcctggg.cg 318O acacatctaa togcaaagatc agccaccittt ttctgtaaag gatctgatgg taaac attitt 324 O ccacttgaga gctatoctot to cagotact cagctctgct attgcagtgc aaaag cagot 33OO aaaggcaacg gtaaaggaat gacggaagga gccittagttt atttacaata aagctittatt 3360 tgcaaaag.ca gatgcaa.gcc agacittagtt togctgat citc tdatctacag to agaataca 342O cagaga agga gagattittgc cqtataattit aaaatactitc. tctittgcaaa agcagtc.cat 3480 aaaaaaagtg aggacaacaa act gagaaaa attattoaca acatgtctga ttgatagagc 354. O actaatatto ttaattcaaa aag acattitt atcacaaaag aag acaaata cittagaaaat 3600 tgttgcaaaag acttitccatt ttgttgcata acgtaggaag ctittggittitt acttittccta 3660 tdatctittct aactitccagt accagoctaa ttttgttatt tittattatta totatttatt 372 O ttgagacaga gtc.ttgctct gtc.tc.ccagg citggagtgca gtgacct gac gatagottac 378 O. aacago citct accitcc cagg ttcaagaaat cittct cacct tagct tcc.cg agtagctggg 384 O

US 2005/0287530 A1 Dec. 29, 2005 34

-continued <213> ORGANISM: Homo sapiens <400 SEQUENCE: 42 Met Glu Pro Arg Val Thr Glin Arg Lys Arg Pro Leu Asp Gly Cys Met 1 5 10 15 Gly Lys Ile Thr Gly Ile Thr Ser Asp Ile Leu Lys Tyr Asp His Lys 2O 25 3O Cys Phe Lys Lieu Ser Lieu Pro Ala Lys Phe Pro Glu Val Cys Gly Ser 35 40 45 Asp Glu Val Phe Pro Asp Pro Asp Lieu Lieu. His Val Lieu Pro Val Ala 5 O 55 60 Gly Ser Lieu Glin Glin Ser Ile Asp Glin Cys Cys Lieu Gln Leu Glu Ser 65 70 75 8O Lieu. Cys Arg Pro Gly Lieu Lleu. Cys Ala His Pro Thr Lieu Lleu Phe Lys 85 90 95 Leu. His Ser Ser Met Lys Asn Arg Pro Phe Phe Ser Leu Ile Tyr Thr 100 105 110 Tyr Val Lys Lys Thr Glin Glin Val Arg Lys Arg Asp Arg Llys Pro Arg 115 120 125 Gly Glin Val Ala Ala Gly Pro Asn Pro Thr Ser Val 130 135 1 4 0

<210> SEQ ID NO 43 &2 11s LENGTH 208 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 43 citcc caaagt gctgggatta caggcgtgag ccaccatgcc cqg to accitt totcc tdttt 60 totacaggitt agaa.gcaagg cacaagttcat acctacattcaaggagaggg gaccatgcaa. 120 aggataaact gcagagagac alacagatttg catcc cagac acattcacac toggccalaga 18O gcag ctg.cca ttgcag attc gag to acg to cittittittcct tccttctoct tcttggagtt 240 acaacagaag Ctggggaggt gaga.gtgcag aaaggacgtg gatgaag cag agagg Cacgt 3OO gcctgtc.t.ca titcgc.citctg gatctgctgc atcCagg act gtgc.ca.gcac aaagtgg gag 360 cc.cgataaat atttattgaa gaaatgaaag gtgacaaaag goaga aggag g gaga.ccc.ca 420 aaaaga gacc ccaggctcca gaagcagatg aggagacaaa cagaagagca gaagaatggc 480 agaatggcac agcagagaga agaaaaggaa catctgaatg ttgagaggaa tittggctggg 540 ggcggttgga gaggagatta gcc actogac agccaaacto caggggaaga totatottcc c 600 actccatccc cctitccagot coccatccat cotgctgaga gccaccitcca ccacticagtg 660 aalaccactgc attcatccitt Caagtttatgtctgacctga ttctitcc togg acattggaca 720 aag accitagg to cagtgaac totctaacac ttalag to atc. cacggacago aaggctaa.ca 78O gaatgctgta acatgtgc.cc acttggactic toggagttgc agacacccac cogtaggtoc 840 tgcc atgggit coggagcc.ca gaaagtgctt gcc citggctc. citgcaccitgc cc.gtotgcat 9 OO gctocc ccto cqgtaagggg tittgaggatg togcggctga acatalagagc tatgc.ccctg 96.O to acacatca toggacaggg gtcagggaac totcc cattt cactittcaaa tatto Caggit 1020 cc ccttitttgttcgggagcc agagtictagt ggagacagag ataga aacco toaaatagga 1080 to caggcaat ataaacctac acgct gtaag ccaagtcaga gtgaggctga tigtgataa.ca 1140 US 2005/0287530 A1 Dec. 29, 2005 35

-continued aaagttctgaa tacaaggtgg gag acaatta ttctgactga aagggaggca agagttctgc 200 cataggcttg atgctgtgtg toccitggctt cottaact cat ttgcaaatgg aaaatacitat 260 to cattgtag galacct tott titt coatttgtctggggaag gagaaagaat ggctgggctg 320 aatgaattta toccittgg to citctitccaac aaagcto agc tatgaaagat aaatccagot 38O citcc ccaccc cctotcagtg gagctgggga gaaatcaaaa gocccitctgc caataatgag 4 40 accaaagttt gcaagggcag gacgagc.ccg to cita acaga gaaagtgttg titt cotcaat 5 OO ttggttittag actgtc.ttgt Cotatggggg agaaaagatc tocccttggg agaggtgc.ca 560 actittataga totattaata aaagaactgg caggottaca gttcttgcca atgaggaaac 62O ttgaatgaga gaagcc aggc tica accittgg cca acagact ggagcc.catc accotaactt 680 cacccc.gctt citccttaccc aaccatcaaa gqctagg cag cacco acco a gcagottcca 740 cctggctgaa goctocacct gcttcagacc aagggittaga tiggaaatttg goatgggaag 800 agagggctica cct gtgggca ggatagactic tatccaagaa goagaactga aaaatgaaaa 860 Cctatagac a aggggtgat Cctgaaggca ggCaggagala agggctggag ggagagg CaC 920 tggggaattt titcctggtga atactgaagt tactagatgt tttgtc.ttgc aaaactcaag 98O ggaaaactict caaactictaa totttgtcta ttctgttgtcc aaactgtcct tittgaaacgg 20 40 acco acctitt cagtaaagaa acttgcattg gcc tocco 2O78

<210> SEQ ID NO 44 <211& LENGTH: 110 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 44 Met Pro Gly. His Leu Cys Pro Val Phe Tyr Arg Leu Glu Ala Arg His 1 5 10 15 Lys Ser Tyr Lieu. His Ser Arg Arg Gly Asp His Ala Lys Asp Llys Lieu 2O 25 3O Glin Arg Asp Asn Arg Phe Ala Ser Glin Thr His Ser His Trp Ala Lys 35 40 45 Ser Ser Cys His Cys Arg Phe Glu Ser Arg Pro Phe Phe Leu Pro Ser 5 O 55 60 Pro Ser Trp Ser Tyr Asn Arg Ser Trp Gly Gly Glu Ser Ala Glu Arg 65 70 75 8O Thir Trp Met Lys Glin Arg Gly Thr Cys Leu Ser His Ser Pro Leu Asp 85 90 95 Leu Lieu. His Pro Gly Lieu. Cys Glin His Lys Val Gly Ala Arg 100 105 110

<210> SEQ ID NO 45 &2 11s LENGTH 4509 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 45 gagcttgcto tdgtgctagg gtcagottaga caggtoggca ggagaccago togctotagaa 60 gag cct ggac aggatgaatt gottctocitt coacgtgcat cittgcatcc c toccagccaag 120 gatttgaacc caggtotcgt citgacitccaa atgcaccaag aggatggagc ccagagtgac 18O

US 2005/0287530 A1 Dec. 29, 2005 39

-continued actaaaggct cotggagcta ttcacagtgc tiggggaacaa ccagaggagg aggaggacag 228O agg gagagaa tacttitttitt aaaaatcc.ca aacaaaacca aattictatac atgtatatot 234. O cgagtgagta aatgtgtaac acticagoagt citcct acact toccaggttt totggcttca 24 OO tgcagtaata taaaatgtgc titcacaaag.c taggagttgg cacagaatat agg gttctga 2460 ttcatattitt agttttacitt ttttgtagag atggg atccc actatattgc acaggctggit 252O citcaaacticc tdagctcaag cqatccitcct gcc ttggcct cocacggtgc tigagacitaca 258O ggtgtgagcc accacticcca gcctdattta tattittataa agaggcaacg atggagtaca 264 O cagotgagca atctottgca tttctggacc ttgag catca ggaagct tag g gagggtgaa 27 OO aacticagaag aaagcticaga gttgagacct ttgattittitt gcc.ca.gagag to cagctaac 276 O. acactittata accoaaaagg ccttacaaat atttcagagc agagg cago a tttittgcact 282O ggataaacaa aggtacggta atttcttcat agtttattitt cattacaagt aattcttittc 2880 citgaagaaaa atttatattt Cagatttatt toccotcitca aaggggaggg gagittataat 2.940 attctgtttc catago acca titcct caaga toaaaccoag goagg cattt ttacago aca 3OOO cactitccata totcattgct aatcc togca accitt gagga aacto aggtg caaaggggitt 3060 gaggaatgtg titcaccatcc caaag.ccaac gaagtgc.cag aacco aaatc caagcttggc 312 O tgtctdatcc ccaaggacgt actictittatg cc.gttccitta tdacttittca tag cagdaggc 318O gcaagg cago aagaatttgg Ctcttctgac atttatcagg Cacctggitat gtgcc aggCC 324 O tact gtaaac cccaagatga catggtocat gttctato cat tdag catcag agagacitgtt 33OO atcagg actt gaggcctgag acacagocct gaatcagatg caggc citctg. gtgtttgcaa. 3360 aggatgcgtg agtatgtgta totgitatgcg to cattgttgt gtaggggtgt gtgitatgttg 342O ggggttgggg gcagaaaatg citc.cctgaac tottgg tatt aaggcagatc tittgatggag 3480 ggactggggt gtggagacitt galacc ctagt titcgaagtgg ggaccitc gaa cactagtatg 354. O gccagoagac aggctggagt togctgaattg to agctaggit ctittaatcca citc.ccctitct 3600 citaaatcagg gtggaattgg acticagggct g gttcagggc tigaggtttcc gggggatgaa 3660 tgatto.ca.ga cagtgggaag gggtogatgga tigatgaacct ggcaagttcac agg actato.g 372 O acttggctgt atgttgcatg tatgttgtaag atctgagttg agacctttga tttittgcc.ca 378 O. gaaagtccag citaacatact atataagcca gtgagaccag ccagtgtctg. titcattgcct 384 O citgc cittgcc cctatoccitg ccc.gc.citcct atago agg to cotatagoat aatgaaaatg 39 OO cgaacaacct tittctotgga cactggggaa totagtotgg toggataaaga tiggatggctg 396 O ttaaacgatc acatcttggg citctgtgtgt toagtgaagc citcatttacc agatctotct 4020 gatgagaaag gtgaatggtg gggacaaaat acatgctgtt atcagaagtC togg C9ggggc 408 O cctgtgcagt ggcaag cacc totagtcaca gcc actoggg agg cagaggt gggaagatcg 414 O cittgagcc.ca ggaggttgag gotgcattga gctatoatgg caccattgta citccagoctd 4200 gttgacagag tdag accotg totaaaaaag gaaaagaagt ttagggggala agcttittagg 4260 aggtagatga tiggittaattt taatticcacc talacattitta aactittggta totcaaaagg 4320 tgagaattgc cagtgtcaag goatggaaac caaaggaaaa ccagagggitt gagaaggtgg 4.380 gaagggcc.gc agtgtggcag goggcagttt citgggatggc titctact aaa goaactcatc 4 440 atccaccacc titcaggaagg aggcticcitgg ggcc.ctittaa gagag cagtt toagtggagg 4500

US 2005/0287530 A1 Dec. 29, 2005 43

-continued

<213> ORGANISM mouse

<400 SEQUENCE: 49 gtoc tocc cc totctgaacc to atttalaga agg cacgcaa ccggacggcc to attacitat 60 ggctgattica gttcatttitc cct ggatgcc attcc cacct c gottcc togg totgcacaag 120 agatgacatc tatgaggatgaaaatggtag acagtgggta gttgcaaaag tagaaacttic 18O to gttcacca tatggcagca ggattgaaac atgtata act gtgcacttgc agcatatgac 240 cacaatcc cc caggag ccta citc.cccagca gcc cattaat aacaact citc. tcc ccac gat 3OO gtggagatta gag to catga acacatacac gggaacagat gggacatact g gagattact 360 ggat cattcc cagatggg.cg acacattgca act gatcc to gacatagitaa tatgc gaggit 420 tgactogaatg atggaagctd atgggaaggit cittgatagoc toggctggctic citgcc cctot 480 gcc.cittgggg tag actittgc accoaccoct citgtttgttc citcaagtttc tagttctaca 540 gcaccitcctg. ccatgattag acctgcttitt tag attatct actittctgtg agcctggcct 600 aactctgtcc td accgttct coctagotgg agacaaactg catcttctot tactic attitt 660 gtgcctctgg ccctgaccitt atagatggct tittcttaaga cccctccitcc attcaaactic 720 citccaccitgg gottctagtg ccttgttgttgg caggagattt gtc.cc cattt accittaggat 78O gtotacaggit gccitctggag gaggtacaga taggg actot atgcc citgt g g g c citctgag 840 Ccatcactgg Caagaagttc gactggtgct g g g tagaggc Cactcagtgc ticagtag Ctt 9 OO gaaggcacag tdtgagacitt acagagaa.ca totggagtac ttgtcCaggit gcticagttcc 96.O ttggcticcag gaagtgaatc to accotagg ccc citcc caa ccc.ca acttg tttittaacta 1020 attaaactitt attgtttitta cittaaaaaaa aaaaaaaa 1058

<210 SEQ ID NO 50 &2 11s LENGTH 1040 &212> TYPE DNA <213> ORGANISM mouse

<400 SEQUENCE: 50 gtoatt.cccc tdtctgaccg catttaagaa gqcaggcaac Cagacagott cattacitat g 60 gctgattdag titcgtttitcc citgitatgcca titcccaccitt gctitcctggit citgcacaaga 120 gatgacatct atgaggatga acatggtaga cagtgggtag citgcaaaagt ggaaactitct 18O totcattcac catattgcag caagattgaa acctgtgtaa citgtc. cactt gtggcagatg 240 accacactct tccaggagcc tag toccgac totctdaaga citttcaactt totacco agg 3OO acatggagat tagagtccag galacacatac cqgggag cag atgcc atgca citggagatta 360 gtgaatcatt Cocagttitta togg cacagag gaactggtoc to atgctgga ttcaaggtaa 420 citgactaatg gaagctgctg g galaggtott gatagoctogg citggctocto coccitgtgcc 480 cittgggg.cag actittgcacc caccitctotg cittgttcctd aagtttctag titctacaaca 540 cctoct9.cca taggtotttitt acatgatctt citttctgttga gcctggccta totgttggctt 600 gacggttcto cotagotgga ggcaaactgc atcttct citt gct cactittg togc ctittgac 660 actg accitta tagatggctc ttaag accoc tottctottcaaaatcctac acctgggctt 720 ntagtgccitt gtgtgg cagg agatttgtcc ccatttacct taggagttct agaggtgcc.c 78O citaaag gagg tacagataga gactictatoc cqtgtgg gcc totgagc.cat cactggcaag 840 US 2005/0287530 A1 Dec. 29, 2005 44

-continued aagttcgcct ggtgctgggit agaggacact cagtgcticag tagcttgaag goacagtggg 9 OO agacittacag agaacatctg gaggacittgt coaggtogcto agttccttgg citcCaggaag 96.O tgatgctgac cotag accoc toccaaccoc aacttgtttt taactaatta aactttactg 1020 ttitt tactita aaaaaaaaaa. 1040

<210 SEQ ID NO 51 &2 11s LENGTH 1050 &212> TYPE DNA <213> ORGANISM mouse

<400 SEQUENCE: 51 gtoatt.cccc tdtctgaccg catttaagaa gqcaggcaac Cagacago:ct cattacitat g 60 gctgctgttgt citgttgatcc ccagaggcca citcc.ca.gtoc toctogtotc tottagcctg 120 ggcatctato aggatgaaca totatagagtg toggatagotg taaacgtgga aacttct cat 18O toat cacatg gcaa.caggat tdaaacatgt gta actotgc acttgcago a tatgaccaca 240 citcc cc cagg agcc tactico coagcagocc attaataa.ca act citcticcic cac gatgttgg 3OO agattagagt coaggaacac atacacggga acagatggga catactggag attactggat 360 cattcc caga tiggg.cgacac agtgcaactg accotggaca taataatagg cqaggatgac 420 tgaatgatgg gagctgctgg galaggtottgatagoctogc tiggct cotgc ccctgtgcc.c 480 togggg taga citttgcaccc aaccotctgc titgttccitca agtttctagt totaccacac 540 citcctgcc at gactag acct gctttittaga ttatctactt totgtgagcc ttgccitatct 600 citgtc.ctgac cattctocct agctggagac aaactgcatc ttctottgct cactttgtgc 660 citctgacact gaccttatag atggctttitc ttaag accoc tocto cattcaaacticcitcc 720 acctgggctt citagtgccitt gtgtggcagg agatttgtcc ccatttacct taggatgitat 78O acaggtgcct citggaggagg tacagatagg gactictatoc cct gtgggcc totgagc cat 840 cactggcaag aagttcg act ggtgctgggit agaggacact acgtgcticag tagcttgaag 9 OO gcacagtggg agacittacag agaatatotg gagtacttgt coaggtgctic agttccttgg 96.O citccaggaag tdaatctgac cotagg.cccc toccaaccoc aacttgttitt taactaatta 1020 aactttactg tttittactta aaaaaaaaaa 105 O

<210> SEQ ID NO 52 &2 11s LENGTH 6921 &212> TYPE DNA <213> ORGANISM mouse

<400 SEQUENCE: 52 gtoatt.cccc tdtctgaccg catttaagaa gqcaggcaac toggaaggctt cattacitat g 60 gctgctgcag cittittgatcc cct ggggcca citt.ccagtct acct ggtoto C gttagacitg 120 ggcatctato aggatgaaca totatagagtg toggatagttg caaatgtgga aacttct cac 18O toat cacatg gcaa.cagggit gagtttcagg gcticcagaca ggctattocc caact cittac 240 ccct gtgagc tagggtgtgt ttacgtgttt gtgtgttgttgt gtgttgttgttg tagg tatgta 3OO cittittattac cqtgtcagaa aatagotgat acaggtotct gaatagalacc ttgttgtatac 360 citaggcc.cat accotggagc ticcicttggag accitcacagg cccaactaat acacaggtoc 420 agagcc ctag cattgagaac toc gttacag atagt cagag agaac atgct gagagagctt 480 US 2005/0287530 A1 Dec. 29, 2005 45

-continued tgttaggctg. g.gctttgttgg gatgagtagg agttctacag gtcagaaaaa aaaaaaaaag 540 cactctggtg ccaggcagat toccitggaag gag caaatgt gagtgcc agc act acttitcc 600 gcatttctgc aaatcata at tdatctgctg. citgttgctoc cagacitt gag togcctdaag 660 aagttgtttga cagacaaggg atccttacac taggagctga citacagg act togggggaagt 720 cctaatgggit totgttgat cit gcaggtgatg tagaga.ccca cactgctgga aacct gctot 78O ggaccatggc tittagt coac agcaggacct togggggctaa aac actotag toggaaagctic 840 tggg cattaa aactgctocc cagggtogtg gaaaagacita toggccacca ccaccaccc.c 9 OO cactg.ccctg g g g g totgga ggacittgggg gcattagg to tctittagct g c cagg gttitt 96.O cctgcacacc caccocctgt cataacctcc agcc.ca.gcag coctitccct g cagotcc cct O20 gacticcctga atacct cago cattttggca atggg actot cittggtoccc cccitcccctc O8O totccittgac ttacaaggcc cagatcaagg to atgttcac totgg actot cocaaatgtc 14 O tgtttittgcc tatgttctoc tittittaattt actataaaac toctotttct cotcc.ccctt 200 ttcttitcc cc tocagtcact tatcc cctot cotcc.ccctd to citccctac citaggag cag 260 ttctg.ccatc cittittccatt cottgttitta tatto atctg gagaaaactt tdatattitta 320 aggata catc agc catttitt toggatttgag accoaacaag atagtaattic acctgtc.ca.g 38O tdatgagaaa aaaacticttcaagttgataa gtgttttgtt tttittaaaaa acttcaatat 4 40 ttatttactt attittagtat atgagtatac totagctgta tacatggitta tag cctitca 5 OO tgtagttgtt go gaattaga ttittaggaco totgcct gct citccitctgcc citgcttgatc 560 taatctattt atggttgtaa atagg tacac totagotgtc. titcagatgaa ccagaagagg 62O gcatcagatc acattacagg togttgtgag goaccatgttg attgctggga tittgaactca 680 gtacct tcag aagagcagtc agtactcitta ccaccitgagc catcttgcca ggtocgtaag 740 agttittctitt aatcccatac caaataaagt gtaaaatagt ttittgaccaa atattittcag 800 taggaagaaa galaggalagg aagga aggga aggga aggga aggaaggaala ggaaagalaga 860 aatggaagga agaaaagaat gaaaggaagg gagatacaga aac gaattct gttagggitat 920 aaatgaaaga gctgcaaagg aaataatgtg tagtcaatta aatticaaaca aaa.ca catgt 98O citcc cago.ca ttctgcttct gggctgggct totgttgaag cago cattgg toccacgtgt 20 40 gcatagaggg gcaggtgcag caaccaaatg agotccittgt acaactgagt gtaaagggga 2100 gggtgcaggg aagggtctga tdgcatttgt aaaaggagga acctgaggat C Cagg cagag 216 O citgtct citaa gogata catc. caccagaggit gcactgtgga gtgcc tacac agagacagaa 2220 agCacaatgg togggagggag ggggagttcc aag accCtat ggttgccata ggtttggagc 228O aggtgagtgg acagaaagac totgg gatgg gatggggcca totcagg acc ccc cittacca 234. O cc cctotgca ccctgaaatc acgacagtgg ccagggaaag gttccataga aatagaaaga 24 OO ttggagaaat ttaggcacag gtggccct ga attatttccc taattaaggc aatcc cc cct 2460 citgtct citct gtctgtcaac citatagatta cagtact gag gactittgcaa catgctagoa 252O aaatgttcta caactgaggc acattccago cotcc togcta citttittatgt togcaa.caggg 258O tgtcactaaa ttgcc.catgttggtottcaa citgtagtgca ggtgggtotg gaacttgttga 264 O to citcc tocc toagccitcct gagtag totg ggatt.cc agg ttgcaac act aggcc.ca.gca 27 OO ttgagcaact citttittctitc titcctgcttt tottccctitt cocactcittg gttagtatac 276 O. US 2005/0287530 A1 Dec. 29, 2005 46

-continued taagaaatag gttgcactgt tacattctdt citctotcitct citctdtctot citctotcitct 282O citct citctict cacacacaca cacacacaca cacacaccca gcagaaa.cag gga catctaa 2880 attcaagggc agattggcct ataaag.ccag ttgcagticta ggcagggcta cacagagaga 2.940 to citgtctica aataag caca tagctagata gataaataaa totctaaag.c aaa.catalacc 3OOO citgttgaacct gagittaggat coctogaataa aaagttcaggit ttgactittat toccitcaact 3060 to agggctgt ggggagttta gtcatagagg agaaattgat agg acagata totagagtc.ct 312 O cctittggtoc tittcagg cat atatatacac acacacacac acacacacac acacatatat 318O attatatatat acatacacac acacacacac acacacacac acacatatat atatatatat 324 O atagtacatg agtgcataca cacgcatgca cacatataca cccataaa.ca citcatgcaca 33OO gata cataca tatacccaca tacacacaca totacacaca catgtacaca tacacactca 3360 tacacatata tacatacata cacatgcaca cacacagdac toaaacatat acacccacaa 342O gcacactgtg tatgtcagag ttcaaacaga ccattgcttg cittggttgac agcaccittct 3480 cactgggctt tttcaatcac cqg cctocaa gagaagctta totttctgca ggcactgagg 354. O accitaggaac goccttctgt gccaccittag ggcaaaag.ca toc goagaca acagoagttt 3600 tgaatgaaac tittatgtact acaaaag.cag gtggcaa.gcc tacttgggcc cittgtcc citt 3660 citttgctato coccittctgt coaccctgac atcacaacac aag cagaagg gacagactitt 372 O gag.accatag gatggcagcc aggaaaatga Citc.cccacag togt gaccitat Caaagtgtgt 378 O. gtotttagct caacaaaa.ca aaggcatgga totagg tagac aggaggacgc tigg taggaag 384 O totgtgacac actttgtggit ttgttgg to at gcttgg tatt toggittatggc titt tagg acc 39 OO totcittacitt tatgactittctgtgttggat caggtttatt catcaataaa tagg tacatga 396 O agttcaccat caacagat.ca atggatttct tittagtaaag ccattgc.ccc tdgtgtctgt 4020 gtgttgttctg tdtgtttgcg tacatgactg. ittaatgtgtg catgtgttgttg tdtttgttctg 408 O aaggtgttitt gtgtgtttgt gtatgtacct gttcatctgt gcatctgtgt gtgttgttgttgt 414 O gtgtatacaa gagtgtgcat titc catgttgt gtgtgtaaaa cattcaggtg agctg.ccctg 4200 tgactittctt attcccttga taaaacatcc titccctaaat citgcagtacc agtgg cagoc 4260 agccaa.cccc agtgatcc to citgccitcago tocccaag.ca citggtgcaaa tataact gta 4320 gcttitt tact toggtgctgg to attcaaac ttaggggcto citgcctgtgc ggcaggtgct 4.380 cittatctato aaggcatcto cocactcitct gtcttactitt totgaagaga aatttgataa 4 440 citctacaaat ggccaagaca aagttccaaag g g g c gattta gag cotgtga aaggcactda 4500 CagcCtggag acagtggcta Ctttgttggga gatactctgg ggittaag cag gggtggtgga 45 60 agaga.gtgcc agagaggcag aagag cagca gaCaccC cag gCCCtgagga aaaag accta 462O ggag cagoaa acgagcaagt caatagoatg gct gatggaa ccctd citcct aatgtactgc 4680 totcitctatt coctitcagag gagaacco at gta actotcc acttgttggaa gttaatc.ccc 474. O cago aggtta titc.ccttcaa ccc cittgaat tacgactittc tac coacgac gtggaaatta 4800 gagtcc agga acatatact g g g caacagat ggg acto act ggagattact g gatcattcc 4860 caggcaagtg tittggtaccg caagataaaa gotgcagoag gtgtc.ccatg cittitt cattc 4920 tgacco acco ttgg gaggitt cittctoccitc. tctitccccac agtctag coc ttggtocatt 4.980 tittagtggac agttgggaag tatgg tatga gaaaacttitt cittggggaga tigaga caact 5040

US 2005/0287530 A1 Dec. 29, 2005 48

-continued ggcttctato aggatgaaca totatagacitg tdgatggttg caaaactgga aacttgttct 18O cattcaccat attgcaacaa gotgagtttc agtgcto cag acaggcgatc ccc.ca actot 240 tacccittgtg to citaggcto tdttgttgttgt gtgttgttgttgt gtttaggtag gtgctgttat 3OO tatcgtgtca gaatagatga tacaggtaat tdaatagaaa catgtcc cta taccctggag 360 cittgcttgga ggcttcacag gtc.calactaa tag acagatg aagagcc cta gcagtgagga 420 cgctgttaca gatagagaga acatgctgag aagggitttgg taagctgggc tittgttgg gat 480 gagtaggagt totacagg to agaaaaaaac aaaacaaaac aaaacaaaca aataaccaac 540 totggit gcca ggcagattitc. citggaaggag caaatgtaag togc.cago act actitt.ccgca 600 tittctgcaaa toataattica totgctgctg ttgcticcicag gottgagtgg cct gaagaag 660 tgtttgacag agaagg gatc. cittacactag gagctgacta cqgg acttgg gggaaagtc.c 720 taatgg gttctgttgatctgc aggtogatgta gaga.cccaca citgctggaaa cct gctotgg 78O accatggttt tagtccacag caggtoctitg ggggctaaaa cactcitctaa toggaaggctic 840 tgggitatcaa aactgctocc cagggttgttg gaaaagacita toggcc.ccct coccitggcct 9 OO ggaggtggag agaacttggg g g c cittaagt citctttggct gcc agggittt toctoca acc 96.O ccaaccoct9 toctaaccitt titccagocca gtag ccctitc cct gcagotc ccctgacticc O20 citgaatactt cagdcattitt ggcaatgggg citctdttggit toccc.ccitcc cctoccitcct O8O tgacittgcaa gg.cccagatc aaggtoatgttcactctgga Citctoccaaa tagtctgttitt 14 O tgccitatgtt cittccttitta totactatga accitcctcitt totccitcccc cctoctocto 200 cccatcctct cacttctocc cctoctocto agcctccitca cittctocccc toc gocaaac 260 ccttctocco citcctottcc tdtacctdct coataccaag gag catttat ggcttcattt 320 ttcattccitt gttittatatt catctaaaga tigactittgat attittaatag acaccitcago 38O catgtc.tttg gtttgaga.cc taacaa.cagg gtaattic acc tottcaatta tatatatata 4 40 tatatatata tatatatata tatatatata tatatatato ttcaagttga taagttgttitt 5 OO citttittittct cittittcatta aaaaaactcc aatatttatt tatttattitt aatatatgag 560 tacact gtac totata catg gttgtgagcc titcatatggit totttggaat toggattittag 62O gaccitctgcc td citctdatc toccotgctt gctctgatct atttattatt ataaataggit 680 acacggtagc tigtottcaga tigaaccagaa taggacgtoa gatctoatta caggtggttg 740 tgaagcacca tttggittact gggatttgaa cittagttcct ttggaagagc agt cagtgct 800 cittacccact gagctatott gccago.ccca ataag agittt toctitaacco tatactatac 860 aaagtgtaga atagttittga accaaatatt ttcagtagaa acaaagaaag aaagaaaaga 920 aggaaggaag galaggaagga aggaaggaala aaagaaagaa agaaagaaag galaggaagga 98O agaaaggaag aaaggaagaa aggacgaaag gaCaaaagga agaaaggacg alaaggaagaa 20 40 aggacaaaag galagaaagga C gaaaggaag aaaggacgala aggaagaaag gatgaaagga 2100 agaaagaaag aaagaaagaa agaaagaaag aaagaaaaaa aagagaaaat amnaaaaaga 216 O gagagaaaag gnmggaagaa aagaagaacg galaggaagat C Cagaaaaga attctgttag 2220 ggtataaatgaaatgagctg caaaggaaat aatgtgtaat caattaaatt caaatagaat 228O nacatgtc.tc. ccago cattc agctotgggc tigggcttgtg ttgaagcago coattggtoc 234. O cacgtgtgca tagaggggca ggtgcago aa ccaaatgagc ticcittgtaca actgagtgta 24 OO US 2005/0287530 A1 Dec. 29, 2005 49

-continued aaggggaggg ggCagg galag ggtctgatgg catttgtaaa aggaggalacc taggatgca 2460 ggcagagctg totnta aggg attacatccac Cagaggtgca citgtggggtg cct tcc.caga 252O gacagaaag.c acalatggtgg gagggagggg gagttccaag accotiatggit toccataggit 258O ttggag Cagg tgagtggaca gaaag actict gggatgggat ggggc catct caggaccc.cc 264 O cittaccacct citgtgcaccc tgaaatcacc acagtggcca gggaaaggtt C Cataggggt 27 OO agaaagattg gagaaattta ggCacaggtg gcc ctgaatt attitcccitaa ttaaggcaat 276 O. ccccitctotg totctatgtc tgttcaaccta tagattacag taccgaggac tittgcaa.cat 282O gctagoaaaa tgttctacaa citgaggcaca titccagocct cctgctactt tittatgttgc 2880 alacagggtgt cactaaattg cc catgttgg tottcaactg tagtgcaggt gggtotggaa 2.940 cittgttgatcc toct9.cctca gcc toctoag tag totggga titcCaggttg caactictagg cc.cago attg agcaactcitt titt cittct to citgcttittct toccttitccc acticttggitt 3060 agtatactaa gaaataggitt gcactgttac attcticitotic tottctic to totcitc.tc.tc. 312 O totnitotic wo a CaCaCaCaC a CaCaCaCaC a CaCaCaCaC mcasccagaa acagggacat 318O citaa attcaa. ggg Cagattg gcctataaag ccagttgcag totaggcagg gctacacaga 324 O gagatcct gt citcaaataag cacatagota gatagataaa taaatctota aag caaacat aaccotgttga acctgagtta ggaccc.ca.ga ataaaaagtc aggtgtgact ttgtttgctg 3360 aactitcatgg citgtggggag tittagtcaca caggaaaaag tgataggaca gacatctgag 342O tacticcitttg gccttittcag gctggtatat attacatatac tgitatgtgag to cacagaca 3480 cacaccidata tacatgcata cacacgtaca tacacatgta cacacacacc catacacaca 354. O caag cacaca cacatatgtg cacacataca caccacacao acacatacac gaatacacac 3600 acaggcatac acacgtgcac attataccctic atacacacac acagg cacac acacatgcac 3660 acatacccac acatacacac ccatatacac acatgcacat acacatacat gtgcacacat 372 O gtgcacacac agaaac acac acaaaaac at gcacacatgc acacacatat gcacacacala 378 O. atgcacgcac a CaCaCaCaC acacatatgt ggatcaattg gctatataca gacagottgg 384 O totccagtgg agttgagaga tgaaaacctg citgaaac coa gtgttgg to a ttittcaccita 39 OO

Catgggacag aaggcatt.cc cc catctoct ggaCCCCtgg citcctgtcga agttaccacc 396 O occaca acco CCaCaggaga ggctggtggC tittcagtctt gtag acaatig ccc.caag citt 4020 ctg.ccctitta ggctagacitc citcccagtta cctagoaa.ca acaggataac aaggggctgt 408 O ttggcc.ccac citcacticitct coctottact citctaatcto ttactictata agctttacct 414 O citctictittaa. gctttcttag totctagoct titattittctic totcotttitc. tcc tittatct 4200 cctdttggcc atggtoagtc totcitc.ttitt accittittctic aatcc.ccct g cctittctaca 4260 ataaagctict aaaag catag actgtctotg ttcatcaagg accagagitta aaact citc.gc 4320

Ctgcgtggga accitc.tc.tct citctt.cccac tittctgtatc to Caaggcct ggtgctgctic 4.380 cggggcctict attctgttct taitotoctitt gcactatoca gCgtgggata Cagalagactg 4 440 agaact tagt atctgtgact gcc ccttgtc caccc.ctggc agtgtggagt cagtggcttic 4500 acacago cag atgcccacco aggcc caagt ggaaagcatc cgittagtcct cocto atcta 45 60 cctg.cccaga gCaCaggaac totggccaaa cacaagctdt citccctg.ccc cctoattccc citacacccac agcaccacac a CaCaCaCaC a CaCaCaCaC acacacacgg totatgtaag 4680

US 2005/0287530 A1 Dec. 29, 2005 58

-continued caaccocaac ttgtttittaa citaattaaac tttactgttt ttactt 4786

<210 SEQ ID NO 57 &2 11s LENGTH 116 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 57 Met Ala Ala Ala Ala Phe Asp Pro Leu Gly Pro Leu Pro Val Tyr Lieu 1 5 10 15 Val Ser Val Arg Lieu Gly Ile Tyr Glu Asp Glu His His Arg Val Trp 2O 25 3O Ile Val Ala Asn Val Glu Thir Ser His Ser Ser His Gly Asn Arg Arg 35 40 45 Arg Thr His Val Thr Val His Leu Trp Llys Leu Ile Pro Glin Glin Val 5 O 55 60 Ile Pro Phe Asin Pro Leu Asn Tyr Asp Phe Leu Pro Thr Thr Trp Lys 65 70 75 8O Leu Glu Ser Arg Asn. Ile Tyr Trp Ala Thr Asp Gly Thr His Trp Arg 85 90 95 Leu Lieu. Asp His Ser Glin Leu Gly Asp Thr Glu Glin Lieu. Ile Leu Met 100 105 110 Leu Val Lieu Gly 115

<210 SEQ ID NO 58 &2 11s LENGTH 129 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 58 Met Ala Ala Ala Ala Phe Asp Pro Leu Gly Pro Leu Pro Val Tyr Lieu 1 5 10 15 Val Ser Val Arg Lieu Gly Ile Tyr Glu Asp Glu His His Arg Val Trp 2O 25 3O Ile Val Ala Asn Val Glu Thir Ser His Ser Ser His Gly Asn Arg Arg 35 40 45 Arg Thr His Val Thr Val His Leu Trp Llys Leu Ile Pro Glin Glin Val 5 O 55 60 Ile Pro Phe Asin Pro Leu Asn Tyr Asp Phe Leu Pro Thr Thr Trp Lys 65 70 75 8O Leu Glu Ser Arg Asn. Ile Tyr Trp Ala Thr Asp Gly Thr His Trp Arg 85 90 95 Leu Lieu. Asp His Ser Glin Val Lieu. Ile Ala Trp Leu Ala Pro Ala Pro 100 105 110 Val Pro Ser Gly Arg Leu Cys Thr His Pro Ser Val Cys Ser Ser Ser 115 120 125

Phe

<210 SEQ ID NO 59 &2 11s LENGTH 117 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 59 US 2005/0287530 A1 Dec. 29, 2005 59

-continued Met Ala Ala Ala Gly Phe Tyr Pro Pro Arg Leu Leu Pro Glin Val Leu 1 5 10 15 Ile Ser Thr Gly Pro Gly Phe Tyr Glu Asp Glu His His Arg Leu Trp 2O 25 3O Met Val Ala Lys Leu Glu Thir Cys Ser His Ser Pro Tyr Cys Asn Lys 35 40 45 Ile Glu Thr Cys Val Thr Val His Leu Trp Gln Met Thr Arg Tyr Pro 5 O 55 60 Gln Glu Pro Ala Pro Tyr Asn Pro Met Asn Tyr Asn Phe Leu Pro Met 65 70 75 8O Thir Trp Arg Leu Ala Ser Met Asn Thr Tyr Arg Gly Thr Asp Ala Met 85 90 95 His Trp Arg Lieu Lleu. Asn His Ser Glin Val Gly Asp Thr Val Glin Lieu 100 105 110

Ile Leu Met Leu Glu 115

<210 SEQ ID NO 60 <211& LENGTH 122 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 60 Met Ala Asp Ser Val His Phe Pro Trp Met Pro Phe Pro Pro Arg Phe 1 5 10 15 Leu Val Cys Thr Arg Asp Asp Ile Tyr Glu Asp Glu Asn Gly Arg Glin 2O 25 3O Trp Val Val Ala Lys Val Glu Thir Ser Arg Ser Pro Tyr Gly Ser Arg 35 40 45 Ile Glu Thr Cys Ile Thr Val His Leu Gln His Met Thr Thr Ile Pro 5 O 55 60

Glin Glu Pro Thr Pro Glin Glin Pro Ile Asn Asn Asn. Ser Leu Pro Thr 65 70 75 8O Met Trp Arg Leu Glu Ser Met Asn Thr Tyr Thr Gly Thr Asp Gly Thr 85 90 95 Tyr Trp Arg Lieu Lleu. Asp His Ser Gln Met Gly Asp Thr Lieu Gln Leu 100 105 110 Ile Leu Asp Ile Val Ile Cys Glu Val Asp 115 120

<210> SEQ ID NO 61 &2 11s LENGTH 107 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 61 Met Arg Lieu Ser Gly. His Arg Gly Lieu Gln Trp Ala Ser Lieu Arg Phe 1 5 10 15 Ser Gly His Arg Ala Leu Glin Arg Ala Ser Lieu Lys Lieu Ser Gly His 2O 25 3O Leu Ile Glu Thr Cys Ile Thr Val His Leu Gln His Met Thr Thr Ile 35 40 45

Pro Glin Glu Pro Thr Pro Glin Glin Pro Ile Asn Asn Asn. Ser Leu Pro 5 O 55 60 US 2005/0287530 A1 Dec. 29, 2005 60

-continued Thr Met Trp Arg Leu Glu Ser Met Asn Thr Tyr Thr Gly Thr Asp Gly 65 70 75 8O Thr Tyr Trp Arg Lieu Lieu. Asp His Ser Gln Met Gly Asp Thr Lieu Glin 85 90 95 Lieu. Ile Lieu. Asp Ile Val Ile Cys Glu Val Asp 100 105

<210> SEQ ID NO 62 <211& LENGTH: 112 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 62 Met Pro Phe Pro Pro Cys Phe Leu Val Cys Thr Arg Asp Asp Ile Tyr 1 5 10 15 Glu Asp Glu His Gly Arg Glin Trp Val Ala Ala Lys Val Glu Thir Ser 2O 25 3O Ser His Ser Pro Tyr Cys Ser Lys Ile Glu Thr Cys Val Thr Val His 35 40 45 Leu Trp Gln Met Thr Thr Leu Phe Glin Glu Pro Ser Pro Asp Ser Leu 5 O 55 60 Lys Thr Phe Asin Phe Leu Pro Arg Thr Trp Arg Lieu Glu Ser Arg Asn 65 70 75 8O Thr Tyr Arg Gly Ala Asp Ala Met His Trp Arg Leu Val Asn His Ser 85 90 95 Gln Phe Tyr Gly Thr Glu Glu Leu Val Leu Met Leu Asp Ser Arg Ser 100 105 110

<210 SEQ ID NO 63 <211& LENGTH 121 &212> TYPE PRT <213> ORGANISM mouse

<400 SEQUENCE: 63 Met Ala Ala Val Ser Val Asp Pro Glin Arg Pro Leu Pro Val Leu Leu 1 5 10 15 Val Ser Val Ser Leu Gly Ile Tyr Glu Asp Glu His His Arg Val Trp 2O 25 3O Ile Ala Val Asn Val Glu Thir Ser His Ser Ser His Gly Asn Arg Ile 35 40 45 Glu Thr Cys Val Thr Val His Leu Gln His Met Thr Thr Leu Pro Glin 5 O 55 60

Glu Pro Thr Pro Glin Glin Pro Ile Asn Asn Asn. Ser Leu Pro Thr Met 65 70 75 8O Trp Arg Leu Glu Ser Arg Asn Thr Tyr Thr Gly Thr Asp Gly Thr Tyr 85 90 95 Trp Arg Lieu Lieu. Asp His Ser Glin Met Gly Asp Thr Val Glin Lieu. Thr 100 105 110 Leu Asp Ile Ile Ile Gly Glu Asp Asp 115 120

What is claimed is: 2. The isolated nucleic acid of claim 1, wherein Said 1. An isolated nucleic acid comprising a nucleotide nucleotide Sequence encodes a human Tcl-1b protein having Sequence encoding a Tcl-1b protein, wherein Said nucleotide an amino acid sequence of SEQ ID NO:39 from amino acid Sequence is a cDNA sequence. number 1 to 128. US 2005/0287530 A1 Dec. 29, 2005

3. An isolated nucleic acid of not more than 50 kilobases or centromeric, preferably from a T-cell receptor C/Ö which contains at least an 18 nucleotide portion encoding a locus, to Said Tcl-1b gene, and Tcl-1b protein fragment. 4. An isolated nucleic acid of not more than 50 kilobases b) detecting any resulting amplified target Sequence in which contains at least an 18 nucleotide portion of the which the presence of Said amplified target Sequence is sequence depicted in SEQ ID NO: 40. indicative of Said chromosome 14 abnormality. 5. The isolated nucleic acid of claim 1, comprising a 21. The method of claim 20, wherein said chromosome 14 nucleotide sequence of SEQ ID NO:38 from nucleotide abnormality is in a Tcl-1b locus and comprises a number 1 to 1152. t(14:14)(q11:Q32) translocation or an inv (14)(q11:g32) 6. A Tcl-1b protein. inversion. 7. The isolated Tcl-1b protein of claim 6, comprising an 22. A host cell that contains a recombinant vector com amino acid sequence of SEQ. ID. NO: 39 from amino acid prising a cDNA sequence that encodes a human Tcl-1b 1-128. protein having the amino acid sequence of SEQ ID NO:39 from amino acid number 1 to 128. 8. An isolated nucleic acid, comprising a Sequence encod 23. A host cell that contains a recombinant vector com ing a fragment of a protein having an amino Sequence of prising a nucleic acid that is capable of hybridizing under SEQ ID NO.39 from amino acid number 1 to 128, which Stringent conditions to a nucleotide Sequence that is comple fragment can be specifically bound by an antibody to a mentary to a cDNA sequence that encodes a Tcl-1b protein, Tcl-1b protein. which protein has the amino acid sequence of SEQ ID 9. A recombinant DNA vector, comprising a nucleotide NO:39, and said nucleic acid containing at least an 25 Sequence that encodes a Tcl-1b protein, wherein Said nucle nucleotide portion of SEQ ID NO:38. otide Sequence is a cDNA sequence. 25. A pharmaceutical composition, comprising Said anti 10. A host cell that contains said recombinant DNA vector Sense molecule of claim 15 or 16 in a pharmaceutically of claim 7. acceptable carrier. 11. The recombinant DNA vector of claim 7, wherein the 26. A pharmaceutical composition, comprising Said anti nucleotide Sequence encodes a human Tcl-1b protein having body of claim 18 in a pharmaceutically acceptable carrier. an amino acid sequence of SEQ ID NO:39 from amino acid 27. A method for detecting a target nucleotide Sequence number 1 to 128. indicative of a chromosome 14 abnormality in a nucleic acid 12. An isolated nucleic acid of not more than 50 kilobases Sample, comprising the Steps of which contains at least a 50 nucleotide portion of SEQ ID NO: 40. a) hybridizing said Sample with a nucleic acid probe of not 13. An isolated nucleic acid that is capable of hybridizing more than 10 kilobases, comprising in the range of under Stringent conditions to a nucleotide Sequence that is 15-1152 nucleotides complementary to Said nucleotide complementary to the cDNA sequence of SEQ ID NO:38, sequence of SEQ. ID. NO:38; and Said nucleic acid containing at least an 25 nucleotide portion b) detecting or measuring an amount of any resulting of SEO ID NO:38. hybridization between Said probe and Said target 14. An isolated nucleic acid that is capable of hybridizing Sequence within Said Sample. under Stringent conditions to a nucleotide Sequence that is 28. The method of claim 27, wherein said chromosome 14 complementary to a cDNA sequence that encodes a Tcl-1b abnormality is in a Tcl-1b locus and comprises a protein, which protein has an amino acid Sequence of SEQ t(14:14)(q11:Q32) translocation O inv ID NO:39, and said nucleic acid containing at least an 25 (14)(q11:q32)inversion. nucleotide portion of SEQ ID NO:38. 15. An antisense molecule, comprising a nucleotide 29. A method for detecting a Tcl-1b protein in a patient Sequence complementary to at least a part of a coding Sample, preferably a human Sample, comprising: sequence of a Tcl-1b protein, which is hybridizable to a a) contacting said patient Sample with an anti-Tcl-1b Tcl-1b mRNA. antibody under conditions Such that immunospecific 16. The antisense molecule of claim 15, wherein said binding occurs, and nucleotide Sequence is complementary to a least a part of the b) detecting or measuring an amount of any immunospe sequence depicted in SEQ. ID. NO: 38. cific binding by Said antibody. 17. A fusion protein comprising a Tcl-1b protein Sequence 30. A diagnostic kit, comprising in one or more contain of at least 10 amino acids linked to a non-Tcl-1b protein ers, a pair of primers, each having at least 15-25 nucleotides, Sequence. in which at least one of said primers is hybridizable to SEQ. 18. An antibody which binds to an epitope of a Tcl-1b ID. NO: 38 or it complement and wherein said primers are protein. capable of priming DNA synthesis in a nucleic acid ampli 19. An isolated protein comprising an amino acid fication reaction. Sequence having at least 70% amino acid Sequence identity 31. A method for treating a disease State associated with to an amino acid sequence depicted in SEQ. ID. NO: 39, a chromosome 14 abnormality in a mammal, preferably a over a contiguous Sequence of at least 25 amino acids. human, Suffering from Said disease State associated with Said 20. A method for detecting a target Sequence indicative of chromosome 14 abnormality, comprising administering a a chromosome 14 abnormality in a Sample, comprising the therapeutically effective amount of a Tcl-1b antisense mol Steps of: ecule or an anti-Tcl-1b antibody to Said mammal. a) amplifying said target Sequence in Said Sample using a 32. The method of claim 31, wherein said disease state first primer of 18 to 25 nucleotides complementary to comprises a T-cell leukemia or lymphoma and Said chro a TCL-1b nucleotide sequence of SEQ. ID. NO:38, and mosome 14 abnormality comprises a to 14:14)(q11:g32) a Second primer complementary to a region telomeric translocation or an inv (14)(q11:g32) inversion. US 2005/0287530 A1 Dec. 29, 2005 62

33. An isolated nucleic acid comprising a nucleotide 52. A method for detecting a target Sequence indicative of Sequence encoding a Tng1 protein, wherein Said nucleotide a chromosome 14 abnormality in a Sample, comprising the Sequence is a cDNA sequence. Steps of 34. The isolated nucleic acid of claim 33, wherein said nucleotide Sequence encodes a human Tng1 protein having a) amplifying Said target Sequence in Said Sample using a an amino acid sequence of SEQ ID NO:42 from amino acid first primer of 18 to 25 nucleotides complementary to number 1 to 141 a TNG1 nucleotide sequence of SEQ. ID. NO: 41, and 35. An isolated nucleic acid of not more than 50 kilobases a Second primer complementary to a region telomeric which contains at least an 18 nucleotide portion encoding a or centromeric, preferably from a T-cell receptor C/Ö Tng1 protein fragment. locus, to Said Tng1 gene, and 36. An isolated nucleic acid of not more than 50 kilobases b) detecting any resulting amplified target Sequence in which contains at least an 18 nucleotide portion of the which the presence of Said amplified target Sequence is sequence depicted in SEQ ID NO: 45. indicative of Said chromosome 14 abnormality. 37. The isolated nucleic acid of claim 33, comprising a 53. The method of claim 52, wherein said chromosome 14 nucleotide sequence of SEQ ID NO:41 from nucleotide abnormality is in a Tng1 locus and comprises a number 1 to 1500. t(14:14)(q11:Q32) translocation or an inv (14)(q11:g32) 38. A Tng 1 protein. inversion. 39. The isolated Tng1 protein of claim 38, comprising an 54. A host cell that contains a recombinant vector com amino acid sequence of SEQ. ID. NO: 42 from amino acid prising a cDNA sequence that encodes a human Tng1 1-141. protein having the amino acid sequence of SEQ. ID. NO: 42 40. An isolated nucleic acid, comprising a Sequence from amino acid number 1 to 141. encoding a fragment of a protein having an amino Sequence 55. A host cell that contains a recombinant vector com of SEQ ID NO:42 from amino acid number 1 to 141, which prising a nucleic acid that is capable of hybridizing under fragment can be specifically bound by an antibody to a Tng 1 Stringent conditions to a nucleotide Sequence that is comple protein. mentary to a cDNA sequence that encodes a Tng1 protein, 41. A recombinant DNA vector, comprising a nucleotide which protein has the amino acid sequence of SEQ. ID. NO: Sequence that encodes a Tng1 protein, wherein Said nucle 42, and Said nucleic acid containing at least an 25 nucleotide otide Sequence is a cDNA sequence. portion of SEQ. ID. NO: 41. 42. A host cell that contains said recombinant DNA vector 56. A pharmaceutical composition, comprising said anti of claim 39. Sense molecule of claim 47 or 48 in a pharmaceutically 43. The recombinant DNA vector of claim 39, wherein the acceptable carrier. nucleotide Sequence encodes a human Tng1 protein having 57. A pharmaceutical composition, comprising Said anti an amino acid sequence of SEQ ID NO:42 from amino acid body of claim 50 in a pharmaceutically acceptable carrier. number 1 to 141. 58. A method for detecting a target nucleotide Sequence 44. An isolated nucleic acid of not more than 50 kilobases indicative of a chromosome 14 abnormality in a nucleic acid which contains at least a 50 nucleotide portion of SEQ ID Sample, comprising the Steps of NO:45. 45. An isolated nucleic acid that is capable of hybridizing a) hybridizing said Sample with a nucleic acid probe of not under Stringent conditions to a nucleotide Sequence that is more than 10 kilobases, comprising in the range of complementary to the cDNA sequence of SEQ ID NO:41, 15-1500 nucleotides complementary to said nucleotide Said nucleic acid containing at least an 25 nucleotide portion sequence of SEQ. ID. NO: 41; and of SEO ID NO:41. b) detecting or measuring an amount of any resulting 46. An isolated nucleic acid that is capable of hybridizing hybridization between Said probe and Said target under Stringent conditions to a nucleotide Sequence that is Sequence within Said Sample. complementary to a cDNA sequence that encodes a Tng 1 59. The method of claim 58, wherein said chromosome 14 protein, which protein has an amino acid Sequence of SEQ. abnormality is in a Tng1 locus and comprises a ID. NO: 42, and Said nucleic acid containing at least an 25 t(14:14)(q11:Q32) translocation or an inv (14)(q11:g32) nucleotide portion of SEQ. ID. NO: 41. inversion. 47. An antisense molecule, comprising a nucleotide 60. A method for detecting a Tng1 protein in a patient Sequence complementary to at least a part of a coding Sample, preferably a human Sample, comprising: Sequence of a Tng1 protein, which is hybridizable to a Tng 1 mRNA. a) contacting, said patient Sample with an anti-Tng1 48. The antisense molecule of claim 47, wherein said antibody under conditions Such that immunospecific nucleotide Sequence is complementary to a least a part of the binding occurs, and sequence depicted in SEQ. ID. NO:41. b) detecting or measuring an amount of any immunospe 49. A fusion protein comprising a Tng1 protein Sequence cific binding by Said antibody. of at least 10 amino acids linked to a non-Tng1 protein 61. A diagnostic kit, comprising in one or more contain Sequence. ers, a pair of primers, each having at least 15-25 nucleotides, 50. An antibody which binds to an epitope of a Tng1 in which at least one of said primers is hybridizable to SEQ. protien. ID. NO: 41 or it complement and wherein said primers are 51. An isolated protein comprising an amino acid capable of priming DNA synthesis in a nucleic acid ampli Sequence having at least 70% amino acid Sequence identity fication reaction. to an amino acid sequence depicted in SEQ. ID. NO: 42, 62. A method for treating a disease State associated with over a contiguous Sequence of at least 25 amino acids. a chromosome 14 abnormality in a mammal, preferably a US 2005/0287530 A1 Dec. 29, 2005

human, Suffering from Said disease State associated with Said 80. A fusion protein comprising a Tng2 protein Sequence chromosome 14 abnormality, comprising administering a of at least 10 amino acids linked to a non-Tng2 protein therapeutically effective amount of a Tng1 antisense mol Sequence. ecule or an anti-Tng1 antibody to Said mammal. 81. An antibody which binds to an epitope of a Tng2 63. The method of claim 62, wherein said disease state protien. comprises a T-cell leukemia or lymphoma and Said chro 82. An isolated protein comprising an amino acid mosome 14 abnormality comprises a to 14:14)(cq11:g32) Sequence having at least 70% amino acid Sequence identity translocation or an inv (14)(q11:g32) inversion. to an amino acid sequence depicted in SEQ. ID. NO: 44, 64. An isolated nucleic acid comprising a nucleotide over a contiguous Sequence of at least 25 amino acids. Sequence encoding a Tng2 protein, wherein Said nucleotide 83. A method for detecting a target Sequence indicative of Sequence is a cDNA sequence. a chromosome 14 abnormality in a Sample, comprising the 65. The isolated nucleic acid of claim 64, wherein said Steps of nucleotide Sequence encodes a human Tng2 protein having an amino acid sequence of SEQ. ID. NO: 44 from amino a) amplifying Said target Sequence in Said Sample using a acid number 1 to 110. first primer of 18 to 25 nucleotides complementary to 66. An isolated nucleic acid of not more than 50 kilobases a TNG2nucleotide sequence of SEQ. ID. NO: 43, and which contains at least an 18 nucleotide portion encoding a a Second primer complementary to a region telomeric Tng2 protein fragment. or centromeric, preferably from a T-cell receptor C/Ö 67. An isolated nucleic acid of not more than 50 kilobases locus, to Said Tng2 gene, and which contains at least an 18 nucleotide portion of the b) detecting any resulting amplified target Sequence in sequence depicted in SEQ. ID. NO: 46. which the presence of Said amplified target Sequence is 68. The isolated nucleic acid of claim 64, comprising a indicative of Said chromosome 14 abnormality. nucleotide sequence of SEQ ID NO: 43 from nucleotide 84. The method of claim 83, wherein said chromosome 14 number 1 to XXX. abnormality is in a Tng2 locus and comprises a 69. A Tng2 protein. t(14:14)(q11:Q32) translocation or an inv (14)(q11:g32) 70. The isolated Tng2 protein of claim 69, comprising an inversion. amino acid sequence of SEQ. ID. NO: 44 from amino acid 85. A host cell that contains a recombinant vector com 1-110. prising a cDNA sequence that encodes a human Tng2 71. An isolated nucleic acid, comprising a sequence protein having the amino acid sequence of SEQ ID NO: 44 encoding a fragment of a protein having an amino Sequence from amino acid number 1 to 110. of SEO. ID. NO:44 from amino acid number 1 to 110, which 86. A host cell that contains a recombinant vector com fragment can be specifically bound by an antibody to a Tng2 prising a nucleic acid that is capable of hybridizing under protein. Stringent conditions to a nucleotide Sequence that is comple 72. A recombinant DNA vector, comprising a nucleotide mentary to a cDNA sequence that encodes a Tng2 protein, Sequence that encodes a Tng2 protein, wherein Said nucle which protein has the amino acid sequence of SEQ ID NO: otide Sequence is a cDNA sequence. 44, and Said nucleic acid containing at least an 25 nucleotide 73. A host cell that contains said recombinant DNA vector portion of SEQ ID NO: 43. of claim 70. 87. A pharmaceutical composition, comprising Said anti 74. The recombinant DNA vector of claim 70, wherein the sense molecule of claim 78 or 79 in a pharmaceutically nucleotide Sequence encodes a human Tng2 protein having acceptable carrier. an amino acid sequence of SEQ ID NO:44 from amino acid 88. A pharmaceutical composition, comprising Said anti number 1 to 110. body of claim 80 in a pharmaceutically acceptable carrier. 75. An isolated nucleic acid of not more than 50 kilobases 89. A method for detecting a target nucleotide Sequence which contains at least a 50 nucleotide portion of SEQ ID indicative of a chromosome 14 abnormality in a nucleic acid NO:46. Sample, comprising the Steps of 76. An isolated nucleic acid that is capable of hybridizing under Stringent conditions to a nucleotide Sequence that is a) hybridizing said Sample with a nucleic acid probe of not complementary to the cDNA sequence of SEQ ID NO:43, more than 10 kilobases, comprising in the range of Said nucleic acid containing at least an 25 nucleotide portion 15-2000 nucleotides complementary to said nucleotide of SEO ID NO:43. sequence of SEQ. ID. NO: 43; and 77. An isolated nucleic acid that is capable of hybridizing b) detecting or measuring an amount of any resulting under Stringent conditions to a nucleotide Sequence that is hybridization between Said probe and Said target complementary to a cDNA sequence that encodes a Tng2 Sequence within Said Sample. protein, which protein has an amino acid Sequence of SEQ 90. The method of claim 89, wherein said chromosome 14 ID NO: 44, and said nucleic acid containing at least an 25 abnormality is in a Tng2 locus and comprises a nucleotide portion of SEQ ID NO:43. t(14:14)(q11:Q32) translocation or an inv (14)(q11:g32) 78. An antisense molecule, comprising a nucleotide inversion. Sequence complementary to at least a part of a coding 91. A method for detecting a Tng2 protein in a patient Sequence of a Tng2 protein, which is hybridizable to a Tng2 Sample, preferably a human Sample, comprising: mRNA. 79. The antisense molecule of claim 78, wherein said a contacting Said patient Sample with an anti-Tng2 anti nucleotide Sequence is complementary to a least a part of the body under conditions Such that immunospecific bind sequence depicted in SEQ. ID. NO: 43. ing occurs, and US 2005/0287530 A1 Dec. 29, 2005 64

c) detecting or measuring an amount of any immunospe human, Suffering from Said disease State associated with Said cific binding by Said antibody. chromosome 14 abnormality, comprising administering a 92. A diagnostic kit, comprising in one or more contain therapeutically effective amount of a Tng2 antisense mol ers, a pair of primers, each having at least 15-25 nucleotides, ecule or an anti-Tng2 antibody to Said mammal. in which at least one of said primers is hybridizable to SEQ. 94. The method of claim 93, wherein said disease state ID. NO: 43 or it complement and wherein said primers are comprises a T-cell leukemia or lymphoma and Said chro capable of priming DNA synthesis in a nucleic acid ampli mosome 14 abnormality comprises a to 14:14)(q11:g32) fication reaction. translocation or an inv (14)(q11:g32) inversion. 93. A method for treating a disease State associated with a chromosome 14 abnormality in a mammal, preferably a k k k k k