CA2245783A1 - Fhit proteins and nucleic acids and methods based thereon - Google Patents

Abstract

The present invention relates to nucleotide sequences of FHIT genes and amino acid sequences of their encoded proteins, as well as derivatives and analogs thereof, and antibodies thereto. The FHIT gene sequence is mutated in diseases involving cell overproliferation, particularly malignancies of the digestive tract. The present invention further relates to the use of FHIT genes and their encoded proteins as diagnostic and therapeutic reagents for the detection and treatment of disease states associated with cell overproliferation.

Description

W O 97/29119 PCT~US97~01937 F~IT PRO~ ND NUC~EIC ACIDS ~ND l~h~ S BASED ~H~ON

This application is a continuation-in--part of copending ~ application Serial No. 08/598,873 filed February 9, 1996, 5 which is incorporated by reference herein in its entirety.
This invention was made in part with government support under Grant numbers CA51083, CA39860, CA21124, and CA56336 awardèd by the National Cancer Institute. The government has certain rights in the invention.

1. INTRODUCTION
The present invention relates to nucleotide se~uences of the tumor suppressor FHIT genes and amino acid se~uences of their encoded proteins, as well as derivatives and analogs 5 thereof and antibodies thereto. The present invention relates to the use of nucleotide sequences of FHIT genes and amino acid sequences of their encoded proteins, as well as derivatives and analogs thereof and antibodies thereto, as diagnostic and therapeutic reagents for the detection and 20 treatment of cancer. The present invention also relates to therapeutic compositions comprising Fhit proteins, derivatives or analogs thereof, antibodies thereto, nucleic acids encoding the Fhit proteins, derivatives or analogs, and FHIT antisense nucleic acids.

2. BACKGROUND OF THE I~v~llON
Cancer remains one of the most severe health problems in America, accounting for substantial fatality and health costs in society. Tumorigenesis in humans is a complex process 30 involving activation of oncogenes and inactivation of tumor suppressor genes (Bishop, 1991, Cell 64:235-248). Tumor suppressor genes in humans have been identified through studies of genetic changes occurring in cancer cells (Ponder, 1990, Trends Genet. 6:213-218; Weinberg, 1991, Science 35 254:1138-1146).
There is a close association between particular chromosomal abnormalities, e~g., chromosomal translocations, WO 97/29119 PCTrUS97/01937 inversions, and deletions, and certain types of malignancy, indicating that such abnormalities may have a causative role in the cancer process. Chromosomal abnormalities may lead to ~ ~ gene fusion resulting in chimeric oncoproteins, such as is 5 observed in the majority of the tumors involving the myeloid lineage. Alternatively, chromosomal abnormalities may lead to deregulation of protooncogenes by their juxtaposition to a regulatory element active in the hematopoietic cells, such as i6 observed in the translocation occurring in the lymphocytic 10 lineage (Virgilio et al., 1993, Proc. Natl. Acad. Sci. ~SA
90:9275-9279). Deletions may cause loss of tumor suppressor genes, leading to malignancy.
Nonrandom chromosomal translocations are characteristic of most human hematopoietic malignancies (Haluska et al., 15 1987, Ann. Rev. Genet. Z1:321-345) and may be involved in some solid tumors (Croce, 1987, Cell 49:155-156). In B and T
cells, chromosomal translocations and inversions often occur as a consequence of mistakes during the normal process of recombination of the genes for immunoglobulins (Ig) or T-cell 20 receptors (TCR). These rearrangements juxtapose enhancer elements of the Ig or TCR genes to oncogenes whose expression is then deregulated (Croce, 1987, Cell 49:155-156). In the majority of the cases, the rearrangemen~s observed in lymphoid malignancies occur between two different chromosomes.
The TCL-1 locus on chromosome 14 band q32.1 is frequently involved in the chromosomal translocations and inversions with the T-cell receptor genes observed in several post-thymic types-of T-cell leukemias and lymphomas, including ~-prolymphocytic leukemias (~-PLL) (Brito-Babapulle and 30 Catovsky, 1991, C~ncer Genet. Cytogenet. 55:1-9), acute and chronic leukemias associated with the immunodeficiency syndrome-ataxia-telangiectasia (AT) (Russo et al., 1988, Cell 53:137-144; Russo et al., 1989, Proc. Natl. Acad. Sci. USA
86:602-606), and adult T-cell leukemia (Virgilio et al., 1993, 35 Proc. Natl. Acad. Sci. USA 90:9275-9279).
In 1979, a large Italian-American family in Boston was observed to be transmitting a constitutional reciprocal -PCT~S97101937 t~3;8)(pl4.2;q24) chromosome translocation (Cohen et al., 1979, N. Engl. J. Med. 301:592-595; Wang and Perkins, 1984, Cancer Genet. Cytogenet. 11:479-481) which segregated in the family with e~rly onset, bilateral and multifocal clear cell 5 renal carcinoma (RCC). Follow-up cytogenetic studies in several familial tumors demonstrated that the tumors had lost the derivative 8 chromosome carrying the translocated 3pl4-pter region; consequently, the tumors were homozygous for all loci telomeric to the 3pl4.2 break (Li et al., 1993, ~nn~l~$ of 10 Internal Medicine 118:106-111). It was suggested that the translocation affects expression of a tumor suppressor gene (Cohen et al., 1979, N. Engl. ~. Med. 301:592-595) and several investigators have sought candidate suppressor genes. We had suggested the protein tyrosine phosphatase gamma gene (PTPRG) 15 as a candidate tumor suppres~or gene (~aForgia et al., 1991, Proc. Natl. Acad. Sci. USA 88:5036-5040), and that the majority of clear cell RCCs exhibit loss of heterozygosity of a 0.5 Mb region flanking the translocation (~ubinski et al., 1994, cancer Res. 54:3710-3713; Druck et al., 1995, Cancer 20 Res. 55:5348-535S~, although we did not observe aberrations in the remaining P~PRG gene. The 3pl4.2 region is also included in deletions in numerous other tumor types, including nasopharyngeal carcinomas (Lo et al., 1994, Int. J. Oncol.
4:1359-1364).
The t(3;8) translocation breakpoint was cloned and a 3 kb transcript of a candidate tumor suppressor gene was detected using a probe from near the breakpoint (Boldog et al., 1993, Proc. Natl. Acad. Sci. USA 9~:8509-8513); further details concerning this transcript have not been reported in spite of 30 a later publication from this group relating to this subject, and reporting a YAC contig of approximately 6 Mb DNA spanning the 3pl4.2 3;8 translocation breakpoint (Boldog et al., 1994, Genes, Chromosomes & Cancer 11:216-221). It has also been suggested that there may not be a suppressor gene at 3pl4.2, 35 that in fact the t(3;8) translocation was a mechanism for losing the von Hippel-Lindau gene, a tumor suppressor gene at 4 3p25 (Gnarra et al., 1994, Nature Genet. 7:85-90).

Wo 97/29119 CA 02245783 1998-08-06 PCT/US97/olg37 , Another cytogenetic landmark in chromosome region 3pl4.2 is the most common of the constitutive aphidicolin inducible -fragile sites, F~A3B, which is cytogenetically indisting~ h hle from the t(3;8) translocation (Glover et S al., 1988, Cancer Genet. Cytogenet. 31:69-73). Fragile sites, of which over 100 have been described in human (for review, see Sutherland, 1991, Genet. Anal. Tech. Appl. 8:1616-166), are regions of the human genome which reveal cytogenetically detectable gaps when e~G_cd to specific reagents or culture 10 conditions; several folate sensitive, heritable, X-linked and autosomal fragile sites have been localized to unstable CCG or CGG repeats tYu et al., 1991, Science 252:1179--1181; Kremer et al., 1991, Science 252, 1711-1714; Verkerk et al., 1991, Cell 65:905-914; Fu et al., 1991, Cell 67:1047-1058), and ~or one 15 of these, the FRAllB at llq23.3, the CCG repeat is within the 5 ' untranslated region of the CBL2 gene, a known protooncogene (Jones et 81., 1995, Nature 376: 145-149) . Also this fragile site, FRAllB, is associated with Jacobsen (llq-) syndrome, showing a direct link between a fragile site and in 20 vivo chromosome breakage (Jones et al., 1994, Hum. Mol. Genet.

3: 2123--2130) . Because the induced fragile sites resemble gaps or breaks in chromosomes, it has frequently been speculated that fragile sites could be sites of chromosomal rearrangement in cancer (Yunis and Soreng, 1984, Science 226:119g-1204).
25 Previously identified fragile sites have also been shown to be hypermethylated (Knight et al., 1993, Cell 74:127-134); thus methylation of a fragile site in a tumor suppressor gene regulatory region might cause loss of transcription of the suppressor gene, serving as one "hit" in the tumorigenic 30 process, as pointed out previously (Jones et al., 1995, Nature 376:145-149~. These authors also suggested that an important contribution of fragile site expression in tumorigenesis might be to increase the incidence of chromosome deletion during tumorigenesis.
The FRA3B region has been delineated by studies of sQveral groups using rodent-human hybrids; hybrid cells retaining human chromosome 3 or 3 and X, on a hamster W O 97(29~9 PCTn~S97/~1g37 background, were treated with aphidicolin or 6-thioguanine (to se}ect hy~rids which had lost the X chromosome) and subclones ~elected. Subclones ret~i~ing portions of chromosome 3 with apparent breaks in region 3pl4-p21 were characterized for loss or retention of specific 3p markers to determine the position of 3pl4-21 breaks (LaForgia et al., 1991, Proc. Natl. Acad.
Sci. USA 88:5036-5040, LaForgia et al., 1993, Cancer Res.
53:3118-3124; Paradee et al., 1995, Genomics 27:358-361).
r Alterations in oncogenes and tumor suppressor genes in 10 small cell lung cAnce~ (SCLC) and non-small cell lung cancer ~NSCLC) have been described, the most frequent target being alterations of p53 (Tak~h~h; et al., 1989, Science 246:491-494; Chiba et al., 1990, Oncogene 5:1603-1610; Mitsudomi et al., 1992, Oncogene 7:171-180) and retinoblastoma (~arbour et 15 al., 1988, Science 241:353-357; Xu et al., 1994, J. Natl.
Cancer Inst. 86:695-699) genes and allelic deletions of the short arm of chromosome 3 (3p) (Kok et al., 1987, Nature 330:578-581; ~aylor et al., 1987, Nature 329:451-454; Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105). In addition to 20 cytogenetically visible deletions (Whang-Peng et al., 1982, Science 215:181-182; Testa et al., 1994, Genes Chrom. Cancer 11:178-194), loss of heterozygosity (LOH) at loci on 3p has been reported in nearly 100% of SCLC (Kok et al., 1987, Nature 330:578-581; Naylor et al., 1987, Nature 329:451-454; Brauch 25 et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota et al., 1987, Proc. Natl. Acad. Sci. USA. 84:9252-9256) and in 50% or more o~ NSCLC (Brauch et al., 1987, N. Engl. J. Med. 317:1109-1113; Yokota et al., 1987, Proc. Natl. Acad. Sci. USA.
84:9252-9256; Rabbitts et al., 1990, Genes Chrom. Cancer 30 2:231-238; Hibi et al., 1992, Oncogene 7:445-449; Yo~oyama et al., 1992, Cancer Res. 52:873-877; Horio et al., 1993, Cancer Res. 53:~-4), strongly suggesting the presence of at least one tumor suppressor gene in this chromosomal region.
However, the observation that allelic losses often 35 involve most of the 3p has hampered the isolation of the involved gene~s). Candidate loci have been identified such as the von-Hippel Lindau gene, located at 3pZ5, which was WO 97/29119 CA 0 2 2 4 5 7 8 3 l 9 9 8 - O 8 - O 6 PCT/US97/01937 subsequently found to ~e rarely mutated in lung cancer cell lines tSekido et al., 1994, Oncogene 9:1599-1604). Other loci located in a region within 3p21 were reported to be sites of recurrent homozygous deletions in SCLC (Daly et al., 1993, .
5 Oncogene 8:1721-1729; Kok et al., 1993, Proc. Natl. Acad. Sci.
USA 90:6071-6075; Kok et al., 1994, Cancer Res. 54:4183-4187).
In addition, transfer of subchromosomal fragments of the region 3p21.3-p21.2 to tumor cell lines has suggested tumor ~re~sor activity (Killary et al., 1992, Proc. Natl. Acad.
10 Sci.USA 89:10877-10881; Daly et al., 1993, Oncogene 8:1721-1729). More proximal deletions in the 3pl2-14 region have also been reported (Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105; Rabbitts et al., 1990, Genes Chrom. Cancer 2:231-238; Daly et al., 1991, Genomics 9:113-119).
Lung cancer is a major cause of mortality worldwide and the overall survival rate has not improved significantly in the last 20 years. Despite the success achieved by primary prevention, lung cancer is still an overwhelming medical and social problem. Even in the cohort of ex-smokers lung cancer 20 incidence remains high for several years, as a consequence of the accumulated damage, and there is an objective need for strategies aimed at reducing cancer mortality in individuals who have stopped smoking.
There remains an unfulfilled need to isolate and 25 characterize the genes associated with digestive tract and other cancers for use as a diagnostic and therapeutic/
prophylactic reagent in the detection, treatment, and prevention of such cancers.
Citation of a reference hereinabove shall not ~e 30 construed as an admission that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION
The present invention relates to nucleotide sequences of 35 FHIT genes, and amino acid sequences of their encoded FHIT
proteins, as well as derivatives (e.g., fragments) and analogs thereof, and anti hoA i es thereto. The present invention -W O 97~29119 PCTnUS97/01937 .

further relates to nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences as well as equivalent nucleic acid sequences encoding a FHIT protein. ~n a specific embs~ nt, the FHI~ genes and proteins are human s genes and proteins.
Mutations (in particular, deletions) of FHIT gene sequences are associated with esophageal, gastric, colon, kidney, and other cancers.
The present invention also relates to expression vectors 10 encoding a FHIT protein, derivative or analog thereof, as well as host cells containing the expression vectors encoding the FHIT protein, derivative or analog thereof. As used herein, "FHI~" shall be used with reference to the FWIT gene, whereas "Fhit" shall be used with reference to the protein product of 15 the FHI~ gene.
The present invention further relates to the use of nucleotide sequences of F~I~ genes and amino acid sequences of their encoded Fhit proteins as diagnostic reagents or in the preparation of diagnostic agents useful in the detection 20 of cancer or precancerous conditions or hyperproliferative disorders, in particular those associated with chromosomal or molecular abnormalities, in particular at 3pl4.2, and/or decreased levels of expression, or expression of dysfunctional forms, of the Fhit protein. The invention further relates to 25 the use of nucleotide seguences of FHIT genes and amino acid ~e~uences of their encoded Fhit proteins as therapeutic/
prophylactic agents in the treatment/prevention of cancer, in particular, associated with chromosomal or molecular abnormalities at 3pl4.2, and/or decreased levels of 30 expression, or expression of dysfunctional forms, of the Fhit protein.
The~invention also relates to Fhit derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional 35 activities associated with a full-length (wild-type) Fhit protein. Such functional activities include but are not limited to antigenicity ~ability to bind (or compete with Fhit CA 0224~783 1998-08-06 W O 97/29119 PCTrUS97/01937 -for binding) to an anti-Fhit antibody], immunogenicity (ability to generate antibody which binds to Fhit), and ability to bind (or compete with Fhit for binding) to a receptor/ligand or substrate for Fhit, and ability to 5 multmerize with Fhit.
The invention further relates to fragments (and derivatives and analogs thereof) of Fhit which comprise one or more domains of a Fhit protein, e.g., the histadine triad, and/or retain the antigenicity of a Fhit protein (i.e., are 10 able to be bound by an anti-Fhit antibody).
The FNI~ gene and protein sequences disclosed herein, and antibodies to such protein sequences, may be used in assays to diagnose cancers, e.g., digestive tract and airway tumors, associated with chromosomal or molecular abnormalities at 15 3pl4.2, and/or decreased Fhit protein levels or activity by detecting or measuring a decrease in FHIT wild-type mRNA from a patient sample or by detecting or measuring a decrease in levels or activity of Fhit protein from a patient sample, or by detecting an aberrant Fhit DNA, mRNA, or protein.
The ~hit protein, or derivatives or analogs thereof, disclosed herein, may be used for the production of anti-Fhit antibodies which antibodies may be used diagnostically in immunoassays ~or the detection or measurement of Fhit protein in a patient sample. Anti-Fhit antibodies may be used, for 2~ example, for the diagnostic detection or measurement of Fhit protein in biopsied cells and tissues.
The present invention also relates to therapeutic compositions comprising Fhit proteins, derivatives or analogs thereof, antibodies thereto, and nucleic acids encoding the 30 Fhit proteins, derivatives or analogs, and FHIT antisense nucleic acids.
The ~resent invention also relates to therapeutic and diagnostic methods and compositions based on Fhit proteins and nucleic acids. Therapeutic compounds of the invention include 35 but are not limited to Fhit protei~ns and analogs and derivatives (including fragments) thereof; antibodies thereto;

-CA 02245783 l998-08-06 W O 97/29119 PCTnUS97/0193 nucleic acids encoding the Fhit proteins, analogs, or derivatives, and F~IT anti~ense nucleic acidc.
The invention provides methods for prevention or treatment of disorders of overproliferation (e.g., cancer and 5 hyperproliferative disorders) by administering compounds that promote Fhit activity (e.g., Fhit, an agonist of Fhit; nucleic -~ acids that encode Fhit).
~ he invention also provides methods of prevention and treatment of disorders of overproliferation, wherein the 10 patient is hemizygous for a dominant-negative FHIT mutation, by ~ ;stering compounds that specifically antagonize the F~IT mutant nucleic acid or protein (e.g., antibodies or antisense nucleic acids specific to the mutant).
Animal models, diagnostic methods and screening methods 15 for predisposition to disorders, and methods to identify Fhit agonists and antagonists, are also provided by the invention.
The present invention also relates to methods of production o~ the Fhit proteins, derivatives and analogs, such as, for example, by recombinant means.
In a particular embodiment of the invention described by way of example in Section 6, a human FHIT sequence is disclosed and shown to be mutated in various cancers.

4. DESCRIPTION OF THE FIGURES
Figures 1~-lB. Organization of the FHIT gene relative to the 3pl4.2 FRA3B and translocation sites. A scheme of the normal 3pl4.2 region is shown (A) with the chromosomal region (not ,to scale) represented by the top line with positions of STS markers tposition of D3S1234 relative to the gene is not 30 known) ! the FRA3B represented by the hybrid c13 break and the t~3;8) translocation break point shown. The ~she~ portion represents the region involved in the homozygous deletions in tumor cell lines. Three of the YAC clones used in developing the above markers, map and cosmid contig are shown with the 35 cosmid contig below and the distribution of exons in the FHIT
transcript shown below the contig. Black and dotted boxes represent coding and noncoding exons, respectively; asterisks CA 0224~783 l998-08-06 W O 97/29119 PCTrUS97/01937 indicate exons with start and stop codons. One exon (E5) falls within the def ined homozygously deleted region. Exons 1-(E1), 2 (E2) and 3 (E3) fall centromeric to the t(3;8) translocation break and exon 4 (E4) and 6-10 E6-E10) flank the 5 homozygously deleted region on the centromeric and telomeric sides, respectively. organization of types of aberrant transcripts from tumor cell lines are illustrated in part B, with zigzag regions representing insertions of new sequence, usually repetitive, into the aberrant transcripts. CCL234 and 10 235 are colon carcinoma-derived cell lines in which homozygous deletion in the fragile region was not detected. In CCL234 RNA, only an abnormal-sized FHIT transcript was detected by RT-PCR amplification and se~uencing; the shorter transcript was shown to result from splicing of exon 3 to exon 5, with 15 omission of the noncoding exon 4, leaving the coding region intact. With CCL235 RNA as template, apparently normal and aberrant RT-PCR products were amplified, with the aberrant product resulting from splicing of exon 4 to exon 8 with a repetitive insert of 140 bp (contributing an in frame Met 20 codon) between E4 and E8. RT-PCR amplification of RNA from HeLa cells, a cervical carcinoma-derived cell line which exhibited a deletion or a rearrangement of DNA near the t(3;8) transloca~ion, revea}ed normal and aberrant-sized products, the smallest product resulting from splicing of exon 4 to exon 25 9. RT-PCR amplification of RNA from KatoIII, a gastric carcinoma-derived cell line with discontinuous deletions involving the D3S1481 locus and an ~50 kbp region between exons 5 and 6, apparently leaving all FHIT exons intact, resulted in only an aberrant-sized product which is missing 30 exons 4 through 7, with an 86 bp repeat, inserted downstream of exon 3, contributing an in frame Met codon. Amplification of the RT product from HT29, a colon carcinoma-derived cell line with a large deletion (~200 kbp, about the size of the 648D4 YAC3, which included exon 5, gave only an aberrant-sized 3S product resulting from splicing of exon 3 to exon 7. Numerous other tumor-derived cell lines from lung carcinoma (1/3 tested), osteoC~rcoma (1/1), NPC (3/33, ovarian carcinoma =

W O 971~9119 PCTnUS97501937 ~.
(2/2), and hematopoietic (4/5~ tumors, exhibited aberrant F~I~
transcription products. ~he RF~8 cell line, from a stomach carcinoma witl~out deletion, showed a normal-sized product, as did a lymphoblastoid line with the t(3;8) translocation, a 5 melanoma (WM1158) and a kidney carcinoma (RC17)-derived cell line. Other colon and stomach carcinoma-derived lines with deletion (AGS~ LS180, LoVo), or without deletion (Colo320), showed aberrant reverse transcriptase-polymerase chain reaction ~RT-]?CR~ products (not shown).
Figur~s 2A-2B. Structure of normal and aberrant FHIT
cDNAs. The nucleotide (SEQ ID N0:1) and predicted amino acid (SEQ ID NO:2) seguences of the FHI~ gene are shown ~A) with positions of exons indicated by arrowheads above the sequence 15 and positions of primers used in nested PCR and RACE reactions indicated by arrows below the sequence. A schematic presentation of some of the aberrant transcripts observed in uncultured ~umor tissues of digestive organs is shown in B.
Only transcripts which showed deletion of coding sequence in 20 Table 3 are presented. The top line in B shows the intact FHIT cDNA mapO The thick and thin bars show the coding and untranslated regions, respectively. The positions of splice sites are shown by downward arrows, according to the nucleotide numbers shown above in A. The class I transcripts 25 lack exon 5 while class II transcripts retain exon 5 but generally lose exon 8. In the transcripts with asterisks, insertions of various lengths were observed downstream of exon 4. El-10 indicate exons 1-10.

Figures 3A-3C. Expression of the FHI~ gene in normal tissues and tumors. Northern blot (A, B) and RT-PCR analysis (C) of normal and tumor-derived FHIT mRNA. Panel A shows a northern blot of normal mRNAs (2 ~g/lane) from spleen (lane 1), thymus (lane 2), prostate (lane 3), testis (lane 4), ovary 3s (lane 5), small intestine (lane 6)~, colon (mucosal lining) (lane 7), and peripheral blood leukocytes (lane 8), hybridized with the FHI~ cDNA probe. Panel B shows a northern blot of W O97/29119 PCT~US97/01937 mRNAs (2 ~g/lane) from normal small intestine (lane 1) and mRNAs from tumor-derived cell lines: KatoIII (lane 2), HK1 (lane 3~, LoVo (lane 4), CNE2 (lane 5), CNE1 (lane 6), Colo320 (lane 7), LS180 (lane 8), hybridized with the F~IT cDNA probe 5 (panel B, upper). The same blot was hybridized with a ~-actin cDNA probe (panel B, lower). Panel C shows amplified products observed after nested RT-PCR amplification of mRNAs from matched uncultured tumor (T) and normal (N) tissue6 of the ~ame patients (J4, 9625, 5586, E37, E32, E3), or mRNAs from 10 tumor tissues only (J9, J7, J3, J1, E3). Arrowheads show the positions of amplified products with abnormal DNA sequence.
The details of the DNA sequences of corresponding transcripts are shown in Table 2, and Figure 2B. White dots in the tumor lanes show the position of transcripts with normal DNA
1~ sequence.

F~gures ~A-~B. (A) Alignment of amino acid sequences of HIT family proteins and translation of FHIT cDNAs. Alignment was performed using BOXS~ADE version 3Ø Black shading 20 indicates two or more identical residues at a position; gray ~h~;ng indicates similarity. The PAPH1 (SEQ ID NO:3) (accession ~U32615) and CAPH1 (SEQ ID NO:4) (accession ~U28374) designate the S. pombe and S. cerevisiae diadenosine S',5'''-P1, P4 tetraphosphate asymmetric hydrolases (aphl ) .
25 PHIT (SEQ ID NO:6) indicates the HIT family member from the cyanobacterian Synechococcus Sp. (accession #P32084), BHIT
(SEQ ID NO:5), the protein kinase C inhibitor from B. Taurus (bovine; accession #P16436)), MHIT (SEQ ID NO:7) from M.
hyorhinis (mycoplasma, accession #M37339), YHIT (SEQ ID NO:8) 30 from S. cerevisiae (accession #Q04344); the Fhit protein is 69% similar to the S. pombe (PAPHl) gene over a length of 109 amino acids. (B) Tn vitro translation products from recombinant plasmids cont~;ning different alleles of the FHIT
gene: pFHIT1 with a deletion of noncoding exon 4 (lane 1);
3S pFHIT2 with an insertion of 72 bp between exons 4 and 5 (lane 2); pFHIT3 with a wildtype FHIT lacking exon 1 (lane 3); the pFHIT full-length wildtype gene in Bluescript (lane 4);

W O 97/29119 ~CTnUS9~10193~

control reaction, in vitro translation from the pBCAH vector, carrying a portion of the extracellular region of the P~PRG
gene (predicted molecular weight 40 kDa) (lane 5).

Figure 5. Organization of the FNIT gene relative to documented chromosome breaks in the 3pl4.2 fragile region.
One F~I~ allele i8 disrupted in all the translocation carriers of the t(3;8) family, with exons 1, 2 and 3 r~r-ining on the derivative 3 chromosome and exons 4-10, including the entire 10 ~o~in~ region, being translocated to the derivative 8 chromosome, as illustrated above. The hybrid cell line, c13, with a de novo FfiA3B brea~ just telomeric to exon 5, has lost most of the FHIT coding region. The KatoIII cells apparently retain all F~IT exons but encode only an abnormal transcript 15 which lacks exons 4-7 and thus cannot produce Fhit protein.
The MB436 and HT29 cells have both lost exon 5 through deletion of different segments of the fragile region.

FigurQ 6. Hydrophilicity plot of the Fhit deduced 20 protein sequence (SEQ ID NO:2), plotted using the PEPPLOT
program of the Wisconsin GCG software for DNA and protein analysis.

Figure 7. Printout of R50713 nucleotide sequence (SEQ ID
25 NO:9) aligned with the FHIT cDNA sequence (cDNA 7F1) (SEQ ID
NO:l), and the R11128 nucleotide sequence (SEQ ID NO:77). The ~HIT coding region starts at nucleotide 363 and ends at nucleotide 812.

Figure 8. Translation in all three reading frames, both 5' and 3' dire~tions, of the R50713 EST sequence. 5'3' Frame 1: SEQ ID NOS:10-15 and 76; 5'3' Frame 2: SEQ ID NOS:16-19;
5'3' Frame 3: SEQ ID NOS:20-25; 3'5' Frame 1: SEQ ID
NOS:26-31; 3~5~ Frame 2: SEQ ID NOS:32-36; 3'5' Frame 3: SEQ
35 ID NOS:37-40.

WO97/29119 CA 02245783 1998-08-06 PCT~S97/01937 Figure 9. Translation in all three reading frames, both 5' and 3' directions, of the Rl1128 EST se~uence. 5'3' Frame-l: SEQ ID NOS:41-44; 5'3' Frame 2: SEQ ID NOS:45-48; 5'3' Frame 3: SEQ ID NOS:49-56; 3'5' Frame l: SEQ ID NOS:57-58;
5 3'5' Frame 2: SEQ ID NOS:59-64; 3'5' Frame 3: SEQ ID
NOS:65-68.

F~gures l0A-lOB. ~A) Alignment of yeast (S. pombe) Ap4A
hydrolase sequence (U32615) (SEQ ID NO:69) with F~IT cDNA
10 ~cDNA 7~1) se~uence (SEQ ID NO:l). (B) Result of search for homology stretches between U32615 and cDNA 7Fl.

Figures l~A-llB. Expression of the FHIT gene in small cell lung cancer (SCLC). (A) Expression of the FHIT gene by 15 nested RT-PCR analysis in SCLC tumors tT) and matched normal (N) tissues. Case 83L indicates a cell line established from the tumor 83T. Sizes of the amplified products are shown at the right. (B) A schematic presentation of the aberrant transcripts of types I and II observed in tumor tissue of 20 SCLCs. The top line shows the intact FHIT cDNA sequence. The thick and thin bars show the coding and untranslated regions~
respectively. The positions of splice sites are shown by downward arrows, according to the nucleotide numbers. Type I
transcripts lack exons 4 to 6, while type II transcripts lack 25 exons 4 to 8.

Figures 12A-12B. Expression of the FHIT gene in small cell lung cancer and sequences of FHIT transcripts. (A) FHIT
amplified products observed after nested RT-PCR of mRNA from 30 tumor (T) and normal (N) tissues of case 45 and from tumor (T), normal (N) and cell line (L) samples of case 83.
Arrowheads show the sizes of the amplified products.
(B) Sequences of the type I and II abnormal transcripts observed in SCLCs. Arrows indicate junctions between exons 3 35 and 4 in the wild-type transcript~(WT), between exons 3 and 7 in the abnormal transcripts of type I and between exons 3 and 9 in the abnormal transcripts of type II. WT sequence: SEQ ID

WO97/29119 PCT~S97/01937 NO:78. Type I sequence: SEQ ID No:7s. Type II sequence:
SEQ ID NO:80.

Figures 13A-13B. Expression of the F~IT gene in non small 5 cell lung c~cer (NSCLC) and sequences of FHIT transcripts.
(A) Expression of the FHIT gene by nested RT-PCR analysis in NSCLC tumors (T) and paired normal (N) tissues. Arrowheads indicate the ampli~ied abnormal products. (B) Sequences of the abnormal transcripts observed in NSCLC cases 2, 3 and 17.
10 Arrows indicate the junctions of exons 4 to 5 in the wild-type products of cases 2 and 17 (2WT, 17WT) and of exon 3 to 4 in the wild type product of case 3 (3WT). 2A shows the junction between exons 4 and 9 in the abnormal product of case 2, 3A
shows the junction between exons 3 and 8 in the abnormal 15 product of case 3, and 17A shows the junction between exons 4 and 8 in the abnormal product of case 17. WT sequence: SEQ
ID NO:81. 3WT se~uence: SEQ ID NO:82. 17WT sequence: SEQ
ID NO:83. 2A ~equence: SEQ ID NO:84. 3A sequence: SEQ ID
NO:85. 17A sequence: SEQ ID NO:86.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to nucleotide sequences of FHIT genes and amino acid sequences of their encoded Fhit proteins, as well as derivatives and analogs thereof, and 25 antibodies thereto.
As described by way of example infra, the present inventors have isolated and characterized a human FHIT gene, that is involved in esophageal, gastric, colon, kidney, and other c~ncers. Mutations in F~IT gene sequences leading to 30 loss of FHIT gene function are associated with cancer.
The present invention further relates to the use of FHIT
genes and related nucleic acids and their encoded proteins or derivatives or analogs thereof, and antibodies thereto, in assays for the detection and in treatment/prevention of 35 ~ ~fie states associated with chromosomal or mole~ r abnormalities and/or increased expression of FHI~, such as cancer. The present invention also relates to therapeutic compositions comprising Fhit proteins, derivatives or analogs thereof, ant; ho~i es thereto, nucleic acids encoding the Fhit proteins, derivatives or analogs, and FHIT antisense nucleic acids.
s The FHIT gene sequence can be from one of many different species, including but not limited to, vertebrate, mammalian, bovine, ovine, porcine, eguine, rodent and human, in naturally occurring-sequence or in variant form, or from any source, whether natural, synthetic, or recombinant. In a specific 10 embodiment described herein, the F~IT gene sequence is a human sequence. The Fhit protein can be that present in one of many different species, including but not limited to, mammalian, bovine, ovine, porcine, equine, rodent and human, in naturally occurring or variant form, or from any source, whether 15 natural, synthetic, or recombinant. In specific embodiment described herein, the Fhit protein is a human protein.
As defined herein, a Fhit derivative may be a fragment or amino acid variant (e.g., an insertion, substitution and/or deletion derivative) of the Fhit sequence shown in Figure 2A
20 aC long as the fragment or amino acid variant is capable of displaying one or more functional activities associated with a full-length Fhit protein. Such functional activities include but are not limited to antigenicity, i.e., the ability to bind to an anti-Fhit antibody, immunogenicity, i.e., the ability to 25 generate an antibody which is capable of binding a Fhit protein; the ability to inhibit cell proliferation or inhibit tumor growth; the ability to bind (or compete with Fhit for binding) to a substrate for Fhit; ability to multimerize with Fhit; and, possibly, Ap4A or other diadenosine hydrolase 30 activity. The invention provides fragments of a Fhit protein consisting of at least 1o amino acids, or of at least 25 amino acids, or- of at least 50 amino acids, or of at least 100 amino acids. Nucleic acids encoding such derivatives or analogs are al~o within the scope of the invention. A preferred Fhit 3~ protein variant is one sharing at ~least 70% amino acid sequence homology, a particularly preferred Fhit protein variant i5 one sharing at least 8096 amino acid sequence -W~97/29119 PCT~S97101937 homology and another particularly preferred Fhit protein variant is one sharing at least 90% amino acid sequence homology to the naturally occurring Fhit protein over at least 25, at least 50, at least 75, at least lOo, or at least 147 .
5 (full-length) contiguous amino acids of the FHIT amino acid sequence. As used herein, amino acid sequence homology refers ~ to amino acid se~uences having identical amino acid residues or amino acid seqll~nceC contA; n ing conservative changes in -~ amino acid residues. In another embodiment, a FHIT homologous lO protein i8 one that shares the foregoing percentages of sequences identical with the naturally occurring FHIT protein over the recited lengths of amino acids. Proteins encoded by nucleic acids hybridizable to a FHIT gene under non-stringent, moderately stringent, or stringent conditions are also 15 provided.
The present invention also relates to therapeutic and diagnostic methods and compositions based on Fhit proteins and nucleic acids and anti-Fhit antibodies. The invention provides for treatment or prevention of disorders of 20 overproliferation (e.g~, cancer and hyperproliferative disorders) by administering compounds that promote Fhit activity (e.g., Fhit proteins and functionally active analogs and derivatives (including fragments) thereof; nucleic acids encoding the Fhit proteins, analogs, or derivatives, agonists 25 of Fhit).
The inven~ion also provides methods o~ treatment or prevention of disorders of overproliferation in which the subject is hemizygous for a Fhit dominant-negative mutation by adminis~ering compounds to-the subject that specifically 30 antagonize, or inhibit, the dominant-negative function of the Fhit mutant gene or protein (e.g., antibodies or Fhit antisense nucleic acids specific to the mutant~.
Animal models, diagnostic methods and screening methods for predisposi~ion to disorders a~e also provided by the 35 invention.

-W o 97/29119 pcTruss7lol937 5.1. THE FHII' CODING SEOU~:NCES
FHI~ cDNA, genomic sequences and sequences complementary-thereto are FHIT nucleic acids provided by the present invention. In a specific embodiment herein, a FHIT cDNA
5 sequence is provided, thus lacking any introns. Sequences hybridizable thereto, pref~rably lacking introns, are also provided. Nucleic acids comprising FNIT DNA or RNA exon sequences are also provided; in various emho~; ~nts, at least 15, 25 or 50 contiguous nucleotides of FHIT exon sequences are 10 in the nucleic acid. Also included within the scope of the present invention are nucleic acids comprising FHIT cDNA or RNA consisting of at least 8 nucleotides, at least 15 nucleotides, at least 25 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, or at 15 leas~ 350 nucleotides. In various embodiments, nucleic acids are provided that are less than 2,000, less than 500, less than 275, less than 200, less than 100, or less than 50 bases (or bp, if double-stranded). In various emho~;ments, the nucleic acids are less than 300 kb, 200 kb, 100 kb, 50 kb, or 20 10 kb. Nucleic acids can be single-stranded or double-stranded. In specific embodiments, isolated nucleic acids are provided that comprise at least 15 contiguous nucleotides of F~IT coding sequences but which do not comprise all or a portion of any FHIT intron. In a specific embodiment, the 25 nucleic acid comprises at least one FHIT coding exon (exon 5, 6, 7, 8 or 9). In another embodiment, the nucleic acid substantially lacks the FHIT intron between exon 5 and 6, yet contains exon 5 and at least one other FHIT coding exon selected from among exon 6, exon 7, exon 8, and exon 9. In 30 yet another embodiment, the nucleic acid comprises at least one FHIT exon selected from among exon 1, 2, 3, 4 and 5, and cont~;n~-at least one FHIT exon sele¢ted from among exon 6, 7, 8, 9 and 10, and is preferably less than lo kb in size. In a preferred embodiment the FHIT exon sequences appear in the 35 nucleic acid in the order in which they appear in the genome;
in an alternative ~ ho~; ment, the exon se~uences do not appear in the same order. In another embodiment, the nucleic acid W097/29119 PCT~S97101937 comprises all the FHI~ exons ~exons l-lO) or all the F~IT
coding exons (exons 5-93 in contiguous fashion, and thus lacks introns. In yet another specific embodiment, the nucleic acid comprising F~r~ gene exon sequences does not contain sequences S of a genomic ~l~nk;n~ gene (i.e., 5' or 3' to the F~I~ gene in the genome). In a specific embodiment herein, a FHIT genomic sequence is provided, thus cont~;ning introns.
The invention also provides single-stranded oligonucleotides for use as primers in PCR that amplify a F~IT
10 se~uence-cont~;n;ng fragment, e.g., an oligonucleotide having the sequence of a hybridizable portion (at least -8 nucleotides) of a F~IT gene, and another oligonucleotide having the reverse complement of a downstream sequence in the same strand of the FHIT gene, such that each oligonucleotide 15 primes synthecis in a direction toward the other. The oligonucleotides are preferably in the range of 10-35 nucleotides in length.
The full length cDNA seguence for human FHIT is depicted in Figure 2A (SEQ ID NO: l), with the coding region thereof 20 spanning nucleotide numbers 1-441 of Figure 2A. Sequence analysis of the FHIT cDNA of Figure 2A reveals an open reading frame of 441 nucleotides, encoding a protein of 147 amino acids (SEQ ID N0:2).
In accordance with the present invention, any 25 polynucleotide sequence which encodes the amino acid sequence of a FHIT gene product can be used to generate recombinant molecules which direct the expression of Fhit. Included within the scope of the present invention are nucleic acids consisting of at least 8 nucleotides that are useful as probes 30 or primers (i.e., a hybridizable portion) in the detection or amplification of FHIT.
In-a specific embodiment disclosed herein, the invention relates to the nucleic acid sequence of the human F~IT gene.
In a preferred, but not limiting, aspect of the invention, a 35 human FHIT cDNA sequence is that ~resent in plasmid p7Fl as deposited with the ATCC and assigned ATCC Accession Number 69977. Such a sequence can be cloned and sequenced, for W O97/29119 PCTrUS97/01937 example, as described in Section 6, in f ra . The invention also relates to nucleic acid sequences hybridizable or complementary to the foregoing sequences or equivalent to the foregoing sequences in that the equivalent nucleic acid S sequences also encode a protein product displaying Fhit functional activity.
Nucleic acids encoding fragments and derivatives of FHIT
are additionally described infra.
The invention also relates to nucleic acids hybridizable 10 to or complementary to the above-described nucleic acids comprising FHI~ sequences. In specific aspects, nucleic acids are provided which comprise a se~uence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a FHI~ gene. In a specific embodiment, a nucleic 15 acid which is hybridizable to a FHIT nucleic acid, or to a nucleic acid encoding a Fhit derivative, under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl.
20 Acad. Sci. USA 78:6789-6792): Filters containing DNA are pretreated for 6 h at 40~C in a solution containing 35%
formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1%
PVP, O.1% Ficoll, 1% BSA, and 500 ~g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with 25 the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2%
BSA, 100 ~g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40~C, and then washed for 1.5 h at 55~C in a solution contAining 2X SSC, 30 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60~C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68~C and reexposed *o film. Other conditions 35 of low stringency which may be used are well known in the art r (e.g., as employed for cross-species hybridizations).

WO 97129119 PCTIUS9'7101~37 In another specific embodiment, a nucleic acid which is hybridizable to a F~IT nucleic acid under conditions o~ high-stringency is provided (see infra).
In a preferred aspect, polymerase chain reaction (PCR) is 5 used to amplify a desired nucleic acid sequence in a library or from a t;~ e source by using oligonucleotide primers ~ representing known FHIT sequences. Such primers may be used to amplify se~lenc~s of interest from an RNA or DNA source, preferably a cDNA li~rary. PCR can be carried out, e.g., by lo use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp ). The DNA being amplified can include mRNA or cDNA
or genomic DNA from any eukaryotic species. one can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency 15 of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleotide sequence homology between the FHIT gene being cloned and the known FN~T
gene. Other means for primer dependent amplification of nucleic acids are known to those of skill in the art and can 20 be used.
After successful amplification of a segment of a F~TT
gene (e.g., an allelic or polymorphic variant or species homolog of a known FNIT gene) that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a 25 complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra.
In this fashion, additional genes encoding Fhit proteins may 30 be identified. Alternatively, the FHIT gene of the present invention may be isolated through an exon trapping system, using genomic DNA (Nehls et al., 1994, Oncogene 9(8):2169-2175; Verna et al., 1993, Nucleic Acids Res. 21(22):5198:5202;
Auch et al., 1990, Nucleic Acids Res. 18(22):6743-6744).
Potentially, any eukaryotic cell can serve as the nucleic acid source for the molecular cloning of the F~IT gene. The nucleic acid equences encoding FHIT can be isolated from, for CA 0224~783 1998-08-06 W O 97/29119 ~CTrUS97/01937 example, human, porcine, bovine, feline, avian, e~uine, canine, rodent, as well as additional primate sources. The DNA may be obtained by st~n~A~d procedures known in the art from, for exa~ple, cloned DNA (e.g., a DNA "library"), by 5 chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from a desired cell. (See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D . M.
10 (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions while clones derived from cDNA will contain only FHIT exon sequences. Whatever the source, the gene 15 should be molecularly cloned into a suitable vector for propagation of the gene. In a particular embodiment, a preferred source of nucleic acid for the isolation of FHIT
gene sequences is from kidney or stomach or lung cells.
In the molecular cloning of the gene from genomic DNA, 20 DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA
can be physically sheared, as for example, by sonication. The 25 linear DNA fragments can then be separated according to size by standard t~chn;ques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
Once the DNA fragments are generated, identification of 30 the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, a FHIT gene of the present invention or its specific RNA, or a fragment thereof, such as a probe or primer, may be isolated and labeled and then used in hybridization assays to detect a 35 generated FHTT gene (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness, D., 1975, Proc. Natl.
Acad. Sci. USA 72:3961). Those DNA fragments sharing W O 97/29119 PCTnUS97101937 substantial sequence homology to the probe will hybridize under high stringency conditions. The phrase "high stringency conditions" as used herein refers to those hybridizing conditions that (1) employ low ionic strength and high S temperature for washing, for example, 0.015 M NaCl/0.0015 M
sodium citrate/0.1% SDS at 50~C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium 10 phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42~C; or (33 employ 50% formamide, 5 x SSC (0.75 M
NaCl, 0.075 M sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1~ SDS, and 10~
dextran sulfate at 42~C, with washes at 42~C in 0.2 x SSC and 15 0.1% SDS.
It is also possible to identify the appropriate fragment by restriction enzyme digestion(s~ and comparison of fragment sizes with those expected according to a known restriction map. Further selection can be carried out on the basis of the 20 properties of the gene. Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or genomic DNA clones which hybrid-select the proper mRNAs, can be selected which 25 produce a protein that has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, binding activity or antigenic properties as known for FHIT. Alternatively, the FHIT protein may be identified by binding of labeled antibody to the 30 putatively FHIT expressing clones, e.g., in an ELISA ~enzyme-linked immunosorbent assay)-type procedure.
The F~I~ gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation.
In this procedure, fragments are used to isolate complementary 7 35 mRNAs by hybridization. Such DNA fragments may represent available, purified F~IT DNA of another FHIT gene.
Immunoprecipitation analysis or functional assays of the in W O 97/29119 PCTrUS97/01937 vitro translation products of the isolated products of the i~olated mRNAs identifies the mRNA and, therefore, the complemèntary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by s adsorption of polysomes isolated from cells to immobilized ant;ho~ies specifically directed against FHIT protein. A
radiolabelled FHIT cDN~ can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA may then be used as a probe to 10 identify the F~IT DNA fragments from among other genomic DNA
~ragments.
Alternatives to isolating the FHI~ genomic DNA include, but are not limited to, chemically synthesizing the gene ~equence itself from a known sequence or making cDNA to the 15 mRNA which encodes the FHIT protein. For example, RNA useful in cDNA cloning of the FHIT gene can be isolated from cells which express FHIT, e.g., kidney or stomach or lung cells.
Other methods are known to those of skill in the art and are within the scope of the invention.
The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell 25 used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives or the Bluescript vector tStratagene). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a 30 cloning vector which has complementary cohesive termini.
However, if the complementary restriction sites used to ~ragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified.
Alternatively, any site desired may be produced by ligating 35 nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition WO97/29119 PCT~S97~01~37 sequences. In an alternative method, the cleaved vector and F~IT gene may be modified by homopolymeric tailing.
Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, or s other methods known to those of skill in the art, so that many copies of the gene sequence are generated.
In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a ~'shot gun" approach. Enrichment for the lo desired gene, for example, ~y size fractionization, can be done before insertion into the cloning vector.
In specific embodiments, transformation o~ host cells with recombinant DNA molecules that incorporate the isolated F~IT gene, cDNA, or synthesized DNA sequence enables 15 generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when n~ceficAry, retrieving the inserted gene from the isolated recombinant DNA.
Oligonucleotides contAin;ng a portion of the F~IT coding or non-coding sequences, or which encode a portion of the FHIT
protein te.g., primers for use in PCR) can be synthesized by stAn~Ard methods commonly known in the art. Such oligonucleotides preferably have a size in the range of 8 to 25 25 nucleotides. In a specific embodiment herein, such oligonucleotides have a size in the range of ~5 to 25 nucleotides or 15 to 35 nucleotides.
The FHIT sequences provided by the instant invention include those nucleotide sequences encoding substantially the 30 same amino acid sequences as found in native Fhit proteins, and those encoded amino acid sequences with functionally equivalent amino acids, as well as those encoding other Fhit derivatives or analogs, as described infra for Fhit derivatives and analogs.

W O 97/29119 PCTrUS97/01937 5.2. EXPRESSION OF THE FHIT GENE
In accordance with the present invention, nucleotide sequences coding for a FHIT protein, derivative, e.g.
fragment, or analog thereof, can be inserted into an 5 appropriate expression vec~or, i.e., a vector which contains the n~cecc~ry elements for the transcription and translation of the inserted protein-coding sequence, for the generation of recombinant DN~ molecules that direct the expression of a FHIT
protein. Such F~IT polynucleotide sequences, as well as other 10 polynucleotides or their complements, may also be used in nucleic acid hybridization assays, Southern and Northern blot analysis, etc. In a specific embodiment, a human FNIT gene, or a sequence encoding a functionally active portion of a human F~IT gene is expressed. In yet another embodiment, a 15 derivative or fragment of a human FHIT gene is expressed.
Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent FHIT amino acid sequence, is within the scope of the invention. Such DNA sequences include those 20 which are capa~le of hybridizing to the human F~l~ sequence under stringent conditions.
Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a 25 sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within an F~IT sequence, which result in a silent change thus producing a functionally equivalent FHIT protein. Such amino acid 30 substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include 35 lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; g}ycine, alanine;

, WOg7/29119 PCT~S97/~937 asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
The DNA se~uences of the invention may be engineered in order to alter a F~IT coding sequence for a variety of ends 5 including but not limited to alterations which modify proce~sing and expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert - new restriction sites, etc.
In anoth~r emho~; -nt of the invention, a F~I~ gene sequence or a derivative thereof is li~ated to a non-FHIT
sequence to encode a chimeric fusion protein. A fusion protein may also be engineered to contain a cleavage site located between a FHIT sequence and the non-F~rT protein 15 sequence, so that the FHIT protein may be cleaved away from the non-FHIT moiety. In a specific embodiment, the FHIT amino acid sequence present in the fusion protein consists of at least 10 contiguous amino acids, at least 25 contiguous amino acids, at lea~t 50 contiguous amino acids, at least 75 20 contiguous amino acids, at least 100 contiguous amino acids, or at least 147 amino acids (full-length) of the Fhit protein sequence.
In an alternate embodiment of the invention, the coding sequence of a FHIT is synthesized in whole or in part, using 25 chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233;
Crea and Horn, 1980, Nuc. Acids Res. 9(10):233~; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817. Alternatively, 30 the protein itself could be produced using chemical methods to synthesize an FHIT amino acid seguence in whole or in part.
For exa~ple, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography. (e.g., 35 see Creighton, 1983, Proteins Structures And Molec~ r Principles, W.H. Freeman and Co., N.Y. pp. 50-~0). The c~ _ocition of the synthetic peptides may be confirmed by -, W O 97/29119 CA 02245783 1998-08-06 PCT~US97/01937 amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49.
In order to express a biologically active FHIT protein or - 5 derivative thereof, a polynucleotide sequence encoding a FHIT
protein, or a derivative thereof, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation o~ the inserted coding sequence. The F~IT gene products as 10 well as host cells or cell lines transfected or transformed with recombinant FHIT expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that immunospecifically bind a FHIT protein. Anti-FHIT antibodies 15 can be used in detecting or measuring levels of a FHIT protein in patient samples.

5.2.1. EXPRESSION SYSTEMS
Methods which are well known to those skilled in the art 20 can be used to construct expression vectors containing a FHIT
coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic tec~iques and in vivo recombination/genetic recombination. See, for example, the 25 techniques described in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual 2d ed., Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.
A variety of host-expression vector systems may be utilized to express a FNIT coding sequence. These include but are not limited to microorg~n; r ~ such as bacteri~ transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
expression vectors con~; n; ng an F~IT coding sequence; yeast 35 transformed with recombinant yeast expression vectors r containing an FHIT coding sequence, insect cell systems infected with recombinant virus expression vectors (e.g., CA 02245783 l998-08-06 W O97/29119 PCTnUS97101g37 baculovirus) containing an FHI~ coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression 5 vectors (e.g., Ti plasmid) cont~ining an F~IT coding sequence;
or animal cell ~ystems. The expression elements of these systems vary in their strength and specificities. Depen~;nq on the host/vector system utilized, any of a number of suitable transcription and translation elements, including 10 constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage A, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such 15 as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISC0; the promoter for the chlorophyll a/b binding protein3 or from plant viruses 20 (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) 25 may be used; when generating cell lines that contain multiple copies of an FHIT DNA, SV~0-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for 30 the FHIT protein expressed. For example, when large quantities of FHIT protein are to be produced for the generatisn of antibodies, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not 35 limited to the E. col i expression vector pUR278 ~Ruther et al., 1983, EMB0 J. 2:1791), in which the FHIT coding sequence may be ligated into the vector in frame with the lac Z coding -W O 97129119 PCT~US97/01937 region so that a hybrid AS-lac Z protein is produced; pIN
vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 198g, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express 5 foreign polypeptides as fusion proteins with glutathione S-transferase (GST) (Smith and Johnson, 1988, Gene 7:31-40). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
10 The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.
In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review see, Current 15 Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13;
Grant et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, 1987, Acad. Press, N.Y. 153:516-544; Glover, 1986, DNA Cloning, Vol.
20 II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. 152:673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds.
Strathern et al., Cold Spring Harbor Press, Vols. I and II.
In cases where plant expression vectors are used, the expression of an F~I~ coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and l9S RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV
30 (Takamatsu et al., 1987, ~MB0 J. 6:307-311) may be used;
alternatively, plant promoters such as the small subunit of RUBISC0 (Coruzzi et al., 1984, EMB0 J. 3:1671-1680; Broglie et al., 1984, Science 224:838-843); or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al., 1986, 35 Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, W O 971~9119 PCTnUS97J~lg37 microinjection, electroporation, etc. For reviews of such te~hn;ques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant 5 Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
An alternative expression system which could be used to express a FHI~ gene is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in 10 Spodopter~ frugiperd;~ cells. A FHIT coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example, the polyhedrin promoter). Successful insertion of a FHI~ coding sequence will result in 15 inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.
20 (e.g., see Smith et al., 1983, J. Virol. 46:584; 5mith, U.S~
Patent No. 4,215,051).
In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a FHIT coding 2 s sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader se~uence. This chimeric gene ~ay then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of 30 the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a FHlT in -infected hosts. (e.g., see Logan & Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:3655-3659). Alternatively, the vaccinia 7.5 K promoter may be used. (See, e.g., Mackett et _ 35 al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc.
Natl. Acad. Sci. USA 79:4927-4931).

W O 97/29119 PCTrUS97/01937 Specific initiation signals may also be required for e~ficient translation of an inserted FHIT coding sequences.
These signals include the ATG initiation codon and adjacent sequenceG. In cases where an entire FHIT gene, including its 5 own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be nee~. However, in cases where only a portion o~ a FHIT ~oA i ng sequence is inserted, lacking the 5 end, exogenous translational control signals, including the 10 ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of a FHIT coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural 15 and synthetic. The efficiency of expression may be enhanced by the inclusion of a~ iate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).
In addition, a host cell strain may be chosen which 20 modulates the expression of the inserted sequences, or modifies and proc~C~Q the gene product in the specific fashion desired. Such modifications (e.g., phosphorylation) and processing (e.g., cleavage) of protein products may be importan~ for the function of the protein. Different host Z5 cells have characteristic and specific mechanisms for the post-t~anslational processing and modification of proteins.
Appropriate cell~ lines or host systems can be chosen to ensure the CV~Le~ modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which 30 possess the cellular machinery for proper processing of the primary transcript, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
For long-term, high-yield production of recombinant 35 proteins, stable expression is preferred. For example, cell lines which stably express a FHIT protein may be engineered.
Rather than using expression vectors which contain viral WOg7/29119 PCT~S97101937 origins of replication, host cells can be transformed with F~IT D~A controlled by appropriate expression control elements (~.g., promoter, enhancer, sequences, transcription termina-tors, polyadenylation sites, etc.), and a selectable marker.
5 Following the introduction of foreign DNA, engineered cells may be allowed to grow ~or 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their 10 chromosomes and grow to form foci which in turn can be cloned and ~r~n~ed into cell line5. This method may advantageously be used to engineer cell lines which express a FHIT protein.
The present invention provides a method for producing a recombinant FHIT protein comprising culturing a host cell 15 transformed with a recombinant expression vector encoding a FHIT protein such that the FHIT protein is expressed by the cell and recovering the expressed FHIT protein.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase 20 tWigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska ~ Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, 25 respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA
77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. ~SA
78:1527); gpt, which confers resistance to mycophenolic acid 30 (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072);
neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Recently, additional selectable 35 genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman &

CA 0224~783 l998-08-06 W O 97/29119 PCTrUS97/01937 Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC
(ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, ~., 1987, In: Current 5 Communications in Molecular Biology, Cold Spring Harbor Laboratory, Ed.).

5.2.2. IDENTIFICATION OF TRAN~ ANTS OR
TRANSFORMANTS THAT EXPRESS FHIT
The host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker"
gene functions; (c) assessing the level of transcription as 15 measured by the expression of F~IT mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.
In the first approach, the presence of the FHIT coding sequence inserted in the expression vector can be detected by 20 DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the FHIT coding sequence, respectively, or portions or derivatives thereof.
In the second approach, the recombinant expression vector/host system can be identified and selected based upon 25 the presence or absence of certain "mar~er" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the human FHIT coding sequence is inserted within a marker 30 gene sequence of the vector, recombinant cells containing the FHIT coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in t~n~r~ with a FHIT sequence under the control of the same or different promoter used to control the expression of 35 the FHIT coding sequence. Expression of the marker in response to induction or selection indicates expression of the FNI~ coding se~uence.
-W O97119119 PCTnUS97l01g37 In the third approach, transcriptional activity of a FHIT
gene can be assessed by hybridization assays. For example, ~NA can be isolated and analyzed by Northern blot using a probe having seguence homology to a FNIT coding sequence or 5 transcribed noncoding sequence or particular portions thereof.
Alternatively, total nucleic acid of the host cell may be extracted and ~uantitatively assayed for hybridization to such probes.
In the fourth approach, the levels of a FNIT protein 10 product can be A~ses~ed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.

5.3. PURIFICATION OF THE EXPRESSED GENE PRODUCT
Once a recombinant which expresses the FHIT gene sequence is identified, the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, 20 immunoassay, or other detection methods known to those of skill in the art.
Once the FHIT protein is identified, it may be isolated and purified by s~n~d methods including chromatography (e.g., ion exchange, affinity, and sizing column 25 chromatography), centrifugation, differential solubility, or by any other st~n~rd t~chnique for the purification of proteins. The functional properties may be evaluated using any suitable assay.
Alternatively, once a FHIT protein produced by a 30 recombinant is identified, the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric-gene contained in the recombinant. As a result, the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al., 1984, Nature _ 35 310:105-111).
In a specific embodiment of the present invention, such FHIT proteins, whether produced by recombinant DNA tec~n;ques W O 97/29119 PCT~US97/01937 or by chemical synthetic methods, include but are not limited to those contAini ng, as a primary amino acid sequence, all or-part of the amino acid sequence substantially as depicted in Figure 2A (SEQ ID NO:2), as well as fragments and other 5 derivatives, and ana~ogs thereof.

5.4. G~NERATION OF ANTIBODIES TO Fhit According to the invention, Fhit protein, its fragments or other derivatives, or analogs thereof, may be used as an 10 immunogen to generate antibodies which recognize such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a specific embodiment, antibodies to a human Fhit protein are produced.
Various procedures known in the art may be used for the production of polyclonal antibodies to a Fhit protein or derivative or analog. For the production of antibody, various host animals can be immunized by injection with the native Fhit protein, or a synthetic version, or derivative (e.g., 20 fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete~, mineral gels such as aluminum hydroxide, surface 25 active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
For preparation of monoclonal antibodies directed toward a Fhit protein sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma t~chnique originally developed by Kohler and 35 Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma te~hni que (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma WO97J2gll9 PCT~S971019~7 t~chrl;que to produce human monoclonal antibodies (Cole et al., 198~, in Monoclonal Ant;hoAies and Cancer Therapy, Alan R.
Li8s, Inc., pp. 77-96). In an additional ~ ho~iment of the invention, monoclonal an~; ho~; es can be produced in germ-free S animals utilizing recent technology tPCT/US90/02545).
According to the invention, human antibodies may be used and can be obt~in~ by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030) or by trans~orming human ~ cells with EBV virus in vitro (Cole et al., 1985, in lO Monoclonal An~Jbo~;es and Cancer Therapy, Alan R. Liss, pp.
77-96). In fact, according to the invention, t~-hn;ques developed for lthe production of ~ch; --~ic antibodies"
(Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et 15 al., 1985, Nature 314:~52-454) by splicing the genes from a mouse antibody molecule specific for FHIT together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this inventionO
According to the invention, techniques described for the production of single chain antibodies (U.S. Patent No.
4,946,778) can be adapted to produce Fhit-specific single chain antibodies. An additional P~ho~iment of the invention utilizes the te~hni~ues described for the construction of Fab 25 expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for Fhit proteins, derivatives, or analogs.
In a specific embodiment, a molecule comprising a 30 fragment of the Fhit protein is used as an immunogen. For example, since hydrophilic regions are believed most likely to contain sntigenic determinants, a peptide corresponding to or con~; ni ng a hydrophilic portion of a Fhit protein is preferably used as immunogen.
3S Antibody ~ragments which contain the idiotype of the molecule can be generated by known t~chn; ques. For example, such fragments include ~ut are not limited to: the F(ab') 2 -W O 97/29119 PCTrUS97tO1937 fragment which can be produced by pepsin digestion o~ the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab' )2 frag~ent, and the Fab fragments which can be generated by treating the 5 antibody molecule with papain and a reducing agent.
In the production of anti hoA; es, ~;creening for the desired antibody can be accomplished by techlli ques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which r~co~n;ze a specific 10 domain of a Fhit protein, one may assay generated hybridomas for a product which binds to a Fhit fragment cont~i n; ng such domain. For selection of an antibody specific to human Fhit, one can select on the basis o~ positive binding to human Fhit and a lack of binding to, for example, mouse Fhit.
The foregoing antibodies can be used in methods known in the art relating to the localization and activity o~ the protein sequences of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
5~5. STRUC~URE OF THE F~IT GENE AND PROTEIN
The structure of the FH~T gene and protein can be analyzed by various methods known in the art.

5.5.1. GENETIC ANALYSIS
The cloned DNA or cDNA corresponding to the F~I~ gene can be analyzed by methods including but not limited to Southern hybridization (Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see, e.g., Freeman et al., 1983, 30 Proc. Natl. Acad. Sci. USA 80:4094-4098), restriction endonuclease mapping (Maniatis, T., 1982, Molecl~lA~ Cloning, A
Laboratory, Cold Spring Harbor, New York), and DNA se~uence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos.
4,683,202, 4,683,19~, and 4,889,818; Gyllenstein et al., 1988, 35 Proc. Natl. Acad. Sci. USA 85:7652 7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989, Science 243:217-220~
followed by Southern hybridization with a FHI~-specific probe -W O g71~9119 PC~rUS97JO1937 can allow the detection of the F~IT gene in DNA ~rom various cell types. In one ~ ho~i ~nt, Southern hybridization may be used to determine the genetic linkage of F~IT. PC~ followed by hybridization assay can also be used to detect or measure S F~IT P~A or 3pl4 ~ 2 ahr ~ or molec~ ~ abnormalities.
Northern hybridization analysis can be used to determine the expression lev~ls of the FHI~ gene. Other assays are described in Section 5.11. Various cell types, at various states of deve~opment or activity can be tested for FHIT
10 expression. l'he stringency of the hybridization conditions for both Southern and Northern hybridization, or dot blots, can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the speci~ic F~IT probe used.
Restriction endonuclease mapping can be used to roughly determine the genetic structure of the FHTT gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis.
DNA sequence analysis can be performed by any t~-hn; ques 20 known in the art, including but not limited to the method of Maxam and Gilbert ~1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl. Acad.
Sci. USA i4:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), or use of an automated 25 DNA seguenator (e.g., Applied Biosystems, Foster City, CA).
The cDNA sequence of a representative FHIT gene comprises the sequence substantially as depicted in Figure 2A (SEQ ID NO:
1), and described in Section 6, in~ra.

5~5.2. PROTEIN ANALYSIS
The amino acid sequence of the Fhit protein can be derived b~ deduction from the DNA seguence, or alternatively, by direct seguencing of the protein, e.g., with an automated amino acid segl~nc~r. The amino acid seguence of a _ 35 representative Fhit protein comprises the seguence substantially as depicted in Figure 2A ~SEQ ID NO: 2), and CA 02245783 l998-08-06 detailed in Section 6, in~ra, with the representative mature protein that is shown by amino acid numbers 1-147.
The Fhit protein sequence can be further characterized by a hydrophilicity analysis ~Hopp, T. and Woods, K., 1981, Proc.
S Natl. Acad. Sci. USA 78:3824). A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the Fhit protein and the corresponding regions of the gene sequence which encode such regions.
S~con~ry structural analysis ~Chou, P. and Fasman, G., 10 1974, Biochemistry 13:222) can also be done, to identify regions of the Fhit protein that assume specific secondary structures.
Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and 15 plotting, can also be accomplished using computer software programs available in the art.
Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, A., 1974, Biochem. Exp. Biol. 11:7-20 13) and computer modeling (Fletterick, R. and Zoller, M.(eds.), 198~, Computer Graphics and Molecular Modeling, in Current r~ -nications in Mol~c~ r Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

5.6. Fhit PROl~l~S DERIVA~IVES AND ANALOGS
The invention further relates to Fhit proteins, and derivatives (including but not limited to fragments~ and analogs of Fhit proteins. Nucleic acids encoding Fhit protein derivatives and protein analogs are also provided. Molecules 30 comprising Fhit proteins or derivatives are also provided. In one embodiment, the Fhit proteins are encoded by the Fhit nucleic acids described in Section 5.1 supra. In particular aspects, the proteins, derivatives, or analogs are of Fhit proteins of animals.
The production and use of derivatives and analogs related to Fhit are within the scope of the pre~ent invention. In a specific embodiment, the derivative or analog is functionally W O 47l291~9 PCTAUS9~10193~

active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type Fhit protein. As one example, such derivatives or analogs which have the desired immunogenicity or antigenicity can be used, 5 for example, in immunoa~ays, for ; ln;zation, for inhibition of Fhit activity, etc. As another example, such derivatives or analogs which have hydrolase activity are provided.
Derivatives or analogs that retain, or alternatively lack or inhibit, a desired Fhit property of interest (e.g., inhibition 10 of cell prolifQration~ tumor inhibition), can be used as inducers, or inhibitors, respectively, of such property and its physiological correlates. A specific embodiment relates to a Fhit fragment that can be bound by an anti-Fhit antibody.
Derivatives or analogs of Fhit can be tested for the desired 15 activity by procedures known in the art, including but not limited to the assays described infra.
In particular, Fhit derivatives can be made by altering FHIT sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the 20 degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a FHIT gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of FHrT genes which are 25 altered by the substitution of different codons that encode a ~unctionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the Fhit derivatives of the invention include, but are not limited to, those con~A;ning, as a primary amino acid sequence, all or 30 part of the amino acid sequence of a Fhit protein including altered sequences in which functionally equivalent amino acid residues-are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by 35 another amino acid of a similar po~larity which acts as a functional equivalent, resulting in a silent alteration.
Substitutes for an amino acid within the sequence may be CA 0224~783 1998-08-06 W O 97/29119 PCTrUS97/01937 selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino -acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral 5 amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
In a spec~fic emho~iment of the invention, proteins consisting of or comprising a fragment of a Fhit protein consisting of at least 1~ (continuous) amino acids of the Fhit protein is provided. In other embodiments, the fragment consists of at least 20 or 50 amino acids of the Fhit protein.
15 In specific embodiments, such fragments are not larger than 35, 100 or 140 amino acids. Derivatives or analogs of Fhit include but are not limited to those molecules comprising regions that are substantially homologous to Fhit or fragments thereof (e.g., in various embodiments, at least 60% or 70~ or 20 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a coding F~IT sequence, under stringent, 25 moderately stringent, or nonstringent conditions.
The Fhit derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned FHIT gene 30 sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molec~ r Cloning, A
Laboratory Manual, 2d ed., Cold Spring ~arbor Laboratory, Cold Spring Harbor, New York). The se~uence can be cleaved at appropriate sites with restriction endonuclease(s), followed 35 by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of Fhit, care should be taken to ensure .

W O 97/29119 PCTnUS97101937 that the modified gene remains within the same translational reading frame as Fhit, uninterrupted by translational stop signals, in the gene region where the desired Fhit activity is encoded.
Additionally, the Fhit-encod;ng nucleic acid sequence can be ~utated in vitro or in vivo, to create and/or destroy translation, initiation, and/or tel inAtion sequences, or to create variations in coding regions and/or form new restriction ~nAsnllclease sites or destroy preexisting ones, to 10 facilitate further in ~itro modification. Any t~nique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchi~on, C., et al., 1978, J. Biol. Chem 253:6551), etc.
Manipulations of the Fhit sequence may also be made at the protein level. Included within the scope of the invention are Fhit protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, 20 amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modi~ications may be carried out by known te~hn i ques, including but not limited to specific chemical cleavage by cyanogen bromide, 25 trypsin, chymotrypsin, papain, V8 protease, NaBH4;
acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
-In addition, analogs and derivatives of Fhit can be chemically synthesized. For example, a peptide corresponding 30 to a portion of a Fhit protein which comprises the desired domain (see Section 5.6.1), or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a - 35 substitution or addition into the~Fhit sequence. Non-classical amino acids include but are not limited to the D-is- ~rs of the common amino acids, ~-amino isobutyric acid, 4-wog7l2sll9 CA 0224s7s3 1998-08-06 pcT~ss7/ol937 aminobutyric acid, Abu, 2-amino butyric acid, ~-Abu, ~-Ahx, 6-amino h~Anoic acid, Aib, 2-amino isobutyric acid, 3-amino -propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-5 butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ~-alanine, fluoro-amino acids, designer amino acids such as ~-methyl amino acids, C~-methyl amino acids, N~-methyl amino acids, and amino acid analogs in general~ Furthermore, the amino acid can be D (dextrorotary) lO or ~ (levorotary).
In a specific embodiment, the Fhit deri~ative is a chimeric, or fusion, protein comprising a Fhit protein or fragment thereof (preferably consisting of at least a domain or motif of the Fhit protein, or at least lO amino acids of 15 the Fhit protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a c~i ~ric protein is produced by recombinant expression of a nucleic acid Pnco~inq the protein (comprising a Fhit-coding sequence joined in-frame 20 to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by 25 methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic tech~;ques, e.g., by use of a peptide synthesizer. Chimeric genes comprising portions of FHI~ fused to any heterologous protein-encoA ~ ng sequences may be constructed.
In another specific embodiment, the Fhit derivative i~ a molecule comprising a region of homology with a Fhit protein.
By way of example, in various embodiments, a first protein region can be considered "homologous'l to a second protein region when the amino acid sequence of the first region is at 35 least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%
identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in WO97129119 PCT~S97101937 the first region or when compared to an aligned sequence of the ~~-ond region that has been aligned by a computer homology program known :in the art. For example, a molecule can comprise one or more regions homologous to a Fhit ~o ~; n ~see 5 Section 5.6.l) or a portion thereof or a full-length Fhit protein.

5.7. ASSAYS OF Fhit PROl~lN~, D~IVATIV~S AND ANALOGS
The functional activity of Fhit proteins, derivatives and analogs can be assayed by various methods.
For example, in one embodiment, where one is assaying for the ability to bind or compete with wild-type Fhit for binding to anti-Fhit antibody, various immunoassays known in the art 15 can be used, including but not limited to competitive and non-competitive assay systems using ~ohn;~ues such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in 20 sf ~u immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, 25 and immunoelectrophoresis assays, etc. In one ~ hoA; ~t, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the 30 secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In another embodiment, where a Fhit-binding protein is identified, the binding can be assayed, e.g., by means well-3~ known in the art.
In another embodiment, should a Fhit protein havehydrolase activity, hydrolase assays can be used to measure ~, CA 02245783 1998-08-06 PCTrUS97/01937 Fhit hydrolase activity. Such assays can be carried out by methods well known in the art.
In addition, assays known in the art can ~e used to detect or ~ re the ability to inhibit cell proliferation, 5 in vitro or in v~vo.
Other methods will ~e known to the skilled artisan and are within the scope of the invention.

5.8. THERAPEUTIC USES: TR~M~T AND
PR~v~llON OF DISORDERS INVOLVING
OV~PROLIFERATION OF ~rT~
The invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic"). Such "Therapeutics" include but are not limited to: Fhit proteins 15 and analogs and derivatives (including fragments) thereof (e.g., as described hereinabove); antibodies thereto (as described hereinabove); nucleic acids encoding the Fhit proteins, analogs, or derivatives (e.g., as described hereinabove); and Fhit agonists, and antagonists of mutant 20 F~IT genes or proteins (e.g., antibodies or antisense nucleic acids). In a preferred embodiment, disorders involving cell overproliferation are treated or prevented by administration of a Therapeutic that promotes Fhit function. The above is described in detail in the subsections below.
Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, a human Fhit protein, derivative, or analog, or nucleic acid, or an antibody to a human Fhit 30 protein or human FHIT antisense nucleic acid, is therapeutically or prophylactically administered to a human patient.~
Additional descriptions and sources of Therapeutics that can be used according to the invention are found in Sections 35 5.1 through 5.7 herein.

W O 97129119 PCT~US971~1937 A F~I~ polynucleotide and its Fhit protein product can be used for therapeutic/ prophy~actic purposes for diseases involving cell overproliferation, as well as other disorders associated with chromosomal translocations or inversions or S mol~ ~ abnormalities associated with the FHIT locus, and/or decr~ expression of wild-type FHIT RNA or protein and/or expression of a mutant FHIT RNA or protein and/or expression of a mutant F~IT RNA or protein. A FNI~ polynucleotide, and its FHIT protein product, may be used for 10 therapeutic/prophylactic purposes alone or in combination with other therapeutics useful in the treatment of cancer and hyperproliferative or dysproliferative disorders.
Diseases and disorders involving cell overproliferation are treated or prevented by administration of a Therapeutic 15 that promotes (i.e., increases or supplies) Fhit function.
Examples of such a Therapeutic include but are not limited to Fhit proteins, derivatives, or fragments that are functionally active, particularly that are active in inhibiting cell proliferation (e.g., as demonstrated in in vitro assays or in 20 animal ~o~el~), and nucleic acids encoding a Fhit protein or functionally active derivative or fragment thereof (e.g., for use in gene therapy). Other Therapeutics that can be used, e.g., Fhit agonists, can be identified using in vitro assays or A~ i m~ l models, examples of which are described infra .
In specific embodiments, ~herapeutics that promote Fhit function are administered therapeutically (including prophylactically): (1) in diseases or disorders involving an absence or decreased (relative to normal or desired) level of Fhit functional protein or of Fhit function, for example, in 30 patients where Fhit protein is lacking, genetically defective, biologically inactive or underactive, or underexpressed; or (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of Fhit agonist a~m; n; ~tration.
The absence or decreased level in Fhit protein or function can _ 35 be readily detected, e.g., by obtA;n;ng a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or protein levels, structure and/or activi~y of the W O 97/29119 CA 02245783 l998-08-06 PCTnU$97/01937 expressed Fhit RNA or protein (see section 5.11 infra re assays uced in diagnosi~ any methods stA~d~rd in the art-can be thus employed, including ~ut not limited to immunoas~ays to detect and/or visualize Fhit protein (e.g., 5 Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect Fhit expression by detecting and/or vi~ualizing Fhit mRNA or cDNA (e.g., Northern assays, dot blots, in situ 10 hybridization, and preferably those assays described in Section 5.11), etc.
Diseases and disorders involving cell overproliferation that can be treated or prevented include but are not limited to malignancies, premalignant conditions ~e.g., hyperplasia, 15 metaplasia, dysplasia), benign tumors, hyperproliferative disorders, benign dysproliferative disorders, etc. Examples of these are detailed below.

5.8.1. MALIGNANCI~S
Z0 Malignancies and related disorders that can be treated or prevented by administration of a Therapeutic that promotes Fhit function te.g., a full-length Fhit protein or functional derivative thereof or nucleic acid encoding the foregoing) include but are not limited to those listed in Table 1 (for a z5 review of such disorders, see Fishman et al., 1985 , Medicine , 2d Ed., J.B. Lippincott Co., Philadelphia~:

MALIGNANCIES AND R~T~TED DISORDERS
Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic 3~ promyelocytic myelomonocytio monocytic erythroleukemia - ~8 -CA 02245783 l998-08-06 WO 97129119 ~CTnus97JQlg37 chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma - - Ho~k~ n~5 disease non--~oA~kin~s disease Multiple myeloma Waldenstrom's macroglobulinemia Heavy cha~in disease Solid tumors sa$~ and carcinomas fibrosarcoma myxosarcoma liposarcoma ch~l.d~arcoma osteogenic sarcoma osteosarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma colorectal carcinoma pancreatic cancer breast C~n~r ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary ad~oc~cinomas ~ cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile duct carcinoma ~ choriocarcinoma seminoma embryonal carcinoma Wilms' tumor - 3S cervical cancer uterine cancer testicular tumor lung carcinoma W O 97/29119 PCTrUS97/01937 small cell lung carcinoma non small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma -- - astrocytoma S medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma menangioma melanoma neuroblastoma retinoblastoma nasopharyngeal carcinoma esophageal carcinoma In a specific embodiment, digestive tract tumors are treated or prevented, including but not limited to esophageal, stomach, colon, and colorectal cancers. In another specific emho~; -nt, airway c~c-rs such as lung cancers (e.g., small 20 cell lung carcinoma) and nasopharyngeal carcinoma are treated or prevented. In yet other specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented in the head, neck, cervix, kidney, stomach, skin, 25 ovary, bladder, breast, colon, lung, or uterus. In other specific embodiments, sarcoma, or leukemia is treated or prevented. In another particular emho~; ?nt, osteosarcoma or renal cell carcinoma is treated or prevented.

5.8.2. PREMALIGNANT CONDITIONS
The T~erapeutics of the invention that promote Fhit activity can also be administered to treat premalignant conditions and to prevent pLoyLe~sion to a neoplastic or malignant state, including but not limited to those disorders 35 listed in Table 1. Such prophylac~tic or therapeutic use is indicated in conditions known or suspected of prec-~ing o~Lession to neoplasia or cancer, in particular, where non-_ -CA 02245783 l998-08-06 W O 97129119 PCTnUS9710193~

neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has oc~.Led ~for review of ~
such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., PhilA~elr~;~, pp.
s 68-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often ~ precedes endo~etrial cancer. Metaplasia is a form of 10 controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner ~5 of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
20 Dysplasia characteristically occ~rs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
In a preferred embo~i ~nt of the invention, a patient in whose DNA is detected a mutation in the F~I~ gene, 25 particularly a deletion, and most particularly a homozygous mutation, is thereby determined to have a predisposition to cancer and is treated by administration of a Fhit protein or functional derivative thereof or nucleic acid encoding the ~ame (gene therapy).
Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dyspla~ia, the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or di~played in vitro by a cell sample from a patient, 35 can indicate the desirability of ~rophylactic/therapeutic administration of a Therapeutic that promotes Fhit function.
As mentioned s~pra, such characteristics of a transformed W O97/29119 PCT~US97/01937 phenotype include morphology changes, looser su~stratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, 5 disappearance of the 250,000 dalton cell surface protein, etc.
(see also id., at pp. 84-90 for characteristics as~ociated with a transformed or malignant phenotype).
In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or 10 Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
In another embodiment, fibrocystic disease ~cystic hyperplasia, mammary dysplasia, particularly adenosis (benign 15 epithelial hyperplasia)) is indicative of the desirability of prophylactic intervention.
In other embodiments, a patient which exhibits one or more of the following predisposing factors for malignancy i5 treated by A~ tration of an ef~ective amount of a 20 Therapeutic: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia, t(l4;18) for follicular lymphoma, etc.~, familial polyposis or Gardner~s syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy ~a possible 25 forerunner of multiple myeloma3, and a first degree k;n~hip with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e . g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid 30 carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblsstoma, carotid body tumor, cutaneous -l~nocarcinoma, intraocular melanocarcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's 3s aplastic anemia, and Bloom's syndrome; see Robbins and Angell, 1976 , Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.

W~97J29119 PCT~S97101937 In a specific emho~iment~ a Therapeutic of the invention is administered to a human patient to prevent progression to~
breast, colon, lung, stomach or uterine cancer, or melanoma or sarcoma.
5.8.3. HYPE~PROLIFERATIVE AND
~YSPRo~TFE~ATIVE DISORDERS
In another emh~Aiment of the invention, a Therapeutic ~ that promotes Fhit activity is used to treat or prevent lO hyperproliferative or benign dysproliferative disorders.
Specific embodiments are directed to treatment or prevention of benign tumors, fibrocystic conditions, and tis~ue hypertrophy (e.g., prostatic hyperplasia). In specific embodiments, a patient having an intestinal polyp, colon 15 polyp, or esophageal dysplasia is treated by administration of a ~herapeutic.

5.8.4. GE~E THERAPY
In a specific embodiment, nucleic acids comprising a 20 sequence encoding a Fhit protein or functional derivative thereof, are administered to promote Fhit function, by way of gene therapy. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
A F~IT polynucleotide may be used in the treatment of 25 various disease states associated with chromosome 3pl4.2 abnormalitiest such as cancers, and/or decreased expression of wild-type FHI'~ RNA or protein. By introducing F~IT gene sequences into cells, gene therapy can be used to treat conditions associated with under-expression o~ functional FHIT
30 RNA or protein. Accordingly, the present invention provides a method for treating a disease state associated with a chromosome 3pl4.2 abnormality in mammal suffering from a disease state associated with a chromosome 3pl4.2 abnormality comprising administering a therapeutically effective amount of 35 a nucleic acid encoding a functional Fhit protein to a mammal suffering from a disease state associated with a chromosome 3pl4.2 abnormality. In this embodiment of the invention, the ~, W O 97129119 PCTrUS97/01937 nucleic acid pro~ its encoded protein that mediates a therapeutic effect by promoting Fhit function, thereby, e.g.,~
inhibiting tumor or c~ncer appearance or progression.
Any of the methods for gene therapy available in the art S can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev.
10 Pharmacol. Toxicol. 3~:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, 15 Current Protocols in Molecular Biology, John Wiley & Sons, NY;
and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.
In a preferred aspect, the Therapeutic comprises a FHI~
nucleic acid that is part of an expression vector that 20 expresses a Fhit protein or fragment or chimeric protein thereof in a suitable host. In particular, such a nucleic acid has a promoter operably linked to the F~IT coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular 25 embodiment, a nucleic acid molecule is used in which the F~IT
coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a de~ired site in the genome, thus providing for intrachromosomal expression of the F~IT nucleic acid ~Koller and Smithies, 30 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in 35 which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two W O 97/29119 P~TnUS97J019~7 approaches are known, respectively, as in vivo or ex vi~o gene therapy.
In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the 5 enco~d product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other 10 viral vector (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or trans~ecting agents, encapsulation in liposomes, microparticles, or microcapsules, 15 or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), 20 etc. In another embodiment, a nucleic acid-ligand complex can be formed in wl1ich the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake 25 and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al.);
WO 92/22635 da1:ed December 23, 1992 (Wilson et al.);
WO92/20316 dated November 26, 1992 (Findeis et al.);

8 dated July 22, 1993 (Clarke et al.), WO 93/20221 30 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cel~ DNA i~or expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
In a specific embodiment, a viral vector that contains the F~IT nucleic acid is used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.

9 CA 02245783 1998-08-06 PCT~S97/01937 217:581-599). These retroviral vectors have been modified to delete retrovira} sequences that are not n~c~ccAry for packaging of the viral genome and integration into host cell DNA. The FHIT nucleic acid to be used in gene therapy is - 5 cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Ro~n et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem 10 cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, 15 Curr. Opin. in Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild 20 disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present 25 a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991~ Science 30 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol.
Med. 204:289-300. Herpesviruses can also be used.
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated -W~97S29119 PCT~S97l01937 transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the -cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the - 5 ~ransferred gene. Those cells are then delivered to a patient.
In this ~ ho~; -nt, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any 10 method known in the art, including but not limited to transfection, ele~Lr~oration, microinjection, infection with a viral or bacteriophage vector containin~ the nucleic acid se~uences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
15 Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth.
Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance with the present invention, 20 provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The tec-h~; que should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and 25 expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. In a preferred embodiment, epithelial cells are injected, e.g., subcutaneously~ In another - ho~ment~ recombinant skin cells 30 may be applied as a skin graft onto the patient. Recombinant blood cells (e ~., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect! patient state, etc., and can be determined by one skilled in the art.
3~ Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, -W O 97/29119 PCTrUS97/01937 endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes,-monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor 5 cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
In an ~ hoAiment in which recombinant cells are used in gene therapy, a FHIT nucleic acid is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered ~n vivo for therapeutic effect. In a specific embodiment, stem or 15 progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention. Such stem cells include but are not limited to hematopoietic stem cells (HSC), stem cells of epithelial 20 tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (PCT Publication W0 94/08598, dated April 28, 1994), and neural stem cells (Stemple and Anderson, 1992, Cell 71:973-985).
Epithelial stem cells (ESCs) or keratinocytes can be 25 obtained from tissues such as the skin and the lining of the gut by known procedures (Rheinwald, 1980, Meth. Cell Bio.
21A:229). In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells 30 within the lining of the gut provide for a rapid renewal rate of this tissue. ESCs or keratinocytes obtained from the skin or lining of the gut of a patient-or donor can be grown in tissue culture (Rheinwald, 1980, Meth. Cell Bio. 21A:229;
Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771). If the 35 ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody WO 97l29119 PCTfUS97/Ot937 ~t- i n; stration to promote moderate immunosuppression) can also be used.
With respect to hematopoietic stem cells ~HSC), any technique which provides for the isolation, propagation, and 5 maint~Anc~ in vitro of HSC can be used in this emhs~; -nt of the invention. Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the LuLu~e host, or a donor, or (b) the use of previously established long-term HSC
10 cultures, which may be allogeneic or xenogeneic. Non-autologous HSC are used preferably in conjunction with a method of ~uppressing transplantation immune reactions of the future host/patient. In a particular embodiment of the present inventlon, human bone marrow cells can be obtained 15 from the posterior iliac crest by needle aspiration (see, e.g., Kodo et al., 1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of the present invention, the HSCs can be made high-y enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long-20 term culturing, and can be done by any t~c-hni ques known in the art. ~ong-term cultures of bone marrow cells can be established an~ maintained by using, for example, modified Dexter cell culture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques (Witlock 25 and Witte, 1982, Proc. Natl. Acad. Sci. USA 79:3608-3612).
In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by control}ing 30 the presence or absence of the appropriate inducer of transcription.
Additional methods that can be adapted for use to deliver a nucleic acid encoding a Fhit protein or functional derivative thereof are described in Section 5.8.5.

W 097/29119 CA 02245783 1998-08-06 PCT~US97/01937 5.8.5. ANTAGONIZING D~P~-NEGATIVE FHIT
MUTATIONS FOR T~M~T OR PR~v~NllON
OF DIsORDERS OF OV~RPROLIFERATION
The invention al60 provides methods of treating or preventing disorders of overproliferation (e.g.., cancer, 5 hyperproliferative disorders) in which the patient has a hemizygous FHI~ mutation (presumably a dominant-negative FHIT
mutation) by specifically antagonizing tadministering an antagonist to) the mutant F~IT gene or protein (and not wild-type F~I~ or Fhit). Hemizygosity for a FHIT mutation can be 10 detected by observing the presence of both normal and mutant FHIT DNA (e.g., cDNA) or RNA in a sample from a patient, e.g., by methods as described in Sections 5.11 and 6 hereof.
For example, in a specific embodiment, an effective amount of antisense oligonucleotide that inhibits the 15 expression of the mutant FHIT gene, and not the wild-type F~IT
gene, is administered. For example, if the hemizygous FHIT
mutation in the patient is a deletion of at least a portion of one or more FHIT exons, the antisense oligonucleotide can comprise a hybridizable se~uence complementary to the junction 20 formed by the deletion, said junction being present in the mutant F~I ~ gene but not the wild-type FHIT gene. Thus, the antisense oligonucleotide comprises a sequence complementary to conti~uous sequences from two exons not naturally found contiguous in wild-type FHI~ cDNA.
In another specific embodiment, an antibody can be used therapeutically or prophylactically to specifically antagonize the hemizygous Fhit mutant protein. For example, such an antibody can specifically recognize an epitope in a Fhit deletion mutant formed by the ~usion of sequences not 30 naturally contiguous in the wild-type Fhit protein. ~or therapeutic purposes, a Fhit mutant protein can be used as immunogen to make anti-Fhit antibodies that neutralize the activity of the Fhit mutant protein and not wild-type Fhit protein. Accordingly, the present invention provides a method 35 for treating a disease state asso~iated with a FHIT
abnormality in a mammal suffering from a disease state CA 02245783 l998-08-06 Wo97129~19 PCT~S9~101937 associated with a FHIT abnormality comprising administering a therapeutical~y effective amount of an anti-Fhit antibody specific to the abnormal F~I~ gene or protein to a mammal suffering fro~ a ~ ce state associated with a FHIT
5 abnormality.
In another specific embodiment, a recombinant nucleic acid consisting of non - FHIT sequences flanked by FHIT
sequences so aLs to promote homologous recombination specifically with a mutant FHI~ gene in a patient, is O il,LlGduced into the patient, in order to "knock out" (inhibit the effect of) the mutant, particularly where such mutant is believed to be a dominant-negative one.
Antisense oligonucleotides are described in further detail below.
~5 5.8.5.1. ANTISENSE REGUhATION OF
MUTANT FHIT GENE EXPRESSION
In a specific embodiment, mutant function of Fhit or FHIT
is specifically inhibited by use of FHIT antisense nucleic 20 acids. The present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding a mutant Fhit.
A FXIT "antisense1' nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a F~IT RNA
25 (preferably mRNA) or mutant form thereof by virtue of some sequence complementarity (other than to nonspecific sequences such as a polyA tail). The antisense nucleic acid may be complementary to a coding and/or nonco~;ng region of a F~TT
mRNA. Such antisense nucleic acids have utility as 30 Therapeutics that inhibit dominant-negative mutant Fhit function, and can be used in the treatment or prevention of disorders as described supra in Section 5.8 and its subsections.
The antisense nucleic acids of the invention can be 35 oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced W 097/29119 CA 02245783 1998-08-06 PCT~US97/01937 intracellularly by transcription of exogenous, introduced sequences.
The invention further provides pharmaceutical compositions comprising an effective amount of the F~IT
5 antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described in Section 5.10.
In another embodiment, the invention is directed to methods for inhibiting the expression specifically of a FHIT
mutant nucleic acid ~equence in a prokaryotic or eukaryotic 10 cell comprising providing the cell with an effective amount of a composition comprising an FHIT antisense nucleic acid of the invention.
The F~IT antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 15 6 to about 5~ oligonucleotides). In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The oligonucleotides can be DNA or RNA or çhi ~ ic mixtures or derivatives or modified versions thereof, 20 single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, 2s Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.
Wo 88/09810, published Dec~- h~r 15, 1988~ or blood-brain barrier (see, e.g., PCT Publication No. W0 89/10134, published April 25, 1988~, hybridization-triggered cleavage agents (see, 30 e.g., Krol et al., 1988, BioT~chniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).
In a preferred aspect of the invention, a F~IT antisense oligonucleotide is provided, preferably of single-stranded 35 DNA. In a most preferred aspect, such an oligonucleotide comprises a sequence antisense to a junction of two non-normally contiguous sequences in a F~IT gene deletion mutant, WOg7/291~9 PCT~S97J~1937 most preferably, of a human FHIT gene mutant. The oligonucleotide may be modified at any position on its ~tructure with substituents generally known in the art.
The FHIT antisense oligonucleotide may comprise at least 5 one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, S-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcyto~ine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, lO 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, l-methylguanine, l-methylinosine, 2,2-dimethylgll~ n ine~
2-methyladenine, 2-methylguanine, 3-methylcytosine, S-methylcytosine, N6-adenine, 7-methylguanine, 15 5-methylA inl: ?thyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 20 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
In another embodiment, the oligonucleotide comprises at 25 least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
-In yet another embodiment, the oligonucleotide comprises at lea~t one modified phosphate backbone selected from the 30 group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the oligonucleotide is an 35 ~-anomeric oligonucleotide. An ~ anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA
in which, contrary to the usual ~-units, the strands run W O 97129119 CA 02245783 1998-08-06 PCTrUS97/01937 parallel to each other (Gautier et al., 1987, Nucl. Acids Res.
15:6625-6641).
The oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-5 linking agent, transport agent, hybridization-triggered cleavage agent, etc.
oligonucleotides of the invention may be synthesized by s~n~d methods known in the art, e.~. by use of an automated DNA synthesizer (such as are commercially available from 10 Biosearch, Applied Biosystems, etc.). These include techniques for chemically synthesizing oligodeoxyri-bonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. As examples, phosphorothioate oligonucleotides may be synthesized by the 15 method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
Alternatively, RNA molecules may be generated by in vitro and 20 in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA
polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that 25 synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
In a specific emhoA;ment, the FHIT antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, 30 e.g., PCT International Pub}ication WO 90/11364, published October ~, 1990; Sarver et al., 1990, Science 247:1222-12253.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mech~ni~ of ribozyme action involves sequence specific hybridization of the 35 ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that ~pecifically and efficiently catalyze endonucleolytic cleavage of mutant FHIT RNA seguences.
Specific ribozyme cleavage sites within any potential RNA
target are initially identified by ~c~nnin~ the target 5 molecule for ribozyme cleavage ~ites which include the following sequ~nces, GUA, GUU and GUC. Once identified, short RNA se~?~ncefi of between 15 and 20 ribonucleotides corresponding to the region of the target gene cont~; n; ng the cleavage site may be evaluated for predicted structural 10 features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
lS In another embodiment, the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131--6148), or a Ch; ?~iC RNA--DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
In an alternative embodiment, the F~IT antisense nucleic 20 acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the 25 invention. Such a vector would contain a sequence encoding the FHIT antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology 30 methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the F~IT antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such 35 promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3~ long terminal repeat o~ Rous sarcoma virus (Y~ ~ oLo et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.
U.S.A. 78:1441-1445), the regulatory se~uences of the 5 metallothionein gene (Brinster et al., 1982, Nature 296:3~-42), etc.
~ he antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an ~NA
transcript of a FHI~ gene, preferably a human mutant FHIT
10 gene. ~owever, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-15 stranded FHIT antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic 20 acid, the more base mismatches with a F~IT RNA it may contain and still form a stable duplex (o~ triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
The FHIT antisense nucleic acids can be used to treat (or prevent) malignancies or hyperproliferative disorders, of a cell type which has been shown to express mutant FHIT RNA.
Malignant, neoplastic, and pre-neoplastic cells which can be tested for such expression include but are not limited to 30 those described supra in Sections 5.8. In a preferred ~~ hoA;ment, a single-stranded DNA antisense FNIT
oligonucleotide is used.
Malignant (particularly, tumor) cell types which express FHIT RNA can be identified by various methods known in the 3~ art. Such methods include but are not limited to hybridization with a FHIT-specific nucleic acid (e.g., by Northern hybridization, dot blot hybridization, in situ -W O 97129119 . PCT~US971~193 hybridization), observing the ability of RNA ~rom the cell type to be translated in vitro into Fhit protein, etc. (see the assays described for diagnosis in Section 5.11). In a preferred aspect, primary tumor tissue from a patient can be 5 assayed for F~IT expression prior to treatment.
Pharmaceutical compositions of the invention, comprising an effective amount of a F~IT antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a patient having a malignancy which is of a type that expresses 10 mutant FHI~ RNA that i~ specifically antagonized by the antisense nucleic acid.
The amount of FHIT antisense nucleic acid which will be effective in the treatment of a particular disease state or condition will depend on the nature of the disease state or 15 condition, and can be determined ~y standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity of the tumor type to be treated in v~tro, and then in useful animal model systems prior to testing and use in humans.
In a specific embodiment, pharmaceutical compositions comprising FHIT antisense nucleic acids are administered via liposomes, microparticles, or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the FHIT
25 antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable tumor antigens (Leonetti et al., 19gO, Proc. Natl. Acad. Sci. USA 87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).
3Q In a particular embodiment of the invention, antisense FHIT oligonucleotides or anti-Fhit antibodies that specifically antagonize a mutant FNIT gene or protein present in a patient, are al ; n; stered to the patient in combination with administration to the patient of F~IT gene therapy 3s (administration of wild-type Fhit~function) or functional Fhit protein or agonists.

-5 . 9 . DEMONSTRATION OF THERAPE:~IC
OR PROPHYhACTIC UTILITY
The FHIT polynucleotides, FHIT protein products, derivatives and analogs thereof, and antibodies thereto, and 5 antisense nucleic acids of the invention can be tested in vivo for the desired therapeutic or prophylactic activity. For example, such c_ -unds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For 7 n 10 viYo testing, prior to administration to humans, any animal model system known in the art may be used.

5.10. THERAPEUTIC/PROPHYLACTIC
METHODS AND COMPOSITIONS
The invention provides methods of treatment and 15 prophylaxis by administration to a subject of an effective amount of a Therapeutic, i.e., a FHIT nucleic acid, FHIT
protein, derivative or analog thereof, or antibody thereto of the present invention. In a preferred aspect, the Therapeutic is substantial~y purified. The subject is preferably an 20 animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human. The subject can be a fetus, child, or adult.
In a specific embodiment, a non-human ~ ~1 is the sub~ect.
~ ormulations and methods of a~min;~tration that can be employed when the TherapeutiC comprises a nucleic acid are described in Sections 5.8.4 and 5.8.5.1 above; additional appropriate formulations and routes of ~, i ni ~tration can be 30 selected from among those described hereinbelow.
Various delivery systems are known and can be used to adminis~er a Therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recom~inant cells, receptor-mediated endocytosis (see, e.g., 35 Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other CA 0224~783 1998-08-06 WO971~9119 PCT~S9710193~

vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The comrol-n~c may be administered by any convenient route, for 5 example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. A~ i n; ~tration can be systemic or 1ocal. In addition, it may be desira~le to lO introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection;
intraventricular iniection may be facilitated by an intraventricular catheter, for example, attac~ed to a 15 reservoir, such as an Ommaya reservoir.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not ~y way of limitation, local infusion 20 during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic 25 membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
-In a specific emboAi ~nt where the Therapeutic is a nucleic acid encoding a protein therapeutic, the nucleic acid 30 can be ~in;stered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic-acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by 3s use of microparticle bombardment ~e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a _ ~;9 _ W O 97/29119 PCT~US97/01937 homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., ~991, Proc. Natl. Acad. Sci. USA
88:1864-1868), etc. Alternatively, a nucleic acid therapeutic can be intro~c~ intracellularly and incorporated within host S cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but 10 is not limited to ~lin~, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.
The composition, if desired, can also contain minor 15 amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as 20 triglycerides. Oral formulation can include stAn~rd carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
In a preferred embodiment, the composition is formulated 25 in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where nece~s~y, the composition may also include a 30 solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredi~nts are supplied either separately or ~;x~ together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container 35 such a~ an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle CA 0224~783 1998-08-06 W097/2g119 PCT~S97101937 containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to ~r; n;~tration.
The Therapeutics of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl 10 groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or 15 condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also ~ep~n~ on the 20 route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per 25 kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.0l pg/kg body weight to l mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of 3s the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency -WO 97/291l9 CA 0224~783 1998 - 08 - 06 PCTIUS97/01937 regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

5.11. DIAGNOST~C USES
A FHIT polynucleotide and nucleic acids complementary thereto, its Fhit protein product, fragments thereof, and antibodies thereto can be used for diagnostic purposes for disorders involving overproliferation of cells, as well as 10 other disorders associated with chromosomal translocations and inversions or molecular abnormalities associated with the FHIT
gene, and/or decreased expression of wild-type FHIT RNA or protein.
Such molecules can also be used in diagnostic assays, 15 such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders associated with expression of mutant FHIT transcripts or monitor the treatment thereof. Accordingly, in specific embodiments, cancer or premalignant changes or hyperproliferative or benign 20 dysproliferative disorders in tissue is diagnosed by detecting the presence of one or more mutant FHIT transcripts, alone or in combination with a decrease in expression of wild-type FHIT
transcript, in patient samples relative to FHIT expression in an analogous non-diseased sample (from the patient or another 25 person, as determined experimentally or as is known as a st~ ~d level in such samples). For diagnostic purposes, a FHIT polynucleotide may be used to detect mutant FHIT gene expression in disease states.
The subject, or patient, is an animal, e.g., a mammal, 30 and is preferably human, and can be a fetus, child, or adult.
As illustrated infra, the FNIT gene se~uence is associated with cancers, particularly associated with translocations and deletions within the FHIT gene. In specific embodiments, diseases and disorders involving 35 overproliferation of cells can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting decreased WO97/29119 PCT~S97101937 levels o~ wild-type Fhit protein, wild-type FHIT RNA, or Fhit functional acf;ivity, or by detecting mutations in FHIT RNA, DNA, cDNA, or protein (e.g., translocations or deletions in FHIT nucleic acids, truncations in the FHIT gene or protein, 5 changes in nucleotide or amino acid sequence relative to wild-type Fhit~ that cause decreased expression or activity of Fhit or a d,~ nt negative effect. Such diseases and disorders include but are not limited to those described in Section 5.8 and its subsections. By way of example, levels of Fhit 10 protein can be detected by immunoassay, levels of FHIT RNA can be detected by hybridization assays (e.g., Northern blots, dot blots) or RT-PCR, translocations, deletions, and point mutations in FHIT nucleic acids can be detected by Southern blotting, RFLP analysis, PCR of cDNA using primers that 15 preferably generate a fragment spanning at least most of ~he F~IT gene, sequencing of the FNIT genomic DNA or cDNA obtained from the patient, etc.
In a preferred embodiment, levels of FHIT mRNA (or cDNA) or protein in a patient sample are detected or measured or 20 analyzed by size and/or sequence, in which aberrant levels, size or se~uence indicate that the subject has, or has a predisposition to developing, a malignancy or hyperproliferative disorder; in which the decreased levels are relative to the levels present in an analogous sample from a 25 portion of the body or from a subject not having the malignancy or hyperproliferative disorder, as the case may be.
FHIT gene sequences may be used diagnostically for the deteGtion of d;s~ses states resulting from chromosomal or mo~ecular abnormalities, e.g., translocations, inversions and 30 deletions, involving the FHIT gene. Nucleic acids comprising FHIT nucleotide sequences of at least 8 nucleotides, at least 15 nucleotides, at least 25 nucleotides, at least 50 nucleotides, at least lO0 nucleotides, at least 200 nucleotides, at least 300 nucleotides, and preferably less 35 than 500 nucleotides, and the nuc~eic acids described in Section 5.l, may be used as probes in hybridization assays for the detection and measurement of FHIT gene sequences. Nucleic _ WO 97/29119 CA 0 2 2 4 5 7 8 3 l 9 9 8 - O 8 - O 6 PCT/US97/01937 acids of not more than 5 kilobase6, of not more than lo kilobases, not more than 25 kilohAc~c, not more than SQ
kilobases or not more than 70 kilobases which are hybridizable to a FHIT gene, cDNA, or complementary strand can be used as 5 probes in hybridization assays for the detection and measurement of FHIT nucleotide sequences. As an example, the FHIT DNA sequence may be used in hybridization assays, e.g., Southern or Northern analysis, including in sl tu hybridization assays, of patient's samples to diagnose abnormalities of FHIT
10 expression. Hybridization assays can be used to detect, prognose, diagnose, or monitor malignancies, associated wi~h aberrant changes in FHIT expression and/or activity as described supra. In particular, such a hybridization assay is carried out by a method comprising contacting a sample 15 containing nucleic acid with a nucleic acid probe capable of hybridizing to FHIT DNA (e.g., cDNA) or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization. In particular, hybridization assays can be used to detect the presence of abnormalities 20 associated with expression of mutant FHIT mRNA, by hybridizing mRNA or cDNA from a patient sample to a FHIT probe, and analyzing by size and/or sequence the resulting hybridized nucleic acids. For example, assays which can be used include, but are not limited to Northern blots, dot blots, etc. A
25 particular hybridization assay is Northern blot analysis of a patient sample using FHIT gene probes of at least 15 nucleotides up to the full length cDNA sequence shown in Figure 2A. Another hybridization assay is in situ hybridization analysis of a patient sample using anti-FHIT
30 antibodies or FHIT nucleotide hybridization probes. Such techniques are well ~nown in the art, and are in fact the basis of many commercially available diagnostic kits.
In a specific embodiment, cancer or other disorder of cell overproliferation (e.g., those described in Sections 35 5.8.1-5.8.3 above), is diagnosed Qr prognosed ~y detecting a mutation in the F~IT gene or its produced RNA in a sample derived from a patient. The mutation can be a translocation, , CA 02245783 l998-08-06 W O 97/29119 PCTnUS97101937 deletion, insertion or substitution/point mutation. In a preferred ~ ho~i -nt, the mutation is a deletion of all or a -portion of at least one coding exon (i.e., exon 5, 6, 7, 8 or 9), preferably exon 5 or exon 8. In a preferred embodiment, 5 the deletion is a homozygous deletion. In another embodiment, the muta~ion is a mutation that causes a frameshift upstream of exon 8, or otherwise causes a lack of the wild-type open reading frame (ORF) of exon 8 in the patient's FHIT RNA.
In other specific embodiments, the mutation is a deletion 10 o f FHII' exons 4-6 resulting in a fusion of exon 3 seguences to exon 7 sequences in a FHI~ RNA or cDNA, or the mutation is a deletion of F~IT exons 4-8 resulting in a fusion of exon 3 sequences to exon 9 sequences in a FHIT RNA or cDNA.
In another particular embodimen~, the mutation that is 15 detected is an insertion into a coding region of the FNIT gene or an insertion downstream of exon 4, or an insertion in the 5' noncoding region between exon 4 and 5. In a speci~ic embodiment, the mutation in the F~IT gene coding sequence is detected by detecting an aberrant sized FHIT cDNA or mRNA from 20 the subject (i.e., FHIT RNA or cDNA that has a different size than the wild-type FHIT RNA (that is present or expected to be present in normal individuals not having or pre-disposed to a cancer associated with a FHIT mutation, e.g., the -1.1 kb transcript)).
In another ~ ho~; ~nt, diagnosis or prognosis is carried out by detecting an aberrant sized FHIT cDNA or mRNA from the subject as well as the loss of one FHIT allele in the subject.
-Polynucleotide sequences of FHIT consisting of at least 8 to 25 nucleotides that are useful as primers in primer 30 dependent nucleic acid amplification methods may be used for the detection of mutant FHIT genomic or RNA sequences in patient samples. Primer dependent nucleic acid amplification methods useful in the present invention include, but are not limited to, polymerase chain reaction (PCR), competitive PCR, 35 cyclic probe reaction, and ligase chain reaction. Such ~hn;ques are well known by those of skill in the art. A
preferred nucleic acid amplification method of the present CA 0224~783 1998-08-06 W O 97/29119 PCT~US97/01937 invention is reverse transcriptase PCR (RT-PCR) (Siebert et al., 1992, Nature 35g:557-558).
In a particular c h5~ ment of the present invention, each primer of a pair of primers for use in a primer dependent 5 nucleic acid amplif~cation method is selected from a different exon of the genomic F~IT nucleotide seqllence~. For example, if one primer of a pair or primers is selected from exon 1 of the F~IT genomic sequence, the second primer will be selected from exon 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the FHIT genomic 10 sequence. As another example, if one primer of a pair of primers is selected from exon 2 of the F~I~ genomic sequence, the second primer will be selected from exon 1, 3, 4, 5, 6, 7, 8, 9 or 10 of the FHIT genomic sequence. Resulting amplified genomic nucleotide sequences will contain amplified intron 15 sequences and will be of a larger size than amplified cDNA
nucleotide sequences that will not contain amplified intron sequences. Similarly, amplified cDNA sequences having a deletion mutation can be distinguished from amplified wild-type sequences due to the size difference of the resulting 20 amplified sequences (the deletion mutant will generate a shorter amplified fragment). For amplification of cDNA
nucleotide sequences, the primer se~uences should be selected from exons sequences that are sufficiently far enough apart to provide a detectable amplified nucleotide sequence.
In a specific embodiment, cancer or other disorder of cell proliferation or a predisposition thereto is detected or diagnosed in a subject by detecting mutation(s) within the FNIT gene as follows: A sample cont~ining RNA of tissue or cells of a patient is obtained, and the RNA is reverse-30 transcribed into cDNA by methods commonly known in the art;
preferably this step is followed by amplifying fragments comprising F~IT coding sequences within the cDNA, and detecting one or more mutation(s) within the FHIT coding se~uences within the amplified fragment. The amplification 35 can be by any suitable methods known in the art, and is preferably done by polymerase chain reaction (PCR). RT-PCR is preferred due to the great size (>500 kb) of the FHIT gene in CA 02245783 l998-08-06 W 097129119 PCTnUS9710193 the genome, which renders one unable to amplify a single fragment cont~; n; ~g most of the FHIT exons from a genomic sample, whereas amplification of such a fragment is readily accomplished from a cDNA sample. ~he primers for use in PCR
5 are upstream and downstream primers that prime synthesis by a polymerase toward each other, and are preferably in the range of 8-35 nucleotides, preferably separated by in the range of 10-2,000 nucleotides in the FHIT mRNA. In a preferred embodiment, each primer comprises a hybridizable sequence 10 contained within an exon of the FHI~ gene or within 200 nucleotides flanking (5' or 3' to) an exon of the FHIT gene.
In a specific embodiment, the first primer hybridizes 5' to eXon 5 (preferably containing sequences of exon 4 or 5' thereto) and the second primer hybridizes on the other strand 15 5' to the intron between exons 5 and 6 (such that an amplified fragment from wild-type FHIT cDNA would contain exon 5). In another specific embodiment, the second primer hybridizes on the other strand 5' to exon 6. In other specific embodiments, the first and second primers respectively hybridize on 20 opposite strands 5' to the 3' terminus of exon 4 and 5' to exon 8; 5' to the 3' terminus of exon 4 and 5' to exon 9; and 5' to exon 1 and 5' to exon lo, such that the resulting amplified fragment would contain the exon sequences normally present between where the primers hybridize should they be 25 present in the cDNA. Thus, for example, in the foregoing examples, the PCR primer pairs are adapted to amplify a fragment of wild-type FHIT cDNA comprising F~I~ exon 5, exon 5 plus exon 6, sequences between the 3' terminus of exon 4 and exon 8, sequences between the 3' terminus of exon 4 and exon 30 9, and exons 1 through 10, respectively. The presence of one or more mutations in the cDNA can be detected by detecting an aberrantly sized (preferably amplified) fragment (compared to those fragment(s) produced by a wild-type FHI~ transcript), e.g., by subjecting the cDNA to size separation such as by 35 agarose gel electrophoresis or column chromatography. In a preferred embodiment, the presence of one or more mutations in the cDNA is detected by sequencing of the cDNA, or more preferably, of the isolated fragments amplified from the cDNA.
The amplified fragments can be isolated by methods known in the art, e.g., agarose gel electrophoresis and recovery from the gel band and/or column chromatography. Such sequencing 5 can be carried out by standard methods commonly known in the art, and can be automated or manual.
In yet another specific embodiment, mutation(s) in the FHIT gene or mRNA from a patient can be detected by other methods commonly known in the art, e.g., Northern 10 hybridization. By way of example but not limitation, RNA from a patient's tissue is separated by gel electrophoresis, transferred to a filter or other solid phase, and hybridized to labelled DNA probes. The hybridized RNAs are then visualized by detecting the label. Preferably, numerous DNA
15 probes are used, from different portions of the FHI~ cDNA.
In another embodiment, Southern hybridization can be used to detect gross mutations in FNIT DNA. For example, genomic DNA isolated from a patient, separated by gel electrophoresis, transferred to a filter or other solid phase, and hybridized 20 with a ~IT probe (e.g., an oligonucleotide containing a FHIT
gene sequence, affixed to a detectable label). Preferably, a multiplicity of F~I~ probes are used, hybridizable to sequences within each of the coding exons, and particularly preferably, including probe(s) hybridizable to sequences 2S within exon 5.
In another embodiment, a translocation within the FHIT
gene i5 detected by methods commonly known in the art. For example, in a preferred embodiment, a sample comprising FNIT
genomic DNA, or, preferably F~IT cDNA (e.g., cDNA of total 30 polyA mRNA) from a patient is subjected to PCR by use of primers that prime synthesis across the suspected trans~ocation junction. For example, one primer can have a sequence hybridizable to chromosome 3 (preferably within the F~IT gene upstream of exon 4, e.g., a sequence within exon 1, 35 2 or 3) and one primer can have a sequence hybridizable to chromosome 8 (downstream of the translocation event);
amplification of a fragment indicates the presence of a WO 97/~gll9 PCTIUS97101937 translocation between chromosomes 3 and 8. Additionally or alternatively performing PCR by priming with primers, each having a sequence within the FHI~ gene (see e.g., de~cription supra regarding primers for RT-PCR) will result in an 5 amplified ~ragment only if at least one FHIT allele contains the primer sequences undisrupted by a translocation event in between them.
Detection of homozygous mutations (mutations in both alleles) in F~.rT genes are d2~ -~ more severe indicators of 10 the presence of, or a predisposition to, cancer than hemizygous mutations (of one allele) in F~IT genes.
As used herein, patient samples which can be used include, but are not limited to, fresh or frozen tissue samples, which can be used in in situ hybridization assays;
15 cell or tissue from biopsies and, in general, patient samples containing nucleic acid, which can be used in assays that measure or quantitate or analyze FHIT nucleic acid.
The FHIT gene sequences of the present invention may be used diagnostically for the detection of chromosome 3pl4.2 20 abnormalities, in particular, translocations with chromosome 8, and deletions. Accordingly, the present invention provides a process for detecting a target sequence indicative of or including a chromosome 3pl4.2 abnormality in a sample, comprising the steps of amplifying the target sequence in the 25 sample using a first primer of 8 to 25 nucleotides, preferably 18-25 nucleotides, complementary to the nucleotide sequence of SEQ ID N0: 1, and a second primer complementary to a region telomeric or centromeric to the FHIT gene and detecting any resulting amplified target sequence in which the presence of 30 the amplified target sequence is indicative o~ the abnormality. The present invention also provides a method of diagnosing a malignancy associated with chromosome 3pl4.2 abnormalities in a patient comprising, detecting said chromosome 3pl4.2 abnormality according to the method above in 35 which the presence of an amplified target sequence indicative of a mutant F~T transcript indicates the presence of a cancer or precancerous condition in the patient. The resultant -CA 0224~783 1998-08-06 amplified target sequence can be detected on gel electrophoresis and comp~red with a normal sample or st~n~rd that does not contain a chromosome 3pl4.2 abnormality. The - - amplification of genomic DNA target sequences may require 5 generating long PCR products. PCR t~c-hniques for generating long PCR products are described in Science (1994) 263:1564-1565; PCR kits for generating long PCR products are available from Perkin Elmer and Takara Shuzo Co., Ltd. The present invention al80 provides a method for detecting a target 10 nucleotide sequence indicative of or including at least a portion of a chromosome 3pl4.2 abnormality (thereby indicative of the presence of or a predisposition to a disorder of cell overproliferation) in a nucleic acid sample, comprising the steps of hybridizing the sample with a nucleic acid probe of 15 not more than lo kilobases, comprising FHIT cDNA sequences selected from among at least exon 1, 2, 3 or 4 and selected from among at least exon ?, 8 or 9, or a sequence absolutely complementary thereto, and detecting or measuring the amount of any resulting hybridization between the probe and the 20 target sequence within the sample. Alternatively, the probe comprises at least 310 contiguous nucleotides of a FEIT cDNA, or at least 266 contiguous nucleotides of F~IT cDNA coding sequences. The resultant hybridization between the probe and the target sequence within the sample can be detected using 25 gel electrophoresis and can be compared to a target sequence from a normal sample or standard that does not contain the abnormality. The present invention also provides a method of diagnosing a malignancy associated with a FHIT abnormality in a patient comprising detecting said F~IT abnormality according 30 to the method above in which the presence of the amplified target sequence indicates the presence of a malignancy in the patient.- Absolute complementarity between a hybridization probe and a target sequence, although preferred, is not required. A sequence "complementary to at least a portion 35 of", as referred to herein, means~a sequence having sufficient complementarity to be able to hybridize with the nucleic acid, forming a stable hybridization complex. The ability to WOg7129119 PCT~S97J01937 hybridize will depend on both the degree of complementarity and the length of the nucleic acid. Generally, the longer the hybridizing nucleic acid, th~ more base mismatches with a FHIT
RNA it may contain and still form a stable duplex (or triplex, 5 as the case may be). One skilled in the art can ascertain a tolerable degree of ;l ~tch by use of s~An~rd proc~res to determine the melting point of the hybridized complex.
An additional aspect of the present invention relates to diagnostic kits for the detection or measurement of FHIT gene 10 sequences and FHIT protein. Kits for diagnostic use are provided, that comprise in one or more containers an anti-Fhit antibody, and, optionally, a labeled binding partner to the antibody. Alternatively, the anti-Fhit antibody can be labeled (with a detectable marker, e.g., a chemiluminescent, 15 enzymatic, fluorescent, or radioactive moiety). A kit is also provided that comprises in one or more containers a nucleic acid probe capable of hybridizing to FHIT RNA. Accordingly, the present invention provides a diagnostic kit comprising, in a container a compound comprising a probe of not more than l0 20 kilobases and comprising FHI~ cDNA sequences comprising at least one of exon l, 2, 3 or 4 and at least one of exon 7, 8 or 9; or its complement. Alternatively, the probe comprises at least 310 contiguous nucleotides of a FHI~ cDNA, or at least 266 contiguous nucleotides of FHIT cDNA coding 25 sequences. Alternatively, the present invention provides a diagnostic kit comprising, in one or more containers, a pair of primers of at least 8-35, preferably 8-25, nucleotides in which- at least one of said primers is hybridizable to SEQ ID
NO: l or its complement and wherein said primers are capable 30 of priming cDNA synthesis in an amplification reaction. In a specific embodiment, a kit can comprise in one or more containers a pair of primers (e.g., each in the size range of 8-35 nucleotides) that are capable of priming amplification ~e.g., by polymerase chain reaction (see e.g., Innis et al., 35 l990, PCR Protocols, Academic Pres~s, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of Q~ replicase, cyclic probe reaction, or other methods known in the art]

W O 97/29119 PCTrUS97/01937 under appropriate reaction conditions of at least a portion of a FHIT nucleic acid. The present invention also provides a diagnostic kit in which at least one of the primers is hybridizable to SEQ ID No: 1 or its complement and in which 5 one of the primers is hybridizable to a DNA sequence located telomeric or centromeric to the FHIT gene. In another embs~ -nt, the kit comprises a primer pair such as described supra for use in diagnostic assays. In a specific embodiment, one of the foregoing compounds of the contAine~ can be 10 detectably labeled. A kit can optionally further comprise in a container a predetermined amount of a purified Fhit protein or nucleic acid, e.g., for use as a st~ rd or control.
The amplification reaction of the present invention may be a polymerase chain reaction, competitive PCR and 15 competitive reverse-transcriptase PCR (Clementi et al., 1994, Genet Anal Tech Appl 11(1):1-6 and Siebert et al., 1992, Nature 359:557-558); cyclic probe reaction, which allows for amplification of a target sequence using a hybrid RNA/DNA
probe and RNase (ID Biomedical); ligase chain reaction (Wu et 20 al., 1989, Genomics 4:560-569). In a particular e ho~;~~nt, the chromosomal abnormality associated with a FHIT locus can be detected as described in PCT Publication No. W092/19775, dated November 12, 1992. In a specific embodiment, the FHIT
probe used in a hybridization assay is detectably labeled.
25 Such a label can be any known in the art including, but not limited to, radioactive labels, fluorescent labels, biotin, chemiluminescent labels, etc.
-In a specific embodiment in which the assay used employs primers, at least one primer can be detectably labeled. In 30 another embodiment, one of a primer pair is attached to a moiety providing for capture, e.g., a magnetic bead.
Anti-FHIT antibodies may be generated and used diagnostically to detect the presence of mutant Fhit protein in patient samples, and/or the absence of wild-type Fhit 35 protein, thereby identifying disease states associated with chromosome 3pl4.2 abnormalities such as disorders of cell overproliferation.

WO97l2911g PCT~S97/01937 For example, in one embodiment, where one is detecting or measuring mutant Fhit protein by assaying for binding to anti-Fhit antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-5 competitive assay systems using t~r-hniques such as radioimm~noassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel dif~usion precipitin reactions, imm-no~iffusion assays, in s~tu iD unoassays (using colloidal gold, enzyme or lO radioisotope l~helR, for example), western blots, in situ hybridizations, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one lS embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means 20 are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. In particular, such an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-Fhit antibody under conditions such that immunospecific 2~ binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody. In a specific embodiment, antibody to a Fhit protein can be used to assay a patient tissue or serum sample for the presence of a FHIT
protein where an increased level of FHIT protein is an 30 indication of a diseased condition. In one embodiment of the pre~ent invention, the FHIT protein is detected or measured by immunocy~torh~ try of a patient sample. In another embodiment, assay~ to measure the levels of FHIT protein or RNA can be used to monitor therapy of disease associated with 35 increased expression of FHIT. For example, a decrease in levels of FHIT RNA or protein after therapy, relative to the level found before therapy, may be indicative of a favorable -W O 97/29119 PCTrUS97/01937 response to therapy. An increase in Cuch levels after therapy may be indicative of a poor response to therapy.
For detection of Fhit protein sequences, a diagnostic kit of the present invention comprises, in one or more containers, 5 an anti-Fhit antibody which optionally can be detectably labeled. In a different ~ ho~i~ent, the kit can comprise in a container, a labeled specific binding portion of an antibody.
As used herein, the term detectable label refers to any label which provides directly or indirectly a detectable signal and 10 includes, ~or example, enzymes, radiolabelled molecules, ~luorescent molecules, particles, chemiluminecors, enzyme substrates or cofactors, enzyme inhibitors, or magnetic particles. Examples of enzymes useful as detectable labels in the present invention include alkaline phosphatase and horse 1~ radish peroxidase. A variety of methods are available for li nki ng the detectable labels to proteins of interest and include for example the use of a bifunctional agent, such as, 4,4'-di~luoro-3,3'-dinitro-phenylsulfone, for attaching an enzyme, for example, horse radish peroxidase, to a protein of Z0 interest. The attached enzyme is then allowed to react with a substrate yielding a reaction product which is detectable.
The present invention provides a method for detecting a Fhit protein in a patient sample, comprising, contacting the patient sample with an anti-Fhit antibody under conditions 25 such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody. The method preferably also comprises subjecting the protein to size fractionation and/or sequence determination.
Samples can be any sample from a patient containing FHIT
30 protein, e.g., tissue sections.
In diagnosing disease states, the functional activity of Fhit protèins, derivatives and analogs may be assayed by various methods. Accordingly, the present invention also provides a method of diagnosing a malignancy or other disorder 35 associated with chromosome 3pl4.2 ~(F~I~) abnormalities in a patient comprising, detecting expression of a mutant Fhit protein in a sample from the patient, in which the presence of WO97/29119 PCT~S97101937 a mutant Fhit protein indicates the presence of a malignancy or other disorder associated with FHIT abnormalities in the patient.
== In a specific - ho~i~?nt of the invention, prenatal 5 diagnosis of a disorder of cell overproliferation or a predisposition thereto, is carried out. For example, one can first obtain tissue (e.g., blood cells) from an expectant parent. If one or more of the expectant parents have a FHIT
mutation, thus indicating possible inheritance of this l0 mutation by the offspring, amniocentesis or some other method of fetal tissue sampling can then be carried out to obtain fetal cells which can then be tested for the presence of FHIT
mutant DNA or RNA or protein by methods as described above ~e.g., RT-PCR to detect mutant FHIT ~NA) .
In another embodiment, the levels of FHIT protein or RNA
expression may be used to stage or monitor disease, with the appearance of or an increase in mutant Fhit protein or RNA
expression, and/or a decrease of or loss in wild-type Fhit protein or RNA expression, relative to that present in a 20 sample derived from the subject at an earlier time, indicates disease progression.
The ability of antibodies, peptides or other molecules to modulate the effect of Fhit protein on disease states may be monitored. For example, the expression of F~IT gene sequences 25 or Fhit protein sequences may be detected as described, supra, both before and after administration of a therapeutic composition, e.g., comprising a FHIT nucleotide sequence, ~hit protein sequence, derivative or analog thereof, or antibody thereto, or antisense nucleic acid of the present invention.
In another embodiment, presence of FHIT mutation(s), particularly homozygous ones, can be used as indicators of adverse outcome to therapy or recurrence of the disorder in patients with disorders of cell overproliferation.
Other methods will be known to the skilled artisan and 35 are within the scope of the invent~ion.

CA 0224~783 1998-08-06 W O 97/29119 PCTAUSs7/01937 5.12. ~CREENING ~OR Fhit AGONISTS ~D ~iNTAGONISTS
FHIT nucleic acids, proteins, and derivatives also have -uses in scr~enin~ assays to detect molecules that specifically bind to FHI~ nucleic acids, proteins, or derivatives and thus 5 have potential use as agonists or antagonists of Fhit, in particular, molecules that thus affect cell proliferation. In a preferred emh~li ?r~t, such ass ~s are performed to screen for molecules with potential utility as anti-cancer drugs or lead rom~ n~R for drug development. The invention thus 10 provides assay~ to detect molecules that specifically bind to F~IT nucleic acids, proteins, or derivatives. For example, recombinant cells expressing F~TT nucleic acids can be used to recombinantly produce Fhit proteins in these assays, to screen for molecules that bind to a Fhit protein. Molecules (e.g., S5 putative binding partners of Fhit) are contacted with the Fhit protein (or fragment thereo~) under conditions conducive to binding, and then molecules that specifically bind to the Fhit protein are identified. Similar methods can be used to screen for molecules that bind to Fhit derivatives or nucleic acids.
20 Methods that can be used to carry out the foregoing are commonly known in the art.
In a specific embodiment of the present invention, a Fhit protein and/or cell ~ine that expresses a Fhit protein can be used to screen for antibodies, peptides, or other molecules 25 that bind to the FHIT protein and thus may act as agonists or antagonists of FHIT protein. For example, anti-Fhit antibodies capable of neutralizing the activity of a dominant-negative mutant Fhit protein may be used to inhi~it or prevent a disease state associated with cell overproliferation such as 30 cancer.
Screening of organic or peptide libraries with recombinantly expressed mutant Fhit protein may be useful for identification of therapeutic molecules that f~nction to inhibit the activity of mutant Fhit protein. Screening 35 against wild-type Fhit protein ca~ then be carried out to select for antagonists specific to the mutant Fhit protein, i.e., that do not inhibit (or bind) the wild-type Fhit CA 02245783 l998-08-06 W O 97/29119 PCTn~S97JO1937 protein. Synthetic and naturally occurring products can be screened in a - h~r of ways deemed routine to those of skill-in the art.
By way of example, diversity libraries, such as random or - 5 combinatorial peptide or non~ptide libraries can be screened for molecules that specifically bind to Fhit. Many libraries are known in the art that can be used, e.g., chemica}ly synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
Examples of chemically synthesized li~raries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., l9gl, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, lg94, Bio/Technology 12:709-710;
Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;
15 Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA
90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA
91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA
91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA
20 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 19g2, Proc. Natl. Acad. Sci. USA 89:5381-5383.
~ xamples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R.B., et al., 1992, J.
25 Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth.
152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT
Publication No. WO 94/18318 dated August 18, 1994.
Fn vitro translation-based libraries include but are not limited to tho~e described in PCT Publication No. WO 91/OS058 30 dated April 18, 1991; and Mattheakis et al., 1994, Proc. Natl.
Acad. Sci. USA 91:9022-9026.
By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc.
Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use.
3~ Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci.
USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities WO97/29119 CA 02245783 l998-08-06 PCT~S97/01937 in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et-al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
Screening the librari,es can be accomplished by any of a 5 variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries:
Parmley and Smith, 1989, Adv. Exp. Ned. Biol. 251:215-218;
Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc.
10 Natl. Acad. Sci. USA 89:5393-5397; Yu et al., lg94, Cell 76:933-945; Staudt et al., 1988, Science 241:577-58Q; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl.
Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Patent No. 5,096,815, U.S. Patent No.
15 5,223,409, and U.S. Patent No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and PCT
Publication No. WO 94/18318.
In a specific embodiment, screening can be carried out by contacting the library members with a Fhit protein (or nucleic 20 acid or derivative) immobilized on a solid phase and harvesting those library members that bind to the protein (or nucleic acid or derivative). Examples of such scr~; nq methods, termed "panning" t~c~n;ques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes 25 et al., 1992, BioTechniques 13:422-427; PCT Publication No.
WO 94/18318; and in references cited hereinabove.
In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340-245-246; Chien et al., 1991, Proc. Natl.
30 Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to a Fhit protein or derivative.
.
5.13. ANIMAL MODE~S
The invention also provides animal models.
3S In one embodiment, animal models for diseases and disorders involving cell overproliferation (e.g., as described in Section 5.8.1) are provided. Such an animal can be WO 97/2gll9 PCTIUS97/01937 initially produced by promoting homologous recombination between a FHIT gene in its chromosome and an exogenous FHI~ -gene that has been rendered biologically inactive (preferably by insertion o~ a heterologous sequence, e.g., an antibiotic 5 resistance gene). In a pre~erred aspect, this homologous recombination is carried out by transforming embryo-derived stem (ES) cell~ with a vector cont~i n; ng the insertionally inactivated FHl'T gene, such that homologous recombination occurs, followed by injecting the ES cells into a blastocyst, 10 and implanting the blastocyst into a foster mother, followed by the birth of the chimeric ~n;~l ("knockout animal") in which a F~IT gene has been inactivated (see Capecchi, 1989, Science 244:12~8-1292). The chimeric animal can be bred to produce additional knockout animals. Such animals can be ~5 mice, hamsters, sheep, pigs, cattle, etc., and are preferably non-human mammals. In a specific embodiment, a knockout mouse is produced.
Such knockout Ani~-ls are expected to develop or be predisposed to developing diseases or disorders involving cell 20 overproliferation (e.g., malignancy) and thus can have use as animal models of such diseases and-disorders, e.g., to screen for or test molecules (e.g., potential anti-cancer therapeutics) for the ability to inhibit overproliferation (e.g., tumor formation) and thus treat or prevent such 2S ~ Res or disorders.
In a different embodiment of the invention, transgenic animals that have incorporated and express a dominant-negative mutant FHIT gene have use as animal models of diseases and disorders involving cell overproliferation. Such An; ~l s can 30 be used to screen for or test molecules for the ability to speci~ically inhibit the dominant-negative mutant and thus treat or prevent such diseases and disorders.

--WO97/29119 CA 02245783 1998-08-06 PCT~S97/01937 6. THE HUMAN FHIT GENE, SPANNING THE
CHROMOSOME 3pl4.2 FRAGILE SITE AND
RENAL CARCINOMA ASSOCIATED TRANSLOCATION
BREAKPOINT, IS ABNORMAL IN DIGESTIVE
TRA~T C~NCERS

As described herein, we have isolated and characterized a human gene involved in ~orh~geal, gastric, colon, kidney, and other cancers. A 200-300 kilobase (kb) region of chromosome 3pl4.2, including the fragile site locus, FRA3B, is involved in homozygous deletions in multiple tumor-derived cell lines 10 and in hemizygous deletions in ~sQr~geal, gastric, colon, kidney and other cancers. Exon amplification from a cosmid contig covering this 200-300 kilobase region allowed identification of the human FHIT gene, a member of the zinc-binding histidine triad gene family, which encodes a 15 ubiquitous 1.1 kilobase transcript and a 16.8 kDa protein with homology to a protein kinase c inhibitor gene, another member of the HIT family.
The FHIT locus i5 composed of 10 small exons distributed over at least 500 kilobases, with the three 5' most 20-untranslated exons mapping centromeric to the clear cell renal carcinoma associated 3pl4.2 translocation breakpoint; the remaining exons map telomeric to this translocation breakpoint with exon 5, the first amino acid coding exon, falling within the homozygously deleted fragile region, FhA3B, and exons 6-10 25 mapping telomeric to the tumor cell common deleted region and the FRA3B region. Aberrant transcripts of the FHIT locus were found in approximately 50% o~ esophageal, stomach and colon carcinomas, and the familial t(3;8) renal carcinomas have lost one FHIT allele due to disruption by the translocation.
The aberrant F~IT transcripts usually resulted from abnormal splicing, which often deleted exon S or 8, resulting in transcripts which could not encode Fhit protein. Thus, chromosome abnormalities at 3pl4.2 and FRA3B, resulting in loss of the Fhit protein, are involved in initiation and/or 35 progression of several important types of human cancer.

-WQ97r29119 PCT~S971~1937 6.1. R~SULTS
Th~ c08~id contig From the 648D4 cosmid library, clones were selected initially using the BE758-6, A6URA, A3, and 1300E3, probes, S which were distributed across the homozygously deleted region a~ shown in Fig. lA. Cosmid end-clones were then isolated and used for the next round of cosmid screening. The cosmid map was as~embled by PCR-amplification of the starting STSs (DNA
~ seguence tags) and new ones developed from cosmid ends, using 10 cosmid DNA templates. Additionally, each new STS was tested against the YAC contig (also shown in Fig. lA), against cell lines with homozygous deletions and rodent-human hybrids ret~i~in~ portions of chromosome 3 (LaForgia et al., 1993, Cancer Res. 53 3118-3124; Druck et al., 1995, Cancer Res.
15 55:5348-5355; Bullrich et al., 1995, Cytogenet. Cell Genet.
70:250-254). Six cosmids were assembled into a contig which covered the homozygously deleted region.
To define more precisely the homozygously deleted region, which we will refer to as the fragile region, 42 STS markers, 20 epAnn;ng the chromosomal region from the P~PRG locus to D3S1234, derived from cosmid walking and exon trapping, were tes~ed by PCR-amplification for presence in eleven cancer-derived cell lines which had been tested previously with a subset of markers (data not shown; and Lisitsyn et al., 1995, 25 Proc. Natl. Acad. Sci. USA 92:151-155).
Colon carcinoma-derived LoVo, HT29 and SW480 and gastric carcinoma-derived AGS cell lines showed similar large deletions such as depicted by the dotted portion of the top line in Fig. lA. Colon carcinoma-derived LS180 and breast 30 carcinoma-derived MDA-MB436 cells exhibited discontinuous deletions, covering this same region, with most markers lost but some retained. The gastric carcinoma-derived KatoIII
cells appeared to have lost the D3Sl481 marker and the telomeric portion of the fragile region, from AP4/5 to D3S2977 35 (see Fig. lA). The HKI cells, derived from a nasopharyngeal carcinoma (NPC), had lost the region between D3S1481 and the AP4/S marker, while CNE2, another NPC-derived cell line had a W O 97/2gll9 PCT~US97/01937 discontinuous deletion which included a region near the t(3j8) and the region between D3S1481 and D3S2977. HeLa cells also -exhibited discontinuous deletions with one deleted region near the t(3;8) and between D3S1481 and AP4/5. The NPC-derived 5 CNEl cells were tested with most markers without detection of a deletion. Thus, there are many different tumor associated 3pl4.2 chromosome breakpoints surrounding the t(3;8), the F~A3B locus and the homozygously deleted region covered by the cosmid contig.

Isol~tion of cDNA~
The six cosmids covering the homozygous deletion, shown in Fig. lA, were used in exon trapping experiments aimed at identifying genes within the deleted region. Putative trapped 15 exons were sequenced and sequences analyzed using GRAIL 2 of the ORNL GRAIL server. Several trapped exons were recognized as exons by Grail 2 and were used as probes on northern blots of poly A+ RNA from a spectrum of human tissues. Addi-tionally, se~uences of trapped exons were comp~red against 20 nucleotide sequence databases. One exon, trapped from a cosmid 76 subclone (c76, Fig. lA) matched a number of cDNA
sequences from breast (G~nh~nk accession #R53187 and #R86313) and fetal liver and spleen (#R11128) libraries submitted by the Washington University-Merck EST Project. A 23 basepair 25 (bp) oligonucleotide primer designed from this sequence (Fig.
2A, primer X8) was used in primer extension to obtain a 5' extended product of the cDNA by a RACE (Rapid _mplification of cDNA ends) reaction (Marathon~ cDNA amplification ~it, Clontech). The longest product (37Q bp) from the RACE
30 reaction detected a ubiquitously expressed 1.1-kb mRNA by northern blot analysis of mRNAs from various normal tis~ues.
The size-was similar to the length of the largest cDNA clone isolated from a normal colon cDNA library using the same DNA
fragment as a probe. The DNA sequence analysis of this full 35 length clone (Fig. 2A) revealed a long 5' untranslated region of more than 350 bp followed by an initial methionine codon and surrounding sequence which fitted Kozak's rule, an open W~97129119 PCT~S97101937 reading frame (ORF) of 147 amino acids, a 3' untranslated region, a polyadenylation consensus sequence and a poly A
tail. Exon sizes varied widely, e.g., exon 5 having 120 nucleotides, exon 6 having 146 nucleotides, and exon 7 having 5 30 nucleotides. With reference to Figure 2A, exon 1 consists of nucleotide numbers -362 to -213; exon 2 consists of nucleotide numbers -212 to -164; exon 3 consists of nucleotide numbers -163 to -111; exon 4 consists of nucleotide numbers -110 to -18; exon 5 consists of nucleotide numbers -17 to 103;
10 exon 6 consists of nucleotide numbers 104 to 249; exon 7 consists of nucleotide numbers 250 to 279; exon 8 consists of nucleotide numbers 280 to 348; exon 9 consists of nucleotide numbers 349 to 449; and exon 10 consists of nucleotide numbers 450 to 73~.
A hydrophilicity plot for the Fhit protein was carried out and is shown in Figure 6.
This F~IT cDNA, as well as the matching sequences from the EST database, were translated and open reading frame (ORF) amino acid seguences (Fig. 2A) compared to the protein 20 databases. The longest EST in the 5' direction was R50713 (which contained sequence found in the 3' end of FHIT exon 7, exon 8, and exon 9). The longest EST in the 3' direction was R11128 (which contained sequence found in half of exon 2, and in exons 3-6). EST R53187 had the longest span of sequences 2S corresponding to the F~IT cDNA, including 297 nucleotides identical to the FHIT cDNA sequence from exon 2 through a portion of exon 5. Among the best matches in the database retrieved by co~uLer searches, this 297 nucleotide sequence was the longest stretch of identity with the FHIT cDNA
3~ sequence. The next longest stretch of identity was found in EST 11128, with 287 nucleotides identical to the FHIT cDNA
sequence starting within exon 2 until 3 bases before the end of exon 6. A printout of the R5~713 nucleotide se~uence aligned with the FHIT cDNA sequence (cDNA 7F1) and the R11128 35 nucleotide seguence is shown in Figure 7. As will be noted, neither of the R53187 nor R11128 nucleotide sequences, or any of numerous other EST sequences, span the full FHIT protein WO 97t29119 PCTnUS97/01937 coding region. Also, the translations of the R50713, and R11128 sequences in all three reading frames, in both orientations, are shown in Figures 8 and 9, respectively, and from none of the translated sequences shown in Figures 8 or 9 5 can the Fhit protein seguence be deduced.
The full length F~IT cDNA probe was then hybridized to northern blots carrying mRNA from a spectrum of tissues. As shown in Fig. 3A, the cDNA detected the ubiquitously expressed l.l-kb transcript.

Rel~tion~hip of the cDNA to the gsnomic map of ~he region Oligonucleotide primers from the initially trapped exon were used to generate intron sequences from cosmid 76; these sequences were used in turn to prepare primers and probes to 15 map the exon (E5 in Fig. lA) on the cosmids, YACs and DNA from cancer cell lines with deletions, as illustrated in Fig. 1A.
Using cDNA as template, oligonucleotide primer pairs bracketing the exons upstream and downstream of exon 5 were then used to amplify cDNA fragments to serve as probes for 20 mapping the 5' and 3 ~ flAnk; ng exons on the cosmid contig;
these probes demonstrated that the cDNA se~uences 5' and 3' of exon 5 were not within the 648D4 cosmid contig covering the homozygous deletions. Thus, cosmid libraries from YACs 850A6 and 750Fl, which extend centromeric and telomeric to the 25 fragile region deletions, respectively, as shown in Fig. lA, were prepared and screened with the 5' and 3' cDNA probes flanking exon 5. Cosmids contAin;ng the remaining exons were then used to derive intron seguences using cDNA primers, and the structure of the gene determined as shown in Fig. lA. The 30 cDNA consisted of 10 exons which were distributed among 3 YAC
clones (Fig. lA); exons 1 through 4 mapped to YAC clone 850A6, exon 5 was present in all three YAC clones, and exons 6 through 10 mapped to YAC clone 750F1. Only exon 5 fell within the region of homozygous deletion in tumor-derived cell lines, 35 i.e. within YAC clone 648D4. The coding region of the ORF
began in exon 5 and ended in exon 9, as shown in detail in Figs. 2A and 2~.

CA 02245783 l998-08-06 WO 97/29119 P~TnUS97J01937 Mo~t interestingly, the first three exons (El, E2 and E3) of the gene mapped centromeric to the t(3;8) break, between the t(3;8) break and the 5' end of the PTPRG gene, as determined by amplification of these exons from the YAC DNAs 5 and DNAs derived from hybrids carrying portions of chromosome 3, derived from the t(3;8) break and a FRA3B break (data not shown). Thus, this gene became a strong candidate ~or involvement in initiation of the familial ~CCs, because one copy of the gene is disrupted by the translocation.
The homology 5earch in amino acid s~quence databases showed a significant homology to a group of proteins which have a histidine triad motif, designated ~IT proteins (Seraphin, 1992, J. DN~ Sequencing & Mapping 3:177-179). The predicted amino acid sequence of the cDNA for the human gene, 15 designated the Fragile Histidine Triad gene or the F~IT gene, is shown in Fig. 4A compared to the other members of the ~IT
family. The highe~t homology o~ the F~I~ protein ( 50~
identity) is to the yeast diadenosine hydrolases (aphl s), shown in Fig. 4A as PAPHl and CAPHl, identified in S. pombe 20 and S. cerevisiae, respectively (Huang et al., 1995, Biochem.
J. 312:925-932). An alignment of the yeast (S. pombe) Ap4A
hydrolase (PAP~l) sequence (U32615) with FHIT (cDNA 7Fl) is shown in Fig. lOA. There is not extensive homology. When we did a computer search for homology stretches between the yeast 25 hydrolase and the FHIT nucleotide sequences, the result was the small region of nucleotide homology shown in Fig. lOB.
The consensus sequence for the HIT family proteins is shown below the amino acid sequences in Fig. 4A.
To recapitulate, the F~IT gene, which may be the human 30 cognate gene for the yeast Ap4A hydrolase gene, spans a >500 kbp region which includes the t(3;8), the FRA3B and a tumor cell-specific commonly deleted region.

WO 97/29119 PCTrUS97/01937 E~pression of the F~Iqr gene We had placed the BB758-6 locus and microsatellite marker, D3S1300, within the region of common loss in a variety of tumor-derived cell lines and our LOH study of gastric and 5 colon tumors detected a high frequency of allelic deletion, often involving D3S1300, in the region between the t(3;8) and the D3S1234 locus ~see Fig. lA). Now, the localization of both the BE758-6 and D3S1300 loci within the FH~T gene locus, close to the first coding exon, exon 5, suggested that the lO FHIT gene was the target of deletion in uncultured tumors, as well as tumor-derived cell lines. To begin an analysis of FHIT transcripts in tumor-derived cells, mRNAs from tumor-derived cell lines and normal tissues was studied by northern analysis.
Poly A+ RNA from normal tissues and a number of NPC, colon and gastric tumor-derived cell lines, with and without apparent deletions in the fragile region, was tested for hybridization to the FHIT cDNA on northern blots (Figs. 3A and B)~
A low level of expression of the F~IT gene occurred in all human tissues tested, as shown in Fig. 3A for spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood lymphocytes. The major transcript was -1.1 kb with a longer transcript at -5 kb, which was barely 25 detectable or undetectable on some blots. Since the 1.1 kb transcript matches the size of the full-length cDNA, the longer transcript may represent a precursor RNA which is not fully pro~s~e~. Similar transcripts were seen in mRNA from brain, heart, lung, liver, skeletal muscle, kidney and 30 pancreas, with the putative unprocessed RNA appearing to be more abundant in lung, small intestine and colon on some northern blots.
mRNAs from tumor-derived cell lines with known homozygous deletions in the fragile region exhibited varying levels of 3S FHI~ transcripts ~Fig. 3B), from ~arely detectable ~Fig. 3B, l~nes 2-4, KatoIII, HKI and LoVo mRNA, respectively) to almost -- g6 ----WO97/2~119 PCT~S97J01937 a normal level (lane 8, LS180), relative to normal small intestine mRNA (lane 1).
Note that the NPC cell lines with (CNE2, HK1; Fig. 3B, lane S, 3) and without (CNE~; Fig. 3B, lane 6) homozygous 5 deletions we had documented expressed barely detectable FHI~
mRNA. ~he NPC-derived cell line, CNE2, exhibited a possible smaller transcript (Fig. 3B, lane 5), while Colo320, a colorectal carcinoma-derived cell line without a deletion, exhibited an apparently normal-sized F~IT transcript (Fig. 3B, 10 lane 7), al~ho~ it should be noted that size alone does not imply presence of a wildtype transcript. The -1.1 kb bands could harbor transcripts with one or more small exons missing, since several exons are very small, e.g. exon 7, 30 nucleotides, exon 2, 49 nucleotides, exon 3, 53 nucleotides.
15 One conclusion of the northern ana~ysis is that there was no direct relationship between size or abundance of transcript and detection of homozygous deletions in specific tumor-derived cell lines, suggesting that there may be small deletions in some tumor cell lines which have not been 20 detected with the available markers.

RT-PCR ~n~ cDNA s~qu~n¢~ ~n~lysi~ of tumor-d~rived mRNA
In order to look for abnormalities in FNIT transcripts from deleted and nondeleted tumor cell lines, we reverse-25 transcribed mRNAs with (dT3 17 primer, amplified the cDNA with5' and 3' primers and then reamplified using primers inside the original primers (nested PCR), as described in methods.
Positions of the primers are shown in Fig. 2A. The amplified products were separated on agarose gels and normal-sized and 30 aberrant fragments were cut from the gels and sequenced (examples of aberrant bands are shown for mRNAs of uncultured tumors in Fig. 3C; RT-PCR products from the tumor cell lines were very similar). The tumor-derived cell lines exhibited a pattern of products ranging from only one apparently normal-35 sized amplified transcript to numerous aberrant bands withouta normal-sized band. Some tumor-derived cell lines exhibited both an apparently normal-sized and one or more aberrant W O 97129119 PCT~US97/01937 bands. The sequencing of the aberrant bands revealed numerous abnormal products, some examples of which are illustrated in ~
Fig. lB. Colon tumor-derived CCL235 and CCL234 cell lines did not show deletion of the STS markers tested, but both showed 5 aberrant transcripts, as illustrated, with CCL235 exhibiting a normal-sized product in addition. HT29 and KatoIII cell lines both showed homozygous deletion, but the KatoIII cell line exhibited a deletion of the telomeric portion of the homozygously deleted region and not the region containing 10 exons 4 and 5, nor the region of exon 6, exons which are all missing in the aberrant RT-PCR product, as illustrated in Fig.
lB. Numerous other tumor-derived cell lines also exhibited aberrant RT-PCR products similar to those shown schematically in Fig. lB (data not shown). Detailed descriptions of similar 15 aberrant products from uncultured tumors (Fig. 3C) are given in Table 2 and Fig. 2B.
Ten case~ of uncultured esophagus, nine of stomach and eight of colon tumors were analyzed, and aberrant transcripts were observed in 5, 5 and 3 cases, respectively (summarized in 20 Tables 2 and 3 and illustrated in Fig. 2B.

Table 2 Derivation of F~IT RT-PCR ~m~ Pd Products and cDNA Se(l-lenres From Uncultured Tumors of Gastric Organs Cases with abe ~~l tl~sc - No. of cases Origin of Tumors analyzed Number of cases Codes ' l~so~ba~ 10 5 E3*, E12*, E13*
E32*, E37*
St~m~h 9 5 Jl*, J3, J4*, J7, J9*
Colon 8 3 9625*, 5586*, 9575*
In cases with asterisks (*), normal tissues from the same organs were analyzed and did not exhibit alt~,~dliol~s in the coding region sP~UPnr~p~.

WO 97129t 19 PCT/lJSg7101g37 Table 3 Aberrant T~ ,ri~ls Observed in Uncultured Tumors Insertion~
Tumor~erived nel~i~ n Putative protein~
,L;~L'' (posilio~ Size (bp) Homology Effect coded in frame 1O *E3 a 28~348 72 NS Ex 8 loss H~(-) *E12 a 28~348 - - Ex 8 loss H~(-) - b 122-516 - - FS after EX 6 HIT(-) *E13 a -17-249 - - Ex 5 & 6 loss - b -17-348 - - Ex 5-8 loss 15 E32 ~ 280-449 ~ ~ Ex 8 & 9 loss HlT(-) *E37 a - 72 NS none intact b -73-173 - Ex S loss HlT(+) *9625 a 28û-348 - - Ex 8 loss H~(-) ~ b -17-279 87 Alu Ex 5-7 loss - c -110-204 - - Ex 4 & 5 loss HlT(+) *5586 a -17-349 135 Alu Ex 5-8 loss - b -17-279 37 NS Ex 5-7 loss -*9575 a 280-348 - - Ex 8 loss ~IlT(-) ~ 60-181 - - FS after Ex S H~(-) - c -110-348 - . - Ex 4-8 loss J1 a -110-(-17) - - none intact - b -17-279 - - Ex 5-7 loss 30 J3 - -17-279 173 Alu Ex 5-7 loss HlT(+) J4 - -17-457 305 Alu Ex 5-9 loss *J7 - a -110-249 - - Ex 4~ loss *~9 a 280-348 - - Ex 8 loss H~(-) 35 ' All the ~f ,~ ansc~i~ which involve the~coding se~uenre of the FHIT gene are shown in Figure 3B. Alu, Alu repeat, FS, fr~rn~shi~; NS, no signific~nt homology; Ex, e~on.
_ 99 _ -W 097/29119 CA 02245783 l998-08-06 PCTrUS97/01937 2 In tumors wi~ ~5,~ (*3, normal l-a.,sc.i~ without alteration of coding ~egion se~lPn~,e were also observed.
s The positions of ~e ~Irst and t~t nucleotides of ~e deletions are shown acco~ing to ~e nucleotide n~ b". ~ in Figure 2A.
S ~ The position of all i~ ,,lions was dow,~ n of exon 4.
~ protein coded in frame with the Fhit protein is shown: HlT(~), protein wi~
HIT motif; HlT(-3, protein wi~out HIT motif; -, no protein in frame.

The sequence analyses of the aberrant cDNAs revealed absence of various regions between exons 4 and 9 (Table 3 and Figure 2B), while the RT-PCR and cDNA sequence analyses of normal tissue mRNAs from the same organs did not exhibit any alterations of the coding region sequence (~able 2, E3, ~12, E113, E37, Jl, J4, Js, 9625, 5586, 9575). In 8 of 13 cases with aberrant transcripts, normal-sized transcripts were also observed (Fig. 3C; E3, E12, E13, E37, 9625, 9575, J7 and J9;
E12 and 9575, not shown~, while in 5 of 13 cases normal-sized transcripts were not detected (Fig. 3C, J3, J4), or were barely detected (Fig. 3C, E32, 5586, Jl). In most of the aberrant transcripts, the beginning and the end of the deleted portions of the transcripts coincided with splice sites (Fig.
2B), suggesting that the cDNA deletions resulted from the loss of genomic regions containing or surrounding the relevant lost FHIT exons. The aberrant transcripts can be classified into two groups (class I and II, Figure 2B): class I transcripts lack exon 5, which has the initial methionine codon of the FHIT ORF, resulting in the locs of the ORF; class II
transcripts have an intact initial methionine codon but do not include exon 8, except for 9575b, which exhibited a frameshift after exon 6. Thus, in all the class II transcripts, the wildtype ORF of exon 8, the histidine triad contain;ng domain, is not present. Moreover, some of the class II transcripts exhibited loss only of exon 8 (Fig. 2B; E3, E12a, 9625a, W O 97129119 PCTnUS97/01937 .
9575a, J9a), suggesting that exon 8 was the target of deletion. Since exon 8 encodes the histidine triad motif, it-is likely that neither class I nor class II transcripts, constituting the major fraction of aberrant transcripts, can 5 encode a fully functional protein. However, there is an in frame methionine (Met) codon in exon 6 (~ee Fig. 2B), and in some cases insertions contribute an in frame Met (not shown~;
thus, the majority of aberrant transcripts could encode ~ partial proteins with or without the HIT domain as indicated 10 in Table 3. Insertions of various lengths, of DNA not derived from the F~IT gene, were observed in some transcripts;
insertions were found only downstream of exon 4 (Table 3, 5586a, 5586b, 9625, J3, J4). A minor group of aberrant transcripts retained intact full length ORFs, but were missing 15 exon 4 (Table 3, Jla), or had insertion of 72 bp of DNA
sequence in the 5' noncoding region between exon 4 and 5 (Table 3, E37a, and Fig. lB). It ~s possible that such insertions affect translation of the ORF.
In order to determine if the wildtype FHTT cDNA and 20 various cDNAs derived from tumor specific transcripts, which retained the entire coding region, could be translated 7n vitro, several recombinant plasmids were constructed, each con~;n;ng a F~IT gene downstream from the T7 promoter and lacking the first noncoding exon. The pFHIT1 plasmid carried 25 an aberrant cDNA, missing exon 4, from the CC~234 colon cancer cell line. Plasmid pFHIT2 carried a cDNA from esophageal tumor E37 with an insertion of 72 bp between exon 4 and exon 5. The pFHIT3 plasmid contained the wildtype F~I~ gene lacking exon 1. The constructs were used for in vi ~ro 30 translation by rabbit reticulocyte lysate. Analysis of translation products (Fig. 4B) showed the predicted 16.8 kDa protein translated from each cDNA construct.

Th~ FHIT protein } 35 The protein se~uence predict~d by the FHrT cDNA is very similar (57/10~ amino acid identities; 76/log or 69%, ~imilarities, as calculated by the NCBI BLAST server) to the W O 97/29119 CA 02245783 1998-08-06 PCT~US97/01937 S. pombe diadenosine 5l,5~PI, ~ tetrapho~phate hydrolase, aphl (Huang et al., ~995, Biochem. J. 312:925-932), as shown -in the amino acid alignment in Fig. 4A, where PAPH1 represents the S. pombe sequence.
The S. pombe aphl enzyme was cloned by purification o f the enzyme, amino acid sequencing of the N-terminus and decign of primers to amplify a partial cDNA; the full length genomic and a cDNA of 1.2 kbp were then cloned, sequenced and translated (Huang et al., 1995, Biochem. J. 312:925-932). By 10 similar methods, a human hydrolase (APW1) has been cloned, sequenced and translated (~horne et al., 1995, Biochem. J.
311:717-721) and, surprisingly, does not resemble the S. pombe apAl gene nor the FNIT gene. Since higher eukaryotes appear to possess a single 16-21 kDa Ap4A asymmetrical 15 pyrophosphohydrolase (cited in Thorne et al., 1995, Biochem.
~. 311:717-721), it is thus not clear if the FHIT gene is a human APNl enzyme, although it may be a human cognate of the S. pombe aphl enzyme.
The FHIT gene is also very similar to the S. cerevisiae 20 aphl gene product (CAPH1 in Fig. 4A) with 40% identity and 62%
similarity in the 50 amino acids between 49 and 102 of the FHIT amino acid sequence, and higher similarity in the HIT
domain. The other proteins or hypothetical proteins in Fig.
4A are all members of this ~IT gene family, a family of 25 proteins present in prokaryotes, yeast and mammals, described by Seraphin, 1992, DNA Sequencing & Mapping 3:177-179. The signature feature of the family is the histidine triad (most commonly HVHVH, amino acids 94-98 of the FHIT protein, Fig.
4A), which for the case of BHIT (Fig. 4A), the bovine 30 inhibitor of protein kinase C (PRCIl) has been shown to be a zinc-binding site (Pearson et al., 1990, J. Biol. Chem.
265:4583-4591; Mozier et al., l9gl, FEBS 279:14-18). The Fhit protein product is only 39~ similar to the bovine PKCI1 protein over Fhit amino acids 12-lOo, as calculated by NCBI
35 BLAST. Thus, the FHIT gene is not~likely to be the human PRCIl gene. Functions of the other ~IT genes are thus far not known. Furthermore, structural features of family members WO 97/29119 PCT/US97101g37 have not been studied extensively. The PKCI1 protein has a predicted content of 23% ~ helix and 42% ~ conformation (31%
sheet and 11% ~ turn) (Pearson et al., l99o, J. Biol. Chem.
265:4583-4591); the conserved region, including the histidine 5 triad and upstream region were predicted to be mostly random coil alternating with ~ sheet conformation, with the HIT
~o~a~n ~ sheet. This conformation may be preserved in the F~it protein. Also, the HI~ domain consists of basic and ~ hydrophobic amino acids and might be expected to be buried 10 inside the protein, as suggested for the PKCIl protein (Pearson et al., 1990, J. Biol. Chem. 265:4583-4591).

6.2. DISCUSSION
The ~Aning of fragile sites for cancer has been a 15 subject of speculation for years and the near coincidence of the chromosomal position of the FRA3B and the t(3;8) translocation at 3pl4.2 has been especially intriguing. The FRA3B is constitutive; that is, after treatment of peripheral blood lymphocytes with ~0.4 ~M aphidicolin, which interferes 20 with the action of DNA polymerase ~, the characteristic gaps in chromosome region 3pl4.2 are observed in ~70% of metaphases from all individuals. So the structural basis for the induction of gaps is present in all individuals. It is also known that within the 3pl4.2 band, some of the induced gaps 25 represent chromosome breaks, which occur possibly at several sites in the chromatin of an ~200-300 kilobase region (Paradee et al., 1995, ~enomics 27:358-361). Thus, the sequences involved in gaps and breaks may occur in more than one site within the fragile region. At other fragile sites such as the 30 folate-sensitive fragile sites on X, FRAXA, FRAXE, FRAXF, the structural basis for the gaps seems to be variable lengths of CCG or CGG triplet repeats and imperfect repeats are more stable than perfect repeats (Chung et al., 1993, Nature Genet.
5:254-258); these fragile sites seem to be single sites of 35 fragility. Perhaps the FRA3B appe~ars to be the most common fragile site because it actually represents a collection of different fragile sites in a small chromosomal region. The CA 0224~783 1998-08-06 specific sequences responsible for the breaks at FRA3B in ~y~rid cells have not been described but we have observed that-many tumor-derived cell lines exhibit apparent discontinuous homozygous deletions. Fig. 5 diagrams the relationship 5 between the various types of chromosome breaks in 3pl4.2 and the organization of the FNI~ gene relative to the breaks.
Note that in Fig. ~, the chromosome breaks and deletions in the KatoIII gastric carcinoma-derived cells leave the ~o~;n~
region intact, but we have observed only aberrant FHI~
10 transcript in this cell line. Thus, inapparent chromosomal abnormalities must account for the lack of normal transcription in KatoIII and other tumor cells; one possibility is that two F~IT alleles are present in KatoIII
with hemizygous alterations in the portions of the FHIT genes 15 not homozygously deleted. Another possibility is that alteration near an exon affects splicing. Additionally, some cancer-derived cell lines and uncultured tumors showed transcripts with alterations to noncoding regions of the FHIT
transcript. These transcripts were transcribed and translated 20 into full length protein in a coupled system using a reticulocyte lysate for translation (Fig. 4B), but perhaps in the tumor cells from which they were derived, the lack of exon 4 or insertion of new sequences would affect expression of the Fhit protein. Another puzzle, if the FHIT gene acts as a 25 classical suppressor gene with inactivation of both alleles, is the presence of normal-sized transcripts along with aberrant products in the RT-PCR amplification products of tumor-derived cell lines such as CCL235 (colon), A549 (lung) and HeLa (cervical). It is possible that the aberrant 30 transcripts, which in most cases might encode partial Fhit proteins, could interfere with the function of a normal Fhit protein.- The normal-sized products from these cell lines have not yet been fully se~uenced so it is possible that they do not, in fact, represent normal transcripts. A number of the 35 uncultured tumors also exhibited a~berrant and normal-sized products, and sequencing showed that some of these normal-sized products were indeed wildtype products. In these cases, CA 02245783 l998-08-06 W O 97129119 PCTnUS97/01937 the normal transcripts could have derived from admixed normal cells .
We have not yet observed point mutations within the coding region of any FHIT transcripts, perhaps suggesting that 5 aberrant FNIT yenes usually are the result of deletions.
Aphidicolin, which inhibits the action of DNA polymerase ~, induces the gaps and breaks observed in the FRA3B region in normal met~phA~es; thus in the digestive tract tumors and tumor cell lines we have studied, the genomic deletions 10 resulting in aberrant transcription and loss of functional Fhit protein, could have been induced by exposure of these organs to other agents which interfere with DNA replication, such as nicotine, caffeine, possibly alcohol and other known carcinogens. Interestingly, zinc deficiency is associated 15 with a high fre~uency of esophageal tumors in man (Yang, lg80, Cancer Res. 40:2633-2644) and rat (Fong et al., 1978, J. Natl.
Cancer Inst. 61:145-150); zinc deficiency may cause proliferation of the epithelial cells lining the esophagus (Yang et al., 1987, J. Natl. Cancer Inst. 79:1241-1246), so 20 perhaps zinc deficiency mimics loss of the Fhit protein, which may require bound zinc for its function. It is, therefore, interesting that F~IT gene exon 8, carrying the HIT motif, the presumptive zinc binding site, is a target of deletion in numerous digestive tract tumors.
Whether or not this region of 3pl4.2 contains repeated CCG or CGG triplets is not yet known, but because there are differences between the rare, inherited folate-sensitive fragile sites which have been characterized, and the common, constitutive, aphidicolin fragile sites, perhaps a different 30 basis for the fragility should be expected. Thus far, we have noted that there are many Alu repeats in the telomeric portion of the fragile region (not shown) and there is a (TAA)I5 repeat in this same commonly deleted region for which the nt h~r of repeats is highly variable. Perhaps other triplet repeats of 35 this type exist in the region. Also in ~9 kilobase pairs of sequenced portions of the cosmid S8 (telomeric portion of the fragile region, see Fig. lA), several Alu repeats and a LINE

WO97/29119 CA 02245783 lggs-os-06 PCT~S97/01937 element were encountered; the nucleotide content of the ~equenced region was 57.4% A and T residues, while the FHI~
cDNA nucleotide content was 48% A and T. A high A and T
content is characteristic of some characterized origins of DNA
5 replication, especially in yeast and, in fact, although higher eukaryotic origins of replication have not been identi~ied, it has been speculated that Alu repeats may be connected with replication. Another notable feature of the FHIT gene itself is that nearly all the exons end with the sequence AG, the 10 usual sequence for splice acceptor sites. Based on our observation of frequent aberrant splicing in this fragile region, it is tempting to speculate that the region is especially rich in sequences resembling splice acceptor sites.
Interestingly, we have previously observed a homozygous 15 deletion in mouse ~ cells, which involves several N-terminal exons of the murine Ptprg gene (Wary et al., 1993, Cancer Res.
53:1498-1502), and Pathak et al. (1995, Cancer Genet.
Cytogenet. 83:172-173) have shown that mouse colon and mammary tumors as well as melanomas have abnormalities in the proximal 20 region of mouse chromosome 14 where Ptprg (Wary et al., 1993, Cancer Res. 53:1498-1502) and probably Fhit loci map.
Studies of FHIT gene RT-PCR products from RNA of numerous cell lines suggested that FHIT gene abnormalities could be important not only in airway and digestive tract tumors such 25 as nasopharyngeal, esophageal, stomach and colorectal carcinomas, but possibly also in ovarian, cervical and lung tumors, osteosarcoma, and some leukemias; also a bladder and breast carcinoma cell line exhibited homozygous deletions in the fragile region (Lisitsyn et al., 1995, Proc. Natl. Acad.
30 Sci. USA ~2:151-155; and our data). Thus, uncultured tumors of these types should be tested for FHIT gene abnormalities.
Cl~ar cell RCCs might also be expected to involve FHIT
gene aberrations because the FHIT gene is disrupted by the familial RCC translocation break in 3pl4.2 and the 35 translocation/FRA3B region is the~target of allelic loss in most sporadic clear cell RCCs (Druck et al., 1995, Cancer Res.
55:5348-5355). Since the FHIT ORF is contained in exons 5 CA 02245783 l998-08-06 W O97/29119 PCT~US97JOlg37 through 9, translocated to chromosome 8 in the t~3;8) family, it is possible that both alleles could still be expressed in -some or all tissues; we have found a few polymorphisms within the ORF but none yet which distinguishes the two allelic FHIT
5 transcripts in the t(3;8) lymphoblastoid cell lines (data not shown). If the FNIT gene disruption is the first "hit" to a suppressor gene in this family, then the second FHIT allele should be altered in the tt3j8) tumors. Since we have not yet detected point mutations in the FHIT gene, the best way to 10 look for alterations of the FHT~ gene in t(3;8) RCCs would be to amplify the FNIT reverse transcript, as done for uncultured tumors in this study. We have done this experiment for RNA
from two RCc cell lines and two uncultured RCCs, all from sporadic tumors, and have observed normal-sized products, 15 which have not yet been cloned and sequenced. Nor have we yet observed homozygous deletions in RCCs using a subset of STS
markers in the fragile region. Nevertheless, it would be surprising if the FHIT gene is not involved in some sporadic RCCs.
Since the FHIT gene is probably ubiquitously expressed, it may not be surprising if it can serve as a tumor suppressor gene for specific tissues of many different organs, apparently predominantly of the digestive tract, or maybe predominantly organs with epithelial cell linings. Another common 25 denominator of the types of tumor exhibiting aberrant F~IT
alleles might be that they are predominantly organs dirsctly exposed to environmental carcinogens; some of the types of tumors exhibiting FHIT gene aberrations occur very frequently in restricted regions of the globe, NPC in China, gastric 30 cancer in southeast Asia, and often there are environmental factors at play. A possible role for EBV in promotion of Chinese ~PCs might be through viral ~NA integration into the FRA3B region, suggested by the previous experiments of Rassool et al. ~1992, Am. J. Hum. Genet. 50:1243-1251), showing 35 apparent preferential integration~of exogenous DNA into induced fragile sites in cultured cells. Similarly human papillomaviruses associated with cervical carcinomas might CA 0224~783 1998-08-06 W O97/29119 PCTrUS97/01937 promote induction of the F~A3B, contributing to the loss of heterozygosity on 3p in uterine cancers (Yokota et al., 1989,-Cancer Res. 49:3598-3601), and possibly to inactivation of the -- - FHIT gene. Perhaps the t(3;8) family members, carrying one 5 disrupted FHIT gene succumb to kidney tumors rather than colon or esophageal tumors due to specific types of environmental agents to which they are exposed.
We observed strong similarity of the FHIT gene to S.
pombe and S. cerevisiae Ap4A hydrolases. Specific roles for 10 the diadenosine, Ap4A, have not been defined (Huang et al., 1995, Biochem. J. 312:925-932) and it is not clear that the Ap4A hydrolase activity is the only or even the major in vivo function of these proteins. Expression of the S. pombe aphl in S. cerevisiae did not inhibit growth, but for unknown 15 reasons the S. pombe enzyme was not expressed at a high level (Euang et al., 1995, Biochem. J. 312:925-932). Very little is known of the function of the other members of the ~IT family.
If indeed the FHIT gene is the cognate of the s. pombe aphl gene identified as an Ap4A hydrolase, then the strong 20 conservation (69% similarity) between the yeast and human gene suggests important functions. Whether the FHIT gene does or does not encode an Ap4A hydrolase, it is likely that the study of the S. pombe and S. cerevisiae hydrolase knockouts and other types of mutations will be useful in understAn~;ng the 25 functions of the Fhit protein.
There is some suggestion that as an intracellular regulatory molecule, the Ap4A diadenosine may regulate ability of cells to adapt to metabolic stress such as heat, oxidation, and ~NA damage; thus deviation from a normal level of Ap4A may 30 result in inability of cells to adapt to environmental stresses imposed by carcinogens or viruses which cause genetic damage.~

WO97529119 PCT~S97101937 6.3. MATE~IA~ AND METHODS
Tissues and cell lines Matched normal and cancerous tissues ~rom patients with primary esophageal, colon and stomach carcinomas were obtA; netg 5 immediately after surgery. Tumors were dissected to eliminate normal tissue before preparation of DNA. Many cell lines were obtained from the ATCC. The RC kidney cell lines were kindly provided by E. Lattime.

10 RNA extr~ction an~ reverse tr~nscriptio~
Total and poly A~ mRNA was extracted from cell lines and tissues using the RNAzol kit (TelTest, Inc., Texas) or the FastTrack Kit (Invitrogen), respectively. To obtain mRNA from tissues, fresh specimens were frozen immediately after 15 excision, and stored at -85~C or in liquid nitrogen until extraction of mRNA. RNA was stored as a pellet under ethanol or solubilized in RNAse-free water and kept at -70~C. Reverse transcription was performed in 3Q ~l final volume of 50 mM
tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, l0 mM DTT, 2 ~M dNTPs, 20 500 ng oligo-dT, 600 units MM~V-RT (BRL), 40 units RNasin (Promega), and 2 ~g RNA. This reaction was incubated at 37OC
for 90 min and boiled for 5 min.

DNA ~esruence ~ aly8i8 2S cDNA, genomic clones and putative exons were sequenced using primers specific for vector flanking sequences (T3, T7 etc.) and various synthetic oligonucleotides. RT-PCR products were directly sequenced after isolation of bands from low melt agarose and purification by column chromatography (Qiagen, 30 Chatsworth, CA). Sequencing of double-stranded plasmids, PCR
products and phage or cosmid genomic clones was performed using Taq DyeDeoxy Terminator Cycle Sequencing Kits (Applied Biosystems, Inc. (ABI)); reaction products were electrophoresed and recorded on the 373 or 377 DNA sequencer ~ 35 (ABI3. Sequences were analyzed using GCG, BLAST, and GRAIL
software.

CA 02245783 l99X-08-06 W O 97/29119 pcTrus97l~l937 PCR 2~nplific~tion The oligonucleotides for generating probes, PCR products-and RT-PCR products were designed using the computer program Oligo 4.0 (National Biosciences). For Southern blots, probes 5 were produced by PCR amplification using various FHIT specific primers, as indicated in results. Sequences and positions of some primers are shown in Fig. 2A.
PCR reactions were carried out in 12.5 or 25 ~1 final volume with 1-100 ng of template, 20-40 ng primers, 10 mM
10 tris-HCl pH 8.3, 50 mM KCl, 0.1 mg/ml gelatin, 15 mM MgCl2, 200-600 mM dNTPs and 0.5-2.5 units Taq polymerase (ABI). The amplifications were performed in a Perkin-~lmer Cetus thermal cycler for 30 cycles of 94~C for 30 s (for denaturation), 60~C
(varied for specific primer pairs) for 30 s (for annealing~, 15 and extending at 72~C for 30-45 s. The PCR products were visualized in ethidium bromide stained low melting agarose gels. The bands of amplified DNA were excised from gels and purified for labeling or sequencing.

20 DNA prep~r~tion and 8Outhern blot hybridiz~tion Cellular DNAs were isolated and Southern blots prepared using conventional methods. Probes were labeled by random-priming with t32P]-dCTP (NEN) and hybridized to membranes in 0.75 M NaCl, 50% formamide at 42~C overnight. Final washes of 25 membranes were in 0.1 x SSC and 0.1% SDS at 65~C for 30 min.
Hybridized filters were exposed to XAR-2 X-ray film (Kodak) with intensifying screens for times varying from 1 to 72 h.

Identification of YACs We and others (Boldog et al., 1993, Proc. Natl. Acad.
Sci. USA 90:8509-8513; Boldog et al., 1994, Genes Chrom.
CAnc~r 11:216-221; Wilke et al., 1994, Genomics 22:319-326;
Michaelis et al., 1995, Cancer Genet. Cytogenet. 81:1-12;
Kastury et al., 1996, Genomics, in press) had previously 35 identified the 850A6 clone from the Genethon mega YAC library as containing the D3S1300 and D3S1481 markers (Roche et al., W O 97/29119 PCTnUS97~193 1994, ~um. Mol. Genet. 3:215). Overlapping YACs were identified by analysis of the Genethon database information. -Identificatio~ of region sp~cific 8TSs A number of STSs were available from our work with the 850A6 YAC. The A6URA marker was from the 850A6 URA end, A3 was from an Alu-vectorette amplified fragment of 850A6; BE758-6 and D3S1480 amplified fragments were used as probes to select phage genomic clones from which end se~uences were 10 obtained and ~equence tagged. A phage genomic clone for D3S1300 was selected from the 850A6 phage library and end clone D1300E3 isolated. Other D3S and WI marker primer pairs were obtained from Research Genetics or were synthesized from sequences provided in the WI database. From sequencing of a 15 phage genomic subclone from the 648D4 YAC, a (TA~) l5 trinucleotide repeat was found and designated locus phl3; the AP4/5 STS was derived from partial sequencing of a cosmid subclone of the 648D4 YAC.

20 Co~mi~ mappinS
High molec~ r weight YAC containing yeast DNA in agarose plugs was partially restricted with the Sau3AI enzyme, and subcloned into a cosmid vector. This cosmid library was initially screened with DNA probes derived from STSs 25 previously mapped to this region. The ends of the insert DNAs flanking the cosmid vector were sequenced to find new STSs, which were used as probes to rescreen the cosmid libraries.

Exon trapping ana cDNA cloning The cosmid DNAs were partially restricted with Sau3AI
enzyme, run on a 1.~% agarose gel, and fragments larger than 2 kbp cut out and subcloned into the pSPL vector and transfected into COS-7 cells, according to the manufacturer' 5 instructions. The DNA inserts trapped between the splice t 35 sites of the vector were sequenced~ by a primer supplied with the vector (GIBCO/BRL). The cDNA was extended in the 5' direction by PCR-amplification of a total human fetal brain W 097/29119 CA 02245783 1998-08-06 PCT~US97/01937 cDNA using an exon-specific primer (X8, nucleotide num~ers -l9 to -41) and a RACE reaction kit (Clontech). The normal colon -cDNA library was purchased from Clontech.

S F~I~ Qxon mapping The genomic sequences of exon-intron junctions of the F~IT gene were determined by sequencing the positive cosmids with primers derived from the cDNA. Localization of each exon of the FHIT gene was determined by PCR amplification using 10 primers derived from each exon with YAC and chromosome 3 hybrid DNAs as templates. The primer sequences used to obtain cDNA probes flanking exon 5 were: 5'TCTGCTCTGTCCGGTCACA3' (SEQ ID NO:70) (nuc. #-355 to -337) with primer X8 (shown in Fig. 2A) for 5' flanking exons; 5'ATGTCCTTGTGTGCCCGCT3' (SEQ
15 ID NO:71) (nuc. #105 to 123) with 3D2 (see Fig. 2A) for 3' flanking exons.

Nort~er~ blot and hybridization Two ~g of mRNAs were electrophoresed through 1.5% agarose 20 gel in 2.2 M formaldehyde and 1 x MOPS buffer and blotted to a positively charged membrane by standard procedures. Northern blot filters of multiple normal tissue mRNAs were purchased (Clontech, Palo Alto, CA). The F~IT cDNA probe for hybridization was obtained using the FHIT cDNA as template for 25 PCR-amplification with the following primer pair:
5'TGAGGACATGTCGTTCAGATTTGG3' (SEQ ID NO:72), nuc. #-7 to 17;
and 5'CTGTGTCACTGAAAGTAGACC3' (SEQ ID NO:73), nuc. #449 to 429. Probes were labeled by random-priming with ~32P]-dCTP and 2 x lQ6 cpm/ml was hybridized to each filter. Hybridizations 30 were at 42~C for 16 hours in SSPE buffer (5x SSPE, 10x Denhardt's solution, 0.1 mg/ml carrier DNA, 50% formamide, 2%
SDS). Final washes were in 0.1x SSC, 0.1% SDS at 50~C for 30 min before exposure at -80~C to XAR-2 films (Kodak) with intensifying screens on both sides.
t WO 97/~911g PCTIUS97101937 Ne~stQd RT--PCR and seguencing of cDNAs First strand cDNAs were synthesized and 1 ~l of each product was subjected to a first round of PCR amplification with 30 cycles of 95~C for 20 sec, 60~C for 30 sec, and 72~C
5 for 1 min with 5% dimethylsulfoxide and 0.5 mM spermidine in 10 ~l reaction volume under stAn~Ard conditions, using primers 5U2 and 3D2, indicated in Fig. 2A. One ~l of the reaction products, after 20-fold dilution, was subjected to a second round of PCR amplification using nested primers 5U1 and 3D1 10 (shown in Fig. 2A), under the conditions noted above, except the reaction volume was 30 ~l. The PCR products were run on 1.5% agarose gels, stained with ethidium bromide, purified and 2.5 ng sequenced using the 5Ul primer.

15 In vitro transcription and translation Three different fragments of DNA, containing the FHIT
gene were obtained by PCR, using oligonucleotides UR5(5'CTGTAAAGGTCCGTAGTG3' (SEQ I~ NO:74), nuc. #-171 to -154 in Fig. 2A) and 06 (5'CTGTGTCACTGAAAGTAGACC3' (SEQ ID NO:75), 20 the reverse complement of nuc. #429-449). Amplifications were performed in 100 ~l final volume of 10 mM Tris-HCI (pH 8.9), 50 mM KCl, 1.5 mM MgCl2, 200 ~M deoxynucleotide triphosphates, 10 ng RT-PCR products and 2.~ U Taq polymerase using an Omni Gene Thermal Cycler. 25 PCR cycles consisted of 94~C 1 min, 25 52~C 1 min and an extension step at 72~C 45 sec. PCR products were separately inserted in a PCRII plasmid using the TA
cloning system (Invitrogen). Recombinant vectors, containing the normal FHl T and aberrant genes under the control of the T7 promoter, were sequenced and used for in vitro transcription 30 and translation.
The in vitro transcription and translation reactions were performed by TnT Coupled reticulocyte systems (Promega) in a final volume of 50 ~l contAin;ng l/2 volume rabbit reticulocyte lysate, 1 ~g recombinant plasmid DNA, 10 U T7 ~ 35 polymerase, 20 ~M amino acid mixture without methionine, 40 ~M
35S-methionine (Amersham) and 40 U RNasin ribonuclease inhibitor. Reactions were carried out for 90 min at 30~C.

W097/29119 CA 02245783 1998-08-06 PCT~S97/01937 The synthesized proteins were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.

7. THE F~I~ GENE AT 3pl4.2 IS
ABNOR~AL IN LI~G CANCER
The FHIT gene is disrupted by the t(3;8) chromosomal translocation observed in a family with renal cell carcinoma and contains the FRA3B fragile site and the target of homozygous deletions in various human cancer derived cell lO lines. The study in Section 6 hereof indicates that FHIT gene abnormalities often occur in primary digestive tract cancers.
Deletions of the short arm of chromosome 3 occur at a very high frequency and in early phases of lung carcinogenesis suggesting that this chromosomal region contains crucial genes 15 for lung cancer development. We isolated the FHIT gene, located at the chromosomal band 3pl4.2, and found that it contains the FRA3B fragile site. The gene is disrupted in the t(3;8) translocation observed in a family with renal cell carcinoma and resides in a region which shows allelic losses 20 in various human malignancies. In this study in Section 7 hereof, we have analyzed the role of the FHIT gene in cancers associated with carcinogenic exposure, that is lung cancer of the small-cell and non-small-cell type. Analysis of 5g tumors and paired normal lung tissues was performed by reverse 25 transcription of FHIT transcripts followed by PCR
amplification and sequencing of products; allelic losses affecting the gene were evaluated by microsatellite polymorphisms analysis. About 80% of small-cell lung tumors and 40~ of non small-cell lung cancers showed abnormalities in 30 RNA transcripts of FHIT and 88% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. All the cases showing abnormal transcripts also had loss of one allele of the FHIT gene. The results indicate abnormalities of this gene in nearly all SCLC
35 and in a high proportion of NSCLC, suggesting a critical role for the F~IT gene in lung carcinogenesis.

CA 02245783 l998-08-06 WO97J2~119 PCT~S97J01937 7.1. RESULTS
RT-PCR AND cDN~ 8EQUENCE ANA~Y8I~ ~
8mall Cell ~ung C~n~ 8C~C) In order to study abnormalities in FHIT transcripts from S tumors and normal tissues, we reverse-transcribed mRNAs and amplified the cDNAs by nested PCR as described in methods.
Fourteen primary tumor samples and one cell line (83L) were studied. In two cases matched normal lung parenchyma was also analyzed. Eleven of the 14 cases (79~) analyzed by RT-PCR
10 showed the presence of abnormal transcripts (Fig. 113. The analysis of the amplified transcripts from the primary tumors consistently revealed the presence of two abnormal bands of -360 bp (type ~:) and ~250 bp (type II). Seven cases displayed ~oth type I and II abnormal transcripts whereas four cases 15 showed only the type I band. In two samples (Fig. llA, case 107; Fig. 12A, case 45) as well as in the tumor-derived cell line (Fig. 12A, case 83L) the normal sized transcript was undetectable while in the other nine cases a normal-sized band of varying intensity was observed.
In one patient (Fig. 12A, case 83) we examined the primary tumor, a tumor derived-cell line and a normal lung specimen. Whereas in the normal lung only the normal transcript was detected, the primary tumor exhibited the type I and II abnormal transcripts together with a normal sized 25 transcript and the tumor-derived cell line displayed the type I abnormal transcript and a novel band of -420 bp (Fig. 12), probably generated following in vitro subculturing.
Accordingly, cytogenetic analysis of this cell line revealed extensive chromosomal instability resulting in the presence of 30 dicentric and tricentric chromosomes, telomeric associations and double minutes. FISH analysis with a painting probe of chromosome 3 showed the occurrence of several structural rearrangements of this chromosome including a translocation of the 3p arm with a breakpoint in 3pl4-21 (data not shown).
35 Interestingly, in the cell line, the normal-sized transcript was undetectable suggesting that the normal-sized product r ~ 115 ~

W097/29119 CA 02245783 1998-08-06 PCT~S97/01937 observed in the primary tumor reflected the presence of normal cells infiltrating the tumor specimen.
Abnormal and normal-sized ~ands were separated on agarose gel, cut and sequenced (Fig. 12B). Sequence analysis of the 5 aberrant bands revealed that the type I transcript corresponded to absence of exons 4 to 6 (nt -111 to 249) of the FHIT cDNA sequence (to be accorded G~R~nk accession # u46922), resulting in a junction between exons 3 and 7 of the FHIT cDNA. A }oss of exons 4 to 8 (nt -111 to 348), 10 resulting in fusion of exons 3 and 9 was found in the type II
transcript. In each aberrant transcript the fusion junctions coincided with splice sites.
Sequence analysis of the normal-sized bands revealed that they contained the normal F~IT cDNA, possibly reflecting the 15 contribution of normal cells infiltrating the tumor specimens.
Since both types of aberrant transcripts lacked exon 5, containing the initial methionine codon of the FHIT open reading frame (see Section 6), and type II transcripts also lacked nearly the entire coding region, including exon 8, 20 containing the highly conserved HIT motif (see Section 6), it is likely that the results of these aberrant fusion transcripts would lead to loss of FEIT function.
Sequencing of the normal-sized RT-PCR product amplified from normal tissues (cases 45 and 83) revealed presence of the 25 wildtype F~I T transcript.

Non ~mall Cell Lung Cancer ~NSC~C) -RNA from 45 primary NSCLC tumors and matching normal lung parenchyma samples were similarly studied; 18 of 45 tumors 30 (40~) displayed aberrant RT-PCR products. A detailed descriptlon of these results is summarized in Table 4.

wos7nsll9 PCT~S~710193~

~able 4 RT-PCR, SEQUENCING AND LOH RESULTS IN NSCLC
S CASE/TYPE RT-PCR- SEQUENCE LOH~
1~ N Normal YES
Adel ex3~7 (nt ~ 249) 2~ N Normal NE
Adel ex4~8 ~ (nt -17~279) Adel ex4~9 (nt -17~348) 3~MU~ N Normal NE
Adel ex3--8 (nt -111-279) 4~ N Normal YES
Adel ex4-8 (nt -17-279) 5~ N Normal YES
Adel ex3~9 (nt -111~348) 6~ N Normal YES
Adel ex3-8 (nt -111-279) 7~ N Normal YES
Adel ex3-9 (nt -111~348) 8~ N Normal YES
Adel ex3~7 (nt -111-249) 9~ N Normal NE
Adel ex3~9 (nt -111-348) 10~ N NE YES
A

11~ N NE YES
12A~ N NE YES
A

13~ N NE YES
A

14~ N Normal YES
A~el ex3-9 (nt -111-348) 15~ N NE NI
Adel ex3-s (nt -111~348) 16~ N NE NE
A
17~ N NE
Adel ex4~8 (nt -17-279) -W O 97/29119 PCT~US97/01937 18~DC N NE NE
A
N = Normal Transcript ~ ~A - Abnormal Transcript ~ In Normal Lung Parenchyma: del ex3~9 oLoci analyzed: D3S4103, (phl3), D3S1234, D3S1313, D3S1312 NE: Not Evaluated NI: Non Informative The RT-PCR amplified products from the transcripts present in these tumors consisted of one or two abnormal bands, always accompanied by a normal-sized transcript (Fig.
13A). All the paired normal lung RNAs from the same lung cancer cases showed the presence of the normal FHI~ product 15 only, except one case (case 3 in Table 4) which displayed an aberrant product differing in size from the aberrant product observed in the corresponding tumor. Sequence analysis of the aberrant fragments revealed a range of RT-PCR products with losses of various exons from 4 to 9 (Table 4 and Fig. 13B), 20 including an RT-PCR product missing exons 4 to 8, resulting in a junction of exons 3 and 9 (nt -111 to 348), products missing exons 4 to 6 or 7, fusing exon 3 to exon 7 or 8, respectively, and products missing exon 5 to exon 7 or 8, resulting in junctions between exon 4 and 8 or exons 4 and 9, respectively.
25 All the types of abnormal transcripts observed lacked exon 5, the first coding exon, and half (6/12) of the abnormal transcripts characterized by sequence analysis also showed loss of exon 8 containing the HIT domain. Sequence analysis of the normal-sized transcript amplified from RNA of the 30 normal tissue of these patients revealed a normal FHIT c~NA
sequence. A small alternatively spliced region at the beginning of exon 10 from nucleotides 450 to 460, outside the open reading frame of the FHI~ gene, was observed in the normal-sized transcript present in the tumor and in the 35 corresponding normal tissue of seyeral patients.

CA 02245783 l998-08-06 W O 97/29119 PCT~US97l~1g37 ~08S of ~8tQroZygosity ILOH) ~Ln~lysis To look for allelic losses in tumor samples, a PC~-based~
approach was used using primers which amplify polymorphic microsatellite markers internal and flanking the FHIT gene.
5 DNA from tumor and corresponding normal tissues from 28 NSCLC
and 7 SCLC cases were analyzed for allelic losses at locus D3S4103 (ph 13) (see Section 6), internal to the FHIT gene, and at loci located in flanking regions, centromeric (D3S1312 at 3pl4.2) (Druck et al., 1995, Cancer Res. 55:5348-5353) and 10 telomeric (D3S1234 at 3pl4.2 and D3Sl313 at 3pl4.3) to the FHIT gene. (See Table 5).

Table 5 15 LOH Frequency at 3pl4.2 Loci in SCLC and NSCLC

20/25 (80~) 20/25 (80%) 12/15 (80~) In NSCLC samples LOH at loci D3S4103 and D3S1234 was found in 17 of the 21 informative cases (81%). The combined frequency of losses affecting these two loci was 88% (23/26 of the informative cases). Ten of 13 (76%) and 9 of 11( 81%) 25 tumors also showed LOH at D3S1312 and at D3S1313 loci, respectively, indicating a large deletion in this genomic region. In SCLC cases, 3 of 4 informative patients showed LOH
at D3S4103 and D3S1234, and 4 of 5 informative cases had lost at least one of these loci. Overall 12 of 12 (100%~ of the 3~ informative tumors which exhibited abnormal FHIT transcripts, showed allelic losses at one or more of the loci tested.

.
7.2. DISCUSSION
Three distinct chromosomal regions of 3p, which include 35 3p25, 3p21.3-p21.2 and 3pl2-pl4.1, are believed to harbour gene(s) involved in lung cancer on the basis of the high frequency of allelic loss in primary tumors and defined W O 97/29119 CA 02245783 l998-08-06 PCT~US97/01937 homozygous deletions in lung cancer derived cell lines;
extensive efforts have been made to define small common region of loss in order to isolate tumor suppressor genes in these chromosomal regions.
Deletions of 3p constitute particularly useful genetic markers since several studies have reported that they occur in the early stages of lung carcinogenesis, such as bronchial dysplasia and metaplasia (Sundaresan et al., 1992, Oncogene 7:1989-1997; Sozzi et al., 1991, Cancer Res. 51:400-404; Hung 10 et al., 1995, JAMA 273:558-563). Moreover allelic loss on chromosome 3p in primary SCLC has been suggested to represent an unfavorable prognostic factor (Horio et al., 1993, Cancer Res. 53:1-4).
The study disclosed herein describes the occurrence of 15 abnormalities in transcripts of the F~I~ gene, located at 3pl4.2, in at least 80% of SCLC and 40% of NSCLC with 88% of the cases also exhibiting loss of one FHIT allele. Since the RT-P~R nested ampli~ication would detect only internal alterations in the FHIT gene transcripts, this is a 20 conservative estimate of the involvement of the ~HIT gene in SCLC and NSCLC.
The lung tumor transcripts were missing two or more exons of the FHIT gene. While in NSCLCs a varying pattern of abnormal transcripts was detected, in SCLCs the amplified 25 transcripts were either missing exons 4 to 6 or exons 4 to 8.
Both types result in loss of exon 5, containing the initial methionine codon (see Section 6), with the second type also showi-ng loss of exon 8 containing the highly conserved HIT
domain (see Section 6). The consequence of the loss of these 30 exons is that no in-frame Fhit protein could be produced.
Two cases of primary SC~C and a tumor cell line lacked the normal-sized transcript, which was also underrepresented in the remaining cases. In addition, one primary tumor exhibited two abnormal transcripts and a normal transcript, 35 while a normal-sized product was not amplified from the cell line established from this tumor. These observations suggest W ~ 97/29119 PCTnUSg7fO1937 that in the SCLC RNA the wild-type transcript could have derived from a~ i~eA normal cells.
In RNAs from NSCLCs the abnormal RT-PCR amplified products were sometimes less ~hlln~Ant than the normal-sized 5 RT-PCR amplified products. A possible explanation could be the heterogeneous, often multifocal nature of these neoplasms, which arise as a consequence of the chronic exposure of the entire bronchial "field" to carcinogens, resulting in the presence of di~ferent cell clones carrying different genetic 10 changes (Kim et al., 1993, Am. J. Pathol. 142:307-317; Barsky et al., 1994, Moa. Pathol. 7:633-640; Ebina et al., 1994, Cancer Res. 54:2496-2503). In addition, the tumor samples contained variable amounts of normal stromal tissue (stromal infiltration i~ known to occur in non small-cell tumors) 15 ~Rabbitts et al., 1989, Genes Chrom. Cancer 1:95-105). The complete allele loss seen in the SCLC cell line and in several SCLC primary specimens and lack of complete loss of alleles in NSCLC supports this interpretation. It is also possible that the abnormal transcripts are less stable then the wild type 20 product.
In the corresponding normal tissue of the patients showing abnormal tumor transcripts we have observed a normal FHIT product by PCR and se~uence analysis.
Of particular interest was one NSCLC patient (case 3 of 25 Table 4) with a mucoepidermoid carcinoma of the lung who subse~uently developed a renal cell carcinoma. In the normal lung parenchyma of this patient an abnormal transcript ~issing exons 4 to 8 was detected. ~his finding raises the possibility that a constitutional alteration within the FHIT
30 gene could be associated with a predisposition to develop both lung and renal cancer or other types of multiple primary tumors. -~owever this alteration could have been somatically acquired because carcinogen exposure càn induce transformation in several fields of the bronchial epithelium through the 3S induction of different genetic changes which are also detectable in early preneoplastic lesions.

W O 97/29119 PCTrUS97/01937 The high frequency (88%) of loss of one FHIT allele observed in lung tumors of both smalI cell and non small cell ~
type is noteworthy. Although we did not determine a minimal region of loss in our cases, these findings support the idea 5 that inactivation of the F~IT gene could have occurred by a mechanism of loss of one allele and altered expression of the remaining one.
This model is consistent with the observation that the F~IT gene spans a common fragile region, FRA3B, where 10 abnormalities such as deletions could be more frequent than point mutations. Tumors associated with carcinogen exposure, such as cancers of the aerodigestive tract, could be particularly susceptible to alterations of the FHIT gene. Due to its etiology, lung cancer is the likely to be strongly and 15 directly associated with the effects of agents which interfere with DNA replication, such as nicotine and mutagens like benzo(a)pyrene contained in cigarette smoke. Breakage in a fragile site containing gene as a conse~uence of physical, chemical and biological agents can thus be expected.
20 Expressivity of the FRA3B fragile site in peripheral blood lymphocytes of patients with cancer has been investigated; the expression of FRA3B appeared to be influenced by habitual tobacco oking and signi~icantly higher expression was reported in lung cancer patients (Murata et al., 1992, 3pn. J.
25 Hum. Genet. 37:205-213).
High levels of intracellular diadenosine 5',5'''-Pl,P4 tetraphosphate (Ap4A) have been detected at the G1-S boundary (Weinmann-Dorsch et al., 1984, Eur. J. Biochem. 138:179-185) and a role for Ap4A in the stimulation of DNA polymerase 30 activity has been proposed (Baxi et al., 1994, Bioc-he~;~try 33:14601-14607). It seems plausible that loss of function of the FHIT-gene could result in the constitutive accumulation of AP4A and in the stimulation of DNA synthesis and proliferation. Thus loss of FHIT function could initiate the 35 malignant process by stimulating the proliferation of the cells that are the precursors of digestive tract cancer and lung cancer.

-W0471~119 PCT~S97101937 7.3. ~XPERTM~TAL PROCEDURES
Tumor~
The 59 tumors, including 25 cases of adenocarcinomas, l9 squamous cell carcinomas, l mucoepidermoid carcinoma and 14 5 small cell lung carcinomas, were obtained from surgically resected lung cancer patients at Istituto Nazionale Tumori (Milano, Italy). A cell line (83L) was established from one small-cell tumor (83T). Twenty-nine NSC~Cs were in stage I, 9 in stage II and 6 in stage III. The tumors were classified 10 histologically according ~o the Histological Typing of Lung Tumors by the World Health Organization (1987) and staged according to the TNM classification of malignant tumors defined by the International Union Against Cancer (1987).
Most cases (54 out of 59) were from male patients and the mean 15 age of cases at presentation was 63 years. Matched normal lung parenchyma tis~ue samples were taken at a most distant site from the tumor or in a different segment or lobe, as a source for the normal RNA and DNA.

20 RNA Extraction and ReversQ Transcription Tumor and normal specimens were frozen immediately after surgical resection and stored at -80~C. Total mRNA was extracted from frozen tumor and normal lung tissues using guanidinium-LiCl separation (Sambrook et al., 1989, Molecular 25 cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press, Plainview, NY) or the RNA-STAT kit (Tel TEST, Inc., Texas).
cDNA was synthesized from 1 ~g of total RNA. Reverse transcription was performed in a 20 ~l volume of lx first strand buffer (GIBCO), ~0 mM DTT (GIBCO), 500 ~M dNTPs, 50 30 ng/~l oligo-dT, 0.3 ~g/~l random primers, 16.5 U RNAsin (PROMEGA), 300 U Superscript II (GIBCO). The samples were firct denatured for 5 min at 95~C and incubated at 37~C for 60 min. The reaction was stopped by inactivating the enzyme at 94~C for 5 min. The reaction was diluted to 30 ~l and l ~l was ~ 35 used for subsequent PCR amplification.

W O 97/29119 PCT~US97/Olg37 RT-PCR and cDNA 8eguen¢ing 1 ~l of cDNA was used for a first PCR amplification performed in a volume o~ 25 ~l containing 0.8 ~M of primers 5U2 and 3D2 (see Section 6), 50 ~M of each dNTP (TAKARA3, lx S PCR buffer and 1.25 U Taq Polymerase (TAKARA). The PCR
reaction consisted of an initial denaturation at 95~ for 3 min and 25 cycles of 15 sec at 94~, 30 sec at 62~, 45 sec at 72~
and a final extension of 5 min at 72~, using a Perkin Elmer PCR Thermocycler. The amplified product was diluted 20-fold 10 in TE buffer and 1 ~l of the diluted reaction product was subjected to a second round of PCR amplification using nested primers 5U1 and 3D1 (see Section 6) for 30 cycles under the above conditions. The PCR products were resolved on 1.5%
ethidium-bromide stained Metaphor gel (FMC). Bands were cut 15 from gels and DNA was purified using a QIA quick gel extraction Kit (QIAGEN). 5-50 ng of cDNA, depending on the size of the PCR products, were sequenced using primers 5~1 and 3D1 by the dideoxynucleotide termination reaction chemistry for sequence analysis on the Applied Biosystems Models 373A
20 and 377 DNA sequencing systems.
.

LO~ Analy8i8 DNAs from frozen tumor and normal tissues were extracted using standard methods (Sambrook et al., 1989, Molecular 25 cloning: A Laboratory Manual. Cold Spring Harbor Lab. Press, Plainview, NY). Analysis of allelic losses was performed using a PCR-based approach. Primers which amplify polymorphic microsatellite markers were used for the following loci:
D3S4103 (phl3) (3pl4.2) internal to the FHIT gene (see Section 30 6), D3S1234 (3pl4.2), D3S1313 (3pl4.3) and D3S1312 (3pl4.2) flanking the gene. The sequence of all oligonucleotide primers will be available through the Genome Data Base.
Twenty cycles of amplification were carried out at 550-60~C
annealing temperature as appropriate for each primer.
For informative cases, allelic loss was scored if the autoradiographic signal of one allele was approximately 50%
reduced in the tumor DNA, compared to the corresponding normal -W ~ 971~9119 PCTnUSg7101937 allele. The loci displaying microsatellite instability were not scored for allelic 108s.

8. DEPOSIT OF MICROORGANISMS
S E . col i strain DH5~ carrying plasmid p7F1, containing a full-length FHIT cDNA as a BamHI-XbaI insert into the pBluescript SK+ vector (Stratagene) was deposited on January 30, 1996, with the Ame~ican Type Culture Collection, 1201 Parklawn Drive, Rockville, Maryland 20852, under the 10 provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures, and assigned accession number 69977.

The present invention is not to be limited in scope by 15 the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the 20 appended claims.
Various publications are c~ted herein, the disclosures of which are incorporated by reference in their entireties.

CA 02245783 l998-08-06 ~'yUL.._~ LISTING

(l) r-~AT INFORMATION: ~
(i) APPLICANT: Croce, Carlo M.
~ nsr, Kay (ii) TITLE OF lNv~wLION: FHIT PROTEINS AND NUCLEIC ACIDS AND
METHODS BASED ~n~_ '~
( iii ) NUMk~K OF ~u~S: 86 (iv) COR~SPONDENCE AnDR~SS:
~A~ AnDp~s~ Penni~ & r~ -- 'n ,B STREET: 1155 Avenue of the America~
,C~ CITYs New York ~D STATE: New York ~EJ COu.~ : U.S.A.
~FJ ZIP: 10036-2711 (v) COMPUTER R~n~RT~ FORM:
'A) MEDIUM TYPE: Floppy di~k ~B) COMPUTER: IBM PC compatible ,'C~ OPERATING SYSTEM: PC-DOS/MS-DOS
~D) SOFTWARE: PatentIn Release ~1.0, Version ~1 30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 22-FEB-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Friebel, Thoma~ E
(B) REGISTRATION NUMBER: 29,258 (C) k~K~N~/DOCKET NUMBER: 8666-005 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 790-9090 (B) TELEFAX: (212) 869-9741/8864 (C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
Al LENGTH: 1095 base pairs ~B TYPE: nucleic acid CJ STRANDEDNESS: single DJ TOPOLOGY: unknown (~i) MOLECULE TYPE: DNA

(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 363 803 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCCCCGCTCT G~l~ ~CCG GTCACAGGAC TTTTTGCCCT ~.~.. CCCGG ~lCC~lCAGG 60 CGGC~CC~ GTGGGCACAC TCCCAGGCGG CGCTCCGGCC CCGCGCTCCC ~CC~.~lGCC 120 TTTCATTCCC AGCTGTCAAC ATCCTGGAAG CTTTGAAGCT CAG~-~A~-~A GAGAAATCCA 180 CA 02245783 l998-08-06 .
CTr-A~-~CA~ TCTGTAAAGG TCCGTAGTGC TATCTACATC CAGACGGTGG AAGG~ GA 240 A~r-A~A~A~A AGGTATCCTA Gr-AATA~cTG CCTGCTTAGA CC~lATAA AAG~-~ G 300 CATCCTGCCA CTGAGGACTC C~-~A~-~-GTA-GCAG-~ AAr-~CTTC AACTGTGAGG 360 - - Met Ser Phe Arg Phe Gly Gln His Leu Ile Ly~ Pro Ser Val Val Phe Leu Ly~ Thr Glu Leu Ser Phe Ala Leu Val Aqn Arg Ly~ Pro Val 7 Val Pro Gly Hi-q Val Leu Val Cy8 Pro Leu Arg Pro Val G}u Arg Phe ' 35 40 45 Hin A~p Leu Arg Pro anp Glu Val Ala ABP Leu Phe Gln Thr Thr Gln AGA GTC GGG ACA GTG GTG GAA AaA CAT TTC CAT GGG ACC TCT CTC ACC 599 Arg Val Gly Thr Val Val Glu LYQ HLQ Phe Hiq Gly Thr Ser Leu Thr Phe Ser Met Gln ARP Gly Pro Glu Ala Gly Gln Thr Val Ly~ Hi5 Val Hi~ Val Hi~ Val Leu Pro Arg Ly~ Ala Gly A~p Phe HiQ Arg A~n A~p Ser Ile Tyr Glu Glu Leu Gln Ly~ Hiq A~p Lyq Glu Aqp Phe Pro Ala Ser Trp Arg Ser Glu Glu Glu Met Ala Ala Glu Ala Ala Ala Leu Arg GTC TAC TTT CAG T~A~G~T ~lL~ ~. AGA TCCTGAATTC CAGCA~AAGA 843 Val Tyr Phe Gln CATGAAAATG CA~ . C ATCTCACCAT CCTGTATTCT T~C~A~-TG ATCCCC~CC 963 TC ~ TCACTC CAA~CC~L~ A~A~TAccTA GACCTAAACG GCT~ ~G GCAGATTTGA 1023 o~..~ccccc ~ .C~1A TTCGGCAGCC TTATGATTAA A~. C~l~l CTGCTGCAAA 1083 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 147 amino acid~
B) TYPE: amino acid ~D) TOPOLOGY: I~nknc..,~
( ii ) MoT.~cuT~ TYPE: protein t (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

W O 97/29119 PC~US97/01937 Met Ser Phe Arg Phe Gly Gln His Leu Ile Lys Pro Ser Val Val Phe Leu Lys Thr Glu Leu Ser Phe Ala Leu Val Asn Arg Lys Pro Val Val Pro Gly Hiu Val Leu Val cys Pro Leu Arg Pro Val Glu Arg Phe His Asp Leu Arg Pro Asp Glu Val Ala Asp Leu Phe Gln Thr Thr Gln Arg Val Gly Thr Val Val Glu Lys HiB Phe His Gly Thr Ser Leu Thr Phe Ser Met Gln Asp Gly Pro Glu Ala Gly Gln Thr Val Lys His Val Hi~.

Val H~ B Val Leu Pro Arg Lys Ala Gly Asp Phe ~Ls Arg A~n Asp Ser Ile Tyr Glu Glu Leu Gln Lys His Asp Lys Glu A8p Phe Pro Ala Ser Trp Arg Ser Glu Glu Glu Met Ala Ala Glu Ala Ala Ala Leu Arg Val Tyr Phe Gln (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 168 amino acids (B) TYPE: amino acid (D) TOPOLOGY: ~lnk~t.,~
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Pro Lys Gln Leu Tyr Phe Ser Ly~ Phe Pro Val Gly Ser Gln Val Phe Tyr Arg Thr Ly~ Leu Ser Ala Ala Phe Val Asn Leu Lys Pro Ile L~u Pro Gly His Val Leu Val Ile Pro Gln Arg Ala Val Pro Arg Leu Lys Asp Leu Thr Pro Ser Glu Leu Thr Asp Leu Phe Thr Ser Val Arg Ly~ Val Gln Ser Ala Ser Ala Ser Asn Ile Gly Ile Gln Asp Gly Val - 70 . 75 80 A~p Ala Gly Gln Thr Val Pro Hi~ Val His Val His Ile Ile Pro Arg Ly~ Lys Ala Asp Phe Ser Glu Asn Asp Leu Val Tyr Ser Glu Leu GLu lOO 105 ' 110 Ly~ A~n Glu Gly Asn Leu Ala Ser Leu Tyr Leu Thr Gly Asn Glu Arg Tyr Ala Gly Asp Glu Arg Pro Pro Thr Ser Met Arg Gln Ala Ile Pro Arg Thr Leu Glu Glu Met Glu Ly~ Glu Ala Gln Trp Leu Ly~ Gly Tyr Phe Ser Glu Glu Gln Glu Lys Glu (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE Cl~aRACTERISTICSs (A) LENGT]~: 203 amino acLds (B) TYPE: amino acid (D) TOPOLOGY: tlnkn. ."
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ile Leu Ser Lys Thr Ly~ Ly~ Pro Lya Ser Met A~n Lya Pro Ile Tyr Phe Ser Ly~ Phe Leu Val Thr Glu Gln Val Phe Tyr Lys Ser Ly~

Tyr Thr Tyr Ala Leu Val Asn Leu Lys Pro Ile Val Pro Gly Hi~ Val Leu Ile Val Pro Leu Arg Thr Thr Val Leu A~n Leu Ser A~p Leu Thr Met Pro Glu Ser Gln A~p Tyr Phe Ly~ Thr Leu Gln Leu Ile Hi~ Ly~

Ala Asp Ser Ile A~n Val Ala Ile Gln A~p Gly Pro Glu Ala Gly Gln Ser Val Pro Hi~ Leu Hi~ Thr Hi~ Ile Ile Pro Arg Tyr Ly~ Ile A~n Ann Val Gly Asp Leu Ile Tyr A~p Ly~ Leu A~p Hi~ Trp Asp Gly A~n Gly Thr Leu Thr A~p Trp Gln Gly Arg Arg Asp Glu Tyr Leu Gly Val ~30 135 140 Gly-Gly Arg Gln Ala Arg Lys Ann Ann Sèr Thr Ser Ala Thr Val Anp Gly A~p Glu Leu Ser Gln Gly Pro A~n Val Leu Val Arg Ala Leu Thr Glu Met Lys Ly~ Glu Ala Glu Anp Leu Gln Ala Arg Leu Glu Glu Phe Val Ser Ser Asp Pro Gly Leu Thr Gln Trp Leu (2) INFORMAT}ON FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 119 amino acids r ( B) TYPE: amino acid , WO 97/29119 PCTrUS97/01937 (D) TOPOLoGy 11 n ~n~
(ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ala Asp Glu Ile Ala Lys Ala Gln Val Ala Arg Pro Gly Gly Asp Thr Ile Phe Gly Lys Ile Ile Arg Lys Glu Ile Pro Ala Lys Ile Ile Tyr Glu Asp Asp Gln Cys Leu Ala Phe HiB A8p Ile Ser Pro Gln Ala Pro Thr His Phe Leu Val Ile Pro Lys Lys Tyr Ile Ser Gln Ile Ser Ala Ala Glu Asp Asp Asp Glu Ser Leu Leu Gly His Leu Met Ile Val Gly 7~ 80 Lys Lys Cys Ala Lys Gly Tyr Arg Met Val Val Asn Glu Gly Ser Asp Gly Gly Gln Ser Val Tyr His Val His Leu Hi~ Val Leu Gly Gly Arg lOO 105 llO
Gln Met Asn Trp Pro Pro Gly (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 108 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: lln~n~t~.~
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ser Glu Asp Thr Ile Phe Gly Lys Ile Ile Arg Arg Glu Ile Pro 1 5 lO 15 Ala-Asp Ile Val Tyr Glu Asp Asp Leu Cys Leu Ala Phe Arg Asp Val Ala Pro Gln Ala Pro Val His Ile Leu Val Ile Pro Ly~ Gln Pro Ile Ala Asn Leu Leu Glu Ala Thr Ala Glu His Gln Ala Leu Leu Gly His Leu Leu Leu Thr Val Lys Ala Ile Ala Glu Gly Tyr Arg Thr Val Ile Asn Thr Gly Pro Ala Gly Gly Gln Thr Val Tyr His Leu His Ile Hi~ ~
. 85 9O' 95 Leu Leu Gly Gly Arg Ser Leu Ala Trp Pro Pro Gly lOO 105 CA 02245783 l998-08-06 (2) lNrOk~ATION FOR SEQ ID NO:7:
( i ) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 104 amino acids (B) TYPE: amino acid (D) TOPOLo&y: l~nk~,~ l, -- - (ii) MOLECULE TYPE: prote$n (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met A~n Ann Trp Gln Glu Glu Le~ Phe Leu Lys Ile Ile Lys Arg Glu 1 5 lO 15 Glu Pro Ala Thr Ile Leu Tyr Glu A~p A~p Ly8 Val Ile Ala Phe Leu Anp Ly~ Tyr Ala Hi~ Thr Lyn Gly ~i~ Phe Leu Val Val Pro Ly~ AQn Tyr Ser Arg A~n Leu Phe Ser Ile Ser A~p Glu A~p Leu Ser Tyr Leu Ile Val Lys Ala Arg Glu Phe Ala Gly Ala Thr Gly Phe LYQ Leu Leu Ile Asn A~n Glu Pro Asp Ala Glu Gln Ser Ile Phe HiQ Thr His Val His Ile Ile Pro Tyr Tyr LYQ Ly~

(2) INFORMATION FOR SEQ ID NO:8:
(L) SEQUENCE C}I~RACTERISTICS:
(A) LENGT~: 151 amino acLd~
(B) TYPE: amino acid (D) TOPOLOGY: unknown (iL) MOLECULE TYPE: protein (xi) ~QD~N~ DESCRIPTION: SEQ ID NO:8:
Met Glu Pro Leu Ile Ser Ala Pro Tyr Leu Thr Thr Thr Ly~ Met Ser 1 - 5 lO 15 Ala Pro Ala Thr Leu Asp Ala Ala Cy~ Ile Phe Cy~ Ly~ Ile Ile Lys Ser Glu Ile Pro Ser Phe Lyn Leu Ile Glu Thr Lys Tyr Ser Tyr Ala Phe Leu Asp Ile Gln Pro Thr Ala Glu Gly Hi~ Ala Leu Ile Ile Pro Ly~ Tyr Hi~ Gly Ala Ly~ Leu Hi~ A~p Ile Pro A~p Glu Phe Leu Thr A~p Ala Met Pro Ile Ala Ly~ Leu A~p Thr Tyr A~n Val Leu Gln Asn r -W O 97/29119 PCTrUS97/01937 A~n Gly Lys Ile Ala Hi~ Gln Glu Val Asp His Val Hi~ Phe Hi~ Leu l00 105 llO
Ile Pro Ly~ Arg Asp Glu Ly~ Ser Gly Leu lle Val Gly Trp Pro Ala Gln Glu Thr Anp Phe A~p Ly~ Leu Gly Lyu Leu HLs Lyn Glu Leu Leu . - 130 135 140 Ala Lyn Leu Glu Gly Ser Asp (2) INFORMATION FOR SEQ ID NO:9s (i~ x~Qu~ CHARACTERISTICSs ,'Aj LENGTH: 455 base pair~
B TYPE: nucleic acid ~CJ STRP~ n~Qs: ~ingle ~D;, TOPOLOGYs unkn,~
(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
A~r-~AA~cG GANAGACTTT GAAGCACGTT CACGTCCACG .~ CCCGG GAAGGCTGGA 60 AAACTTTCAC AGGAATGACA GCATCTATGA GGAGCTCCCA ~-~AANATGAC AAGGAGGACT 120 TACTTTCAGT GA~-~ Q G~TC CTGAATTCCA Gr-~AAAGAGC TATTGCCAAC CAGTTTGAAN 240 ACCGCCCCCC CGC~l~.CCC CA~,AG~-PAC TGAATCAGCA TGAAAATGCA Gi,~.l~AT 300 CTCACCATCC TGTANTCTTC AACCAGTGAT CCCC~ACCTC GGTCACTCCA ACTCCCTTAA 360 AA~AccTAGA CCTAAACGGC TCAGACAGGC AGATTTGAGG ~l~CCCC~G lClC~l~ATT 420 (2) INFORMATION FOR SEQ ID NO:l0:
tL) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE.DESCRIPTION: SEQ ID NO:l0:
Ala Ala-Glu Xaa Glu Val l 5 (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: llnkn. a~

-, CA 02245783 l998-08-06 ( ii ) ~T~CuT~ TYPE: peptide (xL) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- - Gly Cy~ Arg Ile Arg Arg Gln Gly Glu Thr Ser A~n Leu Pro Val 1 5 lO 15 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acidn (B) TYPE: amino ac~d (D) TopoLoGy ~Inkn~
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Gly Ser Trp Ser A~p Arg Gly Gly Gly Ser Leu Val Glu Xaa Tyr Arg l 5 lO 15 Met Val Arg ~2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY llnk~nr.,,l (ii) MOLECULE TYPE: peptide (xL) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Arg AQn Cys Ile Phe Met Leu Ile Gln Phe Leu Leu Gly Arg Gly Gly 1 5 lO 15 Gly Ala Xaa Phe Ly~ Leu Val Gly Asn Ser Ser Phe Ala Gly Ile Gln A~p -Leu Cyn His (2) INFORMATION FOR SEQ ID NO:14:
(i) ~Qu~ CH~RACTERISTICS:
(A) LENGTH: 13 amino acids (B)~ TYPE: amino acid (D) TopoLoGy n n kn~
(ii) ~OT.~CULE TYPE: peptide -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
r CA 02245783 l998-08-06 W O 97/29119 PCT~US97/01937 Thr Arg Arg Ala Ala Ala Phe Cy~ Cys His Phe Leu Leu (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids ~ - (B) TYPE: amino acid (D) TOPOLOGy unl--(ii) Mnr~T~ TYPE: peptide (xi) ~gu~ DESCRIPTION: SEQ ID NO:15:
Ser Pro Arg Gly Arg Ly~ Val Leu Leu Val Xaa Phe Leu Gly Ala Pro 1 5 lO 15 Hi~ Arg Cy8 Cy~ Hi~ Ser Cy~ Glu Ser Phe Pro Ala Phe Pro Gly Xaa Thr Trp Thr (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Gln Gln Xaa Xaa Ly~ Phe A~n Hi~ Ly~ Ala Ala Glu 1 5 lO
~2) INFORNATION FOR SEQ ID NO:17:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acidn (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Gly A~p-Arg Gly Ly~ Pro Gln Ile Cy~ Leu Ser Glu Pro Phe Arg Ser Arg Tyr Phe Ly~ Gly Val Gly Val Thr Glu Val Gly A~p Hi~ Trp Leu Ly~ Xaa Thr Gly Trp (2) INFORMATION FOR SEQ ID NO:18:

CA 02245783 l998-08-06 W O 97/29119 PCTn~S97J01937 ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids ~ (B) TYPE:. amino acid (D) TOPOLoGy: 1lnkn~
~ T-~CuT-~ TYPE: peptide (xi) ~:QD~ DESCRIPTION: SEQ ID NO:18:
A~p Glu Glu Thr Ala Phe Ser CYB
l 5 (2) INFORMATION FOR SEQ ID NO:l9:
(i) ~QD~n~ CHARACTERISTICS:
(A) LENGTH: 9l amino acidu ~B) TYPE: amLno acid (D) TOPOLOGY: unknown (~i) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Phe Ser Ser Ser Trp Gly Glu Ala Gly Gly Arg Xaa Ser Asn Trp Leu l 5 lO 15 Ala Ile Ala Leu Leu Leu Glu Phe Arg Ile Cy~ Val Thr Glu Ser Arg Pro Ala &lu Leu Arg Leu Ser Ala Ala Ile Ser Ser Ser Asp Leu Gln ~ 35 40 45 Glu Ala Gly Ly~ Ser Ser Leu Ser Xaa Phe Trp Glu Leu Leu Ile A~p Ala Val Ile Pro Val Lys Val Phe Gln Pro Ser Arg Glu Xaa Arg Gly Arg Glu Arg Ala Ser Lys Ser Xaa Arg Phe Ser (2) INFOR~ATION FOR SEQ ID NO:20:
(i) SEQUENCE CI1~RACTERISTICS:
- (A) LENGTH: 36 amino acid~
(8) TYPE: amino acid (D) TOPOLOGY: ~lnkn~"
(iL) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No:20:
Ser Arg Xaa Gly Ser Leu Ile Ile Arg Leu Pro Asn LYB Glu Thr Gly l 5 l0 15 Gly A~n Leu Lys Ser Ala Cy~ Leu Ser Arg Leu Gly Leu Gly Ile Leu W 097t29119 CA 02245783 1998-08-06 PCT~US97/01937 Arg Glu Leu Glu (2) INFO~MATION FOR SEQ ID NO:21: ~
(i) S~QD~NCE CHARACTERISTICS:
(A) LENGTH: 7 amino acid~
(B) TYPE: smino acid (D) TOPOLOGy: l1nknl ~
(iL) MOLECULE TYPE: peptide (xi) SEQUENCE D~eCPTPTION: SEQ ID NO:21:
Pro Arg Trp Gly Il~ Thr Gly l 5 ~2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Arg Xaa Gln A~p Gly Glu Met Lys Lys Leu Hi~ Phe His Ala Asp Ser l 5 lO 15 Val Pro Leu Gly Glu Arg Arg Gly Gly Gly Xaa Gln Thr Gly Trp Gln (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCR}PTION: SE~ ID NO:23:
Leu Phe Cy~ Trp A~n Ser Gly Ser Val Ser Leu Ly~ Val A~p Pro Gln l 5 lO 15 Ser Cy8 Gly Phe Leu Leu Pro Phe Pro Pro Leu Ile Ser Ly~ Arg Gln Glu Ser Pro Pro Cy~ ~is Xaa Ser Gly Ser Ser Ser (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

WO 97/29119 PCTlU~97Jû1937 (A) LENGTH: 5 amino acid~
( B ) TYPE: amino acid (D) TOPOLOGY: 1-n'-- .."
(ii) MOT-T~'CULE l'YPE: peptide (xL) SEQUENCE D~C~TpTIoN: SEQ ID NO:24:
Met Leu Ser Phe Leu (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGl'H: 22 amino acidq (B) TYPE: amino acid (D) TOPOLOGY unknr 1"
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Lys Phe Ser Ser Leu Pro Gly Xaa A~n Val A~p Val Asn Val Leu Gln 1 5 lO 15 Ser Xaa Ser Gly Phe Leu . 20 (2) INFORMATION FOR SEQ ID No:26:
(i) ~QD~NCE CHARACTERISTICS:
(A) LENGTH: 25 amino acLd~
(B) TYPE: amino acid (D) TOPOLOGY: I7nkn~ .1,, (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ly~ Ly~ Thr Gly Xaa Thr Leu Ly~ Hiq Val ~L~ Val ~i~ Val Xaa Pro 1 5 lO 15 Gly Ly~ Ala Gly Ly~ Leu Ser Gln Glu (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) T- _ln: 33 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: I~nl~
( ii ) MOT.~CUT.T' .TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

G}y Ala Pro Arg Xaa Met Thr Arg Arg Thr Phe Leu Pro Leu Gly A~p Gln Arg Arg Ly~ Trp Gln Gln Ly~ Ala Ala Ala Leu Arg Val Tyr Phe Gln (2) INFORMATION FOR SEQ ID NO:28:
( i ) S~U~NC~ CHARACTERISTICS:
(A~ T~ 22 amino acids (B) TYPE: am~ no acid (D) TOPOLOGY: unknown (lL) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Ile Pro Ala Lys Glu Leu Leu Pro Thr Ser Leu Xaa Thr Ala Pro Pro Pro Leu Pro Lyq Arg A~n (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acid~
( B ) TYPE: amino acid (D) TOPOLOGY: I~nk~
(iL) MOLECULE TYPE: peptide (xL) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ile Ser Met Lys Met Gln Phe Leu Hi~ Leu Thr Ile Leu Xaa Ser Ser Thr Ser A~p Pro Pro Pro Arg Ser Leu Gln Leu Pro (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acid~
(8) TYPE: amLno acLd (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) ~:Qu~NCE DESCRIPTION: SEQ ID NO:30:
Thr Ala Gln Thr Gly Arg Phe Glu Val Ser Pro Cy~ Leu Leu Ile Arg Gln Pro Tyr A~p CA 02245783 l998-08-06 WO 97J29119 PCT/US971~1937 (2) INFORMATION FOR SEQ ID NOs31:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acid~
(B) TYPE: amLno acLd - - ~D) TOPOLOGYs Itnknl ~"
(iL) MOL~CULE TYPE: peptLde (xi) ~h~u~N~ D~Cc~TpTIoN: SEQ ID NO:31:
Thr Ser Xaa Ser Ala (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acLd~
(8) TYPE: amino acid (D) TOPOLOGy: ~ln~n_,, (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Arg Ly~ Pro Xaa Arg Leu S
(2) INFORMATION FOR SEQ ID No:33:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 28 amino acidR
(B) TYPE: amino acid ~D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Ser Thr Phe Thr Ser Thr Xaa Phe Pro Gly Arg Leu Glu Ann Phe Hi~
1 5 lO 15 Arg A~n ARP Ser Ile Tyr Glu Glu Leu Pro Glu Xaa (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acLd~
(B) TYPE: amino acid (D) TOPOLOGY: lln~n.
(ii) MOLECULE TYPE: peptide (xL) SEQUENCE DES~R~PTION: SEQ ID NO:34:
Gln G}y Gly Leu Ser Cy~ Leu Leu Glu lle Arg Gly Gly A~n Gly Ser l 5 lO 15 Arg Lys Pro Gln Leu CYB Gly Ser Thr Phe Ser Asp Thr Asp Pro Glu Phe Gln Gln LYB Ser Tyr Cy8 Gln Pro Val (2) INFORMATION FOR SEQ ID NO:35:
(L) ~:QDI~CE CHARACTERISTICS:
(A) r~ 14 amino acid~
(B) TYPE: amino acLd (D) TOPOLOGY llnl-- )U
(lL) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Xaa Pro Pro Pro Arg Leu Ser Pro Arg Gly Thr Glu Ser Ala (2) INFORMATION FOR SEQ ID NO:36:
(L) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acid~
(8) TYPE: amino acid (D) TOPOLOGY: llnknot.,~
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Lys Cys Ser Phe Phe Ile Ser Pro Ser Cys Xaa Leu Gln Pro Val Ile l 5 lO 15 Pro Hi~ Leu Gly Hi~ Ser A~n ser Leu Lys Ile Pro Arg Pro Ly~ Arg Leu Arg Gln Ala A~p Leu Arg Phe Pro Pro Val Ser Leu Phe Gly Ser Leu Met Ile Ly~ Leu Xaa Xaa Leu Leu (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE C~ARACTERISTICS:
(A)-LENGTH: 108 amLno acLds (B) TYPE: amLno acid (D) TOPOLOGY: ~~ nl-- 2 .J~I
(iL) MOLECULE TYPE: peptide (xl) SEQUENCE DESCRIPTION: SEQ ID NO:37:

CA 02245783 l998-08-06 WO 97129119 PCTlUS9~1û193 Glu Asn Arg Xaa Asp Phe Glu Ala Arg Ser Arg Pro Arg Xaa Ser Arg 1 5 lO 15 Glu Gly Trp Lys Thr Phe Thr Gly Met Thr Ala Ser Met Arg Ser Ser Gln Lys Xaa A~p Lys Glu A~p Phe Pro Ala Ser Trp Arg Ser Glu Glu Glu Met Ala Ala Glu Ser Arg Ser Ser Ala Gly Leu Leu Ser Val Thr Gln Ile Leu Asn Scr Ser Lys Arg Ala Ile Ala A8n Gln Phe Glu Xaa Arg Pro Pro Ala S~r Pro Gln Glu Glu Leu Asn Gln His Glu A~n Ala 85 9O g5 Val Ser Ser Ser Hi8 His Pro Val Xaa Phe Asn Gln lOO 105 (2) INFOR~ATION FOR .SEQ ID NO:38:
~QU~N~ CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xL) SEQUENCE D~SC~TPTION: SEQ ID No:38:
Ser Pro Thr Ser Val Thr Pro Thr Pro Leu.Lys Tyr Leu A~p Leu A~n Gly Ser Asp Arg Gln Ile (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: nnknt ,l (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Gly Phe Pro Leu Ser Pro Tyr Ser Ala Ala Leu 1 5 lO
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY llnk~,"

(ii) MOrECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Leu A~n Phe Xaa Leu Cys Cys l 5 - - (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQ~d~: CHARACTERISTICS:
(A) LENGTH: 48 amino acids (B) TYPE: amino acLd (D) TOPOLOGY l-nl-- -( ii ) ~~'T~T~Cur~ TYPE: peptide (xi) S~Y7~N~ DESCRIPTION: SEQ ID NO:41:
Glu Gln Ser Val LYB Val Arg Ser Ala Ile Tyr Ile Gln Thr Val Glul 5 lO 15 Gly Arg Glu Arg Glu Arg Arg Tyr Pro Arg Asn Thr Cys Leu Leu Arg Pro Ser Ile Lys Ala Leu Cy8 Ile Leu Pro Leu Arg Thr Pro Lys Arg (2) INFORMATION FOR SEQ ID No:42:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 8 amino acid~
(B) TYPE: amino ac$d (D) TOPOLOGY: l~n~n~ .
(i$) MOLECULE TYPE: peptide (x$) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Gln Ser Ser Glu Arg Leu Gln Leu l 5 (2) INFORMATION FOR SEQ ID No:43:
(i) ~Q~NCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: amino acLd (D) TOPOLOGY: l7nkn~
(ii) MOT~C'JLE TYPE: peptide (x$) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Gly Hi~ Val Val Gln Ile Trp Pro Thr Ser Hi~ Gln Ala Leu Cyq Ser l 5 lO ' 15 Val Ser Gln Asn Arg Thr Val Leu Arg Ser Cys Glu _ -142-W 097/29119 PCTnU~97J~1937 (~) INFORMATION FOR SEQ ID Nos44:
(i) ~Qu~: CHARACTERISTICS:
(A) LENGTH: 50 amino acid~
( B ) TYPE: amino acid (D) TOPOLOGY: llnknr ~"
(ii) MOLECULE TYPE: peptide (xi~ SEQUENCE DRc~TpTIoN: SEQ ID NO:44:
Glu Thr Cy~ Gly Thr Arg A~p Met Ser Leu Cy8 Ala Arg Cys Gly Gln t l 5 lO 15 Trp Glu Arg Phe Hi~ Aup Leu Arg Pro Asp Glu Val Gly Arg Phe Val Ser Asp A~p Pro Glu Ser Ser Gly Gln Trp Leu Xaa Ly~ Hi~ Phe Pro Gly A~p (2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acid~
~B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Ser Thr Glu A~n Ser Leu l 5 (2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acid~
( B ) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Arg Ser Val Val Leu Ser Thr Ser Arg Arg Trp Lys Gly Glu Lys Glu Ly~ Glu Gly Ile Leu Gly Ile Pro Ala Cy~ Leu A~p Pro Leu (2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 7 amino acid~
i~

W O 97/29119 PCTrUS97/01937 (B) TYPE: amino acLd (D) TOPOLOGY ~nkn~
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Lys Leu Cy~ Ala Ser Cy~ Hi~

(2) INFORMATION FOR SEQ ID NO:48:
(L) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 amino acids (B) TYPE: amino acid (D) TOPOLOGY: ~no~m (ii) MO~.ECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Gly Leu Arg Arg Gly Ser Ser Leu Leu Lys Asp Phe Asn CYB Glu Asp 1 5 lO 15 Met Ser Phe Arg Phe Gly Gln Hi~ Leu Ile Lys Pro Ser Val Val Phe Leu Lys Thr Glu Leu Ser Phe Ala Leu Val Asn Arg Lys Pro Val Val Pro Gly Thr Cys Pro Cys Val Pro Ala Ala Ala Ser Gly Ser Ala Ser ~et Thr Cys Val Leu Met Ly~ Trp Ala ABP Leu Phe Gln Thr Thr Gln Arg Val Arg ABP Ser Gly Trp Xaa Asn Ile Phe Leu Gly (2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) ToPoLoaY: l~nlrnç,l"
(ii) MOLECULE TYPE: peptide (x~) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Pro Leu Arg Thr Val Cys Lys Gly Pro (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENaTH: 17 amino acids ~ (8) TYPE: amino acid ~D) TOPOLOGy ~lnkn~, ~ii) MO~ECULE TYPE: peptide (xi) SEQ~ D~-~r-RTPTION: SEQ ID NO:50:
Cy~ Tyr Leu Hia Pro Asp Gly Gly Arg Glu Arg Ly~ Arg Ly~ Ly~ Val Ser t ( 2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE ~AR~-TERISTICS:
(A) LENGTH: 5 ~mLno acid~
(B) TYPE: amino acLd (D) TOPOLOGY: I~knct~, (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Glu Tyr Leu Pro Ala (2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) r~ LA 20 amino acids (B) TYPE: amLno acid (D) TOPOLOGY: tlnknc ,l (ii) MOLECULE TYPE: peptLde (x~) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Thr Leu Tyr Lys Ser Ser Val His Pro Ala Thr Glu A~p Ser Glu Glu Val Ala Val Phe (2) INFORMATION FOR SEQ ID NO:53:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 20 amino acLds (B~ TYPE: amino acLd (D) TOPOLOGY: l~nkn~
(iL) MOLECULE TYPE: peptide (xL) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Lys Thr Ser Thr Val Arg Thr Cyq Arg Ser A8p Leu Ala A~n Ile Ser W O 97/29119 PCTnUS97/01937 l 5 lO 15 Ser Ser Pro Leu (2) INFORMATION FOR SEQ ID NO:54-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ll amino acid~
(8) TYPE: amino acid (D) TOPOLOGY: ~lnkn _ ~"
(ii) MOLECULE TYPE: pep~ide (xi) SEQUENCE D~fi~TPTION: SEQ ID NO:54:
Cy~ Phe Ser Ly~ Gln Asn Cyu Pro Ser Leu Leu ~2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERIST}CS:
(A) LENGTH: 22 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi,) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Ile Gly A~n Leu Trp Tyr Gln Gly Hi~ Val Leu Val Cy~ Pro Leu Arg l 5 lO 15 Pro Val Gly Ala Leu Pro (2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amLno acid~
(B) TYPE: amino acid ~D) TOPOLOGY: ~nkn~
(ii) ~OLECULE TYPE: peptide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Ser Gly Pro Ile Cy~ Phe Arg Arg Pro Arg Glu Phe Gly Thr Val Val l 5 lO l5 Gly Xaa Thr Phe Ser Trp Gly ~2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l7 amino acid~
(B) TYPE: amino acid CA 02245783 l998-08-06 (D) TOPOLOGY: .. n ~n~ , ~
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Val Pro Arg Ly~ Met Phe.Xaa Gln Pro Leu Ser Arg Thr Leu Trp Val 1 5 lO 15 V~l (2) INFORMATION FOR SEQ ID NO:58:
( i) ~h'Q~ ; ~U~RP~TT;~RTsTIcs (A) LENGTH: 122 amino acid~
(B) TYPE: nmino acid (D) TOPoLo~y: l~nkn~ J
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
A~n Lyq Ser Ala His Phe Ile Arg Thr Gln Val Met Glu Ala Leu Pro 1 5 lO 15 Leu Ala Ala Ala Gly Thr Gln Gly Hiq Val Pro Gly Thr Thr Gly Phe Leu Phe Thr Arg Ala Ly~ A~p Ser Ser Val Leu Arg A~n Thr Thr GLu Gly Leu Met Arg Cy~ Trp Pro A~n Leu A~n A~p Met Ser Ser Gln Leu Ly~ Ser Phe Arg Arg Leu Leu Pro Leu Arg Ser Pro Gln Trp Gln A~p Ala Gln Ser Phe Tyr Arg Gly Ser Ly~ Gln Ala Gly Ile Pro Arg ILe . 85 9O 95 Pro Ser Phe Ser Phe Ser Pro Phe Hi~ Arg Leu ARP Val A~p Ser Thr lOO 105 llO
Thr Asp Leu Tyr Arg Leu Phe Ser Val Asp ~115 120 (2) INFORMATION FOR S~Q ID NO:59:
~i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 60 amino acid~
~B) TYPE: amino acid (D) TOPOLOGY: l~n~n~
(ii) ~OLBCULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
" .

-W O 97/29119 CA 02245783 1998-08-06 PCT~US97/01937 8er Pro Gly Lys Cy~ Xaa Ser ~n His Cys Pro Glu Leu Ser Gly Ser 1 5 lO 15 8er alu Thr A~n Arg Pro Thr Ser Ser Gly Arg Arg Ser Trp Lys Arg Ser His Trp Pro Gln Arg Ala HLs Ly~ A~p Met Ser Leu Val Pro Gln Val Ser Tyr Ser Gln Glu Arg Arg ~hr Val Leu Phe (2) INFORMATION FOR SEQ ID NO:60:
(i) S~;QUL..--P' ~AR~C~TSTICS
(A) LENGTH: 6 amino acids (B) TYPE: amlno acid (D) TOPOLOGY: l~nknrsl..
( ii ) M~T ~CUT ~ TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Glu Thr Leu Gln Arg Ala (2) INFORMATION FOR SEQ ID NO:61:
(it SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: llnk-~ m (ii) ~OT.~CULE TYPE: pept~de (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Asp Val Gly Gln Ile (2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 6 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: ~l rkr _ h .1 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No:62:
Thr Thr Cy~ Pro His Ser (2) INFORMATION FOR SEQ ID No:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 amino acid~
-~48-WO 97/29119 PCTnUS97/01g37 , (B) TYPE: a~ino acid (D) TopoLoGy l~n~nl ( ii ) Y - T ~CUT-~ TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
Ser Leu Ser Glu A~p Cys Tyr Leu Phe Gly Val Leu Ser Gly Arg ~et l 5 lO 15 ~is Arg Ala Phe Ile Glu Gly Leu Ser Arg Gln Val Phe Leu Gly Tyr Leu Leu Ser Leu Ser Leu Pro Ser Thr Val Trp Met ~2) INFORMATION FOR SEQ ID NO:64:
(i) s~yu~ CHARACTERISTICS:
(A) T~ ~: 12 amino acid~
(B) TYPE: amino acid (D) TOPOLoGY ~lnk~t,,l ~ii) MOLECULE TYPE: peptide .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Ile Ala Leu Arg Thr Phe Thr ABP Cys Ser Gln Trp l 5 lO

~2) INFOR~ATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 amino acids (B) TYPE: amino acid (D) TOPOLOGY: u~kn~
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No:65:
Pro Gln Glu Ann Val Xaa Pro Thr Thr Val Pro Asn Ser Leu Gly Arg l 5 . lO 15 Leu Lys Gln Ile Gly Pro Leu His Gln Asp Ala Gly His Gly Ser Ala Pro Thr Gly Arg Ser Gly His Thr Arg Thr Cy~ Pro Trp Tyr His Arg Phe Pro Ile His Lys Ser Glu Gly Gln Phe Cys Phe Glu LYB His Tyr Arg Gly Leu A~p Glu Met Leu Ala Lys Ser Glu Arg His Val ~eu Thr - 65 70 ~75 80 Val Glu Val Phe Gln Lys Thr Ala Thr Ser Ser Glu Ser Ser Val Ala W O 97/29119 CA 02245783 l998-08-06 PCTrUS97/01937 Gly Cy~ Thr Glu Leu Leu (2) INFORMATION FOR SEQ ID No:66: _ (L) SEQUENCE CHARACTERISTICS:
(A) LENGTHs 5 amino acid~
(B) TYPE: amLno acLd (D) TOPOLOGY: unknown ( ii ) ~OT-~cur~ TYPE: peptLde L) ~QU~:N~ ~ RTPTION: SEQ ID NO:66:
Ala Gly Arg Tyr Ser (2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUEWCE DESCRIPTION: SEQ ID NO:67:
A~p Thr Phe Phe Leu Phe Leu Ser Leu Pro Pro Ser Gly Cy~ Arg (2) INFORMATION FOR SEQ ID No:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: ~ln~n~ ~"
(ii) MOLECULE TYPE: peptLde (xL) SEQUENCE DESCRIPTION: SEQ ID NO:68:
His Tyr Gly Pro Leu Gln Thr Val Leu Ser Gly l 5 10 (2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
'! LENGTH: 1409 ba~e pairs B) TYPE: nucleLc acid 'C, STRANDEDNESS: nlngle ~DJ TOPOLOGY: unknown (Li) MOLECULE TYPE: DNA

-CA 02245783 l998-08-06 W O 97/29119 PCTnJS~7J01937 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
GAaACTTGTG TGr~TArr~ TAATAAAATT CrATAATATT GGAATTTTTA GTCCGCTTTA 60 1~V1~C~AT GATACTGTTA CTTA~TATA TG~AAr.ArGC TAL11~ ~ ~ A TAVL~1V~ 20 V~ AAG TATATCAATC .-.~..ATTA TATTCCATAG ACACTTTCGC ACATGACTCT 180 CCAGGGACTC CGCGATATGG GTTGTGAGCA TCGTGAAGCT GAATTCAACC AAC~CTTAG 240 ATTCTTACAA TATTCGTAAG Cr-ArAATGCC AAAACAGCTA TAL~ C~A AG~L*C~ V~ 300 TGGAAGTCAA V~1 L ~ ~ ~ ATC GTACTAAGGT AAGTTAACGG TCTCATGTGT GT~GATATTG 3 60 .~11.VCAA A~.i..~... GTCATTCTTA TTTATTCTAT AACGGCAGAC A~-~V~VAT 420 ~L' I~L~ GG TTGAGGTCAG CTGCTAACGA TTTTAGTTAT CTGCCGCGTT TGTAAACCTG 480 AAACCAATTT TACCAGGTCA ~ GV1A ATTCCGCAAC GGGCGV~CCC TAGATTGAAA 540 ACGCTAACTA ATGAAAACTT AGTTGACGGA ~ ACT i~ ~ V--CGCA AAGTGCAACA 660 GGTAATCGAA AAGV1V.~-- CGGCATCTGC AT~AAA~ATT GGTATTCAAG TAAGTACTTT 720 GATAGTCAAG GAATAAATAA APAAArATAT ~C~1~AC ATT~AAAATA AAAAATCGTT 780 TTAATTTAGA AGCTGACATT TTGCTTTTAA CTCAATAGGA ~5 ~1AGAC GCTGGTCAAA 840 CA~L1C~.CA TGTACATGTT CACATTATCC CTCGTAA~AA GGCAGATTTT TCAr-AAAA~G 900 ATCTAGTCTA CAGTGAGTTG GPAAAAAACG AAGGAAATCT TG~1-CC~ TATCTTACGG 960 GAAATGAGCG GTATGCAGGA GATr~GArAC CGCr~ACrAr TATGAGGCAA GCTATTCCTA 1020 AGrACrA~GA TCGTAAGCCA AGAArACTTG Ar-~-AAATGGA AAAGGAAGCT CAGTGGTTGA 1080 AAGGGTACTT TTCCr-AAr-AG rAArArTAAr~G AATAAA~rT TGAAGTACCT r~TACrArA 1140 GGGGTAGTGT TTACGTATGA ATTAAGCTAA ATATTATATG ACC~111.1- TTTATTTCAC 1200 CCAAGGTTAC AAGAPAAATT TC~ 11C TCTCTACCCT GCTTACATTG CATCTGTCTG 1260 CTGAGCTTTA GCAArACAAr- GTAACrATAC ATATTGTGAT GAACC~.. ~ ACAATTCGAT 1320 CGAATTAGCT TCAG1~ CC~1 ATTTTGATTT TG~.~-~-1 CTTTCATCCT TTCCTCATAA 1380 (2) INFORMATION FOR SEQ ID NO:70:
(1) SEQUENCE CHARACTERISTICS:
~A LENGTH: 19 base pair~
,8, TYPE: nucleic acid ,C, STRANnEnNESS: ~ingle !D~ TOPOLOGY: linear (iL) MOLECULE TYPE: DNA

(xL) SEQUENCE DESCRIPTION: SEQ ID NO:70:

c -15l-W O 97/29119 PCT~US97/01937 (2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
,'A' LENGTH: 19 ba~e pair~ _ ,BI TYPE: nucleic acid (C, STRANDEDNESS. ~ingle ~DI TOPOLOGY: line~r (ii) MOLBCULE TYPE: DNA

~Xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
AL~LC~L~G1 GTGCCCGCT 19 (2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A'l LENGTH: 24 base pair~
B~ .TYPE: nucleic acid C, sTRANnEnNEss: single ,,D,, TOPOLOGY: 1 inear (ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
TGAGGACATG LC~IL~AGAT TTGG 24 (2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 21 base pairs .BJ TYPE: nucleic acid ~CI STRANDEDNESS: single D i TOPOLOGY: 1 inear (ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
~L ACT GAAAGTAGAC C 21 (2) INFORMATION FOR SEQ ID NO:74:
(L) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 18 ba#e .pairs B TYPE: nucleic acid ,C~ STRANDEDNESS: ~ingle lD~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

CA 02245783 l998-08-06 (2) INFORMATION FOR SEQ ID NO:75:
(i) ShQu ~ CHARACTERISTICS:
'A' LENGTH: 21 ba~e pair~ -IBI TYPE: nucleic acid ,C ST~ANn~nNESS: ~ingle ~D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(Xi) S~Q~KN~ DESCRIPTION: SEQ ID NO:75:
~AcT GAAAGTAGAC C 21 ~2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acid~
(B) TYPE: ~mino acid (D) TOPOLOGy: ln~n~ "
(ii) MOLECUT~ TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
Cys Phe Ly~ Val Xaa Pro Val Phe (2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CEARACTERISTICS:
,AI LENGTH: 420 ba~e pair~
8, TYPE: nucleic acid Cl STRANDEDNESS: ~Lngle ,,D, TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:

At~At~AAArAI~. AAA-'AA~,GTA, TCCTAG~.AAT ACCTGCCTGC TTAGACCCTC TA'rAAAA~.CT 120 CTGTGCATCC TGCCACTGAG GACTCCGAAG AGGTAGCAGT ~.,~.vAAAG ACTTCAACTG 180 TGAGGA~A~G ~L ~AGAT TTGGCCAACA TCTCATCAAG CC~l~l~.AG ~~ ~AA 240 ~A~Ar-AAcTG .C~.CGCTC TTGTGAATAG GAAACCTGTG GTACCAGGGA CA~,C~..G 300 TGTGCCCGCT GCGGCCAGTG GGAGCGCTTC CATGACCTGC ~.C~lGATGA AGTGGGCCGA 360 ~ AG ~C~-ACCCA~-A GAGTTCGGGA CAGTGGTTGG ANAAACATTT TCCTGGGGAC 420 - (2 ) INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 ba~e pair~

W O 97/29119 PCT~US97/01937 (B) TYPE: nucleic acid (C) STRPNn~nN~CS: single (D) TOPOLOGY: ~Inkn~t."
(ii) MOLECULE TYPE: DNA

(xL) s~Qu~ DESCRIPTION: SEQ ID NO:78:
GAAGGGAGAG AAA,~ArAAAr AAGaTACCTa GGTAATACCT G 41 (2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 46 base pairs 'B, TYPE: nucleic acid ' C STRAI~DF!nN~::S: single ~DJ TOPOLOGY: llnl~
(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
AGGG~r-Ar-Ac AGAGAAAGAA AGATGGCCCC GAAGCCGGAC AGACTG 46 (2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
'A'l LENGTH: 21 ba~e pairs ~B TYPE: nucleic acid 'C STRANDEDNESS: ~ingle ,;D,, TOPOLOGY: I~nknr.l., (Li) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
AAr-~GAAAr~A AcTc~Ar-AAA C 21 (2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
~A'I LENGTH: 24 base pairs IB~ TYPE: nucleic acid ,C, STRANDEDNESS: single -,,D, TOPOLOGY: ~Inkn~t,~
(ii) MOLECULE TYPE: DNA

(xi) SEQU,ENCE DESCRIPTION: SEQ ID NO:81:
AGCAGTCTTC TGAAAr~AcTT CAAC 24 (2) INFORMATIoN FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 ba~e pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 02245783 l998-08-06 ~D) TOPOLOGY: l~n~n~....
(ii) Mnr-~CUT.~ TYPE: DNA

(xi) SEQUENCE D~SC~TPTION: SEQ ID NO:82:
G~AAA~, Ar~AAr-AA~r ACC~A~,r-AA~ ACC 33 (2) INFOR~ATION FOR SEQ ID NO:83:
~i) SEQUENCE ~ARA~T~TsTIcs 'A'i LENGTH: 31 ba~e pair~
~B TYPE: nucleic acid C STRANn~nN~-CS 8Lngle ~D~ TOPOLOGY: ~nkn~
(ii) MOLECULE TYPE: DNA

~xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
AGCAGTCTTC TGA~r.ACTT CAACTGTGAG G 31 (2~ INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
'A~ LENGTH: 26 base pairs B~ TYPE: nucleic acid Cl STRANDEDNESS: Ringle ~Dl TOPOLOGY: 1-nkn~ .1,, (ii) ~OLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:

(2) INFORMATION FOR SEQ ID NO:85:
(i) ~QU~:NCE CHARACTERISTICS:
'A'l LENGTH: 21 ba~e pair~
B TYPE: nucleic acid .IC STRANDEDNESS: single ~D, TOPOLOGY: ~nkn (ii) MOLECULE TYPE: DNA

(Xi) SEQUENCE D~SCRTPTION: SEQ ID NO:85:
A~A~AA~.AA CACGTTCACG T 21 (2) INFORMATION FOR SEQ ID NO:86:
(i) S~Qu~:~CE CHARAC,TERISTICS:
(A) LENGTH: 31 ba~e pair~
(B) TYPE: nucleic acid -W O 97/29119 CA 02245783 1998-08-06 PCT~US97/01937 (C) STRANDEDNESS: ~Lngle (D) TOPOLOGY: 1~nk--,1"
(ii) M~r-r~CULE TYPE: DNA

(xi) ~Q~N~: DESCRIPTION: SEQ ID NO:86:
A~ GA AAGCACGTTC ACGTCCATGT T 31 W ~97129119 PCTnU~g71~1937 T ' Arrlir ~ir~-~ No: PCT/
MICROORGANISMS
OptionDI SherJt in connection with the .. ~ al rrJferred to on pllge 125, lines 1-23 of thu dcsc~iption A. IDENTIFICATION OF DEPOSIT I
Further drJposits ~re identifbd on ~n ~ddition~l sheet Name of depositary institution ~
~terieau Type Culture Co~lectio~t Address of depositary institution (includin~ postal code and country~
12301 Parklawn Driv~
Rockville, MD 208B2 US

Date of deposit ~ Januarv 30. 1 g96 Accession Numb~r 69977 B. ADb H ~ r ~ INDICATIONS ae ve bbnlc if nr~l p,olic ble). This inforrn~lion is continued r,n ~ ~epnru~e ~tL cbed sb~et C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ~ id~u.~.ud~a~

D. SEPARATE FURNISHINC OF INDICATIONS a ~ bbnlr if r~ ~pplir bl~) Th- Indic~tions li~tcd bolow will b- riubmlttcd to tho International suro~u later ~ (Sp8clfv the pcneral naturo of thr indlc~tion5 ~.o, ~Acc~sion Numbor of Deposlt~) E. I~his sheet was receivcd with the r ~P ~ ~ be checkcd by the rec7,~ g Offlce~
~n (Au~orlzed Officer) Cl The date of receipt (from the applicar,lt) by the T Bureau was (Autborized Officer) Form PCT/RO/134 (January 1g81) ,, , ,, , , , ~ ~

Claims (112)

WHAT IS CLAIMED IS:

1. A purified Fhit protein.

2. The protein of claim 1 which is a human protein.

3. The protein of claim 1 having the amino acid sequence depicted in Figure 2A (SEQ ID NO:2).

4. A purified protein encoded by a nucleic acid hybridizable to the FHIT DNA sequence in plasmid p7F1 as deposited with the ATCC and assigned accession number 69977.

5. A purified protein encoded by a nucleic acid hybridizable to a DNA having a nucleotide sequence consisting of the coding region of SEQ ID NO:1.

6. The protein of claim 2 which is encoded by plasmid p7F1 as deposited with the ATCC and assigned accession number 69977.

7. A purified derivative of the protein of claim 3, which comprises at least 10 contiguous amino acids of the Fhit protein sequence depicted in Figure 2A (SEQ ID NO:2) and displays one or more functional activities of a Fhit protein.

8. The derivative of claim 7 which is able to be bound by an antibody directed against a Fhit protein.

9. A molecule comprising the derivative of claim 7.

10. A protein comprising an amino acid sequence that has at least 70% identity to the Fhit protein of claim 3, in which the percentage identity is determined over an amino acid sequence of 147 amino acids.

11. The derivative or analog of claim 7, which inhibits proliferation of a cell.

12. A chimeric protein comprising a fragment of a Fhit protein consisting of at least 10 amino acids fused via a covalent bond to an amino acid sequence of a second protein, in which the second protein is not a Fhit protein.

13. The chimeric protein of claim 12 in which the fragment of a Fhit protein is a fragment capable of being bound by an anti-Fhit antibody.

14. An antibody which is capable of binding a Fhit protein.

15. The antibody of claim 14 which is monoclonal.

16. A molecule comprising a fragment of the antibody of claim 14, which fragment is capable of binding a Fhit protein.

17. An isolated nucleic acid of less than 100 kb, comprising a nucleotide sequence encoding a Fhit protein.

18. The nucleic acid of claim 17 in which the Fhit protein has the sequence depicted in Figure 2A (SEQ ID NO:2).

19. The nucleic acid of claim 17 which is a cDNA.

20. An isolated nucleic acid comprising a nucleotide sequence absolutely complementary to the nucleotide sequence of claim 18.

21. The nucleic acid of claim 17 which lacks introns of the FHIT gene.

22. The nucleic acid of claim 17 in which the Fhit protein is a human Fhit protein.

23. An isolated nucleic acid comprising the FHIT
coding sequence contained in plasmid p7F1 as deposited with the ATCC and assigned accession number 69977.

24. An isolated nucleic acid encoding a Fhit protein, and hybridizable to the FHIT DNA sequence in plasmid p7F1 as deposited with the ATCC and assigned accession number 69977.

25. An isolated nucleic acid of less than 100 kb, hybridizable to a DNA having a nucleotide sequence consisting of at least exon 6 or exon 7 of the coding region of SEQ ID NO:1, under stringent conditions.

26. An isolated nucleic acid comprising at least 310 contiguous nucleotides of the FHIT cDNA sequence of SEQ
ID NO:1, said nucleic acid consisting of less than 2,000 nucleotides; and further comprising at least exon 7 of SEQ ID
NO:1.

27. An isolated nucleic acid of less than 100 kb, comprising at least 266 contiguous nucleotides of the FHIT
cDNA coding sequence of SEQ ID NO:1; and further comprising at least exon 7 of SEQ ID NO:1.

28. An isolated nucleic acid of less than 100 kb, comprising (a) the nucleotide sequence of one or more FHIT
exons selected from among exon 1, exon 2, exon 3, and exon 4;
and (b) the nucleotide sequence of one or more FHIT exons selected from among exon 7, exon 8, and exon 9.

29. An isolated nucleic acid comprising a nucleotide sequence encoding a fragment of a Fhit protein that is able to be bound by an anti-Fhit antibody.

30. An isolated nucleic acid comprising a nucleotide sequence encoding the chimeric protein of claim 12.

31. An isolated nucleic acid comprising a nucleotide sequence encoding the protein of claim 10.

32. A recombinant cell containing a nucleic acid vector comprising the nucleic acid of claim 17.

33. A cell containing a recombinant nucleic acid of claim 17.

34. A recombinant cell containing a nucleic acid vector comprising the nucleic acid of claim 28.

35. A recombinant cell containing a nucleic acid vector comprising the nucleic acid of claim 10.

36. A method of producing a Fhit protein comprising growing the recombinant cell of claim 32 such that the encoded Fhit protein is expressed by the cell, and recovering the expressed Fhit protein.

37. A method of producing a Fhit protein comprising growing the recombinant cell of claim 33 such that the encoded Fhit protein is expressed by the cell, and recovering the expressed Fhit protein.

38. A method of producing a Fhit protein comprising growing the recombinant cell of claim 34 such that the encoded protein is expressed by the cell, and recovering the expressed protein.

39. The product of the process of claim 36.

40. The product of the process of claim 37.

41. A pharmaceutical composition comprising a therapeutically effective amount of a Fhit protein; and a pharmaceutically acceptable carrier.

42. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 3;
and a pharmaceutically acceptable carrier.

43. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 5;
and a pharmaceutically acceptable carrier.

44. A pharmaceutical composition comprising a therapeutically effective amount of the protein of claim 7;
and a pharmaceutically acceptable carrier.

45. A pharmaceutical composition comprising a therapeutically effective amount of the chimeric protein of claim 12; and a pharmaceutically acceptable carrier.

46. A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of claim 17; and a pharmaceutically acceptable carrier.

47. A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of claim 29; and a pharmaceutically acceptable carrier.

48. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant cell of claim 33; and a pharmaceutically acceptable carrier.

49. A pharmaceutical composition comprising a therapeutically effective amount of an antibody that immunospecifically binds to a mutant Fhit protein and not to a wild-type Fhit protein; and a pharmaceutically acceptable carrier.

50. A pharmaceutical composition comprising a therapeutically effective amount of a fragment or derivative of an antibody that immunospecifically binds to a Fhit protein and not to a wild-type Fhit protein, said fragment or derivative containing the binding domain of the antibody; and a pharmaceutically acceptable carrier.

51. A method of treating or preventing a disease or disorder associated with a chromosome 3p14.2 mutation in a subject comprising administering to a subject in which such treatment or prevention is desired a therapeutically effective amount of a molecule that promotes Fhit function.

52. The method according to claim 51 in which the disease or disorder involves cell overproliferation.

53. The method according to claim 52 in which the disease or disorder is a malignancy.

54. The method according to claim 53 in which the malignancy is a malignancy of the digestive tract.

55. The method according to claim 53 in which the disease or disorder is selected from the group consisting of esophageal cancer, stomach cancer, renal carcinoma, bladder cancer, breast cancer, colon cancer, lung cancer, melanoma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, and uterine cancer.

56. The method according to claim 52 in which the subject is a human.

57. The method according to claim 52 in which the disease or disorder is selected from the group consisting of premalignant conditions, benign tumors, hyperproliferative disorders, and benign dysproliferative disorders.

58. The method according to claim 52 in which the molecule that promotes Fhit function is selected from the group consisting of a Fhit protein, a Fhit derivative or analog that is active in inhibiting cell proliferation, a nucleic acid encoding a Fhit protein, and a nucleic acid encoding a Fhit derivative or analog that is active in inhibiting cell proliferation.

59. The method according to claim 52 in which the molecule that promotes Fhit function is the protein of claim 3.

60. A method of treating or preventing a disease or disorder involving cell overproliferation in a subject having a hemizygous FHIT mutation comprising administering to a subject having a hemizygous FHIT mutation and in which such treatment or prevention is desired a therapeutically effective amount of a molecule that specifically antagonizes a mutant FHIT gene or protein in the subject, but does not substantially antagonize wild-type FHIT gene or protein.

61. The method according to claim 60 in which the molecule is an antibody or a derivative of the antibody containing the binding region thereof, that specifically binds to a mutant Fhit protein in the subject.

62. The method according to claim 60 in which the molecule is a FHIT antisense nucleic acid that specifically inhibits the expression of a mutant FHIT RNA in the subject.

63. The method according to claim 60 which further comprises administering to the subject a molecule that promotes Fhit function.

64. The method according to claim 60 in which the molecule is a nucleic acid comprising a heterologous nucleotide sequence flanked by FHIT gene portions so as to promote homologous recombination with a genomic mutant FHIT
gene but not a wild-type FHIT gene.

65. The method according to claim 60 in which the molecule is an oligonucleotide which (a) consists of at least six nucleotides; (b) comprises a sequence complementary to at least a portion of an RNA transcript of a FHIT gene; and (c) is hybridizable to the RNA transcript under moderately stringent conditions.

66. A pharmaceutical composition comprising an isolated oligonucleotide consisting of at least six nucleotides, and comprising a sequence complementary to at least a portion of an RNA transcript of a FHIT gene, which oligonucleotide is specifically hybridizable to the RNA
transcript; and a pharmaceutically acceptable carrier.

67. An isolated first nucleic acid comprising a sequence absolutely complementary to a second nucleic acid, said second nucleic acid comprising (a) the nucleotide sequence of one or more FHIT exons selected from among exon 1, exon 2, exon 3, and exon 4; and (b) the nucleotide sequence of one or more FHIT exons selected from among exon 7, exon 8, and exon 9.

68. A method of inhibiting the expression of a nucleic acid sequence encoding a Fhit protein in a cell comprising providing the cell with an effective amount of a nucleic acid comprising at least 10 nucleotides, and comprising a sequence specifically hybridizable to an RNA
transcript of a FHIT gene in the cell.

69. A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving cell overproliferation in a subject comprising detecting or measuring the level of Fhit protein, FHIT RNA or Fhit functional activity in a sample derived from the subject, in which a decrease in the level of Fhit protein, FHIT RNA, or Fhit functional activity in the sample, relative to the level of Fhit protein, FHIT RNA, or Fhit functional activity found in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder, indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.

70. A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving cell overproliferation in a subject comprising detecting one or more mutations in FHIT DNA, RNA
or protein derived from the subject in which the presence of said one or more mutations indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.

71. The method according to claim 70 in which a mutation in an exon of a FHIT DNA or RNA is detected.

72. The method according to claim 71 in which a mutation in a FHIT DNA or RNA coding sequence is detected.

73. The method according to claim 71 in which the mutation is a deletion of at least a portion of the FHIT
coding sequence.

74. The method according to claim 73 in which the mutation is a deletion of at least all or a portion of FHIT
exon 5.

75. The method according to claim 71 in which the mutation causes a lack of the wild-type open reading frame of exon 8 in a FHIT RNA in the subject.

76. The method according to claim 71 in which the mutation is detected by detecting the presence of a FHIT RNA
of aberrant size or sequence when compared to wild-type FHIT
RNA.

77. The method according to claim 76 in which the presence of a FHIT RNA of aberrant size or sequence is detected by a method comprising reverse-transcribing RNA from the subject into cDNA.

78. The method according to claim 77 in which the cDNA is subjected to polymerase chain reaction using oligonucleotide primers adapted to amplify a fragment of wild-type FHIT cDNA comprising FHIT exon 5.

79. The method according to claim 77 in which the cDNA is subjected to polymerase chain reaction using oligonucleotide primers adapted to amplify a fragment of wild-type FHIT cDNA comprising FHIT exon sequences between the 3' terminus of exon 4 and exon 8.

80. The method according to claim 71 in which the subject is a human fetus.

81. The method according to claim 71 in which the subject is a human child or adult.

82. The method according to claim 71 in which the mutation is detected by a method comprising contacting RNA or cDNA made therefrom from the subject with a nucleic acid probe capable of specifically hybridizing to FHIT RNA or cDNA
under conditions such that hybridization can occur, and detecting or measuring the amount of any hybridization between the RNA or cDNA and the nucleic acid probe.

83. The method according to claim 72 in which the mutation is detected by a method comprising contacting a sample containing protein from the subject with an anti-Fhit antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.

84. The method according to claim 72 in which the mutation is detected by a method comprising subjecting FHIT
RNA from the subject to in vitro translation.

85. A kit comprising in one or more containers a purified molecule selected from the group consisting of an anti-Fhit antibody that specifically binds to a mutant Fhit protein, a nucleic acid probe capable of specifically hybridizing to a mutant FHIT gene or RNA, a nucleic acid encoding a Fhit protein or a derivative thereof active in inhibiting cell proliferation, and a Fhit protein or derivative thereof active in inhibiting cell proliferation.

86. A kit comprising in one or more containers a pair of nucleic acid primers capable of priming amplification of at least a portion of a FHIT cDNA or mRNA sequence.

87. The kit according to claim 86 in which the primers are adapted so as to be able to amplify a fragment of wild-type FHIT cDNA comprising exon 5.

88. The kit according to claim 86 in which the primers are adapted so as to be able to amplify a fragment of wild-type FHIT cDNA comprising exons 5 and 6.

89. The kit according to claim 86 in which the primers are adapted so as to be able to amplify a fragment of wild-type FHIT cDNA comprising sequences between the 3' terminus of exon 4 and exon 8.

90. The kit according to claim 86 in which the primers ars adapted so as to be able to amplify a fragment of wild-type FHIT cDNA comprising exons 1 through 10.

91. A kit comprising in a container a therapeutically effective amount of a Fhit protein.

92. A method of identifying a molecule that specifically binds to a ligand selected from the group consisting of a Fhit protein, a fragment of a Fhit protein that is functionally active, and a nucleic acid encoding the protein or fragment, comprising (a) contacting said ligand with a plurality of molecules under conditions conductive to binding between said ligand and the molecules;
and (b) identifying a molecule within said plurality that specifically binds to said ligand.

93. A recombinant non-human animal that is the product of a process comprising introducing a nucleic acid encoding a Fhit protein or functionally active derivative thereof into the animal.

94. A progeny of the animal of claim 93 in which the nucleic acid is heritably contained within its genome.

95. A diagnostic kit comprising in a container a compound comprising a nucleic acid of not more than 2,000 nucleotides and comprising at least 10 nucleotides of the nucleotide sequence of SEQ ID NO:1 or its complement.

96. A diagnostic kit comprising in one or more containers, a pair of primers, each consisting of 15-35 nucleotides, in which at least one of said primers is hybridizable to SEQ ID NO: 1 or its complement and wherein said primers are capable of priming DNA synthesis in a nucleic acid amplification reaction.

97. Plasmid p7F1 as deposited with the ATCC and having accession number 69977.

98. A method of monitoring a disease involving cell overproliferation in a subject comprising detecting or measuring the amount of FHIT RNA or cDNA or protein in a sample derived from the subject, in which an increase in mutant FNIT RNA or cDNA or protein expression, or a decrease or loss in wild-type FHIT RNA or cDNA or protein relative to that present in a sample derived from the subject at an earlier time, indicates disease progression.

99. A method of detecting a predisposition toward an adverse therapeutic outcome or toward recurrence of a disorder involving cell overproliferation in a subject comprising detecting one or more mutations in a FHIT gene in the subject.

100. The method according to claim 69 or 70 in which the disease or disorder is a malignancy of the digestive tract.

101. The method according to claim 69 in which the disease or disorder is lung cancer.

102. The method according to claim 70 in which the disease or disorder is lung cancer.

103. The method according to claim 102 in which the disease or disorder is small cell lung carcinoma.

104. The method according to claim 102 in which the disease or disorder is non small cell lung carcinoma.

105. The method according to claim 72 which further comprises detecting the loss of one allele of a FHIT gene, and in which the disease or disorder is lung cancer.

106. The method according to claim 73 in which the mutation is a deletion of two or more exons in a FHIT DNA or RNA.

107. The method according to claim 102 in which the mutation is a deletion of at least all of FHIT exon 5.

108. The method according to claim 102 in which the mutation is a deletion of FHIT exons 4-6 resulting in a fusion of exon 3 sequences to exon 7 sequences in a FHIT RNA
or cDNA.

109. The method according to claim 102 in which the mutation is a deletion of FHIT exons 4-8 resulting in a fusion of exon 3 sequences to exon 9 sequences in a FHIT RNA
or cDNA.

110. A cell containing a recombinant nucleic acid of less than 100 kb, said recombinant nucleic acid comprising a nucleotide sequence encoding a Fhit protein having the amino acid sequence depicted in Figure 2A (SEQ ID NO:2); and a promoter operatively linked to said nucleotide sequence.

111. An isolated RNA transcript of the isolated nucleic acid of claim 26.

112. An isolated RNA transcript of the isolated nucleic acid of claim 27.