| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 01.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 25.01.00 |
| PATENT TITLE |
Human thymosin .beta.15 gene, protein and uses thereof |
| PATENT ABSTRACT |
The present inventors have now discovered that humans have a gene that encodes a novel protein of the thymosin .beta. family. This novel protein, herein referred to as thymosin .beta.15, has the ability to bind and sequester G-actin, like other members of the thymosin .beta. family, but unlike what is known about other members it also directly regulates cell motility in prostatic carcinoma cells. The present invention is direct to an isolated cDNA encoding the human thymosin .beta.15 gene (SEQ ID NO: 1) and have deduced the amino acid sequence (SEQ ID NO: 2). |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 29.04.98 |
| PATENT REFERENCES CITED |
Nachmiar, V., Current Opinion in Cell Biology, 1993, 5:56. Safer, et al., Proc. Natl. Acad. Sci USA 1990 87:2536-2540. Safter, et al., J. Biol. Chem., 1991, 268:4029-4032). D. Safer, J. Muscle Res. Cell Motil, 1992. 13:269-271). Weber, et al., Biochemistry 1992, 31:6179-6185). Yu, et al., J. Biol. Chem., 1993, 268:502-509. Cassimeris, et al., J. Cell Biol., 1992, 119:1261-1270. Low, et al., Arch. Biochem. Biophys., 1992, 293:32-39. Low, et al., Proc. Natl. Acad. Sci., USA 1981, 78:1162-1166. Rebar, et al., Science 1981, 214:669-671. Gomez-Marquez, et al., J. Immunol. 1989, 143:2740-2744. Bao, et al., The American Association for Cancer Research annual meeting (Mar. 18-22, 1995), Abstract. Clauss, et al., Genomies 1991, 9:75-180. Sanders, et al., Proc. Natl. Acad. Sci. USA 1992, 89:4678-4682. Bao, et al., Proceedings of the American Association For Cancer Research, Annual Meeting, vol. 36, p. 85 XP002041934, see abstract A505 (1995). K. Kwiatowska, et al., Molecular Biology of the Cell, Supplement, vol. 6, p. 141A XP002041935, see abstract 816 (1995). Sun, et al., J. Bio. Chem., vol. 271, No. 16, pp. 9223-9230 XP002041936 (1996). S. Varghese, et al., J. Bio. Chem., vol. 266, No. 22, pp. 14256-14261 XP002041937 (1991). Lin, et al., J. Bio. Chem, vol. 266, No. 35, pp. 23347-23353, XP002041938 (1991). Bao, et al., Proceedings of the American Association For Cancer Research, Annual Meeting, vol. 38, pp. 492-493, XP002041939 (1997). B. Zetter, vol. 51, No. 4, p. 185 XP002041940 (1997). Bao, et al., Nature Medicine, vol. 2, No. 12, pp. 1322-1328 XP002041941 (1996). D. Coffey, Nature Medicine, vol. 2, No. 12, pp. 1305-1306 XP002041942 (1996). European Search Report for PCT/US97/10315, mailed Oct. 22, 1997. |
| PATENT GOVERNMENT INTERESTS | The work described herein was supported, in part, by National Institutes of Health grant CA37393. The U.S. Government has certain rights to this invention |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method for inhibiting thymosin .beta.15 expression in a cell comprising administering to the cell an effective amount of a antibody or fragments therof, that inhibits thymosin .beta.15 expression. 2. The method of claim 1 wherein said fragment is a Fab, Fab', F(ab')2 or Fv fragment. 3. The method of claim 1, wherein said antibody is a single chain antibody. 4. The method of claim 1, wherein said antibody is humanized. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION The present invention provides novel genes, proteins, and uses thereof including, methods for diagnosing and treating cancer, particularly metastatic cancer. Most eukaryotic cells (execptions include red blood cells and adult muscles) contain high concentrations, i.e., up to .about.250 .mu.mol/l, of momomeric actin. How such actin remains unpolymerized in the cytoplasm has remained a problem in cell biology (Nachmiar, V., Current Opinion in Cell Biology, 1993, 5:56). Profilin, originally thought to be the actin-sequestering protein, is not present in sufficient amounts to account for more than part of the monomeric actin levels observed. Recently, an actin-sequestering 5 kD peptide was discovered in high concentration in human platelets (Safer, et al., Proc. Natl. Aced. Sci USA 1990 87:2536-2540) and shown to be identical to a previously known peptide (Safter, et al., J. Biol. Chem., 1991, 268:4029-4032) originally thought to be the thymic hormone, thymosin .beta..sub.4 (T.beta..sub.4) (D. Safer, J. Muscle Res. Cell Motil, 1992. 13:269-271). A detailed kinetic study of the interaction of T.beta..sub.4 and actin (Weber, et al., Biochemistry 1992, 31:6179-6185)), together with other studies (Yu, et al., J. Biol. Chem., 1993, 268:502-509 and Cassimelds, et al., J. Cell Biol., 1992, 119:1261-1270) support the hypothesis that T.beta..sub.4 and T.beta..sub.10 function primary as G-actin buffers. Unpublished data (E. Hannappel) extend the function to several other .beta. thymosins. T.beta..sub.4 has also been shown to inhibit nucleotide exchange by actin, whereas profilin increases the rate of exchange (Coldschmidt-Clermont, et al., Mol Cell Biol, 1992, 3:1015-1025). All vertebrates studied contain one or often two .beta.-thymosins. Thus, the members of the .beta.-thymosin family are believed to be important in all species. Three new family members (Low, et al., Arch. Biochem. Biophys., 1992, 293:32-39 and Schmid, B., Ph.D Thesis, University of Tubingen 1989) have been found in perch, trout and in sea urchin, the first non-vertebrate source. The sequences are well conserved suggesting :hat actin sequestration is probably a property of all .beta.-thymosins. However, when T.beta..sub.4 was discovered and its sequence first determined in 1981 (Low, et al., Proc. Natl. Acad. Sci., USA 1981, 78:1162-1166), data were presented that suggested two extracellular functions (Low, et al. supra and Rebar, et al., Science 1981, 214:669-671). Two recent papers indicate a different and unexpected effect of a tetrapeptide which may be derived from the amino terminus of T.beta..sub.4. Several reports demonstrate regulation of T.beta..sub.4 or T.beta..sub.10 synthesis at the transcriptional or translational level. An interferon-inducible gene (Cassimelds, et al., J. Cell. Biol. 1992, 119:1261-1270 and Sanders, et al., Proc. Natl. Acad. Sci. USA 1992, 89:4678-4682) is identical to the cDNA of human T.beta..sub.4, and there are several genes for T.beta..sub.4 in humans. (Clauss, et al., Genomies 1991, 9:75-180 and Gomez-Marquez, et al., J. Immunol 1989, 143:2740-2744) It would be desirable to identify new members of the .beta.-thymosin family, particularly in humans. Bao and Zetter reported in an abstract presented at the American Association for Cancer Research annual meeting (Mar. 18-22, 1995) the differential expression of a novel mRNA expressed in high-metastatic rat tumor cell lines, but not in a low metastatic variant. cDNA was isolated and was reported to encode a protein with 68% identity to the rat thymosin .beta.4. However, the nucleotide sequence and the deduced amino acid sequence were not reported. SUMMARY OF THE INVENTION We have now discovered that humans have a gene that encodes a novel protein of the thymosin .beta. family. This novel protein, herein referred to as thymosin .beta.15, has the ability to bind and sequester G-actin, like other members of the thymosin .beta. family, but unlike what is known about other members it also directly regulates cell motility in prostatic carcinoma cells. We have isolated a cDNA of the human thymosin .beta.15 gene (SEQ ID NO: 1) and have deduced the amino acid sequence (SEQ ID NO: 2). We have shown that enhanced transcripts (mRNA) and expression of the thymosin .beta.15 gene in non-testicular cells has a high correlation to disease state in a number of cancers, such as prostate, lung, melanoma and breast cancer, particularly metastatic cancers. Accordingly, discovering enhanced levels of transcript or gene product in non-testicular tissues can be used in not only a diagnostic manner, but a prognostic manner for particular cancers. The present invention provides isolated nucleic acids (polynucleotides) which encode thymosin .beta.15 having the deduced amino acid sequence of SEQ ID. NO: 2 or a unique fragment thereof. The term "unique fragment" refers to a portion of the nucleotide sequence or polypeptide of the invention that will contain sequences (either nucleotides or amino acid residues) present in thymosin .beta.15 (SEQ ID NO: 2) but not in other member of the thymosin family. This can be determined when the hybridization profile of that fragment under stringent conditions is such that it does not hybridize to other members of the thymosin family. Such fragments can be ascertained from FIG. 3. A preferred set of unique fragments are those that contain, or contain polynucleotides that encode, amino acid 7 to 12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ ID NO: 2. Preferably, the unique nucleotide sequence fragment is 10 to 60 nucleotides in length, more preferably, 20 to 50 nucleotides, most preferably, 30 to 50 nucleotides. Preferably, the unique polypeptide sequence fragment is 4 to 20 amino acids in length, more preferably, 6 to 15 amino acids, most preferably, 6 to 10 amino acids. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptides may be identical to the coding sequence shown in SEQ ID NO: 1 or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same protein as the DNA of SEQ ID NO: 1. The polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in SEQ ID NO: 1. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded protein. The present invention also provides an isolated polynucleotide segment which hybridize under stringent conditions to a unique portion of the hereinabove-described polynucleotides, particularly SEQ ID NO:1. The segment preferably comprises at least 10 nucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. These isolated segments may be used in nucleic acid amplification techniques, e.g., PCR, to identify and/or isolate polynucleotides encoding thymosin .beta.15. As used herein a polynucleotide "substantially identical" to SEQ ID NO:1 is one comprising at least 90% homology, preferably at least 95% homology, most preferably 99% homology to SEQ ID NO: 1. The reason for this is that such a sequence can encode thyfnosin .beta.15 in multiple mammalian species. The present invention further provides an isolated and purified human thymosin .beta.15 having the amino acid sequence of SEQ ID NO: 2, or a unique fragment thereof, as well as polypeptides comprising such unique fragments, including, for example, amino acid 7 to 12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ ID NO: 2. In accordance with yet another aspect of the present invention, there are provided isolated antibodies or antibody fragments which selectively binds human thymosin .beta.15. The antibody fragments include, for example, Fab, Fab', F(ab')2 or Fv fragments. The antibody may be a single chain antibody, a humanized antibody or a chimeric antibody. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotides or polypeptides present in a living animal is not isolated, but the same polynucleotides or DNA or polypeptides, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. The present invention further provides a method of treating a neoplastic cell expressing human thymosin .beta.15 by administering to the cell an effective amount of a compound which suppresses the activity or production of the human thymosin .beta.15. Preferably, the compound interferes with the expression of the human thymosin .beta.15 gene. Such compounds include, for example, antisense oligonucleotides, ribozymes, antibodies, including single chain antibodies and fragments thereof. DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B show differential mRNA display and Northern analysis of Dunning R-3327 rat prostatic adenocarcinoma variants. Total RNA from AT2.1 (lane 1), AT3.1 (lane 2) and AT6.1 (lane 3) cells were reverse-transcribed and amplified by PCR with a primer set, T.sub.11 AG and a 10 mer AGGGAACGAG (SEC ID NO:3) in the presence of [.alpha.35-S]dATP. The PCR fragments were displayed on a 6% polyacrylamide gel and autoradiographed. The differentially expressed band is indicated by arrowhead. B. Northern blot analysis of thymosin .beta.15 gene. Two .mu.g of poly (A) RNA was isolated from Dunning R-3327 variants AT2. I (lane 1), AT3.1 (lane 2), AT6.1 (lane 3), and Mat Lylu (lane 4), fractionated on a 1.1% formaldehyde-agarose gel, transferred to Hybond-N+ nylon membrane (Amersham) and hybridized with a random primed (Grillon C, et al., FEBS 1990, 274:30-34).sup.32 P-labeled T.beta.15 cDNA fragment. The same blot was hybridized with a rat .beta.-actin probe to demonstrate that equivalent amounts of RNA were loaded in each lane. FIG. 2 is the nucleotide sequence (SEQ ID NO.: 1) of T.beta.15 cDNA and the predicted amino acid sequence (SEQ ID NO.: 2) (single-letter code). The sequence numbers of the nucleotides and amino acids are indicated on the right side of the sequences. The translationinitiation codon ATG is underlined, and the termination codon TAA is marked with an asterisk. A putative actin binding region is underlined. These sequence data are available from GenBank under accession number U25684. FIG. 3 shows the alignment of the deduced T.beta.15 protein sequence and some of the other .beta. thymosin isoforms. Regions of amino acid identity are represented by white leters boxed in black. Unboxed black letters correspond to nonidentical regions. Dots correspond to gaps introduced in the sequence to optimize alignment. FIG. 4 shows expression of T.beta.15 in various rat tissues. The multiple-tissue blot was obtained from Clontech. The blot was hybridized with the T.beta.15 cDNA probe. Rat GAPDH is a loading control. FIGS. 5A and 5B show in situ hybridization with antisense riboprobe for T.beta.15 on prostatic adenocarcinoma patients. FIG. 5A shows differential expression in tumors. The small arrow shows positive staining. The large arrow shows negative staining. FIG. 5B shows that in poorly differentiated and invasive prostate carcinoma, single cells invading stroma display intense staining (arrow). FIGS. 6A, 6B and 6C show the effect of T.beta.15 on actin polymerization. FIG. 6A. 3 .mu.M of pyrene-labeled G-actin was polymerized in the presence of various amounts of GST-T.beta.4 fusion peptide (.tangle-soliddn.), GST-T.beta.15 (.tangle-solidup.) or GST alone (.smallcircle.). The final extent of polymerization was determined from the final levels of pyrene-labeled actin (fluorescence). All solutions contained 5.5 mM Tris, pH7.6, 167 .mu.M CaCl.sub.2, 0.5 mM glutathione, 167 .mu.M DTT, and 420 .mu.M ATP. Polymerization was induced by addition of 2 mM MgCl.sub.2 and 150 mM KCl. Error bars denote the range of duplicate measurements made from separate dilutions of the fusion proteins. FIG. 6B. 2 .mu.M of pyrene-labeled G-actin was polymerized in the presence of various amounts of monomeric T.beta.15 that had been cleaved from GST by thrombin. The relative rates of polymerization were derived from the maximal rate of fluorescence increase in the initial phase of polymerization. FIG. 6C. The final extent of actin assembly was determined by the same methods used for the thymosin GST fusion peptides. Experimental conditions are those described for FIG. 6B. FIGS. 7A, 7B and 7C show serum stimulated migration of control transfected and T.beta.15 transfected Dunning R-3327 variants and their growth rate. FIG. 7A. Vector control transfected (.smallcircle.,.gradient.) and T.beta.15 antisense (.circle-solid.,.tangle-soliddn.) transfected AT3.1 cell clones. FIG. 7B. Vector control transfected (.smallcircle.,.gradient.) and T.beta.15 sense transfected (.circle-solid.,.tangle-soliddn.) AT2.1 cell clones. Data are expressed as the mean .+-.SE (n=4). FIG. 7C. Growth curves of control transfected and T.beta.15 (sense or antisense) transfected Dunning R-3327 clones. Cells from vector control transfected AT2.1 (.smallcircle.), T.beta.15 sense transfected AT2.1 (.circle-solid.), vector control transfected AT3.1 (.gradient.) and T.beta.15 antisense transfected AT3.1 (.tangle-soliddn.) were plated at initial 10.sup.4 cells/well in RPMI 1640 with 10% FBS and 250 nM dexamethasome in 12-well plates. Cells were harvested and counted at indicated times. Points represent the mean .+-.SE (n=3). FIGS. 8A and 8B show Western analysis of thymosin .beta.-GST fusion protein. FIG. 8A is a Coomasie staining of GST-T.beta. fusion proteins. FIG. 8B is a Western analysis of GST-T.beta. fusion proteins with affinity purified anti-T.beta.15 C-terminal peptide antibody. Lane 1: GST-T.beta.4; Lane 2: GST-T.beta.15; Lane 3: GST only FIG. 9 shows a Northern analysis of thymosin .beta.15 in mouse lung tumor cells. LA-4: mouse lung adenoma cell line; M27 and H59: metastatic variants derived from mouse Lewis lung adenocarcinoma cell line. Northern blot analysis revealed that the probe detected the thymosin .beta.15 mRNA expression in M27 cells, less expression in H59 cells, but no expression in LA-4 cells. FIGS. 10A, 10B, 10C and 10 show immunohistochemical staining of human prostatic carcinoma tissues with an affinity purified polyclonal antibody to thymosin .beta.15. A. Nonmalignant prostatic epithelia (large arrow) and high-grade prostatic intraepithelial neoplasia (PIN) (small arrow). B. Moderately differentiated prostatic carcinoma showing heterogeneoue immunostaining (small arrow, positive; large arrow, negative). C. Poorly differentiated prostatic carcinoma. D. Single cells invading stroma showing intense staining. FIG. 11 is a 1.4% agarose gel electrophoresis of RT-PCR amplified .beta. thymosins from the rat prostatic cell lines. Lane1, weakly metastatic AT2.1; lane 2, 3 and 4, highly metastatic AT3.1, AT6.1 and Mat Lylu; lane 5 and 6, nonmetastatic NbE and MC2; lane 7, weakly metastatic Fb2. .beta.-actin PCR was used as internal control of each sample. DETAILED DESCRIPTION OF THE INVENTION A well characterized series of cell lines that show varying metastatic potential has been developed from the Dunning rat prostatic carcinoma (Isaacs, et al., Prostate 9, 261-281 and Bussebakers, et al., Cancer Res. 52,2916-2922 (1992)). Coffey and colleagues previously showed a direct correlation between cell motility and metastatic potential in the Dunning cell lines (Mohler, et al., Cancer Res. 48, 4312-4317 (1988), Parin, et al., Proc. Natl. Acad. Sci, USA 86, 1254-1258 (1989) and Mohler, et al., Cancer Metast. Rev 12, 53-67 (1993)). We compared gene expression in poorly metastatic and highly metastatic cell lines derived from Dunning rat prostate carcinoma using differential mRNA display. The results of these studies revealed the expression of a novel member of the thymosin beta family of actin-binding molecules, thymosin .beta.15. Using this information, we isolated and sequenced a cDNA encoding human thymosin .beta.15. Although members of the thymosin .beta. family have been shown to bind and sequester G-actin, they have not previously been demonstrated to alter cell motility. Our studies, however, reveal that this new member, thymosin .beta.15, directly regulates cell motility in prostatic carcinoma cells. We have shown that expression of thymosin .beta.15 is upregulated in highly metastatic prostate cancer cell lines relative to poorly metastatic or nonmetastatic lines. In addition, thymosin .beta.15 was expressed in human prostate carcinoma specimens but not in normal human prostate. Although not wishing to be bound by theory, this indicates that .beta.15 plays a role in the process of metastatic transformation. The present invention provides a polynucleotide sequence encoding all or part of thymosin .beta.15 having the deduced amino acid sequence of SEQ ID NO:2 or a unique fragment thereof. A nucleotide sequence encoding human thymosin .beta.15 is set forth as SEQ ID NO:1. The sequences of the invention may also be engineered to provide restriction sites, if desired. This can be done so as not to interfere with the peptide sequence of the encoded thymosin .beta.15, or may interfere to any extent desired or necessary, provided that the final product has the properties desired. Where it is desired to express thymosin .beta.15 or a unique fragment thereof, any suitable system can be used. The general nature of suitable vectors, expression vectors and constructions therefor will be apparent to those skilled in the art. Suitable expression vectors may be based on phages or plasmids, both of which are generally host-specific, although these can often be engineered for other hosts. Other suitable vectors include cosmids and retroviruses, and any other vehicles, which may or may not be specific for a given system. Control sequences, such as recognition, promoter, operator, inducer, terminator and other sequences essential and/or useful in the regulation of expression, will be readily apparent to those skilled in the art, and may be associated with the natural thymosin .beta.15 or with the vector used, or may be derived from any other source as suitable. The vectors may be modified or engineered in any suitable manner. Correct preparation of nucleotide sequences may be confirmed, for example, by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463-7 (1977)). A CDNA fragment encoding the thymosin .beta.15 of the invention may readily be inserted into a suitable vector. Ideally, the receiving vector has suitable restriction sites for ease of insertion, but blunt-end ligation, for example, may also be used, although this may lead to uncertainty over reading frame and direction of insertion. In such an instance, it is a matter of course to test transformants for expression, 1 in 6 of which should have the correct reading frame. Suitable vectors may be selected as a matter of course by those skilled in the art according to the expression system desired. By transforming a suitable organism or, preferably, eukaryotic cell line, such as HeLa, with the plasmid obtained, selecting the transformant with ampicillin or by other suitable means if required, and adding tryptophan or other suitable promoter-inducer (such as indoleacrylic acid) if necessary, the desired thymosin .beta.15 may be expressed. The extent of expression may be analyzed by SDS polyacrylamide gel electrophoresis-SDS-PAGE (Lemelli, Nature 227:680-685 (1970)). Suitable methods for growing and transforming cultures etc. are usefully illustrated in, for example, Maniatis (Molecular Cloning, A Laboratory Notebook, Maniatis et al. (eds.), Cold Spring Harbor Labs, N.Y. (1989)). Cultures useful for production of thymosin .beta.15, or a peptide thereof, may suitably be cultures of any living cells, and may vary from prokaryotic expression systems up to eukaryotic expression systems. One preferred prokaryotic system is that of E. coli, owing to its ease of manipulation. However, it is also possible to use a higher system, such as a mammalian cell line, for expression of a eukaryotic protein. Currently preferred cell lines for transient expression are the HeLa and Cos cell lines. Other expression systems include the Chinese Hamster Ovary (CHO) cell line and the baculovirus system. Other expression systems which may be employed include streptomycetes, for example, and yeasts, such as Saccharomyces spp., especially S. cerevisiae. Any system may be used as desired, generally depending on what is required by the operator. Suitable systems may also be used to amplify the genetic material, but it is generally convenient to use E. coli for this purpose when only proliferation of the DNA is required. Standard detection techniques well known in the art for detecting RNA, DNA, proteins and peptides can readily be applied to detect thymosin .beta.15 or its transcript to diagnose cancer, especially metastatic cancer or to confirm that a primary tumor has, or has not, reached a particular metastatic phase. In one such technique, immunohistochemistry, anti-thymosin .beta.15 antibodies may be used to detect thymosin .beta.15 in a biopsy sample. Anti-thymosin .beta.15 antibodies may also be used for imaging purposes, for example, to detect tumor metastasis. Suitable labels include radioisotopes, iodine (.sup.125 I ,.sup.121 I), carbon (.sup.14 C), sulphur (.sup.35 S), tritium (.sup.3 H), indium (.sup.112 In), and technetium (.sup.99m Tc), fluorescent labels, such as fluorescein and rhodamine, and biotin. However, for in vivo imaging purposes, the position becomes more restrictive, as antibodies are not detectable, as such, from outside the body, and so must be labelled, or otherwise modified, to permit detection. Markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow external detection. Suitable markers may include those that may be detected by X-radiography, NMR or MIR. For X-radiographic techniques, suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the patient, such as barium or caesium, for example. Suitable markers for NMR and MIR generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labelling of nutrients for the relevant hybridoma, for example. In the case of in vivo imaging methods, an antibody or antibody fragment which has been labelled with an appropriate detectable imaging moiety, such as a radioisotope (for example, .sup.131 I,.sup.112 In, .sup.99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the subject (such as a human) to be examined. The size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium-99m. The labelled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain thymosin .beta.15. The labelled antibody or antibody fragment can then be detected using known techniques. The antibodies may be raised against either a peptide of thymosin .beta.15 or the whole molecule. Such a peptide may be presented together with a carrier protein, such as an KLH, to an animal system or, if it is long enough, say 25 amino acid residues, without a carrier. Preferred peptides include regions unique to thymosin .beta.15, such as amino acid 7 to 12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ ID NO: 2. Polyclonal antibodies generated by the above technique may be used direct, or suitable antibody producing cells may be isolated from the animal and used to form a hybridoma by known means (Kohler and Milstein, Nature 256:795. (1975)). Selection of an appropriate hybridoma will also be apparent to those skilled in the art, and the resulting antibody may be used in a suitable assay to identify thymosin .beta.15. Antibodies, or their equivalents, may also be used in accordance with the present invention for the treatment or prophylaxis of cancers. Administration of a suitable dose of the antibody may serve to block production, or to block the effective activity of thymosin .beta.15, and this may provide a crucial time window in which to treat the malignant growth. Prophylaxis may be appropriate even at very early stages of the disease, as it is not known what actually leads to metastasis in any given case. Thus, administration of the antibodies, their equivalents, or factors which interfere with thymosin .beta.15 activity, may be effected as soon as cancer is diagnosed, and treatment continued for as long as is necessary, preferably until the threat of the disease has been removed. Such treatment may also be used prophylactically in individuals at high risk for development of certain cancers, e.g., prostate. A method of treatment involves attachment of a suitable toxin to the antibodies which then target the area of the tumor. Such toxins are well known in the art, and may comprise toxic radioisotopes, heavy metals, enzymes and complement activators, as well as such natural toxins as ricin which are capable of acting at the level of only one or two molecules per cell. It may also be possible to use such a technique to deliver localized doses of suitable physiologically active compounds, which may be used, for example, to treat cancers. It will be appreciated that antibodies for use in accordance with the present invention, whether for diagnostic or therapeutic applications, may be monoclonal or polyclonal as appropriate. Antibody equivalents of these may comprise: the Fab' fragments of the antibodies, such as Fab, Fab', F(ab')2 and Fv; idiotopes; or the results of allotope grafting (where the recognition region of an animal antibody is grafted into the appropriate region of a human antibody to avoid an immune response in the patient), for example. Single chain antibodies may also be used. Other suitable modifications and/or agents will be apparent to those skilled in the art. Chimeric and humanized antibodies are also within the scope of the invention. It is expected that chimeric and humanized antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody. A variety of approaches for making chimeric antibodies, comprising for example a non-human variable region and a human constant region, have been described. See, for example, Morrison et al., Proc. Nati. Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314,452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication EP 171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B. Additonally, a chimeric antibody can be further "humanized" such that parts of the variable regions, especially the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such altered immunoglobulin molecules may be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and are preferably made according to the teachings of PCT Publication WO92/06193 or EP 0239400. Humanized antibodies can be commercially produced by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain. Another method of generating specific antibodies, or antibody fragments, reactive against thymosin .beta.15 is to screen phage expression libraries encoding immunoglobulin genes, or portions thereof, with a protein of the invention, or peptide fragment thereof. For example, complete Fab fragments, V H regions and V-region derivatives can be expressed in bacteria using phage expression libraries. See for example Ward, et al., Nature 341,544-546: (1989); Huse, et al., Science 246, 1275-1281 (1989); and McCafferty, et al., Nature 348, 552-554 (1990). The antibody can be administered by a number of methods. One preferred method is set forth by Marasco and Haseltine in PCT WO94/02610, which is incorporated herein by reference. This method discloses the intracellular delivery of a gene encoding the antibody, in this case the thymosin .beta.15 antibody. One would preferably use a gene encoding a single chain thymosin .beta.15 antibody. The antibody would preferably contain a nuclear localization sequence, for example Pro-Lys-Lys-Lys-Arg-Lys-Val (SEQ ID NO:4) [Lawford, et al. Cell 46:575 (1986)]; Pro-Glu-Lys-Lys-lle-Lys-Ser (SEQ ID NO:5) [Stanton, et al., Proc. Natl. Aced. Sci. USA 83:1772 (1986)], Gln-Pro-Lys-Lys-Pro (SEQ ID NO:6) [Harlow, et al., Mol. Cell. Biol. 5:1605 (1985)]; Arg-Lys-Lys-Arg (SEQ ID NO:7) for the nucleus. One preferably uses an SV40 nuclear localization signal. By this method one can intracellularly express a thymosin .beta.15 antibody, which can block thymosin .beta.15 functioning in desired cells. In addition to using antibodies to inhibit thymosin .beta.15, it may also be possible to use other forms of inhibitors. Inhibitors of thymosin .beta.15 may manufactured, and these will generally correspond to the area of the substrate affected by the enzymatic activity. It is generally preferred that such inhibitors correspond to a frozen intermediate between the substrate and the cleavage products, but it is also possible to provide a sterically hindered version of the binding site, or a version of the binding site which will, itself, irreversibly bind to thymosin .beta.15. Other suitable inhibitors will be apparent to the skilled person. The invention also provides for the treatment of a cancer by altering the expression of the thymosin .beta.15. This may be effected by interfering with thymosin .beta.15 production, such as by directing specific antibodies against the protein, which antibodies may be further modified to achieve the desired result. It may also be possible to block the thymosin .beta.15 receptor, something which may be more easily achieved by localization of the necessary binding agent, which may be an antibody or synthetic peptide, for example. Affecting thymosin .beta.15 gene expression may also be achieved more directly, such as by blocking of a site, such as the promoter, on the genomic DNA. Where the present invention provides for the administration of, for example, antibodies to a patient, then this may be by any suitable route. If the tumor is still thought to be, or diagnosed as, localized, then an appropriate method of administration may be by injection direct to the site. Administration may also be by injection, including subcutaneous, intramuscular, intravenous and intradermal injections. Formulations may be any that are appropriate to the route of administration, and will be apparent to those skilled in the art. The formulations may contain a suitable carrier, such as saline, and may also comprise bulking agents, other medicinal preparations, adjuvants and any other suitable pharmaceutical ingredients. Catheters are another preferred mode of administration. Thymosin .beta.15 expression may also be inhibited in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. An antisense nucleic acid molecule which is complementary to a nucleic acid molecule encoding thymosin .beta.15 can be designed based upon the isolated nucleic acid molecules encoding thymosin .beta.15 provided by the invention. An antisense nucleic acid molecule can comprise a nucleotide sequence which is complementary to a coding strand of a nucleic acid, e.g. complementary to an mRNA sequence, constructed according to the rules of Watson and Crick base pairing, and can hydrogen bond to the coding strand of the nucleic acid. The antisense sequence complementary to a sequence of an mRNA can be complementary to a sequence in the coding region of the mRNA or can be complementary to a 5' or 3' untranslated region of the mRNA. Furthermore, an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element. Preferably, an antisense nucleic acid complementary to a region preceding or spanning the initiation codon or in the 3' untranslated region of an mRNA is used. An antisense nucleic acid can be designed based upon the nucleotide sequence shown in SEQ ID NO: 1. A nucleic acid is designed which has a sequence complementary to a sequence of the coding or untranslated region of the shown nucleic acid. Alternatively, an antisense nucleic acid can be designed based upon sequences of a .beta.15 gene, which can be identified by screening a genomic DNA library with an isolated nucleic acid of the invention. For example, the sequence of an important regulatory element can be determined by standard techniques and a sequence which is antisense to the regulatory element can be designed. The antisense nucleic acids and oligonucleotides of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. The antisense nucleic acid or oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used. Alternatively, the antisense nucleic acids and oligonucleotides can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e. nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). The antisense expression vector is introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews--Trends in Genetics, Vol. 1 (1)1986. In addition, ribozymes can be used to inhibit in vitro expression of thymosin .beta.15. For example, the nucleic acids of the invention can further be used to design ribozymes which are capable of cleaving a single-stranded nucleic acid encoding a .beta.15 protein, such as a thymosin .beta.15 mRNA transcript. A catalytic RNA (ribozyme) having ribonuclease activity can be designed which has specificity for an mRNA encoding thymosin .beta.15 based upon the sequence of a nucleic acid of the invention (e.g., SEQ ID NO: 1 ). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in a thymosin .beta.15-encoding mRNA. See for example Cech, et al., U.S. Pat. No. 4,987,071; Cech, et al., U.S. Pat. No. 5,116,742. Alternatively, a nucleic acid of the invention could be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and Szostak, J. W. Science 261,1411-1418 (1993). Methods for the diagnosis and prognosis of cancer using the polynucleotides and antibodies of the present invention are set forth in copending application (Docket No. 46403) Express Mail No. TB338582354US, the disclosure of which is herein incorporated by reference. All references cited above or below are herein incorporated by reference. The following Examples serve to illustrate the present invention, and are not intended to limit the invention in any manner. |
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