# Patent

## Title
Genes differentially expressed in breast cancer

## Abstract

A polynucleotide sequence as shown in SEQ ID NO:1 is associated with metastatic potential of cancer cells, especially breast cancer cells. Methods are provided for determining the risk of metastasis of a tumor, by determining whether a tissue sample from a tumor expresses a polypeptide or mRNA encoded by a polynucleotide as shown in SEQ ID NO:1. Also provided are therapeutic methods and compositions.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 10/200,026, filed Jul. 18, 2002, now allowed, which is a continuation-in-part of U.S. patent application Ser. No. 09/758,575, filed Jan. 9, 2001, now pending, and claims priority from U.S. Patent Application No. 60/175,462, filed Jan. 10, 2000, which applications are incorporated herein by reference in their entirety.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods for predicting the behavior of tumors. More particularly, the invention relates to methods in which tumor samples (primary and metastases) are examined for expression of a specified gene.
2. Description of the Related Art
Breast cancer is one of the most common malignant diseases with about 1,000,000 new cases per year worldwide. Despite use of a number of histochemical, genetic, and immunological markers, clinicians still have a difficult time predicting which tumors will metastasize to other organs. Some patients are in need of adjuvant therapy to prevent recurrence and metastasis and others are not. However, distinguishing between these subpopulations of patients is not straightforward, and course of treatment is not easily charted. There is a need in the art for new markers for distinguishing between tumors which will or have metastasized and those which are less likely to metastasize.
There is also a need in the art for markers of tumors, particularly markers that may also be found in metastatic tumors.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide markers for detecting tumors, particularly those having a tendency to metastasize. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides an isolated and purified human protein having an amino acid sequence which is at least 85% identical to an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1 or the complement thereof.
Another embodiment of the invention provides a fusion protein which comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond. The first protein segment consists of at least six contiguous amino acids selected from an amino acid sequence encoded by a nucleotide sequence SEQ ID NO:1 or the complement thereof, and the second protein segment comprises an amino acids sequence not found adjacent to the first protein segment in the native protein encoded by SEQ ID NO:1.
Yet another embodiment of the invention provides an isolated and purified polypeptide consisting of at least six contiguous amino acids of a human protein having an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
Still another embodiment of the invention provides a preparation of antibodies which specifically bind to a human protein which comprises an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
Even another embodiment of the invention provides an isolated and purified subgenomic polynucleotide comprising at least 11 contiguous nucleotides of a nucleotide sequence which is at least 95% identical to a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
Another embodiment of the invention provides an isolated and purified polynucleotide which comprises a coding sequence comprising a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
Yet another embodiment of the invention provides a method for identifying metastasis in a tissue sample. An expression product of a gene which comprises a coding sequence of SEQ ID NO:1 is measured in a non-primary tumor tissue sample. A tissue sample which expresses the product at a higher level than in a control sample is categorized as being metastatic.
Yet a further embodiment of the invention provides a method for detecting a human gene encoding SEQ ID NO:2, the method comprising obtaining in computer-readable format SEQ ID NO:1, comparing the sequence with polynucleotide sequences of a human genome, and identifying one or more human genome sequences having at least 95% sequence identity to SEQ ID NO:1 as determined by the Smith-Waterman algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1 as parameters.
The invention further provides a population of antibodies that can be used to detect breast cancer, wherein the antibodies are contacted with primary breast cancer tissue, metastatic breast cancer tissue, and/or a body fluid of a person suspected of having breast cancer, thereby detecting a protein encoded by SEQ ID NO:1.
The invention also provides a kit for use in diagnosing breast cancer, comprising at least one ligand, such as an antibody, capable of binding to a protein encoded by SEQ ID NO:1, wherein the ligand is detectably labeled.
The invention thus provides the art with a number of polynucleotides and polypeptides, which can be used as markers of metastasis. These are useful for more rationally prescribing the course of therapy for breast cancer patients.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 illustrates the polynucleotide sequence of human Out at First (SEQ ID NO:1).
FIG. 2 illustrates the amino acid sequence encoded by SEQ ID NO:1 (SEQ ID NO:2).
FIG. 3 illustrates the putative signal peptide (SEQ ID NO:3).
FIG. 4 illustrates the translation of SEQ ID NO:1 (SEQ ID NO:1, polynucleotide; SEQ ID NO:2, amino acid sequence).
FIG. 5 illustrates the expression of hsOAF relative to β-Actin in tumor cell lines and tumor tissues from SCID mice developed from the cell lines. “PT” refers to primary tumor.
FIG. 6 illustrates the growth of colonies by MDA-MB-435 cells in soft agar following treatment with antisense oligo SEQ ID NO:4 (66-2 as) or reverse control SEQ ID NO:5 (66-2rc), relative to untreated cells (WT).
FIG. 7 is an alignment of the human OAF amino acid sequence (SEQ ID NO: 2) with the Drosophila OAF amino acid sequence (SEQ ID NO: 7).
FIG. 8. FIG. 8A illustrates the expression of hsOAF protein in COS-7 and MCF-7 cell lines. FIG. 8B illustrates the expression of hsOAF protein in mammory carcinoma cell lines.
FIG. 9 illustrates the expression of hsOAF in normal human tissues.
FIG. 10. FIG. 10A illustrates the morphological changes seen in MDA-MB-435 cells following treatment with antisense oligo (SEQ ID NO:4). AS=antisense; RC=reverse control (SEQ ID NO:5); M=conditioned medium. FIG. 10 B illustrates cell invasion following treatment of MDA-MB-435 cells with AS, RC and RC+M.
FIG. 11 illustrates the predicted signal sequence of human OAF (SEQ ID NO: 9) (double underline), DNA sequence (SEQ ID NO:8).
FIG. 12. FIGS. 12A and 12 B illustrate the secretion of hsOAF by MDA-MB-435 cells treated with antisense oligo (SEQ ID NO:4) or reverse control oligo (SEQ ID NO:5).



DETAILED DESCRIPTION OF THE INVENTION
Metastasis of breast carcinomas and their proliferation at distant loci (lung and bone, mainly) is one of the more severe developments in patients with breast cancer. Metastasis is a multistep process by which tumor cells emigrate from the primary tumor, disseminate through blood and lymph vessels, and then are deposited in specific target organs where they re-colonize. Schirrmacher, V., Adv. Cancer Res. 43:1-73, 1985 and Liotta, L. A. et al., Cell 64(2):327-36 (1991). During this process the invasiveness of tumor cells is crucial since they must encounter and pass through numerous basement membranes. Liotta, L. A., Am. J. Pathol. 117(3):339-48 (1984) and Fidler, I. J., Cancer Res. 38(9):2651-60 (1978). Therefore the elucidation of the molecular causes of tumor cell invasion and metastasis is essential for the development of efficient treatment procedures for breast cancer patients. Genes expressed in breast tumor metastasis are potential targets that play critical roles during metastasis. Identification of such genes and their biological function will significantly contribute to the development of therapy and diagnosis for breast cancer.
Some important genes involved in breast tumor metastasis have been discovered. Loss of estrogen receptor and presence of vimentin have been associated with human breast tumor invasiveness and poor prognosis, and also correlate with the invasiveness and metastatic potential of human breast cancer cell lines. Aamdal S., et al., Cancer 53(11):2525-9 (1984); Clark, G. M., et al., Semin Oncol., 2 Suppl 1:20-5 (1988); Raymond, W. A. et al., J. Pathol. 157(4):299-306 (1989); Raymond, W. A., et al., J. Pathol. 158(2):107-14 (1989); and Thompson, E. W. et al., J. Cell Physiol. 150(3):534-44 (1992). E-cadherin underexpression has been implicated in mammary tumor invasiveness. Vleminckx, K., et al., Cell 66(1):107-19 (1991) and Oka, H., et al., Cancer Res. 53(7):1696-701 (1993). Maspin, a protease inhibitor expressed in normal mammary epithelial cells but not in most breast carcinoma cell lines, was able to suppress MDA-MB-435 cells' ability to induce tumors and metastasize in mice and to invade basement membrane in vitro. Loss of maspin expression occurred most frequently in advanced cancers. Zou, Z., et al., Science 263(5146):526-9 (1994) and Seftor, R. E., et al., Cancer Res. 58(24):5681-5 (1998).
Overexpression of TIMP-4 (tissue inhibitor of metalloproteinases-4) or CLCA2 (Ca2+-activated chloride channel-2) in MDA-MB-435 cells by transfection inhibited the tumorigenicity, invasiveness and metastasis ability of the cells. Wang, M., et al., Oncogene 14(23):2767-74 (1997) and Gruber, A. D., et al., Cancer Res. 59(21):5488-91 (1999). Overexpression of the growth factor receptors IGF-IR and p185ErbB-2 has been found to be involved in breast cancer metastasis. Surmacz, E., et al., Breast Cancer Res. Treat 47(3):255-67 (1998); Dunn, S. E., et al., Cancer Res. 58(15):3353-61 (1998); Tan, M., et al., Cancer Res. 57(6):1199-205 (1997); Dhingra, K., et al., Semin Oncol. 23(4):436-45 (1996); and Revillion, F., et al., Eur. J. Cancer 34(6):791-808 (1998).
The aspartyl protease cathepsin D has been reported to be a marker of poor prognosis for breast cancer patients and there is a significant correlation between high cathepsin D concentration in the cytosol of primary breast cancer and development of metastasis, though no correlation was found between cathepsin D secretion and invasion ability of breast cancer cell lines. Rochefort, H., Breast Cancer Res Treat 16(1):3-13 (1990); Johnson, M. D., et al., Cancer Res. 53(4):873-7 (1993); and Rochefort, H., et al., Clin Chim Acta. 291(2):157-70 (2000). Osteopontin, a secreted integrin-binding glycoprotein that is thought to be involved in bone resorption and bone formation, can induce migration and invasion of mammary carcinoma cells. Osteopontin levels (tumor cell or plasma levels) have been associated with enhanced malignancy of breast cancer. Denhardt, D. T., et al., FASEB J. 7(15): 1475-82 (1993); Denhardt, D. T., et al., J. Cell Biochem Suppl., 30-31:92-102 (1998); Tuck, A. B., et al., J. Cell Biochem. 78(3):465-75 (2000); Tuck, A. B., et al., Oncogene 18(29):4237-46 (1999); and Singhal, H., et al., Clin Cancer Res. 3(4):605-11 (1997).
The invention relates to the cloning of a novel gene first identified as being expressed in highly metastatic human breast cancer cell lines. Antibodies to the protein were raised and immunohistochemical staining of breast tumor samples was performed. The protein was strongly expressed in 44/45 primary breast tumors, and in 26/26 metastasis. Thus, the protein is a marker for primary and metastatic breast cancer. It may also play a role particular to tumors with a tendency to metastasize. Because of its expression in primary and metastatic breast cancer, the protein is useful in detecting such cancer in body fluids including blood, which is consistent with the secretory nature of the protein.
The gene encodes a secreted protein and its protein secretion has been confirmed to be much greater in highly metastatic human breast cancer cell lines than in low metastatic/nonmetastatic cell lines. Knockout of the secretion of this protein of the aggressive MDA-MB-435 cell line by antisense oligo technology resulted in significant morphological alteration along with reduced invasiveness and proliferation rate of the cells. The gene is named hsOAF based on its homology with the Drosophila gene OAF (out at first). Bergstrom, D. E., et al., Genetics 139(3):1331-46 (1995) and Merli, C., et al., Genes Dev. 10(10):1260-70 (1996).
This information can be utilized to make diagnostic reagents specific for the expression products of the expressed gene. It can also be used in diagnostic and prognostic methods which will help clinicians in planning appropriate treatment regimes for cancers, especially of the breast.
The polynucleotide is shown in FIG. 1 (SEQ ID NO:1), and the predicted open reading frame (ORF) encodes a polypeptide shown in FIG. 2 (SEQ ID NO:2). The first 30 amino acid residues (SEQ ID NO:3) comprise a putative signal peptide, with a predicted protease cleavage site indicated by “*”: APLLG*TGAPA (SEQ ID NO: 10) (between amino acids at positions 25 and 26 of SEQ ID NO:3).
The polynucleotide sequence of the invention shares some homology with a Drosophila gene known as “Out at First” (oaf). Transcription of oaf results in three classes of alternatively polyadenylated RNAs, the expression of which is developmentally regulated. All oaf transcripts contain two adjacent ORFs separated by a single UGA stop codon. Suppression of the UGA codon during translation could lead to the production of different proteins from the same RNA molecule. During oogenesis, oaf RNA is expressed in nurse cells of all ages, and is maternally contributed to the egg.
During embryonic development, zygotic transcription of the oaf gene occurs in small clusters of cells in most or all segments at the time of germband extension and later in a segmentally repeated pattern in the developing central nervous system. The oaf gene is also expressed in the embryonic, larval and adult gonads of both sexes. (Bergstrom, D. E. et al., Genetics 139:1331-1346, 1995.)
The polynucleotide of the invention was differentially expressed in seven pairs of high metastatic versus non-metastatic or low metastatic breast cancer cell lines. The cell lines used are MDA-MB-361 (derived from human breast adenocarcinoma), MDA-MB-231 (human breast cancer cells metastatic to bone and/or lung); MDA-MB-468 (derived from human estrogen receptor-negative breast cancer cells); MCF-7 (non-metastatic human breast cancer cells); ZR-75-1 (derived from estrogen receptor-positive human breast carcinomas, Engle et al., Cancer Res. 38:3352-64 (1978)); and MDA-MB-435 (derived from estrogen receptor-negative human breast carcinoma cells, Rishi et al., Cancer Res. 56:5246-5252 (1996)).
The expression profile is as follows:










TABLE 1








Ratio of



Cell Line Pair
Expression









MDA-MB-361/MDA-MB-231
0.11



MDA-MB-468/MDA-MB-231
0.44



MCF-7/MDA-MB-231
0.17



ZR-75-1/MDA-MB-231
0.12



MDA-MB-361/MDA-MB-435
0.06



MDA-MB-468/MDA-MB-435
0.36



MCF-7/MDA-MB-435
0.03










The upregulation of the mRNA expression was confirmed by Northern blot analysis using total RNA from the cell lines (FIG. 5).
The cell lines in which expression of the polynucleotide of the invention was compared represent human breast cancers of varying metastatic potential. Cell line ZR-75-1 cultures were derived from malignant ascitic effusion of a breast cancer patient. The cell lines grown in vitro closely resembled the morphology seen in biopsies or cell preparations from the donors of the original cells. ZR-75-1 cells are specifically stimulated by estrogen, and have been used as a model system for studying estrogen responsiveness. Engel, L. W. et al., Cancer Res. 38:3352-3364, 1978.
Cell line MDA-MB-435 is an estrogen receptor-negative cell line that has been studied as a model for human breast cancer, for example, for studying the mechanism of action of growth inhibition in the presence of retinoic acid. Rishi, A. K. et al., Cancer Res. 56:5246-5252, 1996. Growth inhibition by retinoids has also been studied in MCF-7 cells and MDA MB 468 cells. Tin-U, C. K. et al., Am. Soc. Clin. One. Proceedings, Vol. 17, 2125, 1998.
Cell line MDA-MB-361 was derived from a human breast adenocarcinoma, specifically from a malignant site. ATCC Number HTB-27. Differential expression of human Wnt genes has been studied in this cell line. Huguet, E. L. et al., Cancer Res. 54:2615-2621, 1994.
Once metastasis occurs, mammary primary tumor cells invade basement membranes and spread to other organs of the body and the survival chance of patients with breast cancer is reduced. It is critical to identify genes participating in breast cancer invasion and metastasis on behalf of clinical diagnosis and therapy. Such genes are potential markers for diagnosis or candidate targets for therapeutic drug development. For instance, presence of vimentin in human breast tumor has been associated with lack of estrogen receptor and tumor invasiveness as a marker of poor prognosis. Raymond, W. A. et al., J. Pathol. 157(4):299-306 (1989); Raymond, W. A., et al., J. Pathol. 158(2):107-14 (1989); and Thompson, E. W. et al., J. Cell Physiol. 150(3):534-44 (1992). Increased activities of matrix metalloproteinases are related with the metastatic phenotype of carcinomas, especially breast cancer. Basset, P., et al., Nature 348(6303):699-704 (1990) and Basset, P., et al., Cancer 74(3 Suppl):1045-9 (1994). Osteopontin, a secreted integrin-binding glycoprotein, is able to induce increased invasiveness of human mammary epithelial cells and has been associated with enhanced malignancy in breast cancer. Tuck, A. B., et al., J. Cell Biochem. 78(3):465-75 (2000); Tuck, A. B., et al., Oncogene 18(29):4237-46 (1999); and Singhal, H., et al., Clin Cancer Res. 3(4):605-11 (1997).
The invention relates to identification of a novel secreted protein (hsOAF) expressed in primary breast cancer and related metastasis. The human breast cancer cell lines used in elucidating the role of the protein are divided into three groups according to their metastatic abilities: highly metastatic, low metastatic, and nonmetastatic. Taking advantage of different metastatic potentials among these cell line groups and utilizing the advanced microarray technology, genes were identified which are differentially expressed between highly metastatic human breast cancer cell lines and low metastatic/nonmetastatic ones. hsOAF gene is the focus of this invention as it encodes a novel secreted protein, and is expressed in breast cancer tissue and metastatic breast cancer tissue.
To investigate the potential role of secreted hsOAF protein in breast cancer metastasis, antisense oligo technology was used to specifically knock out hsOAF expression. Antisense oligo technology is an efficient, fast way to dramatically reduce gene expression for gene functional studies. Stein, C. A., et al., Science 261(5124):1004-12 (1993); Defacque, H. et al., J. Cell Physiol. 178(1):109-19 (1999). Knockout of hsOAF protein secretion of highly metastatic MDA-MB-435 cells resulted in cell shape change, reduced cell invasiveness and slower cell proliferation. Treatment of cells with the conditioned medium (culture medium of normal MDA-MB-435 cells) led to recovery of all those phenotypic alterations caused by the knockout of hsOAF protein secretion to some degree. Although the inventors are not bound by a specific mechanism, the secreted hsOAF protein is believed to be involved in the invasiveness and proliferation of MDA-MB-435 cells. However, knockout of hsOAF protein secretion of another highly metastatic cell line, MDA-MB-231, by antisense oligo technology did not cause any significant cellular changes. MDA-MB-435 and MDA-MB-231 are quite different metastatic cell lines and MDA-MB-435 shows much stronger hsOAF protein secretion than does MDA-MB-231.
hsOAF gene is located at chromosome 11q23 region where loss of heterozygosity occurs frequently in human breast tumors. Negrini, M., et al., Cancer Res 55(14):3003-7 (1995) and Tomlinson, I. P., et al., J. Clin. Pathol. 48(5):424-8 (1995). Loss of heterozygosity at 11q23 in primary human breast tumors has been reported to be associated with poor survival after metastasis. Winqvist, R., et al., Cancer Res. 55(12):2660-4 (1995). 11q23 also contains loci such as ATM (Ataxia-telangiectasia, mutated), and MLL (which is frequently disrupted by chromosomal rearrangement in acute leukemia). Rasio, D., et al., Cancer Res. 55(24):6053-7 (1995) and Rubnitz, J. E., et al., Leukemia 10(1):74-82 (1996). The relationship between mutation at chromosome 11q23 and hsOAF gene expression in breast cancer metastasis remains unclear.
Secreted hsOAF protein may be a suitable target for drug development against breast cancer and a good diagnostic marker for the malignancy of breast tumor. SEQ ID NO:1 and polynucleotides comprising this sequence are therefore useful as hsOAFs. Reference to hsOAF nucleotide or amino acid sequences includes variants which have similar expression patterns in high metastatic relative to non-metastatic or low metastatic cells. HsOAF polypeptides can differ in length from full-length hsOAF proteins and contain at least 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 265, 270 or 271 or more contiguous amino acids of a hsOAF protein. Exemplary polynucleotides include those encoding amino acids from about 1 to about 273; from 1 to 273; from about 2 to about 273; from 2 to 273; from about 26 to about 273; and from 26 to 273 of SEQ ID NO:2.
Variants of marker proteins and polypeptides can also occur. HsOAF protein or polypeptide variants can be naturally or non-naturally occurring. Naturally occurring hsOAF protein or polypeptide variants are found in humans or other species and comprise amino acid sequences which are substantially identical to a protein encoded by a gene corresponding to the nucleotide sequence shown in SEQ ID NO:1 or its complement. Non-naturally occurring hsOAF protein or polypeptide variants which retain substantially the same differential expression patterns in high metastatic relative to low-metastatic or non-metastatic breast cancer cells as naturally occurring hsOAF protein or polypeptide variants are also included here. Preferably, naturally or non-naturally occurring hsOAF protein or polypeptide variants have amino acid sequences which are at least 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to amino acid sequences encoded by the nucleotide sequence shown in SEQ ID NO:1. More preferably, the molecules are at least 96%, 97%, 98% or 99% identical. Percent sequence identity between a wild-type protein or polypeptide and a variant is determined by aligning the wild-type protein or polypeptide with the variant to obtain the greatest number of amino acid matches, as is known in the art, counting the number of amino acid matches between the wild-type and the variant, and dividing the total number of matches by the total number of amino acid residues of the wild-type sequence.
Preferably, amino acid changes in hsOAF protein or polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
It is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological properties of the resulting hsOAF protein or polypeptide variant. Properties and functions of hsOAF protein or polypeptide variants are of the same type as a hsOAF protein or polypeptide comprising amino acid sequences encoded by the nucleotide sequence shown in SEQ ID NO:1, although the properties and functions of variants can differ in degree. Whether an amino acid change results in a hsOAF protein or polypeptide variant with the appropriate differential expression pattern can readily be determined. For example, nucleotide probes can be selected from the marker gene sequences disclosed herein and used to detect marker gene mRNA in Northern blots or in tissue sections, as is known in the art. Alternatively, antibodies which specifically bind to protein products of hsOAF genes can be used to detect expression of hsOAF proteins or variants thereof.
HsOAF variants include glycosylated forms, aggregative conjugates with other molecules, and covalent conjugates with unrelated chemical moieties. HsOAF variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions which do not affect the differential expression of hsOAF genes are also hsOAF variants. Covalent variants can be prepared by linking functionalities to groups which are found in the amino acid chain or at the N- or C-terminal residue, as is known in the art.
It will be recognized in the art that some amino acid sequence of the polypeptide of the invention can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there are critical areas on the protein which determine activity. In general, it is possible to replace residues that form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein. The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors. Thus, the polypeptides of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
The invention further includes variations of the disclosed polypeptide which show comparable expression patterns or which include antigenic regions. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).
Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the disclosed protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev Therapeutic Drug Carrier Systems 10:307-377 (1993)).
Amino acids in the polypeptides of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein. Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.
Full-length hsOAF proteins can be extracted, using standard biochemical methods, from hsOAF protein-producing human cells, such as metastatic breast cancer cells. An isolated and purified hsOAF protein or polypeptide is separated from other compounds which normally associate with a hsOAF protein or polypeptide in a cell, such as certain proteins, carbohydrates, lipids, or subcellular organelles. A preparation of isolated and purified hsOAF proteins or polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure.
A human gene encoding SEQ ID NO:2 can be identified and isolated using methods know in the art. According to one method, SEQ ID NO:1 is prepared in a computer-readable format. The sequence is compared with polynucleotide sequences of a human genome, and one or more human genome sequences having at least 95% sequence identity to SEQ ID NO:1 are identified, for example by using the Smith-Waterman algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1 as parameters. Probes based on the regions of homology between SEQ ID NO:1 and the human genome sequences are prepared and used to isolate polynucleotides from human genomic DNA, using methods known in the art. As of the filing date a human polynucleotide corresponding to the full polynucleotide of SEQ ID NO:1 was not identified in the public databases. Thus, the invention includes human genomic DNA comprising the coding region of SEQ ID NO:1 and any untranslated regions which do not share homology with SEQ ID NO:1 but which are contiguous with homologous regions. Such genomic DNA includes but is not limited to introns, promoters, and other regulatory regions functionally associated with a human gene having a region encoding SEQ ID NO:2.
hsOAF proteins and polypeptides can also be produced by recombinant DNA methods or by synthetic chemical methods. For production of recombinant hsOAF proteins or polypeptides, coding sequences selected from the nucleotide sequences shown in SEQ ID NO:1, or variants of those sequences which encode hsOAF proteins, can be expressed in known prokaryotic or eukaryotic expression systems (see below). Bacterial, yeast, insect, or mammalian expression systems can be used, as is known in the art.
Alternatively, synthetic chemical methods, such as solid phase peptide synthesis, can be used to synthesize a hsOAF protein or polypeptide. General means for the production of peptides, analogs or derivatives are outlined in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS—A SURVEY OF RECENT DEVELOPMENTS, Weinstein, B. ed., Marcell Dekker, Inc., publ., New York (1983). Moreover, substitution of D-amino acids for the normal L-stereoisomer can be carried out to increase the half-life of the molecule. hsOAF variants can be similarly produced.
Non-naturally occurring fusion proteins comprising at least 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 265, 270 or 271 or more contiguous hsOAF amino acids can also be constructed. Human hsOAF fusion proteins are useful for generating antibodies against hsOAF amino acid sequences and for use in various assay systems. For example, hsOAF fusion proteins can be used to identify proteins which interact with hsOAF proteins and influence their functions. Physical methods, such as protein affinity chromatography, or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can also be used for this purpose. Such methods are well known in the art and can also be used as drug screens.
A hsOAF fusion protein comprises two protein segments fused together by means of a peptide bond. The first protein segment comprises at least 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 265, 270 or 271 or more contiguous amino acids of a hsOAF protein. The amino acids can be selected from the amino acid sequences encoded by the nucleotide sequence shown in SEQ ID NO:1 or from variants of the sequence, such as those described above. The first protein segment can also comprise a full-length hsOAF protein.
In one preferred embodiment, the first protein segment comprises the polypeptide shown in SEQ ID NO:2. In a variation of this embodiment, the first protein segment consists of amino acids 31-287 of SEQ ID NO:2. This fusion protein lacks the signal peptide of SEQ ID NO:2 and would be suitable for retention of the expressed fusion protein inside the cell. In some embodiments the first protein segment comprises amino acids 20-30 of SEQ ID NO:2. In some embodiments the first protein segment comprises amino acids 26-287 of SEQ ID NO:2.
The second protein segment can be a full-length protein or a protein fragment or polypeptide not found adjacent to the first protein segment in the native protein encoded by SEQ ID NO:1. The fusion protein can be labeled with a detectable marker, as is known in the art, such as a radioactive, fluorescent, chemiluminescent, or biotinylated marker. The second protein segment can be an enzyme which will generate a detectable product, such as β-galactosidase. The first protein segment can be N-terminal or C-terminal, as is convenient.
Techniques for making fusion proteins, either recombinantly or by covalently linking two protein segments, are also well known. Recombinant DNA methods can be used to prepare hsOAF fusion proteins, for example, by making a DNA construct which comprises coding sequences of SEQ ID NO:1 in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as described below. The open reading frame of SEQ ID NO:1 is shown in FIG. 4.
Isolated and purified hsOAF proteins, polypeptides, variants, or fusion proteins can be used as immunogens, to obtain preparations of antibodies which specifically bind to a hsOAF protein. The antibodies can be used, inter alia, to detect wild-type hsOAF proteins in human tissue and fractions thereof. The antibodies can also be used to detect the presence of mutations in hsOAF genes which result in under- or over-expression of a hsOAF protein or in expression of a hsOAF protein with altered size or electrophoretic mobility.
Preparations of polyclonal or monoclonal antibodies can be made using standard methods. Single-chain antibodies can also be prepared. A preferred immunogen is a polypeptide comprising SEQ ID NO:2. Single-chain antibodies which specifically bind to hsOAF proteins, polypeptides, variants, or fusion proteins can be isolated, for example, from single-chain immunoglobulin display libraries, as is known in the art. The library is “panned” against hsOAF protein amino acid sequences of SEQ ID NO:2, and a number of single chain antibodies which bind with high-affinity to different epitopes of hsOAF proteins can be isolated. Hayashi et al., 1995, Gene 160:129-30. Single-chain antibodies can also be constructed using a DNA amplification method, such as the polymerase chain reaction (PCR), using hybridoma cDNA as a template. Thirion et al., 1996, Eur. J. Cancer Prev. 5:507-11.
HsOAF-specific antibodies specifically bind to epitopes present in a full-length hsOAF protein having an amino acid sequence encoded by a nucleotide sequence shown in SEQ ID NO:1, to hsOAF polypeptides, or to hsOAF variants, either alone or as part of a fusion protein. Preferably, hsOAF epitopes are not present in other human proteins. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
Antibodies which specifically bind to hsOAF proteins, polypeptides, fusion proteins, or variants provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays. Preferably, antibodies which specifically bind to hsOAF epitopes do not detect other proteins in immunochemical assays and can immunoprecipitate a hsOAF protein, polypeptide, fusion protein, or variant from solution. In a preferred method, hsOAF protein expression is detected using an immunohistochemical staining kit, such as that of BioGenex Laboratories, Inc. (San Ramon, Calif.).
Subgenomic polynucleotides contain less than a whole chromosome. Preferably, the polynucleotides are intron-free. In a preferred embodiment, the polynucleotide molecules comprise a contiguous sequence of 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300 or 2350 nucleotides from SEQ ID NO:1 or the complements thereof. The complement of a nucleotide sequence shown in SEQ ID NO:1 is a contiguous nucleotide sequence which forms Watson-Crick base pairs with a contiguous nucleotide sequence shown in SEQ ID NO:1.
Degenerate nucleotide sequences encoding amino acid sequences of hsOAF protein or variants, as well as homologous nucleotide sequences which comprise a polynucleotide at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the coding region of the nucleotide sequence shown in SEQ ID NO:1, are also hsOAF subgenomic polynucleotides. Typically, homologous hsOAF subgenomic polynucleotide sequences can be confirmed by hybridization under stringent conditions, as is known in the art. Percent sequence identity between wild-type and homologous variant sequences is determined by aligning the wild-type polynucleotide with the variant to obtain the greatest number of nucleotide matches, as is known in the art, counting the number of nucleotide matches between the wild-type and the variant, and dividing the total number of matches by the total number of nucleotides of the wild-type sequence. A preferred algorithm for calculating percent identity is the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty of 12, and gap extension penalty of 1.
A hsOAF subgenomic polynucleotide comprising hsOAF protein coding sequences can be used in an expression construct. Preferably, the hsOAF subgenomic polynucleotide is inserted into an expression plasmid (for example, the Ecdyson system, pIND, In vitro Gene). HsOAF subgenomic polynucleotides can be propagated in vectors and cell lines using techniques well known in the art. HsOAF subgenomic polynucleotides can be on linear or circular molecules. They can be on autonomously replicating molecules or on molecules without replication sequences. They can be regulated by their own or by other regulatory sequences, as are known in the art.
A host cell comprising a hsOAF expression construct can then be used to express all or a portion of a hsOAF protein. Host cells comprising hsOAF expression constructs can be prokaryotic or eukaryotic. A variety of host cells are available for use in bacterial, yeast, insect, and human expression systems and can be used to express or to propagate hsOAF expression constructs (see below). Expression constructs can be introduced into host cells using any technique known in the art. These techniques include transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, and calcium phosphate-mediated transfection.
A hsOAF expression construct comprises a promoter which is functional in a chosen host cell. The skilled artisan can readily select an appropriate promoter from the large number of cell type-specific promoters known and used in the art. The expression construct can also contain a transcription terminator which is functional in the host cell. The expression construct comprises a polynucleotide segment which encodes all or a portion of the hsOAF protein, variant, fusion protein, antibody, or ribozyme. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter. The expression construct can be linear or circular and can contain sequences, if desired, for autonomous replication.
Bacterial systems for expressing hsOAF expression constructs include those described in Chang et al., Nature (1978) 275:615, Goeddel et al., Nature (1979) 281:544, Goeddel et al., Nucleic Acids Res. (1980) 8:4057, EP 36,776, U.S. Pat. No. 4,551,433, deBoer et al., Proc. Nat'l Acad. Sci. USA (1983) 80:21-25, and Siebenlist et al., Cell (1980) 20:269.
Expression systems in yeast include those described in Hinnen et al., Proc. Nat'l Acad. Sci. USA (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen. Microbiol. (1986) 132:3459, Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302) Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt et al., J. Bacteriol. (1983) 154:737, Van den Berg et al., Bio/Technology (1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg et al., Mol. Cell. Biol. (1985) 5:3376, U.S. Pat. No. 4,837,148, U.S. Pat. No. 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr Genet. (1985) 10:380, Gaillardin et al., Curr. Genet (1985) 10:49, Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289; Tilburn et al., Gene (1983) 26:205-221, Yelton et al., Proc. Nat'l Acad. Sci. USA (1984) 81:1470-1474, Kelly and Hynes, EMBO J. (1985) 4:475479; EP 244,234, and WO 91/00357.
Expression of hsOAF expression constructs in insects can be carried out as described in U.S. Pat. No. 4,745,051, Friesen et al. (1986) “The Regulation of Baculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476, and Vlak et al., J. Gen. Virol. (1988) 69:765-776, Miller et al., Ann. Rev. Microbiol. (1988) 42:177, Carbonell et al., Gene (1988) 73:409, Maeda et al., Nature (1985) 315:592-594, Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Nat'l Acad. Sci. USA (1985) 82:8404, Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al., Bio/Technology (1988) 6:47-55, Miller et al., in GENETIC ENGINEERING (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al., Nature, (1985) 315:592-594.
Mammalian expression of hsOAF expression constructs can be achieved as described in Dijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Nat'l Acad. Sci. USA (1982b) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S. Pat. No. 4,399,216. Other features of mammalian expression of hsOAF expression constructs can be facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.
Subgenomic polynucleotides of the invention can also be used in gene delivery vehicles, for the purpose of delivering a hsOAF mRNA or oligonucleotide (either with the sequence of native hsOAF mRNA or its complement), full-length hsOAF protein, hsOAF fusion protein, hsOAF polypeptide, or hsOAF-specific ribozyme or single-chain antibody, into a cell, preferably a eukaryotic cell. According to the present invention, a gene delivery vehicle can be, for example, naked plasmid DNA, a viral expression vector comprising a hsOAF subgenomic polynucleotide, or a hsOAF subgenomic polynucleotide in conjunction with a liposome or a condensing agent.
The invention provides a method of detecting hsOAF gene expression in a biological sample. Detection of hsOAF gene expression is useful, for example, for identifying metastases or for determining metastatic potential in a tissue sample, preferably a tumor. Appropriate treatment regimens can then be designed for patients who are at risk for developing metastatic cancers in other organs of the body.
The body sample can be, for example, a solid tissue or a fluid sample. The native polypeptide encoded by SEQ ID NO:1 is a putative secreted protein, and is detected in body fluids including blood and lymphatic fluid, particularly those draining from tumor sites in the body. Protein or nucleic acid expression products can be detected in the body sample. In one embodiment, the body sample is assayed for the presence of a hsOAF protein. A hsOAF protein comprises a sequence encoded by a nucleotide sequence shown in SEQ ID NO:1 or its complement and can be detected using the hsOAF protein-specific antibodies of the present invention. The antibodies can be labeled, for example, with a radioactive, fluorescent, biotinylated, or enzymatic tag and detected directly, or can be detected using indirect immunochemical methods, using a labeled secondary antibody. The presence of the hsOAF proteins can be assayed, for example, in tissue sections by immunocytochemistry, or in lysates, using Western blotting, as is known in the art.
In another embodiment, the body sample is assayed for the presence of marker protein mRNA. A sample can be contacted with a nucleic acid hybridization probe capable of hybridizing with the mRNA corresponding the selected polypeptide. Still further, the sample can be subjected to a Northern blotting technique to detect mRNA, indicating expression of the polypeptide. For those techniques in which mRNA is detected, the sample can be subjected to a nucleic acid amplification process whereby the mRNA molecule or a selected part thereof is amplified using appropriate nucleotide primers. Other RNA detection techniques can also be used, including, but not limited to, in situ hybridization.
Marker protein-specific probes can be generated using the cDNA sequence disclosed in SEQ ID NO:1. The probes are preferably at least 15 to 50 nucleotides in length, although they can be at least 8, 10, 11, 12, 20, 25, 30, 35, 40, 45, 60, 75, or 100 or more nucleotides in length. A preferable region for selecting probes is within nucleotide positions 446-1173 of SEQ ID NO:1. The probes can be synthesized chemically or can be generated from longer polynucleotides using restriction enzymes. The probes can be labeled, for example, with a radioactive, biotinylated, or fluorescent tag.
Optionally, the level of a particular hsOAF expression product in a body sample can be quantitated. Quantitation can be accomplished, for example, by comparing the level of expression product detected in the body sample with the amounts of product present in a standard curve. A comparison can be made visually or using a technique such as densitometry, with or without computerized assistance. For use as controls, body samples can be isolated from other humans, other non-cancerous organs of the patient being tested, or non-metastatic breast cancer from the patient being tested. As indicated by the results herein, expression of SEQ ID NO:1 in low-metastatic or non-metastatic breast cancer cells is between 3% and 44% of the expression levels in highly-metastatic breast cancer cells. If expression in a test sample is at least 2-fold greater than in a suitable control sample, this is indicative of metastatic cells.
Polynucleotides encoding hsOAF-specific reagents of the invention, such as antibodies and nucleotide probes, can be supplied in a kit for detecting marker gene expression products in a biological sample. The kit can also contain buffers or labeling components, as well as instructions for using the reagents to detect the marker expression products in the biological sample.
Expression of a hsOAF gene can be altered using an antisense oligonucleotide sequence. The antisense sequence is complementary to at least a portion of the coding sequence (nucleotides 365-1173) of a hsOAF gene having a nucleotide sequence shown in SEQ ID NO:1. Preferably, the antisense oligonucleotide sequence is at least six nucleotides in length, but can be at least about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Longer sequences can also be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into cells whose division is to be decreased. Such cells include highly-metastatic breast cancer cells.
Antisense oligonucleotides can comprise deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5′ end of one nucleotide with the 3′ end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux, 1994, Meth. Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev. 90:543-583.
Antibodies of the invention which specifically bind to a hsOAF protein can also be used to alter hsOAF gene expression. By antibodies is meant antibodies and parts or derivatives thereof, such as single chain antibodies, that retain specific binding for the protein. Specific antibodies bind to hsOAF proteins and prevent the proteins from functioning in the cell. Polynucleotides encoding specific antibodies of the invention can be introduced into cells, as described above.
Marker proteins of the present invention can be used to screen for drugs which have a therapeutic anti-metastatic effect. The effect of a test compound on hsOAF protein synthesis can also be used to identify test compounds which modulate metastasis. Test compounds which can be screened include any substances, whether natural products or synthetic, which can be administered to the subject. Libraries or mixtures of compounds can be tested. The compounds or substances can be those for which a pharmaceutical effect is previously known or unknown.
Synthesis of hsOAF proteins can be measured by any means for measuring protein synthesis known in the art, such as incorporation of labeled amino acids into proteins and detection of labeled hsOAF proteins in a polyacrylamide gel. The amount of hsOAF proteins can be detected, for example, using hsOAF protein-specific antibodies of the invention in Western blots. The amount of the hsOAF proteins synthesized in the presence or absence of a test compound can be determined by any means known in the art, such as comparison of the amount of hsOAF protein synthesized with the amount of the hsOAF proteins present in a standard curve.
The effect of a test compound on hsOAF protein synthesis can also be measured by Northern blot analysis, by measuring the amount of hsOAF protein mRNA expression in response to the test compound using hsOAF protein specific nucleotide probes of the invention, as is known in the art.
Typically, a biological sample is contacted with a range of concentrations of the test compound, such as 1.0 nM, 5.0 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 mM, 10 mM, 50 mM, and 100 mM. Preferably, the test compound decreases expression of a hsOAF protein by 60%, 75%, or 80%. More preferably, a decrease of 85%, 90%, 95%, or 98% is achieved.
The invention provides compositions for decreasing expression of hsOAF protein. These compositions comprise polynucleotides encoding all or at least a portion of a hsOAF protein gene expression product. Preferably, the therapeutic composition contains an expression construct comprising a promoter and a polynucleotide segment encoding at least a portion of the hsOAF protein which is effective to decrease metastatic potential. Portions of hsOAF genes or proteins which are effective to decrease metastatic potential of a cell can be determined, for example, by introducing portions of hsOAF genes or polypeptides into metastatic cell lines, such as MDA-MB-231, MDA-MB-435, Km12C, or Km12L4, and assaying the division rate of the cells or the ability of the cells to form metastases when implanted in vivo, as is known in the art. Non-metastatic cell lines can be used to assay the ability of a portion of a hsOAF protein to increase expression of a hsOAF gene.
Typically, a therapeutic hsOAF composition is prepared as an injectable, either as a liquid solution or suspension; however, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. A hsOAF composition can also be formulated into an enteric coated tablet or gel capsule according to known methods in the art, such as those described in U.S. Pat. No. 4,853,230, EP 225,189, AU 9,224,296, and AU 9,230,801.
Administration of the hsOAF therapeutic agents of the invention can include local or systemic administration, including injection, oral administration, particle gun, or catheterized administration, and topical administration. Various methods can be used to administer a therapeutic hsOAF composition directly to a specific site in the body.
For treatment of tumors, including metastatic lesions, for example, a therapeutic hsOAF composition can be injected several times in several different locations within the body of tumor. Alternatively, arteries which serve a tumor can be identified, and a therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor.
A tumor which has a necrotic center can be aspirated and the composition injected directly into the now empty center of the tumor. A therapeutic hsOAF composition can be directly administered to the surface of a tumor, for example, by topical application of the composition. X-ray imaging can be used to assist in certain of the above delivery methods. Combination therapeutic agents, including a hsOAF proteins or polypeptide or a hsOAF subgenomic polynucleotide and other therapeutic agents, can be administered simultaneously or sequentially.
Alternatively, a hsOAF therapeutic composition can be introduced into human cells ex vivo, and the cells then replaced into the human. Cells can be removed from a variety of locations including, for example, from a selected tumor or from an affected organ. In addition, a therapeutic composition can be inserted into non-affected cells, for example, dermal fibroblasts or peripheral blood leukocytes. If desired, particular fractions of cells such as a T cell subset or stem cells can also be specifically removed from the blood (see, for example, PCT WO 91/16116). The removed cells can then be contacted with a hsOAF therapeutic composition utilizing any of the above-described techniques, followed by the return of the cells to the human, preferably to or within the vicinity of a tumor or other site to be treated. The methods described above can additionally comprise the steps of depleting fibroblasts or other non-contaminating tumor cells subsequent to removing tumor cells from a human, and/or the step of inactivating the cells, for example, by irradiation.
Both the dose of a therapeutic composition and the means of administration can be determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. Preferably, a therapeutic composition of the invention decreases expression of the hsOAF genes by 50%, 60%, 70%, or 80%. Most preferably, expression of the hsOAF genes is decreased by 90%, 95%, 99%, or 100%. The effectiveness of the mechanism chosen to alter expression of the hsOAF genes can be assessed using methods well known in the art, such as hybridization of nucleotide probes to mRNA of the hsOAF genes, quantitative RT-PCR, or detection of the hsOAF proteins using specific antibodies of the invention.
If the composition contains the hsOAF proteins, polypeptide, or antibody, effective dosages of the composition are in the range of about 5 μg to about 50 μg/kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg.
Therapeutic compositions containing hsOAF subgenomic polynucleotides, such as antisense oligonucleotides, can be administered in a range of about 100 ng to about 200 mg of DNA for local administration. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the hsOAF subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of hsOAF subgenomic polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, can be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.
Expression of an endogenous hsOAF gene in a cell can also be altered by introducing in frame with the endogenous hsOAF gene a DNA construct comprising a hsOAF protein targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site by homologous recombination, such that a homologously recombinant cell comprising the DNA construct is formed. The new transcription unit can be used to turn the hsOAF gene on or off as desired. This method of affecting endogenous gene expression is taught in U.S. Pat. No. 5,641,670, which is incorporated herein by reference.
A hsOAF subgenomic polynucleotide can also be delivered to subjects for the purpose of screening test compounds for those which are useful for enhancing transfer of hsOAF subgenomic polynucleotides to the cell or for enhancing subsequent biological effects of hsOAF subgenomic polynucleotides within the cell. Such biological effects include hybridization to complementary hsOAF mRNA and inhibition of its translation, expression of a hsOAF subgenomic polynucleotide to form hsOAF mRNA and/or hsOAF protein, and replication and integration of a hsOAF subgenomic polynucleotide. The subject can be a cell culture or an animal, preferably a mammal, more preferably a human.
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
EXAMPLES
Materials and Methods
Human tissues. Normal human tissues were obtained as Human Total RNA Panels, Clontech. Tissue samples were also obtained from breast cancer patients, and included primary breast tumors and metastases.
Cell culture. MDA-MB-435, MDA-MB-231, ALAB, MDA-MB468, MDA-MB-361, ZR-75-1, MCF-7, MDA-MB-453 and SK-BR-3 human breast cancer cell lines (obtained from Chiron Master Culture Collection, Chiron Corporation) were grown at 37° C. in 5% CO2 in DMEM+HAM'S F-12 (1:1) (Bio*Whittaker, Walkersville, Md.) containing 2 mM L-Glutamine, 1 mM Sodium Pyruvate, 100 U/ml Penicillin and 100 μg/ml Streptomycin (Bio*Whittaker, Walkersville, Md.), 1× Vitamin Solution, 1× Non-Essential Amino Acids (Irvine Scientific, Santa Ana, Calif.), and 10% heat-inactivated fetal bovine serum (Life Technologies, Rockville, Md.). COS-7 cells were obtained from ATCC and grown at 37° C. in 5% CO2 in DMEM with 10% heat-inactivated fetal bovine serum (Life Technologies).
Concentration of Opti-MEM1 supernatant. Opti-MEM1 (Life Technologies) culture media were concentrated through Centricon YM-10 and/or Microcon YM-10 columns (Millipore Corporation, Bedford, Mass.). SDS-PAGE sample loading buffer was then added and the samples were boiled.
Northern blot hybridization. Total RNAs were prepared from cultured breast cancer cell lines and tumor tissues of SCID mice transplanted with breast cancer cell lines with RNeasy Maxi Kit (Qiagen, Valencia, Calif.). Approximately 20 μg of total RNA per lane was loaded onto a formaldehyde/agarose gel for electrophoresis, then transferred to a Hybond-N+ nylon membrane (Amersham Life Science, Little Chalfont, England). The blot was fixed by UV irradiation. Rapid-Hyb buffer (Amersham Life Science) with 5 mg/ml denatured single stranded sperm DNA was pre-warmed to 65° C. and the blot was pre-hybridized in the buffer with shaking at 65° C. for 30 minutes. A hsOAF cDNA fragment or a β-actin cDNA fragment as probe labeled with [α-32P]dCTP (3000 Ci/mmol, Amersham Pharmacia Biotech Inc., Piscataway, N.J.) (Prime-It RmT Kit, Stratagene, La Jolla, Calif.) and purified with ProbeQuantTM G-50 Micro Column (Amersham Pharmacia Biotech Inc.) was added and hybridized to the blot with shaking at 65° C. for overnight. The blot was washed in 2×SSC, 0.1% (w/v) SDS at room temperature for 20 minutes, twice in 1×SSC, 0.1% (w/v) SDS at 65° C. for 15 minutes, then exposed to Hyperfilms (Amersham Life Science).
Immunoblotting. Protein samples were subjected to electrophoresis on 10-20% SDS-PAGE gels then transferred to PVDF membranes (0.2 μm) by electroblotting in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3. Membranes were blocked in TBST (pH 7.5) containing 10% non-fat milk, then blotted in PBS (pH 7.4) containing 1% BSA with a rabbit anti-hsOAF serum (1:1000), followed by probing with a secondary antibody alkaline phosphatase-conjugated goat anti-rabbit IgG (1:2000) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). Protein bands were then visualized by NBT/BCIP reagent (Boehringer Mannheim, Germany).
Transient transfection. The coding region (356-1174) of hsOAF cDNA was cloned into a modified expression vector pRetro-On (Clontech, Palo Alto, Calif.). The pRetro-On vector harboring hsOAF or the control pRetro-On vector with GFP was transfected into COS-7 cells on a 100 mm culture plate using EffecteneTM Transfection Reagent Kit (Qiagen) as instructed in the protocol provided by the manufacturer. Cells were recovered in DMEM with 10% FBS for overnight then switched to Opti-MEM1. After two more days, the supernatant was collected and concentrated for western blot analysis.
Antisense oligo transfection. MDA-MB-435 cells were seeded on 6-well culture plates one day before transfection to yield a 90% density at transfection. 100 μM antisense or reverse control oligo was diluted to 2 μM in Opti-MEM1 for transfection. 0.5 mM sterile lipitoid1 was diluted to a ratio of 1.5 nmol lipitoid1:1 μg oligo in the same volume of Opti-MEM1. The diluted oligo and the diluted lipitoid1 were mixed and immediately added to cells in culture media to a final concentration of 100, 200, or 300 nM oligo. After 6 hrs, the transfection mixture was replaced with normal culture media and cells were incubated for recovery for overnight. The sequence of the antisense oligo is AGCTGCGGATGCCACACTTGTAGG (SEQ ID NO:4) and the sequence of the reverse control oligo is GGATGTTCACACCGTAGGCGTCGA (SEQ ID NO:5).
Matrigel invasion assay. Cells were trypsinized, washed, and resuspended in media for counting. 4×104 cells were washed and resuspended in 100 μl media on ice. 200 μl Matrigel (Collaborative Biomedical Products, Bedford, Mass.) was added to the cells on ice. The Matrigel and the cells were carefully mixed then dispensed into a well of 24-well culture plate and solidified at 37° C. for 30 min. The Matrigel-cell mixture was topped with 0.5 ml medium and incubated at 37° C. in 5% CO2 for 6 days. The medium was replenished every 2 days.
Proliferation assay. Cells were trypsinized, washed, and resuspended in media for counting. Cells were then transferred into 96-well plates (5000 cells/well) for incubation. Cell numbers were measured with QuantosTM Cell Proliferation Assay Kit (Stratagene, La Jolla, Calif.) every day.
Preparation of hsOAF polyclonal antibody. hsOAF antisera were generated in two rabbits immunized against the C-terminal peptide (H-FYVPQ RQLCLWDEDPYPG-OHN, KLH conjugated, SEQ ID NO: 11), and then affinity purification was conducted to obtain the hsOAF polyclonal antibody (ResGen, an Invitrogen Corporation, Huntsville, Ala.). The antibody preparation was titrated by ELISA assay.
Immunohistochemical staining. Immunohistochemical staining was performed to detect hsOAF protein expression in tissues with the hsOAF polyclonal antibody using the immunohistochemical staining kits from BioGenex Laboratories, Inc. (San Ramon, Calif.). All procedures were carried out as instructed in the protocol provided by the manufacturer.
Example 1
Identification of a Human cDNA Sequence
DNA encoding a putative human homologue of the Drosophila Out at First (oaf) gene is shown in SEQ ID NO:1. An alignment of hsOAF and Drosophilia OAF is shown in FIG. 7. The polynucleotide comprises 2366 base pairs, and an open reading frame is identified. A translation of the ORF, a polypeptide of 273 amino acids, is shown in SEQ ID NO:2. FIG. 4 provides the DNA and amino acid sequences, indicating the position of the ORF. The first 30 amino acids form a signal peptide, indicating that the protein may be secreted. The amino acid sequence of the signal peptide is: MRLPGVPLARPALLLLLPLLAPLLG#TGAPA (SEQ ID NO:3). “#” indicates the location of the predicted protease cut site.
Example 2
Expression of hsOAF in Primary and Metastatic Breast Cancer Tissue
To further understand the importance of hsOAF gene expression in breast cancer, immunohistochemical staining in tissue samples from breast cancer patient was conducted using the hsOAF polyclonal antibody (FIG. 5). Strong hsOAF expression was detected not only in all metastases (26/26) but also in almost all primary breast tumors (44/45). Meanwhile, a weak hsOAF positive staining was observed in 8 out of 24 normal breast tissue samples. These results suggest that up-regulation of hsOAF gene expression may play important roles in both mammary tumor formation and development.
Example 3
Differential Expression of SEQ ID NO:1 in Breast Cancer Cell Lines
Expression of SEQ ID NO:1 in the following human breast cancer cell lines was compared:
MDA-MB-361, derived from human breast adenocarcinoma;
MDA-MB-231, derived from human breast cancer cells metastatic to bone and/or lung;
MDA-MB-468, derived from estrogen receptor-negative human breast cancer cells;
MDA-MB-435, derived from estrogen receptor-negative human breast carcinoma cells;
MCF-7, derived from non-metastatic human breast cancer cells; and
ZR-75-1, derived from estrogen receptor-positive human breast carcinoma cells.
Expression of SEQ ID NO:1 was measured in the highly metastatic breast cancer cell lines MDA-MB 231 and MDA-MB-435, and compared with low-metastatic or non-metastatic breast cancer cell lines. Expression in MDA-MB-361 was 11% of the level in MDA-MB-231; expression in MDA-MD-468 was 44% of the level in MDA-MB-231; expression in MCF-7 was 17% of the level in MDA-MB-231; and expression in ZR-75-1 was 12% of the level in MDA-MB-231.
Expression in MDA-MB-361 was 6% of the level in MDA-MB-435; expression in MDA-MB-468 was 36% of the level in MDA-MB-435; and expression in MCF-7 was 3% of the level in MDA-MB-435. Thus, as shown in Table 2, there is a clear trend of increased expression of SEQ ID NO:1 in breast cancer cell lines derived from human tumors with high metastatic potential.








TABLE 2







High
Low Metastatic Cell Lines:


Metastatic
% Expression Relative to High Metastatic Cell Line











Cell Line
MDA-MB-361
MDA-MB-468
MCF-7
ZR-75-1





MDA-MB-231
11%
44%
17%
12%


MDA-MB-435
 6%
36%
 3%
ND









A similar expression pattern of this gene remained in tumor tissue samples from SCID mice transplanted with tumorigenic mammary carcinoma cell lines. (FIG. 6.)
Example 4
hsOAF Encodes a Secreted Protein and hsOAF Protein Secretion Levels are Consistent with hsOAF mRNA Expression Levels of Mammary Carcinoma Cell Lines
A predicted signal peptide sequence is located at the N-terminus of the deduced amino acid sequence of hsOAF gene (FIG. 3). To verify the secretion of hsOAF protein, transient transfection of COS-7 cells and MCF-7 cells was performed with vector pRetro-On harboring hsOAF cDNA. Meanwhile, vector pRetro-On harboring GFP was used as control. Using a hsOAF rabbit antiserum, secreted hsOAF protein was detected in Opti-MEM1 culture media of both cell lines after transfection with hsOAF by immunoblotting (FIG. 8A). Secreted hsOAF protein was probably glycosylated since multiple bands with higher apparent molecular weights were seen (the predicted MW of secreted hsOAF protein is 28 Kda). The same hsOAF antiserum was used to detect the secretion of hsOAF protein by various mammary carcinoma cell lines. The secretion levels of hsOAF protein were consistent with the hsOAF mRNA expression levels among these cell lines overall: highly metastatic cell lines showed much stronger hsOAF secretion than low metastatic/nonmetastatic cell lines (FIG. 8B). MDA-MB-435 had the strongest hsOAF protein secretion.
Example 5
Knockout of hsOAF Expression in MDA-MB-435 Cells by Antisense Oligo Caused Morphological Change, Reduced Cell Invasiveness and Slower Proliferation Rate
To determine if high level of hsOAF gene expression is essential for the metastatic potential of human mammary carcinoma cells, antisense oligo technology was used to knock out hsOAF expression, then the consequent effects were observed. MDA-MB-435 was chosen since this highly metastatic cell line showed the strongest hsOAF protein secretion among all of the breast cancer cell lines examined. Several pairs of hsOAF antisense (AS) and reverse control (RC) oligos were chosen to test for their ability to shut down hsOAF gene expression at the mRNA level. Real-time quantitative RT-PCR analysis in Lightcycler (Roche Diagnostics, Indianapolis, Ind.) was performed to measure hsOAF mRNA levels in cells. Kang, S. et al., Cancer Research 60(18):5296-5302 (2000). The best pair was then selected for the titration of oligo working concentration. Low oligo concentration is preferred to reduce potential oligo toxicity to cells. The results indicated that treatment with 100 nM of the antisense oligo was sufficient to significantly reduce hsOAF protein secretion of MDA-MB-435 cells. (FIG. 12). This pair of oligos (SEQ ID NO:4 (AS) and 5 (RC)) at 100 nM working concentration was used for all the following experiments.
After treatment of MDA-MB-435 cells with hsOAF antisense oligo, dramatic morphological alteration of cells was observed along with reduced hsOAF protein secretion (FIG. 10A). Cells became more spherical and lost their spreading protrusions. Meanwhile, cells treated with reverse control oligo remained similar to the normal tissue cultured MDA-MB-435 cells. Furthermore, culture medium of normal MDA-MB-435 cells containing high level of hsOAF protein as the conditioned medium added to cells treated with antisense oligo was able to prevent this morphological change, though not completely. This alteration of cell shape may be an indication of reduced invasion ability of cells.
Matrigel invasion assay was then performed to estimate the invasiveness of cells. It has been reported that a stellate, invasive morphology of breast cancer cells embedded in matrigel correlates with their metastatic potential (Thompson, E. W., et al, J. Cell Physiol. 150(3):534-44 (1992); Sugiura, T., et al. J. Cell Biol, 146(6):1375-89 (1999); Albini, A., et al., Cancer Res. 47(12):3239-45 (1987); and Kramer, R. H., et al., Cancer Res. 46(4 Pt 2):1980-89 (1986)) and this was confirmed with various breast cancer cell lines grown in matrigel. Cells were trypsinized, counted, and mixed with matrigel. Media were then topped on the cell-matrigel mixture. After 6 days of incubation, cell invasion was examined (FIG. 10B). The results showed that cells treated with hsOAF reverse control oligo formed penetrating, invasive, network-like three-dimensional structures, as the normal MDA-MB-435 cells did; on the other hand, cells treated with hsOAF antisense oligo only formed smooth, spherical colonies. Again, penetrating colonies were also observed in hsOAF antisense oligo-treated cells incubated in the conditional medium. These data demonstrate that secreted hsOAF protein is required for the invasiveness and metastatic potential of MDA-MB-435 cells.
Additional experiments were performed to examine if secreted hsOAF protein was involved in MDA-MB-435 cell growth. Cell proliferation assay results indicated that knockout of hsOAF protein secretion indeed slowed down proliferation rate of MDA-MB-435 cells, though the change was moderate.
Example 6
Northern Blot Analysis of RNA Expression in Human Breast Cancer Cell Lines and in Human Tissues
As shown in FIG. 5, mRNA expression was upregulated in metastatic cell lines MDA-MB-231 and MDA-MB-435. Total RNA was prepared using the RNeasy Kit from Quiagen. Northern blot analysis was performed using 20-30 μg total RNA isolated by guanidinium thiocyanate/phenol chloroform extraction from cell lines, from primary tumors, or from metastases in lung. Primary tumors and lung metastasis were developed from cell lines injected into SCID mice according to methods well known in the art. Plasmids containing partial cDNA clones of hOAF cloned into pCR2.0-TA Vector (In vitrogen) were radiolabeled and hybridized at 65° C. in Express-hyb (Clontech). Among all the tissues examined, liver, pancreas, spleen, ovary, and small intestine showed significant hsOAF expression. HsOAF mRNA expression was also detected in heart, skeletal muscle, kidney, prostate, colon and bone marrow. (FIG. 9).
Table 3 shows the percentage of hsOAF positives in a variety of tumors and normal tissues.







TABLE 3







Immunohistochemistry: Percentage of hsOAF positives 










Tumor
Normal















Pancreas
 9/11
0/9



Esophagus
5/8
0/1



Liver
3/6
 0/13



Stomach
6/7
 6/10



Breast
1/1



Hodgkin's
1/8










Example 7
Soft Agar Assay
Soft Agar Assay: The bottom layer consisted of 2 ml of 0.6% agar in media plated fresh within a few hours of layering on the cells. For the cell layer, MDA-MB-435 cells as described above were removed from the plate in 0.05% trypsin and washed twice in media. Cells were counted in coulter counter, and resuspended to 106 per ml in media. 10 ml aliquots were placed with media in 96-well plates (to check counting with WST1), or diluted further for soft agar assay. 2000 cells were diluted in 800 ml 0.4% agar in duplicate wells above 0.6% agar bottom layer.
Media layer: After the cell layer agar solidified, 2 ml of media was bled on top and antisense or reverse control oligo was added without delivery vehicles. Fresh media and oligos were added every 3-4 days.
Colonies were counted in 10 days to 3 weeks. Fields of colonies were counted by eye. Wst-1 metabolism values were used to compensate for small differences in starting cell number. Larger fields can be scanned for visual record of differences. The results are shown in FIG. 6, in which MDA-MB-435 cells treated with antisense formed fewer colonies compared to cells exposed to the control oligonucleotide.
Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such specific embodiments and equivalents are intended to be encompassed by the following claims.
All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.

## Claims

1. An isolated polypeptide comprising amino acids selected from the group consisting of:
(a) amino acids about 1 to about 273 of SEQ ID NO:2;
(b) amino acids about 2 to about 273 of SEQ ID NO:2; and
(c) amino acids 26 to 273 of SEQ ID NO:2.

2. An epitope-bearing portion of the polypeptide of SEQ ID NO:2, which comprises at least 200 contiguous amino acids of SEQ ID NO:2.

3. The isolated polypeptide of claim 1 comprising amino acids about 1 to about 273 of SEQ ID NO:2.

4. The isolated polypeptide of claim 1 comprising amino acids about 2 to about 273 of SEQ ID NO:2.

5. The isolated polypeptide of claim 1 comprising amino acids 26 to 273 of SEQ ID NO:2.