Patent Application
20050181375
The invention relates to the identification of nucleic acids and proteins identified by expression profiles, and nucleic acids, products, and antibodies thereto that are involved in metastatic cancer; and to the use of such expression profiles and compositions in diagnosis and therapy of metastatic cancer. The invention further relates to methods for identifying and using agents and/or targets that inhibit metastatic cancer.
Metastatic disease can be viewed as two simultaneously occurring diseases; a disease at a primary site, and a related disease at a secondary location distant from the primary site. Each disease may have different mortality rates, for example in cases of metastatic breast or lung cancer to the brain. In such cases untreated brain metastases are rapidly fatal, while primary breast or lung cancer may actually be cureable.
Unfortunately, many cancers metastasize. While there are many variables that determine where metastatic tumors grow, often, the metastatic location is the nearest cluster of small blood vessels found by the circulating cancer cells. Thus, lung cancer commonly metastasizes to the brain; colon cancer commonly metastasizes to the liver. Alternatively, the cancer may have a preferred site of metastasis. For example, the brain is a preferred site for melanoma and small cell lung cancer. A metastasis of a metastasis may develop as well. For example, a colon cancer may metastasize to the liver, which in turn may metastasize to the lung, which may in turn metastasize to the brain.
Without wishing to be bound by theory, it is believed that metastasis occurs when cancer cells from the primary site break away and enter the body's circulatory system through the blood stream, lymph system, or spinal fluid and travel to distant locations. Although cancer metastasis may occur in nearly any organ, brain metastases are one of the most common sites of systemic spread from solid tumors, with an annual incidence of over 100,000 cases. Indeed, metastatic brain tumors occur in about one-fourth of all cancers that metastasize, and primary and metastatic brain tumors kill 15,000 people each year. The most common primary tumors that metastasize to the brain are lung, breast, melanoma, and colon, however almost any cancer has this potential.
Although almost 1 in 4 patients with cancer will develop tumors that spread to the central nervous system (CNS) cancer can metastasize to almost any organ.
Classification of metastatic tumors depends on the tissue type from which they are derived. Unfortunately, it is often difficult or impossible to determine the location of the primary cancer and this can unnecessarily complicate diagnosis and treatment of the metastatic cancer. In addition early diagnosis of metastatic cancer can greatly improve the prognostic outlook for a cancer patient. Often metastatic burden, rather than the primary cancer, is what ultimately kills a patient.
Thus need exists for an efficient and effective method for the identification of metastatic tumor origins, as well as methods for diagnosis, prognosis and treatment of metastatic cancer. The development of successful therapeutic modalities is however, unlikely to follow the conventional approaches of surgery, radiation and cytotoxic chemotherapy. Rather, the best hope lies in the rapidly expanding field of molecular medicine.
Accordingly, provided herein are molecular targets for therapeutic intervention in metastatic breast and lung cancer. Additionally, provided herein are methods that can be used in diagnosis and prognosis of metastatic breast and lung cancer. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate metastatic cancer including metastatic brain tumors.
The present invention therefore provides nucleotide sequences of genes that are up- and down-regulated in metastatic breast or metastatic lung cancer cells. Such genes and the proteins they encode are useful for diagnostic and prognostic purposes, and also as targets for screening for therapeutic compounds that modulate metastatic breast or lung cancer, such as antibodies. The methods of detecting nucleic acids of the invention or their encoded proteins can be used for a number of purposes. Examples include, early detection of breast or lung cancers, monitoring and early detection of relapse following treatment of breast or lung cancers including early detection of metastatic cancer, monitoring response to therapy of breast or lung cancers, determining prognosis of breast or lung cancers, directing therapy of breast or lung cancers, selecting patients for postoperative chemotherapy or radiation therapy, selecting therapy, determining tumor prognosis and the likelihood that a given cancer will metastasize or has metastasized, treatment, or response to treatment, early detection of precancerous conditions and early detection of metastasis. Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.
In one aspect, the present invention provides a method of detecting a metastatic breast or lung cancer-associated transcript in a cell from a patient, the method comprising contacting a biological sample from the patient with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1A-12C.
In one embodiment, the polynucleotide selectively hybridizes to a sequence at least 95% identical to a sequence as shown in Tables 1A-12C. In another embodiment, the polynucleotide comprises a sequence as shown in Tables 1A-12C.
In one embodiment, the biological sample is a tissue sample. In another embodiment, the biological sample comprises isolated nucleic acids, e.g., mRNA.
In one embodiment, the polynucleotide is labeled, e.g., with a fluorescent label.
In one embodiment, the polynucleotide is immobilized on a solid surface.
In one embodiment, the patient is undergoing a therapeutic regimen to treat metastatic breast or lung cancer. In another embodiment, the metastatic breast or lung cancer has metastasized to the brain.
In one embodiment, the patient is a human.
In one embodiment, the method further comprises the step of amplifying nucleic acids before the step of contacting the biological sample with the polynucleotide.
In another aspect, the present invention provides methods of detecting polypeptide encoded by a metastatic breast or lung cancer-associated transcript in a cell from a patient, the method comprising contacting a biological sample from the patient with an antibody that specifically binds a polypeptide encoded by a sequence at least 80% identical to a sequence as shown in Tables 1A-12C.
In another aspect, the present invention provides a method of monitoring the efficacy of a therapeutic treatment of metastatic breast or lung cancer, the method comprising the steps of: (i) providing a biological sample from a patient undergoing the therapeutic treatment; and (ii) determining the level of a metastatic breast or lung cancer-associated transcript in the biological sample by contacting the biological sample with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1A-12C., thereby monitoring the efficacy of the therapy.
In one embodiment, the method further comprises the step of: (iii) comparing the level of the metastatic breast or lung cancer-associated transcript to a level of the metastatic breast or lung cancer-associated transcript in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
In another aspect, the present invention provides a method of monitoring the efficacy of a therapeutic treatment of metastatic breast or lung cancer, the method comprising the steps of: (i) providing a biological sample from a patient undergoing the therapeutic treatment; and (ii) determining the level of a metastatic breast or lung cancer-associated antibody in the biological sample by contacting the biological sample with a polypeptide encoded by a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1A-12C, wherein the polypeptide specifically binds to the metastatic breast or lung cancer-associated antibody, thereby monitoring the efficacy of the therapy.
In one embodiment, the method further comprises the step of: (iii) comparing the level of the metastatic breast or lung cancer-associated antibody to a level of the metastatic breast or lung cancer-associated antibody in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
In another aspect, the present invention provides a method of monitoring the efficacy of a therapeutic treatment of metastatic breast or lung cancer, the method comprising the steps of: (i) providing a biological sample from a patient undergoing the therapeutic treatment; and (ii) determining the level of a metastatic breast or lung cancer-associated polypeptide in the biological sample by contacting the biological sample with an antibody, wherein the antibody specifically binds to a polypeptide encoded by a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1A-12C, thereby monitoring the efficacy of the therapy.
In one embodiment, the method further comprises the step of: (iii) comparing the level of the metastatic breast or lung cancer-associated polypeptide to a level of the metastatic breast or lung cancer-associated polypeptide in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
In one aspect, the present invention provides an isolated nucleic acid molecule consisting of a polynucleotide sequence as shown in Tables 1A-12C.
In one embodiment, an expression vector or cell comprises the isolated nucleic acid.
In one aspect, the present invention provides an isolated polypeptide which is encoded by a nucleic acid molecule having polynucleotide sequence as shown in Tables 1A-12C.
In another aspect, the present invention provides an antibody that specifically binds to an isolated polypeptide which is encoded by a nucleic acid molecule having polynucleotide sequence as shown in Tables 1A-12C.
In one embodiment, the antibody is conjugated to an effector component, e.g., a fluorescent label, a radioisotope or a cytotoxic chemical.
In one embodiment, the antibody is an antibody fragment. In another embodiment, the antibody is humanized.
In one aspect, the present invention provides a method of detecting a metastatic breast or lung cancer cell in a biological sample from a patient, the method comprising contacting the biological sample with an antibody as described herein.
In another aspect, the present invention provides a method of detecting antibodies specific to metastatic breast or lung cancer in a patient, the method comprising contacting a biological sample from the patient with a polypeptide encoded by a nucleic acid comprises a sequence from Tables 1A-12C.
In another aspect, the present invention provides a method for identifying a compound that modulates a metastatic breast or lung cancer-associated polypeptide, the method comprising the steps of: (i) contacting the compound with a metastatic breast or lung cancer-associated polypeptide, the polypeptide encoded by a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1A-12C; and (ii) determining the functional effect of the compound upon the polypeptide.
In one embodiment, the functional effect is a physical effect, an enzymatic effect, or a chemical effect.
In one embodiment, the polypeptide is expressed in a eukaryotic host cell or cell membrane. In another embodiment, the polypeptide is recombinant.
In one embodiment, the functional effect is determined by measuring ligand binding to the polypeptide.
In another aspect, the present invention provides a method of inhibiting proliferation of a metastatic breast or lung cancer-associated cell to treat breast or lung cancer in a patient, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified as described herein.
In one embodiment, the compound is an antibody.
In another aspect, the present invention provides a drug screening assay comprising the steps of: (i) administering a test compound to a mammal having metastatic breast or lung cancer or a cell isolated therefrom; (ii) comparing the level of gene expression of a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1A-12C in a treated cell or mammal with the level of gene expression of the polynucleotide in a control cell or mammal, wherein a test compound that modulates the level of expression of the polynucleotide is a candidate for the treatment of metastatic breast or lung cancer.
In one embodiment, the control is a mammal with metastatic breast or lung cancer or a cell therefrom that has not been treated with the test compound. In another embodiment, the control is a normal cell or mammal.
In another aspect, the present invention provides a method for treating a mammal having metastatic breast or lung cancer comprising administering a compound identified by the assay described herein.
In another aspect, the present invention provides a pharmaceutical composition for treating a mammal having meta static breast or lung cancer, the composition comprising a compound identified by the assay described herein and a physiologically acceptable excipient.
In accordance with the objects outlined above, the present invention provides novel methods for diagnosis and treatment of metastatic breast or metastatic lung cancer. The invention is useful for the treatment of metastatic breast and metastatic lung cancer when the cancer is metastasized to the brain, as well as when the cancer is metastasized to other organs and tissues. The invention also provides methods of screening for compositions which modulate metastatic breast cancer or metastatic lung cancer.
Primary tumors are classified by the type of tissue from which they arise, metastatic tumors are classified by the tissue type from which the cancer cells are derived. Almost any cancer can metastasize. The metastases may occur to any site, however some cancers preferentially metastasize to particular organs. For example lung, breast, head & neck, cervical, and bladder tumors frequently metastasize to particular organs. Specifically, lung cancer metastatisizes to: brain, bone, liver, adrenal glands, lung, pleura, subcutaneous tissue, kidney, lymph nodes, cerebrospinal fluid, pancreas, bone marrow. Breast cancer metastatisizes to: lymph nodes, breast, abdominal viscera, lungs, bones, liver, adrenal glands, brain, meninges, pleura, cerebrospinal fluid. Head and neck cancer metastatisizes to: lung, esophagus, upper aerodigestive tract, lymph nodes, oral cavity, nasal cavity. Cervical cancer metastatisizes to: vagina, paracervical spaces, bladder, rectum, pelvic wall, lymph nodes. Bladder cancer metastatisizes to: prostate, uterus, vagina, bowel, pelvic wall, lymph nodes, and perivesical fat.
Brain metastases are a particular concern because of the deadly nature of brain tumors in general. Because the brain is generally unforgiving in its response to both the tumor and therapy, prognosis in cases metastatic brain tumors is especially poor. This is the case whether or not the primary cancer is treatable or even cured.
Whether or not cancer cells metastasize to the brain or other parts of the body depends on many factors including the type of cancer, stage of cancer, and original location of the cancer. Treatment for secondary (metastatic) tumors depends on where the cancer started and the extent of the spread as well as other factors, including the patient's age, general health, and response to previous treatment.
Knowing the origin of metastatic cancer can greatly improve the probable outcome of treatment for individuals with metastatic disease. Indeed, the earlier metastatic cancer can be detected, the better is the prognosis for the individual since it is often metastatic burden that kills a patient. Because metastatic burden increases with time, early detection is essential for successful treatment.
Thus, in accordance with the objectives of the invention, Tables 1A-12C provide UniGene cluster identification numbers for the nucleotide sequence of genes that exhibit increased or decreased expression in metastasizing breast and lung cancer samples. Tables 1A-12C also provide an exemplar accession number that provides a nucleotide sequence that is part of the UniGene cluster.
Table 1A shows about 461 genes upregulated in breast metastases to the brain relative to normal breast tissues. Table 2A shows about 445 genes upregulated in breast metastases to the brain relative to normal body tissues. Table 3A shows about 216 genes upregulated in breast metastases to the brain relative to primary breast tumors. Table 4A shows about 350 genes downregulated in breast metastases to the brain relative to primary breast tumors. Table 5A shows about 489 genes downregulated in breast metastases to the brain relative to normal breast tissue. Table 6A shows about 1251 genes upregulated in lung metastases to the brain relative to normal lung tissues. Table 7A shows about 381 genes upregulated in lung metastases to the brain relative to normal body tissues. Table 8A shows about 330 genes upregulated in lung metastases to the brain relative to primary lung tumors. Table 9A shows about 252 genes downregulated in lung metastases to the brain relative to primary lung tumors. Table 10A shows about 289 genes downregulated in lung metastases to the brain relative to normal lung tissue. Table 11A shows about 1198 genes upregulated in breast and lung metastases to the brain relative to normal body tissues. Table 12A shows about 2867 genes upregulated in breast and lung metastases to the brain relative to normal breast and lung tissues.
Although the Tables and analysis herein is derived primarily from metastases to the brain, it is expected that markers identified from these samples should also be expressed in metastasis to other organs, particularly metastasis originating from tumors in the lung, breast, head and neck, cervix, and bladder. Indeed, the tumor-specific genes expressed in lung tumors are often also expressed in head and neck, cervical, and bladder tumors Therefore, the genes identified in metastases of primary lung tumors may also be expressed in primary tumors and metastases arising from primary tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oral pharynx, lip, larynx, hypopharynx, salivary glands, paragangliomas, esophagus, cervix, vagina, vulva, or bladder. Thus, the identified metastatic markers should be useful in diagnosis, prognosis, or therapy of metastases from these cancers.
Furthermore, one of skill will recognize that although the sequences identified in Tables 1A-12C exhibited increased or decreased expression in metastasizing breast or lung cancer samples, the sequences of the invention, and their encoded proteins, can also be used to diagnose, treat or prevent cancers in patients with non-metastatic breast cancers or non-metastatic lung cancers. Alteration of gene expression for a gene in Tables 1A-12C may be more likely or less likely to indicate that the subject will progress to metastatic disease. The sequences can also be used to diagnose, treat or prevent precancerous or benign conditions.
Alteration of gene expression for a gene in Tables 1 A-12C may or may not indicate that the subject is more likely to progress to cancer or to metastatic disease. Thus, although the specification focuses primarily on metastasizing breast or lung cancer, the methods described below can also be applied to non-metastasizing breast or lung cancers and precancerous or benign conditions as well.
The term “metastatic breast cancer protein” or “metastatic breast cancer polynucleotide” or “metastatic breast cancer-associated transcript” refers to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a nucleotide sequence of or associated with a UniGene cluster of Tables 1-5, 11, and 12; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence encoded by a nucleotide sequence of or associated with a UniGene cluster of Tables 1-5, 11, and 12, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to a nucleic acid sequence, or the complement thereof of Tables 1-5, 11, and 12 and conservatively modified variants thereof or (4) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acid, to an amino acid sequence encoded by a nucleotide sequence of or associated with a UniGene cluster of Tables 1-5, 11, and 12. A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or other mammal. A “metastatic breast cancer polypeptide” and a “metastatic breast cancer polynucleotide,” include both naturally occurring or recombinant.
The term “metastatic lung cancer protein” or “metastatic lung cancer polynucleotide” or “metastatic lung cancer-associated transcript” refers to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a nucleotide sequence of or associated with a UniGene cluster of Tables 6A-12C; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence encoded by a nucleotide sequence of or associated with a UniGene cluster of Tables 6A-12C, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to a nucleic acid sequence, or the complement thereof of Tables 6A-12C and conservatively modified variants thereof or (4) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acid, to an amino acid sequence encoded by a nucleotide sequence of or associated with a UniGene cluster of Tables 6A-12C. A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or other mammal. A “metastatic lung cancer polypeptide” and a “metastatic lung cancer polynucleotide,” include both naturally occurring or recombinant.
The term “metastasis” refers to the process by which a disease shifts from from one part of the body to another. This process may include the spreading of neoplasms from the site of a primary tumor to distant parts of the body.
The term “metastatic brain tumor” refers to a tumor of the brain and/or its associated bone, blood vessels, meninges etc that has developed as a result of the metastasis of cancer from a primary site to the brain.
The term “secondary brain tumor” refers to a metastatic brain tumor as defined above.
The term “metastatic breast cancer” refers to any cancer in any part of the body which has its origins in breast cancer tissue. Metastatic breast cancer includes, but is not limited to “metastatic brain tumors” that have their origin in a primary breast cancer, and pre- metastatic primary tumor cells in the process of developing a metastatic phenotype.
The term “metastatic lung cancer” refers to any cancer in any part of the body which has its origins in lung cancer tissue. Metastatic lung cancer includes, but is not limited to “metastatic brain tumors” that have their origin in a primary lung cancer, and pre-metastatic primary tumor cells in the process of developing a metastatic phenotype.
A “full length” metastatic breast or lung cancer protein or nucleic acid refers to a metastatic breast or lung cancer polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type metastatic breast or lung cancer polynucleotide or polypeptide sequences. The “full length” may be prior to, or after, various stages of post-translation processing or splicing, including alternative splicing.
“Biological sample” as used herein is a sample of biological tissue or fluid that contains nucleic acids or polypeptides, e.g., of a metastatic breast or lung cancer protein, polynucleotide or transcript. Such samples include, but are not limited to, tissue isolated from primates, e.g., humans, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or other mammal; or a bird; reptile; fish.
“Providing a biological sample” means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of one of the number of contiguous positions selected from the group consisting typically of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, e.g., for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. Log values may be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, etc.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.
A “host cell” is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, and the like (see, e.g., the American Type Culture Collection catalog or web site, www.atcc.org).
The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene. The term “purified” in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Preferably, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. “Purify” or “purification” in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, often silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor & Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that often form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. “Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed, usually by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds., Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g. to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, PNAs are not degraded by cellular enzymes, and thus can be more stable.
The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. “Transcript” typically refers to a naturally occurring RNA, e.g., a pre-mRNA, hnRNA, or mRNA. As used herein, the term “nucleoside” includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, “nucleoside” includes non-naturally occurring analog structures. Thus, e.g. the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
An “effector” or “effector moiety” or “effector component” is a molecule that is bound (or linked, or conjugated), either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, to an antibody. The “effector” can be a variety of molecules including, e.g., detection moieties including radioactive compounds, fluorescent compounds, an enzyme or substrate, tags such as epitope tags, a toxin; activatable moieties, a chemotherapeutic agent; a lipase; an antibiotic; or a radioisotope emitting “hard” e.g., beta radiation.
A “labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. Alternatively, method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.
As used herein a “nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not functionally interfere with hybridization. Thus, e.g., probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence. Diagnosis or prognosis may be based at the genomic level, or at the level of RNA or protein expression.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are often: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1 -2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990).
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement).
The phrase “functional effects” in the context of assays for testing compounds that modulate activity of a metastatic breast cancer or metastatic lung cancer protein includes the determination of a parameter that is indirectly or directly under the influence of the metastatic breast cancer or metastatic lung cancer protein or nucleic acid, e.g., an enzymatic, functional, physical, or chemical effect, such as the ability to decrease metastatic breast cancer or metastatic lung cancer. It includes ligand binding activity; cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cellular proliferation; cellular transformation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis, and other characteristics of metastatic breast cancer or metastatic lung cancer cells. “Functional effects” include in vitro, in vivo, and ex vivo activities.
By “determining the functional effect” is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a metastatic breast cancer or metastatic lung cancer protein sequence, e.g., functional, enzymatic, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the metastatic breast cancer or metastatic lung cancer protein; measuring binding activity or binding assays, e.g., binding to antibodies or other ligands, and measuring cellular proliferation. Determination of the functional effect of a compound on metastatic breast cancer or metastatic lung cancer can also be performed using metastatic breast cancer or metastatic lung cancer assays known to those of skill in the art such as an in vitro assays, e.g., cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cellular proliferation; cellular transformation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis, and other characteristics of metastatic breast cancer or metastatic lung cancer cells. The functional effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels for metastatic breast cancer or metastatic lung cancer-associated sequences, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, calorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
“Inhibitors”, “activators”, and “modulators” of metastatic breast cancer or metastatic lung cancer polynucleotide and polypeptide sequences are used to refer to activating, inhibitory, or modulating molecules or compounds identified using in vitro and in vivo assays of metastatic breast cancer or metastatic lung cancer polynucleotide and polypeptide sequences of the invention. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of metastatic breast cancer or metastatic lung cancer proteins of the invention, e.g., antagonists. Antisense nucleic acids may seem to inhibit expression and subsequent function of the protein. “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate metastatic breast cancer or metastatic lung cancer protein activity. Inhibitors, activators, or modulators also include genetically modified versions of metastatic breast cancer or metastatic lung cancer proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., expressing the metastatic breast cancer or metastatic lung cancer protein in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above. Activators and inhibitors of metastatic breast cancer or metastatic lung cancer can also be identified by incubating metastatic breast cancer or metastatic lung cancer cells with the test compound and determining increases or decreases in the expression of 1 or more metastatic breast cancer or metastatic lung cancer proteins, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more metastatic breast cancer or metastatic lung cancer proteins, such as metastatic breast cancer or metastatic lung cancer proteins encoded by the sequences set out in Tables 1-12.
Samples or assays comprising metastatic breast cancer or metastatic lung cancer proteins that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition of a polypeptide is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation of a metastatic breast cancer or metastatic lung cancer polypeptide is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
The phrase “changes in cell growth” refers to any change in cell growth and proliferation characteristics in vitro or in vivo, such as formation of foci, anchorage independence, semi-solid or soft agar growth, changes in contact inhibition and density limitation of growth, loss of growth factor or serum requirements, changes in cell morphology, gaining or losing immortalization, gaining or losing tumor specific markers, ability to form or suppress tumors when injected into suitable animal hosts, and/or immortalization of the cell. See, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique pp. 231-241 (3rd ed. 1994).
“Tumor cell” refers to precancerous, cancerous, and normal cells in a tumor. “Cancer cells,” “transformed” cells or “transformation” in tissue culture, refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation is associated with phenotypic changes, such as immortalization of cells, aberrant growth control, nonmorphological changes, and/or malignancy (see, Freshney, Culture of Animal Cells a Manual ofBasic Technique (3rd ed. 1994)).
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody or its functional equivalent will be most critical in specificity and affinity of binding. See Paul, Fundamental Immunology.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, e.g., pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab′)2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab′)2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab′)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990))
For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).
A “chimeric antibody” is an antibody molecule in which, e.g, (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
In one aspect, the expression levels of genes are determined in different patient samples for which diagnosis information is desired, to provide expression profiles. An expression profile of a particular sample is essentially a “fingerprint” of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell. That is, normal tissue may be distinguished from cancerous or metastatic cancerous tissue, or metastatic cancerous tissue can be compared with tissue from surviving cancer patients. By comparing expression profiles of tissue in known different metastatic breast cancer or metastatic lung cancer states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained.
The identification of sequences that are differentially expressed in metastatic breast cancer or metastatic lung cancer versus non-metastatic breast cancer or non-metastatic lung cancer tissue allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated: does a chemotherapeutic drug act to down-regulate metastatic breast cancer or metastatic lung cancer, and thus tumor growth or recurrence, in a particular patient. Similarly, diagnosis and treatment outcomes may be done or confirmed by comparing patient samples with the known expression profiles. Metastatic tissue can also be analyzed to determine the stage of metastatic breast cancer or metastatic lung cancer in the tissue. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates with an eye to mimicking or altering a particular expression profile; e.g., screening can be done for drugs that suppress the metastatic breast cancer or metastatic lung cancer expression profile. This may be done by making biochips comprising sets of the important metastatic breast cancer or metastatic lung cancer genes, which can then be used in these screens. PCR methods may be applied with selected primer pairs, and analysis may be of RNA or of genomic sequences. These methods can also be done on the protein basis; that is, protein expression levels of the metastatic breast cancer or metastatic lung cancer proteins can be evaluated for diagnostic purposes or to screen candidate agents. In addition, the metastatic breast cancer or metastatic lung cancer nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or the metastatic breast cancer or metastatic lung cancer proteins (including antibodies and other modulators thereof) administered as therapeutic drugs or as protein or DNA vaccines.
Thus the present invention provides nucleic acid and protein sequences that are differentially expressed in metastatic breast cancer or metastatic lung cancer, herein termed “metastatic breast cancer or metastatic lung cancer sequences.” As outlined below, metastatic breast cancer or metastatic lung cancer sequences include those that are up- regulated (i.e., expressed at a higher level) in metastatic breast cancer or metastatic lung cancer, as well as those that are down-regulated (i.e., expressed at a lower level). In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer sequences are from humans; however, as will be appreciated by those in the art, metastatic breast cancer or metastatic lung cancer sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other metastatic breast cancer or metastatic lung cancer sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc.) and pets (dogs, cats, etc.). Metastatic breast cancer or metastatic lung cancer sequences from other organisms may be obtained using the techniques outlined below.
Metastatic breast cancer or metastatic lung cancer sequences can include both nucleic acid and amino acid sequences. As will be appreciated by those in the art and is more fully outlined below, metastatic breast cancer or metastatic lung cancer nucleic acid sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications; e.g., biochips comprising nucleic acid probes or PCR microtiter plates with selected probes to the metastatic breast cancer or metastatic lung cancer sequences can be generated.
A metastatic breast cancer or metastatic lung cancer sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the metastatic breast cancer or metastatic lung cancer sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
For identifying metastatic breast cancer or metastatic lung cancer-associated sequences, the metastatic breast cancer or metastatic lung cancer screen typically includes comparing genes identified in different tissues, e.g., normal and cancerous tissues, or tumor tissue samples from patients who have metastatic disease vs. non metastatic tissue, or tumor tissue samples from patients who have been diagnosed cancer but have survived vs. metastatic tissue. Other suitable tissue comparisons include comparing metastatic breast cancer or metastatic lung cancer samples with metastatic cancer samples from other cancers, such as gastrointestinal cancers, prostate, ovarian, etc. Samples of, e.g., breast cancer survivor tissue and tissue undergoing metastasis are applied to biochips comprising nucleic acid probes. The samples are first microdissected, if applicable, and treated as is known in the art for the preparation of mRNA. Suitable biochips are commercially available, e.g., GENECHIP® (DNA microarray) technology from Affymetrix, Inc. (Santa Clara, Calif.). Gene expression profiles as described herein are generated and the data analyzed.
In one embodiment, the genes showing changes in expression as between normal and disease states are compared to genes expressed in other normal tissues, preferably normal colon, but also including, and not limited to lung, heart, brain, liver, breast, kidney, muscle, prostate, small intestine, large intestine, spleen, bone and placenta. In a preferred embodiment, those genes identified during the metastatic breast cancer or metastatic lung cancer screen that are expressed in significant amounts in other tissues are removed from the profile, although in some embodiments, this is not necessary. That is, when screening for drugs, it is usually preferable that the target be disease specific, to minimize possible side effects.
In a preferred embodiment, metastatic breast cancer or metastatic lung cancer sequences are those that are up-regulated in metastatic breast cancer or metastatic lung cancer; that is, the expression of these genes is higher in the metastatic tissue as compared to non-metastatic cancerous tissue or normal tissue (see, e.g., Tables 1-3, 6-8, 11-12). “Up-regulation” as used herein means, when the ratio is presented as a number greater than one, that the ratio is greater than one, preferably 1.5 or greater, more preferably 2.0 or greater. All UniGene cluster identification numbers and accession numbers herein are for the GenBank sequence database and the sequences of the accession numbers are hereby expressly incorporated by reference. GenBank is known in the art, see, e.g., Benson, D A, et al., Nucleic Acids Research 26:1-7 (1998) and. Sequences are also available in other databases, e.g., European Molecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ).
In another preferred embodiment, metastatic breast cancer or metastatic lung cancer sequences are those that are down-regulated in the metastatic breast cancer or metastatic lung cancer; that is, the expression of these genes is lower in metastatic tissue as compared to non-metastatic cancerous tissue or normal tissue (see, e.g., Tables 4-5 and 9-10). “Down-regulation” as used herein means, when the ratio is presented as a number greater than one, that the ratio is greater than one, preferably 1.5 or greater, more preferably 2.0 or greater, or, when the ratio is presented as a number less than one, that the ratio is less than one, preferably 0.5 or less, more preferably 0.25 or less.
Informatics
The ability to identify genes that are over or under expressed in metastatic breast cancer or metastatic lung cancer can additionally provide high-resolution, high-sensitivity datasets which can be used in the areas of diagnostics, therapeutics, drug development, pharmacogenetics, protein structure, biosensor development, and other related areas. For example, the expression profiles can be used in diagnostic or prognostic evaluation of patients with metastatic breast cancer or metastatic lung cancer. Or as another example, subcellular toxicological information can be generated to better direct drug structure and activity correlation (see Anderson, Pharmaceutical Proteomics: Targets, Mechanism, and Function, paper presented at the IBC Proteomics conference, Coronado, Calif. (June 11-12, 1998)). Subcellular toxicological information can also be utilized in a biological sensor device to predict the likely toxicological effect of chemical exposures and likely tolerable exposure thresholds (see U.S. Pat. No. 5,811,231). Similar advantages accrue from datasets relevant to other biomolecules and bioactive agents (e.g., nucleic acids, saccharides, lipids, drugs, and the like).
Thus, in another embodiment, the present invention provides a database that includes at least one set of assay data. The data contained in the database is acquired, e.g., using array analysis either singly or in a library format. The database can be in substantially any form in which data can be maintained and transmitted, but is preferably an electronic database. The electronic database of the invention can be maintained on any electronic device allowing for the storage of and access to the database, such as a personal computer, but is preferably distributed on a wide area network, such as the World Wide Web.
The focus of the present section on databases that include peptide sequence data is for clarity of illustration only. It will be apparent to those of skill in the art that similar databases can be assembled for assay data acquired using an assay of the invention.
The compositions and methods for identifying and/or quantitating the relative and/or absolute abundance of a variety of molecular and macromolecular species from a biological sample undergoing metastatic breast cancer or metastatic lung cancer, i.e., the identification of metastatic breast cancer or metastatic lung cancer-associated sequences described herein, provide an abundance of information, which can be correlated with pathological conditions, predisposition to disease, drug testing, therapeutic monitoring, gene-disease causal linkages, identification of correlates of immunity and physiological status, among others. Although the data generated from the assays of the invention is suited for manual review and analysis, in a preferred embodiment, prior data processing using high-speed computers is utilized.
An array of methods for indexing and retrieving biomolecular information is known in the art. For example, U.S. Pat. Nos. 6,023,659 and 5,966,712 disclose a relational database system for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more protein finction hierarchies. U.S. Pat. No. 5,953,727 discloses a relational database having sequence records containing information in a format that allows a collection of partial-length DNA sequences to be catalogued and searched according to association with one or more sequencing projects for obtaining full-length sequences from the collection of partial length sequences. U.S. Pat. No. 5,706,498 discloses a gene database retrieval system for making a retrieval of a gene sequence similar to a sequence data item in a gene database based on the degree of similarity between a key sequence and a target sequence. U.S. Pat. No. 5,538,897 discloses a method using mass spectroscopy fragmentation patterns of peptides to identify amino acid sequences in computer databases by comparison of predicted mass spectra with experimentally-derived mass spectra using a closeness-of-fit measure. U.S. Pat. No. 5,926,818 discloses a multi-dimensional database comprising a functionality for multi-dimensional data analysis described as on-line analytical processing (OLAP), which entails the consolidation of projected and actual data according to more than one consolidation path or dimension. U.S. Pat. No. 5,295,261 reports a hybrid database structure in which the fields of each database record are divided into two classes, navigational and informational data, with navigational fields stored in a hierarchical topological map which can be viewed as a tree structure or as the merger of two or more such tree structures.
See also Mount et al., Bioinformatics (2001); Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids (Durbin et al., eds., 1999); Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins (Baxevanis & Oeullette eds., 1998)); Rashidi & Buehler, Bioinformatics: Basic Applications in Biological Science and Medicine (1999); Introduction to Computational Molecular Biology (Setubal et al., eds 1997); Bioinformatics: Methods and Protocols (Misener & Krawetz, eds, 2000); Bioinformatics: Sequence, Structure, and Databanks: A Practical Approach (Higgins & Taylor, eds., 2000); Brown, Bioinformatics: A Biologist's Guide to Biocomputing and the Internet (2001); Han & Kamber, Data Mining: Concepts and Techniques (2000); and Waterman, Introduction to Computational Biology: Maps, Sequences, and Genomes (1995).
The present invention provides a computer database comprising a computer and software for storing in computer-retrievable form assay data records cross-tabulated, e.g., with data specifying the source of the target-containing sample from which each sequence specificity record was obtained.
In an exemplary embodiment, at least one of the sources of target-containing sample is from a control tissue sample known to be free of pathological disorders. In a variation, at least one of the sources is a known pathological tissue specimen, e.g., a neoplastic lesion or another tissue specimen to be analyzed for metastatic breast cancer or metastatic lung cancer. In another variation, the assay records cross-tabulate one or more of the following parameters for each target species in a sample: (1) a unique identification code, which can include, e.g., a target molecular structure and/or characteristic separation coordinate (e.g., electrophoretic coordinates); (2) sample source; and (3) absolute and/or relative quantity of the target species present in the sample.
The invention also provides for the storage and retrieval of a collection of target data in a computer data storage apparatus, which can include magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays. Typically, the target data records are stored as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, such as an array of cells in a DRAM device (e.g., each cell comprised of a transistor and a charge storage area, which may be on the transistor). In one embodiment, the invention provides such storage devices, and computer systems built therewith, comprising a bit pattern encoding a protein expression fingerprint record comprising unique identifiers for at least 10 target data records cross-tabulated with target source.
When the target is a peptide or nucleic acid, the invention preferably provides a method for identifying related peptide or nucleic acid sequences, comprising performing a computerized comparison between a peptide or nucleic acid sequence assay record stored in or retrieved from a computer storage device or database and at least one other sequence. The comparison can include a sequence analysis or comparison algorithm or computer program embodiment thereof (e.g., FASTA, TFASTA, GAP, BESTFIT) and/or the comparison may be of the relative amount of a peptide or nucleic acid sequence in a pool of sequences determined from a polypeptide or nucleic acid sample of a specimen.
The invention also preferably provides a magnetic disk, such as an IBM-compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive, comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing in a computerized sequence analysis, comparison, or relative quantitation method.
The invention also provides a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable (coax or 1OBaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal transmission medium, whereby at least one network device (e.g., computer, disk array, etc.) comprises a pattern of magnetic domains (e.g., magnetic disk) and/or charge domains (e.g., an array of DRAM cells) composing a bit pattern encoding data acquired from an assay of the invention.
The invention also provides a method for transmitting assay data that includes generating an electronic signal on an electronic communications device, such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like, wherein the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
In a preferred embodiment, the invention provides a computer system for comparing a query target to a database containing an array of data structures, such as an assay result obtained by the method of the invention, and ranking database targets based on the degree of identity and gap weight to the target data. A central processor is preferably initialized to load and execute the computer program for alignment and/or comparison of the assay results. Data for a query target is entered into the central processor via an I/O device. Execution of the computer program results in the central processor retrieving the assay data from the data file, which comprises a binary description of an assay result.
The target data or record and the computer program can be transferred to secondary memory, which is typically random access memory (e.g., DRAM, SRAM, SGRAM, or SDRAM). Targets are ranked according to the degree of correspondence between a selected assay characteristic (e.g., binding to a selected affinity moiety) and the same characteristic of the query target and results are output via an I/O device. For example, a central processor can be a conventional computer (e.g., Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc.); a program can be a commercial or public domain molecular biology software package (e.g., UWGCG Sequence Analysis Software, Darwin); a data file can be an optical or magnetic disk, a data server, a memory device (e.g., DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory, flash memory, etc.); an I/O device can be a terminal comprising a video display and a keyboard, a modem, an ISDN terminal adapter, an Ethernet port, a punched card reader, a magnetic strip reader, or other suitable I/O device.
The invention also preferably provides the use of a computer system, such as that described above, which comprises: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention, which may be stored in the computer; (3) a comparison target, such as a query target; and (4) a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
Metastatic breast cancer or metastatic lung cancer proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins. In one embodiment, the metastatic breast cancer or metastatic lung cancer protein is an intracellular protein. Intracellular proteins may be found in the cytoplasm and/or in the nucleus and/or in the organelles. Proteins containing one or more transmembrane domains that exclusively reside in organelles are also considered intracellular proteins. Intracellular proteins are involved in all aspects of cellular function and replication (including, e.g., signaling pathways); aberrant expression of such proteins often results in unregulated or disregulated cellular processes (see, e.g., Molecular Biology of the Cell (Alberts, ed., 3rd ed., 1994). For example, many intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity and the like. Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are involved in maintaining the structural integrity of organelles.
An increasingly appreciated concept in characterizing proteins is the presence in the proteins of one or more motifs for which defined functions have been attributed. In addition to the highly conserved sequences found in the enzymatic domain of proteins, highly conserved sequences have been identified in proteins that are involved in protein-protein interaction. For example, Src-homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner. PTB domains, which are distinct from SH2 domains, also bind tyrosine phosphorylated targets. SH3 domains bind to proline-rich targets. In addition, PH domains, tetratricopeptide repeats and WD domains to name only a few, have been shown to mediate protein-protein interactions. Some of these may also be involved in binding to phospholipids or other second messengers. As will be appreciated by one of ordinary skill in the art, these motifs can be identified on the basis of primary sequence; thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate. One useful database is Pfam (protein families), which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains. Versions are available via the internet from Washington University in St. Louis, the Sanger Center in England, and the Karolinska Institute in Sweden (see, e.g., Bateman et al., Nuc. Acids Res. 28:263-266 (2000); Sonnhammer et al., Proteins 28:405-420 (1997); Bateman et al., Nuc. Acids Res. 27:260-262 (1999); and Sonnhammer et al., Nuc. Acids Res. 26:320-322-(1998)).
In another embodiment, the metastatic breast cancer or metastatic lung cancer sequences are transmembrane proteins. Transmembrane proteins are molecules that span a phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both. The intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins. For example, the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyrosine kinases have both protein kinase activity and SH2 domains. In addition, autophosphorylation of tyrosines on the receptor molecule itself, creates binding sites for additional SH2 domain containing proteins.
Transmembrane proteins may contain from one to many transmembrane domains. For example, receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl cyclases and receptor serine/threonine protein kinases contain a single transmembrane domain. However, various other proteins including channels, pumps, and adenylyl cyclases contain numerous transmembrane domains. Many important cell surface receptors such as G protein coupled receptors (GPCRs) are classified as “seven transmembrane domain” proteins, as they contain 7 membrane spanning regions. Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted (see, e.g. PSORT web site).
The extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly among various extracellular domains. Conserved structure and/or functions have been ascribed to different extracellular motifs. Many extracellular domains are involved in binding to other molecules. In one aspect, extracellular domains are found on receptors. Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like. For example, growth factors such as EGF, FGF and PDGF are circulating growth factors that bind to their cognate receptors to initiate a variety of cellular responses. Other factors include cytokines, mitogenic factors, hormones, neurotrophic factors and the like. Extracellular domains also bind to cell-associated molecules. In this respect, they mediate cell-cell interactions. Cell-associated ligands can be tethered to the cell, e.g., via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins. Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
Metastatic breast cancer or metastatic lung cancer proteins that are transmembrane are particularly preferred in the present invention as they are readily accessible targets for extracellular immunotherapeutics, as are described herein. In addition, as outlined below, transmembrane proteins can be also useful in imaging modalities. Antibodies may be used to label such readily accessible proteins in situ or in histological analysis. Alternatively, antibodies can also label intracellular proteins, in which case analytical samples are typically permeablized to provide access to intracellular proteins.
It will also be appreciated by those in the art that a transmembrane protein can be made soluble by removing transmembrane sequences, e.g., through recombinant methods. Furthermore, transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
In another embodiment, the metastatic breast cancer or metastatic lung cancer proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or signal sequence that targets the molecule to the secretory pathway. Secreted proteins are involved in numerous physiological events; by virtue of their circulating nature, they often serve to transmit signals to various other cell types. The secreted protein may function in an autocrine manner (acting on the cell that secreted the factor), a paracrine manner (acting on cells in close proximity to the cell that secreted the factor) or an endocrine manner (acting on cells at a distance). Thus secreted molecules find use in modulating or altering numerous aspects of physiology. Metastatic breast cancer or metastatic lung cancer proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, e.g., for blood, plasma, serum, or stool tests.
As described above, metastatic breast cancer or metastatic lung cancer sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology or linkage to the metastatic breast cancer or metastatic lung cancer sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions. Typically, linked sequences on a mRNA are found on the same molecule.
The metastatic breast cancer or metastatic lung cancer nucleic acid sequences of the invention, e.g., the sequences in Tables 1-12, can be fragments of larger genes, i.e., they are nucleic acid segments. “Genes” in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, extended sequences, in either direction, of the metastatic breast cancer or metastatic lung cancer genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Ausubel, et al., supra. Much can be done by informatics and many sequences can be clustered to include multiple sequences corresponding to a single gene, e.g., systems such as UniGene.
Once the metastatic breast cancer or metastatic lung cancer nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire metastatic breast cancer or metastatic lung cancer nucleic acid coding regions or the entire mRNA sequence. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant metastatic breast cancer or metastatic lung cancer nucleic acid can be further-used as a probe to identify and isolate other metastatic breast cancer or metastatic lung cancer nucleic acids, e.g., extended coding regions. It can also be used as a “precursor” nucleic acid to make modified or variant metastatic breast cancer or metastatic lung cancer nucleic acids and proteins.
The metastatic breast cancer or metastatic lung cancer nucleic acids of the present invention are used in several ways. In a first embodiment, nucleic acid probes to the metastatic breast cancer or metastatic lung cancer nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, e.g., for gene therapy, vaccine, and/or antisense applications. Alternatively, the metastatic breast cancer or metastatic lung cancer nucleic acids that include coding regions of metastatic breast cancer or metastatic lung cancer proteins can be put into expression vectors for the expression of metastatic breast cancer or metastatic lung cancer proteins, again for screening purposes or for administration to a patient.
In a preferred embodiment, nucleic acid probes to metastatic breast cancer or metastatic lung cancer nucleic acids (both the nucleic acid sequences outlined in the figures and/or the complements thereof) are made. The nucleic acid probes attached to the biochip are designed to be substantially complementary to the metastatic breast cancer or metastatic lung cancer nucleic acids, i.e. the target sequence (either the target sequence of the sample or to other probe sequences, e.g., in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by “substantially complementary” herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under appropriate reaction conditions, particularly high stringency conditions, as outlined herein.
A nucleic acid probe is generally single stranded but can be partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. In general, the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally complements of ORFs or whole genes are not used. In some embodiments, nucleic acids of lengths up to hundreds of bases can be used.
In a preferred embodiment, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e., have some sequence in common), or separate. In some cases, PCR primers may be used to amplify signal for higher sensitivity.
As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By “immobilized” and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can typically be covalent or non-covalent. By “non-covalent binding” and grammatical equivalents herein is typically meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By “covalent binding” and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
In general, the probes are attached to a biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
The biochip comprises a suitable solid substrate. By “substrate” or “solid support” or other grammatical equivalents herein is meant a material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluoresce. A preferred substrate is described in copending application entitled Reusable Low Fluorescent Plastic Biochip, U.S. application Ser. No. 09/270,214, filed Mar. 15, 1999, herein incorporated by reference in its entirety.
Generally the substrate is planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
In a preferred embodiment, the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. Thus, e.g., the biochip is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred. Using these functional groups, the probes can be attached using functional groups on the probes. For example, nucleic acids containing amino groups can be attached to surfaces comprising amino groups, e.g., using linkers as are known in the art; e.g., homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200). In addition, in some cases, additional linkers, such as alkyl groups (including substituted and heteroalkyl groups) may be used.
In this embodiment, oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5′ or 3′ terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
In another embodiment, the immobilization to the solid support may be very strong, yet non-covalent. For example, biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques are used. In a preferred embodiment, the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the GENECHIP® (DNA microarray) technology from Affymetrix, Inc. (Santa Clara, Calif.).
Often, amplification-based assays are performed to measure the expression level of metastatic breast cancer or metastatic lung cancer-associated sequences. These assays are typically performed in conjunction with reverse transcription. In such assays, a metastatic breast cancer or metastatic lung cancer-associated nucleic acid sequence acts as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the amount of metastatic breast cancer or metastatic lung cancer-associated RNA. Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (1990).
In some embodiments, a TAQMAN® (PCR reagent kit) based assay is used to measure expression. TAQMAN® (PCR reagent kit) based assays use a fluorogenic oligonucleotide probe that contains a 5′ fluorescent dye and a 3′ quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3′ end. When the PCR product is amplified in subsequent cycles, the 5′ nuclease activity of the polymerase, e.g., AMPLITAQO (PCR enzyme reagent), results in the cleavage of the TAQMAN® (PCR reagent kit) probe. This cleavage separates the 5′ fluorescent dye and the 3′ quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, e.g., literature provided by Perkin-Elmer, e.g., www2.perkin-elmer.com).
Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu & Wallace, Genomics 4:560 (1989), Landegren et al., Science 241:1077 (1988), and Barringer et al., Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA 87:1874 (1990)), dot PCR, and linker adapter PCR, etc.
In a preferred embodiment, metastatic breast cancer or metastatic lung cancer nucleic acids, e.g., encoding metastatic breast cancer or metastatic lung cancer proteins, are used to make a variety of expression vectors to express metastatic breast cancer or metastatic lung cancer proteins which can then be used in screening assays, as described below. Expression vectors and recombinant DNA technology are well known to those of skill in the art (see, e.g., Ausubel, supra, and Gene Expression Systems (Fernandez & Hoeffler, eds, 1999)) and are used to express proteins. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the metastatic breast cancer or metastatic lung cancer protein. The term “control sequences” refers to DNA sequences used for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, e.g., include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is typically accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the metastatic breast cancer or metastatic lung cancer protein. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
In general, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.
Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
In addition, an expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art (e.g., Fernandez & Hoeffler, supra).
In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
The metastatic breast cancer or metastatic lung cancer proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a metastatic breast cancer or metastatic lung cancer protein, under the appropriate conditions to induce or cause expression of the metastatic breast cancer or metastatic lung cancer protein. Conditions appropriate for metastatic breast cancer or metastatic lung cancer protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation or optimization. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial cells), THPl cells (a macrophage cell line) and various other human cells and cell lines.
In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer proteins are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral and adenoviral systems. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter (see, e.g., Fernandez & Hoeffler, supra). Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenylation signals include those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
In a preferred embodiment, metastatic breast cancer or metastatic lung cancer proteins are expressed in bacterial systems. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; e.g., the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the metastatic breast cancer or metastatic lung cancer protein in bacteria. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others (e.g., Fernandez & Hoeffler, supra). The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
In one embodiment, metastatic breast cancer or metastatic lung cancer proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
In a preferred embodiment, metastatic breast cancer or metastatic lung cancer protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
The metastatic breast cancer or metastatic lung cancer protein may also be made as a fusion protein, using techniques well known in the art. Thus, e.g., for the creation of monoclonal antibodies, if the desired epitope is small, the metastatic breast cancer or metastatic lung cancer protein may be fused to a carrier protein to form an immunogen. Alternatively, the metastatic breast cancer or metastatic lung cancer protein may be made as a fusion protein to increase expression for affinity purification purposes, or for other reasons. For example, when the metastatic breast cancer or metastatic lung cancer protein is a metastatic breast cancer or metastatic lung cancer peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer protein is purified or isolated after expression. Metastatic breast cancer or metastatic lung cancer proteins may be isolated or purified in a variety of appropriate ways. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the metastatic breast cancer or metastatic lung cancer protein may be purified using a standard anti-metastatic breast cancer or metastatic lung cancer protein antibody colurn. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, Protein Purification (1982). The degree of purification necessary will vary depending on the use of the metastatic breast cancer or metastatic lung cancer protein. In some instances no purification will be necessary.
Once expressed and purified if necessary, the metastatic breast cancer or metastatic lung cancer proteins and nucleic acids are useful in a number of applications. They may be used as immunoselection reagents, as vaccine reagents, as screening agents, etc.
In one embodiment, the metastatic breast cancer or metastatic lung cancer proteins are derivative or variant metastatic breast cancer or metastatic lung cancer proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative metastatic breast cancer or metastatic lung cancer peptide will often contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. The amino acid substitution, insertion or deletion may occur at a particular residue within the metastatic breast cancer or metastatic lung cancer peptide.
Also included within one embodiment of metastatic breast cancer or metastatic lung cancer proteins of the present invention are amino acid sequence variants. These variants typically fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the metastatic breast cancer or metastatic lung cancer protein, using cassette or PCR mutagenesis or other techniques, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant metastatic breast cancer or metastatic lung cancer protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the metastatic breast cancer or metastatic lung cancer protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
While the site or region for introducing an amino acid sequence variation is often predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed metastatic breast cancer or metastatic lung cancer variants screened for the optimal combination of desired activity. Techniques exist for making substitution mutations at predetermined sites in DNA having a known sequence, e.g., M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of metastatic breast cancer or metastatic lung cancer protein activities.
Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be occasionally tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. Larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of a metastatic breast cancer or metastatic lung cancer protein are desired, substitutions are generally made in accordance with the amino acid substitution chart provided in the definition section.
Variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analog, although variants also are selected to modify the characteristics of the metastatic breast cancer or metastatic lung cancer proteins as needed. Alternatively, the variant may be designed or reorganized such that the biological activity of the metastatic breast cancer or metastatic lung cancer protein is altered. For example, glycosylation sites may be altered or removed.
Covalent modifications of metastatic breast cancer or metastatic lung cancer polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a metastatic breast cancer or metastatic lung cancer polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a metastatic breast cancer or metastatic lung cancer polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking metastatic breast cancer or metastatic lung cancer polypeptides to a water-insoluble support matrix or surface for use in the method for purifying anti-metastatic breast cancer or metastatic lung cancer polypeptide antibodies or screening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifinctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-((p-azidophenyl)dithio)propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the γ-amino groups of lysine, arginine, and histidine side chains (Creighton, Proteins: Structure and Molecular Properties, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the metastatic breast cancer or metastatic lung cancer polypeptide encompassed by this invention is an altered native glycosylation pattern of the polypeptide. “Altering the native glycosylation pattern” is intended herein to mean adding to or deleting one or more carbohydrate moieties of a native sequence metastatic breast cancer or metastatic lung cancer polypeptide. Glycosylation patterns can be altered in many ways. For example the use of different cell types to express metastatic breast cancer or metastatic lung cancer-associated sequences can result in different glycosylation patterns.
Addition of glycosylation sites to metastatic breast cancer or metastatic lung cancer polypeptides may also be accomplished by altering the amino acid sequence thereof. The alteration may be made, e.g., by the addition of, or substitution by, one or more serine or threonine residues to the native sequence metastatic breast cancer or metastatic lung cancer polypeptide (for O-linked glycosylation sites). The metastatic breast cancer or metastatic lung cancer amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the metastatic breast cancer or metastatic lung cancer polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the metastatic breast cancer or metastatic lung cancer polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330, and in Aplin & Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the metastatic breast cancer or metastatic lung cancer polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
Another type of covalent modification of metastatic breast cancer or metastatic lung cancer comprises linking the metastatic breast cancer or metastatic lung cancer polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, each of which is hereby incorporated by reference herein.
Metastatic breast cancer or metastatic lung cancer polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a metastatic breast cancer or metastatic lung cancer polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of a metastatic breast cancer or metastatic lung cancer polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino-or carboxyl-terminus of the metastatic breast cancer or metastatic lung cancer polypeptide. The presence of such epitope-tagged forms of a metastatic breast cancer or metastatic lung cancer polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the metastatic breast cancer or metastatic lung cancer polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of a metastatic breast cancer or metastatic lung cancer polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
Various tag polypeptides and their respective antibodies are well known and examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelation tags, the flu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3(6):547-553 (1990)). Other tag polypeptides include the Flag-peptide (Hopp et al., BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science 255:192-194 (1992)); tubulin epitope peptide (Skinner et al., J Biol. Chem. 266:15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA 87:6393-6397 (1990)).
Also included are other metastatic breast cancer or metastatic lung cancer proteins of the metastatic breast cancer or metastatic lung cancer family, and metastatic breast cancer or metastatic lung cancer proteins from other organisms, which are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related metastatic breast cancer or metastatic lung cancer proteins from primates or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include unique areas of the metastatic breast cancer or metastatic lung cancer nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. PCR reaction conditions are well known in the art (e.g., Innis, PCR Protocols, supra).
In a preferred embodiment, when a metastatic breast cancer or metastatic lung cancer protein is to be used to generate antibodies, e.g., for immunotherapy or immunodiagnosis, the metastatic breast cancer or metastatic lung cancer protein should share at least one epitope or determinant with the full length protein. By “epitope” or “determinant” herein is typically meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller metastatic breast cancer or metastatic lung cancer protein will be able to bind to the full-length protein, particularly linear epitopes. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
Methods of preparing polyclonal antibodies are well known (e.g., Coligan, supra; and Harlow & Lane, supra). Polyclonal antibodies can be raised in a mammal, e.g., by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of Tables 1-12 or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Immunogenic proteins include, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Adjuvants include, e.g., Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art.
The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler & Milstein, Nature 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a polypeptide encoded by a nucleic acid of Tables 1-12, or fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (1986)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and primate origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
In one embodiment, the antibodies are bispecific antibodies. Bispecific antibodies are typically monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen. In one embodiment, one of the binding specificities is for a protein encoded by a nucleic acid of Tables 1-12 or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific. Alternatively, tetramer-type technology may create multivalent reagents.
In a preferred embodiment, the antibodies to metastatic breast cancer or metastatic lung cancer protein are capable of reducing or eliminating a biological function of a metastatic breast cancer or metastatic lung cancer protein, as is described below. That is, the addition of anti-metastatic breast cancer or metastatic lung cancer protein antibodies (either polyclonal or preferably monoclonal) to metastatic breast cancer or metastatic lung cancer tissue (or cells containing metastatic breast cancer or metastatic lung cancer) may reduce or eliminate the metastatic breast cancer or metastatic lung cancer. Generally, at least a 25% decrease in activity, growth, size or the like is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
In a preferred embodiment the antibodies to the metastatic breast cancer or metastatic lung cancer proteins are humanized antibodies (e.g., Xenerex Biosciences, Mederex, Inc., Abgenix, Inc., Protein Design Labs, Inc.) Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);. and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567, which is hereby incorporated by reference herein.), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
Human-like antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in virtually all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, each of which is hereby incorporated by reference herein, and in the following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995).
By immunotherapy is meant treatment of metastatic breast cancer or metastatic lung cancer with an antibody raised against a metastatic breast cancer or metastatic lung cancer proteins. As used herein, immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient). Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient). Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised. The antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen, leading to an immune response.
In a preferred embodiment the metastatic breast cancer or metastatic lung cancer proteins against which antibodies are raised are secreted proteins as described above. Without being bound by theory, antibodies used for treatment, bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted metastatic breast cancer or metastatic lung cancer protein.
In another preferred embodiment, the metastatic breast cancer or metastatic lung cancer protein to which antibodies are raised is a transmembrane protein. Without being bound by theory, antibodies used for this treatment typically bind the extracellular domain of the metastatic breast cancer or metastatic lung cancer protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules. The antibody may cause down-regulation of the transmembrane metastatic breast cancer or metastatic lung cancer protein. The antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the metastatic breast cancer or metastatic lung cancer protein. The antibody may be an antagonist of the metastatic breast cancer or metastatic lung cancer protein or may prevent activation of the transmembrane metastatic breast cancer or metastatic lung cancer protein. In some embodiments, when the antibody prevents the binding of other molecules to the metastatic breast cancer or metastatic lung cancer protein, the antibody prevents growth of the cell. The antibody may also be used to target or sensitize the cell to cytotoxic agents, including, but not limited to TNF-α, TNF-β, IL-1, INF-γ and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like. In some instances the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC). Thus, metastatic breast cancer or metastatic lung cancer is treated by administering to a patient antibodies directed against the transmembrane metastatic breast cancer or metastatic lung cancer protein. Antibody-labeling may activate a co-toxin, localize a toxin payload, or otherwise provide means to locally ablate cells.
In another preferred embodiment, the antibody is conjugated to an effector moiety. The effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety. In one aspect the therapeutic moiety is a small molecule that modulates the activity of the metastatic breast cancer or metastatic lung cancer protein. In another aspect the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the metastatic breast cancer or metastatic lung cancer protein. The therapeutic moiety may inhibit enzymatic activity such as protease or collagenase activity associated with metastatic breast cancer or metastatic lung cancer.
In a preferred embodiment, the therapeutic moiety can also be a cytotoxic agent. In this method, targeting the cytotoxic agent to metastatic breast cancer or metastatic lung cancer tissue or cells. results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with metastatic breast cancer or metastatic lung cancer. Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against metastatic breast cancer or metastatic lung cancer proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. Targeting the therapeutic moiety to transmembrane metastatic breast cancer or metastatic lung cancer proteins not only serves to increase the local concentration of therapeutic moiety in the metastatic breast cancer or metastatic lung cancer afflicted area, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
In another preferred embodiment, the metastatic breast cancer or metastatic lung cancer protein against which the antibodies are raised is an intracellular protein. In this case, the antibody may be conjugated to a protein or other entity which facilitates entry into the cell. In one case, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Moreover, wherein the metastatic breast cancer or metastatic lung cancer protein can be targeted within a cell, i.e., the nucleus, an antibody thereto contains a signal for that target localization, i.e., a nuclear localization signal.
The metastatic breast cancer or metastatic lung cancer antibodies of the invention specifically bind to metastatic breast cancer or metastatic lung cancer proteins. By “specifically bind” herein is meant that the antibodies bind to the protein with a Kd of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better. Selectivity of binding is also important.
In one aspect, the RNA expression levels of genes are determined for different cellular states in the metastatic breast cancer or metastatic lung cancer phenotype. Expression levels of genes in normal tissue (i.e., not undergoing metastatic breast cancer or metastatic lung cancer) and in metastatic breast cancer or metastatic lung cancer tissue (and in some cases, for varying severities of metastatic breast cancer or metastatic lung cancer that relate to prognosis, as outlined below) are evaluated to provide expression profiles. An expression profile of a particular cell state or point of development is essentially a “fingerprint” of the state. While two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is reflective of the state of the cell. By comparing expression profiles of cells in different states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. Then, diagnosis may be performed or confirmed to determine whether a tissue sample has the gene expression profile of normal or cancerous tissue. This will provide for molecular diagnosis of related conditions.
“Differential expression,” or grammatical equivalents as used herein, refers to qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue. Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus metastatic breast cancer or metastatic lung cancer tissue. Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states. A qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques. Some genes will be expressed in one state or cell type, but not in both. Alternatively, the difference in expression may be quantitative, e.g., in that expression is increased or decreased; i.e., gene expression is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript. The degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of GENECHIP® (DNA microarray) expression arrays from Affymetrix, Inc. (Santa Clara, Calif.), as described in Lockhart et al., Nature Biotechnology 14:1675-1680 (1996), hereby expressly incorporated by reference. Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, northern analysis and RNase protection. As outlined above, preferably the change in expression (i.e., upregulation or downregulation) is typically at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, with from 300 to at least 1000% being especially preferred.
Evaluation may be at the gene transcript, or the protein level. The amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, e.g., with antibodies to the metastatic breast cancer or metastatic lung cancer protein and standard immunoassays (ELISAs, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc. Proteins corresponding to metastatic breast cancer or metastatic lung cancer genes, i.e., those identified as being important in a metastatic breast cancer or metastatic lung cancer phenotype, can be evaluated in a metastatic breast cancer or metastatic lung cancer diagnostic test.
In a preferred embodiment, gene expression monitoring is performed simultaneously on a number of genes.
The metastatic breast cancer or metastatic lung cancer nucleic acid probes may be attached to biochips as outlined herein for the detection and quantification of metastatic breast cancer or metastatic lung cancer sequences in a particular cell. The assays are further described below in the example. PCR techniques can be used to provide greater sensitivity. Multiple protein expression monitoring can be performed as well. Similarly, these assays may be performed on an individual basis as well.
In a preferred embodiment nucleic acids encoding the metastatic breast cancer or metastatic lung cancer protein are detected. Although DNA or RNA encoding the metastatic breast cancer or metastatic lung cancer protein may be detected, of particular interest are methods wherein an mRNA encoding a metastatic breast cancer or metastatic lung cancer protein is detected. Probes to detect mRNA can be a nucleotide/deoxynucleotide probe that is complementary to and hybridizes with the mRNA and includes, but is not limited to, oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein. In one method the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample. Following washing to remove the non-specifically bound probe, the label is detected. In another method detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-specifically bound probe, the label is detected. For example a digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding a metastatic breast cancer or metastatic lung cancer protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
In a preferred embodiment, various proteins from the three classes of proteins as described herein (secreted, transmembrane or intracellular proteins) are used in diagnostic assays. The metastatic breast cancer or metastatic lung cancer proteins, antibodies, nucleic acids, modified proteins and cells containing metastatic breast cancer or metastatic lung cancer sequences are used in diagnostic assays. This can be performed on an individual gene or corresponding polypeptide level. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes and/or corresponding polypeptides.
As described and defined herein, metastatic breast cancer or metastatic lung cancer proteins, including intracellular, transmembrane or secreted proteins, find use as markers of metastatic breast cancer or metastatic lung cancer. Detection of these proteins in putative metastatic breast cancer or metastatic lung cancer tissue allows for detection or diagnosis of metastatic breast cancer or metastatic lung cancer. In one embodiment, antibodies are used to detect metastatic breast cancer or metastatic lung cancer proteins. A preferred method separates proteins from a sample by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be another type of gel, including isoelectric focusing gels and the like). Following separation of proteins, the metastatic breast cancer or metastatic lung cancer protein is detected, e.g., by immunoblotting with antibodies raised against the metastatic breast cancer or metastatic lung cancer protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
In another preferred method, antibodies to the metastatic breast cancer or metastatic lung cancer protein find use in in situ imaging techniques, e.g., in histology (e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993)). In this method cells are contacted with from one to many antibodies to the metastatic breast cancer or metastatic lung cancer protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected. In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label, e.g., multicolor fluorescence or confocal imaging. In another method the primary antibody to the metastatic breast cancer or metastatic lung cancer protein(s) contains a detectable label, e.g., an enzyme marker that can act on a substrate. In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of metastatic breast cancer or metastatic lung cancer proteins. Many other histological imaging techniques are also provided by the invention.
In a preferred embodiment the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used in the method.
In another preferred embodiment, antibodies find use in diagnosing metastatic breast cancer or metastatic lung cancer from blood, serum, plasma, stool, and other samples. Such samples, therefore, are useful as samples to be probed or tested for the presence of metastatic breast cancer or metastatic lung cancer proteins. Antibodies can be used to detect a metastatic breast cancer or metastatic lung cancer protein by previously described immunoassay techniques including ELISA, immunoblotting (western blotting), immunoprecipitation, BIACORE technology and the like. Conversely, the presence of antibodies may indicate an immune response against an endogenous metastatic breast cancer or metastatic lung cancer protein or vaccine.
In a preferred embodiment, in situ hybridization of labeled metastatic breast cancer or metastatic lung cancer nucleic acid probes to tissue arrays is done. For example, arrays of tissue samples, including metastatic breast cancer or metastatic lung cancer tissue and/or normal tissue, are made. In situ hybridization (see, e.g., Ausubel, supra) is then performed. When comparing the fingerprints between an individual and a standard, the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes.
In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer proteins, antibodies, nucleic acids, modified proteins and cells containing metastatic breast cancer or metastatic lung cancer sequences are used in prognosis assays. As above, gene expression profiles can be generated that correlate to metastatic breast cancer or metastatic lung cancer, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred. As above, metastatic breast cancer or metastatic lung cancer probes may be attached to biochips for the detection and quantification of metastatic breast cancer or metastatic lung cancer sequences in a tissue or patient. The assays proceed as outlined above for diagnosis. PCR method may provide more sensitive and accurate quantification.
In a preferred embodiment members of the three classes of proteins as described herein are used in drug screening assays. The metastatic breast cancer or metastatic lung cancer proteins, antibodies, nucleic acids, modified proteins and cells containing metastatic breast cancer or metastatic lung cancer sequences are used in drug screening assays or by evaluating the effect of drug candidates on a “gene expression profile” or expression profile of polypeptides. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).
In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified metastatic breast cancer or metastatic lung cancer proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the metastatic breast cancer or metastatic lung cancer phenotype or an identified physiological function of a metastatic breast cancer or metastatic lung cancer protein. As above, this can be done on an individual gene level or by evaluating the effect of drug candidates on a “gene expression profile”. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokamik, supra.
Having identified the differentially expressed genes herein, a variety of assays may be applied. In a preferred embodiment, assays may be run on an individual gene or protein level. That is, having identified a particular gene with altered regulation in metastatic breast cancer or metastatic lung cancer, test compounds can be screened for the ability to modulate gene expression or for binding to the metastatic breast cancer or metastatic lung cancer protein. “Modulation” thus includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing metastatic breast cancer or metastatic lung cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in metastatic breast cancer or metastatic lung cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in metastatic breast cancer or metastatic lung cancer tissue compared to normal tissue often provides a target value of a 10-fold increase in expression to be induced by the test compound.
The amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the gene product itself can be monitored, e.g., through the use of antibodies to the metastatic breast cancer or metastatic lung cancer protein and standard immunoassays. Proteomics and separation techniques may also allow quantification of expression.
In a preferred embodiment, gene or protein expression monitoring of a number of entities, i.e., an expression profile, is monitored simultaneously. Such profiles will typically involve a plurality of those entities described herein.
In this embodiment, the metastatic breast cancer or metastatic lung cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of metastatic breast cancer or metastatic lung cancer sequences in a particular cell. Alternatively, PCR may be used. Thus, a series, e.g., of microtiter plate, may be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
Expression monitoring can be performed to identify compounds that modify the expression of one or more metastatic breast cancer or metastatic lung cancer-associated sequences, e.g., a polynucleotide sequence set out in Tables 1-12. Generally, in a preferred embodiment, a test compound is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate metastatic breast cancer or metastatic lung cancer, modulate metastatic breast cancer or metastatic lung cancer proteins, bind to a metastatic breast cancer or metastatic lung cancer protein, or interfere with the binding of a metastatic breast cancer or metastatic lung cancer protein and an antibody, substrate, or other binding partner.
The term “test compound” or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the metastatic breast cancer or metastatic lung cancer phenotype or the expression of a metastatic breast cancer or metastatic lung cancer sequence, e.g., a nucleic acid or protein sequence. In preferred embodiments, modulators alter expression profiles of nucleic acids or proteins provided herein. In one embodiment, the modulator suppresses a metastatic breast cancer or metastatic lung cancer phenotype, e.g., to a normal tissue fingerprint. In another embodiment, a modulator induces a metastatic breast cancer or metastatic lung cancer phenotype. Generally, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
In one aspect, a modulator will neutralize the effect of a metastatic breast cancer or metastatic lung cancer protein. By “neutralize” is meant that activity of a protein and the consequent effect on the cell is inhibited or blocked.
In certain embodiments, combinatorial libraries of potential modulators will be screened for an ability to bind to a metastatic breast cancer or metastatic lung cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9):1233-1251 (1994)).
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514, which is hereby incorporated by reference herein), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083, which is hereby incorporated by reference herein), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3):309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Pat. No. 5,593,853, which is hereby incorporated by reference herein), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like, each of which is hereby incorporated by reference herein).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).
A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. The above devices, with appropriate modification, are suitable for use with the present invention. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).
The assays to identify modulators are amenable to high throughput screening. Preferred assays thus detect modulation of metastatic breast cancer or metastatic lung cancer gene transcription, polypeptide expression, and polypeptide activity.
High throughput assays for evaluating the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins, U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding. Each of the above-cited patents is hereby incorporated by reference herein.
In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.). These systems typically automate procedures, including sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
In one embodiment, modulators are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes or ligands and receptors.
In a preferred embodiment, modulators are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. By “randomized” or grammatical equivalents herein is meant that the nucleic acid or peptide consists of essentially random sequences of nucleotides and amino acids, respectively. Since these random peptides (or nucleic acids, discussed below) are often chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. In a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc.
Modulators of metastatic breast cancer or metastatic lung cancer can also be nucleic acids, as defined above.
As described above generally for proteins, nucleic acid modulating agents may be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. Digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
In a preferred embodiment, the candidate compounds are organic chemical moieties, a wide variety of which are available in the literature.
After a candidate agent has been added and the cells allowed to incubate for some period of time, the sample containing a target sequence is analyzed. If required, the target sequence is prepared using known techniques. For example, the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with Cy-3 or Cy-5.
In a preferred embodiment, the target sequence is labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
Nucleic acid assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. No. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference herein. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc.
These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697, which is hereby incorporated by reference herein. Thus, it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein may be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target.
The assay data are analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
Screens are performed to identify modulators of the metastatic breast cancer or metastatic lung cancer phenotype. In one embodiment, screening is performed to identify modulators that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype. In another embodiment, e.g., for diagnostic applications, having identified differentially expressed genes important in a particular state, screens can be performed to identify modulators that alter expression of individual genes. In an another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product, or evaluate genetic polymorphisms.
Genes can be screened for those that are induced in response to a candidate agent. After identifying a modulator based upon its ability to suppress a metastatic breast cancer or metastatic lung cancer expression pattern leading to a normal expression pattern, or to modulate a single metastatic breast cancer or metastatic lung cancer gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated metastatic breast cancer or metastatic lung cancer tissue reveals genes that are not expressed in normal tissue or metastatic breast cancer or metastatic lung cancer tissue, but are expressed in agent treated tissue. These agent-specific sequences can be identified and used by methods described herein for metastatic breast cancer or metastatic lung cancer genes or proteins. In particular these sequences and the proteins they encode find use in marking or identifying agent treated cells. In addition, antibodies can be raised against the agent induced proteins and used to target novel therapeutics to the treated metastatic breast cancer or metastatic lung cancer tissue sample.
Thus, in one embodiment, a test compound is administered to a population of metastatic breast cancer or metastatic lung cancer cells, that have an associated metastatic breast cancer or metastatic lung cancer expression profile. By “administration” or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent (i.e., a peptide) may be put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used.
Once the test compound has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.
Thus, e.g., metastatic breast cancer or metastatic lung cancer tissue may be screened for agents that modulate, e.g., induce or suppress the metastatic breast cancer or metastatic lung cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on metastatic breast cancer or metastatic lung cancer activity. By defining such a signature for the metastatic breast cancer or metastatic lung cancer phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented in the original expression screening platform, nor does the level of transcript for the target protein need to change.
Measure of metastatic breast cancer or metastatic lung cancer polypeptide activity, or of metastatic breast cancer or metastatic lung cancer or the metastatic breast cancer or metastatic lung cancer phenotype can be performed using a variety of assays. For example, the effects of the test compounds upon the function of the metastatic polypeptides can be measured by examining parameters described above. A suitable physiological change that affects activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as, in the case of metastatic breast cancer or metastatic lung cancer associated with tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGMP. In the assays of the invention, mammalian metastatic breast cancer or metastatic lung cancer polypeptide is typically used, e.g., mouse, preferably human.
To perform assays in intact animals where the breast or lung cancer has metastasized to the brain, it may be necessary to provide special treatments to facilitate crossing of the blood brain barrier by the metastatic cancer modulator or therapeutic. Any method known in the art can be used to achieve this objective.
Assays to identify compounds with modulating activity can be performed in vitro. For example, a metastatic breast cancer or metastatic lung cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the metastatic breast cancer or metastatic lung cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the metastatic breast cancer or metastatic lung cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using the metastatic breast cancer or metastatic lung cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or β-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself can be done. The gene products of differentially expressed genes are sometimes referred to herein as “metastatic breast cancer or metastatic lung cancer proteins.” The metastatic breast cancer or metastatic lung cancer protein may be a fragment, or alternatively, be the full length protein to a fragment shown herein.
In one embodiment, screening for modulators of expression of specific genes is performed. Typically, the expression of only one or a few genes are evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
In a preferred embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more differentially expressed nucleic acids are made. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising the metastatic breast cancer or metastatic lung cancer proteins can be used in the assays.
Thus, in a preferred embodiment, the methods comprise combining a metastatic breast cancer or metastatic lung cancer protein and a candidate compound, and determining the binding of the compound to the metastatic breast cancer or metastatic lung cancer protein. Preferred embodiments utilize the human metastatic breast cancer or metastatic lung cancer protein, although other mammalian proteins may also be used, e.g., for the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative metastatic breast cancer or metastatic lung cancer proteins may be used.
Generally, in a preferred embodiment of the methods herein, the metastatic breast cancer or metastatic lung cancer protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g., a microtiter plate, an array, etc.). The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, TEFLON®, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer protein is bound to the support, and a test compound is added to the assay. Alternatively, the candidate agent is bound to the support and the metastatic breast cancer or metastatic lung cancer protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
The determination of the binding of the test modulating compound to the metastatic breast cancer or metastatic lung cancer protein may be done in a number of ways. In a preferred embodiment, the compound is labeled, and binding determined directly, e.g., by attaching all or a portion of the metastatic breast cancer or metastatic lung cancer protein to a solid support, adding a labeled candidate agent (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as appropriate.
In some embodiments, only one of the components is labeled, e.g., the proteins (or proteinaceous candidate compounds) can be labeled. Alternatively, more than one component can be labeled with different labels, e.g., 1251 for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
In one embodiment, the binding of the test compound is determined by competitive binding assay. The competitor is a binding moiety known to bind to the target molecule (i.e., a metastatic breast cancer or metastatic lung cancer protein), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding between the compound and the binding moiety, with the binding moiety displacing the compound. In one embodiment, the test compound is labeled. Either the compound, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at a temperature which facilitates optimal activity, typically between 4 and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In a preferred embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the metastatic breast cancer or metastatic lung cancer protein and thus is capable of binding to, and potentially modulating, the activity of the metastatic breast cancer or metastatic lung cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the test compound is bound to the metastatic breast cancer or metastatic lung cancer protein with a higher affinity. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the test compound is capable of binding to the metastatic breast cancer or metastatic lung cancer protein.
In a preferred embodiment, the methods comprise differential screening to identity agents that are capable of modulating the activity of the metastatic breast cancer or metastatic lung cancer proteins. In this embodiment, the methods comprise combining a metastatic breast cancer or metastatic lung cancer protein and a competitor in a first sample. A second sample comprises a test compound, a metastatic breast cancer or metastatic lung cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the metastatic breast cancer or metastatic lung cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the metastatic breast cancer or metastatic lung cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to the native metastatic breast cancer or metastatic lung cancer protein, but cannot bind to modified metastatic breast cancer or metastatic lung cancer proteins. The structure of the metastatic breast cancer or metastatic lung cancer protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect the activity of a metastatic breast cancer or metastatic lung cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
Positive controls and negative controls may be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in an order that provides for the requisite binding.
In a preferred embodiment, the invention provides methods for screening for a compound capable of modulating the activity of a metastatic breast cancer or metastatic lung cancer protein. The methods comprise adding a test compound, as defined above, to a cell comprising metastatic breast cancer or metastatic lung cancer proteins. Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes a metastatic breast cancer or metastatic lung cancer protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g., hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts). In another example, the determinations are determined at different stages of the cell cycle process.
In this way, compounds that modulate metastatic breast cancer or metastatic lung cancer agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the metastatic breast cancer or metastatic lung cancer protein. Once identified, similar structures are evaluated to identify critical structural feature of the compound.
In one embodiment, a method of inhibiting metastatic breast cancer or metastatic lung cancer cell division is provided. The method comprises administration of a metastatic breast cancer or metastatic lung cancer inhibitor. In another embodiment, a method of inhibiting metastatic breast cancer or metastatic lung cancer is provided. The method comprises administration of a metastatic breast cancer or metastatic lung cancer inhibitor. In a further embodiment, methods of treating cells or individuals with metastatic breast cancer or metastatic lung cancer are provided. The method comprises administration of a metastatic breast cancer or metastatic lung cancer inhibitor.
A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described below.
Soft Agar Growth or Colony Formation in Suspension
Normal cells require a solid substrate to attach and grow. When the cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, regenerate normal phenotype and require a solid substrate to attach and grow. Soft agar growth or colony formation in suspension assays can be used to identify modulators of metastatic breast cancer or metastatic lung cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A therapeutic compound would reduce or eliminate the host cells' ability to grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft.
Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994), herein incorporated by reference. See also, the methods section of Garkavtsev et al. (1996), supra, herein incorporated by reference.
Contact Inhibition and Density Limitation of Growth
Normal cells typically grow in a flat and organized pattern in a petri dish until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. When cells are transformed, however, the cells are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, the transformed cells grow to a higher saturation density than normal cells. This can be detected morphologically by the formation of a disoriented monolayer of cells or rounded cells in foci within the regular pattern of normal surrounding cells. Alternatively, labeling index with (3H)-thymidine at saturation density can be used to measure density limitation of growth. See Freshney (1994), supra. The transformed cells, when transfected with tumor suppressor genes, regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
In this assay, labeling index with (3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a metastatic breast cancer or metastatic lung cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined autoradiographically. See, Freshney (1994), supra.
Growth factor or Serum Dependence
Transformed cells have a lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Insti. 37:167-175 (1966); Eagle et al., J. Exp. Med. 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. Growth factor or serum dependence of transformed host cells can be compared with that of control.
Tumor Specific Markers Levels
Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, tumor vascularization, and potential interference with tumor growth. in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor angiogenesis factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)).
Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al. , J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980); Angiogenesis, tumor vascularization, and potential intereference with tumor growth. in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney Anticancer Res. 5:111-130 (1985).
Invasiveness into Matrigel
The degree of invasiveness into Matrigel or some other extracellular matrix constituent can be used as an assay to identify compounds that modulate metastatic breast cancer or metastatic lung cancer-associated sequences. Tumor cells exhibit a good correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells.
Techniques described in Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells can be measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125I and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
Tumor Growth in Vivo
Effects of metastatic breast cancer or metastatic lung cancer-associated sequences on cell growth can be tested in transgenic or immune-suppressed mice. Knock-out transgenic mice can be made, in which the metastatic breast cancer or metastatic lung cancer gene is disrupted or in which a metastatic breast cancer or metastatic lung cancer gene is inserted. Knock-out transgenic mice can be made by insertion of a marker gene or other heterologous gene into the endogenous metastatic breast cancer or metastatic lung cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous metastatic breast cancer or metastatic lung cancer gene with a mutated version of the metastatic breast cancer or metastatic lung cancer gene, or by mutating the endogenous metastatic breast cancer or metastatic lung cancer gene, e.g., by exposure to carcinogens.
A DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts will produce invasive tumors in a high proportions of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing a metastatic breast cancer or metastatic lung cancer-associated sequences are injected subcutaneously. After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth. Additionally, human tumor cells expressing the genes of the invention may be injected into immune compromised animals. Growth of these tumors, or xenografts, is compared to growth of similar human tumor cell that do not express the genes of the invention. These animals may also be used to binding assays and efficacy studies for therapeutic compounds that modulate metastatic breast cancer or metastatic lung cancer, such as antibodies or small molecules.
Antisense Polynucleotides
In certain embodiments, the activity of a metastatic breast cancer or metastatic lung cancer-associated protein is downregulated, or entirely inhibited, by the use of antisense polynucleotide, i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a metastatic breast cancer or metastatic lung cancer protein mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprehended by this invention so long as they function effectively to hybridize with the metastatic breast cancer or metastatic lung cancer protein mRNA. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.
Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems (Norwalk, Conn.). The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for metastatic breast cancer or metastatic lung cancer molecules. A preferred antisense molecule is for a metastatic breast cancer or metastatic lung cancer sequence selected from those listed in Tables 1A-12C, or for a ligand or activator thereof. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of metastatic breast cancer or metastatic lung cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0 360 257; U.S. Pat. No. 5,254,678, which is hereby incorporated by reference herein. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad. Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
Polynucleotide modulators of metastatic breast cancer or metastatic lung cancer may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of metastatic breast cancer or metastatic lung cancer may be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of an polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
Thus, in one embodiment, methods of modulating metastatic breast cancer or metastatic lung cancer in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-metastatic breast cancer or metastatic lung cancer antibody that reduces or eliminates the biological activity of an endogenous metastatic breast cancer or metastatic lung cancer protein. Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a metastatic breast cancer or metastatic lung cancer protein. This may be accomplished in any number of ways. In a preferred embodiment, e.g., when the metastatic breast cancer or metastatic lung cancer sequence is down-regulated in metastatic breast cancer or metastatic lung cancer, such state may be reversed by increasing the amount of metastatic breast cancer or metastatic lung cancer gene product in the cell. This can be accomplished, e.g., by overexpressing the endogenous metastatic breast cancer or metastatic lung cancer gene or administering a gene encoding the metastatic breast cancer or metastatic lung cancer sequence, using known gene-therapy techniques. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), e.g., as described in PCT/US93/03868, hereby incorporated by reference in its entirety. Alternatively, e.g., when the metastatic breast cancer or metastatic lung cancer sequence is up-regulated in metastatic breast cancer or metastatic lung cancer, the activity of the endogenous metastatic breast cancer or metastatic lung cancer gene is decreased, e.g., by the administration of a metastatic breast cancer or metastatic lung cancer antisense nucleic acid.
In one embodiment, the metastatic breast cancer or metastatic lung cancer proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to metastatic breast cancer or metastatic lung cancer proteins. Similarly, the metastatic breast cancer or metastatic lung cancer proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify metastatic breast cancer or metastatic lung cancer antibodies useful for production, diagnostic, or therapeutic purposes. In a preferred embodiment, the antibodies are generated to epitopes unique to a metastatic breast cancer or metastatic lung cancer protein; that is, the antibodies show little or no cross-reactivity to other proteins. The metastatic breast cancer or metastatic lung cancer antibodies may be coupled to standard affinity chromatography columns and used to purify metastatic breast cancer or metastatic lung cancer proteins. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the metastatic breast cancer or metastatic lung cancer protein.
Without being bound by theory, expression of various metastatic breast cancer or metastatic lung cancer sequences is correlated with metastatic breast cancer or metastatic lung cancer. Accordingly, disorders based on mutant or variant metastatic breast cancer or metastatic lung cancer genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant metastatic breast cancer or metastatic lung cancer genes, e.g., determining all or part of the sequence of at least one endogenous metastatic breast cancer or metastatic lung cancer genes in a cell. This may be accomplished using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the metastatic breast cancer or metastatic lung cancer genotype of an individual, e.g., determining all or part of the sequence of at least one metastatic breast cancer or metastatic lung cancer gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced metastatic breast cancer or metastatic lung cancer gene to a known metastatic breast cancer or metastatic lung cancer gene, i.e., a wild-type gene.
The sequence of all or part of the metastatic breast cancer or metastatic lung cancer gene can then be compared to the sequence of a known metastatic breast cancer or metastatic lung cancer gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc. In a preferred embodiment, the presence of a difference in the sequence between the metastatic breast cancer or metastatic lung cancer gene of the patient and the known metastatic breast cancer or metastatic lung cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the metastatic breast cancer or metastatic lung cancer genes are used as probes to determine the number of copies of the metastatic breast cancer or metastatic lung cancer gene in the genome.
In another preferred embodiment, the metastatic breast cancer or metastatic lung cancer genes are used as probes to determine the chromosomal localization of the metastatic breast cancer or metastatic lung cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the metastatic breast cancer or metastatic lung cancer gene locus.
In one embodiment, a therapeutically effective dose of a metastatic breast cancer or metastatic lung cancer protein or modulator thereof, is administered to a patient. By “therapeutically effective dose” herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery; Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd, The Art, Science and Technology of pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)). As is known in the art, adjustments for metastatic breast cancer or metastatic lung cancer degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
A “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is human.
The administration of the metastatic breast cancer or metastatic lung cancer proteins and modulators thereof of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, e.g., in the treatment of wounds and inflammation, the metastatic breast cancer or metastatic lung cancer proteins and modulators may be directly applied as a solution or spray.
The pharmaceutical compositions of the present invention comprise a metastatic breast cancer or metastatic lung cancer protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, ftimaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges. It is recognized that metastatic breast cancer or metastatic lung cancer protein modulators (e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc.) when administered orally, should be protected from digestion. It is also recognized that, after delivery to other sites in the body (e.g., circulatory system, lymphatic system, or the tumor site) the metastatic breast cancer or metastatic lung cancer modulators of the invention may need to be protected from excretion, hydrolisis, proteolytic digestion or modification, or detoxification by the liver. In all these cases, protection is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic. hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome or a protection barrier or by modifying the molecular size, weight, and/or charge of the modulator. Means of protecting agents from digestion degradation, and excretion are well known in the art.
The compositions for administration will commonly comprise a metastatic breast cancer or metastatic lung cancer protein modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacologial Basis of Therapeutics (Hardman et al., eds., 1996)).
Thus, a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art, e.g., Remington's Pharmaceutical Science and Goodman and Gillman, The Pharmacologial Basis of Therapeutics, supra.
The compositions containing modulators of metastatic breast cancer or metastatic lung cancer proteins can be administered for therapeutic or prophylactic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease (e.g., a cancer) in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient. An amount of modulator that is capable of preventing or slowing the development of cancer in a mammal is referred to as a “prophylactically effective dose.” The particular dose required for a prophylactic treatment will depend upon the medical condition and history of the mammal, the particular cancer being prevented, as well as other factors such as age, weight, gender, administration route, efficiency, etc. Such prophylactic treatments may be used, e.g., in a mammal who has previously had cancer to prevent a recurrence of the cancer, or in a mammal who is suspected of having a significant likelihood of developing cancer.
It will be appreciated that the present metastatic breast cancer or metastatic lung cancer protein-modulating compounds can be administered alone or in combination with additional metastatic breast cancer or metastatic lung cancer modulating compounds or with other therapeutic agent, e.g., other anti-cancer agents or treatments.
In numerous embodiments, one or more nucleic acids, e.g., polynucleotides comprising nucleic acid sequences set forth in Tables 1A-12C, such as antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo. The present invention provides methods, reagents, vectors, and cells useful for expression of metastatic breast cancer or metastatic lung cancer-associated polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.
The particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific. Many procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Berger & Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 (Berger), Ausubel et al., eds., Current Protocols (supplemented through 1999), and Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd ed., Vol. 1-3, 1989.
In a preferred embodiment, metastatic breast cancer or metastatic lung cancer proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, metastatic breast cancer or metastatic lung cancer genes (including both the full-length sequence, partial sequences, or regulatory sequences of the metastatic breast cancer or metastatic lung cancer coding regions) can be administered in a gene therapy application. These metastatic breast cancer or metastatic lung cancer genes can include antisense applications, either as gene therapy (i.e., for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
Metastatic breast cancer or metastatic lung cancer polypeptides and polynucleotides can also be administered as vaccine compositions to stimulate HTL, CTL and antibody responses. Such vaccine compositions can include, e.g., lipidated peptides (see, e.g.,Vitiello, et al., J. Clin. Invest. 95:341 (1995)), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, (1991); Alonso et al., Vaccine 12:299-306 (1994); Jones et al., Vaccine 13:675-681 (1995)), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875 (1990); Hu et al., Clin Exp Immunol. 113:235-243 (1998)), multiple antigen peptide systems (MAPs) (see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413 (1988); Tam, J. Immunol. Methods 196:17-32 (1996)), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, et al., In: Concepts in vaccine development (Kaufmann, ed., p. 379, 1996); Chakrabarti, et al., Nature 320:535 (1986); Hu et al., Nature 320:537 (1986); Kieny, et al., AIDS Bio/Technology 4:790 (1986); Top et al., J. Infect. Dis. 124:148 (1971); Chanda et al., Virology 175:535 (1990)), particles of viral or synthetic origin (see, e.g., Kofler et al., J. Immunol. Methods. 192:25 (1996); Eldridge et al., Sem. Hematol. 30:16 (1993); Falo et al., Nature Med. 7:649 (1995)), adjuvants (Warren et al., Annu. Rev. Immunol. 4:369 (1986); Gupta et al., Vaccine 11:293 (1993)), liposomes (Reddy et al., J. Immunol. 148:1585 (1992); Rock, Immunol. Today 17:131 (1996)), or, naked or particle absorbed cDNA (Ulmer, et al., Science 259:1745 (1993); Robinson et al., Vaccine 11:957 (1993); Shiver et al., In: Concepts in vaccine development (Kaufinann, ed., p. 423, 1996); Cease & Berzofsky, Annu. Rev. Immunol. 12:923 (1994) and Eldridge et al., Sem. Hematol. 30:16 (1993)). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
Vaccine compositions often include adjuvants. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, e.g., Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
Vaccines can be administered as nucleic acid compositions wherein DNA or RNA encoding one or more of the polypeptides, or a fragment thereof, is administered to a patient. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720, each of which is hereby incorporated by reference herein.; and in more detail below. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687, which is hereby incorporated by reference herein.).
For therapeutic or prophylactic immunization purposes, the peptides of the invention can be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode metastatic breast cancer or metastatic lung cancer polypeptides or polypeptide fragments. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848, which is hereby incorporated by reference herein. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein (see, e.g., Shata et al., Mol Med Today 6:66-71 (2000); Shedlock et al., J Leukoc Biol 68:793-806 (2000); Hipp et al., In Vivo 14:571-85 (2000)).
Methods for the use of genes as DNA vaccines are well known, and include placing a metastatic breast cancer or metastatic lung cancer gene or portion of a metastatic breast cancer or metastatic lung cancer gene under the control of a regulatable promoter or a tissue-specific promoter for expression in a metastatic breast cancer or metastatic lung cancer patient. The metastatic breast cancer or metastatic lung cancer gene used for DNA vaccines can encode full-length metastatic breast cancer or metastatic lung cancer proteins, but more preferably encodes portions of the metastatic breast cancer or metastatic lung cancer proteins including peptides derived from the metastatic breast cancer or metastatic lung cancer protein. In one embodiment, a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a metastatic breast cancer or metastatic lung cancer gene. For example, metastatic breast cancer or metastatic lung cancer-associated genes or sequence encoding subfragments of a metastatic breast cancer or metastatic lung cancer protein are introduced into expression vectors and tested for their immunogenicity in the context of Class I MHC and an ability to generate cytotoxic T cell responses. This procedure provides for production of cytotoxic T cell responses against cells which present antigen, including intracellular epitopes.
In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine. Such adjuvant molecules include cytokines that increase the immunogenic response to the metastatic breast cancer or metastatic lung cancer polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are available.
In another preferred embodiment metastatic breast cancer or metastatic lung cancer genes find use in generating animal models of metastatic breast cancer or metastatic lung cancer. When the metastatic breast cancer or metastatic lung cancer gene identified is repressed or diminished in metastatic tissue, gene therapy technology, e.g., wherein antisense RNA directed to the metastatic breast cancer or metastatic lung cancer gene will also diminish or repress expression of the gene. Animal models of metastatic breast cancer or metastatic lung cancer find use in screening for modulators of a metastatic breast cancer or metastatic lung cancer-associated sequence or modulators of metastatic breast cancer or metastatic lung cancer. Similarly, transgenic animal technology including gene knockout technology, e.g., as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence or increased expression of the metastatic breast cancer or metastatic lung cancer protein. When desired, tissue-specific expression or knockout of the metastatic breast cancer or metastatic lung cancer protein may be necessary.
It is also possible that the metastatic breast cancer or metastatic lung cancer protein is overexpressed in metastatic breast cancer or metastatic lung cancer. As such, transgenic animals can be generated that overexpress the metastatic breast cancer or metastatic lung cancer protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. Also, the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of metastatic breast cancer or metastatic lung cancer and are additionally useful in screening for modulators to treat metastatic breast cancer or metastatic lung cancer.
For use in diagnostic, research, and therapeutic applications suggested above, kits are also provided by the invention. In the diagnostic and research applications such kits may include any or all of the following: assay reagents, buffers, metastatic breast cancer or metastatic lung cancer-specific nucleic acids or antibodies, hybridization probes and/or primers, antisense polynucleotides, ribozymes, dominant negative metastatic breast cancer or metastatic lung cancer polypeptides or polynucleotides, small molecules inhibitors of metastatic breast cancer or metastatic lung cancer-associated sequences etc. A therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
The present invention also provides for kits for screening for modulators of metastatic breast cancer or metastatic lung cancer-associated sequences. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise one or more of the following materials: a metastatic breast cancer or metastatic lung cancer- associated polypeptide or polynucleotide, reaction tubes, and instructions for testing metastatic breast cancer or metastatic lung cancer-associated activity. Optionally, the kit contains biologically active metastatic breast cancer or metastatic lung cancer protein. A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. Diagnosis would typically involve evaluation of a plurality of genes or products. The genes will be selected based on correlations with important parameters in disease which may be identified in historical or outcome data.
Tables 1C-12C shows genomic positioning for those pkeys lacking unigene ID's and accession numbers in tables 1A-12A. For each predicted exon, we have listed the genomic sequence source used for prediction. Nucleotide locations of each predicted exon are also listed.
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of breast metastases to the brain Als divided by the 90th percentile of normal breast tissue Als, where the 15th percentile of all normal body tissue Als was subtracted from the both numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens breast cancer antigen NY-BR
Homo sapiens, Similar to RIKEN cDNA 2010
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens breast cancer antigen NY-BR
Homo sapiens Fanconi anemia complementat
Homo sapiens clone 24416 mRNA sequence
Homo sapiens, clone IMAGE: 4285740, mRNA
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA: FLJ23523 fis, clone L
Homo sapiens, clone IMAGE: 3606519, mRNA,
Homo sapiens cDNA: FLJ23230 fis, clone C
Homo sapiens, clone IMAGE: 3606519, mRNA,
Homo sapiens cDNA FLJ14712 fis, clone NT
Homo sapiens clone N11 NTera2D1 teratoca
Homo sapiens cDNA FLJ10071 fis, clone HE
Homo sapiens cDNA: FLJ23371 fis, clone H
Homo sapiens cDNA: FLJ21238 fis, clone C
Homo sapiens IMAGE: 512024 clone, mRNA
Homo sapiens clone 24416 mRNA sequence
Homo sapiens, Similar to RIKEN cDNA 5730
Homo sapiens, clone IMAGE: 3682908, mRNA
Homo sapiens cDNA FLJ14089 fis, clone MA
Homo sapiens cDNA FLJ13446 fis, clone PL
Homo sapiens cDNA FLJ11381 fis, clone HE
Homo sapiens, clone IMAGE: 3344506, mRNA,
Homo sapiens cDNA FLJ10674 fis, clone NT
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of breast metastases to the brain Als divided by the 90th percentile of normal body tissue Als, where the 15th percentile of all normal body tissue Als was subtracted from the numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens, Similar to DNA segment, Ch
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens cDNA FLJ13446 tis, clone PL
Homo sapiens cDNA: FLJ23230 fis, clone C
Homo sapiens cDNA FLJ14162 fis, clone NT
Homo sapiens cDNA FLJ14089 fis, clone MA
Homo sapiens cDNA FLJ13446 fis, clone PL
Homo sapiens GDNF family receptor alpha
Homo sapiens HSPC074 mRNA, partial cds
Homo sapiens mRNA; cDNA DKFZp761E0611 (f
Homo sapiens mRNA for KIAA1668 protein,
Homo sapiens mRNA; cDNA DKFZp434K0322 (f
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of breast metastases to the brain Ais divided by the 90th percentile of breast tumor Als, where the 15th percentile of all normal body Als was subtracted from the numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens mRNA; cDNA DKFZp586B211 (fr
Homo sapiens clone CDABP0086 mRNA sequen
Homo sapiens mRNA; cDNA DKFZp586M0723 (f
Homo sapiens mRNA; cDNA DKFZp564F053 (fr
Homo sapiens clone TUA8 Cri-du-chat regi
Homo sapiens mRNA; cDNA DKFZp564L0822 (f
Homo sapiens cDNA FLJ11570 fis, clone HE
Homo sapiens mRNA; cDNA DKFZp434F1928 (f
Homo sapiens cDNA: FLJ23123 fis, clone L
Homo sapiens clone HH409 unknown mRNA
Homo sapiens cDNA FLJ20767 fis, clone CO
Homo sapiens cDNA FLJ11469 fis, clone HE
Homo sapiens BAC clone RP11-505D17 from
Homo sapiens mRNA; cDNA DKFZp564B182 (fr
Homo sapiens cDNA FLJ11533 fis, clone HE
Homo sapiens cDNA FLJ12381 fis, clone MA
Homo sapiens cDNA FLJ13613 fis, clone PL
Homo sapiens mRNA; cDNA DKFZp564B176 (fr
Homo sapiens breast cancer antigen NY-BR
Homo sapiens, Similar to RIKEN cDNA 2010
Homo sapiens cDNA: FLJ22290 fis, clone H
Homo sapiens mRNA; cDNA DKFZp586B0220 (f
Homo sapiens cDNA FLJ20812 fis, clone AD
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of breast tumor Als divided by the 90th percentile of breast metastases to the brain Als, where the 15th percentile of all normal body tissue Als was subtracted from the numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens mRNA; cDNA DKFZp586B211 (fr
Homo sapiens pyruvate dehydrogenase kina
Homo sapiens mRNA for KIAA1650 protein,
Homo sapiens mRNA; cDNA DKFZp586N2020 (f
Homo sapiens cDNA FLJ11775 fis, clone HE
Homo sapiens cDNA FLJ20767 fis, clone CO
Homo sapiens mRNA full length insert cDN
Homo sapiens mRNA; cDNA DKFZp564L0822 (f
Homo sapiens clone TUA8 Cri-du-chat regi
Homo sapiens mRNA; cDNA DKFZp434G0827 (f
Homo sapiens cDNA FLJ13613 fis, clone PL
Homo sapiens cDNA: FLJ23173 fis, clone L
Homo sapiens mRNA for KIAA 1657 protein,
Homo sapiens clone 24651 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp434F1928 (f
Homo sapiens (clone MG2-5-12) mucin (MG2
Homo sapiens mRNA; cDNA DKFZp564F053 (fr
Homo sapiens cDNA FLJ10158 fis, clone HE
Homo sapiens mRNA; cDNA DKFZp564B076 (fr
Homo sapiens cDNA FLJ13830 fis, clone TH
Homo sapiens clone 23767 and 23782 mRNA
Homo sapiens mRNA; cDNA DKFZp586K1922 (f
Homo sapiens clone 1 thrombospondin mRNA
Homo sapiens mRNA; cDNA DKFZp586M0723 (f
Homo sapiens mRNA; cDNA DKFZp586N0121 (f
Homo sapiens mRNA; cDNA DKFZp434D0215 (f
Homo sapiens cDNA FLJ14177 fis, clone NT
Homo sapiens cDNA FLJ13555 fis, clone PL
Homo sapiens cDNA FLJ14130 fis, clone MA
Homo sapiens mRNA; cDNA DKFZp434E232 (fr
Homo sapiens mRNA; cDNA DKFZp564B222 (fr
Homo sapiens clone 24617 mRNA sequence
Homo sapiens cDNA: FLJ22849 fis, clone K
Homo sapiens cDNA FLJ12381 fis, clone MA
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of breast metastases to the brain Als divided by the 90th percentile of normal breast tissue Als, where the 15th percentile of all normal body tissue Als was subtracted from the both numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens cDNA: FLJ22256 fis, clone H
Homo sapiens cDNA FLJ14814 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp761G02121 (
Homo sapiens cDNA FLJ13329 fis, clone OV
Homo sapiens cDNA: FLJ22528 fis, clone H
Homo sapiens, clone IMAGE:3507281, mRNA,
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens cDNA: FLJ22785 fis, clone K
Homo sapiens clone 24670 mRNA sequence
Homo sapiens cDNA FLJ10500 fis, clone NT
H. sapiens mRNA for 3'UTR of unknown prot
Homo sapiens clone 24416 mRNA sequence
Homo sapiens cDNA: FLJ23241 fis, clone C
Homo sapiens, Similar to epsin 3, clone
Homo sapiens winged helix/forkhead trans
Homo sapiens clone 24416 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp434H1235 (f
Homo sapiens cDNA: FLJ23165 fis, clone L
Homo sapiens mRNA; cDNA DKFZp566O134 (fr
Homo sapiens cDNA FLJ10570 fis, clone NT
Homo sapiens, Similar to RIKEN cDNA 2010
Homo sapiens cDNA FLJ14872 fis, clone PL
Homo sapiens cDNA FLJ14934 fis, clone PL
Homo sapiens cDNA: FLJ23313 fis, clone H
Homo sapiens clone 25028 mRNA sequence
Homo sapiens cDNA:FLJ21081 fis, clone C
Homo sapiens cDNA:FLJ22785 fis, clone K
Homo sapiens mRNA for KIAA1750 protein,
Homo sapiens mRNA; cDNA DKFZp564B182 (fr
Homo sapiens cDNA FLJ11346 fis, clone PL
Homo sapiens mRNA; cDNA DKFZp761J1112 (f
Homo sapiens cDNA:FLJ21930 fis, clone H
Homo sapiens clone 24505 mRNA sequence
Homo sapiens cDNA FLJ14237 fis, clone NT
Homo sapiens cDNA FLJ13289 fis, clone OV
Homo sapiens clone N11 NTera2D1 teratoca
Homo sapiens cDNA FLJ11654 fis, clone HE
Homo sapiens clone FBD3 Cri-du-chat crit
Homo sapiens mRNA; cDNA DKFZp586L141 (fr
Homo sapiens cDNA: FLJ21814 fis, clone H
Homo sapiens cDNA: FLJ22256 fis, clone H
Homo sapiens clones 24622 and 24623 mRNA
Homo sapiens cDNA FLJ12807 fis, clone NT
Homo sapiens cDNA:FLJ22322 fis, clone H
Homo sapiens cDNA:FLJ22993 fis, clone K
Homo sapiens cDNA:FLJ23070 fis, clone L
Homo sapiens cDNA FLJ14218 fis, clone NT
Homo sapiens cDNA:FLJ23013 fis, clone L
Homo sapiens cDNA FLJ11752 fis, clone HE
Homo sapiens clone 24923 mRNA sequence
Homo sapiens, clone MGC:16169, mRNA, com
Homo sapiens, clone IMAGE:4285740, mRNA
Homo sapiens cDNA FLJ11309 fis, clone PL
Homo sapiens cDNA FLJ13051 fis, clone NT
Homo sapiens cDNA:FLJ21447 fis, clone C
Homo sapiens cDNA FLJ10071 fis, clone HE
Homo sapiens cDNA: FLJ22113 fis, clone H
Homo sapiens, clone MGC:5352, mRNA, comp
Homo sapiens cDNA: FLJ22902 fis, clone K
Homo sapiens mRNA full length insert cDN
Homo sapiens mRNA; cDNA DKFZp566B213 (fr
Homo sapiens keratin 17 (KRT17)
Homo sapiens cDNA FLJ13655 flu, clone PL
Homo sapiens mRNA; cDNA DKFZp761E13121 (
Homo sapiens cDNA FLJ14208 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp667D095 (fr
Homo sapiens, Similar to RIKEN cDNA 2810
Homo sapiens mRNA full length insert cDN
Homo sapiens mRNA; cDNA DKFZp434E199 (fr
Homo sapiens, Similar to RIKEN cDNA 2700
Homo sapiens cDNA:FLJ22930 fis, clone K
Homo sapiens, clone IMAGE:3606519, mRNA,
Homo sapiens cDNA:FLJ23005 fis, clone L
Homo sapiens mRNA:cDNA DKFZp586C1723 (f
Homo sapiens mRNA; cDNA DKFZp434G0972 (f
Homo sapiens mRNA; cDNA DKFZp566P2324 (f
Homo sapiens, clone IMAGE:3162799, mRNA,
Homo sapiens cDNA:FLJ23117 fis, clone L
Homo sapiens cDNA FLJ13663 fis, clone PL
Homo sapiens cDNA:FLJ22044 fis, clone H
Homo sapiens, clone IMAGE:3636299, mRNA,
Homo sapiens cDNA:FLJ21561 fis, clone C
Homo sapiens cDNA FLJ14201 fis, clone NT
Homo sapiens cDNA:FLJ21887 fis, clone H
Homo sapiens mRNA full length insert cDN
Homo sapiens, clone IMAGE:4098694, mRNA,
Homo sapiens cDNA:FLJ22696 fis, clone H
Homo sapiens cDNA:FLJ21238 fis, clone C
Homo sapiens cDNA FLJ10366 fis, clone NT
Homo sapiens cDNA FLJ14752 fis, clone NT
Homo sapiens cDNA: FLJ21260 fis, clone C
Homo sapiens clone 24405 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp761E212 (fr
Homo sapiens cDNA:FLJ22096 fis, clone H
Homo sapiens cDNA:FLJ22115 fis, clone H
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of lung metastases to the brain Als divided by the 90th percentile of normal lung Als, where the minimum in the numerator and denominator was set to 20.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens secretoglobin, family 3A, m
Homo sapiens mRNA; cDNA DKFZp434L1226 (f
Homo sapiens, clone IMAGE:4285740, mRNA
Homo sapiens clone N11 NTera2D1 teratoca
Homo sapiens 14q32 Jagged2 gene, complet
Homo sapiens cDNA: FLJ22044 fis, clone H
Homo sapiens, clone IMAGE:3677194, mRNA,
Homo sapiens, clone IMAGE:3535294, mRNA,
Homo sapiens cDNA: FLJ22322 fis, clone H
Homo sapiens PAK2 mRNA, complete cds
Homo sapiens clone 24505 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp434B0425 (f
Homo sapiens, clone IMAGE:3447394, mRNA,
Homo sapiens quiescin Q6 (QSCN6)
Homo sapiens, Similar to RIKEN cDNA 5430
Homo sapiens clone FLB9213 PRO2474 mRNA,
Homo sapiens cDNA FLJ10674 fis, clone NT
Homo sapiens cDNA: FLJ22785 fis, clone K
Homo sapiens cDNA FLJ14408 fis, clone HE
Homo sapiens cDNA FLJ14162 fis, clove NT
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens cDNA FLJ14105 fis, clone MA
Homo sapiens cDNA FLJ14218 fis, clone NT
Homo sapiens cDNA FLJ14814 fis, clone NT
Homo sapiens, Similar to RIKEN cDNA 2010
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of lung metastases to the brain Als divided by the 90th percentile of normal body tissues Als, where the 15th percentile of all normal body tissue Als was subtracted from the numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et at.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens cDNA FLJ11027 fis, clone PL
Homo sapiens GNAS1 antisense transcript
Homo sapiens mRNA; cDNA DKFZp76111224 (f
Homo sapiens skin-specific protein (xp32
Homo sapiens mRNA for KIAA1870 protein,
Homo sapiens cDNA: FLJ23230 fis, clone C
Homo sapiens cDNA FLJ14218 fis, clone NT
Homo sapiens hair and skin epidermal-typ
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens HRIHFB2063 mRNA, partial cd
Homo sapiens mRNA for KIAA1644 protein,
Homo sapiens mRNA; cDNA DKFZp761J1324 (f
Homo sapiens mRNA for KIAA1769 protein,
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of lung metastases to the brain Als divided by the 90th percentile of lung adenocarcinoma Als, where the 15th percentile of all normal body tissue Als was subtracted from the numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (Gl) numbers. “Dunham l. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham l. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens mRNA; cDNA DKFZp564B2062 (f
Homo sapiens mRNA for KIAA1657 protein,
Homo sapiens mRNA for KIAA1650 protein,
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA: FLJ23123 fis, clone L
Homo sapiens cDNA FLJ14300 fis, clone PL
Homo sapiens cDNA FLJ14130 fis, clone MA
Homo sapiens cDNA FLJ14438 fis, clone HE
Homo sapiens cDNA: FLJ23123 fis, clone L
Homo sapiens mRNA; cDNA DKFZp586N2020 (f
Homo sapiens cDNA FLJ11775 fis, clone HE
Homo sapiens clone 1 thrombospondin mRNA
Homo sapiens cDNA FLJ14130 fis, clone MA
Homo sapiens cDNA FLJ13384 fis, clone PL
Homo sapiens mRNA; cDNA DKFZp564G103 (fr
Homo sapiens cDNA FLJ14832 fis, clone OV
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA FLJ10984 fis, clone PL
Homo sapiens cDNA FLJ12052 fis, clone HE
Homo sapiens mRNA; cDNA DKFZp434O032 (fr
Homo sapiens mRNA; cDNA DKFZp564D2071 (f
Pkey: Unique Eos probeset identifier number
ExAccn: Exemplar Accession number, Genbank accession number
UniGeneID: UniGene number
UniGene Title: UniGene gene title
R1: 90th percentile of lung adenocarcinoma Als divided by the 90th percentile of lung metastases to the brain Als, where the 15th percentile of all normal body tissue Als was subtracted from the numerator and denominator.
SEQ ID NO(s): SEQ ID number(s) for nucleic acid and protein sequences associated with table entry.
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens mRNA; cDNA DKFZp564B2062 (f
Homo sapiens mRNA for KIAA1657 protein,
Homo sapiens mRNA for KIAA1650 protein,
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA: FLJ23123 fis, clone L
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA FLJ14300 fis, clone PL
Homo sapiens cDNA FLJ14130 fis, clone MA
Homo sapiens mRNA; cDNA DKFZp586N0121 (f
Homo sapiens cDNA FLJ14438 fis, clone HE
Homo sapiens cDNA: FLJ23123 fis, clone L
Homo sapiens mRNA; cDNA DKFZp434C1915 (f
Homo sapiens cDNA FLJ13510 fis, clone PL
Homo sapiens mRNA; cDNA DKFZp564B2062 (f
Homo sapiens mRNA; cDNA DKFZp586N2020 (f
Homo sapiens cDNA FLJ11775 fis, clone HE
Homo sapiens HSPC324 mRNA, partial cds
Homo sapiens mRNA, exon 1, 2, 3, 4, clon
Homo sapiens cDNA: FLJ21326 fis, clone C
Homo sapiens clone 1 thrombospondin mRNA
Homo sapiens cDNA FLJ14130 fis, clone MA
Homo sapiens cDNA FLJ13384 fis, clone PL
Homo sapiens cDNA: FLJ23038 fis, clone L
Homo sapiens, Similar to DNA segment, Ch
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens breast cancer antigen NY-BR
Homo sapiens, Similar to RIKEN cDNA 2010
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens cDNA: FLJ22785 fis, clone K
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA FLJ10570 fis, clone NT
Homo sapiens clone N11 NTera2D1 teratoca
Homo sapiens clone 24416 mRNA sequence
Homo sapiens, clone IMAGE: 4285740, mRNA
Homo sapiens cDNA: FLJ22322 fis, clone H
Homo sapiens cDNA FLJ10071 fis, clone HE
Homo sapiens Fanconi anemia complementat
Homo sapiens winged helix/forkhead trans
Homo sapiens clone 24416 mRNA sequence
Homo sapiens cDNA FLJ14208 fis, clone NT
Homo sapiens, Similar to RIKEN cDNA 5730
Homo sapiens, clone IMAGE: 3606519, mRNA,
Homo sapiens cDNA FLJ14814 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp564B083 (fr
Homo sapiens cDNA FLJ13495 fis, clone PL
Homo sapiens, clone IMAGE: 3682908, mRNA
Homo sapiens, clone IMAGE: 4139775, mRNA,
Homo sapiens cDNA FLJ13207 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp761E212 (fr
Homo sapiens cDNA: FLJ22044 fis, clone H
Homo sapiens cDNA FLJ14712 fis, clone NT
Homo sapiens cDNA: FLJ22113 fis, clone H
Homo sapiens clone TCCCTA00142 mRNA sequ
Homo sapiens clone 24505 mRNA sequence
Homo sapiens cDNA FLJ14934 fis, clone PL
Homo sapiens mRNA; cDNA DKFZp761J1324 (f
Homo sapiens mRNA; cDNA DKFZp566B213 (fr
Homo sapiens IMAGE: 512024 clone, mRNA
Homo sapiens cDNA: FLJ22373 fis, clone H
Homo sapiens mRNA; cDNA DKFZp434L1226 (f
Homo sapiens mRNA for KIAA1666 protein,
Homo sapiens cDNA FLJ14218 fis, clone NT
Homo sapiens cDNA: FLJ21260 fis, clone C
Homo sapiens cDNA: FLJ21238 fis, clone C
Homo sapiens similar to Echinoidin (LOC1
Homo sapiens cDNA: FLJ21447 fis, clone C
Homo sapiens secretoglobin, family 3A, m
Homo sapiens cDNA: FLJ22785 fis, clone K
Homo sapiens cDNA FLJ11381 fis, clone HE
Homo sapiens cDNA FLJ10366 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp434B0425 (f
Homo sapiens cDNA: FLJ23523 fis, clone L
Homo sapiens cDNA: FLJ22133 fis, clone H
Homo sapiens cDNA FLJ10674 fis, clone NT
Homo sapiens cDNA FLJ12532 fis, clone NT
Homo sapiens cDNA FLJ14408 fis, clone HE
Homo sapiens mRNA; cDNA DKFZp434K0514 (f
Homo sapiens cDNA: FLJ22044 fis, clone H
Homo sapiens clone 23570 mRNA sequence
Homo sapiens clone HB-2 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp566O134 (fr
Homo sapiens cDNA FLJ13794 fis, clone TH
Homo sapiens clone TA40 untranslated mRN
Homo sapiens cDNA FLJ14872 fis, clone PL
Homo sapiens cDNA: FLJ23371 fis, clone H
Homo sapiens cDNA: FLJ23117 fis, clone L
Homo sapiens cDNA FLJ14934 fis, clone PL
Homo sapiens, Similar to RIKEN cDNA 2310
Homo sapiens cDNA: FLJ22256 fis, clone H
Homo sapiens cDNA: FLJ22096 fis, clone H
Homo sapiens cDNA: FLJ23230 fis, clone C
Homo sapiens, clone IMAGE: 3606519, mRNA,
Homo sapiens mRNA; cDNA DKFZp434I0121 (f
Homo sapiens clone FLB9213 PRO2474 mRNA,
Homo sapiens cDNA FLJ12807 fis, clone NT
Homo sapiens, clone IMAGE: 3344506, mRNA,
Homo sapiens mRNA; cDNA DKFZp434B1620 (f
Homo sapiens, Similar to epsin 3, clone
Homo sapiens DNA from chromosome 19, cos
Homo sapiens, clone IMAGE: 3536432, mRNA,
Homo sapiens PIG-M mRNA for mannosyltran
Homo sapiens mRNA; cDNA DKFZp564E122 (fr
Homo sapiens cDNA FLJ14089 fis, clone MA
Homo sapiens mRNA; cDNA DKFZp564B1264 (f
Homo sapiens clone HB-2 mRNA sequence
Homo sapiens cDNA FLJ11654 fis, clone HE
Homo sapiens mRNA for KIAA1764 protein,
Homo sapiens clone 25218 mRNA sequence
Homo sapiens cDNA FLJ11309 fis, clone PL
Homo sapiens cDNA: FLJ22902 fis, clone K
Homo sapiens cDNA FLJ13051 fis, clone NT
Homo sapiens testis-development related
Homo sapiens mRNA; cDNA DKFZp761C1712 (f
Homo sapiens cDNA FLJ14162 fis, clone NT
Homo sapiens cDNA: FLJ20869 fis, clone A
Homo sapiens mRNA; cDNA DKFZp566E1346 (f
Homo sapiens, Similar to RIKEN cDNA 2700
Homo sapiens, clone MGC: 9658, mRNA, comp
Homo sapiens clone FLB3411 PRO0852 mRNA,
Homo sapiens cDNA: FLJ23241 fis, clone C
Homo sapiens, clone IMAGE: 3507281, mRNA,
Homo sapiens cDNA: FLJ23068 fis, clone L
Homo sapiens mRNA full length insert cDN
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
Homo sapiens cDNA FLJ14814 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp761G02121 (
Homo sapiens cDNA FLJ13458 fis, clone PL
Homo sapiens, Similar to epsin 3, clone
Homo sapiens, clone IMAGE: 3507281, mRNA,
Homo sapiens, Similar to RIKEN cDNA 2010
Homo sapiens clone 24416 mRNA sequence
Homo sapiens winged helix/forkhead trans
Homo sapiens cDNA FLJ10570 fis, clone NT
Homo sapiens clone 24416 mRNA sequence
Homo sapiens, clone IMAGE: 3606519, mRNA,
Homo sapiens cDNA FLJ14872 fis, clone PL
Homo sapiens, Similar to RIKEN cDNA 2810
Homo sapiens cDNA: FLJ22785 fis, clone K
Homo sapiens cDNA: FLJ21260 fis, clone C
Homo sapiens mRNA full length insert cDN
Homo sapiens clone N11 NTera2D1 teratoca
Homo sapiens, clone IMAGE: 4285740, mRNA
Homo sapiens cDNA: FLJ22322 fis, clone H
Homo sapiens clone 24505 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp566B213 (fr
Homo sapiens clone 24405 mRNA sequence
Homo sapiens, clone IMAGE: 3873720, mRNA
Homo sapiens cDNA FLJ12807 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp434E199 (fr
Homo sapiens cDNA FLJ13663 fis, clone PL
Homo sapiens cDNA FLJ14934 fis, clone PL
Homo sapiens cDNA FLJ11309 fis, clone PL
Homo sapiens cDNA: FLJ23371 fis, clone H
Homo sapiens mRNA; cDNA DKFZp586L141 (fr
Homo sapiens Fanconi anemia complementat
Homo sapiens mRNA full length insert cDN
Homo sapiens CDNA: FLJ23020 fis, clone L
Homo sapiens cDNA: FLJ22785 fis, clone K
Homo sapiens IMAGE: 512024 clone, mRNA
Homo sapiens cDNA: FLJ21447 fis, clone C
Homo sapiens, Similar to RIKEN cDNA 5730
Homo sapiens cDNA: FLJ21238 fis, clone C
Homo sapiens clone FBD3 Cri-du-chat crit
Homo sapiens, Similar to RIKEN cDNA 2700
Homo sapiens cDNA FLJ14162 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp761E13121 (
Homo sapiens mRNA; cDNA DKFZp564B1264 (f
Homo sapiens mRNA; cDNA DKFZp564E122 (fr
Homo sapiens mRNA for KIAA1666 protein,
Homo sapiens mRNA; cDNA DKFZp564B083 (fr
Homo sapiens cDNA: FLJ21513 fis, clone C
Homo sapiens cDNA FLJ14934 fis, clone PL
Homo sapiens clone 25218 mRNA sequence
Homo sapiens cDNA FLJ12540 fis, clone NT
Homo sapiens cDNA FLJ13495 fis, clone PL
Homo sapiens, clone IMAGE: 3682908, mRNA
Homo sapiens cDNA FLJ14408 fis, clone HE
Homo sapiens cDNA: FLJ23523 fis, clone L
Homo sapiens, clone MGC: 5352, mRNA, comp
Homo sapiens cDNA FLJ13207 fis, clone NT
Homo sapiens mRNA; cDNA DKFZp761E212 (fr
Homo sapiens cDNA FLJ14712 fis, clone NT
Homo sapiens cDNA FLJ11723 fis, clone HE
Homo sapiens cDNA: FLJ22044 fis, clone H
Homo sapiens cDNA FLJ11654 fis, clone HE
Homo sapiens mRNA full length insert cDN
Homo sapiens cDNA: FLJ22113 fis, clone H
Homo sapiens cDNA FLJ14337 fis, clone PL
Homo sapiens cDNA: FLJ21814 fis, clone H
Homo sapiens cDNA: FLJ21880 fis, clone H
Homo sapiens clone TCCCTA00142 mRNA sequ
Homo sapiens cDNA FLJ12334 fis, clone MA
Homo sapiens cDNA FLJ13794 fis, clone TH
Homo sapiens cDNA: FLJ22993 fis, clone K
Homo sapiens, clone IMAGE: 3867243, mRNA
Homo sapiens cDNA: FLJ23068 fis, clone L
Homo sapiens mRNA; cDNA DKFZp761J1324 (f
Homo sapiens clone FLB9213 PRO2474 mRNA,
Homo sapiens mRNA for FLJ00012 protein,
Homo sapiens cDNA: FLJ22373 fis, clone H
Homo sapiens mRNA; cDNA DKFZp586D0724 (f
Homo sapiens mRNA; cDNA DKFZp434J2235 (f
Homo sapiens cDNA: FLJ22096 fis, clone H
Homo sapiens mRNA: cDNA DKFZp434F1135 (f
Homo sapiens prostein mRNA, complete cds
Homo sapiens clone 24670 mRNA sequence
Homo sapiens cDNA: FLJ23269 fis, clone C
Homo sapiens cDNA: FLJ21909 fis, clone H
Homo sapiens dopamine receptor interacti
Homo sapiens, clone IMAGE: 3357127, mRNA,
Homo sapiens cDNA FLJ10071 fis, clone HE
Homo sapiens cDNA: FLJ22044 fis, clone H
Homo sapiens mRNA; cDNA DKFZp586C1019 (f
Homo sapiens cDNA FLJ11381 fis, clone HE
Homo sapiens cDNA FLJ10366 fis, clone NT
Homo sapiens cDNA: FLJ21410 fis, clone C
Homo sapiens cDNA: FLJ22463 fis, clone H
Homo sapiens cDNA: FLJ22113 fis, clone H
Homo sapiens mRNA; cDNA DKFZp564A026 (fr
Homo sapiens, Similar to transducin (bet
Homo sapiens cDNA FLJ14743 fis, clone NT
Homo sapiens, Similar to RIKEN cDNA 4930
Homo sapiens cDNA FLJ4089 fis, clone MA
Homo sapiens cDNA: FLJ22219 fis, clone H
Homo sapiens cDNA FLJ12231 fis, clone MA
Homo sapiens cDNA: FLJ23117 fis, clone L
Homo sapiens clone 23620 mRNA sequence
Homo sapiens clone HB-2 mRNA sequence
Homo sapiens mRNA: cDNA DKFZp762H106 (fr
Homo sapiens, clone MGC: 9381, mRNA, comp
Homo sapiens glutaminyl-peptide cyclotra
Homo sapiens cDNA: FLJ21784 fis, clone H
Homo sapiens PAC clone RP5-978E18 from 7
Homo sapiens clone 25032 mRNA sequence
Homo sapiens thioredoxin reductase 3 (TR
Homo sapiens cDNA: FLJ21897 fis, clone H
Homo sapiens cDNA: FLJ22696 fis, clone H
Homo sapiens clone 23570 mRNA sequence
Homo sapiens clone HB-2 mRNA sequence
Homo sapiens cDNA FLJ14597 fis, clone NT
Homo sapiens microsomal signal peptidase
Homo sapiens mRNA; cDNA DKFZp566O134 (fr
Homo sapiens cDNA FLJ11632 fis, clone HE
Homo sapiens cDNA FLJ20848 fis, clone AD
Homo sapiens cDNA FLJ11643 fis, clone HE
Homo sapiens cDNA: FLJ23190 fis, clone L
Homo sapiens cDNA FLJ13289 fis, clone OV
Homo sapiens DNA from chromosome 19, cos
Homo sapiens mRNA; cDNA DKFZp566E1346 (f
Homo sapiens cDNA: FLJ22135 fis, clone H
Homo sapiens clone TA40 untranslated mRN
Homo sapiens, clone IMAGE: 3690478, mRNA,
Homo sapiens, clone IMAGE: 4154008, mRNA,
Homo sapiens cDNA: FLJ23156 fis, clone L
Homo sapiens mRNA; cDNA DKFZp586E2317 (f
Homo sapiens cDNA: FLJ21061 fis, clone C
Homo sapiens mRNA; cDNA DKFZp564C0371 (f
Homo sapiens cDNA FLJ10500 fis, clone NT
Homo sapiens cDNA: FLJ21274 fis, clone C
Homo sapiens, Similar to RIKEN cDNA 2310
Homo sapiens cDNA: FLJ21942 fis, clone H
Homo sapiens mRNA full length insert cDN
Homo sapiens, Similar to Bicaudal D (Dro
Homo sapiens cDNA FLJ13207 fis, clone NT
Homo sapiens CATX-2 mRNA, complete cds
Homo sapiens cDNA FLJ14218 fis, clone NT
Homo sapiens cDNA: FLJ22046 fis, clone H
Homo sapiens clone FLB3411 PRO0852 mRNA,
Homo sapiens cDNA: FLJ23230 fis, clone C
Homo sapiens mRNA full length insert cDN
Homo sapiens, clone IMAGE: 3605104, mRNA,
Homo sapiens mRNA; cDNA DKFZp434A2417 (f
Homo sapiens cDNA FLJ12532 fis, clone NT
Homo sapiens cDNA: FLJ22528 fis, clone H
Homo sapiens, Similar to RIKEN cDNA 1110
Homo sapiens cDNA: FLJ20869 fis, clone A
Homo sapiens, clone IMAGE: 3344506, mRNA,
Homo sapiens cDNA FLJ14666 fis, clone NT
Homo sapiens clone 23688 mRNA sequence
Homo sapiens mRNA; cDNA DKFZp564C0962 (f
Homo sapiens cDNA: FLJ21887 fis, clone H
Homo sapiens similar to Echinoidin (LOC1
Homo sapiens, clone IMAGE: 3536432, mRNA,
Homo sapiens, Similar to RIKEN cDNA 5830
Homo sapiens PIG-M mRNA for mannosyltran
Homo sapiens clone 24670 mRNA sequence
Homo sapiens PAK2 mRNA, complete cds
Homo sapiens cDNA: FLJ23070 fis, clone L
Homo sapiens cDNA FLJ14028 fis, clone HE
Homo sapiens cDNA FLJ13692 fis, clone PL
Homo sapiens mRNA; cDNA DKFZp667D095 (fr
Homo sapiens mRNA; cDNA DKFZp434B0425 (f
Homo sapiens killer cell Ig-like recepto
Homo sapiens, clone IMAGE: 3507281, mRNA,
Homo sapiens mRNA for KIAA1764 protein,
Homo sapiens cDNA FLJ12900 fis, clone NT
Homo sapiens clone 25028 mRNA sequence
Homo sapiens breast cancer antigen NY-BR
Homo sapiens cDNA FLJ13271 fis, clone OV
Homo sapiens cDNA FLJ13267 fis, clone OV
Homo sapiens clone 23579 mRNA sequence
Homo sapiens clone 25110 mRNA sequence
Homo sapiens cDNA FLJ13051 fis, clone NT
Homo sapiens testis-development related
Homo sapiens Chromosome 16 BAC clone CIT
Homo sapiens mRNA; cDNA DKFZp761C1712 (f
Homo sapiens, close IMAGE: 3606519, mRNA,
Homo sapiens, clone MGC: 9381, mRNA, comp
Homo sapiens mRNA; cDNA DKFZp434K0514 (f
Homo sapiens C1orf19 mRNA, partial cds
Homo sapiens cDNA: FLJ22431 fis, clone H
Homo sapiens cDNA FLJ13027 fis, clone NT
Homo sapiens quiescin Q6 (QSCN6)
Homo sapiens, clone IMAGE: 3535294, mRNA,
Homo sapiens, Similar to tumor different
Homo sapiens cDNA FLJ13585 fis, clone PL
Homo sapiens cDNA FLJ14459 fis, clone HE
Homo sapiens cDNA: FLJ22133 fis, clone H
Homo sapiens clone IMAGE: 112577 mRNA seq
Homo sapiens cDNA FLJ13485 fis, clone PL
Homo sapiens cDNA: FLJ22783 fis, clone K
Homo sapiens cDNA FLJ12082 fis, clone HE
Homo sapiens mRNA; cDNA DKFZp566P2324 (f
Pkey: Unique Eos probeset identifier number
CAT number: Gene cluster number
Accession: Genbank accession numbers
Pkey: Unique number corresponding to an Eos probeset
Ref: Sequence source. The 7 digit numbers in this column are Genbank Identifier (GI) numbers. “Dunham I. et al.” refers to the publication entitiled “The DNA sequence of human chromosome 22.” Dunham I. et al., Nature (1999) 402: 489-495.
Strand: Indicates DNA strand from which exons were predicted.
Nt_position: Indicates nucleotide positions of predicted exons.
It is understood that the examples described above in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All publications, sequences of accession numbers, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
This application claims priority from U.S. Provisional Application No. 60/439,058 filed Jan. 10, 2003, which is hereby incorporated by reference in its entirety. This application includes a sequence listing on a compact disc submitted with this application. The compact disc includes a 20.724 megabyte ASCII formatted file, created Jan. 9, 2004, entitled, “0188SEQL.txt”. This file lists 5818 sequences. In accordance with 37 C.F.R. § 1.52(e)(5), the sequence listing on the compact disc is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60439058 | Jan 2003 | US |
