WO2006072954A2 - Novel il-6 polynucleotides encoding variant il-6 polypeptides and methods using same - Google Patents

Abstract

Novel IL-6 variant polypeptides and polynucleotides encoding same are provided. Also provided methods and phamaceutical compositions which can be used to treat various disorders such as cancer, immunological-related, blood-related and skin-related disorders using the polypeptides and polynucleotides of the present invention. Also provided are methods and kits for diagnosing, determining predisposition and/or prognosis of various disorders using as diagnostic markers the novel IL-6 variant polypeptides and polynucleotides of the present invention.

Description

NOVEL IL-6 POLYNUCLEOTIDES ENCODING VARIANT IL-6 POLYPEPTIDES AND METHODS USING SAME

FIELD OF THE INVENTION

The present invention relates to novel IL-6 variant polypeptides and polynucleotides encoding same and more particularly, to therapeutic and diagnostic methods and kits utilizing same.

BACKGROUND OF THE INVENTION

Extracellular proteins, including receptors and their corresponding ligands, play active roles in the formation, differentiation and maintenance of multicellular organisms. Any fate of an individual cell including proliferation, migration, differentiation, or interaction with other cells is typically governed by information received from distant cells and/or the immediate environment. This information is often transmitted by secreted polypeptides including but not limited to mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones, which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules are normally transferred through the cellular secretory pathway to reach their site of action at the extracellular environment.

Secreted proteins have various industrial applications, including as pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drags available to date, including thrombolytic polypeptide sequences, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secreted proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic polynucleotide or polypeptide sequences. For example, receptor immunoadhesins can be employed as therapeutic polynucleotide or polypeptide sequences to block receptor-ligand interactions. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.

Non-secreted proteins may also find application as therapeutics or diagnostics. For example, over expression of an intracellular protein (or transcript thereof) which con-elates with a disease may be used to diagnose the presence of a disease or for estimating the risk of developing a disease, by the development of probes which specifically identify the over-expressed transcript or protein. In instances where the individual is at risk of suffering from a disease or other undesirable phenotype as a result of over expression of such transcript, the expression of the protein may be reduced using, for example, antisense or triple helix based strategies,

For these reasons, efforts are being made by both industry and acadeniia to identify new, native, membrane-bound, secreted or non-secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for such proteins. Non-limiting examples of such screening methods and techniques are described in, for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-71 13 (1996); U.S. Pat. No. 5,536,637. These method and techniques allow new proteins to be discovered which can have important therapeutic and diagnostic effects, particularly with regard to disease-related target systems.

One such disease-related target system is that of Interleukin-6 (IL-6). IL-6 is a pleiotropic cytokine with a wide range of biological activities such as regulation of immune responses, hematopoiesis, inflammation, generation of acute-phase reactions, and oncogenesis (Naka T; et al., Research2002, 4:Suppl 3 (S233-S242)). IL-6 was originally identified as an antigen-nonspecific B-cell differentiation factor in the culture supematants of mitogen- or antigen-stimulated peripheral blood mononuclear cells that induced B cells to produce immunoglobulins and was named B-cell stimulatory factor 2 (BSF-2). IL-6 is produced by various types of lymphoid and nonlymphoid cells, such as T cells, B cells, monocytes, fibroblasts, keratinocytes, endothelial cells, mesangial cells, and several tumor cells.

Deregulation of IL-6 production has been implicated in the pathogenesis of a variety of diseases. It has been demonstrated that chronic inflammation of the joint in rheumatoid arthritis (RA) causes IL-6 production by synovial cells, macrophages and lymphocytes in the affected synovium. Overproduction of IL-6 appears to be involved in the pathogenesis of pannus formation, angiogenesis, infiltration of mononuclear cells and destruction of cartilage and bone (Naka T; et al., Research2002. 4:Suppl 3 (S233-S242); Madhok R; et al., Annals of the Rheumatic Diseasesl993, 52:3 (232- 234); Numata Y; et al., American Journal of Hematology 1991, 36:4 (282-284)). IL-6 is also present at very high levels in the serum and/or related tissue from patients with Crohn's disease (CD) (Yamamoto M; et al, Journal of Immunology2000, 164:9 (4878-4882)), Castleman's disease (Nishimoto N; et al., Blood2000, 95:1 (56-61)), multiple myeloma (MM) (Lauta VM, Cytokme2001. 16:3 (79-86)) and systemic lupus erythematosus (SLE) (Mihara M; et al., Clinical and Experimental Immunology 1998, 112:3 (397-402)); it may, therefore, play a crucial role in the pathogenesis of these diseases.

As a result, therapy involving the blocking of IL-6 functions may constitute a new therapeutic strategy for these diseases. The functions of IL-6 are mediated through two different receptors. IL-6R and a 13OkDa common signal transducer- gpl30, which all combine to generate a high-affinity complex of IL-6/ IL-6R/gpl30. It has pathological roles in various disease conditions, including inflammatory- mesangial proliferative glomerulonephritis, autoimmune-RA. psoriasis and malignant cancers, including but not limited to multiple myelonia/plasmacytoma and Kaposi's sarcoma.

Several IL-6 related drugs are currently under clinical investigation. For example, YSIL6, under development of Y's Therapeutics, is a small molecule for the treatment of rheumatoid arthritis and other inflammatory disorders, and is currently in phase II clinical trials. The molecule's modes of action include inhibition of TNF-α and IL-6 production in T-cells and macrophages and inhibition of T-cell migration. Another example is Tocilizuniab (MRA; Atlizumab; Actemra), a recombinant humanized MAb against human IL-6 receptor, under development by Chugai (Roche), which is currently in phase III clinical trials. Tocilizumab is under development for use in treating rheumatoid arthritis (RA), Crohn's disease, Castleman's disease and systemic lupus erythematosus (SLE), An additional example is CNTO-328, a human-mouse anti-IL-6 antibody, under development by Centocor (Johnson & Johnson) for the treatment of myeloma and cachexia associated with cancer. CNTO-328 is currently in phase II clinical trials. Yet another example is ActinoDrug Pharmaceuticals 's AD-GL0002, a fungal small molecule inhibitor of IL-6 that specifically interferes with the transcription factor Stat3, thereby inhibiting the IL-6 induced Stat3/DNA pathway. AD-GL0002 is being developed for the treatment of cancer and inflammation, AD-GL0002 demonstrated preclinical efficacy in an animal model of Parkinson's disease. Anti-IL-6 agents also have potential in autoimmune and inflammatory diseases, such as ulcerative colitis, asthma, psoriasis, bone resoiption due to osteoporosis and RA.

SUMMARY OF THE INVENTION

In view of its critical role in oncogenesis, regulation of immune response, support of hematopoiesis and generation of acute phase reaction in inflammation, there is an unmet need to develop therapies involving blocking of IL-6 function and/or its physiological effects. The pathological roles of IL-6 have been clarified in various disease conditions, such as inflammatory, autoimmune, and malignant diseases. Uncontrolled IL-6 overproduction appeal's to be responsible for the clinical symptoms and abnormal laboratory findings in Rheumatoid arthritis (RA). Because of the B-cell differentiation factor activity of IL-6, overproduction of IL-6 is responsible for the increase in serum γ-globulin and the emergence of rheumatoid factors. IL-6 as a hepatocyte-stimulating factor causes an increase in CRP, serum amyloid A, and erythrocyte sedimentation rate and a decrease in serum albumin. On the other hand, IL-6 as a megakaryocyte differentiation factor causes thrombocytosis. Since IL-6 in the presence of soluble IL-6R activates osteoclasts to induce bone absorption, IL-6 may be involved in the osteoporosis and destruction of bone and cartilage associated with RA. In fact, a large amount of IL-6 has been observed in both sera and synovial fluids from the affected joints of patients with RA. Blockade of the IL-6 signal may thus constitute a new therapeutic strategy for RA. In addition to RA, IL-6 was found to be involved in various diseases such as Castleman's disease, multiple myeloma/plasmacytonia, mesangial proliferative glomerulonephritis, psoriasis and Kaposi's sarcoma. Thus these diseases could be targets of IL-6 inhibitors also.

The present inventors have previously designed algorithms which allow for the mass prediction of new genes and gene products and for annotating these genes and gene products [see for example and without limitation LIS patent No: 6,625,545; U.S. Pat. Appl. No. 10/426,002; and PCT Application No. PCT/IL2005/000106 the teachings of all of which are incorporated herein by reference]. While applying the above-mentioned algorithms, the present inventors uncovered novel naturally occulting valiants of IL-6 gene products, which as described above, play pivotal roles in disease onset and progression. As such these variants can be used in the diagnosis and therapy of a wide range of diseases.

Surprisingly, as discovered by the present inventors, novel naturally occulting splice variants of IL-6 gene products according to the present invention can be used in the therapy and diagnosis of a wide range of variant-detectable diseases and variant- treatable diseases, which are "IL-6-related diseases^". These splice variants of the present invention can be used as valuable therapeutic tools in the treatment of "IL-6- related diseases". Without wishing to be bound by a single theory, the IL-6 splice variants of the present invention can serve as antagonists (i.e., inhibitors), similarly to previously described IL-6 antagonists. As was previously shown (Sporeno et al., Blood 1996 87(l l):4510-9; Savino, R., et al., EMBO J. 1994 13(6): 1357-67), antagonistic activity of IL-6 variant could be achieved by binding the non-signaling subunit (IL-6Rα) of the IL-6 heterodimeric receptor without activating the signaling subunit (gpl30) due to impaired binding to the gpl30 subunit. Therefore, IL-6 splice variants of the present invention can optionally serve as inhibitors of IL-6 functions and/or its physiological effects.

As used herein, the phrase "IL-6-related disease(s)" (also named "variant treatable disease(s)") refers to a disease in which IL-6 activity and/or expression contribute to disease onset and/or progression, such that treating the disease may involve blocking IL-6 activity and/or expression. "Treatment" also encompasses prevention, amelioration, elimination and/or control of the disease and/or pathological condition. Examples of IL-6-related diseases include, but are not limited to, inflammatory disorders, immune disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporosis, RA, Crohn's disease. Castleman's disease, systemic lupus erythematosus, inflammatory-mesangial proliferative glomerulonephritis, autoimmune-RA, Psoriasis, Parkinson's disease, myeloproliferative disorders and cancerous diseases including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer and cachexia associated with cancer. According to preferred embodiments of the present invention, the immune disorders are selected from the group consisting of ulcerative colitis, asthma. psoriasis, bone resorption due to osteoporosis., RA, Crohn's disease, Castleman's disease, systemic lupus erythematosus, inflamniatory-mesangial proliferative glomerulonephritis, autoimmune-RA, and psoriasis. Also according to preferred embodiments of the present invention, the cancerous diseases are selected from the group consisting of malignant diseases-multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer.

Thus, the present invention envisages treatment of the above-mentioned diseases by the provision of polynucleotide or polypeptide sequences of this aspect of the present invention, which are capable of upregulating expression of the polypeptides of the present invention in a subject in need thereof, as is further described hereinbelow. Such polynucleotide or polypeptide sequences of this aspect of the present invention and administration thereof are further described hereinbelow. As meant herein, "variant detectable disease" refers to a disease in which IL-6 expression is altered as compared to the normal level. Examples of variant detectable diseases include, but are not limited to, inflammatory disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporosis, RA, Crohn's disease. Castleman's disease, systemic lupus erythematosus, inflamniatory-mesangial proliferative glomerulonephritis, autoimmune-RA, psoriasis. Parkinson's disease, myeloproliferative disorders and cancerous diseases including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer and cachexia associated with cancer.

Thus, the present invention also envisages detection, diagnosis (including differential diagnosis) and/or deteπnination of prognosis of the above-mentioned diseases by the detection of polynucleotide or polypeptide sequences according to preferred embodiments of the present invention, as is further described hereinbelow. Such polynucleotide or polypeptide sequences of this aspect of the present invention and uses thereof are further described hereinbelow.

It should be noted that the terms "segment", "seg" and "node" are used interchangeably in reference to nucleic acid sequences of the present invention, they refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below. Optionally and preferably, they are examples of oligonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.

As used herein the phrase "disease" includes any type of pathology and/or damage, including both chronic and acute damage, as well as a progress from acute to chronic damage.

The term "marker" in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients (subjects) having one of the herein-described diseases or conditions, as compared to a comparable sample taken from subjects who do not have one the above-described diseases or conditions.

The phrase "differentially present" refers to differences in the quantity of a marker present in a sample taken from patients having one of the herein-described diseases or conditions as compared to a comparable sample taken from patients who do not have one of the herein-described diseases or conditions. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present. Optionally, a relatively low amount of up-regulation may serve as the marker, as described herein, One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided in the description of each individual marker below.

As used herein the phrase "diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity, The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the above.

Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be con-elated with predisposition to, or presence or absence of the disease. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.

As used herein, the term "level" refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention. Typically the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).

Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.

Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g.. brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made. Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.

A "test amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or condition, A test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

A "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition, A control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

"Detect" refers to identifying the presence, absence or amount of the object to be detected.

A "label" includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include "P, S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample. The label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly. For example, the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6:1 165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.

Exemplary detectable labels, optionally and preferably for use with immunoassays, include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the marker in the sample can be detected using an indirect assay, λvherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture. "Immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide (or other epitope), refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example. solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g.. Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

According to certain embodiments of the present invention, the invention provides isolated nucleic acid sequences of IL-6 variants comprising the sequences described herein. According to other embodiments, the present invention provides amino acid sequences of IL-6 variants comprising the sequences described herein.

According to preferred embodiments of the present invention, preferably any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.

According to other embodiments, the present invention provides head, tail, bridge or edge sequence described herein.

According to other embodiments, the present invention provides an antibody capable of specifically binding to an epitope of an amino acid sequence of IL-6 variants comprising the sequences described herein and/or to an epitope of head, tail, bridge, edge or insertion sequence described herein.

According to yet further embodiments, the present invention provides said antibody, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein. According to other embodiments, the invention provides a pharmaceutical composition comprising as an active ingredient any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof.

According to other embodiments, the present invention provides a biomarker capable of detecting variant-detectable disease, comprising any of the above nucleic acid sequences or a fragment thereof, or any of the above amino acid sequences or a fragment thereof. According to other embodiments, the present invention provides a method for treating a variant-treatable disease, comprising administering a therapeutic protein, variant peptide, protein, nucleic acid sequence, antisense and/or antibody to a subject in need of treatment thereof. According to other embodiments, the present invention provides a kit for detecting a variant-detectable disease, comprising a kit detecting specific expression of a splice variant as described herein.

According to further embodiments, the present invention provides the kit for detecting a variant-detectable disease, as above, wherein said kit comprises a NAT- based technology.

According to yet further embodiments, the present invention provides said kit, wherein said kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence as described herein. According to yet further embodiments, the present invention provides said kit, wherein said kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.

According to yet further embodiments, the present invention provides said kit for detecting a variant-detectable disease, as above, wherein said kit comprises an antibody as described herein. According to yet further embodiments, the present invention provides said kit, wherein said kit further comprises at least one reagent for performing an ELISA or a Western blot.

According to other embodiments, the present invention provides a method for detecting a variant-detectable disease, comprising detecting specific expression of a splice variant as described herein.

According to further embodiments, the present invention provides the method for detecting a variant-detectable disease, as above, • wherein said detecting specific expression is performed with a NAT-based technology and/or with an immunoassay.

According to other embodiments, the present invention provides a method for screening for variant-detectable disease, comprising detecting cells affected by a variant-detectable disease with a biomarker or an antibody or a method or assay as described herein. According to other embodiments, the present invention provides a method for diagnosing a marker-detectable disease, comprising detecting cells affected by variant-detectable disease with a biomarker or an antibody or a method or assay as described herein. According to other embodiments, the present invention provides a method for monitoring disease progression and/or treatment efficacy and/or relapse of λ'ariant- detectable disease, comprising detecting cells affected by variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.

According to other embodiments, the present invention provides a method of selecting a therapy for a marker-detectable disease, comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay as described herein and selecting a therapy according to said detection.

According to preferred embodiments of the present invention, there is provided, as listed below, optional but preferred embodiments (although provided as a list, this is for the sake of convenience only and is not intended to indicate a closed list or to otherwise be limiting in any way):

An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) , S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) , S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) , S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) .

An isolated polynucleotide comprising a node having a sequence selected from the group consisting of: S56892_PEA_l_PEA_l_node_0 (SEQ ID NO:5) , S56892_PEA_l_PEA_l_node_10 (SEQ ID NO:6)

S56892_PEA_l_PEA_l_node_18 (SEQ ID NO:7) S56892_PEA_l_PEA_l_node_21 (SEQ ID NO:8) , S56S92_PEAJ_PEA_l_node_3 (SEQ ID NO:9) , S56892_PEA_l_PEA_l_node_4 (SEQ ID NO: 10) , S56892_PEA_l_PEA_l_node_7 (SEQ ID NO: 1 1) , S56892_PEAJ_PEA_l_node_8 (SEQ ID NO: 12) , S56892_PEA_l_PEA_l_node_9 (SEQ ID NO: 13) , S56892_PEA_l_PEA_l_node_12 (SEQ ID NO: 14) S56892_PEA_l_PEA_l_node_13 (SEQ ID NO: 15) S56892_PEA_l_PEA_l_node_14 (SEQ ID NO:16) S56S92_PEA_l_PEA_l_node J 6 (SEQ ID NO: 17) S56892_PEA_l_PEA_l_node_17 (SEQ ID NO:18) S56892_PEA_l_PEA_l_node_19 (SEQ ID N0:19)

S56892_PEA_l_PEA_l_node_20 (SEQ ID NO.20)

S56892_PEA_l_PEA_l_node_22 (SEQ ID N0:21)

S56892_PEA_l_PEA_l_node_23 (SEQ ID NO:22) . An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , S 56892_PEA_1_PEA_1_P 13 (SEQ ID NO:27) .

An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , comprising a first amino acid sequence being at least about 90% homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERID KQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGF NEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKK corresponding to amino acids 1 - 157 of IL6JHUMAN, which also corresponds to amino acids 1 - 157 of S56S92_PEA_1_PEA_1_P8 (SEQ ID NO:24) , and a second amino acid sequence being at least about 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to a polypeptide having the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO: 24) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , comprising a polypeptide being at least about 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) . An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P9

(SEQ ID NO:25) , comprising a first amino acid sequence being at least about 90% homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERID KQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGF

NE corresponding to amino acids 1 - 108 of IL6_HUMAN. which also corresponds to amino acids 1 - 108 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , and a second amino acid sequence being at least about 90% homologous to AKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALR QM corresponding to amino acids 158 - 212 of IL6_HUMAN, which also corresponds to amino acids 109 - 163 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO.25) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order. An isolated chimeric polypeptide encoding for an edge portion of

S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EA, having a structure as follows: a sequence starting from any of amino acid numbers 108-x to 108; and ending at any of amino acid numbers 109+ ((n-2) - x), in which x varies from 0 to n-2.

An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , comprising a first amino acid sequence being at least about 90% homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERID KQIRYILDGISALRKETCNKSN corresponding to amino acids 1 - 76 of IL6_HUMAN, which also corresponds to amino acids 1 - 76 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) . and a second amino acid sequence being at least about 70%. optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P11 (SEQ ID NO: 26) , comprising a polypeptide being at least about 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) . An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P13

(SEQ ID NO:27) , comprising a first amino acid sequence being at least about 90% homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERID KQIRYILDGISALRK corresponding to amino acids 1 - 69 of IL6_HUMAN, which also corresponds to amino acids 1 - 69 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , and a second amino acid sequence being at least about 90% homologous to EETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKN LDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM corresponding to amino acids 108 - 212 of 1L6_HUMAN, which also corresponds to amino acids 70 - 174 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) . wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

An isolated chimeric polypeptide encoding for an edge portion of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KE, having a structure as follows: a sequence starting from any of amino acid numbers 69-x to 69; and ending at any of amino acid numbers 70+ ((n-2) - x), in which x varies from 0 to n-2.

An antibody capable of specifically binding to an epitope of an amino acid sequence as described herein.

An antibody capable of specifically binding to an epitope of an amino acid sequence as described above, optionally wherein said amino acid sequence corresponds to a bridge, edge portion, tail, or head as in any of the previous claims, also optionally wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein. A method for treating a variant-treatable disease, comprising administering a therapeutic protein, variant peptide, protein, nucleic acid sequence, antisense and/or antibody to a subject in need of treatment thereof. Alternatively, the variant-treatable disease is cluster S56892-treatable disease and is selected from the group consisting of inflammatory disorders including but not limited to ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporiosis, RA, Crohn's disease, Castleman's disease, systemic lupus erythematosus, inflammatory-mesangial proliferative glomerulonephritis, autoimmune-RA, Psoriasis, Parkinson's disease, myeloproliferative disorders and cancerous diseases, including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer and cachexia associated with cancer.

According to optional but preferred embodiments of the present invention, there is provided a nucleic acid construct comprising the isolated polynucleotide as described herein. Preferably, the nucleic acid construct further comprises a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation. Also preferably, the nucleic acid construct further comprises positive and negative selection markers for selecting for homologous recombination events. According to other optional but preferred embodiments of the present invention, there is provided a host cell comprising the nucleic acid construct as described herein.

According to preferred embodiments of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as described herein and a pharmaceutically acceptable carrier or diluent.

According to preferred embodiments of the present invention, there is provided a method of treating a variant- related disease in a subject, the method comprising upregulating in the subject expression of a polypeptide as described herein, thereby treating the variant-related disease in a subject. Optionally, upregulating expression of said polypeptide is effected by:

(i) administering said polypeptide to the subject; and/or

(ii) administering an expressible polynucleotide encoding said polypeptide to the subject. All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.

Information given in the text with regard to cellular localization was determined according to four different software programs: (i) tinhmm (from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://www.cbs.dtu.dlc/services/TMHMM/TMHMM2.0b.guide.php) or (ii) tmpred (from EMBnet, maintained by the ISREC Bionformatics group and the LICR Information Technology Office, Ludwig Institute for Cancer Research, Swiss Institute of Bioinformatics, http://www.ch. embnet.org/software/TMPRED_form.html) for transmembrane region prediction; (iii) signalp_hmm and (iv) signalp_nn (both from Center for Biological Sequence Analysis, Technical University of Denmark DTU, http://wλvw,cbs.dtu.dk/senάces/SignalP/background/prediction.php) for signal peptide prediction. The terms "signalpjimm" and "signalp_nn" refer to two modes of operation for the program SignalP: hnim refers to Hidden Markov Model, while mi refers to neural networks. Localization was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor. In some cases for the manual inspection of cellular localization prediction inventors used the ProLoc computational platform [Einat Hazkani-Covo, Erez Levanon, Galit Rotman. Dan Graur and Amit Novik; (2004) Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis. Cell Biology International 2004;28(3): 171-8.], which predicts protein localization based on various parameters including, protein domains (e.g., prediction of trans- membranous regions and localization thereof within the protein), pi, protein length, amino acid composition, homology to pre-armotated proteins, recognition of sequence patterns which direct the protein to a certain organelle (such as, nuclear localization signal. NLS, mitochondria localization signal), signal peptide and anchor modeling and using unique domains from Pfam that are specific to a single compartment.

Information is given in the text with regard to SNPs (single nucleotide polymorphisms). A description of the abbreviations is as follows. "T - > C", for example, means that the SNP results in a change at the position given in the table from T to C. Similarly, "M - > Q", for example, means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frameshift has occurred. A frameshift may also be indicated with a hyphen (-). A stop codon is indicated with an asterisk at the right hand side (*). As part of the description of an SNP, a comment may be found in parentheses after the above description of the SNP itself. This comment may include an FTId, which is an identifier to a SwissProt entry that was created with the indicated SNP. An FTId is a unique and stable feature identifier, which allows construction of links directly from position-specific annotation in the feature table to specialized protein-related databases, The FTId is always the last component of a feature in the description field, as follows: FTId=XXX_number, in which XXX is the 3-letter code for the specific feature key, separated by an underscore from a 6-digit number. In the table of the amino acid mutations of the wild type proteins of the selected splice variants of the invention, the header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence. For each given SNP, it was determined whether it was previously known by using dbSNP build 122 from NCBI, released on August 13, 2004.

Information given in the text with regard to the Homology to the wild type was determined by Smith-Waterman version 5,1.2 Using Special (non default) parameters as follows: -model=sw.model -GAPEXT=O -GAPOP=I 00.0 -MATRIX=blosumlOO Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionaiy of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of

Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.

Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Haiper Collins

Dictionary of Biology (1991). All of these are hereby incoiporated by reference as if fully set forth herein. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings: Figure 1 shows schematic comparison of the domain structure of IL-6 variants to the known or wild-type (WT) proteins. P163, P198, P95 and P174 are

S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) and S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , respectively. The Signal Peptide (SP) and the Helixes A, B, C and D are indicated, Figure 2 shows the optimized nucleotide and protein sequences for IL-6 variants according to the present invention as synthesized (including His-tag and Strep-tag sequences). The known WT IL-6 optimized nucleotide and protein sequence as synthesized (including His-tag and Strep-tag sequences) are shown as well. Figure 3 shows a schematic diagram of an exemplary construct for expressing IL-6 174 protein according to the present invention (using the nucleotide sequence shown in Figure 2).

Figure 4 is a Western blot of purified IL-6 174 protein according to the present invention; IL-6 174 protein itself is in lane 8, and is indicated with an arrow. Lane 10 represents lOOng of a His tagged positive control protein, and lane 1 is the molecular weight marker.

Figure 5 is the PCR analysis results. The high molecular weight PCR band in lane 6 represents the wild type (known) IL-6. The low molecular weight PCR band in lane 7 represents the IL-6 174 variant of the present invention.

Figure 6 shows the results of the 250 niM Imidazole purification step. Lanes 3 and 4 contain the purified IL-6 174. Lane 1 is unpurified material and lane 2 is the molecular weight marker.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of novel IL-6 variant polypeptides and polynucleotides encoding same, which can be used for the diagnosis and treatment of a wide range of diseases, such as cancer and inflammatory diseases.

According to still other preferred embodiments, the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein, including any oligopeptide or peptide relating to such an amino acid sequence or fragment, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges. The present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.

The present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.

In another embodiment, the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides. Such bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.

As used herein a "tail" refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.

As used herein a "head" refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein. As used herein "an edge portion" refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein. An edge may optionally arise due to a join between the above "known protein" portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein. A "bridge" may optionally be an edge portion as described above, but may also include a join between a head and a "known protein" portion of a variant, or a join between a tail and a "known protein" portion of a variant, or a join between an insertion and a "known protein" portion of a variant. Optionally and preferably, a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant, comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a valiant, Also optionally, the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 1 1, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between).

?? It should be noted that a bridge cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself. Furthermore, bridges are described with regard to a sliding window in certain contexts below. For example, certain descriptions of the bridges feature the following format: a bridge between two edges (in which a portion of the known protein is not present in the variant) may optionally be described as follows: a bridge portion of CONTIG-NAME_P1 (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-NAME_P1): a sequence starting from any of amino acid numbers 49-x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) - x) (for example), in which x varies from 0 to n-2. In this example, it should also be read as including bridges in which n is any number of amino acids between 10-50 amino acids in length, Furthermore, the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1, nor 50 + ((n-2) - x) (for example) greater than the total sequence length.

In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such antibodies differentially recognize splice valiants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).

In another embodiment, this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto. In another embodiment, this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention. In another embodiment, this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention. According to preferred embodiments of the present invention, the markers of the present invention, alone or in combination, can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of a marker-detectable disease. For example, optionally and preferably, these markers may be used for staging the disease in patient (for example if the disease features cancer) and/or monitoring the progression of the disease. Furthermore, the markers of the present invention, alone or in combination, can be used for detection of the source of metastasis found in anatomical places other than the originating tissue, again in the example of cancer. Also, one or more of the markers may optionally be used in combination with one or more other disease markers (other than those described herein).

Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease. These markers are specifically released to the bloodstream under conditions of a particular disease, and/or are otherwise expressed at a much higher level and/or specifically expressed in tissue or cells afflicted with or demonstrating the disease. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of a particular disease and/or a condition that is indicative of a higher risk for a particular disease.

The present invention therefore also relates to diagnostic assays for marker- detectable disease and/or an indicative condition, and methods of use of such markers for detection of marker-detectable disease and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.

In another embodiment, this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice valiant in the biological sample.

In another embodiment, this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.

According to the present invention, the splice variants described herein are non-limiting examples of markers for diagnosing marker-detectable disease and/or an indicative condition. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of marker-detectable disease and/or an indicative condition, including a transition from an indicative condition to marker-detectable disease.

According to optional but preferred embodiments of the present invention, any marker according to the present invention may optionally be used alone or combination. Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker. Furthermore, such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker. With regard to such a ratio between any marker described herein (or a combination thereof) and a known marker, more preferably the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene. Panels of markers according to the present invention optionally with one or more known marker(s)

The present invention is of methods, uses, devices and assays for diagnosis of a disease or condition. Optionally a plurality of biomarkers (or markers) may be used with the present invention. The plurality of markers may optionally include a plurality of markers described herein, and/or one or more known markers. The plurality of markers is preferably then correlated with the disease or condition, For example, such correlating may optionally comprise determining the concentration of each of the plurality of markers, and individually comparing each marker concentration to a threshold level. Optionally, if the marker concentration is above or below the threshold level (depending upon the marker and/or the diagnostic test being performed), the marker concentration correlates with the disease or condition. Optionally and preferably, a plurality of marker concentrations correlate with the disease or condition. Alternatively, such correlating may optionally comprise determining the concentration of each of the plurality of markers, calculating a single index value based on the concentration of each of the plurality of markers, and comparing the index value to a threshold level.

Also alternatively, such correlating may optionally comprise determining a temporal change in at least one of the markers, and wherein the temporal change is used in the correlating step.

Also alternatively, such correlating may optionally comprise determining whether at least "X" number of the plurality of markers has a concentration outside of a predetermined range and/or above or below a threshold (as described above). The value of "X" may optionally be one marker, a plurality of markers or all of the markers; alternatively or additionally, rather than including any marker in the count for "X", one or more specific markers of the plurality of markers may optionally be required to correlate with the disease or condition (according to a range and/or threshold). Also alternatively, such correlating may optionally comprise determining whether a ratio of marker concentrations for two markers is outside a range and/or above or below a threshold. Optionally, if the ratio is above or below the threshold level and/or outside a range, the ratio correlates with the disease or condition. Optionally, a combination of two or more these correlations may be used with a single panel and/or for correlating between a plurality of panels.

Optionally, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to normal subjects. As used herein, sensitivity relates to the number of positive (diseased) samples detected out of the total number of positive samples present; specificity relates to the number of true negative (non-diseased) samples detected out of the total number of negative samples present. Preferably, the method distinguishes a disease or condition with a sensitivity of at least 80% at a specificity of at least 90% when compared to normal subjects. More preferably, the method distinguishes a disease or condition with a sensitivity of at least 90% at a specificity of at least 90% when compared to normal subjects. Also more preferably, the method distinguishes a disease or condition with a sensitivity of at least 70% at a specificity of at least 85% when compared to subjects exhibiting symptoms that mimic disease or condition symptoms. A marker panel may be analyzed in a number of fashions well known to those of skill in the art. For example, each member of a panel may be compared to a "normal" value, or a value indicating a particular outcome, A particular diagnosis/prognosis may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers are outside of a normal range, this subset may be indicative of a particular diagnosis/prognosis. The skilled artisan will also understand that diagnostic markers, differential diagnostic markers, prognostic markers, time of onset markers, disease or condition differentiating markers, etc., may be combined in a single assay or device. For example, with stroke as a non-limiting example of a disease or condition, certain markers in a panel may be commonly used to diagnose the existence of a stroke, while other members of the panel may indicate if an acute stroke has occurred, while still other members of the panel may indicate if a non-acute stroke has occulted. Markers may also be commonly used for multiple purposes by, for example, applying a different threshold or a different weighting factor to the marker for the different puipose(s). For example, again with stroke as a non-limiting example of a disease or condition, a marker at one concentration or weighting may be used, alone or as part of a larger panel, to indicate if an acute stroke has occurred, and the same marker at a different concentration or weighting may be used, alone or as part of a larger panel, to indicate if a non-acute stroke has occurred. Preferred panels comprise markers for the following purposes: diagnosis of a disease; diagnosis of disease and indication if the disease is in an acute phase and/or if an acute attack of the disease has occurred; diagnosis of disease and indication if the disease is in a non-acute phase and/or if a non-acute attack of the disease has occurred; indication whether a combination of acute and non-acute phases or attacks has occurred; diagnosis of a disease and prognosis of a subsequent adverse outcome; diagnosis of a disease and prognosis of a subsequent acute or non-acute phase or attack; disease progression (for example for cancer, such progression may include for example occurrence or recurrence of metastasis). The above diagnoses may also optionally include differential diagnosis of the disease to distinguish it from other diseases, including those diseases that may feature one or more similar or identical symptoms.

In certain embodiments, one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s). In other embodiments, threshold level(s) of a diagnostic or prognostic indicator(s) can be established, and the level of the indicator(s) in a patient sample can simply be compared to the threshold level(s). The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test— they also depend on the definition of what constitutes an abnormal result. In practice. Receiver Operating Characteristic curves, or "ROC" curves, are typically calculated by plotting the value of a variable versus its relative frequency in "normal" and "disease" populations, and/or by comparison of results from a subject before, during and/or after treatment. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition.

The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cutoff selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

ROC curves can be used even when test results don't necessarily give an accurate number. As long as one can rank results, one can create an ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (say l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art (see for example Hanley et al., Radiology 143: 29-36 (1982), incorporated by reference as if fully set forth herein).

One or more markers may lack diagnostic or prognostic value when considered alone, but when used as part of a panel, such markers may be of great value in determining a particular diagnosis/prognosis. In preferred embodiments, particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, the present invention may utilize an evaluation of the entire marker profile by plotting ROC curves for the sensitivity of a particular panel of markers versus 1 -(specificity) for the panel at various cutoffs. In these methods, a profile of marker measurements from a subject is considered together to provide a global probability (expressed either as a numeric score or as a percentage risk) that an individual has had a disease, is at risk for developing such a disease, optionally the type of disease which the individual has had or is at risk for, and so forth etc. In such embodiments, an increase in a certain subset of markers may be sufficient to indicate a particular diagnosis/prognosis in one patient, while an increase in a different subset of markers may be sufficient to indicate the same or a different diagnosis/prognosis in another patient. Weighting factors may also be applied to one or more markers in a panel, for example, when a marker is of particularly high utility in identifying a particular diagnosis/prognosis, it may be weighted so that at a given level it alone is sufficient to signal a positive result. Likewise, a weighting factor may provide that no given level of a particular marker is sufficient to signal a positive result, but only signals a result when another marker also contributes to the analysis.

In preferred embodiments, markers and/or marker panels are selected to exhibit at least 70% sensitivity, more preferably at least 80% sensitivity, even more preferably at least 85% sensitivity, still more preferably at least 90% sensitivity, and most preferably at least 95% sensitivity, combined with at least 70% specificity, more preferably at least 80% specificity, even more preferably at least 85% specificity, still more preferably at least 90% specificity, and most preferably at least 95% specificity. In particularly preferred embodiments, both the sensitivity and specificity are at least 75%, more preferably at least 80%, even more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95%. Sensitivity and/or specificity may optionally be determined as described above, with regard to the construction of ROC graphs and so forth, for example.

According to preferred embodiments of the present invention, individual markers and/or combinations (panels) of markers may optionally be used for diagnosis of time of onset of a disease or condition. Such diagnosis may optionally be useful for a wide variety of conditions, preferably including those conditions with an abrupt onset.

The phrase "determining the prognosis" as used herein refers to methods by which the skilled artisan can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition, the chance of a given outcome may be about 3%. In preferred embodiments, a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, and about a 95% chance. The term "about" in this context refers to +/- 1%. The skilled artisan will understand that associating a prognostic indicator with a predisposition to an adverse outcome is a statistical analysis. For example, a marker level of greater than 80 pg/niL may signal that a patient is more likely to suffer from an adverse outcome than patients with a level less than or equal to 80 pg/mL, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 , and 0.0001. Exemplary statistical tests for associating a prognostic indicator with a predisposition to an adverse outcome are described hereinafter. In other embodiments, a threshold degree of change in the level of a prognostic or diagnostic indicator can be established, and the degree of change in the level of the indicator in a patient sample can simply be compared to the threshold degree of change in the level. A preferred threshold change in the level for markers of the invention is about 5%, about 10%, about 15%, about 20%, about 25%. about 30%, about 50%, about 75%, about 100%, and about 150%. The term "about" in this context refers to +/-10%. In yet other embodiments, a "nomogram" can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages.

Exemplary, non-limiting methods and systems for identification of suitable biomarkers for marker panels are now described. Methods and systems for the identification of a one or more markers for the diagnosis, and in particular for the differential diagnosis, of disease have been described previously. Suitable methods for identifying markers useful for the diagnosis of disease states are described in detail in U.S. patent application no. 2004-0126767, entitled METHOD AND SYSTEM FOR DISEASE DETECTION USING MARKER COMBINATIONS, filed Dec. 27, 2002, hereby incoiporated by reference in its entirety as if fully set forth herein. One skilled in the art will also recognize that univariate analysis of markers can be performed and the data from the univariate analyses of multiple markers can be combined to form panels of markers to differentiate different disease conditions.

In developing a panel of markers useful in diagnosis, data for a number of potential markers may be obtained from a group of subjects by testing for the presence or level of certain markers. The group of subjects is divided into two sets, and preferably the first set and the second set each have an approximately equal number of subjects. The first set includes subjects who have been confirmed as having a disease or, more generally, being in a first condition state. For example, this first set of patients may be those that have recently had a disease and/or a particular type of the disease. The confirmation of this condition state may be made through more rigorous and/or expensive testing, preferably according to a previously defined diagnostic standard. Hereinafter, subjects in this first set will be referred to as "diseased".

The second set of subjects are simply those who do not fall within the first set. Subjects in this second set may be "non-diseased;" that is, normal subjects. Alternatively, subjects in this second set may be selected to exhibit one symptom or a constellation of symptoms that mimic those symptoms exhibited by the "diseased" subjects,

The data obtained from subjects in these sets includes levels of a plurality of markers. Preferably, data for the same set of markers is available for each patient. This set of markers may include all candidate markers which may be suspected as being relevant to the detection of a particular disease or condition. Actual known relevance is not required. Embodiments of the methods and systems described herein may be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition. The levels of each marker in the two sets of subjects may be distributed across a broad range, e.g., as a Gaussian distribution. However, no distribution fit is required.

As noted above, a marker often is incapable of definitively identifying a patient as either diseased or non-diseased. For example, if a patient is measured as having a marker level that falls within the overlapping region, the results of the test will be useless in diagnosing the patient. An artificial cutoff may be used to distinguish between a positive and a negative test result for the detection of the disease or condition. Regardless of where the cutoff is selected, the effectiveness of the single marker as a diagnosis tool is unaffected. Changing the cutoff merely trades off between the number of false positives and the number of false negatives resulting from the use of the single marker. The effectiveness of a test having such an overlap is often expressed using a ROC (Receiver Operating Characteristic) curve as described above.

As discussed above, the measurement of the level of a single marker may have limited usefulness. The measurement of additional markers provides additional information, but the difficulty lies in properly combining the levels of two potentially unrelated measurements. In the methods and systems according to embodiments of the present invention, data relating to levels of various markers for the sets of diseased and non-diseased patients may be used to develop a panel of markers to provide a useful panel response. The data may be provided in a database such as Microsoft Access, Oracle, other SQL databases or simply in a data file, The database or data file may contain, for example, a patient identifier such as a name or number, the levels of the various markers present, and whether the patient is diseased or non-diseased. Next, an artificial cutoff region may be initially selected for each marker. The location of the cutoff region may initially be selected at any point, but the selection may affect the optimization process described below. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer, hi a preferred method, the cutoff region is initially centered about the center of the overlap region of the two sets of patients. In one embodiment, the cutoff region may simply be a cutoff point. In other embodiments, the cutoff region may have a length of greater than zero. In this regard, the cutoff region may be defined by a center value and a magnitude of length. In practice, the initial selection of the limits of the cutoff region may be determined according to a pre-selected percentile of each set of subjects. For example, a point above which a pre-selected percentile of diseased patients are measured may be used as the right (upper) end of the cutoff range.

Each marker value for each patient may then be mapped to an indicator. The indicator is assigned one value below the cutoff region and another value above the cutoff region. For example, if a marker generally has a lower value for non-diseased patients and a higher value for diseased patients, a zero indicator will be assigned to a low value for a particular marker, indicating a potentially low likelihood of a positive diagnosis. In other embodiments, the indicator may be calculated based on a polynomial. The coefficients of the polynomial may be determined based on the distributions of the marker values among the diseased and non-diseased subjects.

The relative importance of the various markers may be indicated by a weighting factor. The weighting factor may initially be assigned as a coefficient for each marker. As with the cutoff region, the initial selection of the weighting factor may be selected at any acceptable value, but the selection may affect the optimization process. In this regard, selection near a suspected optimal location may facilitate faster convergence of the optimizer. In a preferred method, acceptable weighting coefficients may range between zero and one, and an initial weighting coefficient for each marker may be assigned as 0.5. In a preferred embodiment, the initial weighting coefficient for each marker may be associated with the effectiveness of that marker by itself. For example, a ROC curve may be generated for the single marker, and the area under the ROC curve may be used as the initial weighting coefficient for that marker.

Next, a panel response may be calculated for each subject in each of the two sets. The panel response is a function of the indicators to which each marker level is mapped and the weighting coefficients for each marker. One advantage of using an indicator value rather than the marker value is that an extraordinarily high or low marker levels do not change the probability of a diagnosis of diseased or non-diseased for that particular marker. Typically, a marker value above a certain level generally indicates a certain condition state. Marker values above that level indicate the condition state with the same certainty. Thus, an extraordinarily high marker value may not indicate an extraordinarily high probability of that condition state. The use of an indicator which is constant on one side of the cutoff region eliminates this concern.

The panel response may also be a general function of several parameters including the marker levels and other factors including, for example, race and gender of the patient, Other factors contributing to the panel response may include the slope of the value of a particular marker over time. For example, a patient may be measured when first arriving at the hospital for a particular marker, The same marker may be measured again an hour later, and the level of change may be reflected in the panel response. Further, additional markers may be derived from other markers and may contribute to the value of the panel response. For example, the ratio of values of two markers may be a factor in calculating the panel response.

Having obtained panel responses for each subject in each set of subjects, the distribution of the panel responses for each set may now be analyzed. An objective function may be defined to facilitate the selection of an effective panel. The objective function should generally be indicative of the effectiveness of the panel, as may be expressed by, for example, overlap of the panel responses of the diseased set of subjects and the panel responses of the non-diseased set of subjects. In this manner, the objective function may be optimized to maximize the effectiveness of the panel by, for example, minimizing the overlap.

In a preferred embodiment, the ROC curve representing the panel responses of the two sets of subjects may be used to define the objective function. For example, the objective function may reflect the area under the ROC curve. By maximizing the area under the curve, one may maximize the effectiveness of the panel of markers. In other embodiments, other features of the ROC curve may be used to define the objective function. For example, the point at which the slope of the ROC curve is equal to one may be a useful feature. In other embodiments, the point at wliich the product of sensitivity and specificity is a maximum, sometimes referred to as the "knee," may be used. In an embodiment, the sensitivity at the knee may be maximized. In further embodiments, the sensitivity at a predetermined specificity level may be used to define the objective function. Other embodiments may use the specificity at a predetermined sensitivity level may be used. In still other embodiments, combinations of two or more of these ROC-curve features may be used. It is possible that one of the markers in the panel is specific to the disease or condition being diagnosed. When such markers are present at above or below a certain threshold, the panel response may be set to return a "positive" test result. When the threshold is not satisfied, however, the levels of the marker may nevertheless be used as possible contributors to the objective function. An optimization algorithm may be used to maximize or minimize the objective function. Optimization algorithms are well-known to those skilled in the ait and include several commonly available minimizing or maximizing functions including the Simplex method and other constrained optimization techniques. It is understood by those skilled in the art that some minimization functions are better than others at searching for global minimums, rather than local minimunis. In the optimization process, the location and size of the cutoff region for each marker may be allowed to vary to provide at least two degrees of freedom per marker. Such variable parameters are referred to herein as independent variables. In a preferred embodiment, the weighting coefficient for each marker is also allowed to van,' across iterations of the optimization algorithm. In various embodiments, any permutation of these parameters may be used as independent variables.

In addition to the above-described parameters, the sense of each marker may also be used as an independent variable. For example, in many cases, it may not be known whether a higher level for a certain marker is generally indicative of a diseased state or a non-diseased state. In such a case, it may be useful to allow the optimization process to search on both sides. In practice, this may be implemented in several ways. For example, in one embodiment, the sense may be a truly separate independent variable which may be flipped between positive and negative by the optimization process. Alternatively, the sense may be implemented by allowing the weighting coefficient to be negative.

The optimization algorithm may be provided with certain constraints as well. For example, the resulting ROC curve may be constrained to provide an area-under- curve of greater than a particular value. ROC curves having an area under the curve of 0.5 indicate complete randomness, while an area under the curve of 1.0 reflects perfect separation of the two sets. Thus, a minimum acceptable value, such as 0.75, may be used as a constraint, particularly if the objective function does not incorporate the area under the curve. Other constraints may include limitations on the weighting coefficients of particular markers. Additional constraints may limit the sum of all the weighting coefficients to a particular value, such as 1.0.

The iterations of the optimization algorithm generally vary the independent parameters to satisfy the constraints while minimizing or maximizing the objective function. The number of iterations may be limited in the optimization process. Further, the optimization process may be terminated when the difference in the objective function between two consecutive iterations is below a predetermined threshold, thereby indicating that the optimization algorithm has reached a region of a local minimum or a maximum. Thus, the optimization process may provide a panel of markers including weighting coefficients for each marker and cutoff regions for the mapping of marker values to indicators. In order to develop lower-cost panels which require the measurement of fewer marker levels, certain markers may be eliminated from the panel. In this regard, the effective contribution of each marker in the panel may be determined to identify the relative importance of the markers. In one embodiment, the weighting coefficients resulting from the optimization process may be used to determine the relative importance of each marker. The markers with the lowest coefficients may be eliminated. Individual panel response values may also be used as markers in the methods described herein. For example, a panel may be constructed from a plurality of markers, and each marker of the panel may be described by a function and a weighting factor to be applied to that marker (as determined by the methods described above), Each individual marker level is determined for a sample to be tested, and that level is applied to the predetermined function and weighting factor for that particular marker to arrive at a sample value for that marker. The sample values for each marker are added together to arrive at the panel response for that particular sample to be tested. For a "diseased" and "non-diseased" group of patients, the resulting panel responses may be treated as if they were just levels of another disease marker. Measures of test accuracy may be obtained as described in Fischer et al..

Intensive Care Med. 29: 1043-51, 2003 (hereby incorporated by reference as if fully set forth herein), and used to determine the effectiveness of a given marker or panel of markers. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. As discussed above, suitable tests may exhibit one or more of the following results on these various measures: at least 75% sensitivity, combined with at least 75% specificity; ROC curve area of at least 0.7, more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0,95; and/or a positive likelihood ratio (calculated as sensitivity/(l -specificity)) of at least 5, more preferably at least 10, and most preferably at least 20, and a negative likelihood ratio (calculated as (1- sensitivity)/specificity) of less than or equal to 0.3, more preferably less than or equal to 0.2, and most preferably less than or equal to 0.1. According to other preferred embodiments of the present invention, a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof, may be featured as a biomarker for detecting marker-detectable disease and/or an indicative condition, such that a biomarker may optionally comprise any of the above.

According to still other preferred embodiments, the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein. Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges. The present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides. The present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.

Non-limiting examples of methods or assays are described below. The present invention also relates to kits based upon such diagnostic methods or assays.

Nucleic acid sequences and Oligonucleotides

Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion. The present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewith, sequences homologous thereto

[e.g., at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75

%, at least 80 %, at least 85 %, at least 95 % or more say 100 % identical to the nucleic acid sequences set forth below], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion. The present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.

In cases where the polynucleotide sequences of the present invention encode previously unidentified polypeptides, the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.

Thus, the present invention provides isolated polynucleotides each encoding a polypeptide which is at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, %, at least 85 %, %, at least 90 %, at least 95 % or more, say 100 % identical to a polypeptide sequence listed in the Examples section or sequence listing, as determined using the LALIGN software of EMBnet Switzerland (http://www.ch.embnet.org/index.html) using default parameters.

A "nucleic acid fragment" or an "oligonucleotide" or a "polynucleotide" are used herein interchangeably to refer to a polymer of nucleic acids. A polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent

DNA polymerase.

As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is composed of genomic and cDNA sequences. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

Preferred embodiments of the present invention encompass oligonucleotide probes.

An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).

Alternatively, an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).

Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988) and "Oligonucleotide Synthesis" Gait, M. J., ed. (1984) utilizing solid phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting and purification by for example, an automated trityl-on method or HPLC. Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases. Preferably, the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.

The oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage. Preferably used oligonucleotides are those modified at one or more of the backbone, intemucleoside linkages or bases, as is broadly described hereinunder.

Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. NOs: 4,469.863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264.423; 5,276,019; 5,278,302; 5,286,717; 5,321,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5.466, 677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,1 11 ; 5,563,253; 5,571,799; 5,587,361; and 5,625,050. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters. methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these_., and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2', Various salts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466.677; 5,470,967; 5,489,677; 5,541 ,307; 5,561 ,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic, includes peptide nucleic acid (PNA). United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.

Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), A- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-niethylguanine and 7-methyladenine, S- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-niethylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁰C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammoniuni l ^-di-O-hexadecyl-rac-glycero-S-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, as disclosed in U.S. Pat. No: 6,303,374.

It is not necessary for all positions in a given oligonucleotide molecule to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide,

It will be appreciated that oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.

Expression of the polynucleotide sequence of the present invention To enable cellular expression of the polynucleotides of the present invention, a nucleic acid construct (or an "expression vector") according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element. As used herein, the phrase "cis acting regulatory element" refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.

Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed. Examples of cell type- specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al, (1987) Genes Dev. 1 :268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473- 5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). The nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom, Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40. In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that cany the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types, Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryotic ieplicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide. The nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication. Preferably, the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice. The construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a vims or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.

Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. For example, bone marrow cells can be targeted using the human T cell leukemia vims type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovims Autographa californica nucleopolyhedrovirus (AcMNPV) as described in Liang CY et al,, 2004 (Arch Virol. 149: 51-60). Recombinant viral vectors are useful for in vivo expression of the polynucleotide sequence of the present invention since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired puipose is to introduce a specified gene into only a localized number of targeted cells.

Various methods can be used to introduce the expression vector of the present invention into stem cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttenvorths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5.464,764 and 5,487,992 for positive-negative selection methods. Preferably, the nucleic acid construct according to the present invention further comprises positive and negative selection markers for selecting for homologous recombination events as is known in the art.

Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses, Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.

Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of the present invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed peptide. For example, the expression of a fusion protein or a cleavable fusion protein comprising Met variant of the present invention and a heterologous protein can be engineered. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein, Where a cleavage site is engineered between the Met moiety and the heterologous protein, the Met moiety can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].

As mentioned hereinabove, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasniid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant vims expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid. containing the coding sequence. Mammalian expression systems can also be used to express the polypeptides of the present invention.

Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).

In yeast, a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447. Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome. In cases where plant expression vectors are used, the expression of the coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al. (1986) MoI. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421- 463.

Other expression systems such as insects and mammalian host cell systems which are well known in the art and are further described hereinbelow can also be used by the present invention.

Recovery of the recombinant polypeptide is effected following an appropriate time in culture. The phrase "recovering the recombinant polypeptide" refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Not withstanding the above, polypeptides of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Hybridization assays

Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non-limiting examples of probes according to the present invention were previously described). Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA). and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.

Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15. 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.

Thus, the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions. Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32P labeled probe, at 65 ⁰C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65⁰C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32P labeled probe, at 65 ⁰C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 ⁰C.

More generally, hybridization of short nucleic acids (below 200 bp in length, e.g. 17-40 bp in length) can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency; (i) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 nig/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 ⁰C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 ⁰C below the Tm; (ii) hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 mg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 ⁰C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 ⁰C below the Tm, final wash solution of 6 x SSC. and final wash at 22 ⁰C; (iii) hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 mg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature. The detection of hybrid duplexes can be carried out by a number of methods.

Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Such labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. A label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.

Probes can be labeled according to numerous well known methods. Non- limiting examples of radioactive labels include 3H, 14C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio-nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.

For example, oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively, when fluorescently-labeled oligonucleotide probes are used, fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.

Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.

It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.

Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods.

As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples of radioactive labels include

3H, 14C. 32P, and 35S.

Those skilled in the ait will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.

It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like, Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA. Amino acid sequences and peptides

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 analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide" and "protein" include glycoproteins, as well as non-glycoproteins. Polypeptide products can be biochemically synthesized such as by employing Standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

Solid phase polypeptide synthesis procedures are well known in the ait and further described by JoIm Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co, N. Y.], after which their composition can be confirmed via amino acid sequencing. In cases where large amounts of a polypeptide are desired, it can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843. Gurley et al. (1986) MoI. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII. pp 421-463.

The present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein. The present invention also encompasses homologues of these polypeptides, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low- complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50. Finally, the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.

It will be appreciated that peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified. Further details in this respect are provided hereinunder.

Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)H-C-O-O-C(R)-N-), ketomethylen bonds (-CO-CH2-), *-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2- ), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retro amide bonds (-NH-CO-), peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

Natural aromatic amino acids, Tip, Tyr and Phe, may be substituted for synthetic non-natural acid such as Phenylglycine, TIC. naphthylelanine (NoI), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o-niethyl-Tyr.

In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.

Table 1 non-conventional or modified amino acids which can be used with the present invention.

Table 1

Table 1 Cont.

Since the peptides of the present invention are preferably utilized in therapeutics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.

The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.

The peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short {i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (L e. , not encoded by a nucleic acid sequence) and therefore involves different chemistry,

Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.

In cases where large amounts of the peptides of the present invention are desired, the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al.. (1987) Methods in Enzymol. 153:516- 544, Studier et al. (1990) Methods in Enzymol. 185:60-89_.. Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3: 1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) MoI. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 and also as described above. Peptide sequences which exhibit high therapeutic activity, such as by competing with wild type signaling proteins of the same signaling pathway, can be also uncovered using computational biology. Software programs useful for displaying three-dimensional structural models, such as RIBBONS (Carson, M., 1997. Methods in Enzymology 277, 25), O (Jones, TA. et al., 1991. Acta Crystallogr. A47, 110), DINO (DINO: Visualizing Structural Biology (2001) http://www.dino3d.org); and QUANTA, INSIGHT. SYBYL. MACROMODE, ICM, MOLMOL, RASMOL and GRASP (reviewed in Kraulis. J., 1991. Appl Crystallogr. 24, 946) can be utilized to model interactions between the polypeptides of the present invention and prospective peptide sequences to thereby identify peptides which display the highest probability of binding for example to a respective ligand (e.g., IL-IO). Computational modeling of protein-peptide interactions has been successfully used in rational drug design, for further details, see Lam et al., 1994, Science 263, 380; Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993. Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993. Perspectives in Drug Discovery and Design 1, 109, and Mauro MJ. et al., 2002. J Clin Oncol. 20, 325-34.

Antibodies

"Antibody" refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsiloii and mu heavy chain constant region genes, and the myriad-immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g._.. Fab' and F(ab)'2 fragments. 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. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHl, CH2 and CH3, but does not include the heavy chain variable region.

The functional fragments of antibodies, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages, are described as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain: two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the ait (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988. incorporated herein by reference). Monoclonal antibody development may optionally be performed according to any method that is known in the art, The method described below is provided for the purposes of description only and is not meant to be limiting in any way.

Step 1 : Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells

Producing mAb requires immunizing an animal, usually a mouse, by injection of an antigen X to stimulate the production of antibodies targeted against X. Antigen X can be the whole protein or any sequence thereof that gives rise to a determinant. According to the present invention, optionally and preferably such antigens may include but are not limited to any variant described herein or a portion thereof, including but not limited to any head, tail, bridge or unique insertion, or a bridge to such head, tail or unique insertion, or any other epitope described herein according to the present invention. Injection of peptides requires peptide design (with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability) and synthesis. The antigen is optionally and preferably prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes optionally used as immunogens. Other immunogens or adjuvants may also optionally be used. In general, mice are immunized every 2-3 weeks but the immunization protocols are heterogeneous. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.

Step 2: Screening of Mice for Antibody Production

After several weeks of immunization, blood samples are optionally and preferably obtained from mice for measurement of serum antibodies, Several techniques have been developed for collection of small volumes of blood from mice

(Loeb and Quimby 1999). Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and/or immunoassays for example (for example a Western blot may optionally be used). If the antibody titer is high, cell fusion can optionally be performed. If the titer is too low, mice can optionally be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneal Iy or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.

Step 3: Preparation of Myeloma Cells

Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are optionally and preferably cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion. The selection growth medium contains the inhibitor aminopterin, which blocks synthetic pathways by which nucleotides are made. Therefore, the cells must use a bypass pathway to synthesize nucleic acids, a pathway that is defective in the myeloma cell line to which the normal antibody-producing cells are fused. Because neither the myeloma nor the antibody-producing cell will grow on its own, only hybrid cells grow. The HAT medium allows only the fused cells to survive in culture. A week before cell fusion, myeloma cells are grown in 8-azaguanine, Cells must have high viability and rapid growth.

The antibody forming cells are isolated from the mouse's spleen and are then fused with a cancer cell (such as cells from a myeloma) to make them immortal, which means that they will grow and divide indefinitely. The resulting cell is called a hybridoma.

Step 4: Fusion of Myeloma Cells with Immune Spleen Cells and antibody screening Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. Alternatively, the cells are centiϊfuged, the supernatant is discarded and PEG is then added. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells (Quinlan and Kennedy 1994). Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells. It is also possible to use murine bone marrow-derived macrophages as feeder cells (Hoffman and others 1996).

Once hybridoma colonies reach a satisfactory cell count, the plates are assayed by an assay, eg ELISA or a regular immunoassay such as RIA for example, to determine which colonies are secreting antibodies to the immunogen. Cells from positive wells are isolated and expanded. Conditioned medium from each colony is retested to verify the stability of the hybridomas (that is, they continue to produce antibody).

Step 5: Cloning of Hybridoma Cell Lines by "Limiting Dilution" or Expansion and Stabilization of Clones by Ascites Production

At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time.

For prolonged stability of the antibody-producing cell lines, it is necessary to clone and then recline the chosen cells. Cloning consists of subcloonng the cells by either limiting dilution at an average of less than one cell in each culture well or by platingout the cells in a thin layer of semisolid agar of methyl cellulose or by single- cell manipulation. At each stage, cultures are assayed for production of the appropriate antibodies. Step 6: Antibody purification The secreted antibodies are optionally purified, preferably by one or more column chromatography steps and/or some other purification method, including but not limited to ion exchange, affinity, hydrophobic interaction, and gel permeation chromatography. The operation of the individual chromatography step, their number and their sequence is generally tailored to the specific antibody and the specific application.

Large-scale antibody production may also optionally and preferably be performed according to the present invention. Two non-limiting, illustrative exemplary methods are described below for the purposes of description only and are not meant to be limiting in any way.

In vivo production may optionally be performed with ascites fluid in mice. According to this method, hybridoma cell lines are injected into the peritoneal cavity of mice to produce ascitic fluid (ascites) in its abdomen; this fluid contains a high concentration of antibody.

An exemplar}' in vitro method involves the use of culture flasks. In this method, monoclonal antibodies can optionally be produced from the hybridoma using gas permeable bags or cell culture flasks.

Antibody Engineering in Phage Display Libraries

PCT Application No. WO 94/18219, and its many US equivalents, including US Patent No. 6096551, all of which are hereby incoiporated by reference as if fully set forth herein, describes methods for producing antibody libraries using universal or randomized immunoglobulin light chains, by using phage display libraries. The method involves inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene for the purpose of producing light chain gene libraries for use in combination with heavy chain genes and gene libraries to produce antibody libraries of diverse and novel immunospecificities. The method comprises amplifying a CDR portion of an immunoglobulin light chain gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide. The resultant gene portions are inserted into phagemids for production of a phage display library, wherein the engineered light chains are displayed by the phages, for example for testing their binding specificity. Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 1 19-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659- 62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker, These single- chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. A scFv antibody fragment is an engineered antibody derivative that includes heavy- and light chain variable regions joined by a peptide linker. The minimal size of antibody molecules are those that still comprise the complete antigen binding site. ScFv antibody fragments are potentially more effective than unmodified IgG antibodies. The reduced size of 27-30 kDa permits them to penetrate tissues and solid tumors more readily. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11 :1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety. Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Laπϊck and Fry [Methods, 2: 106-10 (1991)]. Optionally, there may be 1 , 2 or 3 CDRs of different chains, but preferably there are 3 CDRs of 1 chain. The chain could be the heavy or the light chain.

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') 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, the 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 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)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. 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), wherein substantially less than an intact human valuable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI, Biol., 227:381 (1991); Marks et al., J. MoI. 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, Alan R. Liss, p, 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction 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 all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, 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); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995). Preferably, the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Optionally, a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site. An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination. One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.

Display Libraries According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-17, 15-20, 15-30 or 20-50 consecutive amino acids derived from the polypeptide sequences of the present invention. Since in therapeutic applications it is highly desirable to employ the minimal and most efficacious polypeptide regions, which still exert therapeutic function, identification of such peptide regions can be effected using various approaches, including, for example, display techniques as described herein.

Methods of constructing such display libraries are well known in the art. Such methods are described in, for example, Young AC, et al., "The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes" J MoI Biol 1997 Dec 12;274(4):622-34; Giebel LB et al. "Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities" Biochemistry 1995 Nov 2S;34(47):15430-5; Davies EL et al., "Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes" J Immunol Methods 1995 Oct 12;186(l):125-35; Jones C RT al. "Current trends in molecular recognition and bioseparation" J Cliromatogr A 1995 JuI 14;707(l):3-22; Deng SJ et al. "Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries" Proc Natl Acad Sci U S A 1995 May 23;92(11):4992-6; and Deng SJ et al. "Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display" J Biol Chem 1994 Apr l ;269(13):9533-8, which are incorporated herein by reference.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. A "variant-treatable" disease refers to any disease that is treatable by using a splice variant of any of the therapeutic proteins according to the present invention. "Treatment" also encompasses prevention, amelioration, elimination and control of the disease and/or pathological condition. The diseases for which such valiants may be useful therapeutic agents are described in greater detail below for each of the variants. The variants themselves are described by "cluster" or by gene, as these variants are splice variants of known proteins. Therefore, a "cluster-related disease" or a "protein-related disease" refers to a disease that may be treated by a particular protein, with regard to the description of such diseases below a therapeutic protein variant according to the present invention. The term "biologically active", as used herein, refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" refers to the capability of the natural, recombinant, or synthetic ligand, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The term "modulate", as used herein, refers to a change in the activity of at least one receptor mediated activity. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional or immunological properties of a ligand.

Methods Of Treatment

As mentioned hereinabove the novel therapeutic protein variants of the present invention and compositions derived therefrom {i.e., peptides, oligonucleotides) can be used to treat cluster or protein-related diseases, disorders or conditions.

Thus, according to an additional aspect of the present invention there is provided a method of treating cluster or protein-related disease, disorder or condition in a subject.

The subject according to the present invention is a mammal, preferably a human which is diagnosed with one of the disease, disorder or conditions described hereinabove, or alternatively is predisposed to at least one type of the cluster or protein-related disease, disorder or conditions described hereinabove.

As used herein the term "'treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the above-described diseases, disorders or conditions.

Treating, according to the present invention, can be effected by specifically upregulating or alternatively downregulating the expression of at least one of the polypeptides of the present invention in the subject.

Optionally, upregulation may be effected by administering to the subject at least one of the polypeptides of the present invention (e.g., recombinant or synthetic) or an active portion thereof, as described herein. However, since the bioavailability of large polypeptides may potentially be relatively small due to high degradation rate and low penetration rate, administration of polypeptides is preferably confined to small peptide fragments (e.g., about 100 amino acids). The polypeptide or peptide may optionally be administered in as part of a pharmaceutical composition, described in more detail below.

It will be appreciated that treatment of the above-described diseases according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). Thus, treatment of malignancies using the agents of the present invention may be combined with, for example, radiation therapy, antibody therapy and/or chemotherapy.

Alternatively or additionally, an upregulating method may optionally be effected by specifically upregulating the amount (optionally expression) in the subject of at least one of the polypeptides of the present invention or active portions thereof.

As is mentioned hereinabove and in the Examples section which follows, the biomolecular sequences of this aspect of the present invention may be used as valuable therapeutic tools in the treatment of diseases, disorders or conditions in which altered activity or expression of the wild-type gene product is known to contribute to disease, disorder or condition onset or progression. For example, in case a disease is caused by overexpression of a membrane bound-receptor, a soluble variant thereof may be used as an antagonist which competes with the receptor for binding the ligand, to thereby terminate signaling from the receptor. Examples of such diseases are listed in the Examples section which follows.

It will be appreciated that the polypeptides of the present invention may also have agonistic properties. These include increasing the stability of the ligand (e.g., IL-4), protection from proteolysis and modification of the pharmacokinetic properties of the ligand (i.e. , increasing the half-life of the ligand, while decreasing the clearance thereof). As such, the biomolecular sequences of this aspect of the present invention may be used to treat conditions or diseases in which the wild-type gene product plays a favorable role, for example, increasing angiogenesis in cases of diabetes or ischemia.

Upregulating expression of the therapeutic protein or polypeptide variants of the present invention may be effected via the administration of at least one of the exogenous polynucleotide sequences of the present invention, ligated into a nucleic acid expression construct (as described in greater detail hereinabove) designed for expression of coding sequences in eukaryotic cells (e.g., mammalian cells), as described above. Accordingly, the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding the variants of the present invention or active portions thereof.

It will be appreciated that the nucleic acid construct can be administered to the individual employing any suitable mode of administration including in vivo gene therapy (e.g., using viral transformation as described hereinabove). Alternatively, the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e. , ex-vivo gene therapy).

Such cells (i.e. , which are transfected with the nucleic acid construct of the present invention) can be any suitable cells, such as kidney, bone marrow, keratinocyte, lymphocyte, adult stem cells, cord blood cells, embryonic stem cells which are derived from the individual and are transfected ex vivo with an expression vector containing the polynucleotide designed to express the polypeptide of the present inevntion as described hereinabove.

Administration of the ex vivo transfected cells of the present invention can be effected using any suitable route such as intravenous, intra peritoneal, intra kidney, intra gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural and rectal. According to presently preferred embodiments, the ex vivo transfected cells of the present invention are introduced to the individual using intravenous, intra kidney, intra gastrointestinal track and/or intra peritoneal administrations.

The ex vivo transfected cells of the present invention can be derived from either autologous sources such as self bone marrow cells or from allogeneic sources such as bone marrow or other cells derived from non-autologous sources. Since non- autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and microencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. MoI Biotechnol. 2001. 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylaniine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245- 51.

For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al.. Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002:13: 783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 iim, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T.A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46). It will be appreciated that the present methodology may also be effected by specifically upregulating the expression of the valiants of the present invention endogenously in the subject. Agents for upregulating endogenous expression of specific splice variants of a given gene include antisense oligonucleotides, which are directed at splice sites of interest, thereby altering the splicing pattern of the gene. This approach has been successfully used for shifting the balance of expression of the two isoforms of Bcl-x [Taylor (1999) Nat. Biotechnol. 17:1097-1100; and Mercatante (2001) J. Biol. Chem. 276:16411-16417]; IL-5R [Karras (2000) MoI. Pharmacol. 58:380-387]; and c-myc [Giles (1999) Antisense Acid Drag Dev. 9:213-220],

For example, interleukin 5 and its receptor play a critical role as regulators of hematopoiesis and as mediators in some inflammatory diseases such as allergy and asthma. Two alternatively spliced isoforms are generated from the IL-5R gene, which include {i.e., long form) or exclude {i.e., short form) exon 9. The long form encodes for the intact membrane-bound receptor, while the shorter form encodes for a secreted soluble non-functional receptor. Using 2^*-O-MOE-oligonucleotides specific to regions of exon 9, Karras and co-workers (supra) were able to significantly decrease the expression of the wild type receptor and increase the expression of the shorter isoforms. Design and synthesis of oligonucleotides which can be used according to the present invention are described hereinbelow and by Sazani and KoIe (2003) Progress in Moleclular and Subcellular Biology 31 :217-239.

Treatment can preferably effected by agents which are capable of specifically downregulating expression (or activity) of at least one of the polypeptide variants of the present invention.

Down regulating the expression of the therapeutic protein valiants of the present invention may be achieved using oligonucleotide agents such as those described in greater detail below.

SiRNA molecules - Small interfering RNA (siRNA) molecules can be used to down-regulate expression of the therapeutic protein variants of the present invention. RNA interference is a two-step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA). each with 2-nucleotide 3' overhangs [Hutvagner and Zaniore Curr. Opin. Genetics and Development 12:225- 232 (2002); and Bernstein Nature 409:363-366 (2001)]. In the effector step, the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the niRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2: 110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al. Nat. Rev. Gen. 2: 110-119 (2001), Shaip Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575: 15-25 (2002). Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, the mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245], It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm. nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. Target sites are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

DNAzytne molecules - Another agent capable of downregulating expression of the polypeptides of the present invention is a DNAzyme molecule capable of specifically cleaving an niRNA transcript or DNA sequence of the polynucleotides of the present invention. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997;943:4262) A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM [Curr Opin MoI Ther 4: 119-21 (2002)].

Target sites for DNAzymes are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated.

Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al , 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL,

Aiitisense molecules - Downregulation of the polynucleotides of the present invention can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an niRNA transcript encoding the polypeptide variants of the present invention.

The term "antisense", as used herein, refers to any composition containing nucleotide sequences, which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules also include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand. Antisense oligonucleotides are also used for modulation of alternative splicing in vivo and for diagnostics in vivo and in vitro (Khelifi C. et al., 2002, Current Pharmaceutical Design 8:451-1466; Sazani, P., and KoIe. R. Progress in Molecular and Cellular Biology, 2003, 31 :217-239).

Design of antisense molecules which can be used to efficiently downregulate expression of the polypeptides of the present invention must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated niRNA within cells in a way which inhibits translation thereof.

The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft J MoI Med 76: 75-6 (1998); Kronenwett et al. Blood 91 : 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem Biophys Res Commun 231 : 540-5 (1997)]. In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target niRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)]. Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gpl30) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries. In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374 - 1375 (1998)].

Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin MoI Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin MoI Ther 1 :297-306 (1999)]. More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al., Cancer Res 61 :7855-60 (2001)]. Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation.

Target sites for antisense molecules are selected from the unique nucleotide sequences of each of the polynucleotides of the present invention, such that each polynucleotide is specifically down regulated. Ribozymes - Another agent capable of downregulating expression of the polypeptides of the present invention is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the polypeptide valiants of the present invention. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).

An additional method of regulating the expression of a spcific gene in cells is via triplex forming oligonuclotides (TFOs). Recent studies have shown that TFOs can he designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, L. J., et al., Science,1989;245:725-730; Moser, H. E., et al., Science,1987;238:645-630; Beal, P. A., et al, Science,1992;251 :1360-1363; Cooney, M., et al., Science,198δ;241:456-459; and Hogan, M. E., et al., EP Publication 375408. Modification of the oligonuclotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer. J Clin Invest 2003;l 12:487-94).

In general, the triplex-forming oligonucleotide has the sequence correspondence: oligo 3'--A G G T duplex 5'-A G C T duplex 3'-T C G A

However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Septl2, Epub). The same authors have demonstrated that TFOs designed according to the A-AT and G- GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.

Thus for any given sequence in the gene regulatory region a triplex forming sequence may be devised. Triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puiϊ, et al, J Biol Chem, 2001 ;276:28991-98), and the sequence- and target specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology (Carbone, et al, Nucl Acid Res. 2003;31 :833-43), and the pro-inflammatory ICAM-I gene (Besch et al, J Biol Chem, 2002;277:32473-79). In addition, Vuyisich and Beal have recently shown that sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003:112:487-94). Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn. Alternatively, down regulation of the polypeptide variants of the present invention may be achieved at the polypeptide level using downregulating agents such as antibodies or antibody fragments capabale of specifically binding the polypeptides of the present invention and inhibiting the activity thereof (i.e., neutralizing antibodies). Such antibodies can be directed for example, to the heterodimerizing domain on the variant, or to a putative ligand binding domain. Further description of antibodies and methods of generating same is provided below,

Alternatively, down regulation of the polypeptide variants of the present invention may be achieved using small, unique peptide sequences (e.g., of about 50- 100 amino acids) which are capable of specifically binding to their target molecules (e.g., a receptor subunit) and thus prevent endogenous subunit assembly or association and therefore antagonize the receptor activity. Such peptides can be natural or synthetic peptides which are derived from the polypeptide of the present invention. Pharmaceutical Compositions And Delivery Thereof The present invention features a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent according to the present invention, which is preferably a therapeutic protein variant as described herein. Optionally and alternatively, the therapeutic agent could be an antibody or an oligonucleotide that specifically recognizes and binds to the therapeutic protein valiant, but not to the corresponding full length known protein.

Alternatively, the pharmaceutical composition of the present invention includes a therapeutically effective amount of at least an active portion of a therapeutic protein variant polypeptide.

The pharmaceutical composition according to the present invention is preferably used for the treatment of cluster or protein-related disease, disorder or condition,

"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc, Preferably, the mammal is human.

A "disorder" is any condition that would benefit from treatment with the agent according to the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein are described with regard to specific examples given herein.

The term "therapeutically effective amount" refers to an amount of agent according to the present invention that is effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the agent may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR). The therapeutic agents of the present invention can be provided to the subject per se, or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the preparation accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols,

Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference,

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution. Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the ait. For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such earners enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose_., hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone. carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers, In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration,

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoiOmethane, tiϊchlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran, Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized, The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Pharmaceutical compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Immunogenic Compositions A therapeutic agent according to the present invention may optionally be a molecule, which promotes a specific immunogenic response against at least one of the polypeptides of the present invention in the subject. The molecule can be polypeptide variants of the present invention, a fragment derived therefrom or a nucleic acid sequence encoding thereof. Although such a molecule can be provided to the subject per se, the agent is preferably administered with an immunostimulant in an immunogenic composiiton. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes into which the compound is incoiporated (see e.g., U.S. Pat. No. 4,235,877). λ^accine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995).

Illustrative immunogenic compositions may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems (see below), bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the subject (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette- Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retro vims, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent vims. Suitable systems are disclosed, for example, in Fisher-Hoch et al., PIΌC. Natl. Acad. Sci. USA 86:317-321 , 1989; Flexner et al., Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner et al.. Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651 ; EP 0,345,242; WO 91/02805; Berkner. Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991 : Kolls et al., PIΌC. Natl. Acad. Sci. USA 91 :215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259: 1745-1749, 1993 and reviewed by Cohen, Science 259: 1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

It will be appreciated that an immunogenic composition may comprise both a polynucleotide and a polypeptide component. Such immunogenic compositions may provide for an enhanced immune response. Any of a variety of immunostimulants may be employed in the immunogenic compositions of this invention. For example, an adjuvant may be included. Most 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. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.); 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 or interleukin-2,-7, or -12, may also be used as adjuvants.

The adjuvant composition may be designed to induce an immune response predominantly of the ThI type. High levels of ThI -type cytokines (e.g., IFN-. gamma., TNF. alpha., IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-IO) tend to favor the induction of humoral immune responses. Following application of an immunogenic composition as provided herein, the subject will support an immune response that includes ThI- and Th2-type responses. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffinan, Ann. Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly ThI -type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O- acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, Wash.: see U.S. Pat. Nos. 4,436,727; 4.877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly ThI response. Such oligonucleotides are well known and are described, for example, in WO 96/02555. WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants, For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21 , 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron)_., the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720.

A delivery vehicle may be employed within the immunogenic composition of the present invention to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmernan and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within an immunogenic composition (see Zitvogel et al., Nature Med. 4:594- 600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF. alpha, to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF. alpha., CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are categorized as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which con-elates with the high expression of Fey receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDl 1) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

APCs may generally be transfected with at least one polynucleotide encoding a polypeptide of the present invention, such that variant II, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to the subject, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with a polypeptide of the present inventio, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule) such as described above. Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

Preferred embodiments of the present invention encompass novel naturally occurring secreted (i.e., extracellular) and non-secreted (i.e. , intracellular or membranal) variants of genes and gene products, which, as is described in the Examples section which follows, play pivotal roles in disease onset and progression. As such these variants can be used for a wide range of diagnostic and/or therapeutic uses.

Diagnostic Methods

The term "marker" in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from patients having or predisposed to a cluster or protein-related disease, disorder or condition as compared to a comparable sample taken from subjects who do not have a such a disease, disorder or condition.

The methods for detecting these markers have many applications. For example, one marker or combination of markers can be measured to differentiate between various types of cluster or protein-related disease, disorder or condition, and thus are useful as an aid in the accurate diagnosis of cluster or protein-related disease, disorder or condition in a patient. For example, one marker or combination of markers can be measured to differentiate between various types of lung cancers, such as small cell or non-small cell lung cancer, and further between non-small cell lung cancer types, such as adenocarcinomas, squamous cell and large cell carcinomas, and thus are useful as an aid in the accurate diagnosis of lung cancer in a patient. In another example, the present methods for detecting these markers can be applied to in vitro cluster or protein-related cancers cells or in vivo animal models for cluster or protein-related cancers to assay for and identify compounds that modulate expression of these markers.

The phrase "differentially present" refers to differences in the quantity of a marker present in a sample taken from patients having cluster or protein-related disease, disorder or condition as compared to a comparable sample taken from patients who do not have such disease, disorder or condition. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample, It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present. One of ordinary skill in the art could easily determine such relative levels of the markers; further guidance is provided below.

As used herein the phrase "diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

The phrase "predisposition" used herein refers to the susceptibility to develop a disorder. A subject with a predisposition to develop a disorder is more likely to develop the disorder than a non-predisposed subject. As used herein the phrase "diagnosing" refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The term "detecting" may also optionally encompass any of the above.

Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. As used herein "a biological sample" refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, milk, blood cells, tumors, neuronal tissue, organs, and also samples of in vivo cell culture constituents, It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.

As used herein, the term "level" refers to expression levels of RNA and/or protein or to DNA copy number of a marker of the present invention.

Typically the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual.

Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.

Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g.. brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.

Determining the level of the same variant in normal tissues of the same origin is preferably effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.

A "test amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a cluster or protein-related disease, disorder or condition related cancer or other UbcHlO related disease. A test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

A "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a patient which does not have the cluster or protein-related disease, disorder or condition. A control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).

"Detect" refers to identifying the presence, absence or amount of the object to be detected. A "label" includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P, ³⁵S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample. The label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavidin. The label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly. For example, the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavidin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule, The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6: 1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.

Exemplary detectable labels, optionally and preferably for use with immunoassays, include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g.. horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads, Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.

"Immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with" when referring to a protein or peptide (or other epitope), refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein. This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988). for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

In another embodiment, this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below). In another embodiment, this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice valiant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting the interaction: wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample. In another embodiment, this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.

According to another embodiment of the present invention the detection of the splice variant nucleic acid sequences in the biological sample is effected by detecting at least one nucleic acid change within a nucleic acid material derived from the biological sample; wherein the presence of the at least one nucleic acid change correlates with the presence of a splice variant nucleic acid sequence in the biological sample.

According to the present invention, the splice variants described herein are non-limiting examples of markers for diagnosing the cluster or protein-related disease, disorder or condition. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of such a cancer, disease or pathology.

According to optional but preferred embodiments of the present invention, any marker according to the present invention may optionally be used alone or combination. Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker_., for example a known marker. Furthermore, such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker. With regard to such a ratio between any marker described herein (or a combination thereof) and a known marker, more preferably the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.

According to other preferred embodiments of the present invention, a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof, may be featured as a biomarker for detecting the cluster or protein- related disease, disorder or condiiton, such that a biomarker may optionally comprise any of the above.

Non-limiting examples of methods or assays are described below. The present invention also relates to kits based upon such diagnostic methods or assays. NAT Assays

Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR, or variations thereof (e.g., real-time PCR, RT-PCR and in situ RT-PCR). As used herein., a "primer" defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.

Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non- limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1 173-1177; Lizardi et al., 1988. BioTechnology 6:1197-1202; Malek et al., 1994, Methods MoI. Biol., 28:253-260; and Sambrook et al., 1989, supra). The terminology "amplification pair" (or "primer pair") refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction. Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below. As commonly known in the ait, the oligos are designed to bind to a complementary sequence under selected conditions.

In one particular embodiment, amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid. In one preferred embodiment, RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA. In another preferred embodiment, the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.

The nucleic acid (i.e. DNA or RNA) for practicing the present invention may be obtained according to well known methods. Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. Optionally, the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system. As commonly known in the art, the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition. CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.). It will be appreciated that antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility. Oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.

The polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non-limiting examples of these reactions are described in greater detail below). The pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 ⁰C₅ preferably less than 5 ⁰C, more preferably less than 4 ⁰C, most preferably less than 3 ⁰C, ideally between 3 ⁰C and 0 ⁰C. Polymerase Chain Reaction (PCR): The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683.195 and 4,683,202 to Mullis and Mullis et al., is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification. This technology provides one approach to the problems of low target sequence concentration. PCR can be used to directly increase the concentration of the target to an easily detectable level. This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize, Following hybridization, the primers are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.

The length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are the to be "PCR-aniplified."

Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes referred to as "Ligase Amplification Reaction" (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. W09001069 Al (1990). However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal, The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.

Self-Sustained Synthetic Reaction (3SR/NASBΛ): The self-sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5' end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).

Q-Beta (Qβ) Replicase: In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Qβ replicase, A previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step. However, available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere. A successful diagnostic method must be very specific. A straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Qβ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature {i.e., > 55 degrees C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.

The basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle. The final yield of any such doubling system can be expressed as: (1+X)n = y, where "X" is the mean efficiency (percent copied in each cycle), "n" is the number of cycles, and "y" is the overall efficiency, or yield of the reaction. If every copy of a target DNA is utilized as a template in every cycle of a polymerase chain reaction, then the mean efficiency is 100 %. If 20 cycles of PCR are performed, then the yield will be 220, or 1,048,576 copies of the starting material. If the reaction conditions reduce the mean efficiency to 85 %, then the yield in those 20 cycles will be only 1.8520, or 220,513 copies of the starting material. In other words, a PCR running at 85 % efficiency will yield only 21 % as much final product, compared to a reaction running at 100 % efficiency. A reaction that is reduced to 50 % mean efficiency will yield less than 1 % of the possible product. In practice, routine polymerase chain reactions rarely achieve the theoretical maximum yield, and PCRs are usually run for more than 20 cycles to compensate for the lower yield. At 50 % mean efficiency, it would take 34 cycles to achieve the million-fold amplification theoretically possible in 20, and at lower efficiencies, the number of cycles required becomes prohibitive. In addition, any background products that amplify with a better mean efficiency than the intended target will become the dominant products. Also, many variables can influence the mean efficiency of PCR, including target DNA length and secondary structure, primer length and design, primer and dNTP concentrations, and buffer composition, to name but a few. Contamination of the reaction with exogenous DNA (e.g., DNA spilled onto lab surfaces) or cross- contamination is also a major consideration. Reaction conditions must be carefully optimized for each different primer pair and target sequence, and the process can take days, even for an experienced investigator. The laboriousness of this process, including numerous technical considerations and other factors, presents a significant drawback to using PCR in the clinical setting. Indeed, PCR has yet to penetrate the clinical market in a significant way. The same concerns arise with LCR, as LCR must also be optimized to use different oligonucleotide sequences for each target sequence. In addition, both methods require expensive equipment, capable of precise temperature cycling.

Many applications of nucleic acid detection technologies, such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences. One method of the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer. An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence. This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.

A similar 3 '-mismatch strategy is used with greater effect to prevent ligation in the LCR. Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification. Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory. The direct detection method according to various preferred embodiments of the present invention may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.

When a sufficient amount of a nucleic acid to be detected is available, there are advantages to detecting that sequence directly, instead of making more copies of that target, (e.g., as in PCR and LCR). Most notably, a method that does not amplify the signal exponentially is more amenable to quantitative analysis. Even if the signal is enhanced by attaching multiple dyes to a single oligonucleotide, the correlation between the final signal intensity and amount of target is direct. Such a system has an additional advantage that the products of the reaction will not themselves promote further reaction, so contamination of lab surfaces by the products is not as much of a concern. Recently devised techniques have sought to eliminate the use of radioactivity and/or improve the sensitivity in automatable formats. Two examples are the "Cycling Probe Reaction" (CPR), and "Branched DNA" (bDNA). Cycling probe reaction (CPR): The cycling probe reaction (CPR), uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.

Branched DNA: Branched DNA (bDNA), involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.

The NAT assays of the present invention also include methods of detecting at least one nucleic acid change [e.g., a single nucleotide polymorphism (SNP] in the biological sample of the present invention.

The demand for tests which allow the detection of specific nucleic acid sequences and sequence changes is growing rapidly in clinical diagnostics. As nucleic acid sequence data for genes from humans and pathogenic organisms accumulates, the demand for fast, cost-effective, and easy-to-use tests for as yet mutations within specific sequences is rapidly increasing.

A handful of methods have been devised to scan nucleic acid segments for mutations or nucleic acid changes. One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest. However, specialized equipment and highly trained personnel are required, and the method is too labor-intense and expensive to be practical and effective in the clinical setting.

In view of the difficulties associated with sequencing, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be detennined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs. Restriction fragment length polymorphism (RFLP): For detection of single- base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).

Single point mutations have been also detected by the creation or destruction of RFLPs. Mutations are detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches. Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC). However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.

RFLP analysis suffers from low sensitivity and requires a large amount of sample. When RFLP analysis is used for the detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites. A handful of rare-cutting restriction enzymes with 8 base-pair specificities have been isolated and these are widely used in genetic mapping, but these enzymes are few in number, are limited to the recognition of G+C-iϊch sequences, and cleave at sites that tend to be highly clustered. Recently, endonucleases encoded by group I introns have been discovered that might have greater than 12 base-pair specificity, but again, these are few in number.

Allele specific oligonucleotide (ASO): If the change is not in a recognition sequence, then allele-specific oligonucleotides (ASOs), can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis-match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles. The ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.

With either of the techniques described above (i.e., RFLP and ASO), the precise location of the suspected mutation must be known in advance of the test. That is to say, they are inapplicable when one needs to detect the presence of a mutation within a gene or sequence of interest. Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE):

Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed "Denaturing Gradient Gel Electrophoresis" (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel, In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of mutations in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are "clamped" at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature. Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNAiRNA duplexes.

Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested, Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration. In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE). CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of mutations,

A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient. TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel. Single-Strand Conformation Polymorphism (SSCP): Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other, Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations. The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non- denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.

Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).

In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment, SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments. Although SSCP is reportedly able to detect 90 % of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50 % for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs. The ddF technique, as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.

Reverse dot blot: This technique uses labeled sequence specific oligonucleotide probes and unlabeled nucleic acid samples. Activated primary aniine- conjugated oligonucleotides are covalently attached to carboxylated nylon membranes. After hybridization and washing, the labeled probe, or a labeled fragment of the probe, can be released using oligomer restriction, i.e., the digestion of the duplex hybrid with a restriction enzyme. Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminesceiice after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film.

It will be appreciated that advances in the field of SNP detection have provided additional accurate, easy, and inexpensive large-scale SNP genotyping techniques, such as Pyrosequencing™, Acycloprime™, dynamic allele-specific hybridization (DASH, Howell, W.M. et al. 1999. Dynamic allele-specific hybridization (DASH). Nat. Biotechnol. 17: 87-8), microplate array diagonal gel electrophoresis [MADGE, Day, I.N. et al., 1995. High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE). Biotecliniques. 19: 830-5], , the TaqMan system (Holland, P.M. et al., 1991. Detection of specific polymerase chain reaction product by utilizing the 5'— »3' exonuclease activity of Themius aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 88: 7276-80), as well as various DNA "chip" technologies such as the GeneChip niicroarrays (e.g., Affymetrix SNP chips) which are disclosed in U.S. Pat. Appl. No. 6,300,063 to Lipshutz, et al. 2001, which is fully incorporated herein by reference, Genetic Bit Analysis (GBA™) which is described by Goelet, P. et al. (PCT Appl. No. 92/15712), peptide nucleic acid (PNA, Ren B, et al.. 2004. Nucleic Acids Res. 32: e42) and locked nucleic acids (LNA, Latorra D, et al., 2003. Hum. Mutat. 22: 79-85) probes, Molecular Beacons (Abravaya K, et al., 2003. Clin Chem Lab Med. 41 : 468- 74), intercalating dye [Gerrner, S. and Higuchi, R. Single-tube genotyping without oligonucleotide probes. Genome Res. 9:72-78 (1999)], FRET primers (Solinas A et al., 2001. Nucleic Acids Res. 29: E96), AlphaScreen (Beaudet L, et al.. Genome Res. 2001, 11(4): 600-8), SNPstream (Bell PA, et al., 2002. Biotechniques. SuppL: 70-2, 74, 76-7), Multiplex minisequencing (Curcio M, et al., 2002. Electrophoresis. 23: 1467-72), SnaPshot (Turner D, et al., 2002. Hum Immunol. 63: 508-13), MassEXTEND (Cashman JR, et al., 2001. Drug Metab Dispos. 29: 1629-37), GOOD assay (Sauer S, and Gut IG. 2003. Rapid Commun. Mass. Spectrom. 17: 1265-72), Microarray minisequencing (Liljedahl U, et al., 2003. Pharmacogenetics. 13: 7-17), arrayed primer extension (APEX) (Tonisson N, et al., 2000. Clin. Chem. Lab. Med. 38: 165-70), Microarray primer extension (O'Meara D, et al., 2002. Nucleic Acids Res. 30: e75), Tag arrays (Fan JB, et al., 2000. Genome Res. 10: 853-60), Template- directed incorporation (TDI) (Akula N, et al., 2002. Biotechniques. 32: 1072-8), fluorescence polarization (Hsu TM, et al., 2001. Biotechniques. 31 : 560, 562, 564-8). Colorimetric oligonucleotide ligation assay (OLA, Nickerson DA, et al., 1990. Proc. Natl. Acad. Sci. USA. 87: 8923-7), Sequence-coded OLA (Gasparini P, et al.. 1999. J. Med. Screen. 6: 67-9), Microarray ligation, Ligase chain reaction. Padlock probes, Rolling circle amplification, Invader assay (reviewed in Shi MM. 2001. Enabling large-scale phaπnacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem. 47: 164-72), coded microspheres (Rao KV et al., 2003. Nucleic Acids Res. 31 : e66) and MassArray (Leushner J, Chiu NH, 2000. MoI Diagn. 5: 341-80).

According to a presently preferred embodiment of the present invention the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self-sustained synthetic reaction, Qβ-Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis, dideoxy fingerprinting, Pyrosequencing™, Acycloprime™, and reverse dot blot.

Detection may also optionally be performed with a chip or other such device. The nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group. This reporter group can be a fluorescent group such as phycoerythrin. The labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station. For example, Manz et al. (1993) Adv in Chromatogr 1993; 33: 1-66 describe the fabrication of fluidics devices and particularly niicrocapillary devices, in silicon and glass substrates.

Once the reaction is completed, the chip is inserted into a scanner and patterns of hybridization are detected. The hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.

Preferably, the detection of at least one nucleic acid change and/or the splice variant sequence of the present invention is effected in a biological sample containing RNA molecules using, for example. RT-PCR or in situ RT-PCR. RT-PCR analysis: This method uses PCR amplification of relatively rare

RNAs molecules. First. RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine, Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semiquantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.

//; situ RT-PCR stain: This method is described in Nuovo GJ, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C vims detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction. The reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arctums Engineering (Mountainview, CA).

It will be appreciated that when utilized along with automated equipment, the above described detection methods can be used to screen multiple samples for a disease and/or pathological condition both rapidly and easily.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting,

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons. New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4.666,828; 4,683,202; 4.801 ,531 ; 5.192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook". Volumes I-III Cellis, J. E., ed. (1994): "Current Protocols in Immunology" Volumes I-III Coligan J. E.. ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition). Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3.791,932; 3,839,153; 3,850,752; 3,850.578; 3,853,987; 3,867,517; 3,879,262; 3,901 ,654; 3,935.074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281 ,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J.. eds. (1985); "Transcription and Translation" Hames. B, D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Fresliney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B.. (1984) and "Methods in Enzymology" Vol. 1-317. Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incoiporated herein by reference.

EXAMPLE 1

Description of the methodology undertaken to uncover the biomolecular sequences of the present invention and uses therefor Human ESTs and cDNAs were obtained from GenBank versions 136 (June 15, 2003 ftp://ftp.ncbi.nih.gov/genbank/release.notes/gbl36.release.iiotes) and NCBI genome assembly of April 2003. Novel splice variants were predicted using the LEADS clustering and assembly system as described in US patent No: 6,625,545. U.S. Pat. Appl. No. 10/426,002, both of which are hereby incorporated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins. It then aligns the expressed sequences to the genome taking alternatively splicing into account and clusters overlapping expressed sequences into "clusters" that represent genes or partial genes. These were annotated using the GeneCarta (Compugen, Tel-Aviv, Israel) platform. The GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.

Brief description of the methodology used to obtain annotative sequence information is summarized infra (for detailed description see U.S. Pat. Appl. 10/426,002, published as US20040101876 on May 27 2004).

The ontological annotation approach - An ontology refers to the body of knowledge in a specific knowledge domain or discipline such as molecular biology, microbiology, immunology, virology, plant sciences, pharmaceutical chemistry, medicine, neurology, endocrinology, genetics, ecology, genomics, proteomics, cheminfoπnatics, pharmacogenomics, bioinfomiatics, computer sciences, statistics, mathematics, chemistry, physics and artificial intelligence. An ontology includes domain-specific concepts - referred to, herein, as sub- ontologies. A sub-ontology may be classified into smaller and narrower categories. The ontological annotation approach is effected as follows.

First, biomolecular (/^' e. , polynucleotide or polypeptide) sequences are computationally clustered according to a progressive homology range, thereby generating a plurality of clusters each being of a predetermined homology of the homology range,

Progressive homology is used to identify meaningful homologies among biomolecular sequences and to thereby assign new ontological annotations to sequences, which share requisite levels of homologies. Essentially, a biomolecular sequence is assigned to a specific cluster if displays a predetermined homology to at least one member of the cluster (i.e., single linkage). A "progressive homology range" refers to a range of homology thresholds, which progress via predetermined increments from a low homology level (e.g. 35 %) to a high homology level (e.g. 99 %).

Following generation of clusters, one or more ontologies are assigned to each cluster. Ontologies are derived from an annotation preassociated with at least one biomolecular sequence of each cluster; and/or generated by analyzing (e.g., text- mining) at least one biomolecular sequence of each cluster thereby annotating biomolecular sequences.

The hierarchical annotation approach - "Hierarchical annotation" refers to any ontology and subontology, which can be hierarchically ordered, such as, a tissue expression hierarchy, a developmental expression hierarchy, a pathological expression hierarchy, a cellular expression hierarchy, an intracellular expression hierarchy, a taxonomical hierarchy, a functional hierarchy and so forth.

The hierarchical annotation approach is effected as follows. First, a dendrogram representing the hierarchy of interest is computationally constructed. A "dendrogram" refers to a branching diagram containing multiple nodes and representing a hierarchy of categories based on degree of similarity or number of shared characteristics.

Each of the multiple nodes of the dendrogram is annotated by at least one keyword describing the node, and enabling literature and database text mining, such as by using publicly available text mining software, A list of keywords can be obtained from the GO Consortium (www.geneontlogy.org). However, measures are taken to include as many keywords, and to include keywords which might be out of date. For example, for tissue annotation, a hierarchy is built using all available tissue/libraries sources available in the GenBank, while considering the following parameters: ignoring GenBank synonyms, building anatomical hierarchies, enabling flexible distinction between tissue types (normal versus pathology) and tissue classification levels (organs, systems, cell types, etc.).

In a second step, each of the biomolecular sequences is assigned to at least one specific node of the dendrogram. The biomolecular sequences can be annotated biomolecular sequences, unannotated biomolecular sequences or partially annotated biomolecular sequences.

Annotated biomolecular sequences can be retrieved from pre-existing annotated databases as described hereinabove. For example, in GenBank. relevant armotational information is provided in the definition and keyword fields. In this case, classification of the annotated biomolecular sequences to the dendrogram nodes is directly effected. A search for suitable annotated biomolecular sequences is performed using a set of keywords which are designed to classify the biomolecular sequences to the hierarchy (i.e.. same keywords that populate the dendrogram).

In cases where the biomolecular sequences are unannotated or partially annotated, extraction of additional armotational infoπnation is effected prior to classification to dendrogram nodes. This can be effected by sequence alignment, as described hereinabove. Alternatively, armotational information can be predicted from structural studies. Where needed, nucleic acid sequences can be transformed to amino acid sequences to thereby enable more accurate armotational prediction.

Finally, each of the assigned biomolecular sequences is recursively classified to nodes hierarchically higher than the specific nodes, such that the root node of the dendrogram encompasses the full biomolecular sequence set, which can be classified according to a certain hierarchy, while the offspring of any node represent a partitioning of the parent set.

For example, a biomolecular sequence found to be specifically expressed in "rhabdomyosarcoma", will be classified also to a higher hierarchy level, which is "sarcoma", and then to "Mesenchymal cell tumors" and finally to a highest hierarchy level "Tumor". In another example, a sequence found to be differentially expressed in endometrium cells, will be classified also to a higher hierarchy level, which is "uterus", and then to "women genital system" and to "genital system" and finally to a highest hierarchy level "genitourinary system". The retrieval can be performed according to each one of the requested levels. Annotating gene expression according to relative abundance - Spatial and temporal gene annotations are also assigned by comparing relative abundance in libraries of different origins. This approach can be used to find genes, which are differentially expressed in tissues, pathologies and different developmental stages. In principal, the presentation of a contigue in at least two tissues of interest is determined and significant over or under representation of the contigue in one of the at least two tissues is assessed to identify differential expression. Significant over or under representation is analyzed by statistical pairing. Annotating spatial and temporal expression can also be effected on splice variants. This is effected as follows. First, a contigue which includes exonal sequence presentation of the at least two splice variants of the gene of interest is obtained. This contigue is assembled from a plurality of expressed sequences; Then, at least one contigue sequence region, unique to a portion (i.e., at least one and not all) of the at least two splice variants of the gene of interest, is identified. Identification of such unique sequence region is effected using computer alignment software. Finally, the number of the plurality of expressed sequences in the tissue having the at least one contigue sequence region is compared with the number of the plurality of expressed sequences not-having the at least one contigue sequence region, to thereby compare the expression level of the at least two splice variants of the gene of interest in the tissue.

Data concerning therapies, indications and possible pharmacological activities of the polypeptides of the present invention was obtained from PharmaProject (PJB Publications Ltd 2003 http://www.pjbpubs. com/cms. asp?pageid=340) and public databases, including LocusLink (http://www.genelynx.org/cgi- bin/resource?res=locuslink) and Swissprot

(http://www.ebi. acuk/swissprot/index.htnil). Functional structural analysis of the polypeptides of the present invention was effected using Interpro domain analysis software (Interpro default parameters, the analyses that were run are HMMPfam, HMMSmart, ProfileScan, FprintScan, and BlastProdom). Subcellular localization was analysed using ProLoc software (Einat Hazkani-Covo, Erez Y. Levanon, Galit Rotman, Dan Graur, Amit Novik. Evolution of multicellularity in metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis. Cell Biology International (2004;28(3): 171-8). Identifying gene products by interspecies sequence comparison - The present inventors have designed and configured a method of predicting gene expression products based on interspecies sequence comparison. Specifically, the method is based on the identification of conserved alternatively spliced exons for which there might be no supportive expression data.

Alternatively spliced exons have unique characteristics differentiating them from constitutively spliced ones. Using machine-learning techniques a combination of such characteristics was elucidated that defines alternatively spliced exons with very high probability, Any human exon having this combination of characteristics is therefore predicted to be alternatively spliced. Using this method, the present inventors were able to detect putative splice variants that are not supported by human ESTs. The method is effected as follows. First, alternatively spliced exons of a gene of interest are identified by scoring exon sequences of the gene of interest according to at least one sequence parameter as follows: (i) exon length - conserved alternatively spliced exons are relatively shorter than constitutively spliced ones; (ii) division by 3 - alternatively spliced exons are cassette exons that are sometimes inserted and sometimes skipped; Since alternatively spliced exons frequently contain sequences that regulate their splicing important parameters for scoring alternatively spliced exons include (iii) conservation level to a non-human ortholohgous sequence; (iv) length of conserved intron sequences upstream of each of the exon sequences; (v) length of conserved intron sequences downstream of each of the exon sequences; (vi) conservation level of the intron sequences upstream of each of the exon sequences; and (vii) conservation level of the intron sequences downstream of each of the exon sequences.

Exon sequences scoring above a predetermined threshold represent alternatively spliced exons of the gene of interest. Once alternatively spliced exons are identified, the chromosomal location of each of the alternatively spliced exons is analyzed with respect to coding sequence of the gene of interest to thereby predict expression products of the gene of interest. When performed along with computerized means, mass prediction of gene products can be effected. In addition, for identifying new gene products by interspecies sequence comparison, the expressed sequences derived from non-human species can be used for new human splice variants prediction. EXAMPLE 2

DESCRIPTION FOR CLUSTER S56892

Cluster S56892 features 4 transcript(s) and 18 segment(s) of interest, the names for which are given in Tables 2 and 3, respectively, the sequences themselves are given at the end of the application. The selected protein variants are given in table 4.

Table 2 - Transcripts of interest

Transcript Name

S56892. _PEA_ _1_. _PEA _1_T9 (SEQ I D NO: 1 )

S56892_ _PEA_ _1_ _PEA _l_T10 (SEQ ID NO 2)

S56892. _PEA_ _1_. _PEA J _T 13 (SEQ ID NO 3)

S56892_. _PEA_ 1 PEA _1_T14 (SEQ ID NO 4)

Table 3 - Segments of interest

Segment Name

S56892_ _PEA_ 1 PEA _l_node_0 (SEQ ID NO:5)

S56892. _PEA_ _1_. PEA _l_node_10 (SEQ ID NO:6)

S56892_ _PEA_ 1 _PEA _l_node_18 (SEQ ID

NO:7)

S56892_ _PEA_ 1 _PEA _l_node_21 (SEQ ID NO:8)

S56892_ _PEA_ 1 _PEA _l_node_3 (SEQ ID NO:9)

S56892_ _PEA_ 1 _PEA _l_node_4 (SEQ ID NO: 10)

S56892_ _PEA_ _.1_. _PEA _l_node_7 (SEQ ID NO: 1 1)

S56892_ _PEA_ _.1_. _PEA _l__node_8 (SEQ ID NO:12)

S56892_PEA_l_PEA_l_node_9 (SEQ ID NO:13)

S56892_PEA_l_PEA_l_node_12 (SEQ ID NO:14)

S56892_PEA_l_PEA_l_node_13 (SEQ ID NO:15)

S56892_PEA_l_PEA_l_node_14 (SEQ ID NO:16)

S56892_PEA_l_PEA_l_node_16 (SEQ ID

NO:17)

S56892_PEA_l_PEA_l_node_17 (SEQ ID NO:18)

S56892_PEA_l_PEA_l_node_19 (SEQ ID NO:19)

S56892_PEA_l_PEA_l_node_20 (SEQ ID

NO:20)

S56892_PEA_l_PEA_l_node_22 (SEQ ID NO:21)

S56892_PEA_l_PEA_l_node_23 (SEQ ID NO:22)

Table 4 - Proteins of interest

S56892_PEA_1_PEA_1_P P 174 (also referred to S56892_PEA_1_PEA_1_. 13 (SEQ ID NO:27) herein as IL-6 174) T14 (SEQ ID NO:4)

These sequences are variants of the known protein Interleukin-6 precursor (SEQ ID NO:23) (SwissProt accession identifier IL6_HUMAN; known also according to the synonyms IL-6; B-cell stimulatory factor 2; BSF-2; Interferon beta-2; Hybridoma growth factor; CTL differentiation factor; CDF). referred to herein as the previously known protein.

Protein Interleukin-6 precursor (SEQ ID NO:23) is known or believed to have the following function(s): IL-6 is a cytokine with a wide variety of biological functions: it plays an essential role in the final differentiation of B-cells into Ig- secreting cells, it induces myeloma and plasmacytoma growth, it induces nerve cells differentiation, in hepatocytes it induces acute phase reactants. The sequence for protein Interleukin-6 precursor (SEQ ID NO:23) is given at the end of the application, as "Interleukin-6 precursor (SEQ ID NO:23) amino acid sequence". Known polymorphisms for this sequence are as shown in Table 5,

Table 5 - Amino acid mutations for Known Protein

Protein Interleukin-6 precursor (SEQ ID NO:23) localization is believed to be Secreted.

The previously known protein also has the following indication(s) and/or potential therapeutic use(s): Chemotherapy-induced injury; Cancer, sarcoma, Kaposi's; Cancer, myeloma; Chemotherapy-induced injury, bone marrow, thrombocytopenia; Thrombocytopenia; Infection, HIV/AIDS; Chemotherapy- induced injury, bone marrow, neutropenia; Cancer, breast; Cancer, colorectal; Cancer, leukaemia, acute myelogenous; Cancer, melanoma; Myelodysplastic syndrome; Hepatic dysfunction. It has been investigated for clinical/therapeutic use in humans, for example as a target for an antibody or small molecule, and/or as a direct therapeutic; available information related to these investigations is as follows. Potential pharmaceutically related or therapeutically related activity or activities of the previously known protein or of drugs directed against this protein are as follows: Interleukin 6 modulator. A therapeutic role for a protein represented by the cluster has been predicted. The cluster was assigned this field because there was information in the drug database or the public databases (e.g., described herein above) that this protein, or part thereof, is used or can be used for a potential therapeutic indication: Radio/chemoprotective; Anticancer; Cytokine; Haematological; Anti- inflammatory; Antianaemic; Antiviral, interferon; Anabolic; Hepatoprotective; Antiarthritic, immunological.

The following GO Annotation(s) apply to the previously known protein. The following annotation(s) were found: skeletal development; acute-phase response; humoral defense mechanism; cell surface receptor linked signal transduction; cell-cell signaling; developmental processes; cell proliferation; positive control of cell proliferation; negative control of cell proliferation, which are annotation(s) related to Biological Process; cytokine; interleukin-6 receptor ligand, which are annotation(s) related to Molecular Function; and extracellular space, which are annotation(s) related to Cellular Component. The GO assignment relies on information from one or more of the

SwissProt/TremBl Protein knowledgebase, available from

<http://www.expasy.cli/sprot/>; or Locuslink, available from

^lttp^Avwav.ncbi.nlm.nih.gov/piOJects/LocusLinl^.

Interleukin-6 is a pleiotropic cytokine with a wide range of biological activities in immune regulation, hematopoiesis, inflammation and oncogenesis. It acts through a combination of two different receptors, IL-6R and a 130IcDa common signal transducer-gpl30, to generate a high-affinity complex of IL-6/ IL-6R/gpl30. It has pathological roles in various disease conditions, including but not limited to inflanimatory-tnesaiigial proliferative glomerulonephritis, autoimmune-RA, Psoriasis, Parkinson's disease and cancers, including but not limited to multiple myeloma/plasmacytoma, Kaposi's sarcoma.

As noted above, cluster S56892 features 4 transcript(s), which were listed in Table 1 above. These transcript(s) encode for protein(s) which are variant(s) of protein Interleukin-6 precursor (SEQ ID NO:23) . A description of each variant protein according to the present invention is now provided.

Variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) . An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:

Comparison report between S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) and IL6_HUMAN:

1.An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , comprising a first amino acid sequence being at least 90 % homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSE RIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKM AEKDGC FQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQ FLQKK corresponding to amino acids 1 - 157 of IL6 HUMAN, which also corresponds to amino acids 1 - 157 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order. 2.An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P8 (SEQ ID NO.24) , comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.

The glycosylate sites of variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , as compared to the known protein Interleukin-6 precursor (SEQ ID

NO:23) , are described in Table 6 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the valiant protein; and the last column indicates whether the position is different on the variant protein).

Table 6 - Glycosylation site(s)

The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 7:

Table 7 - InterPro domain(s)

Variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) is encoded by the following transcript(s): S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) , for which the sequence(s) is/are given at the end of the application. The coding portion of transcript S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) is shown in bold; this coding portion starts at position 458 and ends at position 1051, The transcript also has the following SNPs as listed in Table 8 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P8 (SEQ ID NO: 24) sequence provides support for the deduced sequence of this variant protein according to the present invention).

Table 8 - Nucleic acid SNPs

Variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) . An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application, A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:

Comparison report between S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) and IL6_HUMAN:

1. An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a first amino acid sequence being at least 90 % homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSE RIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKM AEKDGC FQSGFNE corresponding to amino acids 1 - 108 of IL6_HUMAN, which also corresponds to amino acids 1 - 108 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , and a second amino acid sequence being at least 90 % homologous to AKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLR ALRQM corresponding to amino acids 158 - 212 of IL6_HUMAN, which also corresponds to amino acids 109 - 163 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

2.An isolated chimeric polypeptide encoding for an edge portion of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise EA, having a structure as follows: a sequence starting from any of amino acid numbers 108-x to 108; and ending at any of amino acid numbers 109+ ((n-2) - x), in which x varies from 0 to n-2.

The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.

Variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 9, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) sequence provides support for the deduced sequence of this variant protein according to the present invention).

Table 9 - Amino acid mutations

The glycosylation sites of valiant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , as compared to the known protein Interleukin-6 precursor (SEQ ID

NO:23) , are described in Table 10 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).

Table 10 - Glycosylation site(s)

The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 11:

Table 11 - InterPro domain(s)

Variant protein S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) is encoded by the following transcript(s): S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) , for which the sequence(s) is/are given at the end of the application. The coding portion of transcript S56892_PEA_l_PEA_l_T10 (SEQ ID NO:2) is shown in bold; this coding portion starts at position 113 and ends at position 601. The transcript also has the following SNPs as listed in Table 12 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein

S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) sequence provides support for the deduced sequence of this variant protein according to the present invention).

Table 12 - Nucleic acid SNPs

Ul

Variant protein S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transαϊpt(s) S56892_PEA_1_PEA_1_T13 (SEQ ID

NO:3) . An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:

Comparison report between S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) and IL6JHUMAN:

1.An isolated chimeric polypeptide encoding for

S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , comprising a first amino acid sequence being at least 90 % homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSE

RIDKQIRYILDGISALRKETCNKSN corresponding to amino acids 1 - 76 of

IL6 HUMAN, which also corresponds to amino acids 1 - 76 of

S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order. 2.An isolated polypeptide encoding for a tail of

S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

The location of the valiant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.

The glycosylation sites of variant protein S56892_PEA_1_PEA_1_P11 (SEQ

ID NO:26) , as compared to the known protein Interleukin-6 precursor (SEQ ID

NO:23) , are described in Table 13 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the variant protein; and the last column indicates whether the position is different on the variant protein).

Table 13 - Glycosylation site(s)

The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 14: Table 14 - Inter Pro domain(s)

Variant protein S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) is encoded by the following transcript(s): S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) , for which the sequence(s) is/are given at the end of the application. The coding portion of transcript S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) is shown in bold; this coding portion starts at position 459 and ends at position 739. The transcript also has the following SNPs as listed in Table 15 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) sequence provides support for the deduced sequence of this variant protein according to the present invention).

Table 15 - Nucleic acid SNPs

Variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) according to the present invention has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) , An alignment is given to the known protein (Interleukin-6 precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:

Comparison report between S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) and IL6_HUMAN:

1.An isolated chimeric polypeptide encoding for

S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , comprising a first amino acid sequence being at least 90 % homologous to

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSE RIDKQIRYILDGISALRK corresponding to amino acids 1 - 69 of IL6JHUMAN, which also corresponds to amino acids 1 - 69 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , and a second amino acid sequence being at least 90 % homologous to

EETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKK AKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLR ALRQM corresponding to amino acids 108 - 212 of IL6 HUMAN, which also corresponds to amino acids 70 - 174 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order. 2.An isolated chimeric polypeptide encoding for an edge portion of

S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KE, having a structure as follows: a sequence starting from any of amino acid numbers 69-x to 69; and ending at any of amino acid numbers 70+ ((n-2) - x), in which x varies from 0 to n-2. The location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs. The variant protein is believed to be located as follows with regard to the cell: secreted. The protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans-membrane region prediction program predicts that this protein has a trans-membrane region.

Variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 16, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) sequence provides support for the deduced sequence of this valiant protein according to the present invention).

Table 16 - Amino acid mutations

The glycosylation sites of variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , as compared to the known protein InterIeukin-6 precursor (SEQ ID

NO:23) , are described in Table 17 (given according to their position(s) on the amino acid sequence in the first column; the second column indicates whether the glycosylation site is present in the valiant protein; and the last column indicates whether the position is different on the valiant protein).

Table 17 - Glycosylation site(s)

The variant protein has the following domains, as determined by using InterPro. The domains are described in Table 18:

Table 18 - InterPro domain(s)

Variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) is encoded by the following transcript(s): S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) , for which the sequence(s) is/are given at the end of the application, The coding portion of transcript S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) is shown in bold; this coding portion starts at position 458 and ends at position 979. The transcript also has the following SNPs as listed in Table 19 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) sequence provides support for the deduced sequence of this variant protein according to the present invention).

Table 19 - Nucleic acid SNPs

Figure 1 shows a schematic comparison of the domain structure of IL-6 valiants to various known or wild-type (WT) IL-6 proteins. In Figure 1 the known IL-6 antagonist P163 is given as S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) ; it lacks or ^uskips" exon 4 of the IL-6 gene. The IL-6 variant P 198 according to the present invention is S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) ; it features intron 4 retention of the IL-6 gene. The IL-6 variant P95 according to the present invention is

S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) and it contains a truncated exon 3.

The IL-6 variant Pl 74 according to the present invention is S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) . and it lacks or "skips" exon 3 of the

IL-6 gene. The Signal Peptide (SP) and the Helixes A, B, C and D are indicated.

Variant protein alignment to the previously known protein:

Sequence name : IL6_HU11A1J

Sequence documentation :

Alignment of : S56892_PEA_1_PEA_1_P8 ( SEQ ID 110 : 24 ) x IL6_HUMAtl

Alignment segment 1 /1 :

Quality : 1526.00 Escore : 0

Matching length : 157 Total length : 157

Matching Percent Similarity : 100.00 Matching Percent Identity : 100.00 Total Percent Similarity : 100.00 Total Percent Identity : 100.00

Gaps : 0

Alignment :

1 MUSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50

I I I I I I I I I I I I I I I I I l I I I I I I I I I I I I I I I I I I I I I I I I I ! I I I I I I 1 MMSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50 51 ERIDKQIRYILDGISALRKETCNKStlMCESSKEALAEUNLNLPKMAEKDG 100

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 51 ERIDKQIRYILDGISALRKETCUKSUMCESSKEALAEΠULΠLPKMAEKDG ioo

101 CFQSGFMEETCLVKI ITGLLEFEVYLEYLQtJRFESSEEQARAVQMSTKVL 150

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 101 CFQSGFNEETCLVKIITGLLEFEVYLEYLQtIRFESSEEQARAVQMSTKVL 150

151 IQFLQKK 157

I I I I I I I

151 IQFLQKK 157

Sequence name : IL6_HUMA1)

Sequence documentation :

Alignment of : S56892 PEA 1 PEA 1 F9 ( SEQ ID UO : 25 ) x IL6 HUt-LAN

Alignment segment 1 / 1 :

Quality : 1490.00 Escore : 0

[latching length : 163 Total length : 212

Matching Percent Similarity : 100.00 Matching Percent Identity : 100.00

Total Percent Similarity : 76.89 Total Percent Identity : 76.89

Gaps : 1

Alignment :

1 MtISFSTSAFGPVAFSLGLLLVLPA-AFPAPVPPGEDSKDVAAPHRQPLTSS 50

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50

51 ERI DKQIRYILDGISALRKETCNKSNMCESSKEALAEtlMLMLPKMAEKDG 100

I I I I I I I I I I I I I I I I I I I I I ! I I I I I I I I I I I ! I l I I I I I I I I I I I I I I 51 ERI DKQIRYILDGISALRKETCNKStJMCESSKEALAEtJNLtJLPKMAEKDG 100

101 CFQSGFtIE . . . 108 I I I I I I I I 101 CFQSGFNEETCLVKIITGLLEFEVYLEYLQtIRFESSEEQARAVQMSTKVL 150

109 AKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKE 151

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

151 IQFLQKKAKtILDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKE 200

152 FLQSSLRALRQM 163

I I I I I I I I I I I I

201 FLQSSLRALRQM 21:

Sequence name : IL6 HUMAN

Sequence documentation :

Alignment of : S56892 PEA 1 PEA 1 PI l ( SEQ ID NO : 26 ) x IL6 HUMAN

Alignment segment 1/1 :

Quality : 733.00 Escore : 0 Matching length : 77 Total length : 77

Matching Percent Similarity : 100.00 Matching Percent Identity : 98.70

Total Percent Similarity : 100.00 Total Percent Identity : 98.70

Gaps : 0

Alignment :

1 Mt)SFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 MtISFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50

51 ERIDKQIRYILDGISALRKETCNKStJ 76

I I I I I I I I I I I I I I I I I I I I I I I I I I

51 ERIDKQIRYILDGISALRKETCNKSN 76 Sequence name : IL6_HUMAN

Sequence documentation :

Alignment of : S56892_PEA_1_PEA_1_P13 ( SEQ ID NO : 27 ) x IL6_HUMAN

Alignment segment 1 / 1 :

Quality : 1572.00 Escore : 0 Matching length : 174 Total length : 212

Matching Percent Similarity : 100.00 Matching Percent Identity : 100.00

Total Percent Similarity : 82.08 Total Percent Identity : 82.08

Gaps : 1

Aliqnment :

1 MtiSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50

I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I

1 MtISFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSS 50

51 ERIDKQIRYILDGI SALRK 69

I I I I I I I I I I I I I I I I I I I 51 ERI DKQIRYILDGI SALRKETCtIKSNMCESSKEALAEt)ULHLPKMAEKDG 100

70 EETCLVKI ITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVL 112

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 101 CFQSGFHEETCLVKIITGLLEFEVYLEYLQtlRFESSEEQARAVQMSTKVL 150

113 IQFLQKKAKNLDAITTPDPTTUASLLTKLQAQMQWLQDMTTHLILRSFKE 162

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ! I I I I I I 151 IQFLQKKAKNLDAITTPDPTTUASLLTKLQAQNQWLQDMTTHLILRSFKE 200

163 FLQSSLRALRQM 174

I I I I I I I I I I I I

201 FLQSSLRALRQM 212

Example 3 Validation of IL-6 variants The expression of IL-6 variants according to the present invention was validated at the level of niRNA expression in renal cell carcinoma tissue. The IL-6 174 transcript was validated using a junction forward primer (primer sequences are given below), The transcript was found in cDNA prepared from RNA extracted from RCC (renal cell carcinoma). The experimental method used is as follows.

RCC (renal cell carcinoma) RNA was obtained from Ichilov. Total RNA samples were treated with DNaseI (Ambion Cat # 1906).

RTPCR - Purified RNA (1 μg) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 μM dNTP in a total volume of 15.6 μl. The mixture was incubated for 5 min at 65 ⁰C and then quickly chilled on ice. Thereafter, 5 μl of 5X SuperscriptII first strand buffer (Invitrogen), 2.4μl 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25 ⁰C, followed by further incubation at 42 ⁰C for 2 min. Then, 1 μl (200units) of SuperscriptII (Invitrogen) was added and the reaction (final volume of 25μl) was incubated for 50 min at 42 ⁰C and then inactivated at 70 ⁰C for 15min. The resulting cDNA was diluted 1 :20 in TE buffer (10 mM Tris pH=8. 1 mM EDTA pH=S).

Table 20 shows primers for the reaction and PCR conditions. Orientation for the primers is given as F (forward) or R (reverse).

Table 20

PCR amplification and analysis cDNA (5ul), prepared as described above (RT PCR), was used as a template in

PCR reactions. The amplification was done using AccuPower PCR PreMix (Bioneer, Korea, Cat# K2016), under the following conditions: IuI - of each primer (lOuM) plus 13ul - H₂O were added into AccuPower PCR PreMix tube with a reaction program of 5 minutes at 94⁰C; 35 cycles of: [30 seconds at 94⁰C, 30 seconds at 55⁰C, 60 seconds at 72⁰C] and 10 minutes at 72⁰C. At the end of the PCR amplification, products were analyzed on agarose gels stained with ethidium bromide and visualized with UV light. The PCR reaction yielded two bands (data not shown). Each band served as a template for a further PCR reaction and at the end of this PCR reaction, the products were analyzed again on agarose gel (Figure 5) and the PCR products were extracted from the gel using QiaQuick™ gel extraction kit (Qiagen, Cat #28706). The extracted DNA products were sequenced by direct sequencing using the gene specific primers described above (Hy-Labs, Israel). The high molecular weight band resulted in WT (known) IL-6 (lane 6 in Figure 5; SEQ ID NO:42) while the low molecular weight band resulted in the expected sequence of IL-6 174 variant (lane 7 in Figure 5; SEQ ID NO:43). The sequence of the primers is shown in bold. The forward primer in the high molecular weight PCR product, representing the known wild type protein, is a junction forward primer used for the PCR of the low molecular weight product (GCCCTGAGAAAGGAGGAGAC: SEQ ID NO:40), which was not supposed to anneal to the WT transcript since this junction is not in the WT sequence: however, as in many PCR reaction, the primer did anneal and gave rise to the WT product as an artifact of the PCR reaction.

High molecular weight band PCR product sequence (SEQ ID NO:42)

ATGTAACAAGAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAA AACAACCTGAACCTTCCAAAGATGGCTGAAAAAGATGGATGCTTCCAATC TGGATTCAATGAGGAGACTTGCCTGGTGAAAATCATCACTGGTCTTTTGGA GTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGG AACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCCAGTTCCTG CAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCCTGACCCAACCA CAAATGCCAGCCTGCTGACGAAGCTGCAGGCACAGAACCAGTGGCTGCA GGACATGACAACTCATCTCATTCTGCGCAGCTTTAAGGAGTTCCTGCAGTC CAGCCTGAGGGCTCTTCGGCAAATGTAGCATGGGCACCTCAGATTGTTGTT GTTAATGGGCATTCCTTCTTCTGGTCAGAAACCTGTCCACTGGGCACAGAA CTTATGTTGTTCTCTATGGAGAACTAAAAGTATGAGCGTTAGGACACTATT TT AATTATTTTT AATTTATT AATATTT AAATATGTGAAGCTGAGTT AATTTA TGTAAGTCATATTTATATTTTTAAGAAGTACCACTTGAAACATTTTATGTA TTAGTTTTGAAATAATAATGGAAAGTGGCTATGCAGTTTGAATATCCTTTG TTTCAGAGCCAGATCATTTCTTGG

Low molecular weight band PCR product sequence (SEQ ID NO:43) GCCCTGAGAAAGGAGGAGACTTGCCTGGTGAAAATCATCACTGG TCTTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTGAGAG TAGTGAGGAACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCC AGTTCCTGCAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCCTGAC CCAACCACAAATGCCAGCCTGCTGACGAAGCTGCAGGCACAGAACCAGT GGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTTTAAGGAGTTCC TGCAGTCCAGCCTGAGGGCTCTTCGGCAAATGTAGCATGGGCACCTCAGA TTGTTGTTGTTAATGGGCATTCCTTCTTCTGGTCAGAAACCTGTCCACTGG GCACAGAACTTATGTTGTTCTCTATGGAGAACTAAAAGTATGAGCGTTAG GACACTATTTTAATTATTTTTAATTTATTAAT ATTT AAATATGTGAAGCTGA GTTAATTTATGTAAGTCATATTTATATTTTTAAGAAGTACCACTTGAAACA TTTTATGTATTAGTTTTGAAATAATAATGGAAAGTGGCTATGCAGTTTGAA TATCCTTTGTTTCAGAGCCAGATCATTTCTTGG

Interestingly, significant expression of IL-6 174 variant was only found in RCC. Other tissues/ cell lines tested by the above method include: blood; lymph nodes; fibroblasts; lymphocytes; and thymus. However, significant expression was not found in these other tissues/cell lines (data not shown). Therefore, it is believed that measurement of IL-6 174 levels, particularly overexpression in RCC, may be detected in any relevant samples such as kidney tissue, blood or any other suitable diagnostic sample and may optionally be used for diagnosis, prognosis, differential diagnosis and so forth of RCC.

Example 4 Cloning, Expression and Purification of IL-6 variants Cloning of IL-6 variants

The IL-6 174 sequence was codon optimized to boost protein expression in mammalian system. The optimized gene was synthesized by Gene Art (Germany) by using their proprietary gene synthesis technology with the addition of DNA sequences encoding the StrepII and His tags at the 5' of the DNA fragment. The gene synthesis technology is a proprietary robust nucleic acid manufacturing platform that makes double stranded DNA molecules. The resultant sequences are shown in Figure 2. For the sequences in Figure 3, the bold part of the nucleotide sequence shows the relevant ORF (open reading frame) including the tag sequence, while the bold part of the amino acid sequence is the His tag (8 His residues- HHHHHHHH; SEQ ID NO:44) and Strep tag (Strep II tag: WSHPQFEK; SEQ ID NO:45) sequences. These protein tag sequences were added to all sequences so that the expressed protein can be more easily purified. The DNA fragment was cloned into EcoRI/Notl sites (underlined portions of the nucleotide sequence shown in Figure 2) in pRIESpuro3 (Clontech, cat # PT3646- 5) and the sequence was verified. Figure 3 shows a schematic diagram of the resultant construct.

Expression of IL-6 174 variant

The construct was transfected to HEK-293T cells (ATCC catalog number CRL-1 1268) as follows. One day prior to transfection, one well from a 6 well plate was plated with 500,000 cells in 2 ml DMEM. At the day of transfection, the FuGENE 6 Transfection Reagent (Roche, Cat#: 1-814-443) was warmed to ambient temperature and mixed prior to use. 6 μl of FuGENE Reagent were diluted into 100 μl DMEM (Dulbecco's modified Eagle's medium; Biological Industries, Cat#: 01- 055-1 A). Next, 2 micrograms of construct DNA were added. The contents were gently mixed and incubated at room temperature (RT) for 15 minutes. 100 μl of the complex mixture was added dropwise to the cells and swirled, The cells were incubated overnight at 37⁰C with 5% CO2. Following about 48 h, transfected cells were split and subjected to antibiotic selection with 5 micrograin/ml puromycin The surviving cells were propagated for about three weeks. Expression of the desired protein was verified by Western Blot (lane 8 of Figure 4) according to the following method.

The supernatants of the puromycin resistant cells were concentrated 16 fold with TCA (1 ml conditioned medium was concentrated into 6OuI). 25 ul of the solution was loaded on a 12% SDS-PAGE gel. Following electrophoresis, proteins on the gel were transferred to nitrocellulose membranes for 60 min at 35 V using Invitrogen's transfer buffer and X-CeIl II blot module. Following transfer, the blots were blocked with 5% skim milk in wash buffer (0.05% Tween-20 in PBS) for at least 60 min. at room temperature with shaking. Following blocking, the blots were incubated for 60 min at room temperature with a commercially available anti His antibody (Serotec, Cat. # MCAl 396) diluted in 1/5 blocking buffer, followed by washing with wash buffer and incubating for another 60 min at room temperature with respective peroxidase-conjugated antibodies. Next, the blots were washed again with wash buffer, followed by ECL (Enhanced Chemiluminescence) detection performed according to the manufacturer's instructions (Amersham; Cat # RPN2209) The results are shown in Figure 4 lane 8. Lane 10 in Figure 4 represents lOOng of a His tagged positive control protein, and lane 1 is the molecular weight marker.

In order to produce sufficient amounts of the protein, the cells expressing IL-6 174 according to the present invention are taken from a T-80 flask containing serum supplemented medium after trypsinization, and are transferred into shake flasks containing serum free medium (EX-CELL293. JRH) supplemented with 4 mM glutamine and selection antibiotics (5 ug/ml puromycin). Cells are propagated in suspension at 37⁰C, 100-120 rpm agitation and culture volume is increased by sequential passages until the desired volume is reached to produce enough protein. Production-phase growth is carried out in suspension in shake flasks, spinner flasks or a stirred-tank bioreactor.

Protein purification IL-6 174 protein according to the present invention can be purified by two different approaches for affinity chromatography in sequential order. The first approach uses Ni-NTA (nickel-nitrilotriacetic acid) resin, This type of chromatography is based on the interaction between a transition Ni²⁺ ion immobilized on a matrix and the histidine side chains of His-tagged proteins. His-tag fusion proteins can be eluted from the matrix by adding free imidazole for example, as described below. The second approach takes advantage of the Biotin-Streptavidin interaction principle, by using a streptavidin analog (streptactin) that is attached to the column resin, which interacts with the engineered tag StrepII. Thus, the purification method used for the variants according to the present invention preferably uses the Strep/όxHistidine system (double-tag) to ensure purification of recombinant proteins at high purity under standardized conditions.

IL-6 174 variant protein according to the present invention, carrying the 6xHistidine-tag and the Strep-tag II at the C - terminus, were efficiently expressed in mammalian cells. After concentration of the supernatant, IL-6 174 protein is initially purified using IMAC (Immobilized metal ion affinity chromatography) based on the 6xHistidine-tag-Ni-NTA interaction. After elution from the Ni-NTA matrix with imidazole, the recombinant protein (which also carries the Strep-tag II epitope) is loaded directly onto a Strep-Tactin matrix. No buffer exchange is required. After a short washing step, the recombinant protein is eluted from the Strep-Tactin matrix using desthiobiotin. A more detailed description of the protocol is provided below; additional information about the resin and its use, as well as the resin itself, is available from IBA GmbH (Germany; http://www.iba-go.com). Since the IL6 174 variant protein initially did not bind the streptactin column, the IMAC column was repeated (1 ml column) with the same buffers. The elution was in 3 Imidazol steps (50, 100 and 250 mM). Most of the IL6 174 protein was eluted in 250 mM Imidazole. 20 ul of the solution was loaded on a 4-12% SDS-PAGE gel. Figure 6 shows the results of the 250 mM Imidazole purification step. Lanes 3 and 4 contain the purified IL-6 174. Lane 1 is unpurified material and lane 2 is the molecular weight marker. This gel shows that optionally the IL-6 174 valiant protein according to the present invention may be purified by using two columns that rely on the His-tag for purification, rather than a first column relying on the His-tag followed by a second column relying on the strep-tag.

MATERIALS

Reagents: D-Desthiobiotin; 5 g {IBA, Cat# 2-1000-005}; Dulbecoo's Phosphate Buffered Saline (PBS), concentrate XlO {Biological Industries, Cat # 020235A}; Sodium Phosphate {Sigma, Cat # S7907) ; Strep-tag® regeneration buffer with HABA; 100 ml {IBA, Cat# 2-1002-100}; Millipore filters_.. 0.22μm (Cat# SCGP UI l RE); WFI (Water For Irrigation) - Teva Medical #AWF71 14; Resins:2-1206- 025 Strep-Tactin® Superflow®; Ni-NTA Superflow® . Buffers: Ni-NTA Binding & Wash Buffer (Buffer A): 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0; Ni-NTA Elution Buffer (Buffer B): 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0 ; Streptactin wash buffer (Buffer A): 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0; Strepactin elution buffer (Buffer D): 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 2.5 mM desthiobiotin, pH 8.0.

PURIFICATION METHOD

IL-6 174 protein according to the present invention is purified by affinity chromatography using Ni-NTA resin, according to the following protocol. 2L of culture is concentrated to 200 ml by ultrafiltration. Imidazole is added to the sample to final concentration of 10 mM and the sup is filtered through a 0.22 um filter (Millipore). The supernatant is transferred to a 250 ml centrifuge tube. Four ml of Ni-NTA Superflow beads are equilibrated with 10 column volumes of WFI and 10 column volumes of Buffer A (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0). The beads are added to the filtered supernatant, and the tube is incubated overnight on a rocking platform at 4⁰C.

The next day 1 ml Streptactin Superflow resin is equilibrated with 10 column volumes of WFI and 10 column volumes of Buffer A. The Streptactin beads are packed in a 1 ml Tricorn column and stored at 4⁰C. The Ni-NTA beads in the 250 ml centrifuge tube are separated from the supernatant and packed in a 4 ml column of Ni-NTA Superflow. Beads are washed with buffer A at a flow rate of 1 column volume per minute, until O.D280nm is lower than 0.005. A 1 ml Streptactin Superflow column is connected directly to the Ni-NTA Column. The IL-6 174 protein is eluted with buffer B (50 mM NaH₂PO₄. 300 mM NaCl, 250 mM imidazole, pH 8.0) at a flow rate not higher than 0.5ml/min through both columns. After 30-35ml have completely entered the Streptactin column, the Ni- NTA column is disconnected and washed with Buffer A at a flow rate of 1 column volume per minute with least 5 column volumes, until O.D280 nm is less then 0.005. The IL-6 174 protein is eluted with buffer D (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole. 2.5 mM Desthiobiotin, pH 8.0) at a flow rate of 0.2 column volumes per mill. Desthiobiotin and imidazole are removed from the purified protein by dialysis against IxPBS at 4⁰C.

Example 5

In vitro assessment of biological activity of IL-6 variants on different IL-6 responsive cell lines 7TDl proliferation assay

The in vitro biological activity of IL-6 valiants is assessed in a cell proliferation assay using the 7TDl cell line (DSMZ cat # ACC23). an IL-6-dependent cell line which originated in murine myeloma cells fused to murine B-cells and which has been used for the development of IL-6 antagonists (Manfredini et al., Peptides 2003, 24:1207-1220). Sant7, a known mutein and potent antagonist of IL-6 activity (Sporeno et al.. Blood, 1996, 87: 4510-4519), serves as positive control for antagonist activity. The activity of each variant is tested alone (i.e. as an agonist) or in the presence of commercial human IL-6 (i.e. as an antagonist). The cells are plated in 96- well plates, and the IL-6 174 variant or the positive control are added in different concentrations_., in the presence or absence of 50 pg/ml human IL-6. Proliferation is assessed 72 hrs later by MTT assay or by BrdU incorporation assay.

U266 proliferation assay

In multiple myeloma, the variety of genetic changes and oncogene mutations that characterize myeloma may give rise to different cells, whose sensitivity to IL-6 diverges in both quantity and quality, while still maintaining their IL-6 response.

Thus, it is important to study IL-6 putative antagonist in several in-vitro systems of multiple myeloma. In addition, assays for evaluation of Ig secretion will shed light on the relevance of IL-6 174 splice variant according to the present invention for the treatment of RA.

U266 (ATCC cat no.: TIB- 196) is a human meyeloma cell line that produces endogenous IL-6 which stimulates cell proliferation via an autocrine loop. These cells have been previously used as in-vitro model for studying IL-6 antagonists (Alberti et al., Cancer Res 2005; 65:2-5; Sporeno et al., Blood, 1996, 87: 4510-4519). Cells are maintained in RPMI, 15% FCS. For proliferation assay, the cells (105/ml) are washed and resuspended in low serum medium (1% FCS) and plated in a 96 microtiter plate in 100 ml. IL-6 174 splice variant or a known antagonistic mutein, such as SANT7 (as positive control), are added to the cells. Proliferation is measured 72 hr later, using BrdU ELISA. Other human multiple myeloma cell lines, such as INA-6 and XG-I, may be used in cell proliferation, cell cyle analysis and/or apoptosis assays, in order to evaluate the potential of IL-6 174 variant to exert an antagonistic effect, as has been described previously (Tassone et al, Clin. Cancer Res., 2005, 11 :4251-4258; Sporeno et al., Blood, 1996, 87: 4510-4519; Petrucci et al 1999. Ann Hematol 78: 13-18).

B9 proliferation assay

B9 (DSMZ cat no.: ACC 211) is a mouse hybridoma cell line which resulted from the fusion of murine myeloma cells with spleen B cells. This cell line is totally dependent on IL-6 for growth, and thus serves as a model system for studying potential IL-6 antagonists (Alberti et al., Cancer Res 2005; Brakenhoff et al., J, Biol Chemistry, 1994, 269:86-93). B9 cells are maintained in RPMI, 10% FBS, 50 μM mercaptoethanol, 100 pg/ml human IL-6. To study the antagonistic effect of IL-6 174 splice variant and a known antagonistic mutein (as positive control) on B9 proliferation, the cells are washed and resuspended (2xlO⁴/ml) in RPMI containing 1% FCS and plated at 100 μl/well in a 96- well microtiter plate in IL-6 174 splice variant or positive control mutein are added from serial dilutions, in the presence or absence of 3pg/ml IL-6, in order to assess their agonistic and antagonistic activities. Proliferation is measured 72 Iu- later, using BrdU incorporation or MTT assay,

A375 cell growth assay

A375 (ATCC cat no.: CRL-1619) are human malignant melanoma cells that respond to IL-6 by growth arrest. These cells have been used to analyze the activity of IL-6 antagonists (Sporeno et al., Blood 1996 87(11):4510-9; Savino, R., et al., EMBO

J. 1994 13(6):1357-67). The cells are maintained in DMED containing 10% FCS. To study the effect of IL-6 174 splice variant on cell survival, it is added from serial dilutions to A375 cells, in a 96-well microtiter plate containing 5000 cells/well_., in the presence or absence of human known (WT) IL-6 as an agonist. Cell survival is evaluated by BrdU incorporation or MTT assays.

IgGl secretion by CESS cells

CESS (ATCC cat no.: TIB- 190), a human myelomonocytic leukemia cell line, is used for this assay. Stimulation of CESS cells with IL-6 results in increased IgGl secretion, and has been used to assess IL-6 antagonist activity (Brakenhoff 1994,). The effect of IL-6 174 splice variant on IgGl secretion is studied by incubating these cells with serial dilutions of the splice variant or a known antagonistic mutein, such as SANT7 (as positive control), in the presence or absence of human IL-6. Levels of IgGl secretion are assessed using an ELISA, as is well known in the ait.

IgM secretion by SKW6.4 cells SKW6.4 (ATCC cat no.: TIB-215) is a human EBV transformed B cell line with plasmacytoid morphology. These cells respond to IL-6 stimulation by increased IgM secretion (10-30 fold) and have been used to analyze IL-6 antagonists (Shiao, et al. Leukemia and Lymphoma, 1995, 17:485-494; Peppard et al J. Biol. Chem., 1996, 271 : 7281-7284). The effect of IL-6 174 splice variant on IgM secretion is studied by incubating the cells (10000 cells/well in 96-well plate) with serial dilutions of the IL-6 174 splice variant or a known antagonist_., such as SANT7 (as positive control), in the presence or absence of IL-6 for a total of 3 days. IgM secretion is assessed using an ELISA as is well known in the art.

Example 6 In vivo biological activity of IL-6 variants

Blocking of IL-6 functions following a delivery of a therapeutic amount of IL- 6 variants of the present invention in the cynomolgus monkey is assessed by inhibition of two functional parameters in vitro: T-cell proliferation stimulated by phytohemaglutinin and human IL-6, and IgG production evoked by Staphylococcus aureus Cowan- 1- and human IL-6-stimulated B-lymphocytes. Inhibition of IL-6- induced typical responses (such as elevation of blood platelet counts and serum C- reactive protein (CRP) levels) in cynomolgus monkeys is assessed (Imazeki I; et al, International Journal of Immunopharmacology 1998, 20:7 (345-357); Shinkura H; et al., Anticancer Research 1998, 18:2A (1217-1221)).

Example 7

Treatment of Rheumatoid Arthritis (RA) by Administering IL-6 Splice Variant Protein According to the Present Invention

Experimental model

The in vivo effect of IL-6 valiants of the present invention on the development of collagen-induced arthritis is examined in cynomolgus monkeys (the same model as for overall assessment of the effect of IL-6 174 variant on IL-6 functionality in vivo). Inhibition of arthritis symptoms is measured following delivery of therapeutic amount of proteins of the IL-6 variants of the present invention. Inhibition of the elevation of serum CRP and fibrinogen levels, and inhibition of erythrocyte sedimentation rate (ESR) are measured as well. Furthermore, radiographic and histological examination is carried out, showing that IL-6 variant treatment suppresses joint destruction.

The effect of IL-6 variant treatment, on severe combined immunodeficiency (SCID) mice in which human RA synovial tissue is grafted, suggest that IL-6 variants of the present invention are an attractive agent for the treatment of RA as they decrease the number of inflammatory cells and metalloproteinase-positive cells in the implanted tissues. Preferably, the IL-6 174 variant is used.

Clinical Model - Rheumatoid Arthritis

A subject diagnosed with RA is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 8 Treatment of Castleman's disease by Administering IL-6 Variant Protein

According to the Present Invention

A subject diagnosed with Castleman's disease is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 9

Treatment of Crohn's disease by Administering IL-6 Variant Protein According to the Present Invention A subject diagnosed with Crohn's disease is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 10

Treatment of Systemic Lupus Erythematosus (SLE) by Administering IL-6 Variant Protein According to the Present Invention

Experimental model - BWFl Mice The effect of IL-6 variant treatment on suppression of the development of autoimmune disease in BWFl mice as a model of human SLE suggests a therapeutic potential of IL-6 variants of the present invention in the treatment of human SLE.

Clinical Model - SLE A subject diagnosed with SLE is treated with an IL-6 valiant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 11

Treatment of Colitis by Administering IL-6 Variant Protein According to the Present Invention

Experimental model - BALB/c Mice The effect of IL-6 variant treatment on inhibition of the average colitis score in the murine colitis model induced by transfer of CD45Rbhigh CD4+ T-cells from

BALB/c mice, ip injection of rat anti-murine IL-6R antibody (2 mg at the time of colitis induction and 1 mg weekly for up to 8 weeks) suggests a therapeutic potential of IL-6 variants of the present invention in the treatment of colitis.

Clinical Model - Colitis

A subject diagnosed with colitis is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 12

Treatment of Multiple Myeloma Malignancies by Administering IL-6 Variant Protein According to the Present Invention

Experimental model - Inhibition of human multiple myeloma The effect of IL-6 variant treatment on inhibition of the growth of myeloma cells, suppression of elevation of serum M-protein and development of the tumor- associated abnormalities and increasing the life span in a SCID mice subcutaneously inoculated with solid tumors of the myeloma cell line, S6B45 or in SCID mice xenograft MM model induced by iv injection of the human MM cell line, KPMM2, suggest IL-6 valiants of the present invention is effective in the treatment of Multiple Myeloma. A novel murine model of human multiple myeloma, in which IL-6-dependent INA-6 multiple myeloma cells are directly injected into human bone marrow implants in severe combined immunodeficient (SCID) mice (SCID-hu), is used to assess the effect of IL-6 variant treatment on inhibition of the growth of myeloma cells. The effect of in vivo drug treatments on multiple myeloma cell growth is monitored by serial determinations of serum levels of soluble IL-6 receptor (shuIL-6R), which is released by INA-6 cells and serves as a marker of tumor growth. In SCID-hu mice engrafted with INA-6 cells, treatment with IL-6 variant of the present invention, alone or in combination with dexamethasone, results in a reduction in serum shuIL-6R levels after 6 consecutive days of treatment. The combined treatment results in a synergistic effect. Sant7, a known IL-6 antagonist previously shown to be effective as antitumor agent in this model of human multiple myeloma (Tassone et al, Clin. Cancer Res., 2005, 11 :4251-4258), is used as a positive control, alone or in combination with dexamethasone.

Clinical Model - Multiple Myeloma

A subject diagnosed with multiple myeloma is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 13

Treatment of Leukemia and/or Lymphoma by Administering IL-6 Variant Protein According to the Present Invention A subject diagnosed with Leukemia and/or Lymphoma is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 14 Treatment of Renal Cell Carcinoma by Administering IL-6 Variant Protein

According to the Present Invention

A subject diagnosed with renal cell carcinoma is treated with an IL-6 variant according to the present invention, preferably with an IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g. , height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 15 Treatment of Tumor-Related Cachexia by Administering IL-6 Variant Protein

According to the Present Invention

Experimental model - Nude Mice Serum levels of IL-6 in RCC patients with paraneoplastic fever and weight loss are higher than those in RCC patients without paraneoplastic symptoms. Hence the role of IL-6 as a major mediator of tumor-related cachexia and a potential therapeutic target has been suggested. Blocking of IL-6 functions following a delivery of a therapeutic amount of IL-6 variants of the present invention in two tumor-related cachexia mouse models is performed to demonstrate that the cachexia cand be inhibited by IL-6 variants. The effect of IL-6 variant treatment on weight loss in the melanoma model female nude mice, inoculated with human melanoma cells, and in a prostate tumor model suggests that IL-6 valiants of the present invention, particular IL-6 174, are potential therapeutic agents for the treatment of tumor-related cachexia.

Clinical Model - Cachexia

A subject diagnosed with tumor-related cachexia is treated with an IL-6 variant according to the present invention, preferably IL-6 174 splice variant protein, to reduce the symptoms associated with the disease. An IL-6 174 splice variant protein is suspended in a suitable buffer for subcutaneous or intravenous delivery of the variant to the subject. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, the suspended protein is delivered in a dose ranging from about 1 mg/kg to 100 mg/kg by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 16

Treatment of Rheumatoid Arthritis (RA) by Gene Therapy with IL-6 Variant

According to the Present Invention A subject diagnosed with RA is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct. The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g. , height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 17

Treatment of Castleman's disease by Gene Therapy with IL-6 Variant According to the Present Invention A subject diagnosed with Castleman's disease is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct, The sequences encoding one or more of the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g. , height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 18 Treatment of Crohn's disease by Gene Therapy with IL-6 Variant According to the Present Invention

A subject diagnosed with Crohn's disease is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct, The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g. , height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 19

Treatment of Systemic Lupus Erythematosus (SLE) by Gene Therapy with IL-6 Variant According to the Present Invention

A subject diagnosed with SLE is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed //; vivo from the expression construct. The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 20

Treatment of Colitis by Gene Therapy with IL-6 Variant According to the

Present Invention

A subject diagnosed with colitis is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct. The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable vims containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g. , height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 21 Treatment of Multiple Myeloma by Gene Therapy with IL-6 Variant According to the Present Invention

A subject diagnosed with Multiple Myeloma is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice valiant proteins of the present invention are expressed in vivo from the expression construct. The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 22

Treatment of Leukemia and/or Lymphoma by Gene Therapy with IL-6 Variant

According to the Present Invention

A subject diagnosed with Leukemia and/or Lymphoma is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease, The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct. The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable vims containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly. Example 23

Treatment of Renal Cell Carcinoma by Gene Therapy with IL-6 Variant According to the Present Invention A subject diagnosed with renal cell carcinoma is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct. The sequences encoding the splice variant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable vims containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g. , height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

Example 24 Treatment of Tumor-Related Cachexia by Gene Therapy with IL-6 Variant

According to the Present Invention A subject diagnosed with tumor-related cachexia is treated by administering a gene therapy construct capable of expressing an IL-6 174 splice variant protein to reduce the symptoms associated with the disease. The IL-6 174 splice variant proteins of the present invention are expressed in vivo from the expression construct. The sequences encoding the splice valiant proteins of the present invention are cloned into an appropriate gene therapy vector downstream of an operable promoter. A suitable virus containing the vector construct is suspended at a concentration that results in a sufficient level of gene expression. Depending on the physical characteristics of the subject, e.g., height, weight, and severity of disease, a dose containing a particular concentration of vector is delivered by intravenous injection. The subject is periodically monitored by observing the change in physical symptoms; optionally, additionally or alternatively, one or more biomarkers are examined for a change to determine the effect of the treatment. Depending on the physical characteristics of the subject and/or the symptoms, additional doses are monitored from about daily to about weekly.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the ait. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a polynucleotide having a sequence selected from the group consisting of: S56892_PEA_1_PEA_1_T9 (SEQ ID NO:1) S56S92_PEA_l_PEA_l_T10 (SEQ ID NO:2) S56892_PEA_1_PEA_1_T13 (SEQ ID NO:3) S56892_PEA_1_PEA_1_T14 (SEQ ID NO:4) , or a polynucleotide at least 70% homologous thereto.

2. The isolated polynucleotide of claim 1, comprising a polynucleotide having a sequence at least 80% homologous thereto,

3. The isolated polynucleotide of claim 1, comprising a polynucleotide having a sequence at least 85% homologous thereto.

4. The isolated polynucleotide of claim 1, comprising a polynucleotide having a sequence at least 90% homologous thereto.

5. The isolated polynucleotide of claim 1. comprising a polynucleotide having a sequence at least 95% homologous thereto.

6. An isolated polynucleotide comprising a node having a sequence selected from the g grroouupp ccoonnssiissttiinngg of: S56892_PEA_l_PEA_l_node_0 (SEQ ID NO: 5) S56892_PEA_l_PEA_l_node_10 (SEQ ID NO:6) S56892_PEA_l_PEA_l_node_l 8 (SEQ ID NO:7) S56892_PEA_l_PEA_l_node_21 (SEQ ID NO:8) S56892_PEA_l_PEA_l_node_3 (SEQ ID NO:9) S56892_PEA_l_PEA_l_node_4 (SEQ ID NO: 10) S56892_PEA_l_PEA_l_node_7 (SEQ ID NO:11) S56892_PEA_l_PEA_l_node_8 (SEQ ID NO:12) S56892_PEA_l_PEA_l_node_9 (SEQ ID NO:13) S56892_PEA_l_PEA_l_node_12 (SEQ ID NO:14) S56892_PEA_l_PEA_l_node_l 3 (SEQ ID NO:15) S56892_PEA_l_PEA_l_node_14 (SEQ ID NO: 16) S56892_PEA_l_PEA_l_node_16 (SEQ ID NO: 17) S56892_PEA_l_PEA_l_node_l 7 (SEQ ID NO: 18) S56892_PEA_l_PEA_l_node_19 (SEQ ID NO: 19) S56892_PEA_l_PEA_l_node_20 (SEQ ID NO:20) S56892_PEA_l_PEA_l_node_22 (SEQ ID NO:21) S56892_PEA_l_PEA_l_node_23 (SEQ ID NO:22) .

7. An isolated polypeptide comprising a polypeptide having a sequence selected from the group consisting of : S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) .

8. An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , comprising a first amino acid sequence being at least about 90% homologous to MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHR QPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENN LNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRF ESSEEQARAVQMSTKVLIQFLQKK corresponding to amino acids 1 - 157 of IL6_HUMAN, which also corresponds to amino acids 1 - 157 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , and a second amino acid sequence being at least about 70% homologous to a polypeptide having the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) . wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

9. The polypeptide of claim 4, wherein the second amino acid sequence being at least about 80% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

10. The polypeptide of claim 4, wherein the second amino acid sequence being at least about 85% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

11. The polypeptide of claim 4, wherein the second amino acid sequence being at least about 90% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

12. The polypeptide of claim 4, wherein the second amino acid sequence being at least about 95% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) corresponding to amino acids 158 - 198 of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

13. An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) , comprising a polypeptide being at least about 70% homologous to the sequence VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

14. The polypeptide of claim 9, wherein the tail comprising a polypeptide being at least about 80% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

15. The polypeptide of claim 9, wherein the tail comprising a polypeptide being at least about 85% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :3S) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

16. The polypeptide of claim 9, wherein the tail comprising a polypeptide being at least about 90% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

17. The polypeptide of claim 9, wherein the tail comprising a polypeptide being at least about 95% homologous to a polypeptide having the sequence

VGVSSFPQLGVGEDRLKDSVLDNSGMQCHFQKRRLHVNKRV (SEQ ID NO :38) in S56892_PEA_1_PEA_1_P8 (SEQ ID NO:24) .

18. An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a first amino acid sequence being at least about 90% homologous to MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHR QPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENN LNLPKMAEKDGCFQSGFNE corresponding to amino acids 1 - 108 of IL6JHUMAN, which also corresponds to amino acids 1 - 108 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , and a second amino acid sequence being at least about 90% homologous to AKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKE FLQSSLRALRQM corresponding to amino acids 158 - 212 of IL6_HUMAN, which also corresponds to amino acids 109 - 163 of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) . wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

19. An isolated chimeric polypeptide encoding for an edge portion of S56892_PEA_1_PEA_1_P9 (SEQ ID NO:25) , comprising a polypeptide having a length "n", wherein n is at least about 50 amino acids in length, wherein at least two amino acids comprise EA, having a structure as follows: a sequence starting from any of amino acid numbers 108-x to 108; and ending at any of amino acid numbers 109+ ((n-2) - x), in which x varies from 0 to n-2.

20. The polypeptide of claim 15, wherein n is at least about 40 amino acids in length.

21. The polypeptide of claim 15, wherein n is at least about 30 amino acids in length.

22. The polypeptide of claim 15, wherein n is at least about 20 amino acids in length.

23. The polypeptide of claim 15, wherein n is at least about 10 amino acids in length.

24. An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) , comprising a first amino acid sequence being at least about 90% homologous to MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHR QPLTSSERIDKQIRYILDGISALRKETCNKSN corresponding to amino acids 1 - 76 of IL6_FfUMAN, which also corresponds to amino acids 1 - 76 of S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) , and a second amino acid sequence being at least about 70% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

25. The polypeptide of claim 20, wherein the second amino acid sequence being at least about 80% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

26. The polypeptide of claim 20, wherein the second amino acid sequence being at least about 85% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

27. The polypeptide of claim 20, wherein the second amino acid sequence being at least about 90% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

28. The polypeptide of claim 20, wherein the second amino acid sequence being at least about 95% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) corresponding to amino acids 77 - 95 of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

29. An isolated polypeptide encoding for a tail of S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) , comprising a polypeptide being at least about 70% homologous to the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) .

30. The polypeptide of claim 25, wherein the tail comprising a polypeptide being at least about 80% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

31. The polypeptide of claim 25, wherein the tail comprising a polypeptide being at least about 85% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) .

32. The polypeptide of claim 25, wherein the tail comprising a polypeptide being at least about 90% homologous to a polypeptide having the sequence IWLKICMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P1 1 (SEQ ID NO:26) .

33. The polypeptide of claim 25, wherein the tail comprising a polypeptide being at least about 95% homologous to a polypeptide having the sequence IWLKKMDASNLDSMRRLAW (SEQ ID NO :39) in S56892_PEA_1_PEA_1_P11 (SEQ ID NO:26) .

34. An isolated chimeric polypeptide encoding for S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , comprising a first amino acid sequence being at least about 90% homologous to MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHR QPLTSSERIDKQIRYILDGISALRK corresponding to amino acids 1 - 69 of 1L6JHUMAN, which also corresponds to amino acids 1 - 69 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , and a second amino acid sequence being at least about 90% homologous to

EETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQ FLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLIL

RSFKEFLQSSLRALRQM corresponding to amino acids 108 - 212 of IL6_HUMAN, which also corresponds to amino acids 70 - 174 of S56892_PEA_1_PEA_1_P13 (SEQ ID NO:27) , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.

35. An isolated chimeric polypeptide encoding for an edge portion of S56892_PEA_1__PEA_1_P13 (SEQ ID NO:27) , comprising a polypeptide having a length "n", wherein n is at least about 50 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KE, having a structure as follows: a sequence starting from any of amino acid numbers 69-x to 69; and ending at any of amino acid numbers 70+ ((n-2) - x), in which x varies from 0 to n-2.

36. The polypeptide of claim 31 , wherein n is at least about 40 amino acids in length.

37. The polypeptide of claim 31, wherein n is at least about 30 amino acids in length.

38. The polypeptide of claim 31, wherein n is at least about 20 amino acids in length.

39. The polypeptide of claim 31, wherein n is at least about 10 amino acids in length.

40. An antibody capable of specifically binding to an epitope of an amino acid sequence of claim 3.

41. The antibody of claim 36. wherein said amino acid sequence corresponds to a bridge, edge portion, tail, or head as in any of the previous claims.

42. The antibody of claim 36 or 37, wherein said antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.

43. A nucleic acid construct comprising the isolated polynucleotide of any of claims 1-5.

44. The nucleic acid construct of claim 43, further comprising a promoter for regulating transcription of the isolated polynucleotide in sense or antisense orientation.

45. The nucleic acid construct of claim 43, further comprising positive and negative selection markers for selecting for homologous recombination events.

46. A host cell comprising the nucleic acid construct of claim 43.

47. A method for treating a variant-treatable disease, comprising administering a therapeutic protein, variant peptide, protein, nucleic acid sequence, antisense and/or antibody to a subject in need of treatment thereof.

48. The method of claim 47, wherein the variant-treatable disease is selected from a group consisting of immune disorders, inflammatory disorders Parkinson's disease, myeloproliferative disorders, cancerous diseases and cachexia associated with cancer.

49. The method of claim 48, wherein said immune disorders are selected from the group consisting of ulcerative colitis, asthma, psoriasis, bone resorption due to osteoporosis, RA, Crohn's disease, Castleman's disease, systemic lupus erythematosus, iriflammatory-mesangial proliferative glomerulonephritis, autoimmune-RA, and psoriasis.

50. The method of claim 48, wherein said cancerous diseases are selected from the group consisting of multiple myeloma/plasmacytoma, Kaposi's sarcoma, breast cancer, gastrointestinal cancer, leukemia, lymphoma, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, bladder cancer.

51. A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide according to any of the above claims and a pharmaceutically acceptable carrier or diluent.

52. A method of treating a variant-related disease in a subject, the method comprising upregulating in the subject expression of a polypeptide as described herein, thereby treating the variant- related disease in a subject.

53. The method of claim 52, wherein said upregulating expression of said polypeptide is effected by i. administering said polypeptide to the subject; and/or ii. administering an expressible polynucleotide encoding said polypeptide to the subject.