JP2007537696A - Interferon gamma-like protein - Google Patents

Abstract

  The present invention relates to a protein, identified as INSP037, identified in the present invention as an interferon γ-like secreted protein of the 4-helix bundle cytokine fold, and said protein in the diagnosis, prevention and treatment of diseases and the same To the use of nucleic acid sequences derived from genes encoding.

Description

The present invention relates to a protein called INSP037 identified as an interferon γ-like secreted protein of a 4-helix bundle cytokine fold in the present invention, and also a nucleic acid derived from the protein and a gene encoding the protein in the diagnosis, prevention and treatment of diseases. Regarding the use of arrays.
All publications, patents and patent applications cited herein are hereby incorporated by reference.

In the drug discovery process, a fundamental revolution is currently underway with the advent of the era of functional genomics. The term “functional genomics” is used in an approach that utilizes bioinformatics tools to assign a function to a protein sequence of interest. Such tools are increasingly needed because the speed of sequence data generation far exceeds the laboratory's ability to assign functions to these protein sequences.
Due to the increasing potential and accuracy of bioinformatics tools, the tools are rapidly being replaced by conventional biochemical characterization techniques. In fact, the advanced bioinformatics tools used to identify the present invention now have the ability to output results with high confidence.
As sequence data becomes available, various research institutes and corporate organizations have investigated them and important discoveries are continually being achieved. However, there continues to be a need to identify and characterize additional genes and the polypeptides they encode as targets for research and drug discovery.

(Introduction section on secreted proteins)
The ability of cells to produce and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signaling molecules are all secreted by the cell. The process is via fusion of secretory vesicles with the plasma membrane. In many, if not all, proteins are directed into the endoplasmic reticulum by a signal peptide and into the secretory vesicle. A signal peptide is a cis-acting sequence that acts to transport a polypeptide chain from the cytoplasm to a membrane-bound compartment such as a secretory vesicle. The polypeptide that is directed to the secretory vesicle is secreted into the extracellular matrix or retained in the plasma membrane. A polypeptide retained in the plasma membrane will have one or more transmembrane domains. Examples of secreted proteins that play a central role in cell function are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors.

(Introduction section on cytokines)
Cytokines are a family of growth factors secreted primarily from leukocytes and messenger proteins that act as potent regulators that can perform a series of intracellular reactions at subnanomolar concentrations. Interleukins, neurotrophins, growth factors, interferons and chemokines are all defined as a family of cytokines that work with cellular receptors to regulate cell growth and differentiation. Their size allows cytokines to be quickly transported around the body and degraded when needed. Their role in regulating a wide range of cellular functions, particularly immune responses and cell proliferation, has been revealed by many studies over the last 20 years (SB Boppana (1996) Indian. J. Pediatr. 63 (4) : 447-52). Cytokines, like other growth factors, are distinguished from classical hormones by the fact that they are produced by many different types of cells rather than one specific tissue or gland, and are also located on target cells It affects a wide range of cells through interaction with specific high affinity receptors.
The communication system of all cytokines is both pleiotropic (one messenger causes multiple actions) and redundant (each action is caused by more than one messenger) (G. Tringali et al. (2000 Therapie. 55 (1): 171-5; L. Tessarollo (1998)) Cytokine Growth Factor Rev. 9 (2): 125-137). The action of individual cytokines on one cell also affects the concentration of said cytokines, the concentration of other cytokines, the temporal order of cytokines, and the internal state of the cells (cell cycle, presence of adjacent cells, cancerous) It will be influenced.

Cytokines are typically small (less than 200 amino acids) proteins, but they are often generated from larger precursors that are spliced post-translationally. In addition to the alternative splicing pathway of mRNA, the splicing provides a wide variety of each cytokine, each of which can differ substantially in biological action. Many membrane and extracellular matrix-bound forms of cytokines have also been isolated (M. Okada-Ban et al. (2000) Int. J. Biochem. Cell Biol. 32 (3): 263-267; SP Atamas (1997) Life Sci. 61 (12): 1105-1112).
Cytokines can be classified into multiple families, but most are irrelevant. Classification is usually based on secondary structure composition since sequence similarity is often very low. The family has been named after prototypical members such as IFN-like, IL2-like, IL1-like, IL6-like and TNF-like (A. Zlotnik et al. (2000) Immunity 12 (2): 121-127).
Cytokines are responsible for many important responses in multicellular organisms such as immune response regulation (J. Nishihira (1998) Int. J. Mol. Med. 2 (1): 17-28), inflammation (PK Kim et al. ( 2000) Surg. Clin. North. Am. 80 (3): 885-894), wound healing (RA Clark (1991) J. Cell Biochem. 46 (1): 1-2), embryogenesis and development, and apoptosis Studies have shown that it is involved in (HD Flad et al. (1999) Pathobiology 67 (5-6): 291-293).
Pathogenic organisms (viruses and bacteria), such as HIV and Kaposi's sarcoma-associated virus, encode anti-cytokine factors and cytokine family analogs that allow the factor and the analogs to interact with cytokine receptors. (S. Sozzani et al. (2000) Pharm. Acta. Helv. 74 (2-3): 305-312; Y. Aoki et al. (2000) J. Hematother Stem. Cell Res. 9 (2): 137-145). The virus-encoded cytokine, virokine, has been shown to be required for the virulence of the virus because of its ability to mimic and destroy the host immune system.

Cytokines can be useful for the treatment, prevention and / or diagnosis of medical conditions and diseases including: immune disorders such as autoimmune diseases, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus and multiple Sclerosis, inflammatory diseases such as allergies, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease, pancreatitis, gastrointestinal inflammation, sepsis, endotoxin shock, septic Shock, epidemic, muscle pain, ankylosing spondylitis, myasthenia gravis, post-virus wasting syndrome, lung disease, respiratory distress syndrome, asthma, chronic embolic lung disease, airway inflammation, wound healing, endometriosis, Skin diseases, Behcet's disease, neoplastic diseases such as melanoma, sarcoma, kidney tumor, colon tumor, blood disease, myeloproliferative disease, Hodgkin's disease, osteoporosis, obesity, diabetes, gout, cardiovascular disease Reperfusion injury, atherosclerosis, ischemic heart disease, heart failure, stroke, liver disease, AIDS, AIDS-related complications, neuropathy, male infertility, aging and infection (protozoal malaria infection, bacterial infection and viral infection including).
Clinical use of cytokines has focused on their role as regulators of the immune system (FH Rodriguez et al. (2000) Curr. Pharm. Des. 6 (6): 665-680), for example, thyroid cancer (C. Schmutzler et al. (2000) 143 (1): 15-24). Cytokines are also anti-cancer targets due to the control of cytokine cell proliferation and cell differentiation (E. Lazar-Molnar et al. (2000) Cytokine. 12 (6): 547-554; K. Gado (2000 24 (4): 195-209). Novel mutations in cytokines and cytokine receptors have been shown to confer resistance to disease in some cases (SJ van Deventer et al. (2000) Intensive Care Med. 26 (Suppl1): S98-S102 ). Creating synthetic cytokines (mutesins) to modulate activity and eliminate potential side effects was also an important research method (AB Shanafelt et al. (1998) 95 (16): 9454-9458 ).
Thus, cytokine molecules have been shown to play roles in diverse physiological functions, many of which can play a role in disease progression. Altering the activity of cytokine molecules is a means to change the phenotype of the disease, and the novel cytokine molecules can also play a role in the treatment of the diseases and other conditions identified above and are useful in the development of the treatment Therefore, identifying new cytokine molecules per se is very suitable.

(Introduction section on interferon)
Interferon is a member of the 4-helix bundle cytokine family. Interferons are classified as type I or type II, depending on their structure and stability in acidic media. Type I interferons are divided into five groups based on their sequence: interferon-alpha (IFN-α), interferon-beta (IFN-β), interferon-omega (IFN-θ), and interferon-tau. (IFN-τ). The only type II interferon identified so far is interferon-gamma (IFN-γ), which is produced by activated T cells and NK cells.
The gene of type I interferon forms a cluster on human chromosome 9. In humans, there are at least 14 IFN-α non-allelic genes and it is estimated that the number of naturally occurring IFN-α proteins is further increased by alleles of the IFN-α gene (Jussain et al, 1996 J. Interferon Cytokine Res. 16: 853-9).
Interferons exert their cellular activity by binding to specific membrane receptors on the cell surface, initiating a complex series of intracellular events. Type I interferons induce a wide variety of biological responses including antiviral, immunomodulatory and antiproliferative effects, and as a result of these effects proved to be effective in the treatment of various diseases and conditions. ing.

Interferon is a potent antiviral substance and α-interferon has been found to be particularly useful for the treatment of various viral infections including human papillomavirus infection, hepatitis B and C infection (Jaeckel et al 2001, 345 (2): 1452-7). Type I interferon also inhibits cell proliferation, and α-interferon has been used clinically for many years to treat a variety of malignant tumors including: ciliary cell leukemia, multiple myeloma, chronic lymphoma Mild lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal cell carcinoma and ovarian cancer. In addition, type I interferons are useful for the treatment of autoimmune diseases and interferon-β is approved for the treatment of multiple sclerosis.
Interferon-τ was first identified in ruminant conceptus homogenates but has since been identified in humans (see WO96 / 35789). Interferon-τ exhibits many activities similar to other type I interferons, but also exhibits several different actions. In particular, interferon-τ has an anti-luteolytic action that promotes the establishment and maintenance of pregnancy (Martal et al, Reprod Fertil Dev, 1997, 9 (3): 355-80). Furthermore, the induction of interferon-α and β by the virus is transient (lasting for several hours), whereas the induction of interferon-τ expression by the virus can last for several days and have antiretroviral activity against HIV-1. (Dereuddre-Bosquet et al, J. Acquir. Immune Defic Syndr. Hum. Retrovirol, 1996, 11 (3): 241-6).

Type II interferons (including interferon gamma) may be useful for the treatment, prevention and / or diagnosis of the following conditions and diseases: immune diseases such as autoimmune diseases, rheumatoid arthritis, osteoarthritis, Psoriasis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, Graves' disease, autoimmune alopecia, scleroderma, psoriasis (Kimball et al., Arch Dermatol 2002 Oct: 138 (10) : 1341-6), graft-versus-host disease (Miura Y., et al., Blood 2002 Oct 1: 100 (7): 2650-8), monocyte and neutrophil dysfunction, attenuation of B cell function Inflammatory diseases such as acute inflammation, septic shock, asthma, anaphylaxis, eczema, dermatitis, allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Sjogren's disease (Anaya et al., J Rheumatol 2002 Sep; 29 (9): 1874-6), Crohn's disease (Schmit A. et al., Eur Cytokine Netw 2002 Jul-Sep: 13 (3): 298 -305), Ulcerative colitis, Inflammatory bowel disease, Pancreatitis, Gastrointestinal inflammation, Ulcerative colitis, Sepsis, Endotoxic shock, Septic shock, Cachexia, Myalgia, Ankylosing spondylitis, Severe Myasthenia, post-virus fatigue syndrome, lung disease, respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, airway inflammation, wound healing, type I and type II diabetes, endometriosis, skin disease, Behcet's disease, Immunodeficiency, chronic lung disease (Oei J et al., Acta Paediatr 2002: 91 (11): 1194-9), invasive and chronic periodontitis (Gonzales JR, et al., J clin Periodontol 2002 Sep: 29 (9): 816-22), cancers such as carcinomas, sarcomas, lymphomas, renal tumors, colon tumors, Hodgkin's disease, metastatic melanoma (Vaishampayan U, Clin Cancer Res 2002 Dec: 8 (12) : 3696-701), mesothelioma, Burkitt lymphoma, neuroblastoma, hematological disease, nasopharyngeal carcinoma, leukemia, myeloma, myeloproliferative disease and other neoplastic diseases, osteoporosis Liver diseases such as obesity, diabetes, gout, cardiovascular disease, reperfusion injury, atherosclerosis, ischemic heart disease, heart failure, stroke, chronic hepatitis (Semin Liver Dis 2002: 22 Suppl 1: 7), AIDS (Dereuddre-Bosquet N., et al., J Acquir Immune Defic Syndr Hum Retroviol 1996 Mar 1: 11 (3): 241-6), AIDS-related syndromes, neurological diseases, fibrotic diseases, male infertility, aging, And infections such as plasmodium infection, bacterial infections, ringworm, histoplasmosis, blastosomiasis, aspergillosis, cryptococcosis, sporotricosis, coccidioidomycosis, paracoccidioidomycosis and candidiasis, diseases with antibacterial immunity ( Bogdan, Current Opinion in Immunology 2000, 12: 419-424), Peony disease (Lacy et al., Int J Impot Res 2002 Oct: 14 (5): 336-9), tuberculosis (Dieli et al., J Infect Dis 2002 Dec 15; 186 (12): 1835-9) and virus infection (Pfeffer LM, Sem in Oncol 1997 Jun 24: S9-63-69).
In summary, secreted proteins that are members of the 4-helix bundle cytokine family have been shown to play a role in diverse physiological functions, many of which can play a role in disease processes. In particular, interferon has been found to play an important role in various physiological processes and as a result has been found to be useful in the treatment of a wide range of diseases. However, there is still a need to identify new interferons that allow the development of new drugs for the treatment and prevention of diseases (including those mentioned above).

The present invention is based on the discovery that the INSP037 protein is an interferon gamma-like secreted protein of the 4-helix bundle cytokine fold.
In one aspect of the first aspect of the invention, the following polypeptides are provided:
(i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(ii) a polypeptide that is an interferon gamma-like secreted protein of a 4-helix bundle cytokine fold or a fragment of (i) that has an antigenic determinant in common with the polypeptide of (i); or
(iii) A polypeptide which is a functional equivalent of (i) or (ii).
According to the second aspect of the first aspect of the invention, the following polypeptides are provided:
(i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2;
(ii) a polypeptide that is an interferon gamma-like secreted protein of a 4-helix bundle cytokine fold or a fragment of (i) that has an antigenic determinant in common with the polypeptide of (i); or
(iii) A polypeptide which is a functional equivalent of (i) or (ii).

The polypeptide having the sequence set forth in SEQ ID NO: 2 is hereinafter referred to as “INSP037 polypeptide”. INSP037 is also referred to herein as IPAAA44548.
Preferably, the INSP037 polypeptide of the first aspect of the invention functions as an interferon gamma-like secreted protein of a 4-helix bundle cytokine fold. Those skilled in the art will understand the term “interferon gamma-like secreted protein of a 4-helix bundle cytokine fold” and use one of a variety of assays known in the art to identify polypeptides in this class. It will be easy to see if it functions as a member of The presence of the 4-helix bundle cytokine fold can be identified by analysis of protein sequence and secondary structure. Interferon activity is often measured as antiviral activity or antiproliferative activity of cancer cells. Examples of such assays can be found in the literature (Schiller JH, J Interferon Res 1986; 6 (6): 615-25, Gibson, UE et al., J Immunol Methods (1989) 20; 125 (1-2 ): 105-13 and Chang et al., J. Biol. Chem. (2002) 277 (9): 7118-7126).

In a second aspect, the invention provides a purified nucleic acid molecule that encodes a polypeptide of the first aspect of the invention.
Preferably, the purified nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 1 (encoding the INSP037 polypeptide). Preferably, the purified nucleic acid molecule consists of the nucleic acid sequence set forth in SEQ ID NO: 1 (encoding the INSP037 polypeptide) or is a redundant equivalent or fragment of that sequence.
In a third aspect, a purified nucleic acid molecule is provided that hybridizes under high stringency conditions with the nucleic acid molecule of the second aspect of the invention.
In a fourth aspect, the present invention provides a vector, such as an expression vector, comprising the nucleic acid molecule of the second or third aspect of the invention. Preferred vectors of the present invention include pDEST14-IPAAA44548-6HIS (see FIG. 10), PCRII-TOPO-IPAAA44548 (see FIG. 11), pDEST14-IPAAA44548-6HIS (see FIG. 12) and pEAK12D-IPAAA44548-6His (see FIG. 13). ) Is included.
In a fifth aspect, the invention provides a host cell transformed with the vector of the fourth aspect of the invention.

In a sixth aspect, the present invention specifically binds to the polypeptide of the first aspect of the present invention, and preferably suppresses secretory protein activity of the polypeptide, more preferably a 4-helix bundle cytokine. Provided is a ligand that suppresses activity, and more preferably suppresses interferon γ-like activity.
In a seventh aspect, the invention alters the expression of a natural gene encoding the polypeptide of the first aspect of the invention or modulates the activity of the polypeptide of the first aspect of the invention To provide effective compounds.
The compound of the seventh aspect of the present invention may increase (agonist action) or decrease (antagonistic action) the gene expression level or activity of the polypeptide.
Importantly, identifying the INSP037 exon polypeptide and the function of the INSP037 polypeptide allows the design of screening methods that can identify compounds that are effective in the treatment and / or diagnosis of disease. The ligands and compounds of the sixth and seventh aspects of the invention can be identified using such methods. These methods are included as features of the present invention. Using these methods, one could readily identify inhibitors or antagonists of INSP037, such as monoclonal antibodies, which may be beneficial for modifying INSP037 activity in vivo in clinical applications. Such compounds would also be useful in counteracting the IFNγ-like activity of INSP037 polypeptide.

  In an eighth aspect, the invention provides a polypeptide of the first aspect of the invention, or a second or third aspect of the invention for use in the treatment or diagnosis of a disease involving interferon. Provide a nucleic acid molecule, or a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention. In particular, IFNγ-like polypeptides are provided. Such diseases include, but are not limited to: immune diseases such as autoimmune diseases, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, multiple sclerosis, severe Myasthenia, Guillain-Barre syndrome, Graves' disease, autoimmune alopecia, scleroderma, psoriasis (Kimball et al., Arch Dermatol 2002 Oct: 138 (10): 1341-6), graft-versus-host disease ( Miura Y., et al., Blood 2002 Oct 1: 100 (7): 2650-8), monocyte and neutrophil dysfunction, attenuation of B cell function, inflammatory diseases such as acute inflammation, septic shock , Asthma, anaphylaxis, eczema, dermatitis, allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Sjogren's disease (Anaya et al., J Rheumatol 2002 Sep; 29 (9): 1874-6), Crohn's disease ( Schmit A. et al., Eur Cytokine Netw 2002 Jul-Sep: 13 (3): 298-305), ulcerative colitis, inflammatory bowel disease, pancreatitis, digestive system inflammation , Ulcerative colitis, sepsis, endotoxic shock, septic shock, cachexia, myalgia, ankylosing spondylitis, myasthenia gravis, post-virus fatigue syndrome, lung disease, respiratory distress syndrome, asthma, chronic obstruction Pulmonary disease, respiratory tract inflammation, wound healing, type I and type II diabetes, endometriosis, skin disease, Behcet's disease, immunodeficiency, chronic lung disease (Oei J et al., Acta Paediatr 2002: 91 ( 11): 1194-9), invasive and chronic periodontitis (Gonzales JR, et al., J clin Periodontol 2002 Sep: 29 (9): 816-22), cancers such as carcinomas, sarcomas, lymphomas, kidneys Tumor, colon tumor, Hodgkin's disease, melanoma such as metastatic melanoma (Vaishampayan U, Clin Cancer Res 2002 Dec: 8 (12): 3696-701), mesothelioma, Burkitt lymphoma, neuroblastoma, blood Disease, nasopharyngeal cancer, leukemia, myeloma, myeloproliferative disease and other neoplastic diseases, osteoporosis, obesity, diabetes, gout, cardiovascular disease, reperfusion injury Liver diseases such as atherosclerosis, ischemic heart disease, heart failure, stroke, chronic hepatitis (Semin Liver Dis 2002: 22 Suppl 1: 7), AIDS (Dereuddre-Bosquet N., et al., J Acquir Immune Defic Syndr Hum Retroviol 1996 Mar 1: 11 (3): 241-6), AIDS-related syndromes, neurological diseases, fibrotic diseases, male infertility, aging, and infections such as plasmodium infection, bacterial infection, fungal disease (ringworm, Histoplasmosis, blastosomiasis, aspergillosis, cryptococcosis, sporotricosis, coccidioidomycosis, paracoccidioidomycosis and candidiasis), diseases with antibacterial immunity (Bogdan, Current Opinion in Immunology 2000, 12: 419-424), Peony disease (Lacy et al., Int J Impot Res 2002 Oct: 14 (5): 336-9), tuberculosis (Dieli et al., J Infect Dis 2002 Dec 15; 186 (12): 1835-9) and viruses Infection (Pfeffer LM, Semin Oncol 1997 Jun 24: S9-63-69).

Some of these first, second, third, fourth, fifth, sixth or seventh features of the present invention can also be used in the manufacture of a medicament for the treatment of the above diseases.
In a ninth aspect, the present invention relates to an expression level of a natural gene encoding the polypeptide of the first feature of the present invention or the level of activity of the polypeptide of the first feature of the present invention. And a method of diagnosing a disease in a patient comprising comparing the expression level or activity to a control level, wherein a level different from the control level is indicative of the disease. Said method will preferably be performed in vitro. Similar methods can be used for monitoring therapeutic treatment of diseases in patients. In this case, a change in the level of expression or activity of the polypeptide or nucleic acid molecule over time, towards the control level, indicates disease remission.
A preferred method for detecting a polypeptide of the first aspect of the invention comprises the following steps: (a) combining a ligand (eg, antibody) of the sixth aspect of the invention with a biological sample, ligand-poly Contacting under conditions suitable for the formation of a peptide complex; and (b) detecting the complex.
One skilled in the art would detect nucleic acid abnormalities using, for example, nucleic acid hybridization methods with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification, and antibodies. It will be apparent that there are a variety of different methods, such as methods. Similar methods can be used on a short-term or long-term basis to allow treatment of the disease being monitored. The present invention also provides a kit useful for the disease diagnosis method.

Preferably, the disease diagnosed by the method of the ninth aspect of the present invention is a disease involving interferon as described above.
In a tenth aspect, the invention provides the use of the polypeptide of the first aspect of the invention as an interferon gamma-like secreted protein of a 4-helix bundle cytokine fold. One suitable use of INSP037 is as an adjuvant in bacterial, fungal or viral infections in combination with well-established therapies. Other possible uses include the use of INSP037 to activate macrophages and the use of INSP037 to increase the expression of MHC molecules and antigen processing components. The experimental results included herein confirm the predicted INFγ-like activity of INSP037. This finding is in that the polypeptides of the present invention can be tested for suitability for use in known applications of INFγ, such as anti-cancer applications (eg Vaishampayan U, Clin Cancer Res 2002 Dec: 8 (12): 3696 -701), pioneering a series of interesting therapeutic uses for the protein itself. Similarly, inhibitors or antagonists of INSP037 (such as monoclonal antibodies) could be readily identified that may be beneficial for further research or clinical application of INSP037 activity in vivo.
In an eleventh aspect, the invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the polypeptide of the first aspect of the invention, the nucleic acid molecule of the second or third aspect of the invention, the invention A vector of the fourth feature of the invention, a host cell of the fifth feature of the invention, a ligand of the sixth feature of the invention or a compound of the seventh feature of the invention in combination with a pharmaceutically acceptable carrier. .
In a twelfth aspect, the present invention provides a polypeptide of the first aspect of the present invention, the second of the present invention for use in the manufacture of a medicament for the diagnosis or treatment of a disease involving interferon. Or a nucleic acid molecule of the third feature, a vector of the fourth feature of the invention, a host cell of the fifth feature of the invention, a ligand of the sixth feature of the invention or a compound of the seventh feature of the invention I will provide a. Such diseases include those described above in connection with the eighth aspect of the invention.

In a thirteenth aspect, the invention provides a method of treating a disease in a patient, the method comprising a polypeptide of the first aspect of the invention, a nucleic acid molecule of the second or third aspect of the invention, Administering to the patient a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, a ligand of the sixth aspect of the invention or a compound of the seventh aspect of the invention.
Affected when the expression of a natural gene encoding the polypeptide of the first aspect of the invention or the activity of the polypeptide of the first aspect of the invention is compared to the level of expression or activity in a healthy subject For a disease that is reduced in a patient who is suffering, the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an agonist. Conversely, for diseases in which the expression of the natural gene or the activity of the polypeptide is elevated in a patient suffering when compared to the level of expression or activity in a healthy subject, it is administered to the patient. Said polypeptide, nucleic acid molecule, vector, host cell, ligand or compound should be an antagonist. Examples of said antagonists include antisense nucleic acid molecules, ribozymes and ligands (eg antibodies).
Preferably, such a disease is a disease as described above involving interferon.
In a fourteenth aspect, the present invention relates to a transgenic or gene knockout non-human animal transformed to express the polypeptide of the first aspect of the present invention at a high level, a low level, or not at all. I will provide a. The transgenic animal is very useful as a model for disease research, and can also be used in screening methods aimed at identifying compounds effective for the treatment or diagnosis of the disease.
Preferably, such a disease is a disease as described above involving interferon.

A summary of standard techniques and methods that can be used to utilize the present invention is provided below. It will be appreciated that the invention is not limited to the particular methodologies, protocols, cell lines, vectors and reagents described. It will also be appreciated that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. The scope of the invention is limited only by the terms of the appended claims.
In this specification, standard abbreviations for nucleotides and amino acids are used.
In the practice of the invention, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology will be used. Such techniques are within the skill of the artisan.
Such techniques are explained fully in the literature. Examples of particularly relevant manuals include: Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Vol. I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. 1984); Transcription and Translation (BD Hames & SJ Higgins eds. 1984); Animal Cell Culture (RI Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press , 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.) Especially Vol. 154 &155; Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos eds 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. 1987, Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer Verlag, NY ); And Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell e ds. 1986).

As used herein, the term “polypeptide” includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. The modified peptide bond is a peptide isostere. The term refers to both short chains (peptides and oligopeptides) and long chains (proteins).
The polypeptide of the present invention may have the form of a mature protein, and is a pre-, pro- or prepro-protein that is activated by cleavage of the pre-, pro- or prepro-moiety and is an active mature polypeptide It may be a protein that produces In such polypeptides, the pre-, pro- or prepro-sequence may be a leader or secretory sequence, or a sequence used for purification of the mature polypeptide sequence.
The polypeptide of the first aspect of the invention can form part of a fusion protein. For example, it is often advantageous to include one or more additional amino acid sequences. Said additional amino acid sequence may comprise a secretory or leader sequence, a pro-sequence, a sequence useful for purification, or a sequence that confers higher protein stability, for example during recombinant formation. Alternatively, or in addition, the mature polypeptide can be fused to another compound, such as a compound that increases the half-life of the polypeptide (eg, polyethylene glycol).

Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified by natural processes (eg, post-translational processing) or by chemical modification techniques well known in the art. Known modifications that are generally present in the polypeptides of the invention include glycosylation, lipidation, sulfurization, γ-carboxylation (eg of glutamic acid residues), hydroxylation and ADP-ribosylation. Other possible modifications include acetylation, acylation, amidation, covalent addition of flavins, covalent addition of heme moieties, covalent addition of nucleotides or nucleotide derivatives, covalent addition of lipid derivatives, covalent attachment of phosphatidylinositol. Bond addition, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bond formation, cysteine formation, pyroglutamate formation, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, protein Degradable processing, phosphorylation, prenylation, racemization, selenoylation, transfer RNA-mediated amino acid addition (eg arginylation) to proteins and ubiquitin binding are included.
The modification may be present anywhere in the polypeptide including the peptide backbone, the amino acid side chain and the amino terminus or carboxy terminus. Indeed, blockage of the amino terminus or carboxy terminus of a polypeptide or both ends thereof by covalent modification is common in naturally occurring and synthetic polypeptides, and such modifications are not limited to the polypeptides of the present invention. Can also exist.

The alterations present in the polypeptide will often be a function of how the polypeptide is produced. For a recombinantly produced polypeptide, the nature and extent of the modification will be determined in large part by the post-translational modification capacity of the particular host cell and the modification signal present in the amino acid sequence of the polypeptide in question. Let ’s go. For example, glycosylation patterns vary between different types of host cells.
The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides (eg, purified from cell culture), recombinantly produced polypeptides (including fusion proteins), synthetically produced Or a polypeptide produced by a combination of the above methods.
The functionally equivalent polypeptide of the first aspect of the invention can be a polypeptide homologous to the INSP037 polypeptide. As used herein, two polypeptides are said to be “homologous” if one sequence of the polypeptides has a sufficiently high identity or similarity to the sequence of the other polypeptide. Is done. “Identity” indicates that amino acid residues are identical between the sequences at any particular location in the aligned sequences. “Similarity” indicates that amino acid residues are of a similar kind between the sequences at any particular location in the aligned sequences. The degree of identity and similarity can be easily calculated (Computational Molecular Biology, AM Lesk ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, DW Smith ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, AM Griffin and HG Griffin eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, G. von Heinje, Academic Press, 1987; and Sequence Analysis Primer, M. Gribskov and J. Devereux eds., M. Stockton Press, New York, 1991). Preferably, the identity ratio referred to herein is the default specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). Determined using BLAST version 2.1.3 with parameters [Blosum 62 matrix; gap open penalty = 11 and gap extension penalty = 1].

Thus, homologous polypeptides include natural biological variants of INSP037 polypeptide (eg, allelic variants or geographical variants in the species from which the polypeptide was derived) and variants (eg, amino acid substitutions, insertions or deletions). Including mutants). Said variant may comprise a polypeptide in which one or more amino acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such Substituted amino acid residues may or may not be encoded by the genetic code. Typical such substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; or Occurs between the aromatic residues Phe and Tyr. Particularly preferred are variants in which several (ie 5 to 10, 1 to 5, 1 to 3, 1 to 2, or just one) amino acids are substituted, deleted or added in any combination. Particularly preferred are silent substitutions, additions and deletions that do not alter the properties and activities of the protein. Also particularly preferred here are conservative substitutions.
The variants also include polypeptides in which one or more amino acid residues contain a substituent.

Typically, greater than 80% identity between two polypeptides is considered an indicator of functional equivalent. Preferably, a functionally equivalent polypeptide of the first aspect of the invention has a degree of sequence identity greater than 80% with an INSP037 polypeptide or an active fragment thereof. More preferred polypeptides have a degree of identity greater than 90%, 95%, 98% or 99%, respectively.
A functionally equivalent polypeptide of the first aspect of the invention may also be a polypeptide identified using one or more techniques of alignment for structure. For example, a PCT application published as WO 01/69507 using the Inpharmatica Genome Threader technology that constitutes one corner of the search tool used to create the Biopendium search database. See), interferon gamma-like secretion of the 4-helix bundle cytokine fold in order to have low sequence identity compared to the INSP037 polypeptide, but to share significant structural homology with the INSP037 polypeptide sequence. A polypeptide that is predicted to be a protein and currently unknown in function can be identified. “Significant structural homology” means that the Informatica = Genome Threader predicts that the two proteins share structural homology with greater than 10% certainty.
Polypeptides of the first aspect of the invention also include fragments of INSP037 polypeptides as well as functional equivalent fragments of these polypeptides, provided that these fragments retain interferon gamma-like activity or these polypeptides And having a common antigenic determinant.

As used herein, the term “fragment” refers to a polypeptide having the same amino acid sequence as part (but not all) of the amino acid sequence of one of INSP037, a polypeptide or one of its functional equivalents. . The fragment should contain at least n consecutive amino acids derived from the sequence, and n is preferably 7 or greater depending on the particular sequence (eg 8, 10, 12, 14, 16, 18, 20 or greater). Small fragments can constitute antigenic determinants.
Such a fragment is “free-standing” (ie, not part of another amino acid or polypeptide, nor fused to part of another amino acid or polypeptide). It may be present or included in a larger polypeptide to form part or region of said polypeptide. When contained within a larger polypeptide, the fragments of the present invention most preferably form a single continuous region. For example, certain preferred embodiments relate to fragments having a pre- and / or pro-polypeptide region fused to the amino terminus of the fragment and / or fragments having an additional region fused to the carboxy terminus of the fragment. However, several fragments may be contained within a single larger polypeptide.
The polypeptide of the present invention or an immunogenic fragment thereof (containing at least one antigenic determinant) can be used to make a ligand immunospecific for the polypeptide, eg, a polyclonal or monoclonal antibody. Such antibodies can be used to isolate or identify clones expressing the polypeptide of the invention, or to purify the polypeptide of the invention by affinity chromatography. The antibody can also be used as a diagnostic or therapeutic aid among other uses, as will be apparent to those skilled in the art.

The term “immunospecific” means that the antibody has a substantially stronger affinity for a polypeptide of the invention than its affinity for other closely related polypeptides in the prior art. As used herein, the term “antibody” refers not only to the complete molecule, but also to fragments thereof that are capable of binding to the antigenic determinant in question, such as Fab, F (ab ′) ₂ and Fv. Accordingly, such antibodies bind to the polypeptide of the first aspect of the invention.
By “substantially strong affinity” is meant that there is a measurable increase in affinity for a polypeptide of the invention compared to the affinity for a known cell surface receptor.
Preferably, the affinity for a polypeptide of the invention is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ than the affinity for a known IFNγ-like polypeptide. Double, 10 ⁶ times or more strong.
If a polyclonal antibody is desired, the selected mammal (eg, mouse, rabbit, goat or horse) can be immunized with the polypeptide of the first aspect of the invention. Polypeptides used to immunize animals may be derived by recombinant DNA technology or may be chemically synthesized. If desired, the polypeptide can be conjugated to a carrier protein. Commonly used carriers that can be chemically conjugated to the polypeptide include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The carrier-bound polypeptide is then used to immunize the animal. Serum is collected from the immunized animal and processed according to known methods (eg, immunoaffinity chromatography).

Monoclonal antibodies against the polypeptide of the first aspect of the invention can also be readily generated by those skilled in the art. The general methodology for producing monoclonal antibodies using hybridoma technology is well known (see, eg, G. Kohler & C. Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., 77-96 “Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss, Inc. (1985)).
Panels of monoclonal antibodies generated against the polypeptide of the first aspect of the invention can be screened for various properties, such as isotype, epitope, affinity and the like. Monoclonal antibodies are particularly useful for the purification of the individual polypeptides from which they are made. Alternatively, the gene encoding the monoclonal antibody of interest can be isolated from the hybridoma, eg, by PCR techniques known in the art, and further cloned and expressed in an appropriate vector.
Also available are chimeric antibodies in which a non-human variable region is linked or fused to a human constant region (eg see Liu et al., Proc. Natl. Acad. Sci. USA, 84: 3439 (1987)). Can be useful.

Antibodies can be modified to reduce immunogenicity in an individual, eg, by humanization (see, eg, Jones et al., Nature, 321: 522 (1986); Verhoeyen et al., Science, 239: 1534 (1988); Kabat et al., J. Immunol., 147: 1709 (1991); Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029 (1989); al., Proc. Natl. Acad. Sci. USA, 88: 34181 (1991); Hodgson et al., Bio / Technology 9: 421 (1991)). As used herein, the term “humanized antibody” refers to CDR amino acids and other selected amino acids in the heavy and / or light chain variable domains of a non-human donor antibody substituted for the equivalent amino acids of a human antibody. Refers to an antibody molecule that has been Thus, a humanized antibody is very similar to a human antibody but has the binding ability of a donor antibody.
In another alternative, the antibody may be a “bispecific” antibody that has two different antigen binding domains, each of which is directed to a different epitope.
Using phage display technology, a gene encoding an antibody having binding activity to the polypeptide of the present invention is subjected to PCR amplified V-gene repertoire of human-derived lymphocytes screened for possession of the relevant antibody, or unrepresented. It can be selected from any of the sensitization libraries (J. McCafferty et al., (1990) Nature 348: 552-554; J. Marks et al., (1992) Biotechnology 10: 779-783). The affinity of the antibody can also be improved by chain shuffling (T. Clackson et al., (1991) Nature 352: 624-628).
Antibodies produced by the above techniques, whether polyclonal or monoclonal, have additional utility in that they can be used as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). Have. In these applications, these antibodies can be labeled with analytically detectable reagents (eg, radioisotopes, fluorescent molecules or enzymes).

Preferred nucleic acid molecules of the second and third aspects of the invention encode the polypeptide sequence set forth in SEQ ID NO: 2 and a functionally equivalent polypeptide. These nucleic acid molecules can be used in the methods and applications described herein. The nucleic acid molecules of the present invention preferably comprise at least n contiguous nucleotides derived from the sequences disclosed herein, where n is 10 or greater depending on the particular sequence (eg 12 , 14, 15, 18, 20, 25, 30, 35, 40 or greater).
The nucleic acid molecules of the invention also include sequences that are complementary to the nucleic acid molecules described above (eg, for antisense or probe purposes).
The nucleic acid molecules of the present invention may be in the form of RNA (eg, mRNA), or DNA (eg, including cDNA, synthetic DNA or genomic DNA). Such nucleic acid molecules can be obtained by cloning, by chemical synthesis, or a combination thereof. The nucleic acid molecule can be prepared by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries, or by separation from organisms. RNA molecules can generally be made by in vitro or in vivo transcription of a DNA sequence.
The nucleic acid molecule may be double stranded or single stranded. Single-stranded DNA may be the coding strand (also known as the sense strand) or the non-coding strand (also referred to as the antisense strand).
The term “nucleic acid molecule” also includes analogs of DNA and RNA (eg, those containing a modified backbone), and peptide nucleic acids (PNA). As used herein, the term “PNA” refers to an antisense molecule or an anti-gene agent and is an oligonucleotide that is at least 5 nucleotides in length and attached to a peptide backbone of amino acid residues. including. The peptide backbone is preferably terminated with lysine, and the terminal lysine confers solubility to the composition. PNA may be PEGylated to extend the lifetime in the cell {in the cell, PNA preferentially binds to complementary single-stranded DNA and RNA to stop transcript elongation. (PE Nielsen et al. (1993) Anticancer Drug Des. 8: 53-63)}.

The nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 can be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO: 1. These molecules may also have sequences that differ from the sequence encoding SEQ ID NO: 2 as a result of the degeneracy of the genetic code. Such nucleic acid molecules include a coding sequence for the mature polypeptide itself; a coding sequence for the mature polypeptide and an additional coding sequence (eg, encoding a leader or secretory sequence), eg, pro-, pre-, or pre-pro -Coding for a polypeptide sequence; maturation with or without additional coding sequences as described above and with additional non-coding sequences (including non-coding 5 'and 3' sequences) A coding sequence for a polypeptide is included, but is not limited to. The non-coding 5 ′ and 3 ′ sequences are, for example, transcribed untranslated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability. The nucleic acid molecule can also include additional sequences that encode additional amino acids, such as amino acids that provide additional functionality.
The nucleic acid molecules of the second and third aspects of the invention may also encode functional equivalents of the polypeptides and fragments of the first aspect of the invention and fragments thereof. Such a nucleic acid molecule may be a naturally occurring variant (eg, a naturally occurring allelic variant) or the molecule may be a variant that is not known to occur naturally. Variants of non-naturally occurring nucleic acid molecules as described above can be achieved by mutagenesis techniques, including techniques applied to nucleic acid molecules, cells or organisms.

Among such variants are those that differ from the aforementioned nucleic acid molecules, particularly by nucleotide substitutions, deletions or insertions. Substitutions, deletions or insertions can involve one or more nucleotides. Variants may vary in the coding region or the non-coding region or both. Changes in the coding region can generate conservative or non-conservative amino acid substitutions, deletions or insertions.
The nucleic acid molecules of the present invention may also be manipulated using methods generally known in the art for a variety of reasons including cloning, processing and processing of gene products (polypeptides). And / or alteration of expression. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are among the techniques that can be used to manipulate nucleotide sequences. Site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, generate splicing variants, introduce mutations, and the like.

The nucleic acid molecule encoding the polypeptide of the first aspect of the invention may be linked to a heterologous sequence such that the binding nucleic acid molecule encodes a fusion protein. Such binding nucleic acid molecules are included in the second or third aspect of the present invention. For example, to screen a peptide library for inhibitors of the activity of the polypeptide of the present invention, it is useful to express a fusion protein that can be recognized by a commercially available antibody using a binding nucleic acid molecule as described above. possible. The fusion protein may also be engineered to contain a cleavage site located between the polypeptide sequence of the invention and the heterologous protein sequence, so that the polypeptide can be purified away from the heterologous protein. Good.
Nucleic acid molecules of the invention also include antisense molecules that are partially complementary to a nucleic acid molecule encoding a polypeptide of the invention and thus hybridize to the encoding nucleic acid molecule (hybridization). Such antisense molecules (eg oligonucleotides) recognize the target nucleic acid encoding the polypeptide of the invention and specifically bind to and transcribe it, as is well known to those skilled in the art. Can be designed to prevent (see for example JS Cohen, Trends in Pharm. Sci., 10: 435 (1989); J. Okano, Neurochem. 56: 560 (1991); O 'Connor, Neurochem. 56: 560 (1991); Lee et al., Nucleic Acids Res. 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); Dervan et al., Science 251: 1360 (1991)).

As used herein, the term “hybridization” refers to the association of two nucleic acid molecules with each other by hydrogen bonding. Typically, one molecule will be immobilized on a solid support and the other will be free in solution. The two molecules can then be contacted with each other under conditions suitable for hydrogen bonding. Factors affecting the binding include: solvent type and volume; reaction temperature; hybridization time; agitation; agents that interfere with non-specific binding of liquid phase molecules to the solid support (Denhardt's reagent) See also: Concentration of molecules; use of compounds that increase the rate of binding of molecules (dextran sulfate or polyethylene glycol); and stringency of wash conditions following hybridization (Sambrook et al., Supra). ).
Inhibition of hybridization between a completely complementary molecule and a target molecule can be examined using hybridization assays known to those skilled in the art (see, eg, Sambrook et al. (Supra)). Thus, substantially homologous molecules, as taught in the literature (GM Wahl and SL Berger, 1987, Methods Enzymol. 152: 399-407; AR Kimmel, 1987, Methods Enzymol. 152: 507-511), It will compete and inhibit the binding of fully homologous molecules to target molecules under various stringency conditions.
“Stringency” refers to conditions for a hybridization reaction that are suitable for the association of molecules that are much more similar than the association of different molecules. High stringency hybridization conditions include a solution (50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate and 20 μg / mL denatured sheared salmon sperm DNA) is defined as overnight incubation at 42 ° C., followed by washing of the filter in 0.1 × SSC at about 65 ° C. Low stringency conditions include hybridization reactions performed at 35 ° C. (see Sambrook et al. (Supra)). Preferably, the conditions used for hybridization are high stringency conditions.

Preferred embodiments of this aspect of the invention include nucleic acid molecules that are at least 70% identical over the entire length of the nucleic acid molecule encoding an INSP037 polypeptide (SEQ ID NO: 2), and nucleic acid molecules that are substantially complementary to such nucleic acid molecules It is. Preferably, the nucleic acid molecule of this aspect of the invention comprises a nucleic acid molecule that is at least 80% identical or complementary thereto over the entire length of the nucleic acid molecule produced by SEQ ID NO: 1. In this regard, nucleic acid molecules that are at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over the entire length of such a nucleic acid sequence are particularly preferred. A preferred embodiment of this feature is a nucleic acid molecule that encodes a polypeptide that substantially retains the same biological function or activity as the INSP037 polypeptide.
The present invention also provides a method for detecting a nucleic acid molecule of the present invention comprising the following steps: (a) a nucleic acid probe of the present invention and a biological sample under hybridization conditions forming a duplex; And (b) detecting all of the formed duplexes.

As discussed further below in connection with assays that can be utilized in accordance with the present invention, full-length cDNAs and genomic clones encoding INSP037 polypeptides using the nucleic acid molecules described above as hybridization probes for RNA, cDNA, or genomic DNA. And a cDNA or genomic clone of a homologous or orthologous gene having a high sequence similarity to the gene encoding INSP037 polypeptide can be isolated.
In this regard, among other techniques known in the art, the following techniques in particular can be utilized. These techniques are discussed below by way of example. Methods for DNA sequencing and analysis are well known and generally available in the art and can be used in practice to practice many of the aspects of the invention discussed herein. . Such methods include Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp., Cleaveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or polymerase and proofreading exonuclease. Such as those found in the ELONGASE amplification system commercially available (Gibco / BRL, Gaithersburg, MD). Preferably, the sequencing process includes, for example, Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler PTC200 (MJ Research, Watertown, MA), ABI Catalyst, and 373 and 377 DNA It can be automated using equipment such as a sequencer (Perkin Elmer).

  One method of isolating a nucleic acid molecule encoding a polypeptide having a function equivalent to that of an INSP037 polypeptide is by using standard techniques known in the art, using natural probes or artificially. Searching genomic or cDNA libraries with designed probes (see, eg, “Current Protocols in Molecular Biology”, Ausubel et al. (Eds). Greene Publishing Association and John Wiley Interscience , New York, 1989, 1992). A particularly useful probe corresponds to a nucleic acid sequence derived from a suitable coding gene (SEQ ID NO: 1) or is complementary to said sequence and is at least 15, preferably at least 30, more preferably at least 50 consecutive It is a probe containing the base to do. Such probes can be labeled with analytically detectable reagents to facilitate identification of the probes. Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes, and enzymes that can catalyze the formation of detectable products. Using these probes, one of skill in the art can isolate a complementary copy of a genomic DNA, cDNA or RNA polynucleotide encoding the protein of interest from a human, mammalian or other animal source and use related sequences such as those described above. The source could be screened for additional members belonging to the same family, type and / or subtype.

  In many cases, the isolated cDNA sequence will be incomplete and the polypeptide coding region will be truncated (usually at the 5 'end). Several methods are available to obtain full length cDNAs or to extend short cDNAs. Such sequences can be extended using partial nucleotide sequences and various methods known in the art for detecting upstream sequences (eg, promoters and regulatory elements). For example, one method that can be used is based on the cDNA end rapid amplification method (RACE; see, eg, Frohman et al., Proc. Natl. Acad. Sci. USA (1988) 85: 8998-9002). Recent modifications of the technology (eg, exemplified by Marathon ™ technology (Clontech Laboratories Inc.)) have significantly simplified the search for longer cDNAs. A slightly different technique called “restriction site” PCR uses universal primers to search for unknown nucleic acid sequences in close proximity to known loci (G. Sarkar (1993) PCR Methods Applic. 2: 318-322). Inverse PCR can also be used to amplify or extend sequences using various primers based on known regions (T. Triglia et al. (1988) Nucleic Acids Res. 16: 8186). Another method that can be used is capture PCR, which involves PCR amplification of DNA fragments in close proximity to known sequences in human and yeast artificial chromosome DNA (M. Lagerstrom et al. (1991) PCR Methods Applic 1: 111-119). Another method that can be used to search for unknown sequences is that of Parker (J.D. Parker et al. (1991) Nucleic Acids Res. 19: 3055-3060). Furthermore, PCR, nested primers, and PromoterFinder ™ library (Clontech, Palo Alto, Calif.) May be used to examine genomic DNA by moving little by little. This method does not require library screening and is useful for finding intron / exon junctions.

When screening full-length cDNAs, it is preferable to use a library that is size-selected to include larger cDNAs. Furthermore, a random-primed library is preferred in that it contains more sequences containing the 5 ′ region of the gene. The use of a random primed library may be particularly preferred in situations where an oligo d (T) library cannot produce a full length cDNA. Genomic libraries can be useful for extending sequences into 5 ′ non-transcribed regulatory regions.
In one embodiment of the present invention, the nucleic acid molecule of the present invention can be used for localization on a chromosome. In this technique, a nucleic acid molecule can be specifically targeted to a specific location on an individual human chromosome and hybridized to a specific location on an individual human chromosome. Mapping of the related sequence of the present invention onto the chromosome is an important step in confirming the correlation of sequences related to gene-related diseases. Once the sequence has been mapped to the exact location of the chromosome, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data can be found, for example, at: V. McKusick, Mendelian Inheritance in Man (available online through Jones Hopkins University, Welch Medical Library). The relationship between the gene mapped to the same chromosomal region and the disease is then identified by linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to researchers searching for disease genes using positional cloning or other gene discovery techniques. Once the location of a disease or syndrome is roughly localized in a particular genomic region by genetic linkage, any sequence mapped to that region can be a related or regulatory gene for further analysis. The nucleic acid molecules can also be used to detect chromosomal location differences due to translocations, inversions, etc. between normal individuals, carrier individuals or affected individuals.

The nucleic acid molecules of the present invention are also valuable for tissue localisation. Such a technique allows the determination of the expression pattern of the polypeptide in the tissue by detection of mRNA encoding the polypeptide. These techniques include in situ hybridization techniques and nucleotide amplification techniques (eg, PCR). The results obtained from these studies suggest the normal function of the polypeptide in the organism. In addition, comparative studies of the expression patterns of mRNA encoded by mutant genes and normal mRNA expression patterns provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may have temporal, positional or quantitative properties.
A gene silencing approach can also be performed to downregulate the endogenous expression of the gene encoding the polypeptide of the invention. RNA interference (RNAi) (SM Elbashir et al. Nature 2001, 411, 494-498) is one method for sequence-specific post-transcriptional gene silencing that can be used. Short dsRNA oligonucleotides are synthesized in vitro and introduced into cells. Sequence specific binding of these dsRNAs initiates target mRNA degradation, reducing or inhibiting target protein expression.
The effectiveness of gene silencing as described above can be assessed by measuring polypeptide expression (eg, by Western blotting) or measuring RNA levels using the TaqMan method.

The vector of the present invention contains the nucleic acid molecule of the present invention, and may be a cloning vector or an expression vector. The host cells of the invention that can be transformed, transfected or transduced with the vectors of the invention can be prokaryotic or eukaryotic cells.
The polypeptide of the present invention can be prepared in a recombinant form by expression of a nucleic acid molecule encoding the polypeptide in a vector contained in a host cell. Such expression methods are well known to those skilled in the art and many are described in more detail in the following literature: Sambrook et al. (Supra) and Fernandez & Hoeffler (1998, eds. “Gene expression”). Using nature for the art of expression ”, Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).

In general, any system or vector suitable for the maintenance, propagation or expression of nucleic acid molecules can be used to produce the polypeptide in the required host. Appropriate nucleotide sequences can be inserted into the expression system by any of a variety of techniques that are well known and routine (eg, as described in Sambrook et al., Supra). In general, a coding gene is placed under the control of control elements (eg, promoters, ribosome binding sites (for bacterial expression), and, in some cases, operators), thereby placing a DNA sequence that encodes the desired polypeptide. It can be transcribed into RNA in transformed host cells.
Examples of suitable expression systems include, for example, chromosomal, episomal and viral-derived systems such as vectors derived from: bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, For example, baculovirus, papovavirus (eg SV40), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, or combinations of the above, eg those derived from plasmid and bacteriophage genetic elements (eg cosmids and phagemids) Including). Human artificial chromosomes (HACs) can also be used to carry larger DNA fragments than are contained and expressed in plasmids.

Particularly suitable expression systems include infection with a microorganism (eg, a bacterium) transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vector; a yeast transformed with a yeast expression vector; a viral expression vector (eg, baculovirus). Insect cell lines; plant cell lines transformed with viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg, Ti or pBR322 plasmid); or animal cell lines. Cell-free translation systems can also be used to produce the polypeptides of the invention.
Introduction of nucleic acid molecules encoding the polypeptides of the present invention into host cells has been described in many standard laboratory manuals (eg, Davis et al., Basic Methods in Molecular Biology (1986) and supra (Sambrook et al. )). Particularly suitable methods include calcium phosphate transfection, DEAE dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or Infections are included (see: Sambrook et al. (1989) supra; Ausubel et al. (1991) supra; Spector, Goldman & Leinwald, (1998)). In eukaryotic cells, the expression system can be transient (eg, episomal) or permanent (chromosomal integration) depending on the requirements of the system.

The encoding nucleic acid molecule includes a sequence that encodes a regulatory sequence, such as a signal peptide or leader sequence, for example, for secretion of the translated polypeptide into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment, if desired. It may or may not be included. These signals may be endogenous to the polypeptide or they may be heterologous signals. The leader sequence can be removed by the bacterial host in post-translational processing.
In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are sequences that increase or decrease gene expression in response to chemical or physical stimuli (including the presence of regulatory compounds) or various temperature or metabolic conditions. Regulatory sequences are the untranslated regions of the vector, such as enhancers, promoters and 5 ′ and 3 ′ untranslated regions. They interact with host cell proteins to perform transcription and translation. Such regulatory sequences can change their strength and specificity. Many suitable transcription and translation elements (including constitutive and inducible promoters) can be used, depending on the vector system and host utilized. For example, when cloning in a bacterial system, an inducible promoter, such as a hybrid lacZ promoter such as Bluescript phagemid (Stratagene, La Jolla, CA) or pSportl ™ plasmid (Gibco BRL) can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from plant cell genomes (eg heat shock, RUBISCO and storage protein genes) or promoters or enhancers derived from plant viruses (eg viral promoters or leader sequences) can be cloned into vectors. In mammalian cell systems, promoters derived from mammalian genes or from mammalian viruses are preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Expression vectors are constructed so that specific nucleic acid coding sequences can be placed in the vector along with appropriate regulatory sequences. The position and orientation of the coding sequence with respect to the regulatory sequence is such that the coding sequence is transcribed under “control” of the regulatory sequence (ie, the RNA polymerase that binds to the DNA molecule at the regulatory sequence is Transcription of the coding sequence). In some cases, it may be necessary to modify the sequence so that it can be attached to the control sequence in the proper orientation (ie, to maintain the reading frame).
Control sequences and other regulatory sequences can be linked to the nucleic acid coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and appropriate restriction sites.
Stable expression is preferred for long-term, high-yield production of recombinant polypeptides. For example, a cell line that stably expresses the polypeptide of interest can be transformed with an expression vector that contains the viral origin of replication and / or endogenous expression elements and a selectable marker gene on the same or separate vectors. . Following the introduction of the vector, the cells can be grown in enriched media for 1-2 days before switching to selective media. The purpose of the selectable marker is to confer resistance to selection, and the presence of the selectable marker allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Mammalian cell lines that can be used as hosts for expression are known in the art and include many immortal cell lines available from the American Type Culture Collection (ATCC). Such cell lines include, for example, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, Bowes. ) But not limited to melanoma cells and human hepatocellular carcinoma (eg, HepG2) cells and numerous other cell lines.
In the baculovirus system, materials for baculovirus / insect cell expression systems are commercially available, particularly in kit form from Invitrogen, San Diego, Calif. (“MaxBac” kit). Such techniques are generally known to those skilled in the art and are fully described in the literature (Summers & Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987)). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 cells and Spodoptera Sf9 cells.
There are many plant cell culture and whole plant gene expression systems known in the art. Examples of suitable plant cell gene expression systems include those described in US Pat. Nos. 5,693,506, 5,659,122 and 5,608,143. Further examples of gene expression in plant cell culture have been described in the literature (Zenk (1991) Phytochemistry 30: 3861-3863).
In particular, any plant that can isolate a protoplast and culture it to form a fully regenerated plant can be utilized, whereby the complete plant containing the transgene can be recovered. In particular, all plants, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, can be regenerated from cultured cells or tissues. .

Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
Examples of particularly suitable host cells for fungal expression include yeast cells (eg, S. cerevisiae) and Aspergillus cells.
Many selection systems that can be used to recover transformed cell lines are known in the art. Examples include herpes simplex virus thymidine kinase (M. Wigler et al. (1977) Cell 11: 223-32) and adenine phosphoribosyltransferase (I. Lowy et al. (1980) Cell 22: 817-23. ), Which can be used in tk− or aprt ± cells, respectively.
Furthermore, antimetabolite resistance, antibiotic resistance or herbicide resistance may be used as selection criteria. For example, dihydrofolate reductase (DHFR) confers resistance to methotrexate (M. Wigler et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-70), npt is resistant to aminoglycoside neomycin and G-418 (F. Colbere-Garapin et al. (1981) J. Mol. Biol. 150: 1-14), and als or pat are for chlorsulfuron and phosphinotricin acetyltransferase, respectively. Gives resistance. Additional selectable genes have been reported and examples of them will be apparent to those skilled in the art.

The presence or absence of marker gene expression suggests that the gene of interest is also present, but it may be necessary to confirm the presence and expression of the gene of interest. For example, if the relevant sequence is inserted within the marker gene sequence, the absence of marker gene function can identify transformed cells containing the appropriate sequence. Alternatively, a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Usually, the expression of a marker gene in response to induction or selection also indicates the expression of a tandem gene.
Alternatively, host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and that express the polypeptide can be identified by a variety of techniques known to those of skill in the art. Such techniques include DNA-DNA or DNA-RNA hybridization and protein bioassays, such as fluorescence activated cell sorting (FACS) or immunoassay techniques (eg, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)). Including, but not limited to, membrane, solution or chip based techniques for the detection and / or quantification of nucleic acids or proteins (see, eg, R. Hampton et al. (1990 ) Serological Methods, A Laboratory Manual, APS Press, St Paul, MN; and DE Maddox et al. (1983) J. Exp. Med. 158: 1211-1216).

A variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. As a means for producing a labeled hybridization probe or PCR probe for detecting a sequence closely related to a nucleic acid molecule encoding the polypeptide of the present invention, an oligo-label, nick translation, end label or PCR using a labeled polynucleotide is used. Amplification is included. Alternatively, a sequence encoding the polypeptide of the present invention can be cloned into a vector to produce an mRNA probe. Such vectors are known in the art and are commercially available and synthesize RNA probes in vitro by adding appropriate RNA polymerase (eg, T7, T3 or SP6) and labeled nucleotides. Can be used for These procedures can be performed using various commercially available kits (Pharmacia & Upjohn (Kalamazoo, MI); Promega (Madison, Wis.); US Biochemical Corp., (Cleaveland, Ohio)). .
Suitable reporter molecules or labels that can be used to facilitate detection include radionuclides, enzymes and fluorescence, chemiluminescent or chromogenic substances, substrates, cofactors, inhibitors, magnetic particles, and the like.

The nucleic acid molecules of the present invention can also be used to create transgenic animals (particularly rodents). Such transgenic animals constitute another feature of the present invention. Such production can be carried out locally by somatic modification or by germline therapy that introduces a genetic modification. Such transgenic animals may be particularly useful for creating animal models for drug molecules that are effective as modulators of the polypeptides of the present invention.
Polypeptides can be recovered and purified from recombinant cell cultures by well-known methods. Said known methods include ammonium sulfate or ethanol precipitation, acidic extraction, anion or cation exchange chromatography, phosphorylated cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. included. High performance liquid chromatography is particularly useful for purification. If the polypeptide is denatured during isolation and purification, the active conformation can be regenerated using techniques well known for protein refolding.

  If desired, specialized vector constructs by linking a sequence encoding a polypeptide of the present invention to a nucleotide sequence encoding a polypeptide domain that facilitates purification of the soluble protein may also facilitate protein purification. Can be used to Examples of such purification facilitating domains include metal chelate peptides (eg, histidine-tryptophan module that allows purification on immobilized metal, protein A domain that allows purification on immobilized immunoglobulin, and FLAGS An elongation / affinity purification system (domain used in Immunex Corp., Seattle, WA) is included. A cleavable linker sequence (eg, specific for factor XA or enterokinase (Invitrogen, San Diego, Calif.)) Is included between the purification domain and the polypeptide of the present invention to facilitate purification. It may be used. One such expression vector provides for the expression of a fusion protein comprising a polypeptide of the invention fused to several histidine residues preceding a thioredoxin or enterokinase cleavage site. Histidine residues facilitate purification by IMAC (fixed metal ion affinity chromatography; J. Porath et al. (1992) Prot. Exp. Purif. 3: 263-281), while thioredoxin or enterokinase cleavage sites Providing means for purifying the polypeptide from the fusion protein. A discussion of vectors containing fusion proteins is provided below: D.J. Kroll et al. (1993) DNA Cell Biol. 12: 441-453).

When expressing a polypeptide for use in a screening assay, it is generally preferred that the polypeptide is secreted into the medium of a host cell that expresses the polypeptide. In this case, the polypeptides of the invention can also be collected using standard protein purification techniques such as gel filtration chromatography, ion exchange chromatography or affinity chromatography prior to use in screening assays. . Examples of suitable methods of protein purification are provided in the examples herein. If the polypeptide is produced intracellularly, the cell must first be lysed before the polypeptide can be recovered.
Alternatively, the polypeptide of the present invention may be preferably expressed as a cell surface fusion protein. In this case, prior to use in the screening assay, the host cells can be harvested using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques.
The polypeptides of the invention can be used to screen compound libraries with any of a variety of drug screening techniques. Such compounds can activate (agonist action) or inhibit (antagonistic action) the gene expression level or activity level of the polypeptides of the invention and form a further feature of the invention. Preferred compounds are effective in altering the expression of the natural gene encoding the polypeptide of the first aspect of the invention or modulating the activity of the polypeptide of the first aspect of the invention. It is valid.
Agonist or antagonist compounds can be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors, or structural or functional mimetics. For a suitable overview of such screening techniques, see: Coligan et al. (1991) Current Protocols in Immunology 1 (2): Chapter 5.

A compound that is likely to be a good antagonist is a molecule that binds to the polypeptide of the invention and does not elicit the biological action of the polypeptide when bound. Potent antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish the activity of the polypeptide of the invention. In such a manner, binding of the polypeptide to normal cellular binding molecules can be inhibited, so that normal biological activity of the polypeptide can be inhibited.
The polypeptide of the present invention used in such screening techniques may be free in solution, immobilized on a solid support, retained on the cell surface, or located within the cell. It may be. In general, such screening methods include using appropriate cells or cell membranes that express the polypeptide, wherein the cells or cell membranes are contacted with a test compound to stimulate binding or functional response or Observe inhibition. The functional response of cells contacted with the test compound is then compared to control cells not contacted with the test compound. Such an assay can be used to assess whether a test compound provides a signal resulting from activation of the polypeptide using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect of activation by the agonist in the presence of the test compound is observed.

A preferred method for identifying a ligand of an IFNγ-like polypeptide of the invention comprises the following steps:
(a) Detection in response to binding of a polypeptide of the invention to a putative binding partner (or in connection with a second component that can provide a detectable signal in response to binding of a polypeptide of the invention) Cells expressing a putative binding partner of an IFN-like polypeptide of the invention that can provide a possible signal on the cell surface are screened under conditions that allow binding to the putative binding partner. Contacting with a polypeptide of the invention; and
(b) by measuring the level of signal resulting from the interaction of the polypeptide of the invention with a putative binding partner, the polypeptide of the invention binds to the putative binding partner and its putative binding Determining whether to activate or inhibit the partner.
A further preferred method for identifying a ligand of an IFNγ-like polypeptide of the invention comprises the following steps:
(a) Detection in response to binding of a polypeptide of the present invention to a putative binding partner (or associated with a second component that can provide a detectable signal in response to binding of a polypeptide of the present invention). Cells expressing a putative binding partner of an IFNγ-like polypeptide of the invention that is capable of providing a possible signal on the cell surface under conditions that allow binding to the putative binding partner of the invention. Contacting with the polypeptide; and
(b) by comparing the level of signal resulting from the interaction of the polypeptide of the invention with a putative binding partner to the level of signal in the absence of the polypeptide of the invention, Determining whether to bind to a putative binding partner and to activate or inhibit the putative binding partner.

In further preferred embodiments, the general methods described above may further comprise performing agonist or antagonist identification in the presence of labeled or unlabeled INSP037 polypeptide.
In another embodiment of the method of identifying an agonist or antagonist of a polypeptide of the invention: a cell expressing a ligand on its surface in the presence of a candidate compound, or in a condition that allows the polypeptide to bind to the ligand or Determining inhibition of binding of a polypeptide of the invention to a cell membrane comprising said ligand; and determining the amount of polypeptide bound to the ligand. A compound that can reduce the binding of a polypeptide of the invention is considered to be an agonist or antagonist. The polypeptide of the present invention is preferably labeled.
More particularly, the method of screening for agonist or antagonist compounds comprises the following steps:
(a) incubating the labeled polypeptide of the present invention and the whole cell expressing the ligand of the present invention on the cell surface or a cell membrane containing the ligand of the present invention;
(b) measuring the amount of the labeled polypeptide bound to the whole cell or to the cell membrane;
(c) adding a candidate compound to the mixture of the labeled polypeptide of step (a) and the whole cell or cell membrane, and allowing the mixture to reach an equilibrium state;
(d) measuring the amount of labeled polypeptide bound to the whole cell membrane or cell membrane after step (c); and
(e) comparing the difference between the labeled polypeptide bound in step (b) and the labeled polypeptide bound in step (d);
A compound that decreases binding in step (d) is considered to be an agonist or antagonist.

The polypeptide will be found to modulate a variety of physiological and pathological processes in a dose dependent manner in the above assay. Thus, a “functional equivalent” of the present invention includes a polypeptide that exhibits any of the same regulatory activities in a dose dependent manner in the above assay. The degree of dose-dependent activity need not be the same as that of the polypeptide of the invention, but preferably said “functional equivalent” is substantially compared to the polypeptide of the invention in a given activity assay. It will show similar dose dependence.
Alternatively, a simple binding assay may be used. In this case, the adhesion of the test compound to the polypeptide-retaining surface is detected by a labeling means coupled directly or indirectly to the test compound, or detected in an assay involving competition with a labeled competitor. In another embodiment, a competitive drug screening assay can be used. In this case, neutralizing antibodies that can specifically bind to the polypeptide compete with the test compound for binding. In this way, the presence of any test compound possessing specific binding affinity for the polypeptide can be detected using the antibody.

An assay can also be designed to detect the effect of added test compounds on the intracellular production of mRNA encoding the polypeptide. For example, an ELISA can be constructed that measures the secretion level or cell binding level of a polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and is appropriately manipulated using said ELISA. For compounds that can inhibit or enhance polypeptide production from isolated cells or tissues. Subsequently, the formation of a binding complex between the polypeptide and the test compound can be measured.
Assay methods within the terminology of the present invention also include those that require the use of the genes and polypeptides of the present invention in overexpression assays or ablation assays. Said assay involves the manipulation of the intracellular level of these genes / polypeptides and the assessment of the effect of this manipulation event on the physiology of said manipulated cells. For example, such experiments reveal details of signaling and metabolic pathways involving specific genes / polypeptides and provide information about the identity of the polypeptide with which the polypeptide under study interacts. In addition, clues about how to regulate related genes and proteins are provided.

Another drug screening technique that can be used provides a rapid high-throughput screening of compounds with appropriate binding affinity for the polypeptide of interest (see International Patent Application WO 84/03564). In said method, a large number of different small test compounds can be synthesized on a solid support and then reacted with a polypeptide of the invention and washed. One way to immobilize polypeptides is to use non-neutralizing antibodies. Subsequently, the bound polypeptide can be detected using methods well known in the art. The purified polypeptide can also be coated directly onto a plate for use in the aforementioned drug screening techniques.
The polypeptides of the present invention can be used to identify membrane bound or soluble receptors by standard receptor binding techniques known in the art. Such standard techniques are, for example, ligand binding assays and cross-linking assays, in which the polypeptide is labeled with a radioisotope, chemically modified, or easy to detect or purify. And is incubated with a putative receptor source (eg, cell composition, cell membrane, cell supernatant, tissue extract or body fluid). The effectiveness of binding can be measured using biophysical techniques such as surface plasmon resonance (supplied by Biacore AB, Uppsala, Sweden) and spectroscopy. Binding assays can be used for receptor purification and cloning, but can also be used to identify agonists and antagonists of the polypeptide that compete for binding of the polypeptide to its receptor. Standard methods for performing screening assays are well understood in the art.

The invention also includes screening kits useful for the methods of identifying agonists, antagonists, ligands, receptors, substrates, enzymes, described above.
The present invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds that are discovered by the methods described above and modulate the activity or antigenicity of the polypeptides of the invention.
The present invention also provides a pharmaceutical composition comprising a polypeptide, nucleic acid, ligand or compound of the present invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as described in detail below.
In accordance with the terminology used herein, a composition comprising a polypeptide, nucleic acid, ligand or compound {X} is an impurity (when X is at least 85% by weight of the sum of X + Y in the composition). As used herein, Y) is “substantially free”. Preferably, X comprises at least about 90%, more preferably at least about 95%, 98% or 99% by weight of the sum of X + Y in the composition.

The pharmaceutical composition should preferably contain a therapeutically effective amount of a polypeptide, nucleic acid molecule, ligand or compound of the invention. As used herein, the term “therapeutically effective amount” refers to a therapeutic agent necessary to treat, alleviate or prevent a target disease or condition, or to exhibit a detectable therapeutic or prophylactic effect. Refers to the quantity. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays (eg, neoplastic cell culture assays) or in animal models (usually mice, rabbits, dogs, or pigs). . The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
The exact effective amount for a human subject is the severity of the disease state, the subject's general health, the subject's age, weight and sex, diet, time and frequency of administration, concomitant medications, response sensitivity and treatment. Will depend on tolerance / responsiveness. This amount can be determined by routine examination and is within the judgment of the clinician. In general, an effective dose will be from 0.01 mg / kg to 50 mg / kg, preferably from 0.05 mg / kg to 10 mg / kg. The composition may be administered individually to the patient or may be administered together with other drugs, pharmaceuticals or hormones.

The pharmaceutical composition can also include a pharmaceutically acceptable carrier for administration of the therapeutic agent. Such carriers include antibodies and other polypeptides, genes, and other therapeutic agents (eg, liposomes) provided that the carrier itself produces antibodies that are harmful to the individual receiving the composition. Provided that it can be administered without inducing and causing adverse toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers and inactive virus particles.
Pharmaceutical compositions include pharmaceutically acceptable salts, such as mineral salts (such as hydrochloride, hydrobromide, phosphate, sulfate, etc.); and salts of organic acids (acetate, propionate) , Such as malonate, benzoate, etc.). A thorough discussion of pharmaceutically acceptable carriers is available in the following text: Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).
Pharmaceutically acceptable carriers in therapeutic compositions can further include liquids such as water, saline, glycerol and ethanol. In addition, auxiliaries such as wetting agents, emulsifying agents, pH buffering substances and the like may be present in the composition. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. so that the patient can take them.

Once formulated, the compositions of the invention can be administered directly to the subject. The subject to be treated can be an animal, especially a human subject.
The pharmaceutical compositions used in the present invention are available in a number of routes (oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intraventricular, percutaneous applications (see eg WO98 / 20734), Subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, intravaginal, or rectal means, but not limited to. Gene guns or hyposprays can also be used to administer the pharmaceutical composition of the present invention. Typically, the therapeutic composition can be prepared as an injectable material (either a liquid solution or a suspension). Solids suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.
Direct delivery of the composition will generally be achieved by injection subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered into the interstitial space of the tissue. The composition may also be administered to a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Several approaches are available when the activity of a polypeptide of the invention is excessive in a particular disease state. One approach involves administering to a subject an inhibitory compound (antagonist) as described above with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide. Inhibition of the function of the polypeptide is accomplished, for example, by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, thereby alleviating abnormal symptoms. Preferably, the antagonist is an antibody. Most preferably, such antibodies are chimeric and / or humanized antibodies that minimize immunogenicity as described above.

In another approach, soluble forms of the polypeptides that retain binding affinity for ligands, substrates, enzymes, receptors can be administered. Typically, the polypeptide can be administered in the form of fragments that retain the relevant portions.
In another approach, expression of the gene encoding the polypeptide is inhibited using expression blocking techniques such as the use of antisense nucleic acid molecules (such as those described above) that are generated internally or separately administered. be able to. Modification of gene expression can be achieved by complementary sequences or antisense molecules (DNA, RNA or PNA) to the regulatory, 5 'or regulatory regions (signal sequences, promoters, enhancers and introns) of the gene encoding the polypeptide. Can be achieved by designing. Similarly, inhibition can be achieved using a “triple helix” base-pairing methodology. Triple helix pairing is useful because it inhibits the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triple helix DNA have been described in the literature (JE Gee et al. (1994) In: BE Huber & BI Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). Complementary sequences or antisense molecules can be designed to block the translation of mRNA by preventing binding to the ribosome and thus preventing transcription. Such oligonucleotides may be administered or generated in situ by in vivo expression.

Furthermore, expression of the polypeptides of the present invention can be prevented by using ribozymes specific for the coding mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see, eg, N. Usman et al., Curr. Opin. Struct. Biol. (1996) 6 (4): 527-. 533). Synthetic ribozymes can be designed to specifically cleave mRNA at selected positions, thereby preventing translation of the mRNA into a functional polypeptide. Ribozymes can be synthesized using a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively, ribozymes may be synthesized using a non-natural backbone (eg 2′-O-methyl RNA) and protected from ribonuclease degradation and may contain modified bases.
RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include the addition of flanking sequences to the 5 ′ and / or 3 ′ ends of RNA molecules, or the use of phosphorothioates or 2′-O-methyls in place of phosphodiester bonds within the backbone of the molecule. However, it is not limited to these. This concept is inherited in the production of PNA and is not easily recognized by endogenous endonucleases as well as unconventional bases such as inosine, gueosine and butosine, and acetyl-, methyl-, thio -And can be extended to all of the PNA molecules by inclusion of similar modified forms of adenine, cytidine, guanine, thymine and uridine.

Several approaches are also available to treat abnormal conditions associated with underexpression of the polypeptides of the invention and their activity. One approach involves administering to the subject a therapeutically effective amount of a compound that activates the polypeptide (ie, an agonist described above) to alleviate the abnormal condition. Alternatively, a therapeutic amount of the polypeptide can be administered in combination with a suitable pharmaceutical carrier to restore the relevant polypeptide physiological balance.
Gene therapy can be used to cause endogenous production of the polypeptide by the relevant cells of the subject. Gene therapy is used to permanently treat improper production of the polypeptide by replacing the defective gene with a modified therapeutic gene.
The gene therapy of the present invention can be performed in vivo or ex vivo. Ex vivo gene therapy requires isolation and purification of patient cells, introduction of therapeutic genes, and introduction of genetically modified cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of patient cells.

  The therapeutic gene is typically “packaged” for administration to a patient. Gene delivery vehicles can be non-viral, such as liposomes, or replication-defective viruses such as adenoviruses described for example in KL Berkner (1992) Curr. Top. Microbiol. Immunol., 158: 39-66 or N Muzyczka (1992) Curr. Top. Microbiol. Immunol., 158: 97-129 and may be the adeno-associated virus (AAV) vector described in US Pat. No. 5,252,479. For example, a nucleic acid molecule encoding a polypeptide of the present invention can be engineered for expression in a replication defective retroviral vector. This expression construct can then be isolated and introduced into a packaged cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide. As a result, the packaged cells can produce infectious virus particles containing the gene of interest. These producer cells can be administered to a subject to manipulate the cells in vivo and to express the polypeptide in vivo (see: Gene Therapy and Other Molecular Genetic-based Therapeutic Approaches, Chapter 20 (and references cited therein), “Human Molecular Genetics” (1996) T. Strachan & AP Read, BIOS Scientific Publishers Ltd.).

Another approach is the administration of “naked DNA”, where the therapeutic gene is injected directly into the bloodstream or muscle tissue.
When the polypeptide or nucleic acid molecule of the present invention is a causative substance that causes a disease, the present invention provides the polypeptide or nucleic acid molecule that can be used as a vaccine for producing an antibody against the causative substance that causes the disease. .
The vaccines of the present invention may be prophylactic (ie prevent infection) or therapeutic (ie treat disease after infection). Such vaccines comprise an antigen, immunogen, polypeptide, protein or nucleic acid that confers immunity, usually in combination with a pharmaceutically acceptable carrier as described above. The carrier includes any carrier that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered. In addition, these carriers may function as immunostimulants (“adjuvants”). Furthermore, the antigen or immunogen may be combined with bacterial toxins (eg, diphtheria, tetanus, cholera, toxins derived from H. pyroli) and other pathogens.
Since polypeptides are degraded in the stomach, vaccines comprising polypeptides are preferably administered parenterally (eg, subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, and aqueous and non-aqueous sterile suspensions. The sterile injectable solution may contain an antioxidant, a buffer, a bacteriostatic agent, and a solute that makes the formulation isotonic with the blood of the recipient, the sterile suspension being a suspension or A thickener may be included.

The vaccine formulations of the present invention may be provided in unit dose or multi-unit dose containers. For example, sealed ampoules and vials can be stored in a lyophilized state requiring only the addition of a sterile liquid carrier just prior to use. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine testing.
For example, genetic delivery of antibodies that bind to the polypeptides of the invention, as described in International Patent Application WO 98/55607 may also be effective.
A technique called jet injection (see, eg, www.powderject.com) may also be useful for the formulation of vaccine compositions.
Some suitable methods and vaccine delivery systems for vaccination are described in International Patent Application WO 00/29428.
The invention also relates to the use of the nucleic acid molecules of the invention as diagnostic agents. Detection of a mutant form of a gene characterized by the nucleic acid molecule of the invention and associated with dysfunction is the diagnosis of a disease resulting from underexpression, overexpression or positional or temporal expression change of said gene, or such Diagnostic tools are provided that can define or add to a diagnosis of susceptibility to a disease. Individuals carrying mutations in the gene can be detected at the DNA level by various techniques.
Nucleic acid molecules for diagnosis can be obtained from a subject's cells, such as blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection, or genomic DNA may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA) or other amplification techniques prior to analysis. (See, eg, Saiki et al., Nature 324: 163-166 (1986); Bej et al., Crit. Rev. Biochem. Molec. Biol., 26: 301-334 (1991). Birkenmeyer et al., J. Virol. Meth., 35: 117-126 (1991); Van Brunt, J., Bio / Technology, 8: 291-294 (1990)).

In certain aspects, this aspect of the invention diagnoses a disease in a patient comprising assessing the expression level of a native gene encoding a polypeptide of the invention and comparing the expression level to a control level. Provide a way to do it. In this case, a level different from the control level indicates a disease. Said method may comprise the following steps:
a) contacting a patient-derived tissue sample with said nucleic acid probe under stringent conditions that allow formation of a hybrid complex between the nucleic acid molecule of the invention and the nucleic acid probe;
b) contacting a control sample with the probe under the same conditions as used in step a); and
c) detecting the presence of a hybrid complex in the sample;
In this case, detection of a hybrid complex level in the patient sample that is different from the hybrid complex level in the control sample is indicative of a disease.
Further features of the invention include diagnostic methods that include the following steps:
a) obtaining a tissue sample from a patient to be tested for disease;
b) isolating a nucleic acid molecule of the invention from the tissue sample; and
c) diagnosing the patient for the disease by detecting the presence of a mutation in the nucleic acid molecule associated with the disease.

To aid in the detection of nucleic acid molecules in the methods described above, the use of an amplification step, such as PCR, can be included.
Deletions and insertions are detected by a change in size in the amplification product as compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled RNA of the present invention or by hybridizing to labeled antisense DNA sequences of the present invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperature. DNA is contacted with a nucleic acid probe that hybridizes with the DNA under stringent conditions to form a hybrid double-stranded molecule (the hybrid double-stranded is in any part corresponding to the mutation associated with the disease). And detecting the presence or absence of a non-hybridized portion of the probe strand as indicating the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand. The presence or absence of mutations in the patient can be detected.
Such diagnoses are particularly useful in prenatal testing and are still useful in newborn testing.

Point mutations and other sequence differences between the reference gene and the “mutant” gene can be achieved by other well-known techniques such as direct DNA sequencing or single-stranded structural polymorphism (Orita et al., Genomics, 5: 874-879 (1989)). For example, sequencing primers can be used with double stranded PCR products or single stranded template molecules generated by modified PCR. Sequencing is performed by conventional methods using radiolabeled nucleotides or by automated sequencing methods using fluorescent tags. Cloned DNA segments can also be used as probes for detecting specific DNA segments.
The sensitivity of this method is greatly enhanced when combined with PCR. In addition, point mutations and other sequence variations (eg, polymorphisms) can be detected as described above, for example, by using allele-specific oligonucleotides for PCR amplification of sequences where only one nucleotide is different. .
DNA sequence differences may also be due to changes in the electrophoretic mobility of DNA fragments in the gel in the presence or absence of denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985) 230 : 1242). Sequence changes at specific positions can also be determined by nuclease protection assays such as RNase and S1 protection or by chemical cleavage methods (Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401. (See below).

Mutations such as microdeletions, aneuploidy, translocations, inversions can be detected by in situ analysis in addition to normal gel electrophoresis and DNA sequencing (see, eg, Keller et al , DNA Probes, 2nd Ed., Stockton Press, New York, NY, USA (1993)).
That is, intracellular DNA or RNA sequences can be analyzed for mutations without the need to isolate and / or immobilize them on the membrane. Fluorescence in situ hybridization (FISH) is the most commonly used method at present, and there are many reviews on FISH (see, eg, Trachuck et al., Science, 250, 559). -562 (1990); and Trask et al., Trends, Genet., 7, 149-154 (1991)).
In another aspect of the invention, an array of oligonucleotide probes comprising the nucleic acid molecules of the invention can be constructed to perform efficient screening for genetic variants, mutations and polymorphisms. Array technology methods are well known and have general applicability and can be used to address various questions in molecular genetics including gene expression, genetic linkage and genetic variability (eg See: M. Chee et al., Science (1996), Vol 274, pp610-613).

  In one embodiment, the array is prepared and used according to the methods described in the following literature (PCT application WO95 / 11995 (Chee et al.); DJ Lockhart et al. (1996) Nat. Biotech. 14: 1675-1680; M. Schena et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairs can range from two to over one million. The oligomer is synthesized in a specified region on the substrate using photoinduced chemistry. The substrate can be paper, nylon or other type of membrane, filter, chip, glass slide or other suitable solid support. In another aspect, oligonucleotides can be synthesized on a substrate surface by using chemical conjugation methods and ink jet application devices, as described in PCT patent application (WO95 / 251116, Baldeschweiler et al.). it can. In another aspect, a “gridded” array, similar to a dot (or slot) blot, is used to generate cDNA fragments on the substrate surface using vacuum systems, thermal bonding methods, UV bonding methods, mechanical or chemical bonding methods. Or it can be used to position and link oligonucleotides. Arrays as described above can be made manually or using available equipment (slot blot or dot blot equipment), materials (all suitable solid supports) and machines (including robotic equipment). , 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number ranging from 2 to over 1 million (this means that the array itself is commercially available) It is intended for effective use of available measuring instruments).

In addition to the methods discussed above, a disease can be diagnosed by a method that includes determining the level of an abnormal increase or decrease in polypeptide or mRNA from a sample derived from a subject. Decreased or increased expression is well known in the art for quantification of polynucleotides such as, for example, nucleic acid amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods, to name a few. Any of the methods can be used to measure at the RNA level.
Assay techniques that can be used to determine levels of a polypeptide of the invention in a sample derived from a host are well-known to those of skill in the art, and are discussed in some detail above (radioimmunoassays, competitive-binding assays). , Including Western blot analysis and ELISA assay). In this aspect of the invention, a diagnostic method is provided comprising the following steps: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex. And (b) detecting the complex.
Protocols for measuring polypeptide levels such as ELISA, RIA and FACS can further provide a basis for diagnosing altered or abnormal levels of polypeptide expression. A normal or standard value for polypeptide expression is obtained by mixing a bodily fluid or cell extract obtained from a normal mammalian subject (preferably human) with an antibody against said polypeptide under conditions suitable for complex formation. Established by The amount of standard complex formation can be quantified by various methods, for example, spectroscopic methods.

An antibody that specifically binds to the polypeptide of the present invention may be used for diagnosis of a condition or disease characterized by expression of said polypeptide, or using the polypeptide, nucleic acid molecule, ligand and other compounds of the present invention. It can be used in assays to monitor patients being treated. Antibodies useful for diagnostic purposes can be prepared in the same manner as described above as therapeutic agents. Diagnostic assays for the polypeptide include methods for detecting the polypeptide in human body fluids or cell or tissue extracts using the antibody and label. The antibodies can be used with or without modification and can be labeled by further covalently or non-covalently binding them to a reporter molecule. A variety of reporter molecules known in the art can be used, some of which are described above.
The amount of polypeptide expressed in the subject, control and disease sample from the biopsy tissue is compared to a standard value. Deviation between standard and subject values establishes parameters for disease diagnosis. Diagnostic assays can be used to identify the presence and excess of polypeptide expression and to monitor the regulation of polypeptide levels during therapeutic treatment. Such assays can also be used to assess the effectiveness of specific therapeutic treatment methods in animal experiments, clinical trials or individual patient therapeutic monitoring.

The diagnostic kit of the present invention may comprise:
(A) the nucleic acid molecule of the present invention;
(B) a polypeptide of the invention; or (c) a ligand of the invention.
In one aspect of the invention, a diagnostic kit includes a first container containing a nucleic acid probe that hybridizes under stringent conditions to a nucleic acid molecule of the invention; a second containing a primer useful for amplifying the nucleic acid molecule. And instructions for the use of the probes and primers to facilitate diagnosis of the disease. The kit may further include a third container holding a drug for digesting unhybridized RNA.
In another aspect of the invention, the diagnostic kit may include an array of nucleic acid molecules, and at least one of the nucleic acid molecules may be a nucleic acid molecule of the invention.
In order to detect the polypeptide of the present invention, the diagnostic kit is useful for detecting one or more antibodies that bind to the polypeptide of the present invention; and a binding reaction between the antibody and the polypeptide. Various reagents.

Such kits are suitable for diseases or susceptibility to diseases, especially immune diseases such as autoimmune diseases, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Guillain-Barre Syndrome, Graves' disease, autoimmune alopecia, scleroderma, psoriasis (Kimball et al., Arch Dermatol 2002 Oct: 138 (10): 1341-6), graft-versus-host disease (Miura Y., et al. , Blood 2002 Oct 1: 100 (7): 2650-8), monocyte and neutrophil dysfunction, attenuation of B cell function, inflammatory diseases such as acute inflammation, septic shock, asthma, anaphylaxis, eczema, Dermatitis, allergy, rhinitis, conjunctivitis, glomerulonephritis, uveitis, Sjogren's disease (Anaya et al., J Rheumatol 2002 Sep; 29 (9): 1874-6), Crohn's disease (Schmit A. et al., Eur Cytokine Netw 2002 Jul-Sep: 13 (3): 298-305), ulcerative colitis, inflammatory bowel disease, pancreatitis, digestive system inflammation, ulcerative colitis, Sepsis, endotoxic shock, septic shock, cachexia, myalgia, ankylosing spondylitis, myasthenia gravis, post-virus fatigue syndrome, lung disease, respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, airway Inflammation, wound healing, type I and type II diabetes, endometriosis, skin disease, Behcet's disease, immunodeficiency, chronic lung disease (Oei J et al., Acta Paediatr 2002: 91 (11): 1194-9 ), Invasive and chronic periodontitis (Gonzales JR, et al., J clin Periodontol 2002 Sep: 29 (9): 816-22), cancer such as carcinoma, sarcoma, lymphoma, renal tumor, colon tumor, Hodgkin Disease, melanoma such as metastatic melanoma (Vaishampayan U, Clin Cancer Res 2002 Dec: 8 (12): 3696-701), mesothelioma, Burkitt lymphoma, neuroblastoma, hematologic disease, nasopharyngeal carcinoma Leukemia, myeloma, myeloproliferative and other neoplastic diseases, osteoporosis, obesity, diabetes, gout, cardiovascular disease, reperfusion injury, atherodynamics Liver disease such as sclerosis, ischemic heart disease, heart failure, stroke, chronic hepatitis (Semin Liver Dis 2002: 22 Suppl 1: 7), AIDS (Dereuddre-Bosquet N., et al., J Acquir Immune Defic Syndr Hum Retroviol 1996 Mar 1: 11 (3): 241-6), AIDS-related syndromes, neurological diseases, fibrotic diseases, male infertility, aging, and infections such as plasmodium infection, bacterial infections, fungal diseases (ringworm, histoplasmosis, Blastomises, aspergillosis, cryptococcosis, sporotricosis, coccidioidomycosis, paracoccidioidomycosis, and candidiasis), diseases with antibacterial immunity (Bogdan, Current Opinion in Immunology 2000, 12: 419-424), Peyronie's disease ( Lacy et al., Int J Impot Res 2002 Oct: 14 (5): 336-9), tuberculosis (Dieli et al., J Infect Dis 2002 Dec 15; 186 (12): 1835-9) and viral infection (Pfeffer LM, Semin Oncol 1997 Jun 24: S9-63-69) or susceptibility to these It will be use.
Various features and aspects of the present invention will now be described in greater detail, particularly through examples relating to INSP037 polypeptides.
It will be understood that modification of detail may be made without departing from the scope of the invention.

(Example 1: Identification of INSP037)
The polypeptide sequence obtained from SEQ ID NO: 2 represents the translation of the exon of INSP037, and the polypeptide sequence was used as a query with the Informatica = Genome Threader tool for the protein structure present in the PDB database. The best match is the structure of a member of the 4-helix bundle cytokine family. The best match showed 84% genome threader confidence in alignment with the query sequence (FIG. 1). Figure 2 shows the INSP037 query sequence and the sequence of bovine interferon gamma, a member of the 4-helix bundle cytokine family (PDB-1d9g) (Randal et al., Acta Crystallogr D Biol Crystallogr 2000 (Jan; 56), (Pt 1 ): Indicates alignment with 14-24). Note that the INSP037 polypeptide sequence is designated “IPAAA445” in FIG. Members of the 4-helix bundle cytokine family protein are therapeutically important.
FIG. 16B shows that INSP037 can be found on chromosome 3 of Homo sapiens. As mentioned above, all type I interferons are clustered on chromosome 9. Therefore, the position of the INSP037 gene on chromosome 3 (3q25.33, chr3: 157121275-157121511 (on hg15 / build 33)) is consistent with the annotation in the present invention that it is an INFγ-like interferon, so II Annotated as type interferon.

(Example 2: Cloning of INSP037 (IPAAA44548) from cDNA library)
{CDNA library}
Human cDNA libraries (contained in bacteriophage lambda (λ) vectors) were purchased from Stratagene or Clontech or prepared in λZAP or λGT10 vectors at the Serono Pharmaceutical Research Institute according to the manufacturer's protocol (Stratagene). . Bacteriophage λ DNA was prepared from small scale cultures of infected E. coli host strains using the Wizard Lambda Preps DNA purification system according to the manufacturer's instructions (Promega, Corporation, Madison WI.). A list of libraries and host strains used is shown in Table I.

{Gene-specific cloning primers for PCR}
To amplify the full length sequence of the virtual cDNA, primer designer software (Scientific & Educational Software, PO Box 72045, Durham, NC 27722-2045, USA) was used, as shown in Table 2 below, 18- A PCR primer pair with a length of 25 bases was designed. PCR primers were optimized to have a Tm close to 55 ± 10 ° C. and a GC content of 40-60%. Primers with high selectivity for the target sequence IPAAA44548 (which show little or no non-specific priming) were selected.

{PCR of virtual cDNA derived from phage library DNA}
A full-length virtual cDNA encoding IPAAA44548 (FIG. 3) was obtained as a 264 bp PCR amplification product (FIG. 4) using gene specific cloning primers (CP1 and CP2, FIG. 3 and Table 2). PCR was performed in a final volume of 50 μL containing 1 × AmpliTaq® buffer, 200 μM dNTPs, 50 pmoles of cloning primers each, 2.5 units of AmpliTaq® (Perkin Elmer) and 100 ng of phage library pool DNA each. The MJ Research DNA Engine was programmed as follows: 94 ° C for 1 minute; 94 ° C for 1 minute, x ° C for y minute, and 72 ° C for 40 cycles (where x Is at least Tm-5 ° C, y is 1 minute per kb of product); followed by 1 minute cycle at 72 ° C followed by a hold cycle at 4 ° C.
Amplification products were visualized on a 0.8% agarose gel in 1 × TAE buffer (Life Technology) and PCR products migrated with the expected molecular weight were purified from the gel using the Wizard PCR Preps DNA purification system (Promega). PCR products eluted in 50 μL of sterile water were either directly subcloned or stored at 20 ° C.
{PCR product subcloning}
The PCR product was subcloned into the topoisomerase I modified cloning vector (pCRII TOPO) using the TOPO TA cloning kit purchased from Invitrogen Corporation using the conditions specified by the manufacturer (catalog number K4575-01, respectively). And K4600-01). Briefly, 4 μL of gel purified PCR product derived from human pituitary library (library number 3) amplification was incubated with 1 μL TOPO vector and 1 μL salt solution for 15 minutes at room temperature. Subsequently, E. coli strain TOP10 (Invitrogen) was transformed with the reaction mixture as follows. A 50 μL aliquot of one-shot TOP10 cells was thawed on ice before adding 2 μL of the TOPO reaction. The mixture was incubated on ice for 15 minutes followed by heat shock by incubation at 42 ° C. for exactly 30 seconds. Samples were returned to ice and 250 μL of warm SOC medium (room temperature) was added. Samples were incubated for 1 hour at 37 ° C. with shaking (220 rpm). Subsequently, the transformation mixture was plated on L-broth (LB) plates containing ampicillin (100 μg / mL) and incubated overnight at 37 ° C. Ampicillin resistant colonies containing the cDNA insert were identified by colony PCR.

{Colony PCR}
The colonies were inoculated into 50 μL of sterile water using sterile toothpicks. Subsequently, a 10 μL aliquot of the inoculum was PCRed in a total reaction volume of 20 μL as described above, except that the primer pair used was SP6 (5 ′) and T7. Cycling conditions were as follows: 94 ° C for 2 minutes; 94 ° C for 30 seconds, 47 ° C for 30 seconds and 72 ° C for 1 minute, 30 cycles; 72 ° C for 7 minutes 1 cycle. The sample was subsequently kept at 4 ° C. (holding cycle) before further analysis.
PCR reaction products were analyzed on 1% agarose gels in 1 × TAE buffer. Colonies showing the expected PCR product size (264 bp cDNA + 187 bp (due to multiple cloning site or MCS)) in 37 ml of L-broth (LB) containing ampicillin (50 μg / mL) at 220 rpm with shaking. Grown overnight.
{Preparation and sequencing of pramide DNA}
Miniprep plasmid DNA was prepared from 5 mL cultures using the Qiaprep Turbo9600 automated system (Qiagen) or Wizard Plus SV Miniprep kit (Promega Cat. # 1460) according to the manufacturer's instructions. Plasmid DNA was eluted in 100 μL of sterile water. The DNA concentration was measured using an Eppendorf BO spectrometer. Plasmid DNA (200-500 ng) was DNA sequenced with T7 and SP6 primers using the BigDye Terminator system (Applied Biosystems Cat. # 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. # LSKS09624) and subsequently analyzed on an Applied Biosystems 3700 sequencer.

(Example 3: Construction of plasmid for expression of INSP037 (IPAAA44548) in HEK293 / EBNA cells)
Subsequently, the pCRII-TOPO clone (Figure 5) containing the complete coding sequence (ORF) of IPAAA44548 identified by DNA sequencing was used to insert the insert by the Gateway® cloning method (Invitrogen). Was subcloned into the mammalian cell expression vector pEAK12d (FIG. 6). The cloned sequence contains only one nucleotide substitution A134G (FIG. 4).
{Generation of gateway compatible IPAAA44548 ORF fused to in-frame 6HIS tag sequence}
The first step of the gateway cloning process requires a two-step PCR reaction that flanks the attB1 recombination site and Kozak sequence at the 5 'end and encodes an in-frame 6 histidine (6HIS) tag at the 3' end. An ORF (gateway compatible cDNA) of IPAAA44548 in which a stop codon and attB2 recombination site flanks is generated. The first PCR reaction (final volume 50 μL) contains: 25 ng pCRII TOPO-IPAAA44548 (plasmid 13124, FIG. 5), 2 μL dNTP (5 mM), 5 μL 10 × Pfx polymerase buffer, 0.5 μL each. Gene-specific primers (100 μM) (EX1 forward and EX2 reverse) and 0.5 μL Platinum Pfx DNA polymerase (Invitrogen). The PCR reaction was performed in 12 cycles of 94 ° C for 15 seconds and 68 ° C for 30 seconds following an initial denaturation step of 2 minutes at 95 ° C. The PCR product was purified directly from the reaction mixture using the Wizard PCR prepDNA purification system (Promega) according to the manufacturer's instructions.
The second PCR reaction (final volume 50 μL) contained: 10 μL purified PCR product, 2 μL dNTP (5 mM), 5 μL 10 × Pfx polymerase buffer, 0.5 μL gateway conversion primer (100 μM each) ) (GCP forward and GCP reverse), and 0.5 μL Platinum Pfx DNA polymerase. The conditions for the second PCR reaction were as follows: 95 ° C for 1 minute; 94 ° C for 15 seconds, 45 ° C for 30 seconds and 68 ° C for 3.5 minutes; 4 cycles; and 94 ° C. 25 cycles of 15 seconds, 55 ° C for 30 seconds and 68 ° C for 3.5 minutes. The PCR product was purified as described above.
Alternatively, to express IPAAA44548 in E. coli, use the first PCR gene-specific primers (EX3-forward and EX2-reverse), and GCPF and GCPR primers, using the same conditions as above, and the methionine start codon An ORF containing a Shine-Dalgarno sequence upstream was prepared. The resulting PCR product was called SD-IPAAA44548.

{Subcloning of gateway compatible IPAAA44548 ORF into gateway entry vector pDONR201 and expression vector pEAK12d}
The second stage of the gateway cloning method involves subcloning of the gateway modified PCR product into the gateway entry vector pDONR201 (Invitrogen, FIG. 7). The subcloning is as follows: 5 μL of purified PCR product, along with 1.5 μL of pDONR201 vector (0.1 μg / μL), 2 μL of BP buffer and 1.5 μL of BP clonase enzyme mix (Invitrogen), Incubated for 1 hour at room temperature. The reaction was stopped by the addition of proteinase K (2 μg) and further incubated at 37 ° C. for 10 minutes. An aliquot (2 μL) of the reaction was transformed into E. coli DH10B cells by electroporation using a Biorad Gene Pulser. Transformants were plated on LB-kanamycin plates. Plasmid mini-prep DNA was prepared from 1-4 of the resulting colonies using Wizard Plus SV Minipreps kit (Promega), followed by 1.5 μL of the plasmid eluate in the recombination reaction. The recombination reaction contained 1.5 μL of pEAK12d vector (FIG. 6) (0.1 μg / μL), 2 μL of LR buffer and 1.5 μL of LR clonase (Invitrogen) in a final volume of 10 μL. The mixture was incubated for 1 hour at room temperature, the reaction was stopped by the addition of proteinase K (2 μg) and incubated for an additional 10 minutes at 37 ° C. An aliquot (1 μL) of this reaction was used to transform E. coli DH10B cells by electroporation.
Clones containing the correct insert were identified by performing colony PCR as described above, except that pEAK12d primers (pEAK12d F and pEAK12d R) were used for PCR. Plasmid mini prep DNA is isolated from clones containing the correct insert using the Qiaprep Turbo 9600 automation system (Qiagen) or manually with the Wizard Plus SV minipreps kit (Promega) and using pEAK12d F and pEAK12d R primers The sequence was confirmed.
CsCl gradient-purified maxi-prep DNA of plasmid pEAK12d-IPAAA44548-6His (plasmid no. 11775, FIG. 8) was prepared from a 500 mL culture of the sequence confirmed clone (J. Sambrook, et al., In Molecular Cloning, a Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press), resuspended in sterile water at a concentration of 1 μg / μL and stored at −20 ° C.

{Construction of expression vector pEAK12d}
Vector pEAK12d is a gateway cloning system compatible type of mammalian cell expression vector pEAK12 (purchased from Edge Biosystems). In pEAK12, the subject cDNA is expressed under the control of the human EF1α promoter. pEAK12d was prepared as follows.
pEAK12 was digested with restriction enzymes HindIII and NotI, blunted using Klenow (New England Biolabs), and dephosphorylated using calf intestinal alkaline phosphatase (Roche). After dephosphorylation, the vector is ligated to a blunt-ended gateway reading frame cassette C (gateway vector conversion system, Invitrogen cat # 11828-019) to allow the growth of E. coli DB3.1 cells (vectors containing the ccdB gene). ) Was transformed. The cassette contains an AttR recombination site flanking the ccdB gene and is resistant to chloramphenicol. With the cassette ligated with the vector, Mini prep DNA was isolated from several resistant colonies using the Wizard Plus SV Minipreps kit (Promega), digested with AseI / EcoRI, and the cassette was inserted in the correct orientation. A colony with a 670 bp fragment was identified, indicating that The resulting plasmid was named pEAK12d (FIG. 6).
{Subcloning of gateway compatible IPAAA44548 ORF into gateway entry vector pDONR201 and E. coli expression vector pDEST14}
A gateway-compatible SD-IPAAA44548 ORF containing an in-frame 3′6HIS tag coding sequence and a 5 ′ upstream Shine-Dalgarno sequence was subcloned into pDONR201 using BP clonase. Next, the obtained plasmid was used for the recombination reaction with the E. coli expression vector pDEST14 (purchased from Invitrogen, FIG. 9) using the above-mentioned LR clonase. The resulting expression plasmid (pDEST14-IPAAA44548-6HIS) (FIG. 10, plasmid 12896) was sequence verified as described above. E. coli host strain BL21 was retransformed with CsCl purified maxi-prep DNA for expression in E. coli. Expression of the inserted cDNA is under the control of the T7 promoter.

(Example 4: Identification of cDNA library containing IPAAA44548)
PCR products obtained using CP1 and CP2 and migrating at the correct size (264 bp) were identified in libraries 3, 8 and 12 (pituitary, brain cortex and fetal kidney, respectively).
(Example 5: Expression of cloned IPAAA44548-S-6HIS (plasmid no. 12118) in mammalian cells)
{Cell culture}
Human embryonic kidney 293 cells (HEK293-EBNA, Invitrogen) expressing Epstein-Barr virus nuclear antigen were maintained in suspension in Ex cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Cells were seeded in two T225 flasks 16-20 hours prior to transfection (Day-1) (2 × 10 ⁵ cells / d in DMEM / F12 (1: 1) with 2% FBS seeding medium (JRH). 50 mL per flask at a density of ml). The next day (transfection day 0), transfection was performed using JetPEI® reagent (plasmid DNA 2 μL / μg, PolyPlus-transfection). For each flask, 113 μg of plasmid number 12118 was cotransfected with 2.3 μg of GFP (fluorescent reporter gene). The transfection mixture was added to the two T225 flasks and incubated at 37 ° C. (5% CO ₂ ) for 6 days. Confirmation of positive transfection was performed by quantitative fluorescence tests on day 1 and 6 (Axiovert 10 Zeiss).

On day 6 (collection date), supernatants (100 mL) from the two flasks were pooled, centrifuged (4 ° C., 400 g) and placed in a pot with a unique identifier.
An aliquot (500 μL) was retained for QC of 6His tagged protein (internal bioprocessing QC).
Scaled-up batches were produced according to a protocol called “suspension cell PEI transfection” (see BP / PEI / HH / 02/04) using polyethylenimine (obtained from Polysciences) as transfection reagent .
This protocol was based on the following ratios:
For 400 mL spinner; 1 × 10 ⁶ HEK293EBNA cells in 200 mL FEME with 1% FBS;
400 μg of plasmid (No. 12118) was diluted in 10 mL of FEME 1%, and 800 μg of PEI was added. Ninety minutes after transfection, 1% FEME medium was added to bring the total volume to 400 mL. Spinners continued to be cultured for 6 days until collection.

{Purification method}
Using a cold buffer A (50 mM NaH ₂ PO ₄ ; 600 mM NaCl; 8.7% (w / v) glucerol; pH 7.5) using a 100 mL or 400 mL culture medium sample containing a recombinant protein having a C-terminal 6His tag And diluted to a final volume of 200 mL and 800 mL, respectively. The sample was filtered through a 0.22 μm sterile filter (Millipore, 500 mL filter unit) and maintained at 4 ° C. in a sterile culture square bottle (Nalgene).
Purification was performed at 4 ° C. on a VISION workstation (Applied Biosystems) connected to an automatic sample addition device (Labomatic). The purification method involved two successive steps: metal affinity chromatography on a Poros 20MC (Applied Biosystems) column (4.6 × 50 mm, 0.83 mL) charged with Ni ions, followed by Sephadex G- Gel filtration on a 25 medium (Amersham Pharmacia) column (1.0 x 10 cm).
For the first chromatography step, the metal affinity column was regenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 M NaCl; pH 8.0) and washed with 15 column volumes of 100 mM NiSO ₄ solution to remove Ni ions. Is recharged and washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (50 mM NaH ₂ PO ₄ ; 600 mM NaCl; 8.7% (w / v) glycerol; 400 mM imidazole; pH 7.5) and finally equilibrated with 15 column volumes of buffer A (containing 15 mM imidazole). Samples were transferred to a 200 mL sample loop with a Labomatic sample adder and subsequently loaded onto a Ni metal affinity column at a flow rate of 10 mL / min. In the case of a 400 mL scale-up sample, the above transfer and loading process was repeated four times. The column was then washed with 12 column volumes of buffer A followed by 28 column volumes of buffer A (containing 20 mM imidazole). During the 20 mM imidazole wash, contaminating proteins that had loosely adhered eluted from the column. Recombinant His-tagged protein was finally eluted with 10 column volumes of buffer B at a flow rate of 2 mL / min, and this eluted protein was collected in 1.6 mL fractions.

For the second chromatography step, a Sephadex G-25 gel filtration column was run with 2 mL of buffer D (1.137 M NaCl; 2.7 mM KCl; 1.5 mM KH ₂ PO ₄ ; 8 mM Na ₂ HPO ₄ ; pH 7.2) followed by 4 column volumes of buffer C (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH ₂ PO ₄ ; 8 mM Na ₂ HPO ₄ ; 20% (w / v) glycerol Equilibrated at pH 7.4). The peak fraction eluted from the Ni column automatically passes through the sample addition device integrated with VISION, loaded onto the Sephadex G-25 column, and the protein is eluted with Buffer C at a flow rate of 2 mL / min. did. The desalted sample was collected in 2.2 mL fractions. The fraction was filtered through a 0.22 μm sterile centrifuge filter (Millipore), frozen and stored at −80 ° C. Sample aliquots were analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) by Coomassie staining and Western blot using anti-His antibody.
Coomassie staining: NuPAGE gel was stained in 0.1% Coomassie Blue R250 staining solution (30% methanol, 10% acetic acid) for 1 hour at room temperature, followed by removal of the background so that the protein band is clearly visible Destained in 20% methanol / 7.5% acetic acid until
Western blot: Following electrophoresis, proteins were electrically transferred from the gel to a nitrocellulose membrane at 290 mA for 1 hour at 4 ° C. The membrane was brought to room temperature with buffer E (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH ₂ PO ₄ ; 8 mM Na ₂ HPO ₄ ; 0.1% Tween 20 (pH 7.4)) containing 5% milk powder. And then incubated overnight at 4 ° C. with two rabbit polyclonal antibody mixtures (G-18 and H-15, 0.2 μg / mL each; Santa Cruz) in buffer E containing 2.5% milk powder . After an additional 1 hour incubation at room temperature, the membrane was washed with buffer E (10 min, 3 times), followed by HRP-conjugated anti-rabbit secondary antibody diluted 1/3000 with buffer E containing 2.5% milk powder (DAKO, HRP0399) and incubated at room temperature for 2 hours. After washing with buffer E (10 minutes, 3 times), the membrane was treated with ECL kit (Amersham Pharmacia) for 1 minute. Subsequently, the membrane was exposed to hyperfilm (Amersham Pharmacia), the film was developed, and Western blot images were visually analyzed.
Protein assay: Protein concentration was determined using the BCA protein assay kit (Pierce) with bovine serum albumin as a standard in a sample showing a protein band detectable by Coomassie staining.

{Expression of IPAAA44548-SEC-6HIS (plasmid no. 12896) in bacterial cells}
The following method describes the use of the E. coli BL-21 DE3 bacterial strain to produce the protein. “BL21 DE3” is part of an expression system that uses T7 RNA polymerase, which is widely used to overexpress recombinant proteins.
{Transformation of bacterial strain BL21 (DE3)}
The TSS procedure was used. The protocol for the method was obtained from the following literature: CT Chug et al., Proc. Natl. Acad. Sci. USA (1989) 86: 2172-2175.
Recombinant plasmid No. 12896 DNA 10-100ng (2 μL) was added to competent BL21 for TSS method and left on ice for 20 minutes. SOC medium (0.8 mL) was added and the tube was incubated at 37 ° C., 200 rpm for 1 hour. 20 μL and 200 μL were collected from this culture, spread on LB plates containing ampicillin (final concentration 40 μg / mL), and allowed to stand at 37 ° C. overnight.
The next day, 3 colonies were isolated and used to prepare the glycerol stock and tested for expression in shake flask experiments before the transformation product was transferred into the fermenter (since the 3 colonies had the same performance in shake flask experiments). , One of the three colonies was selected for large scale).

{Preparation of seed stock for long-term storage of recombinant E. coli strain}
A 5 mL tube containing LB medium containing ampicillin (final concentration 40 μg / mL) was inoculated with a single colony from a fresh agar plate. Bacteria were grown overnight at 37 ° C. and 200 rpm. The next morning, 50 μL of the overnight culture was taken and inoculated into a new 5 mL LB tube (+ antibiotics) and incubated for 2-3 hours at 37 ° C. and 200 rpm to allow the bacteria to enter the exponential growth phase.
Subsequently, 5 mL of 20% glycerol was added to the culture and mixed. Dispensed 1.5 mL each into 5 cryogenic storage vials and stored at −80 ° C. as a seed stock (internal glycerol stock).
{Expression on a 5-liter scale}
The recombinant strain was grown in a 5 liter Biolafitte stirred tank reactor. During operation, the tank reactor is 5 liters of ECPM 1 medium (having the composition shown in Table IV) containing appropriate antibiotics (final concentration 40 μg / mL) and 0.5% glucose to avoid pre-induction of the T7 promoter. Was included. Only Research Grade Run 2464 was prepared and purified.
The inoculum is a 500 mL LB (+ antibiotic, 0.5% glucose) shake flask, starting with one loop of frozen bacteria (scratched from one of the glycerol seed stock vials) and grown for 9 hours prior to automatic inoculation. Prepared. When cells reached OD10 (usually after 7-9 hours of growth), IPTG (final concentration 1 mM) was used to induce protein production. Induction continued for 3 hours.
Fermenter setting conditions until growth and induction were set as follows: dissolved oxygen concentration 50%, 300 to 700 rpm, pH 7.0 depending on oxygen pressure. The oxygen pressure was maintained with air sparging +/- oxygen (25 mL / min). A 5 mL sample was taken every hour and the optical density was measured at 600 nm.
Cells were harvested and centrifuged at 4000 rpm (Sorvall RC 3B). The pellet was stored frozen at −20 ° C. until further processing.
The presence of protein in the cell extract was assessed by Coomassie staining on SDS-PAGE.

{Purification method}
270 mL Buffer A (50 mM NaH ₂ PO ₄ ; It was suspended in 8.7% (w / v) glycerol, pH 7.5). The bacteria were disrupted by passing twice through a Z-plus cell disruptor (Constant Cell Disruption Systems) at 1300 bar.
Subsequently, the sample was centrifuged at 36,000 × g for 30 minutes. The supernatant (300 mL) was loaded onto a Ni-NTA-agarose column (2.5 × 3.0 cm) equilibrated with buffer A at a flow rate of 4 mL / min.
The column was washed with 100 mL of buffer A followed by 85 mL of buffer A containing 20 mM imidazole. The protein was eluted with a 300 mL linear gradient of buffer A containing 20 mM to 250 mM imidazole at a flow rate of 3 mL / min, collecting 7.5 mL fractions. Samples of fractions per second were diluted 1/6 with reducing SDS-sample buffer, loaded with 15 μL per well in a 4-12% NuPage gel (Novex), and after electrophoresis the gel was Coomassie Blue Stained with
Fractions with the highest IPAAA44548 concentration (fractions 36-42) were pooled and their total volume was 53 mL (pool N1). The fractions on both sides of pool N1 with lower purity and concentration (fractions 32-35 and 43-44) were pooled as pool N2 with a volume of 44 mL.

The pool from the Ni-column was further purified on a Q-Sepharose fast flow column (1.5 × 12 cm) equilibrated with buffer B (50 mM Tris-HCl, 1 mM benzamidine, pH 7.5). 52 mL pool N1 was diluted with 300 mL Buffer B and 648 mL H ₂ O to give a final volume of 1000 mL. The sample was loaded onto the column at a flow rate of 5 mL / min, the column was washed with 150 mL of Buffer B, and the protein was eluted using a 160 mL linear gradient of Buffer B containing 0 mM-400 mM NaCl. Fractions were collected in 2 mL aliquots and analyzed by Coomassie-stained SDS-PAGE as described above. Fractions 28-30 (pool Q1) contained one protein band at the expected molecular weight position of 9.6 kDa. Fractions 31-33 (pool Q2) additionally contained a protein band at a position of about 20 kDa indicating dimer formation.
43 mL of pool N2 obtained from the Ni-column was diluted to 1000 mL with 300 mL Buffer B and 657 mL H ₂ O. The sample was loaded onto a Q-Sepharose column, the protein was eluted and the fractions analyzed as described for pool N1. Fraction 28-30 (pool Q3) contained one protein band at the expected molecular weight position of 9.6 kDa. The volume of each Q pool was 5.5 mL.
The pool obtained from the Q-Sepharose column was passed through a Superdex G75 gel filtration column (HiLoad16 / 60, Pharmacia). The column was washed with 0.5M NaOH and equilibrated with PBS. The column was flowed at a flow rate of 1 mL / min and a 5 mL pool was loaded onto the column. Fractions were collected in 2 mL aliquots and analyzed by Coomassie-stained SDS-PAGE as described above.

IPAAA44548 elutes from pool Q1 into fraction 31-35 (9.5 mL) (S1) and from pool Q2 has two peaks, fraction 31-34 (7.5 mL) (S2) and fraction 26-28 ( 5.8 mL) (S3) and eluted from pool Q3 into fractions 32-35 (7.5 mL) (S4). When analyzed by non-reducing SDS-PAGE, pool S3 was shown to contain more than 80% protein as dimers, while the other pools contained only trace amounts of dimer. Pools S1 and S2 had similar purity and concentration and were pooled into one pool S1b (9.5 + 7.5 = 17 mL).
The protein concentration was determined by measuring the absorption at 280 nm and using the calculated molar extinction coefficient of 7,090 and the molecular weight of 9,625. The molecular weight of the protein was determined by mass spectrometry and the molecular weight of the protein was found to be 9,624.6 in pools S1b and S4. The protein molecular weight in pool S3 was determined to be 19,252.2, confirming the presence of dimers with disulfide bridges in this pool. These pools were assayed for LPS and contained 1.1 U / mg to 3.4 U / mg.

(Example 6: Characterization of IPAAA44548 (INSP037) in vivo)
IPAAA44548 (INSP037) protein (IPAAA44548-6-HIS and IPAAA44548-ATT-6HIS) induces IFNγ secretion by human peripheral blood mononuclear cells (hPBMC) stimulated with concanavalin A (ConA) and phytohemagglutinin (PHA) in vitro (Preliminary data, data not shown). Based on these data, it was decided to test the activity of IPAAA44548 (INSP037) by electrotransfer in an in vivo ConA model, as described below.
{Hepatic concanavalin A (ConA) -induced hepatitis}
Addictive liver disease represents a worldwide health problem in mankind because no pharmacological therapy has yet been discovered. For example, acute chronic hepatitis that causes liver cirrhosis is a disease state in which liver parenchymal cells are gradually destroyed by activated T cells. ConA-induced hepatotoxicity is one of three experimental models of T cell-dependent apoptotic and necrotic liver injury in mice. Gal N (D-galactosamine) sensitized mice were exposed to either activated anti-CD3 monoclonal AB or superantigen SEB that showed severe apoptotic and secondary necrotic liver injury (Kusters S, Gastroenterology. 1996 Aug; 111 (2): 462-71). Injecting naive mice with ConA, a T cell mitogenic plant lectin, results in apoptosis of the liver and progresses to necrosis. ConA induces systemic release of TNFα, IFNγ and various other cytokines. Both TNFα and IFNγ are important mediators of liver injury. The injury causes severe liver destruction 8 hours after transaminase release.
Various types of cells have been shown to be involved in liver damage, including CD4 T cells, macrophages and natural killer cells (Kaneko J Exp Med 2000, 191, 105-114). Anti-CD4 antibodies block T cell activation and resulting liver damage (Tiegs et al. 1992, J Clin Invest 90, 196-203). Pretreatment of mice with a monoclonal antibody against CD8 failed to protect, whereas removal of macrophages prevented the induction of hepatitis.

A study was conducted to investigate the role of the IFNγ-like protein IPAAA44548 in liver ConA-induced hepatitis. Various cytokines have been shown to be important in either inducing ConA-induced liver damage or conferring protection from ConA-induced liver damage. For example, TNFα is one of the first cytokines produced after ConA injection, and anti-TNFα antibodies confer protection against disease (Seino et al. 2001, Annals of surgery 234, 681). IFNγ is also considered to be an important mediator of liver injury because anti-IFNγ antiserum significantly protects mice as measured by a decrease in transaminase levels in the blood of ConA-treated animals (see Kusters Et al.). In liver injury, increased IFNγ production was observed in patients with autoimmune or viral hepatitis. In addition, transgenic mice expressing IFNγ in the liver develop liver injury that mimics chronic acute hepatitis (Toyonaga et al. 1994, PNAS 91, 614-618). Since IFNγ causes cell death of mouse hepatocytes in vitro and this event was promoted by TNF, IFNγ may also be cytotoxic to hepatocytes (Morita et al. 1995, Hepatology 21, 1585- 1593).
Other molecules have been described as protective in the ConA model. A single dose of rhIL-6 completely inhibited transaminase release (Mizuhara et al. 1994, J. Exp. Med. 179, 1529-1537).
{Electrical introduction of cDNA into muscle fibers to achieve systemic expression of the protein of interest}
Among the non-viral techniques for in vivo gene transfer, direct injection of plasmid DNA into muscle and subsequent electroporation is simple, inexpensive and safe. Transduced DNA does not normally undergo chromosomal integration, but the post-mitotic properties and long life span of muscle fibers allow stable expression of the transduced gene (Somiari et al. 2000, Molecular Therapy 2,178 ). Various reports indicate that secretion of muscle-produced proteins into the bloodstream is achieved after electroporation of the corresponding cDNA (Rizzuto et al. PNAS, 1996, 6417; Aihara H et al ., 1998, Nature Biotech 16, 867). In addition, in vivo efficacy of muscle-expressed Epo and IL-18 BP has been shown in disease models (Rizzuto, 2000, Human Gene Therapy 41, 1891; Mallat, 2001, Circulation research 89, 41).
In this example, the following materials and methods were used.

{animal}
Female C57 / BL6 (8 weeks old) was used in all studies. In general, 7 animals were used per experimental group. Mice were maintained under normal conditions under a 12 hour light / dark cycle and were given free access to irradiated food and water.
{Muscle electrical introduction}
[Vector selection]
The hIL-6 gene or IPAAA44548 gene tagged with His or StrepII was cloned into gateway compatible CMV promoter-containing pDEST12.2.
[Electroporation protocol]
Mice were anesthetized with gas (isoflurane, Baxter, Ref: ZDG9623). The hind limbs were shaved and echographic gel was applied. Hyaluronidase was injected into the posterior tibial muscle (20 U dissolved in 50 μL sterile NaCl 0.9%, Sigma, Ref. H3631). Ten minutes later, 100 μg of plasmid (50 μg per limb dissolved in 25 L of sterile NaCl 0.9%) was injected into the same muscle. DNA was prepared in PBS-L-glutamate buffer prior to intramuscular injection. For electrical introduction, using an ElectroSquarePorator (BTX, ref ECM830), one pulse is applied to one foot at 75 volts for one second at an interval of 1 second. Ten pulses were subjected to an electric field in a unipolar manner with two circular electrodes (size: 0.5 mm diameter) (Mir LM et al, Proc Natl Acad Sci US A. 1999 Apr 13; 96 (8): 4262 -7 and Haas K et al., Neuron. 2001 Mar; 29 (3): 583-91.

{reading}
[Blood sampling]
1. At different time points of 30 hours, 6 hours and 8 hours, 100 μL of blood was collected from the eye. At the time of slaughter, blood was drawn from the heart.
[Detection of cytokines and transaminases in blood samples]
The TH1 / TH2 CBA assay (BD 551287) was used to measure cytokine levels of IL-2, IL-5, IL-4, TNFα and IFNγ. Blood parameters of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and urea were determined using a COBAS device (Hitachi).
{ConA guidance}
Female C57 / BL6 mice (obtained from IFFA CREDO), 8 week old animals; ConA (purchased from Sigma, ref. C7275). ConA was injected iv at different concentrations (time 0) and blood samples were collected at 1.30 hours, 6 hours or 8 hours after injection. Cytokine, ASAT, and ALAT were measured as described above.
[IL-6 pretreatment with ConA model]
One hour before ConA injection, CHO cells producing hIL-6 were injected.
[Electric introduction of IPAAA44548 and IL-6]
On day 0, IPAAA44548 vector or hIL-6 vector, empty vector (negative control) was electrically introduced (according to the above protocol). On day 5 after electrical introduction, ConA (20 mg / kg) was injected iv and blood was collected at three time points (1.30 hours, 6 hours, 24 hours). Cytokine, ASAT, and ALAT were measured as described above.

{result}
ConA-induced fulminant hepatitis model mice treated with IPAAA44548 using such cDNA electrotransfer showed increased levels of TNFα, IL-2 and IFNγ in blood in vivo (FIGS. 17A- See C). In addition, ASAT and ALAT levels were also increased relative to controls (see FIGS. 17D-E).
The results in FIGS. 18A-F represent a positive control comparative example (rhIL-6 is known to block the pro-inflammatory response induced by ConA). In order to express hIL-6 in blood and subsequently demonstrate protection from ConA-induced liver toxicity, either pDEST12.2hIL-6-STREPII or pDEST12.2 STREPII electrotransfer vector was used.
These examples show that expression of IPAAA44548 protein in serum using electrotransduction increases pro-inflammatory cytokine levels at the systemic level after ConA exposure and is measured by increased transaminase levels. It has been shown to exacerbate liver disease.
These results confirm the predicted IFNγ-like activity of IPAAA44548 and pioneer a series of interesting therapeutic uses for the protein itself. For example, known uses of IFNγ can be readily investigated for compatibility with IPAAA44548 (eg, anticancer activity). It would also be possible to readily identify inhibitors or antagonists of IPAAA44548 that may be beneficial in further studies of IPAAA44548 activity in vivo or clinical applications, such as monoclonal antibodies.

(INSP037 sequence information)
SEQ ID NO: 1 (nucleotide sequence of INSP037)
1 ATGACTTCAC CAAACGAACT AAATAAGCTG CCATGGACCA ATCCTGGAGA
51 AACAGAGATA TGTGACCTTT CAGACACAGA ATTCAAAATA TCTGTGTTGA
101 AGAACCTCAA AGAAATTCAA GATAACACAG AGAAGGAATC CAGAATTCTA
151 TCAGACAAAT ATAAGAAACA GATTGAAATA ATTAAAGGGA ATCAAGCAGA
201 AATTCTGGAG TTGAGAAATG CAGATGGCAC ACTTTAG

SEQ ID NO: 2 (protein sequence of INSP037)
1 MTSPNELNKL PWTNPGETEI CDLSDTEFKI SVLKNLKEIQ DNTEKESRIL
51 SDKYKKQIEI IKGNQAEILE LRNADGTL

The result of the Inpharmatica genome threader search of sequence number 2 is shown. The alignment obtained by the Inpharmatica genome threader between SEQ ID NO: 2 and the nearest neighbor structure is shown. The predicted nucleotide sequence of INSP037 (including SEQ ID NO: 1) is shown along with its translation (SEQ ID NO: 2). The cloned nucleotide sequence of INSP037 (including SEQ ID NO: 1) and its translation (SEQ ID NO: 2) indicate that the sequence predicted for INSP037 is identical to the cloned sequence. The map of PCRII-TOPO-IPAAA44548 is shown. The map of expression vector pEAK12d is shown. The map of plasmid pDONR201 is shown. The map of expression vector pEAK12d-IPAAA44548-6HIS is shown. The map of E. coli expression vector pDEST14 is shown. The map of plasmid pDEST14-IPAAA44548-6HIS is shown. This shows the nucleotide sequence of PCRII-TOPO-IPAAA44548. The nucleotide sequence of pDEST14-IPAAA44548-6HIS is shown. This shows the nucleotide sequence of pEAK12D-IPAAA44548-6HIS. NCBI-NR results for the INSP037 polypeptide (SEQ ID NO: 2) indicate that there is no 100% match, thus indicating that INSP037 is novel. NCBI-month-aa results for the INSP037 polypeptide (SEQ ID NO: 2) show no 100% matching, thus demonstrating that INSP037 is novel. NCBI-month-nt, a database of translated nucleotides for the INSP037 polypeptide (SEQ ID NO: 2), shows no 100% matching, thus indicating that INSP037 is novel. NCBI-nt results for the INSP037 polypeptide (SEQ ID NO: 2) indicate that there is no 100% match, thus indicating that INSP037 is novel. Figure 3 shows the results of an experiment of INSP037 activity in a mouse model of ConA-induced fulminant hepatitis. A positive control showing IL-6 effect is shown against a mouse model of ConA-induced fulminant hepatitis.

Claims (42)

The polypeptide of any of the following (i) to (iii):
(i) a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;
(ii) a polypeptide that is an interferon gamma-like secreted protein of a 4-helix bundle cytokine fold, or a fragment of a polypeptide of (i) that has an antigenic determinant in common with the polypeptide of (i); or
(iii) A polypeptide which is a functional equivalent of (i) or (ii).   The polypeptide according to claim 1, which functions as an interferon γ-like secreted protein of a 4-helix bundle cytokine fold.   A polypeptide which is a functional equivalent of (iii) of claim 1 which is homologous to the amino acid sequence of SEQ ID NO: 2 and is an interferon γ-like secreted protein of a 4-helix bundle cytokine fold.   The amino acid sequence set forth in SEQ ID NO: 2 or an active fragment thereof, having more than 80% sequence identity, preferably more than 90%, 95%, 98% or 99% sequence identity. A fragment or functional equivalent according to any one of the above.   The functional equivalent according to any one of claims 1 to 4, which exhibits a remarkable structural homology with the polypeptide having the amino acid sequence of SEQ ID NO: 2.   The polypeptide of (i) of claim 1, comprising 7 or more (eg, 8, 10, 12, 14, 16, 18, 20 or more) amino acid residues derived from the sequence of SEQ ID NO: 2 The fragment according to any one of claims 1, 2, and 4, which has a common antigenic determinant.   A purified nucleic acid molecule encoding the polypeptide of any one of claims 1-6.   8. The purified nucleic acid molecule of claim 7, having the nucleic acid sequence set forth in SEQ ID NO: 1 or being a surplus equivalent or fragment of the nucleic acid sequence set forth in SEQ ID NO: 1.   9. A purified nucleic acid molecule that hybridizes under high stringency conditions with the nucleic acid molecule of claim 7 or 8.   A vector comprising the nucleic acid molecule according to any one of claims 7 to 9.   A host cell transformed with the vector of claim 11.   A ligand that specifically binds to the polypeptide of any one of claims 1 to 6 and preferably inhibits the interferon γ-like activity of the peptide.   The ligand according to claim 12, which is an antibody.   A compound that increases or decreases the level of expression or activity of the polypeptide of any one of claims 1-6.   15. A compound according to claim 14, which binds to the polypeptide of any one of claims 1 to 6 without inducing any of the biological effects of the polypeptide.   16. A compound according to claim 14 or 15 which is a natural or modified substrate, ligand, enzyme, receptor or structural or functional mimetic.   The polypeptide according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 9, and the vector according to claim 10 for use in the treatment or diagnosis of a disease. A host cell according to claim 11, a ligand according to claim 12 or 13, or a compound according to any one of claims 14-16.   The level of expression of a natural gene encoding the polypeptide according to any one of claims 1 to 6 in a tissue derived from a patient, or the poly according to any one of claims 1 to 6. Assessing the activity of a peptide and comparing the level of expression or activity to a control level, wherein the level is different from the control level is indicative of the disease Method.   19. The method of claim 18, wherein the method is performed in vitro. (A) contacting the ligand of claim 12 or 13 with a biological sample under conditions suitable for formation of a ligand-polypeptide complex; and (b) detecting the complex;
20. A method according to claim 18 or 19, comprising: (A) a patient-derived tissue sample under stringent conditions allowing the formation of a hybrid complex between the nucleic acid probe, the nucleic acid molecule of any one of claims 7-9 and the probe; Contacting with;
(B) contacting a control sample with the probe under the same conditions used in step (a); and (c) detecting the presence of a hybrid complex in the sample; 20. A method according to claim 18 or 19, wherein detection of a hybrid complex level in a patient sample that is different from a hybrid complex level in a control sample is indicative of a disease. (A) a stringent condition that allows a nucleic acid sample from a patient tissue to form a hybrid complex between a nucleic acid primer and the nucleic acid molecule of any one of claims 7 to 9 and the primer; And contacting with;
(B) contacting a control sample with the primer under the same conditions used in step (a);
(C) amplifying the nucleic acid of the sample; and
(D) detecting the level of amplified nucleic acid from both the patient sample and the control sample;
20. A method according to claim 18 or 19, wherein the detection of the level of amplified nucleic acid in the patient sample significantly different from the level of amplified nucleic acid in the control sample is indicative of a disease. (A) obtaining a tissue sample from a patient to be examined for disease;
(B) isolating the nucleic acid molecule according to any one of claims 7 to 9 from the tissue sample; and
(C) diagnosing a patient for a disease by detecting the presence of a disease-related mutation in the nucleic acid molecule as an indicator of the disease;
20. A method according to claim 18 or 19, comprising:   24. The method of claim 23, further comprising amplifying the nucleic acid molecule to produce an amplification product and detecting the presence or absence of a mutation in the amplification product. A nucleic acid molecule is brought into contact with a nucleic acid probe that hybridizes to the nucleic acid molecule under a stringent condition, and an unhybridized portion of the nucleic acid probe strand at an arbitrary portion corresponding to a disease-related mutation is obtained. Forming a hybrid double-stranded molecule having; and
Detecting the presence or absence of a non-hybridized portion of the probe chain as an indicator of the presence or absence of a disease-related mutation;
The method according to claim 23 or 24, wherein the presence or absence of a mutation in the patient is detected by the method.   The disease is an immune disease such as autoimmune disease, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, Graves' disease, autoimmune alopecia, Scleroderma, psoriasis, graft-versus-host disease, monocyte and neutrophil dysfunction, attenuation of B cell function, inflammatory diseases such as acute inflammation, septic shock, asthma, anaphylaxis, eczema, dermatitis, allergy , Rhinitis, conjunctivitis, glomerulonephritis, uveitis, Sjogren's disease, Crohn's disease, ulcerative colitis, inflammatory bowel disease, pancreatitis, gastrointestinal inflammation, ulcerative colitis, sepsis, endotoxic shock, septic Shock, cachexia, myalgia, ankylosing spondylitis, myasthenia gravis, post-virus fatigue syndrome, lung disease, respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, airway inflammation, wound healing, type I and II Type of diabetes Disease, endometriosis, skin disease, Behcet's disease, immunodeficiency, chronic lung disease, invasive and chronic periodontitis, cancer such as carcinoma, sarcoma, lymphoma, renal tumor, colon tumor, Hodgkin's disease, metastatic Melanoma such as melanoma, mesothelioma, Burkitt lymphoma, neuroblastoma, blood disease, nasopharyngeal cancer, leukemia, myeloma, myeloproliferative disease and other neoplastic diseases, osteoporosis, obesity, diabetes, Gout, cardiovascular disease, reperfusion injury, atherosclerosis, ischemic heart disease, heart failure, stroke, liver disease such as chronic hepatitis, AIDS, AIDS related syndrome, neurological disease, fibrotic disease, male infertility, addiction Age, as well as infectious diseases such as plasmodium infection, bacterial infection, fungal disease (ringworm, histoplasmosis, blastomysosis, aspergillosis, cryptococcosis, sporotricosis, coccidioidomycosis, pa Such as coccidioidomycosis and candidiasis), diseases involving antibacterial immunity, is selected from tuberculosis and viral infections, the method according to any one of claims 18 to 25.   Use of the polypeptide according to any one of claims 1 to 6 as an interferon γ-like secreted protein of a 4-helix bundle cytokine fold.   A polypeptide according to any one of claims 1 to 6, a nucleic acid molecule according to any one of claims 7 to 9, a vector according to claim 10, a host cell according to claim 11, a claim 12 Alternatively, a pharmaceutical composition comprising the ligand according to claim 13 or the compound according to any one of claims 14 to 16.   A vaccine composition comprising the polypeptide according to any one of claims 1 to 6 or the nucleic acid molecule according to any one of claims 7 to 9.   Immune diseases such as autoimmune diseases, rheumatoid arthritis, osteoarthritis, psoriasis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, Graves' disease, autoimmune alopecia, scleroderma Psoriasis, graft-versus-host disease, monocyte and neutrophil dysfunction, attenuation of B cell function, inflammatory diseases such as acute inflammation, septic shock, asthma, anaphylaxis, eczema, dermatitis, allergy, rhinitis, Conjunctivitis, glomerulonephritis, uveitis, Sjogren's disease, Crohn's disease, ulcerative colitis, inflammatory bowel disease, pancreatitis, gastrointestinal inflammation, ulcerative colitis, sepsis, endotoxic shock, septic shock, evil Fluid, myalgia, ankylosing spondylitis, myasthenia gravis, post-virus fatigue syndrome, lung disease, respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, airway inflammation, wound healing, type I and type II diabetes The uterus Endometriosis, skin disease, Behcet's disease, immunodeficiency, chronic lung disease, invasive and chronic periodontitis, cancer such as carcinoma, sarcoma, lymphoma, renal tumor, colon tumor, Hodgkin's disease, metastatic melanoma, etc. Melanoma, mesothelioma, Burkitt lymphoma, neuroblastoma, hematological disease, nasopharyngeal carcinoma, leukemia, myeloma, myeloproliferative disease and other neoplastic diseases, osteoporosis, obesity, diabetes, gout, heart Vascular disease, reperfusion injury, atherosclerosis, ischemic heart disease, heart failure, stroke, liver disease such as chronic hepatitis, AIDS, AIDS related syndrome, neurological disease, fibrotic disease, male infertility, aging, and Infectious diseases such as plasmodium infection, bacterial infections, fungal diseases (ringworm, histoplasmosis, blastosomiasis, aspergillosis, cryptococcosis, sporotricosis, coccidioidomycosis, paracoccosis A polypeptide according to any one of claims 1 to 6 for use in the manufacture of a medicament for the treatment of diseases selected from eudessis and candidiasis, diseases with antibacterial immunity, tuberculosis and viral infections. The nucleic acid molecule according to any one of claims 7 to 9, the vector according to claim 10, the host cell according to claim 11, the ligand according to claim 12 or 13, and any one of claims 14 to 16. 29. A compound according to claim 28 or a pharmaceutical composition according to claim 28.   A polypeptide according to any one of claims 1 to 6, a nucleic acid molecule according to any one of claims 7 to 9, a vector according to claim 10, a host cell according to claim 11, a claim 12 Alternatively, a method of treating a disease in a patient comprising administering to the patient a ligand according to claim 13, a compound according to any one of claims 14 to 16, or a pharmaceutical composition according to claim 28.   Polypeptides and nucleic acids administered to a patient against a disease whose natural gene expression or polypeptide activity in a patient suffering from the disease is low when compared to the level of expression or activity in a healthy subject 32. The method of claim 31, wherein the molecule, vector, ligand, compound or composition is an agonist.   Polypeptides and nucleic acids administered to a patient against a disease where the expression of the natural gene or the activity of the polypeptide in the patient suffering from the disease is high when compared to the level of expression or activity in a healthy subject 32. The method of claim 31, wherein the molecule, vector, ligand, compound or composition is an antagonist. The level of expression or activity of the polypeptide according to any one of claims 1 to 6 or the level of expression of a nucleic acid molecule according to any one of claims 7 to 9 in a tissue derived from a patient for a certain period of time. A method of monitoring treatment of a disease in a patient comprising monitoring over
The method wherein the level of expression or activity over the period changes towards a control level is an indication of remission of the disease. The polypeptide according to any one of claims 1 to 6 or the nucleic acid molecule according to any one of claims 7 to 9 is suspected of having binding affinity for the polypeptide or the nucleic acid molecule. Contacting with one or more compounds; and
Selecting a compound that specifically binds to the nucleic acid molecule or the polypeptide; and a method for identifying a compound that is effective in treating and / or diagnosing a disease.   A first container containing a nucleic acid probe that hybridizes with the nucleic acid molecule of any one of claims 7 to 9 under stringent conditions; a second container containing a primer useful for amplification of the nucleic acid molecule; And a kit useful for diagnosis of a disease, comprising instructions for using the probes and primers to facilitate diagnosis of the disease.   37. The kit of claim 36, further comprising a third container holding an agent for digesting non-hybridized RNA.   A kit comprising an array of nucleic acid molecules, wherein at least one of the nucleic acid molecules is a nucleic acid molecule according to any one of claims 7-9.   A kit comprising one or more antibodies that bind to the polypeptide according to any one of claims 1 to 6, and a reagent useful for detecting a binding reaction between the antibody and the polypeptide.   A transgenic or knockout non-human animal that has been transformed to express or not express the polypeptide of any one of claims 1-6 at a high level, a low level, or not.   41. A method of screening for compounds effective to treat a disease comprising contacting the transgenic non-human animal of claim 40 with a candidate compound; and determining the effect of the candidate compound on the animal's disease.   42. The method according to any one of claims 31 to 37 or 41, wherein the disease is any one of the diseases of claim 30.

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